Details of a Researcher - Soga, Tomoyoshi

Papers - Soga, Tomoyoshi

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Elevated glucose metabolism via the hexosamine biosynthesis pathway: A metabolic signature of high-fluorodeoxyglucose-uptake lung adenocarcinoma

Watanabe, H., Makinoshima, H., Kanauchi, N., Kabasawa, T., Suzuki, J., Takamori, S., Sasage, T., Abe, K., Hoshijima, K., Uchida, T., Soga, T., Shiono, S.

Surg. Today 2026.03

Joint Work, Accepted

Purpose
To evaluate hexosamine biosynthetic pathway (HBP) involvement in high-fluorodeoxyglucose (FDG)-uptake lung adenocarcinoma.

Methods
We conducted metabolomic analysis to evaluate the HBP in patients with lung adenocarcinoma, who underwent preoperative 18-FDG positron emission tomography. Capillary electrophoresis-time-of-flight mass spectrometry was done to obtain 511 small-molecule metabolite spectra, and a principal component analysis was performed.

Results
We examined 80 tissue samples: 40 tumor-adjacent non-tumor tissue samples and 40 resected lung adenocarcinomas. The principal component analysis confirmed good clustering between the tumor and non-tumor tissues. The non-tumor tissues comprised uniform materials, whereas the tumor tissues comprised a mixture of materials. Heatmaps for 50 metabolites revealed lower glucose and citrate levels and higher levels of lactate, glycolysis metabolites, succinate, fumarate, adenosine di- and -monophosphate, and all essential amino acids in the tumor tissues than the non-tumor tissues. HBP intermediate and uridine diphosphate N-acetylglucosamine levels were also higher in the tumor tissues. Both lactate and HBP intermediate levels were higher in hypermetabolic tumor tissues (standardized uptake value ≥ 3) than in non-hypermetabolic tumor tissues (standardized uptake value < 3). Low-FDG-uptake cells showed strong expression for glucose transporter SLC2A1 and weak expression for O-linked N-acetylglucosamine, whereas high-FDG-uptake cells showed strong expression for both markers.

Conclusions
Hypermetabolic adenocarcinoma may be associated with intensified glycolysis and HBP activation.

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Global loss of metabolic responsiveness and elevated enzyme in leptin deficient obese mice during starvation

Li, D., Morita, K., Kokaji, T., Hatano, A., Hirayama, A., Soga, T., Suzuki, Y., Matsumoto, M., Tsuchiya, T., Ozaki, H., Ohno, S., Inoue, H., Inaba, Y., Sugimoto, H., Pan, Y., Kuroda, S.

npj Syst. Biol. Appl. online 2026.03

Research paper (scientific journal), Joint Work, Accepted

Starvation induces complex metabolic adaptations in skeletal muscle, a key tissue for maintaining energy homeostasis; however, these adaptations are largely impaired in obesity. How obesity alters global metabolic adaptations to starvation in skeletal muscle remains unclear. Here, we analyzed the metabolic adaptations on a trans-omics scale during starvation in skeletal muscle from wild-type (WT) and leptin-deficient obese (ob/ob) mice. We measured multi-omics data during starvation and constructed global trans-omics networks in WT and ob/ob mice. We found that starvation induces “responsiveness” in WT mice, characterized by increases or decreases in key regulator metabolites, including ATP and AMP, as well as enzyme proteins, leading to global regulation of metabolic pathways, which was lost in ob/ob mice. In contrast, during starvation, ob/ob mice exhibit “difference” in comparison to WT mice, manifested by the persistently elevated expression of metabolic enzymes. These features were similarly found in liver, another key metabolic organ. Thus, global loss of responsiveness and elevated enzyme proteins are systemic features of metabolic dysregulation in ob/ob mice.

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Guanine nucleotides drive ribosome biogenesis and glycolytic reprogramming in acute myeloid leukemia stem cells

Kawano, G., Ikeda, R., Ishihara, D., Shima, T., Sakoda, T., Yamamoto, S., Kochi, Y., Semba, Y., Ashitani, S., Mori, Y., Kato K., Maeda, T., Miyamoto, T., Soga, T., Akashi, K., Kikushige, Y.

Blood 147 ( 7 ) 768 - 782 2026.02

Research paper (scientific journal), Joint Work, Accepted

Therapy resistance in acute myeloid leukemia (AML) remains a major clinical obstacle, particularly due to the persistence of leukemia stem cells (LSCs) capable of metabolic adaptation. While venetoclax (Ven) inhibits oxidative phosphorylation (OXPHOS), we found that Ven-resistant LSCs undergo glycolytic reprogramming to bypass OXPHOS inhibition. This metabolic shift is supported by enhanced ribosome biogenesis, sustained by upregulated de novo guanine nucleotide biosynthesis. Abundant guanine nucleotides suppress the impaired ribosome biogenesis checkpoint (IRBC), leading to TP53 destabilization and persistent MYC expression. Inhibition of inosine monophosphate dehydrogenases (IMPDH1/2) depletes guanine nucleotides, activates IRBC, stabilizes TP53, represses MYC, and impairs the metabolic shift to glycolysis. This metabolic rewiring disrupts LSC stemness and suppresses the reconstitution of human AML cells in xenotransplantation experiments. Notably, the suppression of LSC stemness was observed regardless of Ven resistance or the TP53 mutational status of AML cells. These findings reveal that mutation-independent TP53 inactivation is involved in resistant AML and suggest that targeting guanine nucleotide biosynthesis may offer a clinically actionable strategy to eradicate therapy-resistant LSCs.

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Cross-sectional associations between CES-D scores and the salivary metabolome in nonclinical adults

Kubo, Y., Yamauchi, F., Goshima, N., Ohishi, M., Hasegawa, T., Soga, T., Ikeda, M., Aoki, K.

Discov. Ment. Heatlh 6 ( 1 ) 15 - 15 2026.01

Research paper (scientific journal), Joint Work, Accepted

Background: Depression represents a major global disease burden. While the Center for Epidemiologic Studies Depression Scale (CES-D) is widely used, it relies on subjective self-reporting. Saliva enables non-invasive metabolite analysis, but existing metabolomics studies have focused on clinical depression using blood or urine, with limited investigation of salivary metabolites in community populations. This study investigated associations between CES-D scores and salivary metabolite concentrations in Tokyo residents.

Methods: This cross-sectional study collected CES-D responses and morning saliva samples from Tokyo residents. Salivary metabolites were analyzed using capillary electrophoresis time-of-flight mass spectrometry. Statistical analyses included permutational multivariate analysis of variance (PERMANOVA) for overall metabolite-CES-D associations, pathway enrichment analysis using fast gene set enrichment analysis (FGSEA), and principal component analysis, adjusted for age and sex.

Results: Analysis included 107 participants (40 males, 67 females; mean age 55.3 ± 14.2 years). PERMANOVA revealed significant association between 112 salivary metabolites and CES-D scores (F = 2.870, p = 0.003), with metabolites explaining 7.7% of depressive symptom variance. Pathway enrichment analysis identified significant alterations in amino acid metabolism (normalized enrichment score =-1.750, q = 0.007). Glycolysis and amine/polyamine metabolism showed trend-level enrichment (both q = 0.066). Principal component analysis revealed significant correlations between CES-D scores and pathway activity for amino acid metabolism (r = 0.216, p = 0.027) and amine/polyamine metabolism (r = 0.216, p = 0.027).

Conclusion: This study demonstrates significant associations between salivary metabolite profiles and depressive symptoms in a nonclinical community population, with amino acid metabolism emerging as the most robustly altered pathway. These findings provide preliminary evidence for associations between salivary metabolomic profiles and depressive symptoms in a nonclinical community population.

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Abrogation of aberrant glycolytic interactions eliminates senescent cells and alleviates aging-related dysfunctions

Mikawa, T., Kameda, M., Ikari, S., Shibata, E., Liu, S., Miyagawa, S., Ono, K., Ito, T., Yoshizawa, A., Sugimoto, M., Shibuya, S., Shimizu, T., Almunia, J., Ogiso, N., Revêchon, G., Palazzo, A., Bernard, D., Kanda, H., Soga, T., Takubo, K., Morioka, S., Sasaki, J., Sasaki, T., Itamoto, A., Fujii, T., Seno, H., Inagaki, N., Kondoh, H.

Signal Transduct. Target. Ther. 10 ( 1 ) 402 - 402 2025.12

Research paper (scientific journal), Joint Work, Accepted, ISSN 20959907

Cellular senescence is deeply involved in physiological homeostasis, development, tissue repair, aging, and diseases. Senescent cells (SnCs) accumulate in aged tissues and exert deleterious effects by secreting proinflammatory molecules that contribute to chronic inflammation and aging-related diseases. We revealed that an aberrant interaction between glycolytic PGAM1 and Chk1 kinase is augmented in SnCs associated with increased glycolysis, whose byproduct, lactate, promotes this binding in a noncell autonomous manner. The pseudo-Warburg effect of SnCs with enhanced PPP (pentose phosphate pathway) activity is maintained by HIF-2α phosphorylation by Chk1 and subsequent upregulation of glycolytic enzymes, creating a vicious cycle reprogramming the glycolytic pathway in SnCs. HIF-2α also activates FoxM1 expression, which transcriptionally suppresses proapoptotic profiles, including BIM, and upregulates DNA repair machineries in SnCs. FoxM1 thus supports the genomic integrity and survival capacity of SnCs during their glycolytic changes. Chemical abrogation of PGAM1-Chk1 binding reverts these phenotypes and eliminates SnCs through senolysis. Inhibition of the PGAM1-Chk1 interaction improves physiological parameters during aging and inhibits lung fibrosis in mouse models. Our study highlights a novel pathway contributing to the metabolic reprogramming of SnCs and how the use of a new senolytic molecule that targets the PGAM-Chk1 interaction creates a specific vulnerability of those cells to potentially fight age-related diseases.

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Longitudinal study of plasma metabolites during menopause and their associations with later onset of metabolic syndrome

Miyake, A., Iida, M., Harada, S., Sugiyama, D., Matsumoto, M., Miyagawa, N., Toki, R., Edagawa, S., Hirata, A., Kuwabara, K., Okamura, T., Sato, A., Amano, K., Hirayama, A., Sugimoto, M., Soga, T., Tomita, M., Arakawa, K., Kisu, I., Yamagami, W., Takebayashi, T.

J. Clin. Endocrinol. Metab. online dgaf666 2025.12

Research paper (scientific journal), Joint Work, Accepted

Context: Previous metabolomics studies suggest potential associations between menopausal changes in lipids and an increased risk of metabolic syndrome (MetS). However, longitudinal data on other key metabolites, such as branched-chain amino acids and homocysteine, remain limited, and most studies lack long-term follow-up across the menopause transition.

Objective: This study aimed to investigate longitudinal changes in circulating metabolites during menopause over a mean follow-up of 5 years and assess their associations with subsequent MetS development.

Methods: Premenopausal women from the Tsuruoka Metabolomics Cohort Study who participated in at least one follow-up survey were included. Menopausal status, data on MetS, and plasma metabolites profiled using capillary electrophoresis mass spectrometry were assessed at each visit. Thirty-one metabolites were examined for associations with menopausal status using mixed-effects models. The association of these menopause-related metabolites with MetS development was examined via logistic regression analysis adjusted for follow-up duration.

Results: Among 953 women (43.8 ± 5.4 years old), 316 (33.2%) reached menopause during follow-up (5.0 ± 1.1 years). Eighteen metabolites changed significantly with menopause, particularly those related to BCAA metabolism, urea cycle, and homocysteine metabolism. Of 695 women without MetS at baseline, 65 (9.4%) developed MetS. Glutamate (odds ratio [95% confidence interval]: 1.95 [1.49-2.57]) was associated with higher MetS risk. Higher levels of glutamate, valine, leucine, and cystine were significantly associated with the development of hyperglycemia.

Conclusions: Longitudinal changes in charged metabolites occur across the menopausal transition, with specific metabolites such as glutamate possibly contributing to the metabolic alterations underlying increased MetS risk.

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Context-specific metabolic alterations in HPRT1 knockout cells within a 3D culture system

Tabata, S., Fujimoto, I., Soga, T., Makinoshima, H.

Cancer Med. 14 ( 23 ) e71452 - e71452 2025.12

Research paper (scientific journal), Joint Work, Accepted

Background: Cancer cells reprogram their metabolism to sustain energy production and biosynthesis for malignant proliferation; however, their metabolic phenotypes vary significantly across different growth environments, creating discrepancies between in vitro and in vivo findings. These inconsistencies pose challenges for translating metabolic research into clinical applications. The emergence of 3D culture models as in vitro systems that accurately mimic the in vivo environment can mitigate these challenges by providing conditions that reflect physiological architecture and metabolic interactions. Therefore, we investigated the impact of the purine metabolism enzyme hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1) on the proliferation and metabolism of SCLC cells using 2D and 3D culture models, with the goal of identifying context-specific metabolic regulation not captured in conventional 2D cultures.

Methods: We evaluated cell proliferation and performed metabolomic profiling of HPRT1-knockout (KO) SCLC cells grown in 2D cultures, two 3D culture systems, and mouse xenograft models. Metabolomic profiling was performed using CE-TOFMS, followed by PCA and pathway analysis. The expression of β-alanine (β-Ala) metabolism-related genes, including carnosine synthase 1 (CARNS1), was assessed by RNA-seq and RT-PCR. CARNS1 expression was further evaluated in publicly available lung cancer datasets, including both cell line and clinical tumor cohorts, to determine its association with patient prognosis and its correlation with HPRT1 expression.

Results: The knockout of HPRT1 significantly reduced cell proliferation in 3D cultures and in vivo but had a minimal impact in 2D cultures. Comprehensive metabolomic analyses of HPRT1-KO cells revealed extensive alterations in amino acid and purine metabolism both in vitro and in vivo. Notably, the effect of HPRT1 KO on β-Ala metabolism differed between 2D and 3D cultures. In 3D cultures, HPRT1 KO led to increased expression of the endogenous antitumor metabolite carnosine and its biosynthetic enzyme CARNS1 within the β-Ala metabolic pathway. Furthermore, analysis of clinical databases showed that high CARNS1 expression correlated with improved prognosis in patients with lung cancer and negatively correlated with HPRT1 expression in tumor tissues.

Conclusion: This study highlights the potential of 3D culture systems to elucidate context-specific mechanisms of metabolic regulation, such as the suppressive effect of HPRT1 on carnosine production. Our findings demonstrate that metabolic phenotypes observed in 2D cultures may not fully capture the complexity of in vivo metabolism, whereas 3D models can reveal regulatory pathways that are otherwise overlooked, including context-dependent regulation of carnosine metabolism by HPRT1.

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Decreased Fecal Nicotinamide and Increased Bacterial Nicotinamidase Gene Expression in Ulcerative Colitis Patients

Aoyama, K., Yamamura, R., Katsurada, T., Shimizu, T., Takahashi, D., Kondo, E., Iwasaki, N., Tamakoshi, A., Soga, T., Fukuda, S., Sonoshita, M., Sakamoto, N.

Inflamm. Bowel Dis. 31 ( 10 ) 2815 - 2825 2025.10

Research paper (scientific journal), Joint Work, Accepted, ISSN 10780998

Background/objective: Ulcerative colitis (UC) is significantly linked with gut microbiota, which is essential for maintaining gut health. Their metabolites mitigate gut inflammation and bolster barrier function. Among these metabolites, we focused on vitamin B3, which has been reported to improve the pathogenesis of UC in mice. This study aimed to compare fecal vitamin B3 and gut microbiota between non-UC and UC patients.

Methods: We assessed fecal metabolites and gut microbiota in 71 UC patients (UC group) and 72 non-UC patients (non-UC group) matched by sex and age in 10-year intervals. Fecal samples were collected and metabolites were analyzed using capillary electrophoresis time-of-flight mass spectrometry. Bacterial DNA was extracted for 16S rRNA gene sequencing. We analyzed fecal nicotinamide levels and gut microbiota composition, employing statistical adjustments for confounding factors.

Results: We found that the UC group exhibited significantly lower fecal nicotinamide levels and α-diversity (Shannon index) compared to the non-UC group. The relative abundance of bacterial genera such as Treponema, UCG-002, and Fusicatenibacter was decreased, while Sellimonas, Fournierella, and Oscillospira were increased in the UC group. Moreover, a negative correlation was observed between Sellimonas abundance and fecal nicotinamide levels in the UC group. Additionally, the UC group showed higher expression of a bacterial gene encoding nicotinamidase compared to the non-UC group.

Conclusions: These findings suggest that gut microbiota dysbiosis contributes to reduced vitamin B3 metabolism in UC patients. The study highlights the potential of replenishing vitamin B3 metabolic pathways as a novel therapeutic approach for UC treatment.

Keywords: gut microbiota; inflammatory bowel disease; nicotinamide; ulcerative colitis; vitamin B3.

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Dysregulated Anaerobic Glycolysis in Podocytes is Relevant to the Progression of Focal Segmental Glomerulosclerosis

Sugimura, M., Maeda, K., Shibata, K., Seko, H., Kozaki, Y., Hirayama, A., Soga, T., Ozeki, T., Sato, Y., Kato, N., Kosugi, T., Kadomatsu, K., Maruyama, S.

Kidney Int. Rep. 10 ( 9 ) 3239 - 3254 2025.09

Research paper (scientific journal), Joint Work, Accepted

Introduction
Minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS) are podocytopathies with varying clinical courses and therapeutic responses. FSGS often leads to end-stage renal disease. Consequently, their heterogeneity requires case stratification and pathophysiological elucidation. The involvement of energy metabolism in FSGS pathogenesis and stratification has not been clarified. Therefore, this study aimed to verify whether evaluating energy kinetics can be a new approach to MCD or FSGS stratification and explore the role of energy metabolism in MCD or FSGS.
Methods
Cultured human podocytes were treated with sera from patients with biopsy-confirmed MCD or FSGS. Serum-treated podocytes were analyzed for apoptosis using flow cytometry, metabolomics via mass spectrometry, and real-time adenosine triphosphate (ATP) production rates using an extracellular flux analyzer. Adriamycin-induced nephropathy was induced in podocyte-specific lactate dehydrogenase (LDH) A (LDHA)-deficient and control mice.
Results
The sera from patients with FSGS significantly induced apoptosis in human podocytes compared with those from individuals with MCD. Apoptosis severity was associated with segmental obliteration and corticosteroid resistance. Metabolomic analysis revealed differences in anaerobic glycolysis and tricarboxylic acid cycle (TCA)-related metabolites in podocytes exposed to the sera of patients with MCD and FSGS. In the podocytes treated with sera from patients with FSGS, glycolytic ATP production significantly decreased in cases with high apoptosis rates. The sera from patients with FSGS suppressed LDHA activity, suppressed α-actinin 4 (ACTN4) expression, and promoted actin remodeling of podocytes. Segmental sclerosis was more prominent in podocyte-specific LDHA-deficient mice with adriamycin-induced nephropathy than in control mice.
Conclusion
FSGS progression was associated with decreased anaerobic glycolysis in podocytes.

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EML4–ALK Rearrangement Creates a Distinctive Myeloid Cell–Dominant Immunosuppressive Microenvironment in Lung Cancer

Arai, K., Nishito, Y., Mizuno, H., Motoi, N., Hiraoka, N., Fuse, M., Arai, Y., Shibata, T., Sonobe, Y., Kayukawa, Y., Maruyama, T., Fukuda, H., Mizoguchi, Y., Aikawa, Y., Yoshida, Y., Watanabe, S., Sakamoto, H., Yamashita, M., Kitano, S., Nagata, Y., Mitsumori, R., Ozaki, K., Niida, S., Kanai, Y., Hirayama, A., Soga, T., Yoshida, T., Yasuda, K., Ochiai, A., Tsunoda, H., Aoki, K.

Cancer Immunol. Res. 13 ( 9 ) 1435 - 1452 2025.09

Research paper (scientific journal), Joint Work, ISSN 23266066

Tyrosine kinase inhibitors are initially efficacious against anaplastic lymphoma kinase (ALK) fusion gene-positive lung adenocarcinoma, but acquired resistance inevitably occurs. Therefore, alternative treatment strategies are needed for tyrosine kinase inhibitor-resistant cases. Although the use of immune checkpoint inhibitors (ICI) has improved the prognosis of patients with lung cancer, patients with ALK+ lung adenocarcinoma exhibit little or no response to immunotherapy and the underlying resistance mechanisms remain unknown. In this study, we explored the immunologic status of the tumor microenvironment (TME) in ALK+ lung adenocarcinoma tissues. Tumor-infiltrating leukocyte analysis revealed reduced numbers of effector T cells and increased myeloid-derived suppressor cells (MDSC) relative to ALK- lung adenocarcinoma cases, indicating that ALK+ lung adenocarcinoma has a myeloid cell-dominant immunosuppressive TME. Single-cell RNA sequencing analysis identified a subset of macrophages that expressed most T cell-attractant chemokines (CXCL9, CXCL10, and CXCL11), and the macrophages were inactivated in ALK+ lung adenocarcinoma. In contrast, ALK+ lung adenocarcinoma expressed high levels of MDSC-attractant chemokines (CXCL1 and CXCL8). In addition, ALK+ lung adenocarcinoma showed higher levels of IL6, an MDSC-inducing cytokine, than ALK- lung adenocarcinoma. An IL6R inhibitor transformed the TME in a murine ALK+ lung adenocarcinoma model, shifting it from an immunosuppressive to a T cell-dominant status. Although ICI monotherapy lacked antitumor effects, a combination of ICI and the IL6R inhibitor had significant antitumor effects in mice. Our findings illustrate the molecular basis of fusion gene-mediated immunosuppressive TMEs, providing a rationale for a novel combination immunotherapy for ALK+ lung adenocarcinoma. See related Spotlight by Vitale and Bria, p.1326.

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Lubiprostone in chronic kidney disease: Insights into mitochondrial function and polyamines from a randomized phase 2 clinical trial

Watanabe, S., Nakayama, M., Yokoo, T., Sanada, S., Ubara, Y., Komatsuda, A., Asanuma, K., Suzuki, Y., Konta, T., Kazama, J. J., Suzuki, T., Fukuda, S., Soga, T., Yamada, T., Mizutani, S., Matsumoto, M., Naito, Y., Taguchi, K., Fukami, K., Kashiwagi, H., Kikuchi, K., Suzuki, C., Tokuno, H., Urasato, M., Kujirai, R., Matsumoto, Y., Akiyama, Y., Tomioka, Y., Itai, S., Tongu, Y., Mishima, E., Kawabe, C., Kasahara, T., Ogata, Y., Toyohara, T., Sato, T., Tanaka, T., Abe, T., LUBI-CKD TRIAL Investigators

Sci. Adv. 11 ( 35 ) eadw3934 2025.08

Research paper (scientific journal), Accepted

Chronic kidney disease (CKD) is a life-threatening condition, and constipation is a progressive risk factor. We evaluated changes in uremic toxins, renal function, and the safety of lubiprostone, a selective chloride channel activator, in patients with CKD. In this phase 2, randomized, double-blind, placebo-controlled trial across nine centers in Japan, 150 patients with stage IIIb-IV CKD received lubiprostone (8 or 16 micrograms) or placebo for 24 weeks. The primary end point was change in indoxyl sulfate levels. Secondary end points included other uremic toxins and renal function markers. Lubiprostone did not alter uremic toxin levels but improved or preserved estimated glomerular filtration rate and its slope in the 16-microgram group. Mild-to-moderate gastrointestinal events occurred in the placebo and 16-microgram groups. Multiomics analysis revealed that lubiprostone modulated the gut microbial agmatine pathway and increased spermidine levels, thereby improving renal mitochondrial function. Lubiprostone is a previously unknown and safe therapeutic option to mitigate renal decline in CKD.

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Glucose-activated JMJD1A drives visceral adipogenesis via α-ketoglutarate-dependent chromatin remodeling

Yang, C., Arai, M., Fuji Ariyanto, E., Zhang, J., Mirani Lubis D., Ito, R., Xie, S., Nitta, M., Kawashima, F., Ishitsuka, T., Yang, C., Suzuki, T., Komatsu, T., Sagae, H., Jin, H., Takahashi, H., Kobayashi, E., Wei, Y., Liu, B., Choi, H., Wada, Y., Tanaka T., Osawa, T., Kimura, H., Kodama, T., Aburatani, H., Tachibana, M., Shinkai, Y., Inagaki, T., Soga, T., Osbome, T.F., Yoneshiro, T., Matsumura, Y., Sakai, J.

Cell Rep. 44 ( 8 ) 116060 2025.08

Research paper (scientific journal), Joint Work, Accepted

Adipose tissue remodels via hypertrophy or hyperplasia in response to nutrient status, but the mechanisms governing these expansion modes remain unclear. Here, we identify a nutrient-sensitive epigenetic circuit linking glucose metabolism to chromatin remodeling during adipogenesis. Upon glucose stimulation, α-ketoglutarate (α-KG) accumulates in the nucleus and activates the histone demethylase JMJD1A to remove repressive histone H3 lysine 9 dimethylation (H3K9me2) marks at glycolytic and adipogenic gene loci, including Pparg. JMJD1A is recruited to pre-marked promoter chromatin via nuclear factor IC (NFIC), enabling carbohydrate-responsive element-binding protein (ChREBP) binding and transcriptional activation. This feedforward mechanism couples nutrient flux to chromatin accessibility and gene expression. In vivo, JMJD1A is essential for de novo adipogenesis and hyperplastic expansion in visceral fat under nutrient excess. JMJD1A deficiency impairs hyperplasia, exacerbates adipocyte hypertrophy, and induces local inflammation. These findings define a glucose-α-KG-JMJD1A-ChREBP axis regulating depot-specific adipogenesis and uncover a chromatin-based mechanism by which glucose metabolism governs adaptive adipose tissue remodeling.

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2,3-Pyridinedicarboxylate Is Associated with Shorter Recurrence-Free Survival in Patients with Hypopharyngeal Squamous Cell Carcinoma

Takase, H., Fujisawa, T., Hayashi, R., Makinoshima, H., Suzuki, Y., Soga, T., Fujii, S.

Pathobiology online 1 - 15 2025.08

Research paper (scientific journal), Accepted, ISSN 10152008

Introduction: Metabolites are associated with the biology of cancer; however, no metabolites related to prognosis have been identified in head and neck cancer. This study aimed to identify metabolites associated with prognosis in patients with hypopharyngeal squamous cell carcinoma (HPSCC).

Methods: Fifty-two patients who underwent surgery for HPSCC were included and randomly divided into test and validation cohorts of 26 patients each for further metabolome analysis using capillary electrophoresis/mass spectrometry on tumor and non-tumor tissues of the hypopharynx. Twenty-two patients who received adjuvant therapy after surgery were included. The receiver operating characteristic (ROC) and univariate and multivariate analyses were used to explore the relationship between recurrence-free survival (RFS), clinicopathological factors, and differentiated metabolites.

Results: ROC analysis revealed six metabolites significantly associated with RFS in both cohorts, and multivariate analysis indicated that 2,3-pyridinedicarboxylate was a significantly independent poorer prognostic factor in the cohorts including patients with HPSCC without any adjuvant therapies (p = 0.017).

Conclusion: 2,3-Pyridinedicarboxylate, involved in NAD+ metabolism and genomic stability, suggests the possibility of developing molecular-targeted drugs for the production of metabolites related to prognosis. This study identifies novel prognostic metabolites and their associated metabolic pathways in HPSCC, highlighting potential therapeutic targets for treatment.

Keywords: 2,3-Pyridinedicarboxylate; Hypopharyngeal squamous cell carcinoma; Metabolites; Recurrence-free survival.

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Mitochondria-Homing Drug Mitochonic Acid 5 Improves Barth Syndrome Myopathy in a Human-Induced Pluripotent Stem Cell Model and Barth Syndrome Drosophila Model

Tongu, Y., Kasahara, T., Matsuhashi, T., Oikawa, Y., Akimoto, R., Luo, Y., Sekine, S., Suzuki, M., Kashiwagi, H., Kanno, S., Tanaka, Y., Sato, K., Okubo, Y., Muto, A., Tokuno, H., Suzuki, C., Kawabe, C., Ishikawa, T., Watanabe, S., Kikuchi, K., Itai, S., Sato, T., Suzuki, T., Igarashi, K., Fukuda, S., Soga, T., Murayama, K., Kuranaga, E., Toyohara, T., Abe, T.

FASEB J. 39 ( 12 ) e70739 - e70739 2025.06

Research paper (scientific journal), Joint Work, Accepted

Barth syndrome (BTHS) is a rare disease caused by mutations in the tafazzin gene that affects the heart and muscles; however, to date, no clinically effective drugs are available. In BTHS, mitochondrial function is reduced owing to changes in cardiolipin metabolism. We developed mitochonic acid 5 (MA-5), a small-molecule compound that increases ATP levels, improves mitochondrial dynamics, and is effective in treating mitochondrial and muscle diseases. Therefore, this study examined the effectiveness of MA-5 in treating BTHS. The mitochondrial functions of four isolated BTHS skin fibroblasts were examined. Human BTHS induced pluripotent stem cell (iPSC) were differentiated into myoblasts and cardiolipin metabolism and mitochondrial functions were analyzed. RNA-seq was performed to clarify the metabolic changes. Using a Drosophila melanogaster model of BTHS, the effects of MA-5 on motor performance and cardiac phenotype were examined. MA-5 improved mitochondrial function and reduced cell death due to oxidative stress in skin fibroblasts of patients with BTHS. MA-5 promoted ATP production and reduced oxidative stress in human BTHS iPS cell-derived myoblasts. RNA-seq analysis revealed that MA-5 alleviated endoplasmic reticulum stress in BTHS cells. Administration of MA-5 to BTHS Drosophila improved locomotor ability and tachycardia observed in patients with BTHS. Protein interaction analyses suggested colocalization of ATPase and the MA-5-binding protein mitofilin. These data suggested that MA-5 improves BTHS dysfunction and may serve as a novel therapeutic agent for BTHS.

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Dietary amino acids regulate Salmonella colonization via microbiota-dependent mechanisms in the mouse gut

Pickard, J., Porwollik, S., Caballero-Flores, G., Caruso, R., Fukuda, S., Soga, T., Inohara, N., McClelland, M., Nunez, G.

Nat. Commun. 16 ( 1 ) 4225 2025.05

Research paper (scientific journal), Joint Work

The gut microbiota confers host protection against pathogen colonization early after infection. Several mechanisms underlying the protection have been described, but the contributions of nutrient competition versus direct inhibition are controversial. Using an ex vivo model of Salmonella growth in the mouse cecum with its indigenous microbes, we find that nutrient limitation and typical inhibitory factors alone cannot prevent pathogen growth. However, the addition of certain amino acids markedly reverses the microbiota's ability to suppress pathogen growth. Enhanced Salmonella colonization after antibiotic treatment is ablated by exclusion of dietary protein, which requires the presence of the microbiota. Thus, dietary protein and amino acids are important regulators of colonization resistance.

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MOB1 deletion in murine mature adipocytes ameliorates obesity and diabetes

Nishio, M., Yamaguchi, K., Otani, J., Yuguchi, K., Kohno, D., Sasaki, T., Kitamura, T., Shinohara, M., Soga, T., Kawamura, K., Sasaki, A.T., Oshima, M., Hikasa, H., Woo, M., Sasaki, T., Nishina, H., Nakao, K., Maehama, T., Suzuki, A.

Proc Natl Acad Sci USA 122 ( 17 ) e2424741122 2025.04

Research paper (scientific journal), Joint Work, Accepted, ISSN 00278424

There is currently a global epidemic of obesity and obesity-related diseases such as type 2 diabetes due to decreased physical activity, excessive food intake, and/or genetic predisposition. The Hippo-YAP1 pathway has attracted attention as a potential therapeutic target because YAP1/TAZ activation in murine immature adipocytes in vitro suppresses their differentiation by inhibiting PPARγ activity. However, the role of YAP1 activation in mature adipocytes in vivo remains unclear. MOB1, whose expression is increased in obesity, is the hub of the Hippo core molecule complex and negatively regulates YAP1/TAZ activation. Therefore, we generated aMob1DKO mutant mice, which feature deficiency of Mob1a/b specifically in mature adipocytes. Compared to controls, aMob1DKO mice subjected to a high-fat diet showed beneficial changes consistent with resistance to diet-induced obesity. The mutants exhibited increases in basal lipolysis, "beiging," and energy expenditure, as well as suppression of ROS production and inflammation in white adipose tissue. Insulin sensitivity and glucose tolerance were improved, and ectopic fat accumulation was reduced. Most of these changes were dependent on the YAP1 activation observed in mature white adipose tissue of aMob1DKO mice. FGF21, which improves lipid metabolism, was upregulated directly via YAP1 activation, and many of the phenotypes seen in aMob1DKO mice were also dependent on FGF21. Thus, the aMob1DKO mouse is an interesting model for the study of the metabolic effects of diet-induced obesity and protection against diabetes. Our work suggests that a YAP1-FGF21 axis exists in adipocytes that may be a potential therapeutic target for obesity.

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Genome-wide association study of plasma amino acids and Mendelian randomization for cardiometabolic traits

Toki, R., Fushiki, S., Kojima, S., Sutoh, Y., Otsuka-Yamasaki, Y., Harada, S., Iida, M., Hirata, A., Miyagawa, N., Matsumoto, M., Edagawa, S., Miyake, A., Kuwabara, K., Hirayama, A., Sugimoto, M., Sato, A., Amano, K., Soga, T., Tomita, M., Arakawa, K., Kinoshita, K., Sakurai-Yageta, M., Tamiya, G., Ohmomo, H., Shimizu, A., Okamura, T., Takebayashi, T.

Sci. Rep. 15 ( 1 ) 14569 2025.04

Research paper (scientific journal), Accepted

Plasma amino acids (AAs) have emerged as promising biomarkers for metabolic disorders, yet their causality remains unclear. We aimed to investigate the genetic determinants of AA levels in a cohort of 10,333 individuals and their causal effects on cardiometabolic traits using Mendelian randomization (MR). Plasma levels of 20 AAs were quantified using capillary electrophoresis mass spectrometry. Genome-wide association studies were conducted using BOLT-LMM and heritability estimation via LDSC analysis. Causal effects of AAs on 11 cardiometabolic traits were examined using two-sample MR analyses. We identified 85 locus-metabolite associations across 43 genes for 18 AAs, including 44 novel loci linked to metabolic genes. Heritability for AAs was estimated at 16%. MR analysis demonstrated cystine to positively associate with systolic blood pressure (SBP) (β = 0.056, SE = 0.010), while serine indicated protective effects on SBP (β = - 0.040, SE = 0.011), diastolic BP (β = - 0.044, SE = 0.010), and coronary artery disease (odds ratio 0.888, SE = 0.028). We identified potentially novel genetic loci associated with AA levels and demonstrated robust causal associations between several AAs and cardiometabolic traits. These findings reinforce the importance of AAs as potential biomarkers and therapeutic targets in cardiometabolic health.

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Structural robustness and temporal vulnerability of the starvation-responsive metabolic network in healthy and obese mouse liver

Morita, K., Hatano, A., Kokaji, T., Sugimoto, H., Tsuchiya, T., Ozaki, H., Egami, R., Li, D., Terakawa, A., Ohno, S., Inoue, H., Inaba, Y., Suzuki, Y., Matsumoto, M., Takahashi, M., Izumi, Y., Bamba, T., Hirayama, A., Soga, T., Kuroda, Shinya

Sci. Signal. 18 ( 883 ) eads2547 2025.04

Research paper (scientific journal), Joint Work, Accepted, ISSN 19450877

Adaptation to starvation is a multimolecular and temporally ordered process. We sought to elucidate how the healthy liver regulates various molecules in a temporally ordered manner during starvation and how obesity disrupts this process. We used multiomic data collected from the plasma and livers of wild-type and leptin-deficient obese (ob/ob) mice at multiple time points during starvation to construct a starvation-responsive metabolic network that included responsive molecules and their regulatory relationships. Analysis of the network structure showed that in wild-type mice, the key molecules for energy homeostasis, ATP and AMP, acted as hub molecules to regulate various metabolic reactions in the network. Although neither ATP nor AMP was responsive to starvation in ob/ob mice, the structural properties of the network were maintained. In wild-type mice, the molecules in the network were temporally ordered through metabolic processes coordinated by hub molecules, including ATP and AMP, and were positively or negatively coregulated. By contrast, both temporal order and coregulation were disrupted in ob/ob mice. These results suggest that the metabolic network that responds to starvation was structurally robust but temporally disrupted by the obesity-associated loss of responsiveness of the hub molecules. In addition, we propose how obesity alters the response to intermittent fasting.

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Lysine-arginine imbalance overcomes therapeutic tolerance governed by the transcription factor E3-lysosome axis in glioblastoma

Jing, Y., Kobayashi, M., Shoulkamy, M.I., Zhou, M., Thi Vu, H., Arakawa, H., Sabit, H., Iwabuchi, S., Quang Vu, C., Kasahara, A., Ueno, M., Tadokoro, Y., Kurayoshi, K., Chen, X., Yan, Y., Arai, S., Hashimoto, S., Soga, T., Todo, T., Nakada, M., Hirao, A.

Nat. Commun. 16 ( 1 ) 2876 - 2876 2025.04

Research paper (scientific journal), Joint Work, Accepted

Recent advances in cancer therapy have underscored the importance of targeting specific metabolic pathways. In this study, we propose a precision nutrition approach aimed at lysosomal function in glioblastoma multiforme (GBM). Using patient-derived GBM cells, we identify lysosomal activity as a unique metabolic biomarker of tumorigenesis, controlling the efficacy of temozolomide (TMZ), a standard GBM therapy. Employing combined analyses of clinical patient samples and xenograft models, we further elucidate the pivotal role of Transcription Factor Binding To IGHM Enhancer 3 (TFE3), a master regulator of lysosomal biogenesis, in modulating malignant properties, particularly TMZ tolerance, by regulating peroxisome proliferator-activated receptor-gamma coactivator 1-alpha (PGC1α)-mediated mitochondrial activity. Notably, we find that lysine protects GBM cells from lysosomal stress by counteracting arginine's effects on nitric oxide production. The lysine restriction mimetic, homoarginine administration, significantly enhances the efficacy of anticancer therapies through lysosomal dysfunction. This study underscores the critical role of lysosomal function modulated by amino acid metabolism in GBM pathogenesis and treatment.

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PNPLA6 regulates retinal homeostasis by choline through phospholipid turnover

Ono, T., Taketomi, Y., Higashi, T., Sato, H., Mochizuki-Ono, C., Nagasaki, Y., Ueta, T., Miyai, T., Tokuoka, S., Oda, Y., Nishito, Y., Ono, T., Taya, C., Arata, S., Watanabe, S., Soga, T., Hirabayashi, T., Aihara, M., Murakami, M.

Nat. Commun. 16 ( 1 ) 2221 - 2221 2025.03

Research paper (scientific journal), Joint Work, Accepted

Although mutations in human patatin-like phospholipase PNPLA6 are associated with hereditary retinal degenerative diseases, its mechanistic action in the retina is poorly understood. Here, we uncover the molecular mechanism by which PNPLA6 dysfunction disturbs retinal homeostasis and visual function. PNPLA6, by acting as a phospholipase B, regulates choline mobilization from phosphatidylcholine and subsequent choline turnover for phosphatidylcholine regeneration in retinal pigment epithelial cells. PNPLA6-driven choline is supplied from retinal pigment epithelial cells to adjacent photoreceptor cells to support their survival. Inhibition of this pathway results in abnormal morphology, proliferation, metabolism, and functions of retinal pigment epithelial and photoreceptor cells, and mice with retina-specific PNPLA6 deletion exhibit retinitis pigmentosa-like retinal degeneration. Notably, these abnormalities are entirely rescued by choline supplementation. Thus, PNPLA6 plays an essential role in retinal homeostasis by controlling choline availability for phospholipid recycling and provide a framework for the development of an ophthalmic drug target for retinal degeneration.

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Time and dose selective glucose metabolism for glucose homeostasis and energy conversion in the liver

Pan, Y., Hatano, A., Ohno, S., Morita, K., Kokaji, T., Bai, Y., Sugimoto, H., Egami, R., Terakawa, A., Li, D., Uematsu, S., Maehara, H., Fujita, S., Inoue, H., Inaba, Y., Nagano, A., Hirayama, A., Soga, T., Kuroda, S.

npj Syst. Biol. Appl. 10 ( 1 ) 107 - 107 2024.09

Research paper (scientific journal), Joint Work, Accepted

Hepatic glucose metabolism serves dual purposes: maintaining glucose homeostasis and converting glucose into energy sources; however, the underlying mechanisms are unclear. We quantitatively measured liver metabolites, gene expression, and phosphorylated insulin signaling molecules in mice orally administered varying doses of glucose, and constructed a transomic network. Rapid phosphorylation of insulin signaling molecules in response to glucose intake was observed, in contrast to the more gradual changes in gene expression. Glycolytic and gluconeogenic metabolites and expression of genes involved in glucose metabolism including glucose-6-phosphate, G6pc, and Pck1, demonstrated high glucose dose sensitivity. Whereas, glucokinase expression and glycogen accumulation showed low glucose dose sensitivity. During the early phase after glucose intake, metabolic flux was geared towards glucose homeostasis regardless of the glucose dose but shifted towards energy conversion during the late phase at higher glucose doses. Our research provides a comprehensive view of time- and dose-dependent selective glucose metabolism.

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A Supramolecular Biosensor for Rapid and High-ThroughputQuantification of a Disease-Associated Niacin Metabolite

Ueno, M., Sugiyama, H., Li, F., Nishimura, T., Arakawa, H., Chen, X., Cheng, X., Takeuchi,, S., Takeshita, Y., Takamura, T., Miyagi, S., Toyama, T., Soga, T., Masuo, Y., Kato, Y., Nakamura, H., Tsujiguchi, H., Hara, A., Tajima, A., Noguchi-Shinohara, M., Ono, K., Kurayoshi, K., Kobayashi, M., Tadokoro, Y., Kasahara, A., Shoulkamy, M.I., Maeda, K., Ogoshi, T., Hirao, A.

Anal. Chem. 96 ( 36 ) 14499 - 14507 2024.09

Research paper (scientific journal), Joint Work, Accepted

Metabolic abnormalities play a pivotal role in various pathological conditions, necessitating the quantification of specific metabolites for diagnosis. While mass spectrometry remains the primary method for metabolite measurement, its limited throughput underscores the need for biosensors capable of rapid detection. Previously, we reported that pillar[6]arene with 12 carboxylate groups (P6AC) forms host-guest complexes with 1-methylnicotinamide (1-MNA), which is produced in vivo by nicotinamide N-methyltransferase (NNMT). P6AC acts as a biosensor by measuring the fluorescence quenching caused by photoinduced electron transfer upon 1-MNA binding. However, the low sensitivity of P6AC makes it impractical for detecting 1-MNA in unpurified biological samples. In this study, we found that P6A with 12 sulfonate groups (P6AS) is a specific and potent supramolecular host for 1-MNA interactions even in biological samples. The 1-MNA binding affinity of P6AS in water was found to be (5.68 ± 1.02) × 106 M-1, which is approximately 700-fold higher than that of P6AC. Moreover, the 1-MNA detection limit of P6AS was determined to be 2.84 × 10-7 M, which is substantially lower than that of P6AC. Direct addition of P6AS to culture medium was sufficient to quantify 1-MNA produced by cancer cells. Furthermore, this sensor was able to specifically detect 1-MNA even in unpurified human urine. P6AS therefore enables rapid and high-throughput quantification of 1-MNA, and further improvement of our strategy will contribute to the establishment of high-throughput screening of NNMT inhibitors, diagnosis of liver diseases, and imaging of human cancer cells in vivo.

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Metabolomics Profiles Alterations in Cigarette Smokers and Heated Tobacco Product Users

Harada, S., Ohmomo, H., Matsumoto, M., Sata, M., Iida, M., Hirata, A., Miyagawa, N., Kuwabara, K., Kato, S., Toki, R., Edagawa, S., Sugiyama, D., Sato, A., Hirayama, A., Sugimoto, M., Soga, T., Tomita, M., Shimizu, A., Okamura, T., Takebayashi, T.

J. Epidemiol. 34 ( 9 ) 403 - 410 2024.09

Research paper (scientific journal), Joint Work, Accepted

Background: Heated tobacco products (HTPs) have gained global popularity, but their health risks remain unclear. Therefore, the current study aimed to identify plasma metabolites associated with smoking and HTP use in a large Japanese population to improve health risk assessment.

Methods: Metabolomics data from 9,922 baseline participants of the Tsuruoka Metabolomics Cohort Study (TMCS) were analyzed to determine the association between smoking habits and plasma metabolites. Moreover, alterations in smoking-related metabolites among HTP users were examined based on data obtained from 3,334 participants involved from April 2018 to June 2019 in a follow-up survey.

Results: Our study revealed that cigarette smokers had metabolomics profiles distinct from never smokers, with 22 polar metabolites identified as candidate biomarkers for smoking. These biomarker profiles of HTP users were closer to those of cigarette smokers than those of never smokers. The concentration of glutamate was higher in cigarette smokers, and biomarkers involved in glutamate metabolism were also associated with cigarette smoking and HTP use. Network pathway analysis showed that smoking was associated with the glutamate pathway, which could lead to endothelial dysfunction and atherosclerosis of the vessels.

Conclusions: Our study showed that the glutamate pathway is affected by habitual smoking. These changes in the glutamate pathway may partly explain the mechanism by which cigarette smoking causes cardiovascular disease. HTP use was also associated with glutamate metabolism, indicating that HTP use may contribute to the development of cardiovascular disease through mechanisms similar to those in cigarette use.

Keywords: electronic nicotine delivery devices; heated tobacco products; metabolomics; prevention; smoking-induced disease.

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The role of cytidine 5'-triphosphate synthetase 1 in metabolic rewiring during epithelial-to-mesenchymal transition in non-small-cell lung cancer

Nakasuka, F., Hirayama, A., Mkinoshima, H., Yano, S., Soga, T., Tabata, S.

FEBS Open Bio 14 ( 9 ) 1570 - 1583 2024.09

Research paper (scientific journal), Joint Work, Accepted

Epithelial-to-mesenchymal transition (EMT) contributes to the poor prognosis of patients with cancer by promoting distant metastasis and anti-cancer drug resistance. Several distinct metabolic alterations have been identified as key EMT phenotypes. In the present study, we further characterize the role of transforming growth factor-β (TGF-β)-induced EMT in non-small-cell lung cancer. Our study revealed that TGF-β plays a role in EMT functions by upregulation of cytidine 5'-triphosphate synthetase 1 (CTPS), a vital enzyme for CTP biosynthesis in the pyrimidine metabolic pathway. Both knockdown and enzymatic inhibition of CTPS reduced TGF-β-induced changes in EMT marker expression, chemoresistance and migration in vitro. Moreover, CTPS knockdown counteracted the TGF-β-mediated downregulation of UDP-glucuronate, glutarate, creatine, taurine and nicotinamide. These findings indicate that CTPS plays a multifaceted role in EMT metabolism, which is crucial for the malignant transformation of cancer through EMT, and underline its potential as a promising therapeutic target for preventing drug resistance and metastasis in non-small-cell lung cancer.

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Gut microbial factors predict disease severity in a mouse model of multiple sclerosis

Steimle, A., Neumann, M., Grant, E.T., Willieme, S., Sciscio, A.D., Parrish, A., Ollert, M., Miyauchi, E., Soga, T., Fukuda, S., Ohno, H., Desai, M.S.

Nat. Microbiol. 9 ( 9 ) 2244 - 2261 2024.09

Research paper (scientific journal), Joint Work, Accepted

Gut bacteria are linked to neurodegenerative diseases but the risk factors beyond microbiota composition are limited. Here we used a pre-clinical model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE), to identify microbial risk factors. Mice with different genotypes and complex microbiotas or six combinations of a synthetic human microbiota were analysed, resulting in varying probabilities of severe neuroinflammation. However, the presence or relative abundances of suspected microbial risk factors failed to predict disease severity. Akkermansia muciniphila, often associated with MS, exhibited variable associations with EAE severity depending on the background microbiota. Significant inter-individual disease course variations were observed among mice harbouring the same microbiota. Evaluation of microbial functional characteristics and host immune responses demonstrated that the immunoglobulin A coating index of certain bacteria before disease onset is a robust individualized predictor of disease development. Our study highlights the need to consider microbial community networks and host-specific bidirectional interactions when aiming to predict severity of neuroinflammation.

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FDX2, an iron-sulfur cluster assembly factor, is essential to prevent cellular senescence, apoptosis or ferroptosis of ovarian cancer cells

Miyahara, S., Ohuchi, M., Nomura, M., Hashimoto, E., Soga, T., Saito, R., Hayashi, K., Sato, T., Saito, M., Yamashita, Y., Shimada, M., Yaegashi, N., Yamada, H., Tanuma, N.

J. Biol. Chem. 300 ( 9 ) 107678 - 107678 2024.09

Research paper (scientific journal), Accepted

Recent studies reveal that biosynthesis of iron-sulfur clusters (Fe-Ss) is essential for cell proliferation, including that of cancer cells. Nonetheless, it remains unclear how Fe-S biosynthesis functions in cell proliferation/survival. Here, we report that proper Fe-S biosynthesis is essential to prevent cellular senescence, apoptosis, or ferroptosis, depending on cell context. To assess these outcomes in cancer, we developed an ovarian cancer line with conditional KO of FDX2, a component of the core Fe-S assembly complex. FDX2 loss induced global downregulation of Fe-S-containing proteins and Fe2+ overload, resulting in DNA damage and p53 pathway activation, and driving the senescence program. p53 deficiency augmented DNA damage responses upon FDX2 loss, resulting in apoptosis rather than senescence. FDX2 loss also sensitized cells to ferroptosis, as evidenced by compromised redox homeostasis of membrane phospholipids. Our results suggest that p53 status and phospholipid homeostatic activity are critical determinants of diverse biological outcomes of Fe-S deficiency in cancer cells.

Study Profile of the Tsuruoka Metabolomics Cohort Study (TMCS)

Harada, S., Iida, M., Miyagawa, N., Hirata, A., Kuwabara, K., Matsumoto, M., Okamura, T., Edagawa, S., Kawada, Y., Miyake, A., Toki , R., Akiyama, M., Kawai, A., Sugiyama, D., Sato, Y., Takemura, R., Fukai, K., Ishibashi, Y., Kato, S., Kurihara, A., Sata, M., Shibuki, T., Takeuchi, A., Kohsaka, S., Sawano, M., Shoji, S., Izawa, Y., Katsumata, M., Oki, K., Takahashi, S., Takizawa, T., Maruya, H., Nishiwaki, Y., Kawasaki, R., Hirayama, A., Ishikawa, T., Saito, R., Sato, A., Soga, T., Sugimoto, M., Tomita, M., Komaki, S., Ohmomo, H., Ono, K., Otsuka-Yamasaki, Y., Shimizu, A., Sutoh, Y., Hozawa, A., Kinoshita, K., Koshiba, S., Kumada, K., Ogishima, S., Sakurai-Yageta, M., Tamiya, G., Takebayashi, T.

J. Epidemiol. 34 ( 8 ) 393 - 401 2024.08

Research paper (scientific journal), Joint Work, Accepted, ISSN 09175040

The Tsuruoka Metabolomics Cohort Study (TMCS) is an ongoing population-based cohort study being conducted in the rural area of Yamagata Prefecture, Japan. This study aimed to enhance the precision prevention of multi-factorial, complex diseases, including non-communicable and aging-associated diseases, by improving risk stratification and prediction measures. At baseline, 11,002 participants aged 35-74 years were recruited in Tsuruoka City, Yamagata Prefecture, Japan, between 2012 and 2015, with an ongoing follow-up survey. Participants underwent various measurements, examinations, tests, and questionnaires on their health, lifestyle, and social factors. This study uses an integrative approach with deep molecular profiling to identify potential biomarkers linked to phenotypes that underpin disease pathophysiology and provide better mechanistic insights into social health determinants. The TMCS incorporates multi-omics data, including genetic and metabolomic analyses of 10,933 participants, and comprehensive data collection ranging from physical, psychological, behavioral, and social to biological data. The metabolome is used as a phenotypic probe because it is sensitive to changes in physiological and external conditions. The TMCS focuses on collecting outcomes for cardiovascular disease, cancer incidence and mortality, disability and functional decline due to aging and disease sequelae, and the variation in health status within the body represented by omics analysis that lies between exposure and disease. It contains several sub-studies on aging, heated tobacco products, and women's health. This study is notable for its robust design, high participation rate (89%), and long-term repeated surveys. Moreover, it contributes to precision prevention in Japan and East Asia as a well-established multi-omics platform.

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Dynamics of the gut microbiome in FAP patients undergoing intensive endoscopic reduction of polyp burden

Mizutani, S., Tamaki, A., Shiba, S., Salim, F., Yamada, M., Takamaru, H., Nakajima, T., Yoshida, N., Ikuta, S., Yachida, T., Shibata, T., Soga, T., Saito, Y., Fukuda, S., Ishikawa, H., Yamada, T., Yachida, S.

Gut online 2024.08

Joint Work, Accepted

Prognostic Significance of 18F-FDG PET/CT and Tumor Metabolic Changes in Patients With Pancreatic Ductal Adenocarcinoma

Shimomura, A., Oshima, M., Suto, H., Matsukawa, H., Kondo, A., Ando, Y., Kishino, T., Kumamoto, K., Sato, K., Sugimoto, M., Nagao, M., Miyatake, N., Norikane, T., Soga, T., Okano, K.

Anticancer Res. 44 ( 8 ) 3321 - 3330 2024.08

Research paper (scientific journal), Joint Work, Accepted

Background/aim: 18F-fluorodeoxyglucose positron emission tomography/computed tomography (FDG-PET/CT) is reportedly associated with the malignant potential of cancer. This study aimed to evaluate the association between FDG accumulation and tumor metabolism in pancreatic ductal adenocarcinoma (PDAC).

Patients and methods: A prognostic analysis of data from 131 patients with PDAC who underwent FDG-PET/CT before curative-intent pancreatic surgery was performed. Capillary electrophoresis-mass spectrometry (CE-MS) was used to analyze the metabolome of tumor and non-neoplastic pancreas from 80 patients. These patients were divided into two groups: low SUVmax group (SUVmax <6.09) and high SUVmax group (SUVmax ≥6.09).

Results: Carbohydrate antigen 19-9 (CA19-9), maximum standardized uptake value (SUVmax) of PET, N stage, and postoperative chemotherapy were identified as significant prognostic factors by univariate analysis. SUVmax emerged as an independent prognostic factor for overall survival [hazard ratio (HR)=1.88, p<0.05] and disease-free survival (HR=2.01, p<0.05) in multivariate analysis. Metabolic analyses confirmed that 43 metabolites significantly differed depending on the accumulation of SUV in tumors. Metabolites involved in the removal of reactive oxygen species (e.g., hypotaurine, glutathione, Met), treatment resistance (UDP-N-acetylglucosamine), and proliferation (e.g., choline, leucine, isoleucine) were increased in the high SUVmax group.

Conclusion: FDG accumulation is an important independent prognostic factor reflecting tumor activity associated with metabolic changes in cancer cells.

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Fusobacterium species are distinctly associated with patients with Lynch Syndrome colorectal cancer

Salim, F., Mizutani, S., Shiba, S., Takamaru, H., Yamada, M., Nakajima, T., Yachida, T., Soga, T., Saito, Y., Fukuda, S., Yachida, S., Yamada, T.

iScience 27 ( 7 ) 110181 - 110181 2024.07

Research paper (scientific journal), Joint Work, Accepted

Accumulating evidence demonstrates clear correlation between the gut microbiota and sporadic colorectal cancer (CRC). Despite this, there is limited understanding of the association between the gut microbiota and CRC in Lynch Syndrome (LS), a hereditary type of CRC. Here, we analyzed fecal shotgun metagenomic and targeted metabolomic of 71 Japanese LS subjects. A previously published Japanese sporadic CRC cohort, which includes non-LS controls, was utilized as a non-LS cohort (n = 437). LS subjects exhibited reduced microbial diversity and low-Faecalibacterium enterotypes compared to non-LS. Patients with LS-CRC had higher levels of Fusobacterium nucleatum and fap2. Differential fecal metabolites and functional genes suggest heightened degradation of lysine and arginine in LS-CRC. A comparison between LS and non-LS subjects prior to adenoma formation revealed distinct fecal metabolites of LS subjects. These findings suggest that the gut microbiota plays a more responsive role in CRC tumorigenesis in patients with LS than those without LS.

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Association of Nonalcoholic Fatty Liver Disease with Arterial Stiffness and its Metabolomic Profiling in Japanese Community-Dwellers

Hirata, A., Harada, S., Iida, M., Kurihara, A., Fukai, K., Kuwabara, K., Kato, S., Matsumoto, M., Sata, M., Miyagawa, N., Toki, R., Edagawa, S., Sugiyama, D., Sato, A., Hirayama, A., Sugimoto, M., Soga, T., Tomita, M., Okamura, T., Takebayashi, T.

J. Atheroscler. Thromb. 31 ( 7 ) 1031 - 1047 2024.07

Research paper (scientific journal), Joint Work, Accepted

Aims: Nonalcoholic fatty liver disease (NAFLD) is known to be associated with atherosclerosis. This study focused on upstream changes in the process by which NAFLD leads to atherosclerosis. The study aimed to confirm the association between NAFLD and the cardio-ankle vascular index (CAVI), an indicator of subclinical atherosclerosis, and explore metabolites involved in both by assessing 94 plasma polar metabolites.

Methods: A total of 928 Japanese community-dwellers (306 men and 622 women) were included in this study. The association between NAFLD and CAVI was examined using a multivariable regression model adjusted for confounders. Metabolites commonly associated with NAFLD and CAVI were investigated using linear mixed-effects models in which batch numbers of metabolite measurements were used as a random-effects variable, and false discovery rate-adjusted p-values were calculated. To determine the extent to which these metabolites mediated the association between NAFLD and CAVI, mediation analysis was conducted.

Results: NAFLD was positively associated with CAVI (coefficients [95% Confidence intervals (CI)]=0.23 [0.09-0.37]; p=0.001). A total of 10 metabolites were involved in NAFLD and CAVI, namely, branched-chain amino acids (BCAAs; valine, leucine, and isoleucine), aromatic amino acids (AAAs; tyrosine and tryptophan), alanine, proline, glutamic acid, glycerophosphorylcholine, and 4-methyl-2-oxopentanoate. Mediation analysis showed that BCAAs mediated more than 20% of the total effect in the association between NAFLD and CAVI.

Conclusions: NAFLD was associated with a marker of atherosclerosis, and several metabolites related to insulin resistance, including BCAAs and AAAs, could be involved in the process by which NAFLD leads to atherosclerosis.

Keywords: Atherosclerosis; Branched-chain amino acids; Cardio-ankle vascular index; Insulin resistance; Metabolites; Nonalcoholic fatty liver disease.

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SGLT2 inhibition eliminates senescent cells and alleviates pathological aging

Katsuumi, G., Shimizu, I., Suda, M., Yoshida, Y., Furihata, T., Joki, Y., Hsiao, CL., Jiaqi, L., Fujiki, S., Abe, M., Sugimoto, M., Soga, T., Minamino, T

Nat. Aging 4 ( 7 ) 926 - 938 2024.07

Research paper (scientific journal), Joint Work, Accepted

It has been reported that accumulation of senescent cells in various tissues contributes to pathological aging and that elimination of senescent cells (senolysis) improves age-associated pathologies. Here, we demonstrate that inhibition of sodium-glucose co-transporter 2 (SGLT2) enhances clearance of senescent cells, thereby ameliorating age-associated phenotypic changes. In a mouse model of dietary obesity, short-term treatment with the SGLT2 inhibitor canagliflozin reduced the senescence load in visceral adipose tissue and improved adipose tissue inflammation and metabolic dysfunction, but normalization of plasma glucose by insulin treatment had no effect on senescent cells. Canagliflozin extended the lifespan of mice with premature aging even when treatment was started in middle age. Metabolomic analyses revealed that short-term treatment with canagliflozin upregulated 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside, enhancing immune-mediated clearance of senescent cells by downregulating expression of programmed cell death-ligand 1. These findings suggest that inhibition of SGLT2 has an indirect senolytic effect by enhancing endogenous immunosurveillance of senescent cells.

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The γ-glutamyl cycle serves as an amino acids supply system in colorectal cancer organoids under chronic hypoxia

Tabata, S., Endo, H., Makinoshima, H., Soga, T., Inoue, M.

Biochem. Biophys. Res. Commun. (Biochemical and Biophysical Research Communications) 714 149977 - 149977 2024.06

Research paper (scientific journal), Joint Work, Accepted, ISSN 0006291X

Malignant tumors are characterized by a hypoxic microenvironment, and metabolic reprogramming is necessary to ensure energy production and oxidative stress resistance. Although the microenvironmental properties of tumors vary under acute and chronic hypoxia, studies on chronic hypoxia-induced metabolic changes are limited. In the present study, we performed a comprehensive metabolic analysis in a chronic hypoxia model using colorectal cancer (CRC) organoids, and identified an amino acid supply system through the γ-glutamyl cycle, a glutathione recycling pathway. We analyzed the metabolic changes caused by hypoxia over time and observed that chronic hypoxia resulted in an increase in 5-oxoproline and a decrease in oxidized glutathione (GSSG) compared to acute hypoxia. These findings suggest that chronic hypoxia induces metabolic changes in the γ-glutamyl cycle. Moreover, inhibition of the γ-glutamyl cycle via γ-glutamyl cyclotransferase (GGCT) and γ-glutamyl transferase 1 (GGT1) knockdown significantly reversed chronic hypoxia-induced upregulation of 5-oxoproline and several amino acids. Notably, GGT1 knockdown downregulated the intracellular levels of γ-glutamyl amino acids. Conclusively, these results indicate that the γ-glutamyl cycle serves as an amino acid supply system in CRC under chronic hypoxia, which provides fresh insight into cancer metabolism under chronic hypoxia.

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Molecular subtypes of lung adenocarcinoma present distinct immune tumor microenvironments

Fukuda, H., Arai, K., Mizuno, H., Nishito, Y., Motoi, N., Arai, Y., Hiraoka, N., Shibata, T., Sonobe, Y., Kayukawa, Y., Hashimoto, E., Takahashi, M., Fujii, E., Maruyama, T., Kuwabara, K., Nishizawa, T., Mizoguchi, Y., Yoshida, Y., Watanabe, S., Yamashita, M., Kitano, S., Sakamoto, H., Nagata, Y., Mitsumori, R., Ozaki, K., Niida, S., Kanai, Y., Hirayama, A., Soga, T., Tsukada, K., Yabuki, N., Shimada, M., Kitazawa, T., Natori, O., Sawada, N., Kato, A., Yoshida, T., Yasuda, K., Ochiai, A., Tsunoda, H., Aoki, K

Cancer Sci. (Cancer Science) 115 ( 6 ) 1763 - 1777 2024.06

Research paper (scientific journal), Joint Work, Accepted, ISSN 13479032

Overcoming resistance to immune checkpoint inhibitors is an important issue in patients with non-small-cell lung cancer (NSCLC). Transcriptome analysis shows that adenocarcinoma can be divided into three molecular subtypes: terminal respiratory unit (TRU), proximal proliferative (PP), and proximal inflammatory (PI), and squamous cell carcinoma (LUSQ) into four. However, the immunological characteristics of these subtypes are not fully understood. In this study, we investigated the immune landscape of NSCLC tissues in molecular subtypes using a multi-omics dataset, including tumor-infiltrating leukocytes (TILs) analyzed using flow cytometry, RNA sequences, whole exome sequences, metabolomic analysis, and clinicopathologic findings. In the PI subtype, the number of TILs increased and the immune response in the tumor microenvironment (TME) was activated, as indicated by high levels of tertiary lymphoid structures, and high cytotoxic marker levels. Patient prognosis was worse in the PP subtype than in other adenocarcinoma subtypes. Glucose transporter 1 (GLUT1) expression levels were upregulated and lactate accumulated in the TME of the PP subtype. This could lead to the formation of an immunosuppressive TME, including the inactivation of antigen-presenting cells. The TRU subtype had low biological malignancy and "cold" tumor-immune phenotypes. Squamous cell carcinoma (LUSQ) did not show distinct immunological characteristics in its respective subtypes. Elucidation of the immune characteristics of molecular subtypes could lead to the development of personalized immune therapy for lung cancer. Immune checkpoint inhibitors could be an effective treatment for the PI subtype. Glycolysis is a potential target for converting an immunosuppressive TME into an antitumorigenic TME in the PP subtype.

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Serum Carbohydrate Antigen 19-9 and Metabolite Hypotaurine Are Predictive Markers for Early Recurrence of Pancreatic Ductal Adenocarcinoma

Nagao, M., Oshima, M., Suto, H., Sugimoto, M., Enomoto, A., Murakami, T., Shimomura, A., Wada, Y., Matsukawa, H., Ando, Y., Kishino, T., Kumamoto, K., Kobara, H., Kamada, H., Masaki, T., Soga, T., Okano, K.

Pancreas (Pancreas) 53 ( 4 ) e301 - e309 2024.04

Research paper (scientific journal), Joint Work, Accepted, ISSN 08853177

Objective: A significant number of patients experience early recurrence after surgical resection for pancreatic ductal adenocarcinoma (PDAC), negating the benefit of surgery. The present study conducted clinicopathologic and metabolomic analyses to explore the factors associated with the early recurrence of PDAC.

Materials and methods: Patients who underwent pancreatectomy for PDAC at Kagawa University Hospital between 2011 and 2020 were enrolled. Tissue samples of PDAC and nonneoplastic pancreas were collected and frozen immediately after resection. Charged metabolites were quantified by capillary electrophoresis-mass spectrometry. Patients who relapsed within 1 year were defined as the early recurrence group.

Results: Frozen tumor tissue and nonneoplastic pancreas were collected from 79 patients. The clinicopathologic analysis identified 11 predictive factors, including preoperative carbohydrate antigen 19-9 levels. The metabolomic analysis revealed that only hypotaurine was a significant risk factor for early recurrence. A multivariate analysis, including clinical and metabolic factors, showed that carbohydrate antigen 19-9 and hypotaurine were independent risk factors for early recurrence (P = 0.045 and P = 0.049, respectively). The recurrence-free survival rate 1 year after surgery with both risk factors was only 25%.

Conclusions: Our results suggested that tumor hypotaurine is a potential metabolite associated with early recurrence. Carbohydrate antigen 19-9 and hypotaurine showed a vital utility for predicting early recurrence.

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A population-based urinary and plasma metabolomics study of environmental exposure to cadmium

Ishibashi, Y., Harada, S., Eitaki, Y., Kurihara, A., Kato, S., Kuwabara, K., Iida, M., Hirata, A., Sata, M., Matsumoto, M., Shibuki, T., Okamura, T., Sugiyama, D., Sato, A., Amano, K., Hirayama, A., Sugimoto, M., Soga, T., Tomita, M., Takebayashi, T.

Environ. Health Prev. (Med.) 29 22 2024.03

Research paper (scientific journal), Joint Work

Background: The application of metabolomics-based profiles in environmental epidemiological studies is a promising approach to refine the process of health risk assessment. We aimed to identify potential metabolomics-based profiles in urine and plasma for the detection of relatively low-level cadmium (Cd) exposure in large population-based studies.

Method: We analyzed 123 urinary metabolites and 94 plasma metabolites detected in fasting urine and plasma samples collected from 1,412 men and 2,022 women involved in the Tsuruoka Metabolomics Cohort Study. Regression analysis was performed for urinary N-acetyl-beta-D-glucosaminidase (NAG), plasma, and urinary metabolites as dependent variables, and urinary Cd (U-Cd, quartile) as an independent variable. The multivariable regression model included age, gender, systolic blood pressure, smoking, rice intake, BMI, glycated hemoglobin, low-density lipoprotein cholesterol, alcohol consumption, physical activity, educational history, dietary energy intake, urinary Na/K ratio, and uric acid. Pathway-network analysis was carried out to visualize the metabolite networks linked to Cd exposure.

Result: Urinary NAG was positively associated with U-Cd, but not at lower concentrations (Q2). Among urinary metabolites in the total population, 45 metabolites showed associations with U-Cd in the unadjusted and adjusted models after adjusting for the multiplicity of comparison with FDR. There were 12 urinary metabolites which showed consistent associations between Cd exposure from Q2 to Q4. Among plasma metabolites, six cations and one anion were positively associated with U-Cd, whereas alanine, creatinine, and isoleucine were negatively associated with U-Cd. Our results were robust by statistical adjustment of various confounders. Pathway-network analysis revealed metabolites and upstream regulator changes associated with mitochondria (ACACB, UCP2, and metabolites related to the TCA cycle).

Conclusion: These results suggested that U-Cd was associated with metabolites related to upstream mitochondrial dysfunction in a dose-dependent manner. Our data will help develop environmental Cd exposure profiles for human populations.

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Trans-omic analysis reveals opposite metabolic dysregulation between feeding and fasting in liver associated with obesity

10. Bai, Y., Morita, K., Kokaji, T., Hatano, A., Ohno, S., Egami, R., Pan, Y., Li, D., Yugi, K., Uematsu, S., Inoue, H., Inaba, Y., Suzuki, Y., Matsumoto, M., Takahashi, M., Izumi, Y., Bamba, T., Hirayama, A., Soga, T., Kuroda, S.

iScience 27 ( 3 ) 109121 - 109121 2024.03

Research paper (scientific journal), Joint Work, Accepted

Dysregulation of liver metabolism associated with obesity during feeding and fasting leads to the breakdown of metabolic homeostasis. However, the underlying mechanism remains unknown. Here, we measured multi-omics data in the liver of wild-type and leptin-deficient obese (ob/ob) mice at ad libitum feeding and constructed a differential regulatory trans-omic network of metabolic reactions. We compared the trans-omic network at feeding with that at 16 h fasting constructed in our previous study. Intermediate metabolites in glycolytic and nucleotide metabolism decreased in ob/ob mice at feeding but increased at fasting. Allosteric regulation reversely shifted between feeding and fasting, generally showing activation at feeding while inhibition at fasting in ob/ob mice. Transcriptional regulation was similar between feeding and fasting, generally showing inhibiting transcription factor regulations and activating enzyme protein regulations in ob/ob mice. The opposite metabolic dysregulation between feeding and fasting characterizes breakdown of metabolic homeostasis associated with obesity.

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Genetic mutation in Escherichia coli genome during adaptation to the murine intestine is optimized for the host diet

Tsukimi, T., Obana, N., Shigemori, S., Arakawa, K., Miyauchi, E., Yang, J., Song, I., Ashino, Y., Wakayama, M., Soga, T., Tomita, M., Ohno, H., Mori, H., Fukuda, S.

mSystems (mSystems) 9 ( 2 ) e0112323 - e0112323 2024.02

Research paper (scientific journal), Joint Work, Accepted

The gut microbiota is closely associated with human health and is greatly impacted by the host diet. Bacteria such as Escherichia coli live in the gut all throughout the life of a human host and adapt to the intestinal environment. Adaptive mutations in E. coli are reported to enhance fitness in the mammalian intestine, but to what extent is still poorly known. It is also unknown whether the host diet affects what genes are mutated and to what extent fitness is affected. This study suggests that genetic mutations in the E. coli K-12 strain are selected in response to the intestinal environment and facilitate efficient utilization of abundant nutrients in the germ-free mouse intestine. Our study provides a better understanding of these intestinal adaptation mechanisms of gut microbes.

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Reliability of Time-Series Plasma Metabolome Data over 6 Years in a Large-Scale Cohort Study

Miyake, A., Harada, S., Sugiyama, D., Matsumoto, M., Hirata, A., Miyagawa, N., Toki, R., Edagawa, S., Kuwabara, K., Okamura, T., Sato, A., Amano, K., Hirayama, A., Sugimoto, M., Soga, T., Tomita, M., Arakawa, K., Takebayashi, T., Iida, M.

Metabolites (Metabolites) 14 ( 1 ) 77 - 77 2024.01

Research paper (scientific journal), Joint Work, Accepted

Studies examining long-term longitudinal metabolomic data and their reliability in large-scale populations are limited. Therefore, we aimed to evaluate the reliability of repeated measurements of plasma metabolites in a prospective cohort setting and to explore intra-individual concentration changes at three time points over a 6-year period. The study participants included 2999 individuals (1317 men and 1682 women) from the Tsuruoka Metabolomics Cohort Study, who participated in all three surveys-at baseline, 3 years, and 6 years. In each survey, 94 plasma metabolites were quantified for each individual and quality control (QC) sample. The coefficients of variation of QC, intraclass correlation coefficients, and change rates of QC were calculated for each metabolite, and their reliability was classified into three categories: excellent, fair to good, and poor. Seventy-six percent (71/94) of metabolites were classified as fair to good or better. Of the 39 metabolites grouped as excellent, 29 (74%) in men and 26 (67%) in women showed significant intra-individual changes over 6 years. Overall, our study demonstrated a high degree of reliability for repeated metabolome measurements. Many highly reliable metabolites showed significant changes over the 6-year period, suggesting that repeated longitudinal metabolome measurements are useful for epidemiological studies.

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Metabolic Hallmarks for Purine Nucleotide Biosynthesis in Small-Cell Lung Carcinoma

Tabata, S., Umemura, S., Narita, M., Udagawa, H., Ishikawa, T., Tsuboi, M., Goto, K., Ishii, G., Tsuchihara, K., Ochiai, A., Kobayashi, S., Soga, T., Makinoshima, H.

Mol. Cancer Res. (Molecular Cancer Research) 22 ( 1 ) 82 - 93 2024.01

Research paper (scientific journal), Joint Work, Accepted, ISSN 15417786

Small-cell lung cancer (SCLC) has a poor prognosis, emphasizing the necessity for developing new therapies. The de novo synthesis pathway of purine nucleotides, which is involved in the malignant growth of SCLC, has emerged as a novel therapeutic target. Purine nucleotides are supplied by two pathways: de novo and salvage. However, the role of the salvage pathway in SCLC and the differences in utilization and crosstalk between the two pathways remain largely unclear. Here, we found that deletion of the HPRT1 gene, which codes for the rate-limiting enzyme of the purine salvage pathway, significantly suppressed tumor growth in vivo in several SCLC cells. We also demonstrated that HPRT1 expression confers resistance to lemetrexol (LMX), an inhibitor of the purine de novo pathway. Interestingly, HPRT1-knockout had less effect on SCLC SBC-5 cells, which are more sensitive to LMX than other SCLC cell lines, suggesting that a preference for either the purine de novo or salvage pathway occurs in SCLC. Furthermore, metabolome analysis of HPRT1-knockout cells revealed increased intermediates in the pentose phosphate pathway and elevated metabolic flux in the purine de novo pathway, indicating compensated metabolism between the de novo and salvage pathways in purine nucleotide biosynthesis. These results suggest that HPRT1 has therapeutic implications in SCLC and provide fundamental insights into the regulation of purine nucleotide biosynthesis.

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Human iPSC-derived renal collecting duct organoid model cystogenesis in ADPKD

Mae, S., Hattanda, F., Morita, H., Nozaki, A., Katagiri, N., Ogawa, H., Teranaka, K., Nishimura, Y., Kudoh, A., Yamanaka, S., Matsuse, K., Ryosaka, M., Watanabe, A., Soga, T., Nishio, S., Osafune, K.

Cell Rep. (Cell Reports) 42 ( 12 ) 113431 - 113431 2023.12

Research paper (scientific journal), Joint Work, Accepted

In autosomal dominant polycystic kidney disease (ADPKD), renal cyst lesions predominantly arise from collecting ducts (CDs). However, relevant CD cyst models using human cells are lacking. Although previous reports have generated in vitro renal tubule cyst models from human induced pluripotent stem cells (hiPSCs), therapeutic drug candidates for ADPKD have not been identified. Here, by establishing expansion cultures of hiPSC-derived ureteric bud tip cells, an embryonic precursor that gives rise to CDs, we succeed in advancing the developmental stage of CD organoids and show that all CD organoids derived from PKD1-/- hiPSCs spontaneously develop multiple cysts, clarifying the initiation mechanisms of cystogenesis. Moreover, we identify retinoic acid receptor (RAR) agonists as candidate drugs that suppress in vitro cystogenesis and confirm the therapeutic effects on an ADPKD mouse model in vivo. Therefore, our in vitro CD cyst model contributes to understanding disease mechanisms and drug discovery for ADPKD.

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Niacin restriction with NAMPT-inhibition is synthetic lethal to neuroendocrine carcinoma

Nomura, M., Oh-uchi, M., Sakamoto, Y., Kudo, K., Yaku, K., Soga, T., Sugiura, Y., Morita, M., Hayashi, K., Miyahara, S., Sato, T., Yamashita, Y., Ito, S., Kikuchi, N., Sato, I., Saito, R., Yaegashi, N., Fukuhara, T., Yamada, H., Shima, H., Nakayama, K., Hirao, A., Kawasaki, K., Arai, Y., Akamatsu, S., Tanuma, S., Sato, T., Nakagawa, T., Tanuma, N.

Nat. Commun. (Nature Communications) 14 ( 1 ) 8095 - 8095 2023.12

Research paper (scientific journal), Joint Work, Accepted

Nicotinamide phosphoribosyltransferase (NAMPT) plays a major role in NAD biosynthesis in many cancers and is an attractive potential cancer target. However, factors dictating therapeutic efficacy of NAMPT inhibitors (NAMPTi) are unclear. We report that neuroendocrine phenotypes predict lung and prostate carcinoma vulnerability to NAMPTi, and that NAMPTi therapy against those cancers is enhanced by dietary modification. Neuroendocrine differentiation of tumor cells is associated with down-regulation of genes relevant to quinolinate phosphoribosyltransferase-dependent de novo NAD synthesis, promoting NAMPTi susceptibility in vitro. We also report that circulating nicotinic acid riboside (NAR), a non-canonical niacin absent in culture media, antagonizes NAMPTi efficacy as it fuels NAMPT-independent but nicotinamide riboside kinase 1-dependent NAD synthesis in tumors. In mouse transplantation models, depleting blood NAR by nutritional or genetic manipulations is synthetic lethal to tumors when combined with NAMPTi. Our findings provide a rationale for simultaneous targeting of NAR metabolism and NAMPT therapeutically in neuroendocrine carcinoma.

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Homogeneous luminescent quantitation of cellular guanosine and adenosine triphosphates (GTP and ATP) using QT-LucGTP&ATP assay

Kopra, K., Mahran, R., Yli-Hollo, T., Tabata, S., Vuorinen, E., Fujii, Y., Vuorinen, I., Ogawa-Iio, A., Hirayama, A., Soga, T., Sasaki, AT. Härmä, H.

Anal. Bioanal. Chem. (Analytical and Bioanalytical Chemistry) 415 ( 27 ) 6689 - 6700 2023.11

Research paper (scientific journal), Joint Work, Accepted, ISSN 16182642

Guanosine triphosphate (GTP) and adenosine triphosphate (ATP) are essential nucleic acid building blocks and serve as energy molecules for a wide range of cellular reactions. Cellular GTP concentration fluctuates independently of ATP and is significantly elevated in numerous cancers, contributing to malignancy. Quantitative measurement of ATP and GTP has become increasingly important to elucidate how concentration changes regulate cell function. Liquid chromatography-coupled mass spectrometry (LC-MS) and capillary electrophoresis-coupled MS (CE-MS) are powerful methods widely used for the identification and quantification of biological metabolites. However, these methods have limitations related to specialized instrumentation and expertise, low throughput, and high costs. Here, we introduce a novel quantitative method for GTP concentration monitoring (GTP-quenching resonance energy transfer (QRET)) in homogenous cellular extracts. CE-MS analysis along with pharmacological control of cellular GTP levels shows that GTP-QRET possesses high dynamic range and accuracy. Furthermore, we combined GTP-QRET with luciferase-based ATP detection, leading to a new technology, termed QT-LucGTP&ATP, enabling high-throughput compatible dual monitoring of cellular GTP and ATP in a homogenous fashion. Collectively, GTP-QRET and QT-LucGTP&ATP offer a unique, high-throughput opportunity to explore cellular energy metabolism, serving as a powerful platform for the development of novel therapeutics and extending its usability across a range of disciplines.

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GREB1 isoform 4 is specifically transcribed by MITF and required for melanoma proliferation

Shinzawa, K., Matsumoto, S., Sada, R., Harada, A., Saitoh, K., Kato, K., Ikeda, S., Hirayama, A., Yokoi, K., Tanemura, A., Nimura, K., Ikawa, M., Soga, T., Kikuchi, A.

Oncogene (Oncogene) 42 ( 42 ) 3142 - 3156 2023.10

Research paper (scientific journal), Accepted, ISSN 09509232

Growth regulation by estrogen in breast cancer 1 (GREB1) is involved in hormone-dependent and -independent tumor development (e.g., hepatoblastoma). In this study, we found that a GREB1 splicing variant, isoform 4 (Is4), which encodes C-terminal half of full-length GREB1, is specifically expressed via microphthalmia-associated transcription factor (MITF) in melanocytic melanoma, and that two MITF-binding E-box CANNTG motifs at the 5'-upstream region of GREB1 exon 19 are necessary for GREB1 Is4 transcription. MITF and GREB1 Is4 were strongly co-expressed in approximately 20% of the melanoma specimens evaluated (17/89 cases) and their expression was associated with tumor thickness. GREB1 Is4 silencing reduced melanoma cell proliferation in association with altered expression of cell proliferation-related genes in vitro. In addition, GREB1 Is4 targeting by antisense oligonucleotide (ASO) decreased melanoma xenograft tumor formation and GREB1 Is4 expression in a BRAFV600E; PTENflox melanoma mouse model promoted melanoma formation, demonstrating the crucial role of GREB1 Is4 for melanoma proliferation in vivo. GREB1 Is4 bound to CAD, the rate-limiting enzyme of pyrimidine metabolism, and metabolic flux analysis revealed that GREBI Is4 is necessary for pyrimidine synthesis. These results suggest that MITF-dependent GREB1 Is4 expression leads to melanoma proliferation and GREB1 Is4 represents a new molecular target in melanoma.

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Decreased liver B vitamin-related enzymes as a metabolic hallmark of cancer cachexia

Kojima, Y., Mishiro-Sato, E., Fujishita, T., Satoh, K., Kajino-Sakamoto, R., Oze, I., Nozawa, K., Narita, Y., Ogata, T., Matsuo, K., Muro, K., Taketo, M., Soga, T., Aoki, A.

Nat. Commun. 14 ( 1 ) 6246 - 6246 2023.10

Research paper (scientific journal), Joint Work, Accepted

Cancer cachexia is a complex metabolic disorder accounting for ~20% of cancer-related deaths, yet its metabolic landscape remains unexplored. Here, we report a decrease in B vitamin-related liver enzymes as a hallmark of systemic metabolic changes occurring in cancer cachexia. Metabolomics of multiple mouse models highlights cachexia-associated reductions of niacin, vitamin B6, and a glycine-related subset of one-carbon (C1) metabolites in the liver. Integration of proteomics and metabolomics reveals that liver enzymes related to niacin, vitamin B6, and glycine-related C1 enzymes dependent on B vitamins decrease linearly with their associated metabolites, likely reflecting stoichiometric cofactor-enzyme interactions. The decrease of B vitamin-related enzymes is also found to depend on protein abundance and cofactor subtype. These metabolic/proteomic changes and decreased protein malonylation, another cachexia feature identified by protein post-translational modification analysis, are reflected in blood samples from mouse models and gastric cancer patients with cachexia, underscoring the clinical relevance of our findings.

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Mitotic perturbation is a key mechanism of action of decitabine in myeloid tumor treatment

Yabushita, T. Chinen, T., Nishiyama, A., Asada, S., Shimura, R., Isobe, T., Yamamoto, K., Sato, N., Enomoto, Y., Tanaka, Y., Fukuyama, T., Satoh, H., Kato, K., Saitoh, K., Ishikawa, T., Soga, T., Nannya, Y., Fukagawa, T., Nakanishi, M., Kitagawa, D., Kitamura, T., Goyama, S.

Cell Rep. 42 ( 9 ) 113098 - 113098 2023.09

Research paper (scientific journal), Joint Work, Accepted

Decitabine (DAC) is clinically used to treat myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Our genome-wide CRISPR-dCas9 activation screen using MDS-derived AML cells indicates that mitotic regulation is critical for DAC resistance. DAC strongly induces abnormal mitosis (abscission failure or tripolar mitosis) in human myeloid tumors at clinical concentrations, especially in those with TP53 mutations or antecedent hematological disorders. This DAC-induced mitotic disruption and apoptosis are significantly attenuated in DNMT1-depleted cells. In contrast, overexpression of Dnmt1, but not the catalytically inactive mutant, enhances DAC-induced mitotic defects in myeloid tumors. We also demonstrate that DAC-induced mitotic disruption is enhanced by pharmacological inhibition of the ATR-CLSPN-CHK1 pathway. These data challenge the current assumption that DAC inhibits leukemogenesis through DNMT1 inhibition and subsequent DNA hypomethylation and highlight the potent activity of DAC to disrupt mitosis through aberrant DNMT1-DNA covalent bonds.

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Human acute leukemia uses branched-chain amino acid catabolism to maintain stemness through regulating PRC2 function

Kikushige, Y., Miyamoto, T., Kochi, Y., Semba, Y., Ohishi, M., Irifune, H., Hatakeyama, K., Kunisaki, Y., Sugio, T., Sakoda, T., Miyawaki, K., Kato, K., Soga, T., Akashi, K.

Blood Adv. (Blood Advances) 7 ( 14 ) 3592 - 3603 2023.07

Research paper (scientific journal), Joint Work, Accepted, ISSN 24739529

Cancer-specific metabolic activities play a crucial role in the pathogenesis of human malignancies. To investigate human acute leukemia-specific metabolic properties, we comprehensively measured the cellular metabolites within the CD34+ fraction of normal hematopoietic stem progenitor cells (HSPCs), primary human acute myelogenous leukemia (AML), and acute lymphoblastic leukemia (ALL) cells. Here, we show that human leukemia cells are addicted to the branched-chain amino acid (BCAA) metabolism to maintain their stemness, irrespective of myeloid or lymphoid types. Human primary acute leukemias had BCAA transporters for BCAA uptake, cellular BCAA, α-ketoglutarate (α-KG), and cytoplasmic BCAA transaminase-1 (BCAT1) at significantly higher levels than control HSPCs. Isotope-tracing experiments showed that in primary leukemia cells, BCAT1 actively catabolizes BCAA using α-KG into branched-chain α-ketoacids, whose metabolic processes provide leukemia cells with critical substrates for the trichloroacetic acid cycle and the synthesis of nonessential amino acids, both of which reproduce α-KG to maintain its cellular level. In xenogeneic transplantation experiments, deprivation of BCAA from daily diet strongly inhibited expansion, engraftment and self-renewal of human acute leukemia cells. Inhibition of BCAA catabolism in primary AML or ALL cells specifically inactivates the function of the polycomb repressive complex 2, an epigenetic regulator for stem cell signatures, by inhibiting the transcription of PRC components, such as zeste homolog 2 and embryonic ectoderm development. Accordingly, BCAA catabolism plays an important role in the maintenance of stemness in primary human AML and ALL, and molecules related to the BCAA metabolism pathway should be critical targets for acute leukemia treatment.

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Convergent genomic diversity and novel BCAA metabolism in intrahepatic cholangiocarcinoma

Kitagawa, A., Osawa, T., Noda, M., Kobayashi, Y., Aki, S., Nakano, Y., Saito, T., Shimizu, D., Komatsu, H., Sugaya, M., Takahashi, J., Kosai, K., Takao, S., Motomura, Y., Sato, K., Hu, Q., Fujii, A., Wakiyama, H., Tobo, T., Uchida, H., Sugimachi, K., Shibata, K., Utsunomiya, T., Kobayashi, S., Ishii, H., Hasegawa, T., Masuda, T., Matsui, Y., Niida, A., Soga, T., Suzuki, Y., Miyano, S., Aburatani, H., Doki, Y., Eguchi, H., Mori, M., Nakayama, K., Shimamura, T., Shibata, T., Mimori, K.

Br. J. Cancer 128 ( 12 ) 2206 - 2217 2023.06

Research paper (scientific journal), Joint Work, Accepted, ISSN 00070920

Background: Driver alterations may represent novel candidates for driver gene-guided therapy; however, intrahepatic cholangiocarcinoma (ICC) with multiple genomic aberrations makes them intractable. Therefore, the pathogenesis and metabolic changes of ICC need to be understood to develop new treatment strategies. We aimed to unravel the evolution of ICC and identify ICC-specific metabolic characteristics to investigate the metabolic pathway associated with ICC development using multiregional sampling to encompass the intra- and inter-tumoral heterogeneity.

Methods: We performed the genomic, transcriptomic, proteomic and metabolomic analysis of 39-77 ICC tumour samples and eleven normal samples. Further, we analysed their cell proliferation and viability.

Results: We demonstrated that intra-tumoral heterogeneity of ICCs with distinct driver genes per case exhibited neutral evolution, regardless of their tumour stage. Upregulation of BCAT1 and BCAT2 indicated the involvement of 'Val Leu Ile degradation pathway'. ICCs exhibit the accumulation of ubiquitous metabolites, such as branched-chain amino acids including valine, leucine, and isoleucine, to negatively affect cancer prognosis. We revealed that this metabolic pathway was almost ubiquitously altered in all cases with genomic diversity and might play important roles in tumour progression and overall survival.

Conclusions: We propose a novel ICC onco-metabolic pathway that could enable the development of new therapeutic interventions.

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Tumor-infiltrating Leukocyte Profiling Defines Three Immune Subtypes of NSCLC with Distinct Signaling Pathways and Genetic Alterations

Aoki, K., Nishito, Y., Motoi, N., Arai, Y., Hiraoka, N., Shibata, T., Sonobe, Y., Kayukawa, Y., Hashimoto, E., Takahashi, M., Fujii, E., Nishizawa, T., Fukuda, H., Ohashi, K., Arai, K., Mizoguchi, Y., Yoshida, Y., Watanabe, S., Yamashita, M., Kitano, S., Sakamoto, H., Nagata, A., Mitsumori, R., Ozaki, K., Niida, S., Kanai, Y., Hirayama, A., Soga, T., Maruyama, T., Tsukada, K., Yabuki, N., Shimada, M., Kitazawa, T., Natori, O., Sawada, N., Kato, A., Yoshida, T., Yasuda, K., Mizuno, H., Tsunoda, H., Ochiai, A.

Cancer Res. Commun. (Cancer Research Communications) 3 ( 6 ) 1026 - 1040 2023.06

Research paper (scientific journal), Joint Work, Accepted

Resistance to immune checkpoint blockade remains challenging in patients with non-small cell lung cancer (NSCLC). Tumor-infiltrating leukocyte (TIL) quantity, composition, and activation status profoundly influence responsiveness to cancer immunotherapy. This study examined the immune landscape in the NSCLC tumor microenvironment by analyzing TIL profiles of 281 fresh resected NSCLC tissues. Unsupervised clustering based on numbers and percentages of 30 TIL types classified adenocarcinoma (LUAD) and squamous cell carcinoma (LUSQ) into the cold, myeloid cell-dominant, and CD8+ T cell-dominant subtypes. These were significantly correlated with patient prognosis; the myeloid cell subtype had worse outcomes than the others. Integrated genomic and transcriptomic analyses, including RNA sequencing, whole-exome sequencing, T-cell receptor repertoire, and metabolomics of tumor tissue, revealed that immune reaction-related signaling pathways were inactivated, while the glycolysis and K-ras signaling pathways activated in LUAD and LUSQ myeloid cell subtypes. Cases with ALK and ROS1 fusion genes were enriched in the LUAD myeloid subtype, and the frequency of TERT copy-number variations was higher in LUSQ myeloid subtype than in the others. These classifications of NSCLC based on TIL status may be useful for developing personalized immune therapies for NSCLC.

Significance: The precise TIL profiling classified NSCLC into novel three immune subtypes that correlates with patient outcome, identifying subtype-specific molecular pathways and genomic alterations that should play important roles in constructing subtype-specific immune tumor microenvironments. These classifications of NSCLC based on TIL status are useful for developing personalized immune therapies for NSCLC.

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Tumor cell-derived spermidine is an oncometabolite that suppress TCR clustering for intratumoral CD8+ T cell activation

Hibino, S., Eto, S., Hangai, S., Endo, K., Ashitani, S., Sugaya, M., Osawa, T., Soga, T., Taniguchi, T., Yanai, H.

Proc. Natl. Acad. Sci. USA 120 ( 24 ) e2305245120 - e2305245120 2023.06

Research paper (scientific journal), Joint Work, Accepted

The activation and expansion of T cells that recognize cancer cells is an essential aspect to antitumor immunity. Tumors may escape destruction by the immune system through ectopic expression of inhibitory immune ligands typically exemplified by the PD-L1/PD-1 pathway. Here, we reveal another facet of tumor evasion from T cell surveillance. By secretome profiling of necrotic tumor cells, we identified an oncometabolite spermidine as a unique inhibitor of T cell receptor (TCR) signaling. Mechanistically, spermidine causes the downregulation of the plasma membrane cholesterol levels, resulting in the suppression of TCR clustering. Using syngeneic mouse models, we show that spermidine is abundantly detected in the tumor immune microenvironment (TIME) and that administration of the polyamine synthesis inhibitor effectively enhanced CD8+ T cell–dependent antitumor responses. Further, the combination of the polyamine synthesis inhibitor with anti-PD-1 immune checkpoint antibody resulted in a much stronger antitumor immune response. This study reveals an aspect of immunosuppressive TIME, wherein spermidine functions as a metabolic T cell checkpoint that may offer a unique approach for promoting tumor immunotherapy.

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Quality Control of Targeted Plasma Lipids in a Large-Scale Cohort Study Using Liquid Chromatography–Tandem Mass Spectrometry

Hirayama, A., Ishikawa, T., Takahashi, H., Yamanaka, S., Ikeda, S., Hirata, A., Harada, S., Sugimoto, M., Soga, T., Tomita, M., Takebayashi, T.

Metabolites 13 ( 4 ) 558 - 558 2023.04

Research paper (scientific journal), Joint Work, Accepted

High-throughput metabolomics has enabled the development of large-scale cohort studies. Long-term studies require multiple batch-based measurements, which require sophisticated quality control (QC) to eliminate unexpected bias to obtain biologically meaningful quantified metabolomic profiles. Liquid chromatography–mass spectrometry was used to analyze 10,833 samples in 279 batch measurements. The quantified profile included 147 lipids including acylcarnitine, fatty acids, glucosylceramide, lactosylceramide, lysophosphatidic acid, and progesterone. Each batch included 40 samples, and 5 QC samples were measured for 10 samples of each. The quantified data from the QC samples were used to normalize the quantified profiles of the sample data. The intra- and inter-batch median coefficients of variation (CV) among the 147 lipids were 44.3% and 20.8%, respectively. After normalization, the CV values decreased by 42.0% and 14.7%, respectively. The effect of this normalization on the subsequent analyses was also evaluated. The demonstrated analyses will contribute to obtaining unbiased, quantified data for large-scale metabolomics.

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L-2hydroxyglutaric acid rewires amino acid metabolism in colorectal cancer via the mTOR-ATF4 axis

Tabata, S., Kojima, Y., Sakamoto, T., Igarashi, K., Umetsu, K., Ishikawa, T., Hirayama, A., Kajino-Sakamoto, R., Sakamoto, N., Yasumoto, K., Okano, K., Suzuki, Y., Yachida, S., Aoki, M., Soga, T.

Oncogene 42 ( 16 ) 1294 - 1307 2023.04

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted, ISSN 09509232

Oncometabolites, such as D/L-2-hydroxyglutarate (2HG), have directly been implicated in carcinogenesis; however, the underlying molecular mechanisms remain poorly understood. Here, we showed that the levels of the L-enantiomer of 2HG (L2HG) were specifically increased in colorectal cancer (CRC) tissues and cell lines compared with the D-enantiomer of 2HG (D2HG). In addition, L2HG increased the expression of ATF4 and its target genes by activating the mTOR pathway, which subsequently provided amino acids and improved the survival of CRC cells under serum deprivation. Downregulating the expression of L-2-hydroxyglutarate dehydrogenase (L2HGDH) and oxoglutarate dehydrogenase (OGDH) increased L2HG levels in CRC, thereby activating mTOR-ATF4 signaling. Furthermore, L2HGDH overexpression reduced L2HG-mediated mTOR-ATF4 signaling under hypoxia, whereas L2HGDH knockdown promoted tumor growth and amino acid metabolism in vivo. Together, these results indicate that L2HG ameliorates nutritional stress by activating the mTOR-ATF4 axis and thus could be a potential therapeutic target for CRC.

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Comparison of hepatic responses to glucose perturbation between healthy and obese mice based on the edge type of network structures

Ito, Y., Uda, S., Kokaji, T., Hirayama, A., Soga, T., Suzuki, Y., Kuroda, S., Kuroda, H.

Sci. Rep. 13 ( 1 ) 4758 - 4758 2023.03

Research paper (scientific journal), Joint Work, Accepted

Interactions between various molecular species in biological phenomena give rise to numerous networks. The investigation of these networks, including their statistical and biochemical interactions, supports a deeper understanding of biological phenomena. The clustering of nodes associated with molecular species and enrichment analysis is frequently applied to examine the biological significance of such network structures. However, these methods focus on delineating the function of a node. As such, in-depth investigations of the edges, which are the connections between the nodes, are rarely explored. In the current study, we aimed to investigate the functions of the edges rather than the nodes. To accomplish this, for each network, we categorized the edges and defined the edge type based on their biological annotations. Subsequently, we used the edge type to compare the network structures of the metabolome and transcriptome in the livers of healthy (wild-type) and obese (ob/ob) mice following oral glucose administration (OGTT). The findings demonstrate that the edge type can facilitate the characterization of the state of a network structure, thereby reducing the information available through datasets containing the OGTT response in the metabolome and transcriptome.

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Hepatic phosphatidylcholine catabolism driven by PNPLA7 and PNPLA8 supplies endogenous choline to replenish the methionine cycle with methyl groups

Hirabayashi, T., Kawaguchi1, M., †Harada, S., Mouri, M., Takamiya, R., Yoshimi, M., Sato, H., Taketomi, Y., Yokoyama, K., Kobayashi, T., Tokuoka, S.M., Kita, Y., Yoda, E., Hara, S., Mikami, K., Nishito, Y., Kikuchi, N., Nakata, R., Kaneko, M., Kiyonari, H., Kasahara, K., Aiba, T., Ohsako, S., Ikeda, K., Soga, T., Kurano, M., Yatomi, Y., Murakami, M.

Cell Rep. 42 ( 2 ) 111940 2023.02

Research paper (scientific journal), Joint Work, Accepted

Choline supplies methyl groups for regeneration of methionine and the methyl donor S-adenosylmethionine in the liver. Here, we report that the catabolism of membrane phosphatidylcholine (PC) into water-soluble glycerophosphocholine (GPC) by the phospholipase/lysophospholipase PNPLA8-PNPLA7 axis enables endogenous choline stored in hepatic PC to be utilized in methyl metabolism. PNPLA7-deficient mice show marked decreases in hepatic GPC, choline, and several metabolites related to the methionine cycle, accompanied by various signs of methionine insufficiency, including growth retardation, hypoglycemia, hypolipidemia, increased energy consumption, reduced adiposity, increased fibroblast growth factor 21 (FGF21), and an altered histone/DNA methylation landscape. Moreover, PNPLA8-deficient mice recapitulate most of these phenotypes. In contrast to wild-type mice fed a methionine/choline-deficient diet, both knockout strains display decreased hepatic triglyceride, likely via reductions of lipogenesis and GPC-derived glycerol flux. Collectively, our findings highlight the biological importance of phospholipid catabolism driven by PNPLA8/PNPLA7 in methyl group flux and triglyceride synthesis in the liver.

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Optogenetic decoding of Akt2-regulated metabolic signaling pathways in skeletal muscle cells using transomics analysis

Kawamura, G., Kokaji, T., Kawata, K., Sekine, Y., Suzuki, Y., Soga, T., Ueda, Y., Endo, M., Kuroda, S., Ozawa, T.

Sci Signal 16 ( 773 ) eabn0782 - eabn0782 2023.02

Research paper (scientific journal), Joint Work, Accepted, ISSN 19450877

Insulin regulates various cellular metabolic processes by activating specific isoforms of the Akt family of kinases. Here, we elucidated metabolic pathways that are regulated in an Akt2-dependent manner. We constructed a transomics network by quantifying phosphorylated Akt substrates, metabolites, and transcripts in C2C12 skeletal muscle cells with acute, optogenetically induced activation of Akt2. We found that Akt2-specific activation predominantly affected Akt substrate phosphorylation and metabolite regulation rather than transcript regulation. The transomics network revealed that Akt2 regulated the lower glycolysis pathway and nucleotide metabolism and cooperated with Akt2-independent signaling to promote the rate-limiting steps in these processes, such as the first step of glycolysis, glucose uptake, and the activation of the pyrimidine metabolic enzyme CAD. Together, our findings reveal the mechanism of Akt2-dependent metabolic pathway regulation, paving the way for Akt2-targeting therapeutics in diabetes and metabolic disorders.

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NRF3 activates mTORC1 arginine-dependently for cancer cell viability

Hirose, S., Waku, T., Tani, M., Masuda, H., Endo, K., Ashitani, S., Aketa, I., Kitano, H., Nakada, S., Wada, A., Hatanaka, A., Osawa, T., Soga, T., Kobayashi, A.

iScience 26 ( 2 ) 106045 2023.02

Research paper (scientific journal), Joint Work, Accepted

Cancer cells coordinate the mTORC1 signals and the related metabolic pathways to robustly and rapidly grow in response to nutrient conditions. Although a CNC-family transcription factor NRF3 promotes cancer development, the biological relevance between NRF3 function and mTORC1 signals in cancer cells remains unknown. Hence, we showed that NRF3 contributes to cancer cell viability through mTORC1 activation in response to amino acids, particularly arginine. NRF3 induced SLC38A9 and RagC expression for the arginine-dependent mTORC1 recruitment onto lysosomes, and it also enhanced RAB5-mediated bulk macropinocytosis and SLC7A1-mediated selective transport for arginine loading into lysosomes. Besides, the inhibition of the NRF3–mTORC1 axis impaired mitochondrial function, leading to cancer cell apoptosis. Consistently, the aberrant upregulation of the axis caused tumor growth and poor prognosis. In conclusion, this study sheds light on the unique function of NRF3 in arginine-dependent mTORC1 activation and the pathophysiological aspects of the NRF3–mTORC1 axis in cancer development.

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Mitochondria directly sense osmotic stress to trigger rapid metabolic remodeling via regulation of pyruvate dehydrogenase phosphorylation

Ikizawa, T., Ikeda, K., Arita, M., Kitajima, S., Soga, T., Ichijo, H., Naguro, I.

J. Biol. Chem. 299 ( 2 ) 102837 2023.02

Research paper (scientific journal), Joint Work, Accepted, ISSN 00219258

A high-salt diet significantly impacts various diseases, ilncluding cancer and immune diseases. Recent studies suggest that the high-salt/hyperosmotic environment in the body may alter the chronic properties of cancer and immune cells in the disease context. However, little is known about the acute metabolic changes in hyperosmotic stress. Here, we found that hyperosmotic stress for a few minutes induces Warburg-like metabolic remodeling in HeLa and Raw264.7 cells and suppresses fatty acid oxidation. Regarding Warburg-like remodeling, we determined that the pyruvate dehydrogenase phosphorylation status was altered bidirectionally (high in hyperosmolarity and low in hypoosmolarity) to osmotic stress in isolated mitochondria, suggesting that mitochondria themselves have an acute osmosensing mechanism. Additionally, we demonstrate that Warburg-like remodeling is required for HeLa cells to maintain ATP levels and survive under hyperosmotic conditions. Collectively, our findings suggest that cells exhibit acute metabolic remodeling under osmotic stress via the regulation of pyruvate dehydrogenase phosphorylation by direct osmosensing within mitochondria.

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Metabolomics of small extracellular vesicles derived from isocitrate dehydrogenase 1-mutant HCT116 cells collected by semi-automated size exclusion chromatography

Hayasaka, R., Tabata, S., Hasebe, M., Ikeda, S., Hikita, T., Oneyama, C., Yoshitake, J., Onoshima, D., Takahashi, K., Shibata, T., Uchida, K., Baba, Y.., Soga, T., Tomita, M., Hirayama, A.

Front. Mol. Biosci. 9 1049402 2023.01

Research paper (scientific journal), Joint Work, Accepted

Cancer-derived small extracellular vesicles (sEVs) are multifunctional particles with a lipid bilayer structure that are involved in cancer progression, such as malignant proliferation, distant metastasis, and cancer immunity evasion. The separation protocol used to isolate sEVs is an important process and thus, several have been developed, including ultracentrifugation (UC), size exclusion chromatography (SEC), and affinity purification using antibodies against sEV surface antigens. However, the effects of different separation methods on sEV components have not been adequately examined. Here, we developed a semi-automated system for collecting sEVs by combining SEC and preparative high-performance liquid chromatography and applied it to metabolome analysis. The developed SEC system could recover sEVs more efficiently and non-destructively than UC, suggesting that it is an appropriate recovery method for metabolic analysis and reflects biological conditions. Furthermore, using the developed SEC system, we performed metabolome analysis of sEVs from isocitrate dehydrogenase 1 (IDH)-mutated human colon HCT116 cells, which produce the oncogenic metabolite, 2-hydroxyglutaric acid (2-HG). IDH1-mutated HCT116 cells released significantly more sEVs than wild-type (WT) cells. The metabolomic profiles of IDH1 mutant and WT cells showed distinct differences between the cells and their sEVs. Notably, in IDH mutant cells, large amounts of 2-HG were detected not only in cells, but also in sEVs. These results indicate that the SEC system we developed has wide potential applications in sEVs research.

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Advances in capillary electrophoresis mass spectrometry for metabolomics

Soga, T.

Trends Anal. Chem. 158 116883 2023.01

Research paper (scientific journal), Single Work, Accepted, ISSN 01659936

Capillary electrophoresis mass spectrometry (CE-MS) is now regarded as a powerful tool for the comprehensive analysis of low-molecular-weight charged metabolites. Over the past decades, several CE-MS methods have been established and made significant contributions to metabolomics research in various areas. This review describes the general strategies and principles of the CE-MS-based metabolomics analyses, including sample preparation, CE separation modes, interfacing techniques, and mass spectrometers. Furthermore, new techniques to increase throughput ability in CE-MS for metabolomics are highlighted. Finally, general conclusions and perspectives are provided.

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Interleukin-6 blockade reduces salt-induced cardiac inflammation and fibrosis in subtotal nephrectomized mice

Tanaka, H., Sun, T., Kinashi, H., Kamiya, K., Yamaguchi, M., Nobata, H., Sakata, F., Kim, H., Mizuno, M., Kunoki, S., Sakai, Y., Hirayama, A., Soga, T., Yoshikawa, K., Ishimoto, T., Ito, Y.

Am. J. Physiol. Renal Physiol. 323 ( 6 ) F654 - F665 2022.12

Research paper (scientific journal), Joint Work, Accepted, ISSN 1931857X

Cardiovascular disease is the most common comorbidity in patients with chronic kidney disease (CKD), affecting both their prognosis and quality of life. Cardiac fibrosis is common in patients with CKD with left ventricular diastolic dysfunction, and it is associated with increased risk of heart failure and mortality. Recent evidence suggests that high salt intake activates immune responses associated with local accumulation of sodium. We reported that high salt intake promotes cardiac inflammation in subtotal nephrectomized (Nx) mice. We investigated the effects of administration of MR16-1, a rat anti-mouse monoclonal interleukin (IL)-6 receptor antibody, in Nx mice with salt loading (Nx-salt). Expression of monocyte chemoattractant protein-1, tumor necrosis factor-α, IL-1β, and IL-6 mRNAs and macrophage infiltration was significantly reduced in the heart of Nx-salt mice treated with MR16-1 (Nx-salt-MR16-1) compared with Nx-salt mice treated with control rat rat IgG1 (Nx-salt-rat IgG1). Correspondingly, cardiac fibrosis was significantly attenuated in Nx-salt-MR16-1 mice compared with Nx-salt-rat IgG1 mice. Furthermore, in the heart of Nx-salt-MR16-1 mice, expression of mRNA for nicotinamide adenine dinucleotide phosphate oxidase-2, an oxidative stress marker, was significantly downregulated compared with Nx-salt-rat IgG1 mice. Increases in cardiac metabolites, including histidine and γ-butyrobetaine, were also reversed by IL-6 blockade treatment. In conclusion, IL-6 blockade exerts anti-inflammatory, antifibrotic, and partial antioxidative effects in the heart of Nx-salt mice.NEW & NOTEWORTHY In the present study, IL-6 blockade exerted anti-inflammatory, antifibrotic, and partial antioxidative effects on the hearts of mice with CKD on a high-salt diet. Therefore, IL-6 potentially mediates cardiac fibrosis induced by high salt intake in patients with CKD, a finding with therapeutic implications. Of note, the next therapeutic implication may simply be the reinforcement of low-salt diets or diuretics and further research on the anti-inflammatory effects of these measures rather than IL-6 blockade with high-salt diet.

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SETD1A regulates transcriptional pause release of heme biosynthesis genes in leukemia

Hoshii, T., Perlee, S., Kikuchi, S., Rahmutulla, B, Fukuyo, M., Masuda, T., Ohtsuki, S., Soga, T., Nabet, B., Kaneda, A.

Cell Rep. 41 ( 9 ) 111727 2022.11

Research paper (scientific journal), Joint Work, Accepted

Histone methyltransferase SETD1A is critical for acute myeloid leukemia (AML) cell survival, but the molecular mechanism driving SETD1A gene regulation remains elusive. To delineate the role of SETD1A, we utilize a protein degrader technology to induce rapid SETD1A degradation in AML cell lines. SETD1A degradation results in immediate downregulation of transcripts associated with DNA repair and heme biosynthesis pathways. CRISPR-based functional analyses and metabolomics reveal an essential role of SETD1A to maintain mitochondrial respiration in AML cells. These SETD1A targets are enriched in head-to-head (H2H) genes. SETD1A degradation disrupts a non-enzymatic SETD1A domain-dependent cyclin K function, increases the Ser5P RNA polymerase II (RNAPII) at the transcriptional start site (TSS), and induces the promoter-proximal pausing of RNAPII in a strand-specific manner. This study reveals a non-enzymatic role for SETD1A in transcriptional pause release and provides insight into the mechanism of RNAPII pausing and its function in cancer.

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Relationship between [18 F]FDG PET/CT and metabolomics in patients with colorectal cancer

Imajo, M., Norikane, T., Yamamoto, Y., Maeda, Y., Saitoh, K., Kato, K., Soga, T., Okano, K., Nishiyama, Y.

Metabolomics 18 ( 11 ) 91 2022.11

Research paper (scientific journal), Joint Work, Accepted

Introduction: Advances in metabolomics have significantly improved cancer detection, diagnosis, treatment, and prognosis.

Objectives: To investigate the relationship between metabolic tumor volume (MTV) using 2-deoxy-2-[18F]fluoro-D-glucose (FDG) positron emission tomography (PET)/ computed tomography (CT) and metabolomics data in patients with colorectal cancer (CRC).

Methods: The metabolome in tumor tissues was analyzed using capillary electrophoresis time-of-flight mass spectrometry in 33 patients with newly diagnosed CRC who underwent FDG PET/CT before treatment and had tumor tissue post-surgery. Based on the FDG PET data, MTV was calculated and was dichotomized according to the median value, and tumors were divided into low-MTV and high-MTV tumors. Metabolomics data were compared between the low-MTV and high-MTV tumors.

Results: The levels of most glycolysis-related metabolites were not different between low-MTV and high-MTV tumors. The level of component of the initial part of the tricarboxylic acid (TCA) cycle, citrate, was significantly lower in the high-MTV tumor than in the low-MTV tumor. The TCA intermediate succinate level was significantly higher in the high-MTV tumor than in the low-MTV tumor. In contrast, the TCA intermediate fumarate level was significantly lower in the high-MTV tumor than in the low-MTV tumor. The levels of many amino acids were significantly higher in the high-MTV tumor than in the low-MTV tumor.

Conclusions: Although preliminary, these results suggest that tumors with high FDG metabolism in CRC may obtain more energy by using a reverse reaction of the TCA cycle and amino-acid metabolism. However, further research is required to clarify this relationship.

Keywords: Colorectal cancer; FDG; Metabolome; PET/CT.

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Comprehensive metabolome analysis of intracellular metabolites in cultured cells

Kudo, R., Igarashi, K., Soga, T., Ishikawa, T., Saito, Y.

STAR Protoc. 3 ( 3 ) 101531 2022.09

Research paper (scientific journal), Joint Work, Accepted

Capillary electrophoresis mass spectrometry (CE-MS) can measure the intracellular amount of highly polar and charged metabolites; liquid chromatography mass spectrometry (LC-MS) can quantify hydrophobic metabolites. A comprehensive metabolome analysis requires independent sample preparation for LC-MS and CE-MS. Here, we present a protocol to prepare for sequentially analyzing the metabolites from one sample. Here we describe the steps for breast cancer cell lines, MCF-7 cells, but the protocol can be applied to other cell types.

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Development of Fluorophosphoramidate as a Biocompatibly Transformable Functional Group and its Application as a Phosphate Prodrug for Nucleoside Analogs

Yoshida, Y., Zheng, T., Tanabe, W., Tomoike, F., Hashiya, F., Suzuki, T., Hirota, S., Saiki, Y., Horii, A., Hirayama, A., Soga, T., Kimura, Y., Abe, H.

ChemMedChem 17 ( 17 ) e202200188 2022.09

Research paper (scientific journal), Joint Work, Accepted

Synthetic phosphate-derived functional groups are important for controlling the function of bioactive molecules in vivo. Herein we describe the development of a new type of biocompatible phosphate analog, a fluorophosphoramidate (FPA) functional group that has characteristic P-F and P-N bonds. We found that FPA with a primary amino group was relatively unstable in aqueous solution and was converted to a monophosphate, while FPA with a secondary amino group was stable. Furthermore, by improving the molecular design of FPA, we developed a reaction in which a secondary amino group is converted to a primary amino group in the intracellular environment and clarified that the FPA group functions as a phosphate prodrug of nucleoside. Various FPA-gemcitabine derivatives were synthesized and their toxicity to cancer cells were evaluated. One of the FPA-gemcitabine derivatives showed superior toxicity compared with gemcitabine and its ProTide prodrug, which methodology is widely used in various nucleoside analogs, including anti-cancer and anti-virus drugs.

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Brown adipose tissue dysfunction promotes heart failure via a trimethylamine N-oxide-dependent mechanism

Yoshida, Y., Shimizu, I., Shimada, A., Nakahara, K., Yanagisawa, S., Kubo, M., Fukuda, S., Ishii, C., Yamamoto, H., Ishikawa, T., Kano, K., Aoki, J., Katsuumi, G., Suda, M., Ozaki, K., Yoshida, Y., Okuda, S., Ohta, S., Okamoto, S., Minokoshi, Y., Oda, K., Sasaoka, T., Abe, M., Sakimura, K., Kubota, Y., Yoshimura, N., Kajimura, S., Zuriaga, M., Walsh, K., Soga, T., Minamino, T.

Sci. Rep. 12 14883 2022.09

Research paper (scientific journal), Joint Work, Accepted

Low body temperature predicts a poor outcome in patients with heart failure, but the underlying pathological mechanisms and implications are largely unknown. Brown adipose tissue (BAT) was initially characterised as a thermogenic organ, and recent studies have suggested it plays a crucial role in maintaining systemic metabolic health. While these reports suggest a potential link between BAT and heart failure, the potential role of BAT dysfunction in heart failure has not been investigated. Here, we demonstrate that alteration of BAT function contributes to development of heart failure through disorientation in choline metabolism. Thoracic aortic constriction (TAC) or myocardial infarction (MI) reduced the thermogenic capacity of BAT in mice, leading to significant reduction of body temperature with cold exposure. BAT became hypoxic with TAC or MI, and hypoxic stress induced apoptosis of brown adipocytes. Enhancement of BAT function improved thermogenesis and cardiac function in TAC mice. Conversely, systolic function was impaired in a mouse model of genetic BAT dysfunction, in association with a low survival rate after TAC. Metabolomic analysis showed that reduced BAT thermogenesis was associated with elevation of plasma trimethylamine N-oxide (TMAO) levels. Administration of TMAO to mice led to significant reduction of phosphocreatine and ATP levels in cardiac tissue via suppression of mitochondrial complex IV activity. Genetic or pharmacological inhibition of flavin-containing monooxygenase reduced the plasma TMAO level in mice, and improved cardiac dysfunction in animals with left ventricular pressure overload. In patients with dilated cardiomyopathy, body temperature was low along with elevation of plasma choline and TMAO levels. These results suggest that maintenance of BAT homeostasis and reducing TMAO production could be potential next-generation therapies for heart failure.

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Salivary metabolomics with machine learning for colorectal cancer detection

Kuwabara, H., Katsumata, K., Iwabuthi, A., Udo, R., Tago, T., Kasahara, K., Mazaki, J., Enomoto, M., Ishizaki, T., Soya, R., Kaneko, M., Ota, S., Enomoto, A., Soga, T., Tomita, M., Sunamura, M., Tsuchida, A., Sugimoto, M., Nagakawa, Y.

Cancer Sci. 113 ( 9 ) 3234 - 3243 2022.09

Research paper (scientific journal), Joint Work, Accepted

As the worldwide prevalence of colorectal cancer (CRC) increases, it is vital to reduce its morbidity and mortality through early detection. Saliva-based tests are an ideal noninvasive tool for CRC detection. Here, we explored and validated salivary biomarkers to distinguish patients with CRC from those with adenoma (AD) and healthy controls (HC). Saliva samples were collected from patients with CRC, AD, and HC. Untargeted salivary hydrophilic metabolite profiling was conducted using capillary electrophoresis–mass spectrometry and liquid chromatography–mass spectrometry. An alternative decision tree (ADTree)-based machine learning (ML) method was used to assess the discrimination abilities of the quantified metabolites. A total of 2,602 unstimulated saliva samples were collected from subjects with CRC (n = 231), AD (n = 54), and HC (n = 2,317). Data were randomly divided into training (n = 1,301) and validation datasets (n = 1,301). The clustering analysis showed a clear consistency of aberrant metabolites between the two groups. The ADTree model was optimized through cross-validation (CV) using the training dataset, and the developed model was validated using the validation dataset. The model discriminating CRC + AD from HC showed area under the receiver operating characteristic curves (AUC) of 0.860 (95% confidence interval [CI]: 0.828-0.891) for CV and 0.870 (95% CI: 0.837-0.903) for the validation dataset. The other model discriminating CRC from AD + HC showed an AUC of 0.879 (95% CI: 0.851-0.907) and 0.870 (95% CI: 0.838 - 0.902), respectively. Salivary metabolomics combined with ML demonstrated high accuracy and versatility in detecting CRC.

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In vivo transomic analyses of glucose-responsive metabolism in skeletal muscle reveal core differences between the healthy and obese states

Kokaji, T., Eto, M., Hatano, A., Yugi K., Morita, K., Ohno, S., Fujii, M., Hironaka, K., Ito, Y., Egami, R., Uematsu, S., Terakawa, A., Pan, Y., Maehara, H., Li, D., Bai, Y., Tsuchiya, T., Ozaki, H., Inoue, H., Kubota, H., Suzuki, Y., Hirayama, A., Soga, T., Kuroda, S.

Sci. Rep. 12 13719 2022.08

Research paper (scientific journal), Joint Work, Accepted

Metabolic regulation in skeletal muscle is essential for blood glucose homeostasis. Obesity causes insulin resistance in skeletal muscle, leading to hyperglycemia and type 2 diabetes. In this study, we performed multiomic analysis of the skeletal muscle of wild-type (WT) and leptin-deficient obese (ob/ob) mice, and constructed regulatory transomic networks for metabolism after oral glucose administration. Our network revealed that metabolic regulation by glucose-responsive metabolites had a major effect on WT mice, especially carbohydrate metabolic pathways. By contrast, in ob/ob mice, much of the metabolic regulation by glucose-responsive metabolites was lost and metabolic regulation by glucose-responsive genes was largely increased, especially in carbohydrate and lipid metabolic pathways. We present some characteristic metabolic regulatory pathways found in central carbon, branched amino acids, and ketone body metabolism. Our transomic analysis will provide insights into how skeletal muscle responds to changes in blood glucose and how it fails to respond in obesity.

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Tumor metabolic alterations after neoadjuvant chemoradiotherapy predict postoperative recurrence in patients with pancreatic cancer

Wada, Y., Okano, K., Sato, K., Sugimoto, M., Shimomura, A., Nagao, M., Matsukawa, H., Ando, Y., Suto, H., Oshima, M., Kondo, A., Asano, E., Kishino, T., Kumamoto, K., Kobara, H., Kamada, H., Masaki, T., Soga, T., Suzuki, Y.

Jpn J Clin Oncol. 52 ( 8 ) 879 - 887 2022.08

Research paper (scientific journal), Joint Work, Accepted

Objective: We investigated the metabolic changes in pancreatic ductal adenocarcinoma to identify the mechanisms of treatment response of neoadjuvant chemoradiation therapy.

Methods: Frozen tumor and non-neoplastic pancreas tissues were prospectively obtained from 88 patients with pancreatic ductal adenocarcinoma who underwent curative-intent surgery. Sixty-two patients received neoadjuvant chemoradiation therapy and 26 patients did not receive neoadjuvant therapy (control group). Comprehensive analysis of metabolites in tumor and non-neoplastic pancreatic tissue was performed by capillary electrophoresis-mass spectrometry.

Results: Capillary electrophoresis-mass spectrometry detected 90 metabolites for analysis among more than 500 ionic metabolites quantified. There were significant differences in 27 tumor metabolites between the neoadjuvant chemoradiation therapy and control groups. There were significant differences in eight metabolites [1-MethylnNicotinamide, Carnitine, Glucose, Glutathione (red), N-acetylglucosamine 6-phosphate, N-acetylglucosamine 1-phosphate, UMP, Phosphocholine] between good responder and poor responder for neoadjuvant chemoradiation therapy. Among these metabolites, phosphocholine, Carnitine and Glutathione were associated with recurrence-free survival only in the neoadjuvant chemoradiation therapy group. Microarray confirmed marked gene suppression of choline transporters [CTL1-4 (SLC44A1-44A4)] in pancreatic ductal adenocarcinoma tissue of neoadjuvant chemoradiation therapy group.

Conclusion: The present study identifies several important metabolic consequences and potential neoadjuvant chemoradiation therapy targets in pancreatic ductal adenocarcinoma. Choline metabolism is one of the key pathways involved in recurrence of the patients with pancreatic ductal adenocarcinoma who received neoadjuvant chemoradiation therapy.

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Glutamine deficiency in solid tumor cells confers resistance to ribosomal RNA synthesis inhibitors

Pan, M., Zorbas, C., Sugaya, M., Ishiguro, K., Kato, M., Nishida, M., Zhang, H.F., Candeias, M.M., Okamoto, A., Ishikawa, T., Soga, T., Aburatani, H., Sakai, J., Matsumura, Y., Suzuki, T., Proud, C.G., Lafontaine, D.L.J., Osawa, T.

Nat Commun. 13 ( 1 ) 3706 2022.06

Research paper (scientific journal), Joint Work, Accepted

Ribosome biogenesis is an energetically expensive program that is dictated by nutrient availability. Here we report that nutrient deprivation severely impairs precursor ribosomal RNA (pre-rRNA) processing and leads to the accumulation of unprocessed rRNAs. Upon nutrient restoration, pre-rRNAs stored under starvation are processed into mature rRNAs that are utilized for ribosome biogenesis. Failure to accumulate pre-rRNAs under nutrient stress leads to perturbed ribosome assembly upon nutrient restoration and subsequent apoptosis via uL5/uL18-mediated activation of p53. Restoration of glutamine alone activates p53 by triggering uL5/uL18 translation. Induction of uL5/uL18 protein synthesis by glutamine is dependent on the translation factor eukaryotic elongation factor 2 (eEF2), which is in turn dependent on Raf/MEK/ERK signaling. Depriving cells of glutamine prevents the activation of p53 by rRNA synthesis inhibitors. Our data reveals a mechanism that tumor cells can exploit to suppress p53-mediated apoptosis during fluctuations in environmental nutrient availability.

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Comparative Metabolomics of Small Molecules Specifically Expressed in the Dorsal or Ventral Marginal Zones in Vertebrate Gastrula

Suzuki, Y., Hayasaka, R., Hasebe, M., Ikeda, S., Soga, T., Tomita, M., Hirayama, A., Kuroda, H.

Metabolites 12 ( 6 ) 566 2022.06

Research paper (scientific journal), Joint Work, Accepted

Many previous studies have reported the various proteins specifically secreted as inducers in the dorsal or ventral regions in vertebrate gastrula. However, little is known about the effect on cell fate of small molecules below 1000 Da. We therefore tried to identify small molecules specifically expressed in the dorsal marginal zone (DMZ) or ventral marginal zone (VMZ) in vertebrate gastrula. Small intracellular and secreted molecules were detected using explants and supernatant samples. Hydrophilic metabolites were analyzed by capillary ion chromatography–mass spectrometry and liquid chromatography–mass spectrometry, and lipids were analyzed by supercritical fluid chromatography–tandem mass spectrometry. In total, 190 hydrophilic metabolites and 396 lipids were identified. The DMZ was found to have high amounts of glycolysis- and glutathione metabolism-related metabolites in explants, and the VMZ was richer in purine metabolism-related metabolites. We also discovered some hydrophilic metabolites and lipids differentially contained in the DMZ or VMZ. Our research would contribute to a deeper understanding of the cellular physiology that regulates early embryogenesis.

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AGE/RAGE axis regulates reversible transition to quiescent states of ALK-rearranged NSCLC and pancreatic cancer cells in monolayer cultures

Kadonosono, T., Miyamoto, K., Sasaki, S., Matsuo, Y., Kitajima, S., Wang, Q., Endo, M., Nibori, M., Kuchimaru, T., Soga, T., Hirota, K., Kizaka-Kondoh, S.

Sci. Rep. 12 9886 2022.06

Research paper (scientific journal), Joint Work, Accepted

Cancer recurrence due to tumor cell quiescence after therapy and long-term remission is associated with cancer-related death. Previous studies have used cell models that are unable to return to a proliferative state; thus, the transition between quiescent and proliferative states is not well understood. Here, we report monolayer cancer cell models wherein the human non-small cell lung carcinoma cell line H2228 and pancreatic cancer cell line AsPC-1 can be reversibly induced to a quiescent state under hypoxic and serum-starved (HSS) conditions. Transcriptome and metabolome dual-omics profiles of these cells were compared with those of the human lung adenocarcinoma cell line A549, which was unable to enter a quiescent state under HSS conditions. The quiescence-inducible cells had substantially lower intracellular pyruvate and ATP levels in the quiescent state than in the proliferative state, and their response to sudden demand for energy was dramatically reduced. Furthermore, in quiescence-inducible cells, the transition between quiescent and proliferative states of these cells was regulated by the balance between the proliferation-promoting Ras and Rap1 signaling and the suppressive AGE/RAGE signaling. These cell models elucidate the transition between quiescent and proliferative states, allowing the development of drug-screening systems for quiescent tumor cells.

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Different types of reactions to E7386 among colorectal cancer patient-derived organoids and corresponding CAFs

Imai, T., Naruse, M., Ochiai M., Matsumoto K., Ikeda S., Kani, M., Kato, Y., Hirayama, A., Soga, T., Hori, Y., Yokoi, A., Ochiai, A.

Oncol. Lett. 24 ( 1 ) 221 2022.05

Research paper (scientific journal), Joint Work, Accepted

Colorectal cancer (CRC) harbors genetic alterations in a component of the Wnt signaling pathway in ~90% of cases. In addition, the Wnt signaling pathway has been previously suggested to serve a notable role in the pathophysiology of CRC cells and cancer‑associated fibroblasts (CAFs). In the present study, the possible effects of E7386, a selective inhibitor of the interaction between β‑catenin and the cAMP response element‑binding protein‑binding protein, were evaluated using organoids and the corresponding CAFs derived from patients with CRC. E7386 at 100 nM was revealed to decrease the viability of CRC organoids and CAFs. Analysis of the gene expression profiles revealed marked changes in the expression levels of different types of cancer‑associated genes associated with E7386 concentrations in the organoids and/or CAFs, such as those regulating glucose and amino acid metabolism [phosphoenolpyruvate carboxykinase 2, asparagine synthetase (glutamine‑hydrolyzing), phosphoserine aminotransferase 1 and phosphoglycerate dehydrogenase], stimulation of natural killer cell‑mediated cytotoxicity (UL16‑binding protein 1) and modification of the Wnt/β‑catenin signaling pathway (indicated by very low density lipoprotein receptor). Results of the hydrophilic metabolome analysis in the organoids were consistent with those of the transcriptomic analysis. In vivo experiments used corresponding xenograft models, although changes in volume of tumor tissues could not be observed at 50 mg/kg body weight twice a day for 14 days, results on the protein expression levels partially supported those in the in vitro experiments. In conclusion, different types of reactions, such as alterations in the glucose and amino acid metabolic pathways, stimulation of stress responses and NK‑cell mediated cytotoxicity and components in the Wnt/β‑catenin signaling pathway, to E7386 in the CRC organoids and corresponding CAFs were observed under conditions with decreased cell viability. However, further mechanistic studies to clarify the relationships with Wnt/β‑catenin signaling pathway and these reactions using preclinical models and biomarker studies using clinical human samples are required for verification of the present preclinical biomarkers.

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Differing impact of phosphoglycerate mutase 1-deficiency on brown and white adipose tissue

Yoshida, Y., Shimizu, I., Hsiao, Y.T., Suda, M., Katsuumi, G., Seki, M., Suzuki, Y., Okuda, S., Soga, T., Minamino, T.

iScience 25 ( 5 ) 104268 2022.05

Research paper (scientific journal), Joint Work, Accepted

Brown adipose tissue (BAT) is a metabolically active organ that contributes to the thermogenic response to cold exposure. In addition, other thermogenic cells termed beige adipocytes are generated in white adipose tissue (WAT) by cold exposure. Although activation of brown/beige adipose tissue is associated with mobilization of both glucose and lipids, few studies have focused on the role of glycolytic enzymes in regulating adipose tissue function. We generated mouse models with specific deletion of the glycolytic enzyme phosphoglycerate mutase 1 (PGAM1) from adipose tissue. Deletion of Pgam1 from both BAT and WAT promoted whitening of BAT with beiging of visceral WAT, whereas deletion of Pgam1 from BAT alone led to whitening of BAT without beiging of WAT. Our results demonstrate a potential role of glycolytic enzymes in beiging of visceral WAT and suggest that PGAM1 would be a novel therapeutic target in obesity and diabetes.

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Polarity protein SCRIB interacts with SLC3A2 to regulate proliferation and tamoxifen resistance in ER+ breast cancer

Saito, Y., Matsuda, S., Ohnishi, N., Endo, K., Ashitani, S., Ohishi, M., Ueno, A., Tomita, M., Ueda, K., *Soga, T., *Muthuswamy, S.K

Commun. Biol. 5 ( 1 ) 403 2022.05

Research paper (scientific journal), Joint Work, Accepted

Estrogen receptor (ER) positive breast cancer represents 75% of all breast cancers in women. Although patients with ER+ cancers receive endocrine therapies, more than 30% develop resistance and succumb to the disease, highlighting the need to understand endocrine resistance. Here we show an unexpected role for the cell polarity protein SCRIB as a tumor-promoter and a regulator of endocrine resistance in ER-positive breast cancer cells. SCRIB expression is induced by estrogen signaling in a MYC-dependent manner. SCRIB interacts with SLC3A2, a heteromeric component of leucine amino acid transporter SLC7A5. SLC3A2 binds to the N-terminus of SCRIB to facilitate the formation of SCRIB/SLC3A2/LLGL2/SLC7A5 quaternary complex required for membrane localization of the amino acid transporter complex. Both SCRIB and SLC3A2 are required for cell proliferation and tamoxifen resistance in ER+ cells identifying a new role for the SCRIB/SLC3A2 complex in ER+ breast cancer.

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S-adenosyl-L-homocysteine extends lifespan through methionine restriction effects

Ogawa, T., Masumura, K., Kohara, Y., Kanai, M., Soga, T., Ohya, Y., Blackwell, T.K. Mizunuma, M.

Aging Cell 21 ( 5 ) e13604 2022.05

Research paper (scientific journal), Joint Work, Accepted

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Surgical Treatment for Colorectal Cancer Partially Restores Gut Microbiome and Metabolome Traits

Shiroma, H., Shiba, S., Erawijantari, P.P., Takamaru, H., Yamada, M., Sakamoto, T., Kanemitsu, Y., Mizutani, S., Soga, T., Saito, Y., Shibata, T., Fukuda, S., Yachida, S., Yamada, T.

mSystems 7 ( 2 ) e0001822 2022.04

Research paper (scientific journal), Joint Work, Accepted

Accumulating evidence indicates that the gut microbiome and metabolites are associated with colorectal cancer (CRC). However, the influence of surgery for CRC treatment on the gut microbiome and metabolites and how it relates to CRC risk in postoperative CRC patients remain partially understood. Here, we collected 170 fecal samples from 85 CRC patients pre- and approximately 1 year postsurgery and performed shotgun metagenomic sequencing and capillary electrophoresis-time of flight mass spectrometry-based metabolomics analyses to characterize alterations between pre- and postsurgery. We determined that the relative abundance of 114 species was altered postsurgery (P < 0.005). CRC-associated species, such as Fusobacterium nucleatum, were decreased postsurgery. On the other hand, Clostridium scindens, carcinogenesis-associated deoxycholate (DCA)-producing species, and its biotransformed genes (bai operon) were increased postsurgery. The concentration of 60 fecal metabolites was also altered postsurgery (P < 0.005). Two bile acids, cholate and DCA, were increased postsurgery. We developed methods to estimate postoperative CRC risk based on the gut microbiome and metabolomic compositions using a random forest machine-learning algorithm that classifies large adenoma or early-stage CRC and healthy controls from publicly available data sets. We applied methods to preoperative samples and then compared the estimated CRC risk between the two groups according to the presence of large adenoma or tumors 5 years postsurgery (P < 0.05). Overall, our results show that the gut microbiome and metabolites dynamically change from pre- to postsurgery. In postoperative CRC patients, potential CRC risk derived from gut microbiome and metabolites still remains, which indicates the importance of follow-up assessments. IMPORTANCE The gut microbiome and metabolites are associated with CRC progression and carcinogenesis. Postoperative CRC patients are reported to be at an increased CRC risk; however, how gut microbiome and metabolites are related to CRC risk in postoperative patients remains only partially understood. In this study, we investigated the influence of surgical CRC treatment on the gut microbiome and metabolites. We found that the CRC-associated species Fusobacterium nucleatum was decreased postsurgery, whereas carcinogenesis-associated DCA and its producing species and genes were increased postsurgery. We developed methods to estimate postoperative CRC risk based on the gut microbiome and metabolomic compositions. We applied methods to compare the estimated CRC risk between two groups according to the presence of large adenoma or tumors after 5 years postsurgery. To our knowledge, this study is the first report on differences between pre- and postsurgery using metagenomics and metabolomics data analysis. Our methods might be used for CRC risk assessment in postoperative patients.

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Plant hvu-MIR168-3p enhances expression of glucose transporter 1 (SLC2A1) in human cells by silencing genes related to mitochondrial electron transport chain complex I

Akao, Y., Kuranaga, Y., Heishima, K., Sugito, N., Morikawa, K., Ito, Y., Soga, T., Ito, T.

J. Nutr. Biochem. 101 108922 2022.03

Research paper (scientific journal), Joint Work, Accepted, ISSN 09552863

Diet is a crucial factor for preventing most diseases. Edible plant extracts are known to contain exosome-like nanoparticles, in which food-derived plant microRNAs are included and may serve as a novel functional component in human health. Here, we demonstrated that hvu-MIR168-3p included in the nanoparticles of rice aleurone cells down-regulated the expression of the genes related to mitochondrial electron transport chain complex I in human cells. Subsequently, hvu-MIR168-3p enhanced protein and RNA expression levels of glucose transporter I and caused a decrease in the blood glucose level, which findings were obtained by in vitro and in vivo experiments, respectively. These findings suggest that a cross-kingdom relationship between plants and humans with respect to hvu-MIR168-3p exists and may contribute to preventive medicine for GLUT1-related dysfunctions including glucose metabolism, aging, and tumor immunology.

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Metabolic profiling of prostate cancer in skeletal microenvironments identifies G6PD as a key mediator of growth and survival

Whitburn, J., Rao, S.R., Morris, E.V., Tabata, S., Hirayama, A., Soga, T., Edwards, J.R., Kaya, Z., Palmer, C., Hamdy, F.C., Edwards, C.M.

Sci. Adv. 8 ( 8 ) eabf9096 2022.02

Research paper (scientific journal), Joint Work, Accepted

The spread of cancer to bone is invariably fatal, with complex cross-talk between tumor cells and the bone microenvironment responsible for driving disease progression. By combining in silico analysis of patient datasets with metabolomic profiling of prostate cancer cells cultured with bone cells, we demonstrate the changing energy requirements of prostate cancer cells in the bone microenvironment, identifying the pentose phosphate pathway (PPP) as elevated in prostate cancer bone metastasis, with increased expression of the PPP rate-limiting enzyme glucose-6-phosphate dehydrogenase (G6PD) associated with a reduction in progression-free survival. Genetic and pharmacologic manipulation demonstrates that G6PD inhibition reduces prostate cancer growth and migration, associated with changes in cellular redox state and increased chemosensitivity. Genetic blockade of G6PD in vivo results in reduction of tumor growth within bone. In summary, we demonstrate the metabolic plasticity of prostate cancer cells in the bone microenvironment, identifying the PPP and G6PD as metabolic targets for the treatment of prostate cancer bone metastasis.

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Multi-omics-based label-free metabolic flux inference reveals obesity-associated dysregulatory mechanisms in liver glucose metabolism

Uematsu, S., Ohno, S., Tanaka, K.Y., Hatano, A., Kokaji, T., Ito, Y., Kubota, H., Hironaka, K., Suzuki, Y., Matsumoto, M., Nakayama, K.I., Hirayama, A., Soga, T., Kuroda, S.

iScience 25 ( 2 ) 103787 2022.02

Research paper (scientific journal), Joint Work, Accepted

Glucose homeostasis is maintained by modulation of metabolic flux. Enzymes and metabolites regulate the involved metabolic pathways. Dysregulation of glucose homeostasis is a pathological event in obesity. Analyzing metabolic pathways and the mechanisms contributing to obesity-associated dysregulation in vivo is challenging. Here, we introduce OMELET: Omics-Based Metabolic Flux Estimation without Labeling for Extended Trans-omic Analysis. OMELET uses metabolomic, proteomic, and transcriptomic data to identify relative changes in metabolic flux, and to calculate contributions of metabolites, enzymes, and transcripts to the changes in metabolic flux. By evaluating the livers of fasting ob/ob mice, we found that increased metabolic flux through gluconeogenesis resulted primarily from increased transcripts, whereas that through the pyruvate cycle resulted from both increased transcripts and changes in substrates of metabolic enzymes. With OMELET, we identified mechanisms underlying the obesity-associated dysregulation of metabolic flux in the liver.

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Four features of temporal patterns characterize similarity among individuals and molecules by glucose ingestion in humans

Fujita, S., Karasawa, Y., Fujii, M., Hironaka, K., Uda, S., Kubota, H., Inoue, H., Sumitomo, Y., Hirayama, A., Soga, T., Kuroda, S.

NPJ Syst. Biol. Appl. 8 ( 1 ) 6 2022.02

Research paper (scientific journal), Joint Work, Accepted

Oral glucose ingestion induces systemic changes of many blood metabolites related not only to glucose, but also other metabolites such as amino acids and lipids through many blood hormones. However, the detailed temporal changes in the concentrations of comprehensive metabolites and hormones over a long time by oral glucose ingestion are uncharacterized. We measured 83 metabolites and 7 hormones in 20 healthy human subjects in response to glucose ingestion. We characterized temporal patterns of blood molecules by four features: (i) the decomposability into "amplitude" and "rate" components, (ii) the similarity of temporal patterns among individuals, (iii) the relation of molecules over time among individuals, and (iv) the similarity of temporal patterns among molecules. Glucose and glucose metabolism-related hormones indicated a rapid increase, and citrulline and lipids, which indicated a rapid decrease, returned to fasting levels faster than amino acids. Compared to glucose metabolism-related molecules and lipids, amino acids showed similar temporal patterns among individuals. The four features of temporal patterns of blood molecules by oral glucose ingestion characterize the differences among individuals and among molecules.

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Energy-sparing by 2-methyl-2-thiazoline protects heart from ischemia/reperfusion injury

Nishi, M., Ogata, T., Kobayakawa, K., Kobayakawa, R., Matsuo, T., Cannistraci, C.V., Tomita, S., Taminishi, S., Suga, T., Kitani, T., Higuchi, Y., Sakamoto, A., Tsuji, Y., Soga, T., Matoba, S.

ESC Heart Fail. 9 ( 1 ) 428 - 441 2022.02

Research paper (scientific journal), Joint Work, Accepted

Aims: Cardiac ischaemia/reperfusion (I/R) injury remains a critical issue in the therapeutic management of ischaemic heart failure. Although mild hypothermia has a protective effect on cardiac I/R injury, more rapid and safe methods that can obtain similar results to hypothermia therapy are required. 2-Methyl-2-thiazoline (2MT), an innate fear inducer, causes mild hypothermia resulting in resistance to critical hypoxia in cutaneous or cerebral I/R injury. The aim of this study is to demonstrate the protective effect of systemically administered 2MT on cardiac I/R injury and to elucidate the mechanism underlying this effect.

Methods and results: A single subcutaneous injection of 2MT (50 mg/kg) was given prior to reperfusion of the I/R injured 10 week-old male mouse heart and its efficacy was evaluated 24 h after the ligation of the left anterior descending coronary artery. 2MT preserved left ventricular systolic function following I/R injury (ejection fraction, %: control 37.9 ± 6.7, 2MT 54.1 ± 6.4, P < 0.01). 2MT also decreased infarct size (infarct size/ischaemic area at risk, %: control 48.3 ± 12.1, 2MT 25.6 ± 4.2, P < 0.05) and serum cardiac troponin levels (ng/mL: control 8.9 ± 1.1, 2MT 1.9 ± 0.1, P < 0.01) after I/R. Moreover, 2MT reduced the oxidative stress-exposed area within the heart (%: control 25.3 ± 4.7, 2MT 10.8 ± 1.4, P < 0.01). These results were supported by microarray analysis of the mouse hearts. 2MT induced a transient, mild decrease in core body temperature (°C: -2.4 ± 1.4), which gradually recovered over several hours. Metabolome analysis of the mouse hearts suggested that 2MT minimized energy metabolism towards suppressing oxidative stress. Furthermore, 18F-fluorodeoxyglucose-positron emission tomography/computed tomography imaging revealed that 2MT reduced the activity of brown adipose tissue (standardized uptake value: control 24.3 ± 6.4, 2MT 18.4 ± 5.8, P < 0.05). 2MT also inhibited mitochondrial respiration and glycolysis in rat cardiomyoblasts.

Conclusions: We identified the cardioprotective effect of systemically administered 2MT on cardiac I/R injury by sparing energy metabolism with reversible hypothermia. Our results highlight the potential of drug-induced hypothermia therapy as an adjunct to coronary intervention in severe ischaemic heart disease.

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Group IIA secreted phospholipase A2 controls skin carcinogenesis and psoriasis by shaping the gut microbiota

Miki, Y., Taketomi, Y., Kidoguchi, Y., Yamamoto, K., Muramatsu, K., Nishito, Y., Park, J., Hosomi, K., Mizuguchi, K., Kunisawa, J., Soga, T., Boilard, E., Gowda, S.G.B. Ikeda, K., Arita, M., Murakami, M.

JCI Insight 7 ( 2 ) e152611 2022.01

Research paper (scientific journal), Joint Work, Accepted

Besides promoting inflammation by mobilizing lipid mediators, group IIA secreted phospholipase A2 (sPLA2-IIA) prevents bacterial infection by degrading bacterial membranes. Here, we show that, despite the restricted intestinal expression of sPLA2-IIA in BALB/c mice, its genetic deletion leads to amelioration of cancer and exacerbation of psoriasis in distal skin. Intestinal expression of sPLA2-IIA is reduced after treatment with antibiotics or under germ-free conditions, suggesting its upregulation by gut microbiota. Metagenome, transcriptome, and metabolome analyses have revealed that sPLA2-IIA deficiency alters the gut microbiota, accompanied by notable changes in the intestinal expression of genes related to immunity and metabolism, as well as in the levels of various blood metabolites and fecal bacterial lipids, suggesting that sPLA2-IIA contributes to shaping of the gut microbiota. The skin phenotypes in Pla2g2a-/- mice are lost (a) when they are cohoused with littermate WT mice, resulting in the mixing of the microbiota between the genotypes, or (b) when they are housed in a more stringent pathogen-free facility, where Pla2g2a expression in WT mice is low and the gut microbial compositions in both genotypes are nearly identical. Thus, our results highlight a potentially new aspect of sPLA2-IIA as a modulator of gut microbiota, perturbation of which affects distal skin responses.

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Glucocorticoid imprints a low glucose metabolism onto CD8 T cells and induces the persistent suppression of the immune response

Konishi, A., Suzuki, J., Kuwahara, M., Matsumoto, A., Nomura, S., Soga, T., Yorozuya, T., Yamashita, M.

Biochem. Biophys. Res. Commun. 588 34 - 40 2022.01

Joint Work, Accepted, ISSN 0006291X

Glucocorticoids (GCs), immunosuppressive, and anti-inflammatory agents have various effects on T cells. However, the long-term influence of GCs on the T cell-mediated immune response remain to be elucidated. We demonstrated that the administration of GC during the TCR-mediated activation phase induced long-lasting suppression of glycolysis, even after the withdrawal of GC. The acquisition of the effector functions was inhibited, while the expression of PD-1 was increased in CD8 T cells activated in the presence of GC. Furthermore, adoptive transfer experiments revealed that GC-treated CD8 T cells reduced memory T cell formation and anti-tumor activity. These findings reveal that GCs have long-lasting influence on the T cell-mediated immune response via modulation of T cell metabolism.

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Hao1 Is Not a Pathogenic Factor for Ectopic Ossifications but Functions to Regulate the TCA Cycle In Vivo

Kimura, A., Hirayama, A., Matsumoto, T., Sato, Y., Kobayashi, T., Ikeda, S., Maruyama, M., Kaneko, M., Shigeta, M., Ito, E., Soma, T., Miyamoto, K., Soga, T., Tomita, M., Oya, A., Matsumoto, M., Nakamura, M., Kanaji, A., Miyamoto, T.

Metabolites 12 ( 1 ) 82 2022.01

Research paper (scientific journal), Joint Work, Accepted

Ossification of the posterior longitudinal ligament (OPLL), a disease characterized by the ectopic ossification of a spinal ligament, promotes neurological disorders associated with spinal canal stenosis. While blocking ectopic ossification is mandatory to prevent OPLL development and progression, the mechanisms underlying the condition remain unknown. Here we show that expression of hydroxyacid oxidase 1 (Hao1), a gene identified in a previous genome-wide association study (GWAS) as an OPLL-associated candidate gene, specifically and significantly decreased in fibroblasts during osteoblast differentiation. We then newly established Hao1-deficient mice by generating Hao1-flox mice and crossing them with CAG-Cre mice to yield global Hao1-knockout (CAG-Cre/Hao1flox/flox; Hao1 KO) animals. Hao1 KO mice were born normally and exhibited no obvious phenotypes, including growth retardation. Moreover, Hao1 KO mice did not exhibit ectopic ossification or calcification. However, urinary levels of some metabolites of the tricarboxylic acid (TCA) cycle were significantly lower in Hao1 KO compared to control mice based on comprehensive metabolomic analysis. Our data indicate that Hao1 loss does not promote ectopic ossification, but rather that Hao1 functions to regulate the TCA cycle in vivo.

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SI-MOIRAI: A new method to identify and quantify the metabolic fate of nucleotides

Ikeda, Y., Hirayama, A., Kofuji, S., Hirota, Y., Kamata, R., Osaka, N., Fujii, Y., Sasaki, M., Ikeda, S., Smith, E.P., Bachoo, R., Soga, T., Sasaki, A.T.

J Biochem. 170 ( 6 ) 699 - 711 2022.01

Research paper (scientific journal), Joint Work, Accepted, ISSN 0021924X

Since the discovery of nucleotides over 100 years ago, extensive studies have revealed the importance of nucleotides for homeostasis, health, and disease. However, there remains no established method to investigate quantitively and accurately intact nucleotide incorporation into RNA and DNA. Herein, we report a new method, Stable-Isotope Measure Of Influxed Ribonucleic Acid Index (SI-MOIRAI), for the identification and quantification of the metabolic fate of ribonucleotides and their precursors. SI-MOIRAI, named after Greek goddesses of fate, combines a stable isotope-labeling flux assay with mass spectrometry to enable quantification of the newly synthesized ribonucleotides into r/m/tRNA under a metabolic stationary state. Using glioblastoma U87MG cells and a patient-derived xenograft (PDX) glioblastoma mouse model, SI-MOIRAI analyses showed that newly synthesized GTP was particularly and disproportionally highly utilized for rRNA and tRNA synthesis but not for mRNA synthesis in glioblastoma (GBM) in vitro and in vivo. Furthermore, newly synthesized pyrimidine nucleotides exhibited a significantly lower utilization rate for RNA synthesis than newly synthesized purine nucleotides. The results reveal the existence of discrete pathways and compartmentalization of purine and pyrimidine metabolism designated for RNA synthesis, demonstrating the capacity of SI-MOIRAI to reveal previously unknown aspects of nucleotide biology.

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Stemness and immune evasion conferred by TDO2-AHR pathway are associated with liver metastasis of colon cancer

Miyazaki, T., Chung, S., Sakai, H., Ohata, H., Obata, Y., Shiokawa, D., Mizoguchi, Y., Kubo, T., Ichikawa, H., Taniguchi, H., Aoki, K., Soga, T., Nakagama, H., Okamoto, K.

Cancer Sci. 113 ( 1 ) 170 - 181 2022.01

Research paper (scientific journal), Joint Work, Accepted

The aryl hydrocarbon receptor (AHR) pathway modulates the immune system in response to kynurenine, an endogenous tryptophan metabolite. IDO1 and TDO2 catalyze kynurenine production, which promotes cancer progression by compromising host immunosurveillance. However, it is unclear whether the AHR activation regulates the malignant traits of cancer such as metastatic capability or cancer stemness. Here, we carried out systematic analyses of metabolites in patient-derived colorectal cancer spheroids, and identified high levels of kynurenine and TDO2 that were positively associated with liver metastasis. In a mouse colon cancer model, TDO2 expression substantially enhanced liver metastasis, induced AHR-mediated PD-L1 transactivation, and dampened immune responses; these changes were all abolished by PD-L1 knockout. In patient-derived cancer spheroids, TDO2 or AHR activity was required for not only the expression of PD-L1, but also for cancer stem cell (CSC)-related characteristics and Wnt signaling. TDO2 was coexpressed with both PD-L1 and nuclear β-catenin in colon xenograft tumors, and the coexpression of TDO2 and PD-L1 was observed in clinical colon cancer specimens. Thus, our data indicate that the activation of the TDO2-kynurenine-AHR pathway facilitates liver metastasis of colon cancer via PD-L1-mediated immune evasion and maintenance of stemness.

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Metabolic profiling of charged metabolites in association with menopausal status in Japanese community-dwelling midlife women: Tsuruoka Metabolomic Cohort Study

Watanabe, K., Iida, M., Harada, S., Kato, S., Kuwabara, K., Kurihara, A., Takeuchi, A., Sugiyama, D., Okamura, T., Suzuki, A., Amano, K., Hirayama, A., Sugimoto, M., Soga, T., Tomita, M., Kobayashi, Y., Banno, K., Aoki, D., Takebayashi, T.

Maturitas 155 54 - 62 2022.01

Research paper (scientific journal), Joint Work, Accepted

Background
Emerging evidence has shown that charged metabolites, such as amino acids, may play an important role in the pathogenesis of various metabolic disorders, many of which women in the postmenopausal period are at high risk of developing. This study examined the metabolic profile of middle-aged Japanese women to investigate alterations in charged metabolites induced by menopausal transition.

Methods
The participants were 1193 female residents aged 40–60 at the baseline survey of the Tsuruoka Metabolomics Cohort Study. We investigated the cross-sectional association of menopausal status with 94 metabolomic biomarkers assayed in fasting plasma samples via capillary electrophoresis time-of-flight mass spectrometry using linear regression analysis.

Results
Among the participants, 529 were premenopausal, 132 were in menopausal transition (MT), and 532 were postmenopausal. Significant differences were found in age, blood pressure, glucose and lipid levels, and smoking and drinking habits among the three groups. The concentrations of 5 metabolites in the MT group and 15 metabolites in the postmenopausal group were significantly higher than those in the premenopausal group after adjusting for confounding factors. When classified into pathways, these metabolites were related to the tricarboxylic cycle, urea cycle, and homocysteine metabolism, some of which are linked to arteriosclerosis.

Conclusion
Multiple charged metabolites were associated with women's menopausal status, showing a gradual increase as women shifted from pre-, to peri-, to postmenopause. These findings might reflect the early changes behind the increased risk of dyslipidemia, diabetes, cardiovascular disease, and osteoporosis in later life.

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MEK inhibition preferentially suppresses anchorage-independent growth in osteosarcoma cells and decreases tumors in vivo

Shimizu, T., Kimura, K., Sugihara, E., Yamaguchi-Iwai, S., Nobuse, H., Sampetrean, O., Otsuki, Y., Fukuchi, Y., Saitoh, K., Kato, K., Soga, T., Muto, A., Saya, H.

J. Orthop. Res. 39 ( 12 ) 2732 - 2743 2021.12

Research paper (scientific journal), Joint Work, Accepted

Osteosarcoma is the most common high-grade malignancy of bone, and novel therapeutic options are urgently required. Previously, we developed mouse osteosarcoma AXT cells that can proliferate both under adherent and nonadherent conditions. Based on metabolite levels, nonadherent conditions were more similar to the in vivo environment than adherent conditions. A drug screen identified MEK inhibitors, including trametinib, that preferentially decreased the viability of nonadherent AXT cells. Trametinib inhibited the cell cycle and induced apoptosis in AXT cells, and both effects were stronger under nonadherent conditions. Trametinib also potently decreased viability in U2OS cells, but its effects were less prominent in MG63 or Saos2 cells. By contrast, MG63 and Saos2 cells were more sensitive to PI3K inhibition than AXT or U2OS cells. Notably, the combination of MAPK/ERK kinase (MEK) and PI3K inhibition synergistically decreased viability in U2OS and AXT cells, but this effect was less pronounced in MG63 or Saos2 cells. Therefore, signal dependence for cell survival and crosstalk between MEK-ERK and PI3K-AKT pathways in osteosarcoma are cell context-dependent. The activation status of other kinases including CREB varied in a cell context-dependent manner, which might determine the response to MEK inhibition. A single dose of trametinib was sufficient to decrease the size of the primary tumor and circulating tumor cells in vivo. Moreover, combined administration of trametinib and rapamycin or conventional anticancer drugs further increased antitumor activity. Thus, given optimal biomarkers for predicting its effects, trametinib holds therapeutic potential for the treatment of osteosarcoma.

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Identification of the first highly selective inhibitor of human lactate dehydrogenase B

Shibata, S., Sogabe, S., Miwa, M., Fujimoto, T., Takakura, N., Naotsuka, A., Kitamura, S., Kawamoto, T., Soga, T.

Sci Rep. 11 ( 1 ) 21353 2021.11

Research paper (scientific journal), Joint Work, Accepted

Lactate dehydrogenase (LDH) catalyses the conversion of pyruvate to lactate and NADH to NAD+; it has two isoforms, LDHA and LDHB. LDHA is a promising target for cancer therapy, whereas LDHB is necessary for basal autophagy and cancer cell proliferation in oxidative and glycolytic cancer cells. To the best of our knowledge, selective inhibitors for LDHB have not yet been reported. Here, we developed a high-throughput mass spectrometry screening system using an LDHB enzyme assay by detecting NADH and NAD+. As a result, we identified a small-molecule LDHB selective inhibitor AXKO-0046, an indole derivative. This compound exhibited uncompetitive LDHB inhibition (EC50 = 42 nM). X-ray crystallography revealed that AXKO-0046 bound to the potential allosteric site away from the LDHB catalytic active site, suggesting that targeting the tetramerisation interface of the two dimers is critical for the enzymatic activity. AXKO-0046 and its derivatives can be used to validate LDHB-associated pathways in cancer metabolism.

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Basigin deficiency prevents anaplerosis and ameliorates insulin resistance and hepatosteatosis

Ryuge, A., Kosugi, T., Maeda, K., Banno, R., Gou, Y., Zaitsu, K., Ito, T., Sato, Y., Hirayama, A., Tsubota, S., Honda, T., Nakajima, K., Ozaki, T., Kondoh, K., Takahashi, K., Kato, N., Ishimoto, T., Soga, T., Nakagawa, T., Koike, T., Arima, H., Yuzawa, Y., Minokoshi, Y., Maruyama, S., Kadomatsu, K.

JCI Insight 6 ( 20 ) e142464 2021.10

Research paper (scientific journal), Joint Work, Accepted

Monocarboxylates, such as lactate and pyruvate, are precursors for biosynthetic pathways, including those for glucose, lipids, and amino acids via the tricarboxylic acid (TCA) cycle and adjacent metabolic networks. The transportation of monocarboxylates across the cellular membrane is performed primarily by monocarboxylate transporters (MCTs), the membrane localization and stabilization of which are facilitated by the transmembrane protein basigin (BSG). Here, we demonstrate that the MCT/BSG axis sits at a crucial intersection of cellular metabolism. Abolishment of MCT1 in the plasma membrane was achieved by Bsg depletion, which led to gluconeogenesis impairment via preventing the influx of lactate and pyruvate into the cell, consequently suppressing the TCA cycle. This net anaplerosis suppression was compensated in part by the increased utilization of glycogenic amino acids (e.g., alanine and glutamine) into the TCA cycle and by activated ketogenesis through fatty acid β-oxidation. Complementary to these observations, hyperglycemia and hepatic steatosis induced by a high-fat diet were ameliorated in Bsg-deficient mice. Furthermore, Bsg deficiency significantly improved insulin resistance induced by a high-fat diet. Taken together, the plasma membrane-selective modulation of lactate and pyruvate transport through BSG inhibition could potentiate metabolic flexibility to treat metabolic diseases.

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Urinary Metabolome Analyses of Patients with Acute Kidney Injury Using Capillary Electrophoresis-Mass Spectrometry

Saito, R., Hirayama, A., Akiba, A., Kamei, Y., Kato, Y., Ikeda, S., Kwan, B., Pu, M., Natarajan, L., Shinjo, H., Akiyama, S., Tomita, M., Soga, T., Maruyama, S.

Metabolites 11 ( 10 ) 671 2021.09

Research paper (scientific journal), Joint Work, Accepted

Acute kidney injury (AKI) is defined as a rapid decline in kidney function. The associated syndromes may lead to increased morbidity and mortality, but its early detection remains difficult. Using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS), we analyzed the urinary metabolomic profile of patients admitted to the intensive care unit (ICU) after invasive surgery. Urine samples were collected at six time points: before surgery, at ICU admission and 6, 12, 24 and 48 h after. First, urine samples from 61 initial patients (non-AKI: 23, mild AKI: 24, severe AKI: 14) were measured, followed by the measurement of urine samples from 60 additional patients (non-AKI: 40, mild AKI: 20). Glycine and ethanolamine were decreased in patients with AKI compared with non-AKI patients at 6–24 h in the two groups. The linear statistical model constructed at each time point by machine learning achieved the best performance at 24 h (median AUC: 89%, cross-validated) for the 1st group. When cross-validated between the two groups, the AUC showed the best value of 70% at 12 h. These results identified metabolites and time points that show patterns specific to subjects who develop AKI, paving the way for the development of better biomarkers.

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Upregulation of Thymidylate Synthase Induces Pemetrexed Resistance in Malignant Pleural Mesothelioma

Sato, Y., Tomita, M., Soga, T., Ochiai, A., Makinoshima, H.

Front. Pharmacol. 12 718675 2021.09

Research paper (scientific journal), Joint Work, Accepted

Malignant pleural mesothelioma (MPM) is an invasive malignancy that develops in the pleural cavity, and antifolates are used as chemotherapeutics for treating. The majority of antifolates, including pemetrexed (PMX), inhibit enzymes involved in purine and pyrimidine synthesis. MPM patients frequently develop drug resistance in clinical practice, however the associated drug-resistance mechanism is not well understood. This study was aimed to elucidate the mechanism underlying resistance to PMX in MPM cell lines. We found that among the differentially expressed genes associated with drug resistance (determined by RNA sequencing), TYMS expression was higher in the established resistant cell lines than in the parental cell lines. Knocking down TYMS expression significantly reduced drug resistance in the resistant cell lines. Conversely, TYMS overexpression significantly increased drug resistance in the parental cells. Metabolomics analysis revealed that the levels of dTMP were higher in the resistant cell lines than in the parental cell lines; however, resistant cells showed no changes in dTTP levels after PMX treatment. We found that the nucleic acid-biosynthetic pathway is important for predicting the efficacy of PMX in MPM cells. The results of chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR) assays suggested that H3K27 acetylation in the 5′-UTR of TYMS may promote its expression in drug-resistant cells. Our findings indicate that the intracellular levels of dTMP are potential biomarkers for the effective treatment of patients with MPM and suggest the importance of regulatory mechanisms of TYMS expression in the disease.

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Empagliflozin Maintains Capillarization and Improves Cardiac Function in a Murine Model of Left Ventricular Pressure Overload

Nakao, M., Shimizu, I., Katsuumi, G., Yoshida, Y., Suda, M., Hayashi, Y., Ikegami, R., Hsiao, Y. T., Okuda, S., Soga, T., Minamino, T.

Sci Rep. 11 ( 1 ) 18384 2021.09

Research paper (scientific journal), Joint Work, Accepted

Patients with type 2 diabetes treated with Sodium glucose transporter 2 (SGLT2) inhibitors show reduced mortality and hospitalization for heart failure (HF). SGLT2 inhibitors are considered to activate multiple cardioprotective pathways; however, underlying mechanisms are not fully described. This study aimed to elucidate the underlying mechanisms of the beneficial effects of SGLT2 inhibitors on the failing heart. We generated a left ventricular (LV) pressure overload model in C57BL/6NCrSlc mice by transverse aortic constriction (TAC) and examined the effects of empagliflozin (EMPA) in this model. We conducted metabolome and transcriptome analyses and histological and physiological examinations. EMPA administration ameliorated pressure overload-induced systolic dysfunction. Metabolomic studies showed that EMPA increased citrulline levels in cardiac tissue and reduced levels of arginine, indicating enhanced metabolism from arginine to citrulline and nitric oxide (NO). Transcriptome suggested possible involvement of the insulin/AKT pathway that could activate NO production through phosphorylation of endothelial NO synthase (eNOS). Histological examination of the mice showed capillary rarefaction and endothelial apoptosis after TAC, both of which were significantly improved by EMPA treatment. This improvement was associated with enhanced expression phospho-eNOS and NO production in cardiac endothelial cells. NOS inhibition attenuated these cardioprotective effects of EMPA. The in vitro studies showed that catecholamine-induced endothelial apoptosis was inhibited by NO, arginine, or AKT activator. EMPA activates the AKT/eNOS/NO pathway, which helps to suppress endothelial apoptosis, maintain capillarization and improve systolic dysfunction during LV pressure overload.

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Petasin potently inhibits mitochondrial-complex-I-based metabolism that suppors tumor growth and metastasis

Heishima, K., Sugito, N., Soga, T., Nishikawa, M., Ito, Y., Honda, R., Kuranaga, Y., Sakai, H., Ito, R., Nakagawa, T., Ueda, H., Akao, Y.

Clin. Invest. 131 ( 17 ) e139933 2021.09

Research paper (scientific journal), Joint Work, Accepted

Mitochondrial electron transport chain complex I (ETCC1) is the essential core of cancer metabolism, yet potent ETCC1 inhibitors capable of safely suppressing tumor growth and metastasis in vivo are limited. From a plant extract screening, we identified petasin (PT) as a highly potent ETCC1 inhibitor with a chemical structure distinct from conventional inhibitors. PT had at least 1700 times higher activity than that of metformin or phenformin and induced cytotoxicity against a broad spectrum of tumor types. PT administration also induced prominent growth inhibition in multiple syngeneic and xenograft mouse models in vivo. Despite its higher potency, it showed no apparent toxicity toward nontumor cells and normal organs. Also, treatment with PT attenuated cellular motility and focal adhesion in vitro as well as lung metastasis in vivo. Metabolome and proteome analyses revealed that PT severely depleted the level of aspartate, disrupted tumor-associated metabolism of nucleotide synthesis and glycosylation, and downregulated major oncoproteins associated with proliferation and metastasis. These findings indicate the promising potential of PT as a potent ETCC1 inhibitor to target the metabolic vulnerability of tumor cells.

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High-throughput screening of salivary polyamine markers for discrimination of colorectal cancer by multisegment injection capillary electrophoresis tandem mass spectrometry

Igarashi, K., Ota, S., Kaneko, M., Hirayama, A., Enomoto, M., Katumata, K., Sugimoto M., Soga, T.

J. Chromatogr. A. 1652 462355 2021.08

Research paper (scientific journal), Joint Work, Accepted, ISSN 00219673

Polyamine metabolites provide pathophysiological information on disease or therapeutic efficacy, yet rapid screening methods for these biomarkers are lacking. Here, we developed high-throughput polyamine metabolite profiling based on multisegment injection capillary electrophoresis triple quadrupole tandem mass spectrometry (MSI-CE-MS/MS), which allows sequential 40-sample injection followed by electrophoretic separation and specific mass detection. To achieve consecutive analysis of polyamine samples, 1 M formic acid was used as the background electrolyte (BGE). The BGE spacer volume had an apparent effect on peak resolution among samples, and 20 nL was selected as the optimal volume. The use of polyamine isotopomers as the internal standard enabled the correction of matrix effects in MS detection. This method is sensitive, selective and quantitative, and its utility was demonstrated by screening polyamines in 359 salivary samples within 360 min, resulting in discrimination of colorectal cancer patients from noncancer controls.

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Targeting Amino Acid Metabolic Reprogramming via L-Type Amino Acid Transporter 1 (LAT1) for Endocrine-Resistant Breast Cancer

Shindo, H., Harada-Shoji, N., Ebata, A., Sato, M., Soga, T., Miyashita, M., Tada, H., Kawai, M., Kosaka, S., Onuki, K., Usami, S., Furumoto, S., Hayashi, S., Abe, T., Suzuki, T., Ishida, T., Sasano, H.

Cancers 13 ( 17 ) 4375 2021.08

Research paper (scientific journal), Joint Work, Accepted

The PI3K/Akt/mTOR pathway has been well known to interact with the estrogen receptor (ER)-pathway and to be also frequently upregulated in aromatase inhibitor (AI)-resistant breast cancer patients. Intracellular levels of free amino acids, especially leucine, regulate the mammalian target of rapamycin complex 1 (mTORC1) activation. L-type amino acid transporters such as LAT1 and LAT3 are associated with the uptake of essential amino acids. LAT1 expression could mediate leucine uptake, mTORC1 signaling, and cell proliferation. Therefore, in this study, we explored amino acid metabolism, including LAT1, in breast cancer and clarified the potential roles of LAT1 in the development of therapeutic resistance and the eventual clinical outcome of the patients. We evaluated LAT1 and LAT3 expression before and after neoadjuvant hormone therapy (NAH) and examined LAT1 function and expression in estrogen deprivation-resistant (EDR) breast carcinoma cell lines. Tumors tended to be in advanced stages in the cases whose LAT1 expression was high. LAT1 expression in the EDR cell lines was upregulated. JPH203, a selective LAT1 inhibitor, demonstrated inhibitory effects on cell proliferation in EDR cells. Hormone therapy changed the tumor microenvironment and resulted in metabolic reprogramming through inducing LAT1 expression. LAT1 expression then mediated leucine uptake, enhanced mTORC1 signaling, and eventually resulted in AI resistance. Therefore, LAT1 could be the potential therapeutic target in AI-resistant breast cancer patients.

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An extensive and dynamic trans-omic network illustrating prominent regulatory mechanisms in response to insulin in the liver

Matsuzaki, F., Uda, S., Yamauchi, Y., Matsumoto, M., Soga, T., Maehara, K., Ohkawa, Y., Nakayama, K., Kuroda, S., Kubota, H.

Cell Rep. 36 ( 8 ) 109569 2021.08

Research paper (scientific journal), Joint Work, Accepted

An effective combination of multi-omic datasets can enhance our understanding of complex biological phenomena. To build a context-dependent network with multiple omic layers, i.e., a trans-omic network, we perform phosphoproteomics, transcriptomics, proteomics, and metabolomics of murine liver for 4 h after insulin administration and integrate the resulting time series. Structural characteristics and dynamic nature of the network are analyzed to elucidate the impact of insulin. Early and prominent changes in protein phosphorylation and persistent and asynchronous changes in mRNA and protein levels through non-transcriptional mechanisms indicate enhanced crosstalk between phosphorylation-mediated signaling and protein expression regulation. Metabolic response shows different temporal regulation with transient increases at early time points across categories and enhanced response in the amino acid and nucleotide categories at later time points as a result of process convergence. This extensive and dynamic view of the trans-omic network elucidates prominent regulatory mechanisms that drive insulin responses through intricate interlayer coordination.

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Amino acid transporters as emerging therapeutic targets in cancer

Saito, Y., Soga, T.

Cancer Sci. 112 ( 8 ) 2958 - 2965 2021.08

Research paper (scientific journal), Joint Work, Accepted, ISSN 13479032

Amino acids are indispensable nutrients for both normal and cancer cells. Cancer cells are unable to synthesize essential amino acids as well as some non-essential amino acids adequately to support rapid proliferation, and must take up amino acids from the surroundings. To meet the increased demand for the amino acid needed for proliferation, high levels of amino acid transporters are expressed on the surface of cancer cells. Cancer cells utilize amino acids to synthesize proteins and nucleotides, as well as to obtain energy. In addition, amino acids are known to play pathological roles in cancer cells. Interestingly, breast cancer cells limit the use of amino acids for cell proliferation based on amino acid availability, which depends on estrogen receptor status. Here, we present a summarized literature review of novel amino acid functions in cancer cells. This review organizes the available knowledge on 2 amino acid transporters, SLC7A5 and SLC7A11, which are considered essential for breast cancer cell growth in a cell-dependent manner. In particular, we propose the glutamine recycling model to clarify the mechanism underlying aberrant SLC7A5 activation. Finally, we overview the pathological significances of SLC7A5 and SLC7A11 in cancer tissues.

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The CD44/COL17A1 pathway promotes the formation of multilayered, transformed epithelia

Kozawa, K., Sekai, M., Ohba, K., Ito, S., Sako, H., Maruyama, T., Kakeno, M., Shirai, T., Kuromiya, K., Kamasaki, T., Kohashi, K., Tanaka, S., Ishikawa, S., Sato, N., Asano, S., Suzuki, H., Tanimura, N., Mukai, Y., Gotoh, N., Tanino, M., Tanaka, S., Natsuga, K., Soga, T., Nakamura, T., Yabuta, Y., Saitou, M., Ito, T., Matsuura, K., Tsunoda, M., Kikumori, T., Iida, T., Mizutani,Y., Miyai, Y., Kaibuchi, K., Enomoto, A., Fujita, Y.

Curr. Biol. 31 ( 14 ) 3086 - 3097 2021.07

Research paper (scientific journal), Joint Work, Accepted

At the early stage of cancer development, oncogenic mutations often cause multilayered epithelial structures. However, the underlying molecular mechanism still remains enigmatic. By performing a series of screenings targeting plasma membrane proteins, we have found that collagen XVII (COL17A1) and CD44 accumulate in RasV12-, Src-, or ErbB2-transformed epithelial cells. In addition, the expression of COL17A1 and CD44 is also regulated by cell density and upon apical cell extrusion. We further demonstrate that the expression of COL17A1 and CD44 is profoundly upregulated at the upper layers of multilayered, transformed epithelia in vitro and in vivo. The accumulated COL17A1 and CD44 suppress mitochondrial membrane potential and reactive oxygen species (ROS) production. The diminished intracellular ROS level then promotes resistance against ferroptosis-mediated cell death upon cell extrusion, thereby positively regulating the formation of multilayered structures. To further understand the functional role of COL17A1, we performed comprehensive metabolome analysis and compared intracellular metabolites between RasV12 and COL17A1-knockout RasV12 cells. The data imply that COL17A1 regulates the metabolic pathway from the GABA shunt to mitochondrial complex I through succinate, thereby suppressing the ROS production. Moreover, we demonstrate that CD44 regulates membrane accumulation of COL17A1 in multilayered structures. These results suggest that CD44 and COL17A1 are crucial regulators for the clonal expansion of transformed cells within multilayered epithelia, thus being potential targets for early diagnosis and preventive treatment for precancerous lesions.

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De novo deoxyribonucleotide biosynthesis regulates cell growth and tumor progression in small-cell lung carcinoma

Maruyama, A., Sato, Y., Nakayama, J., Murai, J., Ishikawa, T., Soga, T., Makinoshima, H.

Sci. Rep. 11 ( 1 ) 13474 2021.06

Research paper (scientific journal), Joint Work, Accepted

Deoxyribonucleotide biosynthesis from ribonucleotides supports the growth of active cancer cells by producing building blocks for DNA. Although ribonucleotide reductase (RNR) is known to catalyze the rate-limiting step of de novo deoxyribonucleotide triphosphate (dNTP) synthesis, the biological function of the RNR large subunit (RRM1) in small-cell lung carcinoma (SCLC) remains unclear. In this study, we established siRNA-transfected SCLC cell lines to investigate the anticancer effect of silencing RRM1 gene expression. We found that RRM1 is required for the full growth of SCLC cells both in vitro and in vivo. In particular, the deletion of RRM1 induced a DNA damage response in SCLC cells and decreased the number of cells with S phase cell cycle arrest. We also elucidated the overall changes in the metabolic profile of SCLC cells caused by RRM1 deletion. Together, our findings reveal a relationship between the deoxyribonucleotide biosynthesis axis and key metabolic changes in SCLC, which may indicate a possible link between tumor growth and the regulation of deoxyribonucleotide metabolism in SCLC.

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TGF-β-dependent reprogramming of amino acid metabolism induces epithelial-mesenchymal transition in non-small cell lung cancers

Nakasuka, F., Tabata, S., Sakamoto, T., Hirayama, A., Ebi, H., Yamada, T., Umetsu, K., Ohishi, M., Ueno, A., Goto, H., Sugimoto, M., Nishioka, Y., Yamada, Y., Tomita, M., Sasaki, A.T. Yano, S., Soga, T.

Commun. Biol. 4 ( 1 ) 782 2021.06

Research paper (scientific journal), Joint Work, Accepted

Epithelial-mesenchymal transition (EMT)-a fundamental process in embryogenesis and wound healing-promotes tumor metastasis and resistance to chemotherapy. While studies have identified signaling components and transcriptional factors responsible in the TGF-β-dependent EMT, whether and how intracellular metabolism is integrated with EMT remains to be fully elucidated. Here, we showed that TGF-β induces reprogramming of intracellular amino acid metabolism, which is necessary to promote EMT in non-small cell lung cancer cells. Combined metabolome and transcriptome analysis identified prolyl 4-hydroxylase α3 (P4HA3), an enzyme implicated in cancer metabolism, to be upregulated during TGF-β stimulation. Further, knockdown of P4HA3 diminished TGF-β-dependent changes in amino acids, EMT, and tumor metastasis. Conversely, manipulation of extracellular amino acids induced EMT-like responses without TGF-β stimulation. These results suggest a previously unappreciated requirement for the reprogramming of amino acid metabolism via P4HA3 for TGF-β-dependent EMT and implicate a P4HA3 inhibitor as a potential therapeutic agent for cancer.

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CE-MS-based Identification of Uremic Solutes Specific to Hemodialysis Patients

Akiyama, Y., Kikuchi, K., Toyohara, T., Mishima, E., Suzuki, C., Suzuki, T., Nakayama, M., Tomioka, Y., Soga, T., Abe, T.

Toxins 13 ( 5 ) 324 2021.04

Research paper (scientific journal), Joint Work, Accepted

Uremic toxins are suggested to be involved in the pathophysiology of hemodialysis (HD) patients. However, the profile of uremic solutes in HD patients has not been fully elucidated. In this study using capillary electrophoresis mass spectrometry (CE-MS), we comprehensively quantified the serum concentrations of 122 ionic solutes before and after HD in 11 patients. In addition, we compared the results with those in non-HD patients with chronic kidney disease (CKD) to identify HD patient-specific solutes. We identified 38 solutes whose concentrations were higher in pre-HD than in CKD stage G5. Ten solutes among them did not significantly accumulate in non-HD CKD patients, suggesting that these solutes accumulate specifically in HD patients. We also identified 23 solutes whose concentrations were lower in both pre- and post-HD than in CKD stage G5. The serum levels of 14 solutes among them were not affected by renal function in non-HD patients, suggesting that these solutes tend to be lost specifically in HD patients. Our data demonstrate that HD patients have a markedly different profile of serum uremic solute levels compared to that in non-HD CKD patients. The solutes identified in our study may contribute to the pathophysiology of HD patients.

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A Metabolomic Profile Predictive of New Osteoporosis or Sarcopenia Development

Miyamoto, K., Hirayama, A., Sato, Y., Ikeda, S., Maruyama, M., Soga, T., Tomita, M., Nakamura, M., Matsumoto, M., Yoshimura, N., Miyamoto, T.

Metabolites 11 ( 5 ) 278 2021.04

Research paper (scientific journal), Joint Work, Accepted

The increasing number of patients with osteoporosis and sarcopenia is a global concern among countries with progressively aging societies. The high medical costs of treating those patients suggest that prevention rather than treatment is preferable. We enrolled 729 subjects who attended both the second and third surveys of the Research on Osteoarthritis/Osteoporosis Against Disability (ROAD) study. Blood samples were collected from subjects at the second survey, and then a comprehensive metabolomic analysis was performed. It was found that 35 had newly developed osteoporosis at the third survey performed four years later, and 39 were newly diagnosed with sarcopenia at the third survey. In the second survey, we found that serum Gly levels were significantly higher even after adjustment for age, sex, and BMI in subjects with newly developed osteoporosis relative to those who remained osteoporosis-negative during the four-year follow-up. We also show that serum taurine levels were significantly lower at the second survey, even after adjustment for age, sex, and BMI in subjects with newly developed sarcopenia during the four-year follow-up compared with those not diagnosed with sarcopenia at the second or third surveys. Though our sample size and odds ratios were small, increased Gly and decreased taurine levels were found to be predictive of new development of osteoporosis and sarcopenia, respectively, within four years.

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Methionine restriction breaks obligatory coupling of cell proliferation and death by an oncogene Src in Drosophila

Nishida, H., Okada, M., Yang, L., Takano, T., Tabata, S., Soga, T., Ho, D.M., Chung, J., Minami, Y., Yoo, S.K.

eLife 10 e59809 2021.04

Research paper (scientific journal), Joint Work, Accepted

Oncogenes often promote cell death as well as proliferation. How oncogenes drive these diametrically opposed phenomena remains to be solved. A key question is whether cell death occurs as a response to aberrant proliferation signals or through a proliferation-independent mechanism. Here, we reveal that Src, the first identified oncogene, simultaneously drives cell proliferation and death in an obligatorily coupled manner through parallel MAPK pathways. The two MAPK pathways diverge from a lynchpin protein Slpr. A MAPK p38 drives proliferation whereas another MAPK JNK drives apoptosis independently of proliferation signals. Src-p38-induced proliferation is regulated by methionine-mediated Tor signaling. Reduction of dietary methionine uncouples the obligatory coupling of cell proliferation and death, suppressing tumorigenesis and tumor-induced lethality. Our findings provide an insight into how cells evolved to have a fail-safe mechanism that thwarts tumorigenesis by the oncogene Src. We also exemplify a diet-based approach to circumvent oncogenesis by exploiting the fail-safe mechanism.

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Quality Assessment of Untargeted Analytical Data in a Large-Scale Metabolomic Study

Saito, R., Sugimoto, M., Hirayama, A., Soga, T., Tomita, M., Takebayashi, T.

J. Clin. Med. 10 ( 9 ) 1826 2021.04

Research paper (scientific journal), Joint Work, Accepted

Large-scale metabolomic studies have become common, and the reliability of the peak data produced by the various instruments is an important issue. However, less attention has been paid to the large number of uncharacterized peaks in untargeted metabolomics data. In this study, we tested various criteria to assess the reliability of 276 and 202 uncharacterized peaks that were detected in a gathered set of 30 plasma and urine quality control samples, respectively, using capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS). The linear relationship between the amounts of pooled samples and the corresponding peak areas was one of the criteria used to select reliable peaks. We used samples from approximately 3000 participants in the Tsuruoka Metabolome Cohort Study to investigate patterns of the areas of these uncharacterized peaks among the samples and clustered the peaks by combining the patterns and differences in the migration times. Our assessment pipeline removed substantial numbers of unreliable or redundant peaks and detected 35 and 74 reliable uncharacterized peaks in plasma and urine, respectively, some of which may correspond to metabolites involved in important physiological processes such as disease progression. We propose that our assessment pipeline can be used to help establish large-scale untargeted clinical metabolomic studies.

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Reliability of urinary charged metabolite concentrations in a large-scale cohort study using capillary electrophoresis-mass spectrometry

Ishibashi, Y., Harada, S., Takeuchi, A., Iida, M., Kurihara, A., Kato, S., Kuwabara, K., Hirata, A., Shibuki, T., Okamura, T., Sugiyama, D., Sato, A, Amano, K., Hirayama, A., Sugimoto, M., Soga, T., Tomita, M., Takebayashi, T

Sci. Rep 11 ( 1 ) 7407 2021.04

Research paper (scientific journal), Accepted

Currently, large-scale cohort studies for metabolome analysis have been launched globally. However, only a few studies have evaluated the reliability of urinary metabolome analysis. This study aimed to establish the reliability of urinary metabolomic profiling in cohort studies. In the Tsuruoka Metabolomics Cohort Study, 123 charged metabolites were identified and routinely quantified using capillary electrophoresis-mass spectrometry (CE-MS). We evaluated approximately 750 quality control (QC) samples and 6,720 participants' spot urine samples. We calculated inter- and intra-batch coefficients of variation in the QC and participant samples and technical intraclass correlation coefficients (ICC). A correlation of metabolite concentrations between spot and 24-h urine samples obtained from 32 sub-cohort participants was also evaluated. The coefficient of variation (CV) was less than 20% for 87 metabolites (70.7%) and 20-30% for 19 metabolites (15.4%) in the QC samples. There was less than 20% inter-batch CV for 106 metabolites (86.2%). Most urinary metabolites would have reliability for measurement. The 96 metabolites (78.0%) was above 0.75 for the estimated ICC, and those might be useful for epidemiological analysis. Among individuals, the Pearson correlation coefficient of 24-h and spot urine was more than 70% for 59 of the 99 metabolites. These results show that the profiling of charged metabolites using CE-MS in morning spot human urine is suitable for epidemiological metabolomics studies.

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Metabolomic Analysis of Small Extracellular Vesicles Derived from Pancreatic Cancer Cells Cultured under Normoxia and Hypoxia

Hayasaka, R., Tabata, S., Hasebe, M., Ikeda, S., Ohnuma, S., Mori, M., Soga, T., Tomita, M., Hirayama, A.

Metabolites 11 ( 4 ) 215 2021.04

Research paper (scientific journal), Joint Work, Accepted

Extracellular vesicles (EVs) released from cancer cells contribute to various malignant phenotypes of cancer, including metastasis, cachexia, and angiogenesis. Although DNA, mRNAs, miRNAs, and proteins contained in EVs have been extensively studied, the function of metabolites in EVs remains unclear. In this study, we performed a comprehensive metabolomic analysis of pancreatic cancer cells, PANC-1, cultured under different oxygen concentrations, and small EVs (sEVs) released from them, considering the fact that hypoxia contributes to the malignant behavior of cells in pancreatic cancer, which is a poorly diagnosed cancer. sEVs were collected by ultracentrifugation, and hydrophilic metabolites were analyzed using capillary ion chromatography-mass spectrometry and liquid chromatography-mass spectrometry, and lipids were analyzed by supercritical fluid chromatography-tandem mass spectrometry. A total of 140 hydrophilic metabolites and 494 lipids were detected in sEVs, and their profiles were different from those in cells. In addition, the metabolomic profile of sEVs was observed to change under hypoxic stress, and an increase in metabolites involved in angiogenesis was also detected. We reveal the hallmark of the metabolites contained in sEVs and the effect of tumor hypoxia on their profiles, which may help in understanding EV-mediated cancer malignancy.

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Cardiac Mitofusin-1 is Reduced in Non-responding Patients with Idiopathic Dilated Cardiomyopathy

Hsiao, Y.T., Shimizu, I., Wakasugi, T., Yoshida, Y., Ikegami, R., Hayashi, Y., Suda, M., Katsuumi, G., Nakao, M., Ozawa, T., Izumi, D., Kashimura, T., Ozaki, K., Soga, T., Minamino, T.

Sci. Rep 11 ( 1 ) 6722 2021.03

Research paper (scientific journal), Joint Work, Accepted

Prognosis of severe heart failure remains poor. Urgent new therapies are required. Some heart failure patients do not respond to established multidisciplinary treatment and are classified as "non-responders". The outcome is especially poor for non-responders, and underlying mechanisms are largely unknown. Mitofusin-1 (Mfn1), a mitochondrial fusion protein, is significantly reduced in non-responding patients. This study aimed to elucidate the role of Mfn1 in the failing heart. Twenty-two idiopathic dilated cardiomyopathy (IDCM) patients who underwent endomyocardial biopsy of intraventricular septum were included. Of the 22 patients, 8 were non-responders (left ventricular (LV) ejection fraction (LVEF) of < 10% improvement at late phase follow-up). Electron microscopy (EM), quantitative PCR, and immunofluorescence studies were performed to explore the biological processes and molecules involved in failure to respond. Studies in cardiac specific Mfn1 knockout mice (c-Mfn1 KO), and in vitro studies with neonatal rat ventricular myocytes (NRVMs) were also conducted. A significant reduction in mitochondrial size in cardiomyocytes, and Mfn1, was observed in non-responders. A LV pressure overload with thoracic aortic constriction (TAC) c-Mfn1 KO mouse model was generated. Systolic function was reduced in c-Mfn1 KO mice, while mitochondria alteration in TAC c-Mfn1 KO mice increased. In vitro studies in NRVMs indicated negative regulation of Mfn1 by the β-AR/cAMP/PKA/miR-140-5p pathway resulting in significant reduction in mitochondrial respiration of NRVMs. The level of miR140-5p was increased in cardiac tissues of non-responders. Mfn1 is a biomarker of heart failure in non-responders. Therapies targeting mitochondrial dynamics and homeostasis are next generation therapy for non-responding heart failure patients.

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Trans-Omic Analysis Reveals Obesity-Associated Dysregulation of Inter-Organ Metabolomic Cycles between the Liver and Skeletal Muscle

Egami, R., Kokaji, T., Hatano, A., Yugi, K., Eto, M., Morita, K., Ohno, S., Fujii, M., Hironaka, K., Uematsu, S., Terakawa, A., Bai, Y., Pan, Y., Tsuchiya, T., Ozaki, H., Inoue, H., Uda, S., Kubota, H., Suzuki, Y., Matsumoto, M., Nakayama, K., Hirayama, A., Soga, T., Kuroda, S.

iScience 24 ( 3 ) 102217 2021.03

Research paper (scientific journal), Joint Work, Accepted

Systemic metabolic homeostasis is regulated by inter-organ metabolic cycles involving multiple organs. Obesity impairs inter-organ metabolic cycles, resulting in metabolic diseases. The systemic landscape of dysregulated inter-organ metabolic cycles in obesity has yet to be explored. Here, we measured the transcriptome, proteome, and metabolome in the liver and skeletal muscle and the metabolome in blood of fasted wild-type and leptin-deficient obese (ob/ob) mice, identifying components with differential abundance and differential regulation in ob/ob mice. By constructing and evaluating the trans-omic network controlling the differences in metabolic reactions between fasted wild-type and ob/ob mice, we provided potential mechanisms of the obesity-associated dysfunctions of metabolic cycles between liver and skeletal muscle involving glucose-alanine, glucose-lactate, and ketone bodies. Our study revealed obesity-associated systemic pathological mechanisms of dysfunction of inter-organ metabolic cycles.

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Quantitative and Molecular Similarity Analyses of the Metabolites of Cold- and Hot-Natured Chinese Herbs

Guo, J., Wang, J., Iino, K., Tomita, M., Soga, T.

Evid. Based. Complement. Alternat. Med. 2021 6646507 2021.03

Research paper (scientific journal), Joint Work, Accepted

Background. Based on the theory of traditional Chinese medicine, Chinese herbs possess four different medicinal properties: hot, warm, cold, and cool. These serve as a reference guide for these herbal medicines. However, the molecular mechanisms supporting their relevance remain unclear. Methods. We performed metabolomics based on capillary electrophoresis-time-of-flight mass spectrometry (CE-TOF/MS) and multivariate data analysis for the structural identification of compounds of cold- and hot-natured Chinese herbs. Results. To this end, 30 selected herbs were analyzed and a total of 416 metabolites were identified via CE-TOF/MS, of which 193 compounds were detected in most herbs. The observed profiles offered the potential to understand the mechanism of association between the compounds and nature of the Chinese herbs. Comparison of the similarity in terms of chemical and molecular structures and content revealed that hot-natured herbs contained more nucleotides. In contrast, principal component analysis revealed the presence of more amino acid compounds in cold-natured herbs. Conclusion. Comparing the structural similarities between the samples using the Tanimoto coefficient revealed that a general non-specific structure was observed between cold- and hot-natured herbs; however, the distribution of the molecular groups seemed to contribute more toward the energy properties.

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Reprogramming of glutamine metabolism via glutamine synthetase silencing induces cisplatin resistance in A2780 ovarian cancer cells

Guo, J., Satoh, K., Tabata, S., Mori, M., Tomita, M., Soga, T.

BMC Cancer 21 ( 1 ) 174 2021.02

Research paper (scientific journal), Joint Work, Accepted

Background: Cisplatin (CDDP) significantly prolongs survival in various cancers, but many patients also develop resistance that results in treatment failure. Thus, this study aimed to elucidate the underlying mechanisms by which ovarian cancer cells acquire CDDP resistance.
Methods: We evaluated the metabolic profiles in CDDP-sensitive ovarian cancer A2780 cells and CDDP-resistant A2780cis cells using capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS). We further examined the expression of glutamine metabolism enzymes using real-time PCR and Western blot analyses. Cell viability was accessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
Results: The results showed that levels of glutamine, glutamate, and glutathione (GSH), a key drug resistance mediator synthesized from glutamate, were significantly elevated in A2780cis cells than those in A2780 cells. Furthermore, glutamine starvation decreased the GSH levels and CDDP resistance in A2780cis cells. Interestingly, the expression of glutamine synthetase (GS/GLUL), which synthesizes glutamine from glutamate and thereby negatively regulates GSH production, was almost completely suppressed in resistant A2780cis cells. In addition, treatment of A2780cis cells with 5-aza-2'-deoxycytidine, a DNA-demethylating agent, restored GS expression and reduced CDDP resistance. In contrast, GS knockdown in CDDP-sensitive A2780 cells induced CDDP resistance.
Conclusions: The results indicate that upregulation of GSH synthesis from glutamine via DNA methylation-mediated silencing of GS causes CDDP resistance in A2780cis cells. Therefore, glutamine metabolism could be a novel therapeutic target against CDDP resistance.

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Charged metabolite biomarkers of food intake assessed via plasma metabolomics in a population-based observational study in Japan

Shibutami, E., Ishii, R., Harada, S., Kurihara, A., Kuwabara, K., Kato, S., Iida, M., Akiyama, M., Sugiyama, D., Hirayama, A., Sato, A., Amano, K., Sugimoto, M., Soga, T., Tomita, M., Takebayashi, T.

PLoS One 16 ( 2 ) e0246456 2021.02

Research paper (scientific journal), Joint Work, Accepted

Food intake biomarkers can be critical tools that can be used to objectively assess dietary exposure for both epidemiological and clinical nutrition studies. While an accurate estimation of food intake is essential to unravel associations between the intake and specific health conditions, random and systematic errors affect self-reported assessments. This study aimed to clarify how habitual food intake influences the circulating plasma metabolome in a free-living Japanese regional population and to identify potential food intake biomarkers. To achieve this aim, we conducted a cross-sectional analysis as part of a large cohort study. From a baseline survey of the Tsuruoka Metabolome Cohort Study, 7,012 eligible male and female participants aged 40-69 years were chosen for this study. All data on patients' health status and dietary intake were assessed via a food frequency questionnaire, and plasma samples were obtained during an annual physical examination. Ninety-four charged plasma metabolites were measured using capillary electrophoresis mass spectrometry, by a non-targeted approach. Statistical analysis was performed using partial-least-square regression. A total of 21 plasma metabolites were likely to be associated with long-term food intake of nine food groups. In particular, the influential compounds in each food group were hydroxyproline for meat, trimethylamine-N-oxide for fish, choline for eggs, galactarate for dairy, cystine and betaine for soy products, threonate and galactarate for carotenoid-rich vegetables, proline betaine for fruits, quinate and trigonelline for coffee, and pipecolate for alcohol, and these were considered as prominent food intake markers in Japanese eating habits. A set of circulating plasma metabolites was identified as potential food intake biomarkers in the Japanese community-dwelling population. These results will open the way for the application of new reliable dietary assessment tools not by self-reported measurements but through objective quantification of biofluids.

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Artificial hibernation/life-protective state induced by thiazoline-related innate fear odors

Matsuo, T., Isosaka, T., Tang, L., Soga, T., Kobayakawa, R., Kobayakawa, K.

Commun Biol. 4 ( 1 ) 101 2021.01

Research paper (scientific journal), Joint Work, Accepted

Innate fear intimately connects to the life preservation in crises, although this relationships is not fully understood. Here, we report that presentation of a supernormal innate fear inducer 2-methyl-2-thiazoline (2MT), but not learned fear stimuli, induced robust systemic hypothermia/hypometabolism and suppressed aerobic metabolism via phosphorylation of pyruvate dehydrogenase, thereby enabling long-term survival in a lethal hypoxic environment. These responses exerted potent therapeutic effects in cutaneous and cerebral ischemia/reperfusion injury models. In contrast to hibernation, 2MT stimulation accelerated glucose uptake in the brain and suppressed oxygen saturation in the blood. Whole-brain mapping and chemogenetic activation revealed that the sensory representation of 2MT orchestrates physiological responses via brain stem Sp5/NST to midbrain PBN pathway. 2MT, as a supernormal stimulus of innate fear, induced exaggerated, latent life-protective effects in mice. If this system is preserved in humans, it may be utilized to give rise to a new field: "sensory medicine."

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L-type amino acid transporter 1 is associated with chemoresistance in breast cancer via the promotion of amino acid metabolism

Sato, M., Harada-Shoji, N., Toyohara, T., Soga, T., Itoh, M., Miyashita, M., Tada, H., Amari, M., Anzai, N., Furumoto, S., Abe, T., Suzuki, T., Ishida, T., Sasano, H.

Sci. Rep 11 ( 1 ) 589 2021.01

Research paper (scientific journal), Joint Work, Accepted

18F-FDG PET/CT has been used as an indicator of chemotherapy effects, but cancer cells can remain even when no FDG uptake is detected, indicating the importance of exploring other metabolomic pathways. Therefore, we explored the amino acid metabolism, including L-type amino acid transporter-1 (LAT1), in breast cancer tissues and clarified the role of LAT1 in therapeutic resistance and clinical outcomes of patients. We evaluated LAT1 expression before and after neoadjuvant chemotherapy and examined the correlation of glucose uptake using FDG-PET with the pathological response of patients. It revealed that LAT1 levels correlated with proliferation after chemotherapy, and amino acid and glucose metabolism were closely correlated. In addition, LAT1 was considered to be involved in treatment resistance and sensitivity only in luminal type breast cancer. Results of in vitro analyses revealed that LAT1 promoted amino acid uptake, which contributed to energy production by supplying amino acids to the TCA cycle. However, in MCF-7 cells treated with chemotherapeutic agents, oncometabolites and branched-chain amino acids also played a pivotal role in energy production and drug resistance, despite decreased glucose metabolism. In conclusion, LAT1 was involved in drug resistance and could be a novel therapeutic target against chemotherapy resistance in luminal type breast cancer.

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Relationship between Standard Uptake Values of Positron Emission Tomography/Computed Tomography and Salivary Metabolites in Oral Cancer: A Pilot Study

Ishikawa, S., Hiraka, T., Kirii, K., Sugimoto, M., Shimamoto, H., Sugano, A., Kitabatake, K., Toyoguchi, Y., Kanoto, M., Nemoto, K., Soga, T., Tomita, M., Iino, M.

J Clin. Med. 9 ( 12 ) 3958 - 8 2020.12

Research paper (scientific journal), Joint Work, Accepted

18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET)/computed tomography (CT) is usually used for staging or evaluation of treatment response rather than for cancer screening. However, 18F-FDG PET/CT has also been used in Japan for cancer screening in people with no cancer symptoms, and accumulating evidence supports this application of 18F-FDG PET/CT. Previously, we have observed a correlation between the saliva and tumor metabolomic profiles in patients with oral cancer. Hence, if salivary metabolites demonstrate a significant correlation with PET parameters such as the maximum standardized uptake value (SUVmax), they may have the potential to be used as a screening tool before PET/CT to identify patients with high SUVmax. Hence, in this study, we aimed to explore the relationship between salivary metabolites and SUVmax of 18F-FDG PET/CT using previously collected data. 18F-FDG PET/CT was performed for staging 26 patients with oral cancer. The collected data were integrated and analyzed along with quantified salivary hydrophilic metabolites obtained from the same patients with oral cancer and controls (n = 44). In total, 11 metabolites showed significant correlations with SUVmax in the delayed phases. A multiple logistic regression model of the two metabolites showed the ability to discriminate between patients with oral cancer and controls, with an area under the receiver operating characteristic curve of 0.738 (p = 0.001). This study uniquely confirmed a relationship between salivary metabolites and SUVmax of PET/CT in patients with oral cancer; salivary metabolites were significantly correlated with SUVmax. These salivary metabolites can be used as a screening tool before PET/CT to identify patients with high SUVmax, i.e., to detect the presence of oral cancer.

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Transomics analysis reveals allosteric and gene regulation axes for altered hepatic glucose-responsive metabolism in obesity

Kokaji, T., Hatano, A., Ito, Y. ,Yugi, K., Eto, M., Morita, K., Ohno, S.,Fujii, M., Hironaka, K., Egami, R., Terakawa, A., Tsutchiya, T., Ozaki, H., Inoue, H., Uda, S., Kubota, H., Suzuki, Y., Ikeda, K., Arita, M., Matsumoto, M., Nakayama, K., Hirayama, A., Soga, T., Kuroda, S.

Sci Signal. 13 ( 660 ) eaaz1236 2020.12

Research paper (scientific journal), Joint Work, Accepted

Impaired glucose tolerance associated with obesity causes postprandial hyperglycemia and can lead to type 2 diabetes. To study the differences in liver metabolism in healthy and obese states, we constructed and analyzed transomics glucose-responsive metabolic networks with layers for metabolites, expression data for metabolic enzyme genes, transcription factors, and insulin signaling proteins from the livers of healthy and obese mice. We integrated multiomics time course data from wild-type and leptin-deficient obese (ob/ob) mice after orally administered glucose. In wild-type mice, metabolic reactions were rapidly regulated within 10 min of oral glucose administration by glucose-responsive metabolites, which functioned as allosteric regulators and substrates of metabolic enzymes, and by Akt-induced changes in the expression of glucose-responsive genes encoding metabolic enzymes. In ob/ob mice, the majority of rapid regulation by glucose-responsive metabolites was absent. Instead, glucose administration produced slow changes in the expression of carbohydrate, lipid, and amino acid metabolic enzyme–encoding genes to alter metabolic reactions on a time scale of hours. Few regulatory events occurred in both healthy and obese mice. Thus, our transomics network analysis revealed that regulation of glucose-responsive liver metabolism is mediated through different mechanisms in healthy and obese states. Rapid changes in allosteric regulators and substrates and in gene expression dominate the healthy state, whereas slow changes in gene expression dominate the obese state.

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Gut microbiota depletion by chronic antibiotic treatment alters the sleep/wake architecture and sleep EEG power spectra in mice

Ogawa, Y., Miyoshi, C., Obana, N., Yajima, K., Hotta-Hirashima, N., Ikkyu, A., Kanno, S., Soga, T., Fukuda, S., Yanagisawa, M.

Sci. Rep. 10 ( 1 ) 19554 2020.11

Research paper (scientific journal), Joint Work, Accepted

Dysbiosis of the gut microbiota affects physiological processes, including brain functions, by altering the intestinal metabolism. Here we examined the effects of the gut microbiota on sleep/wake regulation. C57BL/6 male mice were treated with broad-spectrum antibiotics for 4 weeks to deplete their gut microbiota. Metabolome profiling of cecal contents in antibiotic-induced microbiota-depleted (AIMD) and control mice showed significant variations in the metabolism of amino acids and vitamins related to neurotransmission, including depletion of serotonin and vitamin B6, in the AIMD mice. Sleep analysis based on electroencephalogram and electromyogram recordings revealed that AIMD mice spent significantly less time in non-rapid eye movement sleep (NREMS) during the light phase while spending more time in NREMS and rapid eye movement sleep (REMS) during the dark phase. The number of REMS episodes seen in AIMD mice increased during both light and dark phases, and this was accompanied by frequent transitions from NREMS to REMS. In addition, the theta power density during REMS was lower in AIMD mice during the light phase compared with that in the controls. Consequently, the gut microbiota is suggested to affect the sleep/wake architecture by altering the intestinal balance of neurotransmitters.

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Comprehensive Dipeptide Analysis Revealed Cancer-Specific Profile in the Liver of Patients with Hepatocellular Carcinoma and Hepatitis

Ozawa, H., Hirayama, A., Shoji F., Maruyama, M., Suzuki, K., Yamanaka-Okumura, H., Tatano, H., Morine, Y., Soga, T., Shimada, M., Tomita M.

Metabolites 10 ( 11 ) 442 - 11 2020.11

Research paper (scientific journal), Joint Work, Accepted

As the physical properties and functionality of dipeptides differ from those of amino acids, they have attracted attention in metabolomics; however, their functions in vivo have not been clarified in detail. Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer, and its major cause is chronic hepatitis. This study was conducted to explore tumor-specific dipeptide characteristics by performing comprehensive dipeptide analysis in the tumor and surrounding nontumor tissue of patients with HCC. Dipeptides were analyzed by liquid chromatography tandem mass spectrometry and capillary electrophoresis tandem mass spectrometry. Principal component analysis using 236 detected dipeptides showed differences in the dipeptide profiles between nontumor and tumor tissues; however, no clear difference was observed in etiological comparison. In addition, the N- and C-terminal amino acid compositions of the detected dipeptides significantly differed, suggesting the substrate specificity of enzyme proteins, such as peptidase. Furthermore, hepatitis-derived HCC may show a characteristic dipeptide profile even before tumor formation. These results provide insight into HCC pathogenesis and may help identify novel biomarkers for diagnosis.

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Trans-omic Analysis Reveals ROS-Dependent Pentose Phosphate Pathway Activation after High-Frequency Electrical Stimulation in C2C12 Myotubes

Hoshino, D., Kawata, K., Kunida, K., Hatano, A., Yugi, K., Wada, T., Fujii, M., Sano, T., Ito, Y., Furuichi, Y., Manabe, Y., Suzuki, Y., Fujii, N.L., Soga, T., *Kuroda, S.

iScience 23 ( 10 ) 101558 2020.10

Research paper (scientific journal), Joint Work, Accepted

Skeletal muscle adaptation is mediated by cooperative regulation of metabolism, signal transduction, and gene expression. However, the global regulatory mechanism remains unclear. To address this issue, we performed electrical pulse stimulation (EPS) in differentiated C2C12 myotubes at low and high frequency, carried out metabolome and transcriptome analyses, and investigated phosphorylation status of signaling molecules. EPS triggered extensive and specific changes in metabolites, signaling phosphorylation, and gene expression during and after EPS in a frequency-dependent manner. We constructed trans-omic network by integrating these data and found selective activation of the pentose phosphate pathway including metabolites, upstream signaling molecules, and gene expression of metabolic enzymes after high-frequency EPS. We experimentally validated that activation of these molecules after high-frequency EPS was dependent on reactive oxygen species (ROS). Thus, the trans-omic analysis revealed ROS-dependent activation in signal transduction, metabolome, and transcriptome after high-frequency EPS in C2C12 myotubes, shedding light on possible mechanisms of muscle adaptation.

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Quantification of Salivary Charged Metabolites Using Capillary Electrophoresis Time-of-flight-mass Spectrometry

Sugimoto, M., Ota, S., Kaneko, M., Enomoto, A., Soga, T.

Bio Protoc. 10 ( 20 ) e3797 2020.10

Joint Work, Accepted

Salivary metabolomics have provided the potentials to detect both oral and systemic diseases. Capillary electrophoresis time-of-flight-mass spectrometry (CE-TOFMS) enables the identification and quantification of various charged metabolites. This method has been employed to biomarker discoveries using human saliva samples, especially for various types of cancers. The untargeted analysis contributes to finding new biomarkers. i.e., the analysis of all detectable signals including both known and unknown metabolites extends the coverage of metabolite to be observed. However, the observed data includes thousands of peaks. Besides, non-linear migration time fluctuation and skewed peaks are caused by the sample condition. The presented pretreatment protocols of saliva samples enhance the reproducibility of migration time drift, which facilitates the matching peaks across the samples and also results in reproducible absolute concentrations of the detected metabolites. The described protocols are utilized not only for saliva but for any liquid samples with slight modifications.

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Effects of feed crops and boiling on chicken egg yolk and white determined by a metabolome analysis

Ogura T., Wakayama M., Ashino Y., Kadowaki R., Sato M., Soga T., Tomita M.

Food Chem. 327 127077 2020.10

Research paper (scientific journal), Joint Work, Accepted, ISSN 03088146

Compositional analyses of eggs have primarily focused on nutritional components, including large molecules, such as proteins. However, few reports have investigated the effects of heating and hen feed crops on taste components, such as free amino acids and sugars. Herein, water-soluble metabolites in raw and boiled eggs produced from chickens raised with corn- or rice-fed were analyzed. Each egg was separated into yolk and white, and freeze-dried samples were analyzed by CE-MS and LC-MS. Abundant metabolites included amino acids in yolks and sugars in whites. Compared to corn-fed, rice-fed resulted in three times higher betaine and uridine monophosphate concentrations in yolks and whites, respectively. Boiled whites contained more than four times higher concentrations of amino acids and fructose than raw whites. Metabolites in yolks exhibited minimal changes after boiling. Our findings support the use of water-soluble metabolomics to evaluate the effects of heating and feed crops on taste components.

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Proteomic and metabolomic analyses uncover sex-specific regulatory pathways in mouse fetal germline differentiation

Hayashi, Y., Mori, M., Igarashi, K., Tanaka, K., Takehara, A., Ito-Matsuoka, Y., Kanai, A., Yaegashi, N., Soga, T., Matsui, Y.

Biol. Reprod. 103 ( 4 ) 717 - 735 2020.10

Research paper (scientific journal), Joint Work, Accepted, ISSN 00063363

Regulatory mechanisms of germline differentiation have generally been explained via the function of signaling pathways, transcription factors, and epigenetic regulation; however, little is known regarding proteomic and metabolomic regulation and their contribution to germ cell development. Here, we conducted integrated proteomic and metabolomic analyses of fetal germ cells in mice on embryonic day (E)13.5 and E18.5 and demonstrate sex- and developmental stage-dependent changes in these processes. In male germ cells, RNA processing, translation, oxidative phosphorylation, and nucleotide synthesis are dominant in E13.5 and then decline until E18.5, which corresponds to the prolonged cell division and more enhanced hyper-transcription/translation in male primordial germ cells and their subsequent repression. Tricarboxylic acid cycle and one-carbon pathway are consistently upregulated in fetal male germ cells, suggesting their involvement in epigenetic changes preceding in males. Increased protein stability and oxidative phosphorylation during female germ cell differentiation suggests an upregulation of aerobic energy metabolism, which likely contributes to the proteostasis required for oocyte maturation in subsequent stages. The features elucidated in this study shed light on the unrevealed mechanisms of germ cell development.

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Kinetic Trans-omic Analysis Reveals Key Regulatory Mechanisms for Insulin-Regulated Glucose Metabolism in Adipocytes

Ohno, S., Quek, L.E., Krycer, J.R., Yugi, K., Hirayama, A., Ikeda, S., Shoji, F., Suzuki, K., Soga, T., James, D.E., Kuroda, S.

iScience 23 ( 9 ) 101479 2020.09

Research paper (scientific journal), Joint Work, Accepted

Insulin regulates glucose metabolism through thousands of regulatory mechanisms; however, which regulatory mechanisms are keys to control glucose metabolism remains unknown. Here, we performed kinetic trans-omic analysis by integrating isotope-tracing glucose flux and phosphoproteomic data from insulin-stimulated adipocytes and built a kinetic mathematical model to identify key allosteric regulatory and phosphorylation events for enzymes. We identified nine reactions regulated by allosteric effectors and one by enzyme phosphorylation and determined the regulatory mechanisms for three of these reactions. Insulin stimulated glycolysis by promoting Glut4 activity by enhancing phosphorylation of AS160 at S595, stimulated fatty acid synthesis by promoting Acly activity through allosteric activation by glucose 6-phosphate or fructose 6-phosphate, and stimulated glutamate synthesis by alleviating allosteric inhibition of Gls by glutamate. Most of glycolytic reactions were regulated by amounts of substrates and products. Thus, phosphorylation or allosteric modulator-based regulation of only a few key enzymes was sufficient to change insulin-induced metabolism.

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Insulin signaling requires glucose to promote lipid anabolism in adipocytes

Krycer J.R., Quek L.E., Francis D., Zadoorian A., Weiss F.C., Cooke K.C., Nelson M.E., Diaz-Vegas A., Humphrey S.J., Scalzo R., Hirayama A., Ikeda S., Shoji F., Suzuki K., Huynh K., Giles C., Varney B., Nagarajan S.R., Hoy A.J., Soga T., Meikle P.J., Cooney G.J., Fazakerley D.J., James D.E.

J. Biol. Chem. 295 ( 38 ) 13250 - 13266 2020.09

Research paper (scientific journal), Joint Work, Accepted

Adipose tissue is essential for metabolic homeostasis, balancing lipid storage and mobilization based on nutritional status. This is coordinated by insulin, which triggers kinase signaling cascades to modulate numerous metabolic proteins, leading to increased glucose uptake and anabolic processes like lipogenesis. Given recent evidence that glucose is dispensable for adipocyte respiration, we sought to test whether glucose is necessary for insulin-stimulated anabolism. Examining lipogenesis in cultured adipocytes, glucose was essential for insulin to stimulate the synthesis of fatty acids and glyceride-glycerol. Importantly, glucose was dispensable for lipogenesis in the absence of insulin, suggesting that distinct carbon sources are used with or without insulin. Metabolic tracing studies revealed that glucose was required for insulin to stimulate pathways providing carbon substrate, NADPH, and glycerol 3-phosphate for lipid synthesis and storage. Glucose also displaced leucine as a lipogenic substrate and was necessary to suppress fatty acid oxidation. Together, glucose provided substrates and metabolic control for insulin to promote lipogenesis in adipocytes. This contrasted with the suppression of lipolysis by insulin signaling, which occurred independently of glucose. Given previous observations that signal transduction acts primarily before glucose uptake in adipocytes, these data are consistent with a model whereby insulin initially utilizes protein phosphorylation to stimulate lipid anabolism, which is sustained by subsequent glucose metabolism. Consequently, lipid abundance was sensitive to glucose availability, both during adipogenesis and in Drosophila flies in vivo Together, these data highlight the importance of glucose metabolism to support insulin action, providing a complementary regulatory mechanism to signal transduction to stimulate adipose anabolism.

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Comparison of the ischemic and non-ischemic lung cancer metabolome reveals hyper activity of the TCA cycle and autophagy

Kikuchi, N., Soga, T., Nomura, M., Sato, T., Sakamoto, Y., Tanaka, R., Abe, J., Morita, M., Shima, H., Okada, Y., Tanuma, N

Biochem. Biophys. Res. Commun. 530 ( 1 ) 285 - 291 2020.09

Research paper (scientific journal), Joint Work, Accepted, ISSN 0006291X

Recent advances in cancer biology reveal the importance of metabolic changes in cancer; however, less is known about how metabolic pathways in tumors are regulated in vivo. Here, we report analysis of the lung cancer metabolism based on different surgical procedures, namely lobectomy and partial resection. In lobectomy, but not in partial resection, pulmonary arteries and veins are ligated prior to removal of tissues, rendering tissues ischemic. We show that tumors indeed undergo ischemia upon lobectomy and that the tumor metabolome differs markedly from that of tumors removed by partial resection. Comparison of the responses to ischemia in tumor and normal lung tissues revealed that lung cancer tissue exhibits greater TCA cycle and autophagic activity than do normal lung tissues in vivo in patients. Finally, we report that deleting ATG7, which encodes a protein essential for autophagy, antagonizes growth of tumors derived from lung cancer cell lines, suggesting that autophagy confers metabolic advantages to lung cancer. Our findings shed light on divergent metabolic responses to ischemia seen in tumors and normal tissues.

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Pharmacologically targetable vulnerability in prostate cancer carrying RB1-SUCLA2 deletion

Kohno, S., Linn, P. , Nagatani, N., Watanabe, Y., Kumar, S., Soga, T., Takahashi, C.

Oncogene 39 ( 10 ) 5690 - 5707 2020.08

Research paper (scientific journal), Joint Work, Accepted, ISSN 09509232

RB1 gene is often homozygously deleted or mutated in prostate adenocarcinomas following acquirement of castration resistance and/or metastatic ability. We found that SUCLA2 gene is frequently involved in the deletion of the RB1 gene region in advanced prostate cancer. SUCLA2 constitutes the β-subunit of succinate CoA ligase heterodimer that reversibly converts succinyl CoA into succinate. We sought the possibility that deletion of SUCLA2 gives rise to a metabolic vulnerability that could be targeted therapeutically. We found a significant metabolic shift in SUCLA2-deleted prostate cancer cells, including lower mitochondrial respiratory activity. By screening a number of libraries for compounds that induce cell death selectively in SUCLA2-deficient prostate cancer cells, we identified thymoquinone (2-isopropyl-5-methylbenzo-1,4-quinone) and PMA (phorbol-12-myristate-13-acetate) from a natural compound library. These findings indicate that the metabolic vulnerability in SUCLA2-deficient prostate cancer cells is pharmacologically targetable.

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EVI1 triggers metabolic reprogramming associated with leukemogenesis and increases sensitivity to L-asparaginase

Saito, Y., Sawa, D., Kinoshita, M., Yamada, A., Kamimura, S., Suekane, A., Ogoh, H., Matsuo, H., Adachi, S., Taga, T., Tomizawa, D., Osato, M., Soga, T., Morishita, K., Moritake, H.

Haematologica 105 ( 8 ) 2118 - 2129 2020.08

Research paper (scientific journal), Joint Work, Accepted, ISSN 03906078

Metabolic reprogramming of leukemia cells is important for survival, proliferation, and drug resistance under conditions of metabolic stress in the bone marrow. Deregulation of cellular metabolism, leading to development of leukemia, occurs through abnormally high expression of transcription factors such as MYC and Ecotropic Virus Integration site 1 protein homolog (EVI1). Overexpression of EVI1 in adults and children with mixed lineage leukemia-rearrangement acute myeloid leukemia (MLL-r AML) has a very poor prognosis. To identify a metabolic inhibitor for EVI1-induced metabolic reprogramming in MLL-r AML, we used an XFp extracellular flux analyzer to examine metabolic changes during leukemia development in mouse models of AML expressing MLL-AF9 and Evi1 (Evi1/MF9). Oxidative phosphorylation (OXPHOS) in Evi1/MF9 AML cells accelerated prior to activation of glycolysis, with a higher dependency on glutamine as an energy source. Furthermore, EVI1 played a role in glycolysis as well as driving production of metabolites in the tricarboxylic acid cycle. L-asparaginase (L-asp) exacerbated growth inhibition induced by glutamine starvation and suppressed OXPHOS and proliferation of Evi1/MF9 both in vitro and in vivo; high sensitivity to L-asp was caused by low expression of asparagine synthetase (ASNS) and L-asp-induced suppression of glutamine metabolism. In addition, samples from patients with EVI1+MF9 showed low ASNS expression, suggesting that it is a sensitive marker of L-asp treatment. Clarification of metabolic reprogramming in EVI1+ leukemia cells may aid development of treatments for EVI1+MF9 refractory leukemia.

Copyright© 2020 Ferrata Storti Foundation.

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T cell-specific deletion of Pgam1 reveals a critical role for glycolysis in T cell responses

Toriyama, K., Kuwahara, M., Kondoh, H., Mikawa, T., Takemori, N., Konishi, A., Yorozuya, T., Yamada, T., Soga, T., Shiraishi, A., Yamashita, M.

Commun Biol 3 ( 1 ) 394 2020.07

Research paper (scientific journal), Joint Work, Accepted

Although the important roles of glycolysis in T cells have been demonstrated, the regulatory mechanism of glycolysis in activated T cells has not been fully elucidated. Furthermore, the influences of glycolytic failure on the T cell-dependent immune response in vivo remain unclear. We therefore assessed the role of glycolysis in the T cell-dependent immune response using T cell-specific Pgam1-deficient mice. Both CD8 and CD4 T cell-dependent immune responses were attenuated by Pgam1 deficiency. The helper T cell-dependent inflammation was ameliorated in Pgam1-deficient mice. Glycolysis augments the activation of mTOR complex 1 (mTORC1) and the T-cell receptor (TCR) signals. Glutamine acts as a metabolic hub in activated T cells, since the TCR-dependent increase in intracellular glutamine is required to augment glycolysis, increase mTORC1 activity and augment TCR signals. These findings suggest that mTORC1, glycolysis and glutamine affect each other and cooperate to induce T cell proliferation and differentiation.

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Comprehensive Dipeptide Profiling and Quantitation by Capillary Electrophoresis and Liquid Chromatography Coupled with Tandem Mass Spectrometry

Ozawa, H., Hirayama, A., Ishikawa, T., Kudo, R., Maruyama, M., Shoji, F., Doke, T., Ishimoto, T., Maruyama, S., Soga, T., Tomita, M.

Anal. Chem. 92 ( 14 ) 9799 - 9806 2020.07

Research paper (scientific journal), Joint Work, Accepted, ISSN 00032700

Dipeptides have attracted much attention as post-amino acids with physical properties and functions different from those of amino acids. However, a given dipeptide cannot be distinguished by mass spectrometry from its structural isomer with an opposite amino acid binding order unless these isomers are separated before introduction, which complicates the comprehensive analysis of dipeptides. Herein, a novel analytical platform for dipeptide analysis by capillary electrophoresis tandem mass spectrometry and liquid chromatography tandem mass spectrometry is developed. This method is used to quantitate 335 dipeptides and achieves excellent separation of structural isomers with opposite binding orders, high correlation coefficients, and low instrumental detection limits (0.088-83.1 nM). Moreover, acceptable recoveries (70-135%) are observed for most tested dipeptides in chicken liver samples spiked both before and after preparation. The developed method is also applied to the quantitation of dipeptides in the livers of mice fed different diets to detect 236 dipeptides, and the shift from a normal diet to a high-fat diet is shown to increase/decrease (p < 0.05, fold-change < 0.5) the contents of 0/29 dipeptides, respectively. The developed method is expected to facilitate the search for new dipeptide applications such as novel functional components of foods and biomarkers of diseases.

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Local Necrotic Cells Trigger Systemic Immune Activation via Gut Microbiome Dysbiosis in Drosophila

Kosakamoto, H., Yamauchi, T., Akuzawa-Tokita, Y., Nishimura, K., Soga, T., Murakami, T., Mori, H., Yamamoto, K., Miyazaki, R., Koto, A., Miura, M., Obata, F.

Cell Rep. 32 ( 3 ) 107938 2020.07

Research paper (scientific journal), Joint Work

Necrotic cells elicit an inflammatory response through their endogenous factors with damage-associated molecular patterns. Blocking apoptosis in Drosophila wings leads to the necrosis-driven systemic immune response by unknown mechanisms. Here, we demonstrate that immune activation in response to necrotic cells is mediated by commensal gut microbiota. Removing the microbiome attenuates hyperactivation of the innate immune signaling IMD pathway in necrosis-induced flies. Necrotic cells in wings trigger Gluconobacter expansion in the gut. An isolated Gluconobacter sp. strain is sufficient for pathological IMD activation in necrosis-induced flies, while it is not inflammatory for control animals. In addition, bacterial colonization shifts the host metabolome and shortens the lifespan of necrosis-induced flies. This study shows that local necrosis triggers a pathological systemic inflammatory response through interaction between the host and the dysbiotic gut microbiome.

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The use of a double coaxial electrospray ionization sprayer improves the peak resolutions of anionic metabolites in capillary ion chromatography-mass spectrometry

Hirayama, A., Tabata, S., Kudo, R., Hasebe, M., Suzuki, K., Tomita, M., Soga, T.

J. Chromatogr. A. 1619 460914 2020.05

Research paper (scientific journal), Joint Work, Accepted, ISSN 00219673

Recently, ion chromatography coupled with mass spectrometry has been used for the determination of anionic metabolites. However, connection with a mass spectrometer in this method is not straightforward because backpressure produced by the addition of a make-up solution often affects the peak resolutions of the target metabolites. To overcome this problem, we developed a capillary ion chromatography-mass spectrometry method utilizing a double coaxial electrospray ionization sprayer. This method was not affected by backpressure and the number of theoretical plates was about three times that of a conventional sprayer. Under optimized conditions, 44 anionic metabolites, including organic acids, sugar phosphates, nucleotides, and cofactors, were successfully separated and selectively detected with a Q Exactive mass spectrometer. The calibration curves of the tested metabolites showed excellent linearity within the range of 1-100,000 nmol/L and the correlation coefficient was greater than 0.991. The detection limits for these metabolites were between 1 and 500 nmol/L (0.4 and 200 fmol). The developed method was applied to the quantitation of anionic metabolites in cultured cancer cell samples with tumor necrosis factor (TNF)-α stimulation. This allowed for the successful determination of 105 metabolites. The levels of tricarboxylic acid cycle intermediates changed significantly after TNF-α stimulation. These results demonstrate that the developed method is a promising new tool for comprehensive analysis of anionic metabolites.

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Paternal restraint stress affects offspring metabolism via ATF-2 dependent mechanisms in Drosophila melanogaster germ cells

Seong, K.H., Ly, N.H., Katou, Y., Yokota, N., Nakato, R., Murakami, S., Hirayama, A., Fukuda, S., Kang, S., Soga, T., Shirahige, K., Ishii, S.

Commun. Biol. 3 ( 1 ) 208 2020.05

Research paper (scientific journal), Joint Work, Accepted

Paternal environmental factors can epigenetically influence gene expressions in offspring. We demonstrate that restraint stress, an experimental model for strong psychological stress, to fathers affects the epigenome, transcriptome, and metabolome of offspring in a MEKK1-dATF2 pathway-dependent manner in Drosophila melanogaster. Genes involved in amino acid metabolism are upregulated by paternal restraint stress, while genes involved in glycolysis and the tricarboxylic acid (TCA) cycle are downregulated. The effects of paternal restraint stress are also confirmed by metabolome analysis. dATF-2 is highly expressed in testicular germ cells, and restraint stress also induces p38 activation in the testes. Restraint stress induces Unpaired 3 (Upd3), a Drosophila homolog of Interleukin 6 (IL-6). Moreover, paternal overexpression of upd3 in somatic cells disrupts heterochromatin in offspring but not in offspring from dATF-2 mutant fathers. These results indicate that paternal restraint stress affects metabolism in offspring via inheritance of dATF-2-dependent epigenetic changes.

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Effect of blanching on the concentration of metabolites in two parts of Undaria pinnatifida, Wakame (leaf) and Mekabu (sporophyll)

Hamid, S.S., Wakayama, M., Ashino, Y., Kadowaki, R., Soga, T., Tomita, M.

Algal Res. 47 101829 2020.05

Research paper (scientific journal), Joint Work, Accepted, ISSN 22119264

Undaria pinnatifida is a seaweed native to Japan, where it is cultivated and sold commercially as food. Fresh seaweed commonly blanches before consumption. However, the effects of blanching on metabolites present in different parts of U. pinnatifida, namely, Wakame (leaf) and Mekabu (sporophyll), are not well understood. Here, we investigated changes in metabolites in these two seaweed parts after blanching for different periods. The samples were freeze-dried and extracted using methanol-water, and metabolite concentration was quantitated using liquid chromatography mass spectrometry and capillary electrophoresis mass spectrometry. The concentration of most metabolites was the highest in raw samples, decreasing drastically within 20 s of blanching in Wakame and decreasing gradually in Mekabu. In contrast, the concentration of some fatty acids, such as octanoate, increased in both parts. During blanching, seaweed color changed from brown to green, and blanching did not significantly affect amino acid or the sugar composition. Finally, a relationship was found between metabolite and blanching-related characteristics, such as color and drying percentage.

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Adenosine leakage from perforin-burst extracellular vesicles inhibits perforin secretion by cytotoxic T-lymphocytes

Tadokoro, H., Hirayama, A., Kudo, R., Hasebe, M., Yoshioka, Y., Matsuzaki, J., Yamamoto, Y., Sugimoto, M., Soga, T., Ochiya, T.

PLoS One 15 ( 4 ) e0231430 2020.04

Research paper (scientific journal), Joint Work, Accepted

Extracellular vesicles (EVs) in the tumor microenvironment facilitate intercellular communication. Cancer cell-derived EVs act as an immunosuppressor by transporting cargos and presenting transmembrane proteins. By contrast, CD8+ cytotoxic T-lymphocytes (CTLs) exert anti-cancer cytotoxicity via the pore-forming protein perforin. Here, we hypothesize that although EVs are destroyed by perforin, cancer cell-derived EVs might possess mechanisms that enable them to avoid this destruction. We used a breast cancer cell line, MDA-MB-231-luc-D3H2LN (D3H2LN), to generate EVs. Destruction of the EVs by perforin was demonstrated visually using atomic force microscopy. To investigate immunosuppressive metabolites within cancer cell-derived EVs, we performed metabolomic profiling of EVs from D3H2LN cells cultured for 48 h with or without IFN-γ, which induces metabolic changes in the cells. We found that both types of EV from IFN-γ treated D3H2LN cells and non-treated D3H2LN cells contained adenosine, which has immunosuppressive effects. When we exposed cancer cell-derived EVs to CTLs, perforin secretion by CTLs fell significantly. In addition, the decreases in perforin secretion were ameliorated by treatment with adenosine deaminase, which degrades extracellular adenosine. Taken together, these results suggest that after perforin secreted by CTLs disrupts the membrane of EVs, adenosine released from the EVs acts as an immunosuppressive metabolite by binding to the adenosine receptor on the CTL membrane. This mechanism provides a novel survival strategy using cancer cell-derived EVs.

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Sensory properties and metabolomic profiles of dry-cured ham during the ripening process

Sugimoto M., Sugawara T., Obiya S., Enomoto A., Kaneko M., Ota S., Soga T., Tomita M.

Food Res Int. 129 108850 2020.03

Research paper (scientific journal), Joint Work, Accepted, ISSN 09639969

Dry-cured ham with a long ripening period is a valuable product worldwide. Ripening time is a key determinant of the endogenous metabolites that characterize the flavor and taste of ham products. While various studies have analyzed the relationship between ripening duration and sensory characteristics, no studies have evaluated ham products produced in Japan. Here, we conducted time-course metabolomic profiling, taste sensor-based analyses, and sensory evaluations by non-trained consumers during ripening. Capillary electrophoresis-mass spectrometry was used to quantify non-volatile metabolites, such as amino acids, organic acids, and nucleotides. In an analysis of eight time-points during 680 days of ripening, the highest score for the after-taste of umami was observed on day 540, despite subtle changes in the scores for other properties. The concentrations of aspartic acid and glutamic acid relative to those of total amino acids were the highest at this point. This approach can contribute to the understanding of the relationship between the metabolite profile and ripening duration.

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Dynamic 13C Flux Analysis Captures the Reorganization of Adipocyte Glucose Metabolism in Response to Insulin

Quek, L.E., Krycer, J.R., Ohno, S., Yugi, K., Fazakerley, D.J., Scalzo, R., Elkington, S.D., Dai, Z., Hirayama, A., Ikeda, S., Shoji, F., Suzuki, K., Locasale, J.W., Soga, T., James, D.E., Kuroda, S.

iScience 23 ( 2 ) 100855 2020.02

Research paper (scientific journal), Joint Work, Accepted

Cellular metabolism is dynamic, but quantifying non-steady metabolic fluxes by stable isotope tracers presents unique computational challenges. Here, we developed an efficient 13C-tracer dynamic metabolic flux analysis (13C-DMFA) framework for modeling central carbon fluxes that vary over time. We used B-splines to generalize the flux parameterization system and to improve the stability of the optimization algorithm. As proof of concept, we investigated how 3T3-L1 cultured adipocytes acutely metabolize glucose in response to insulin. Insulin rapidly stimulates glucose uptake, but intracellular pathways responded with differing speeds and magnitudes. Fluxes in lower glycolysis increased faster than those in upper glycolysis. Glycolysis fluxes rose disproportionally larger and faster than the tricarboxylic acid cycle, with lactate a primary glucose end product. The uncovered array of flux dynamics suggests that glucose catabolism is additionally regulated beyond uptake to help shunt glucose into appropriate pathways. This work demonstrates the value of using dynamic intracellular fluxes to understand metabolic function and pathway regulation.

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A FLCN-TFE3 Feedback Loop Prevents Excessive Glycogenesis and Phagocyte Activation by Regulating Lysosome Activity

Endoh, M., Baba, M., Endoh, T., Hirayama, A., Nakamura-Ishizu, A., Umemoto, T., Hashimoto, M., Nagashima, K., Soga, T., Lang, M., Schmidt, L.S., Linehan, W.M., Suda, T.

Cell Rep. 30 ( 6 ) 1823 - 1834 2020.02

Research paper (scientific journal), Joint Work, Accepted

The tumor suppressor folliculin (FLCN) suppresses nuclear translocation of TFE3, a master transcription factor for lysosomal biogenesis, via regulation of amino-acid-sensing Rag GTPases. However, the importance of this lysosomal regulation in mammalian physiology remains unclear. Following hematopoietic-lineage-specific Flcn deletion in mice, we found expansion of vacuolated phagocytes that accumulate glycogen in their cytoplasm, phenotypes reminiscent of lysosomal storage disorder (LSD). We report that TFE3 acts in a feedback loop to transcriptionally activate FLCN expression, and FLCN loss disrupts this loop, augmenting TFE3 activity. Tfe3 deletion in Flcn knockout mice reduces the number of phagocytes and ameliorates LSD-like phenotypes. We further reveal that TFE3 stimulates glycogenesis by promoting the expression of glycogenesis genes, including Gys1 and Gyg, upon loss of Flcn. Taken together, we propose that the FLCN-TFE3 feedback loop acts as a rheostat to control lysosome activity and prevents excessive glycogenesis and LSD-like phagocyte activation.

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The guanylate cyclase C agonist linaclotide ameliorates the gut-cardio-renal axis in an adenine-induced mouse model of chronic kidney disease

Nanto-Hara F., Kanemitsu Y., Fukuda S., Kikuchi K., Asaji K., Saigusa D., Iwasaki T., Ho H.J., Mishima E., Suzuki T., Suzuki C., Tsukimi T., Matsuhashi T., Oikawa Y., Akiyama Y., Kure S., Owada Y., Tomioka Y., Soga T., Ito S., Abe T.

Nephrol. Dial. Transplant. 35 ( 2 ) 250 - 264 2020.02

Research paper (scientific journal), Joint Work, ISSN 09310509

Background: Cardiorenal syndrome is a major cause of mortality in patients with chronic kidney disease (CKD). However, the involvement of detrimental humoral mediators in the pathogenesis of cardiorenal syndrome is still controversial. Trimethylamine-N-oxide (TMAO), a hepatic metabolic product of trimethylamine generated from dietary phosphatidylcholine or carnitine derived by the gut microbiota, has been linked directly with progression of cardiovascular disease and renal dysfunction. Thus, targeting TMAO may be a novel strategy for the prevention of cardiovascular disease and chronic kidney disease.
Methods: Linaclotide, a guanylate cyclase C agonist, was administered to adenine-induced renal failure (RF) mice and changes in renal function and levels of gut-derived uremic toxins, as well as the gut microbiota community, were analyzed using metabolomic and metagenomic methods to reveal its cardiorenal effect.
Results: Linaclotide decreased the plasma levels of TMAO at a clinically used low dose of 10 μg/kg in the adenine-induced RF mouse model. At a high concentration of 100 μg/kg, linaclotide clearly improved renal function and reduced the levels of various uremic toxins. A reduction in TMAO levels following linaclotide treatment was also observed in a choline-fed pro-atherosclerotic model. Linaclotide ameliorated renal inflammation and fibrosis and cardiac fibrosis, as well as decreased the expression of collagen I, transforming growth factor-β, galectin-3 (Gal-3) and ST2 genes. Plasma levels of Gal-3 and ST2 were also reduced. Because exposure of cardiomyocytes to TMAO increased fibronectin expression, these data suggest that linaclotide reduced the levels of TMAO and various uremic toxins and may result in not only renal, but also cardiac, fibrosis. F4/80-positive macrophages were abundant in small intestinal crypts in RF mice, and this increased expression was decreased by linaclotide. Reduced colonic claudin-1 levels were also restored by linaclotide, suggesting that linaclotide ameliorated the 'leaky gut' in RF mice. Metagenomic analysis revealed that the microbial order Clostridiales could be responsible for the change in TMAO levels.
Conclusion: Linaclotide reduced TMAO and uremic toxin levels and could be a powerful tool for the prevention and control of the cardiorenal syndrome by modification of the gut-cardio-renal axis.

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Lactate production is a prioritized feature of adipocyte metabolism

Krycer, J.R., Quek, L.E., Francis, D.J., Fazakerley, S.D., Elkington, S., Diaz-Vegas, A., Cooke, K.C., Weiss, F.C., Duan, X., Kurdyukov, S., Zhou, P.X., Tambar, U.,K. Hirayama, A., Ikeda, S., Kamei, Y., Soga, T., Cooney, G.J., James, D.E.

J Biol Chem. 295 ( 1 ) 83 - 98 2020.01

Research paper (scientific journal), Joint Work, Accepted, ISSN 00219258

Adipose tissue is essential for whole-body glucose homeostasis, with a primary role in lipid storage. It has been previously observed that lactate production is also an important metabolic feature of adipocytes, but its relationship to adipose and whole-body glucose disposal remains unclear. Therefore, using a combination of metabolic labeling techniques, here we closely examined lactate production of cultured and primary mammalian adipocytes. Insulin treatment increased glucose uptake and conversion to lactate, with the latter responding more to insulin than did other metabolic fates of glucose. However, lactate production did not just serve as a mechanism to dispose of excess glucose, because we also observed that lactate production in adipocytes did not solely depend on glucose availability and even occurred independently of glucose metabolism. This suggests that lactate production is prioritized in adipocytes. Furthermore, knocking down lactate dehydrogenase specifically in the fat body of Drosophila flies lowered circulating lactate and improved whole-body glucose disposal. These results emphasize that lactate production is an additional metabolic role of adipose tissue beyond lipid storage and release.

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Inosine pranobex enhances human NK cell cytotoxicity by inducing metabolic activation and NKG2D ligand expression

McCarthy, MT., Lin, D., Soga, T., Adam, J., O'Callaghan, CA.

Eur. J. Immunol. 50 ( 1 ) 130 - 137 2020.01

Research paper (scientific journal), Joint Work, Accepted, ISSN 00142980

Inosine pranobex (IP) is a synthetic immunomodulating compound, indicated for use in the treatment of human papillomavirus-associated warts and subacute sclerosing panencephalitis. Previous studies demonstrate that the immunomodulatory activity of IP is characterized by enhanced lymphocyte proliferation, cytokine production, and NK cell cytotoxicity. The activation of NKG2D signaling on NK cells, CD8+ T cells, and γδ T cells also produces these outcomes. We hypothesized that IP alters cellular immunity through the induction of NKG2D ligand expression on target cells, thereby enhancing immune cell activation through the NKG2D receptor. We tested this hypothesis and show that exposure of target cells to IP leads to increased expression of multiple NKG2D ligands. Using both targeted metabolic interventions and unbiased metabolomic studies, we found that IP causes an increase in intracellular concentration of purine nucleotides and tricarboxylic acid (TCA) cycle intermediates and NKG2D ligand induction. The degree of NKG2D ligand induction was functionally significant, leading to increased NKG2D-dependent target cell immunogenicity. These findings demonstrate that the immunomodulatory properties of IP are due to metabolic activation with NKG2D ligand induction.

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Characterization of cancer omics and drug perturbations in panels of lung cancer cells

Suzuki, A., Onodera, K., Matsui, K., Seki, M., Esumi, H., Soga, T., Sugano, S., Kohno, T., Suzuki, Y., Tsuchihara, K.

Sci Rep 9 ( 1 ) 19529 2019.12

Research paper (scientific journal), Joint Work, Accepted

To better understand the disruptions of transcriptional regulations and gene expression in lung cancers, we constructed a multi-omics catalogue of the responses of lung cancer cells to a series of chemical compounds. We generated and analyzed 3,240 RNA-seq and 3,393 ATAC-seq libraries obtained from 23 cell lines treated with 95 well-annotated compounds. To demonstrate the power of the created multi-omics resource, we attempted to identify drugs that could induce the designated changes alone or in combination. The basal multi-omics information was first integrated into co-expression modules. Among these modules, we identified a stress response module that may be a promising drug intervention target, as new combinations of compounds that could be used to regulate this module and the consequent phenotypic appearance of cancer cells have been identified. We believe that the multi-omics profiles generated in this study and the strategy used to stratify them will lead to more rational and efficient development of anticancer drugs.

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Carbonic anhydrase 2 (CAII) supports tumor blood endothelial cell survival under lactic acidosis in the tumor microenvironment

Annan, DA., Maishi, N., Soga T., Dawood, R. Li, C., Kikuchi, H., Hojo, T., Morimoto, M., Kitamura, T., Alam, MT., Minowa, K., Shinohara, N., Nam, JM., Hida, Y., Hida, K.

Cell Commun. Signal. 17 ( 1 ) 169 2019.12

Research paper (scientific journal), Joint Work, Accepted

Background: Tumor endothelial cells (TECs) perform tumor angiogenesis, which is essential for tumor growth and metastasis. Tumor cells produce large amounts of lactic acid from glycolysis; however, the mechanism underlying the survival of TECs to enable tumor angiogenesis under high lactic acid conditions in tumors remains poorly understood.
Methodology: The metabolomes of TECs and normal endothelial cells (NECs) were analyzed by capillary electrophoresis time-of-flight mass spectrometry. The expressions of pH regulators in TECs and NECs were determined by quantitative reverse transcription-PCR. Cell proliferation was measured by the MTS assay. Western blotting and ELISA were used to validate monocarboxylate transporter 1 and carbonic anhydrase 2 (CAII) protein expression within the cells, respectively. Human tumor xenograft models were used to access the effect of CA inhibition on tumor angiogenesis. Immunohistochemical staining was used to observe CAII expression, quantify tumor microvasculature, microvessel pericyte coverage, and hypoxia.
Results: The present study shows that, unlike NECs, TECs proliferate in lactic acidic. TECs showed an upregulated CAII expression both in vitro and in vivo. CAII knockdown decreased TEC survival under lactic acidosis and nutrient-replete conditions. Vascular endothelial growth factor A and vascular endothelial growth factor receptor signaling induced CAII expression in NECs. CAII inhibition with acetazolamide minimally reduced tumor angiogenesis in vivo. However, matured blood vessel number increased after acetazolamide treatment, similar to bevacizumab treatment. Additionally, acetazolamide-treated mice showed decreased lung metastasis.
Conclusion: These findings suggest that due to their effect on blood vessel maturity, pH regulators like CAII are promising targets of antiangiogenic therapy. Video Abstract.

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Mutant IDH1 confers resistance to energy stress in normal biliary cells through PFKP-induced aerobic glycolysis and AMPK activation

Fujiwara, H., Tateishi, K., Misumi, K., Hayashi, A., Igarashi, K, Kato, H., Nakatsuka, T., Suzuki, N., Yamamoto, K., Kudo, Y., Hayakawa, Y., Nakagawa, H., Tanaka, Y., Ijichi, H., Kogure, H, Nakai, Y, Isayama, H., Hasegawa, K., Fukayama, M., Soga, T., Koike, K..

Sci. Rep. 9 ( 1 ) 18859 2019.12

Research paper (scientific journal), Joint Work, Accepted

Metabolism is a critical regulator of cell fate determination. Recently, the significance of metabolic reprogramming in environmental adaptation during tumorigenesis has attracted much attention in cancer research. Recurrent mutations in the isocitrate dehydrogenase (IDH) 1 or 2 genes have been identified in several cancers, including intrahepatic cholangiocarcinoma (ICC). Mutant IDHs convert α-ketoglutarate (α-KG) to 2-hydroxyglutarate (2-HG), which affects the activity of multiple α-KG-dependent dioxygenases including histone lysine demethylases. Although mutant IDH can be detected even in the early stages of neoplasia, how IDH mutations function as oncogenic drivers remains unclear. In this study, we aimed to address the biological effects of IDH1 mutation using intrahepatic biliary organoids (IBOs). We demonstrated that mutant IDH1 increased the formation of IBOs as well as accelerated glucose metabolism. Gene expression analysis and ChIP results revealed the upregulation of platelet isoform of phosphofructokinase-1 (PFKP), which is a rate-limiting glycolytic enzyme, through the alteration of histone modification. Knockdown of the Pfkp gene alleviated the mutant IDH1-induced increase in IBO formation. Notably, the high expression of PFKP was observed more frequently in patients with IDH-mutant ICC compared to in those with wild-type IDH (p < 0.01, 80.9% vs. 42.5%, respectively). Furthermore, IBOs expressing mutant IDH1 survived the suppression of ATP production caused by growth factor depletion and matrix detachment by retaining high ATP levels through 5' adenosine monophosphate-activated protein kinase (AMPK) activation. Our findings provide a systematic understanding as to how mutant IDH induces tumorigenic preconditioning by metabolic rewiring in intrahepatic cholangiocytes.

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Metabolic adaptation to glycolysis is a basic defense mechanism of macrophages for Mycobacterium tuberculosis infection

Osada-Oka, M., Goda, N., Saiga, H., Yamamoto, M., Takeda, K., Ozeki, Y., Yamagchi, T., Soga, T., Tateishi, Y., Miura, K., Okuzaki, D., Kobayashi, K., *Matsumoto, S.

Int. Immunol. 31 ( 12 ) 781 - 793 2019.11

Research paper (scientific journal), Joint Work, Accepted

Macrophages are major components of tuberculosis (TB) granulomas and are responsible for host defenses against the intracellular pathogen, Mycobacterium tuberculosis. We herein showed the strong expression of hypoxia-inducible factor-1α (HIF-1α) in TB granulomas and more rapid death of HIF-1α-conditional knockout mice than wild-type (WT) mice after M. tuberculosis infection. Although interferon-γ (IFN-γ) is a critical host-protective cytokine against intracellular pathogens, HIF-1-deficient macrophages permitted M. tuberculosis growth even after activation with IFN-γ. These results prompted us to investigate the role of HIF-1α in host defenses against infection. We found that the expression of lactate dehydrogenase-A (LDH-A) was controlled by HIF-1α in M. tuberculosis-infected macrophages IFN-γ independently. LDH-A is an enzyme that converts pyruvate to lactate and we found that the intracellular level of pyruvate in HIF-1α-deficient bone marrow-derived macrophages (BMDMs) was significantly higher than in WT BMDMs. Intracellular bacillus replication was enhanced by an increase in intracellular pyruvate concentrations, which were decreased by LDH-A. Mycobacteria in phagosomes took up exogenous pyruvate more efficiently than glucose, and used it as the feasible carbon source for intracellular growth. These results demonstrate that HIF-1α prevents the hijacking of pyruvate in macrophages, making it a fundamental host-protective mechanism against M. tuberculosis.

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A metabolic profile of routine needle biopsies identified tumor type specific metabolic signatures for breast cancer stratification: a pilot study

Harada-Shoji, N., Soga, T., Tada, H., Miyashita, M., Harada, M., Watanabe, G., Hamanaka, Y., Sato, A., Suzuki, T., Suzuki, A., Ishida, T.

Metabolomics 15 ( 11 ) 147 2019.11

Research paper (scientific journal), Joint Work, Accepted, ISSN 15733882

Introduction: Metabolomics has recently emerged as a tool for understanding comprehensive tumor-associated metabolic dysregulation. However, only limited application of this technology has been introduced into the clinical setting of breast cancer.
Objectives: The aim of this study was to determine the feasibility of metabolome analysis using routine CNB/VAB samples from breast cancer patients and to elucidate metabolic signatures using metabolic profiling.
Methods: After breast cancer screenings, 20 consecutive patients underwent CNB/VAB, and diagnosed with benign, DCIS and IDC by histology. Metabolome analysis was performed using CE-MS. Differential metabolites were then analyzed and evaluated with MetaboAnalyst 4.0.
Results: We measured 116-targeted metabolites involved in energy metabolism. Principal component analysis and unsupervised hierarchical analysis revealed a distinct metabolic signature unique to namely "pure" IDC samples, whereas that of DCIS was similar to benign samples. Pathway analysis unveiled the most affected pathways of the "pure" IDC metabotype, including "pyrimidine," "alanine, aspartate, and glutamate" and "arginine and proline" pathways.
Conclusions: Our proof-of-concept study demonstrated that CE-MS-based CNB/VAB metabolome analysis is feasible for implementation in routine clinical settings. The most affected pathways in this study may contribute to improved breast cancer stratification and precision medicine.

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MITF controls the TCA cycle to modulate the melanoma hypoxia response

Louphrasitthiphol P., Ledaki I., Chauhan J., Falletta P., Siddaway R., Buffa FM., Mole DR., Soga T., Goding CR.

Pigment. Cell Melanoma. Res. 32 ( 6 ) 792 - 808 2019.11

Research paper (scientific journal), Joint Work, Accepted, ISSN 17551471

In response to the dynamic intra-tumor microenvironment, melanoma cells adopt distinct phenotypic states associated with differential expression of the microphthalmia-associated transcription factor (MITF). The response to hypoxia is driven by hypoxia-inducible transcription factors (HIFs) that reprogram metabolism and promote angiogenesis. HIF1α indirectly represses MITF that can activate HIF1α expression. Although HIF and MITF share a highly related DNA-binding specificity, it is unclear whether they co-regulate subset of target genes. Moreover, the genomewide impact of hypoxia on melanoma and whether melanoma cell lines representing different phenotypic states exhibit distinct hypoxic responses is unknown. Here we show that three different melanoma cell lines exhibit widely different hypoxia responses with only a core 23 genes regulated in common after 12 hr in hypoxia. Surprisingly, under hypoxia MITF is transiently up-regulated by HIF1α and co-regulates a subset of HIF targets including VEGFA. Significantly, we also show that MITF represses itself and also regulates SDHB to control the TCA cycle and suppress pseudo-hypoxia. Our results reveal a previously unsuspected role for MITF in metabolism and the network of factors underpinning the hypoxic response in melanoma.

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Association between dyslipidemia and plasma levels of branched-chain amino acids in the Japanese population without diabetes mellitus

Fukushima, K., Harada, S., Takeuchi, A., Kurihara, A., Iida, M., Fukai, K., Kuwabara, K., Kato, S., Matsumoto, M., Hirata, A., Akiyama, M., Tomita, M., Hirayama, A., Sato, A., Suzuki, C., Sugimoto, M., Soga, T., Sugiyama, D., Takebayashi, T.

J. Clini. Lipidol 13 ( 6 ) 932 - 939 2019.11

Research paper (scientific journal), Joint Work, Accepted, ISSN 19332874

Background: Branched-chain amino acids (BCAAs) play a key role in energy homeostasis.
Objective: We aimed to investigate the association between plasma BCAA levels and dyslipidemia in the Japanese population without diabetes mellitus.
Methods: This cross-sectional study included 4952 participants without diabetes mellitus, enrolled in the Tsuruoka Metabolomic Cohort Study. Plasma BCAA levels were measured by capillary electrophoresis-mass spectrometry. Correlations between lipid and BCAA profiles were evaluated by sex-stratified multiple linear regression analyses, after adjusting for confounders. Logistic regression was used to identify associations between BCAAs and metabolic dyslipidemia (MD) defined as triglyceride levels ≥150 mg/dL, high-density lipoprotein cholesterol levels ≤40 mg/dL for men and ≤50 mg/dL for women, or low-density lipoprotein cholesterol (LDL-C) levels ≥140 mg/dL.
Results: In both sexes, the levels of individual BCAAs and the total BCAA levels correlated positively with triglyceride levels and negatively with high-density lipoprotein cholesterol levels. Valine, leucine, and total BCAA levels were weakly and positively correlated with LDL-C levels. Increased BCAA levels showed positive associations with MD. However, associations between BCAAs and elevated LDL-C levels were unclear. Furthermore, the associations between BCAA levels and MD regardless of fasting blood sugar (FBS) levels (high or low). Although valine, leucine, and total BCAA levels were weakly associated with elevated LDL-C levels in the high-FBS group, no such association was observed in the low-FBS group.
Conclusions: BCAAs might be associated with MD independently of the FBS level and might play an important role in lipid metabolism and dyslipidemia.

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Metabolome Analysis Reveals Dermal Histamine Accumulation in Murine Dermatitis Provoked by Genetic Deletion of P-Glycoprotein and Breast Cancer Resistance Protein

Hashimoto, N., Nakamichi, N., Nanmo, H., Kimura, K., Masuo, Y., Sakai, Y., Schinkel, A.H., Sato, S., Soga, T., Kato, Y.

Pharm. Res. 36 ( 11 ) 158 2019.11

Research paper (scientific journal), Joint Work, Accepted, ISSN 07248741

Purpose: P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are xenobiotic transporters which pump out variety types of compounds, but information on their interaction with endogenous substrates in the skin is limited. The purpose of the present study was to clarify possible association of these transporters in dermal accumulation of inflammatory mediators. Methods: Dermatitis model was constructed by repeated topical application of oxazolone in wild-type, and P-gp and BCRP gene triple knockout (Mdr1a/1b/Bcrp−/−) mice to observe difference in phenotype. Target metabolome analysis of 583 metabolites was performed using skin and plasma. Results: Dermatitis and scratching behavior in dermatitis model of Mdr1a/1b/Bcrp−/− mice were more severe than wild-type mice, suggesting protective roles of these transporters. This hypothesis was supported by the metabolome analysis which revealed that concentration of histamine and other dermatitis-associated metabolites like urate and serotonin in the dermatitis skin, but not normal skin, of Mdr1a/1b/Bcrp−/− mice was higher than that of wild-type mice. Gene expression of P-gp and BCRP was reduced in oxazolone-treated skin and the skin of patients with atopic dermatitis or psoriasis. Conclusions: These results suggest possible association of these efflux transporters with dermal inflammatory mediators, and such association could be observed in the dermatitis skin.

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Selective inhibition of mutant IDH1 by DS-1001b ameliorates aberrant histone modifications and impairs tumor activity in chondrosarcoma

Nakagawa M., Nakatani F., Matsunaga H., Seki T., Endo M., Ogawara Y., Machida Y., Katsumoto T., Yamagata K., Hattori A., Fujita S., Aikawa Y., Ishikawa T., Soga T., Kawai A., Chuman H., Yokoyama N., Fukushima S., Yahiro K., Kimura A., Shimada E., Hirose T., Fujiwara T., Setsu N., Matsumoto Y., Iwamoto Y., Nakashima Y., Kitabayashi I.

Oncogene 38 ( 42 ) 6835 - 6849 2019.10

Research paper (scientific journal), Joint Work, Accepted, ISSN 09509232

Chondrosarcoma is the second most common malignant bone tumor. It is characterized by low vascularity and an abundant extracellular matrix, which confer these tumors resistance to chemotherapy and radiotherapy. There are currently no effective treatment options for relapsed or dedifferentiated chondrosarcoma, and new targeted therapies need to be identified. Isocitrate dehydrogenase (IDH) mutations, which are detected in ~50% of chondrosarcoma patients, contribute to malignant transformation by catalyzing the production of 2-hydroxyglutarate (2-HG), a competitive inhibitor of α-ketoglutarate-dependent dioxygenases. Mutant IDH inhibitors are therefore potential novel anticancer drugs in IDH mutant tumors. Here, we examined the efficacy of the inhibition of mutant IDH1 as an antitumor approach in chondrosarcoma cells in vitro and in vivo, and investigated the association between the IDH mutation and chondrosarcoma cells. DS-1001b, a novel, orally bioavailable, selective mutant IDH1 inhibitor, impaired the proliferation of chondrosarcoma cells with IDH1 mutations in vitro and in vivo, and decreased 2-HG levels. RNA-seq analysis showed that inhibition of mutant IDH1 promoted chondrocyte differentiation in the conventional chondrosarcoma L835 cell line and caused cell cycle arrest in the dedifferentiated JJ012 cell line. Mutant IDH1-mediated modulation of SOX9 and CDKN1C expression regulated chondrosarcoma tumor progression, and DS-1001b upregulated the expression of these genes via a common mechanism involving the demethylation of H3K9me3. DS-1001b treatment reversed the epigenetic changes caused by aberrant histone modifications. The present data strongly suggest that inhibition of mutant IDH1 is a promising therapeutic approach in chondrosarcoma, particularly for the treatment of relapsed or dedifferentiated chondrosarcoma.

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Phosphoethanolamine Accumulation Protects Cancer Cells under Glutamine Starvation through Downregulation of PCYT2

Osawa T., Shimamura T., Saito K., Hasegawa Y., Ishii N., Nishida M., Ando R., Kondo A., Anwar M., Tsuchida R., Hino S., Sakamoto A., Igarashi K., Saitoh K., Kato K., Endo K., Yamano S., Kanki Y., Matsumura Y., Minami T., Tanaka T., Anai M., Wada Y., Wanibuchi H., Hayashi M., Hamada A., Yoshida M., Yachida S., Nakao M., Sakai J., Aburatani H., Shibuya M., Hanada K., Miyano S., Soga T., Kodama T.

Cell Rep. 29 ( 1 ) 89 - 103.e7 2019.10

Research paper (scientific journal), Joint Work, Accepted

Tolerance to severe tumor microenvironments, including hypoxia and nutrient starvation, is a common feature of aggressive cancer cells and can be targeted. However, metabolic alterations that support cancer cells upon nutrient starvation are not well understood. Here, by comprehensive metabolome analyses, we show that glutamine deprivation leads to phosphoethanolamine (PEtn) accumulation in cancer cells via the downregulation of PEtn cytidylyltransferase (PCYT2), a rate-limiting enzyme of phosphatidylethanolamine biosynthesis. PEtn accumulation correlated with tumor growth under nutrient starvation. PCYT2 suppression was partially mediated by downregulation of the transcription factor ELF3. Furthermore, PCYT2 overexpression reduced PEtn levels and tumor growth. In addition, PEtn accumulation and PCYT2 downregulation in human breast tumors correlated with poor prognosis. Thus, we show that glutamine deprivation leads to tumor progression by regulating PE biosynthesis via the ELF3-PCYT2 axis. Furthermore, manipulating glutamine-responsive genes could be a therapeutic approach to limit cancer progression.

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Fructose Increases the Activity of Sodium Hydrogen Exchanger in Renal Proximal Tubules that is Dependent on Ketohexokinase

Hayasaki, T., Ishimoto, T., Doke, T., Hirayama, A., Soga, T., Furuhashi, K., Kato, N., Kosugi, T., Tsuboi, N., Lanaspa, MA., Johnson, RJ., Maruyama, S., Kadomatsu, K.

J. Nutr. Biochem. 71 ( - ) 54 - 62 2019.09

Research paper (scientific journal), Joint Work, Accepted, ISSN 09552863

High fructose intake has been known to induce metabolic syndrome in laboratory animals and humans. Although fructose intake enhances sodium reabsorption and elevates blood pressure, role of fructose metabolism in this process has not been studied. Here we show that by ketohexokinase - the primary enzyme of fructose - is involved in regulation of renal sodium reabsorption and blood pressure via activation of the sodium hydrogen exchanger in renal proximal tubular cells. First, wild-type and ketohexokinase knockout mice (Male, C57BL/6) were fed fructose water or tap water with or without a high salt diet. Only wild type mice fed the combination of fructose water and high salt diet displayed increased systolic blood pressure and decreased urinary sodium excretion. In contrast, ketohexokinase knockout mice were protected. Second, urinary sodium excretion after intraperitoneal saline administration was reduced with the decreased phosphorylation of sodium hydrogen exchanger 3 in fructose-fed WT; these changes were not observed in the ketohexokinase knockout mice, however. Third, knockdown of ketohexokinase attenuated fructose-mediated increases of NHE activity with decreased cAMP levels in porcine renal proximal tubular cells (LLC-PK1). In conclusion, fructose metabolism by ketohexokinase increases sodium hydrogen exchanger activity in renal proximal tubular cells via decreased intracellular cAMP level, resulting in increased renal sodium reabsorption and blood pressure in mice.
Keywords: Blood pressure; Fructose; Ketohexokinase; Kidney; NHE3; Sodium reabsorption.
Copyright © 2019 Elsevier Inc. All rights reserved.

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BCAA catabolism in brown fat controls energy homeostasis through SLC25A44

Yoneshiro T., Wang, Q., Tajima K., Matsushita M., Maki H., Igarashi K., Dai Z., White PJ., McGarrah RW., Ilkayeva OR., Deleye Y., Oguri Y., Kuroda M., Ikeda K., Li H., Ueno A., Ohishi M., Ishikawa T., Kim K., Chen Y., Sponton CH., Pradhan RN., Majd H., Greiner VJ., Yoneshiro M., Brown Z., Chondronikola M., Takahashi H., Goto T., Kawada T., Sidossis L., Szoka FC., McManus MT., Saito M., Soga T., Kajimura S.

Nature 572 ( 7771 ) 614 - 619 2019.08

Research paper (scientific journal), Joint Work, Accepted, ISSN 00280836

Branched-chain amino acid (BCAA; valine, leucine and isoleucine) supplementation is often beneficial to energy expenditure; however, increased circulating levels of BCAA are linked to obesity and diabetes. The mechanisms of this paradox remain unclear. Here we report that, on cold exposure, brown adipose tissue (BAT) actively utilizes BCAA in the mitochondria for thermogenesis and promotes systemic BCAA clearance in mice and humans. In turn, a BAT-specific defect in BCAA catabolism attenuates systemic BCAA clearance, BAT fuel oxidation and thermogenesis, leading to diet-induced obesity and glucose intolerance. Mechanistically, active BCAA catabolism in BAT is mediated by SLC25A44, which transports BCAAs into mitochondria. Our results suggest that BAT serves as a key metabolic filter that controls BCAA clearance via SLC25A44, thereby contributing to the improvement of metabolic health.

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Metabolomic profiling reveals salivary hypotaurine as a potential early detection marker for medication-related osteonecrosis of the jaw

Yatsuoka, W., Ueno, T., Miyano, K., Uezono, Y., Enomoto, A., Kaneko, M., Ota, S., Soga, T., Sugimoto, M., Ushijima, T.

PLoS ONE 14 ( 8 ) e0220712 2019.08

Research paper (scientific journal), Joint Work, Accepted

Medication-related osteonecrosis of the jaw (MRONJ) is a rare but serious adverse event of bone-modifying agents used to prevent bone complications in cancer patients with bone metastasis. Currently, early treatment is the only way to prevent further progression, as the pathogenesis of MRONJ has not yet been elucidated, and a standard treatment has not been established. The aim of this study was to identify a marker for early detection marker of MRONJ by exploring substances in saliva specific to MRONJ at an early stage. We collected salivary samples from 17 patients with MRONJ and conducted metabolomic analyses using capillary electrophoresis mass spectrometry for non-targeted analysis of hydrophilic metabolites. In the screening cohort, we compared the saliva of patients with stage ≥1 advanced MRONJ (n = 9) with that of controls without MRONJ before chemotherapy (n = 9). The top 5 most elevated salivary metabolites were histamine, 3-(4-hydroxyphenyl)propionate, malonate, carnosine, and hypotaurine. In the validation cohort, we analyzed additional patients with stage ≥1 advanced MRONJ (n = 8) and controls without MRONJ after chemotherapy (n = 9), confirming a significant 2.28-fold elevation in the salivary concentration of hypotaurine. These results revealed elevated salivary hypotaurine concentration as a potential marker for the early detection of MRONJ.

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IMP dehydrogenase-2 drives aberrant nucleolar activity and promotes tumorigenesis in glioblastoma

Kofuji, S., Hirayama, A., Eberhardt, A.O., Kawaguchi R., Sugiura, Y., Sampetrean, O., Ikeda, Y., Warren, M., Sakamoto, N., Kitahara, S., Yohino, H., Yamashita, D., Sumita, K., Wofe, K., Lange, L., Ikeda, S., Shimada, H., Minami, N., Malhotra, A., Morioka, S., Ban, Y., Asano, M., Flanary, V.L., Ramkissoon, A., Chow, L.M.L., Kiyokawa, J., Mahimo, T., Lucey, G., Mareninov, S., Ozawa, T., Onishi, N., Okumura, K., Terakawa, J., Daikoku, T., Wise-Draper, T., Majd, N., Kofuji, K., Sasaki, M., Mori, M., Kanemura, Y., Smith, E.P., Anastasiou, D., Wakimoto, H., Holland, E.C., Yong, W.H., Horbinski, C., Nakano, I., Deberardinis, R.J., Bachoo, R., Mischel, P.S., Yasui, W., Suematsu, M., Saya, H., Soga, T., Grummt, I., Bierhoff, H., Sasaki, AT.

Nat. Cell Biol. 21 ( 8 ) 1003 - 1014 2019.08

Research paper (scientific journal), Joint Work, Accepted, ISSN 14657392

In many cancers, high proliferation rates correlate with elevation of rRNA and tRNA levels, and nucleolar hypertrophy. However, the underlying mechanisms linking increased nucleolar transcription and tumorigenesis are only minimally understood. Here we show that IMP dehydrogenase-2 (IMPDH2), the rate-limiting enzyme for de novo guanine nucleotide biosynthesis, is overexpressed in the highly lethal brain cancer glioblastoma. This leads to increased rRNA and tRNA synthesis, stabilization of the nucleolar GTP-binding protein nucleostemin, and enlarged, malformed nucleoli. Pharmacological or genetic inactivation of IMPDH2 in glioblastoma reverses these effects and inhibits cell proliferation, whereas untransformed glia cells are unaffected by similar IMPDH2 perturbations. Impairment of IMPDH2 activity triggers nucleolar stress and growth arrest of glioblastoma cells even in the absence of functional p53. Our results reveal that upregulation of IMPDH2 is a prerequisite for the occurance of aberrant nucleolar function and increased anabolic processes in glioblastoma, which constitutes a primary event in gliomagenesis.

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Adaptation to HIF1α deletion in hypoxic cancer cells by upregulation of GLUT14 and creatine metabolism

Valli, A., Morotti, M., Zois, CE., Albers, PK., Soga, T, Feldinger, K., Fischer, R., Frejno, M., McIntyre, A., Bridges, E., Haider, S., Buffa, FM., Baban, D., Rodriguezm, M., Yanes, O., Whittington, HJ., Lake, HA., Zervou, S., Lygate, CA., Kessler, BM., Harris, AL.

Mol. Cancer Res 17 ( 7 ) 1531 - 1544 2019.07

Research paper (scientific journal), Joint Work, Accepted, ISSN 15417786

Hypoxia-inducible factor 1α is a key regulator of the hypoxia response in normal and cancer tissues. It is well recognized to regulate glycolysis and is a target for therapy. However, how tumor cells adapt to grow in the absence of HIF1α is poorly understood and an important concept to understand for developing targeted therapies is the flexibility of the metabolic response to hypoxia via alternative pathways. We analyzed pathways that allow cells to survive hypoxic stress in the absence of HIF1α, using the HCT116 colon cancer cell line with deleted HIF1α versus control. Spheroids were used to provide a 3D model of metabolic gradients. We conducted a metabolomic, transcriptomic, and proteomic analysis and integrated the results. These showed surprisingly that in three-dimensional growth, a key regulatory step of glycolysis is Aldolase A rather than phosphofructokinase. Furthermore, glucose uptake could be maintained in hypoxia through upregulation of GLUT14, not previously recognized in this role. Finally, there was a marked adaptation and change of phosphocreatine energy pathways, which made the cells susceptible to inhibition of creatine metabolism in hypoxic conditions. Overall, our studies show a complex adaptation to hypoxia that can bypass HIF1α, but it is targetable and it provides new insight into the key metabolic pathways involved in cancer growth. IMPLICATIONS: Under hypoxia and HIF1 blockade, cancer cells adapt their energy metabolism via upregulation of the GLUT14 glucose transporter and creatine metabolism providing new avenues for drug targeting.
©2019 American Association for Cancer Research.

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Metagenomic and metabolimic analyses reveal distinct stage-specific phenotypes of the gut microbiota in colorectal cancer

Yachida, S., Mizutani, S., Shiroma, H., Shiba, S., Nakajima, T., Sakamoto, T., Watanabe, H., Masuda, K., Nishimoto, Y., Kubo, M., Hosoda, F., Rokutan, H., Matsumoto, M., Takamaru, H., Yamada, M., Matsuda, T., Iwasaki, M., Yamaji, T., Yachida, T., Soga, T., Kurokawa, K., Toyoda, A., Ogura, Y., Hayashi, T., Hatakeyama, M., Nakagama, H., Saito, Y., Fukuda, S., Shibata, T., Yamada, T.

Nat. Med. 25 ( 6 ) 968 - 976 2019.06

Research paper (scientific journal), Joint Work, Accepted

In most cases of sporadic colorectal cancers, tumorigenesis is a multistep process, involving genomic alterations in parallel with morphologic changes. In addition, accumulating evidence suggests that the human gut microbiome is linked to the development of colorectal cancer. Here we performed fecal metagenomic and metabolomic studies on samples from a large cohort of 616 participants who underwent colonoscopy to assess taxonomic and functional characteristics of gut microbiota and metabolites. Microbiome and metabolome shifts were apparent in cases of multiple polypoid adenomas and intramucosal carcinomas, in addition to more advanced lesions. We found two distinct patterns of microbiome elevations. First, the relative abundance of Fusobacterium nucleatum spp. was significantly (P < 0.005) elevated continuously from intramucosal carcinoma to more advanced stages. Second, Atopobium parvulum and Actinomyces odontolyticus, which co-occurred in intramucosal carcinomas, were significantly (P < 0.005) increased only in multiple polypoid adenomas and/or intramucosal carcinomas. Metabolome analyses showed that branched-chain amino acids and phenylalanine were significantly (P < 0.005) increased in intramucosal carcinomas and bile acids, including deoxycholate, were significantly (P < 0.005) elevated in multiple polypoid adenomas and/or intramucosal carcinomas. We identified metagenomic and metabolomic markers to discriminate cases of intramucosal carcinoma from the healthy controls. Our large-cohort multi-omics data indicate that shifts in the microbiome and metabolome occur from the very early stages of the development of colorectal cancer, which is of possible etiological and diagnostic importance.

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Metabolome profiling of various seaweed species discriminates between brown, red, and green algae

Hamid, SS., Wakayama M., Ichihara K., Sakurai K., Ashino Y., Kadowaki R., Soga T., Tomita M.

Planta 249 ( 6 ) 1921 - 1947 2019.06

Research paper (scientific journal), Joint Work, Accepted, ISSN 00320935

Among seaweed groups, brown algae had characteristically high concentrations of mannitol, and green algae were characterised by fructose. In red algae, metabolite profiles of individual species should be evaluated. Seaweeds are metabolically different from terrestrial plants. However, general metabolite profiles of the three major seaweed groups, the brown, red, and green algae, and the effect of various extraction methods on metabolite profiling results have not been comprehensively explored. In this study, we evaluated the water-soluble metabolites in four brown, five red, and two green algae species collected from two sites in northern Japan, located in the Sea of Japan and the Pacific Ocean. Freeze-dried seaweed samples were processed by methanol-water extraction with or without chloroform and analysed by capillary electrophoresis- and liquid chromatography-mass spectrometry for metabolite characterisation. The metabolite concentration profiles showed distinctive characteristic depends on species and taxonomic groups, whereas the extraction methods did not have a significant effect. Taxonomic differences between the various seaweed metabolite profiles were well defined using only sugar metabolites but no other major compound types. Mannitol was the main sugar metabolites in brown algae, whereas fructose, sucrose, and glucose were found at high concentrations in green algae. In red algae, individual species had some characteristic metabolites, such as sorbitol in Pyropia pseudolinearis and panose in Dasya sessilis. The metabolite profiles generated in this study will be a resource and provide guidance for nutraceutical research studies because the information about metabolites in seaweeds is still very limited compared to that of terrestrial plants.

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Synthetic miR-143 Exhibited an Anti-Cancer Effect via the Downregulation of K-RAS Networks of Renal Cell Cancer Cells In Vitro and In Vivo

Takai, T., Tsujino, T., Yoshikawa, Y., Inamoto, T., Sugito, N., Kuranaga, Y., Heishima, K., Soga, T., Hayashi, K., Miyata, K., Kataoka, K., Azuma, H., Akao, Y.

Mol. Ther. 27 ( 5 ) 1017 - 1027 2019.05

Research paper (scientific journal), Joint Work, Accepted, ISSN 15250016

To understand the role of RAS-signaling networks in the pathogenesis of renal cell carcisnoma, we clarified the relationship between miR-143 and RAS. The expression of miR-143 was extremely downregulated in tumor tissues from renal cell carcinoma patients compared with that in the adjacent normal tissues and Caki-1 cells. We developed a synthetic miR-143#12, and we found that the ectopic expression of it inhibited cell growth with autophagy in Caki-1 cells. Also, the expression level of c-Myc was markedly decreased, resulting in the perturbation of cancer-specific energy metabolism by negatively modulating the expression of GLUT1 and the PTBP1/PKMs axis. A partial metabolic shift from glycolysis to oxidative phosphorylation induced autophagy through increasing the intracellular level of reactive oxygen species (ROS). In an in vivo study, the potent anti-tumor activity of polyion complex (PIC)-loaded miR-143#12 (miR-143#12/PIC) was shown by systemic administration of it to Caki-1 cell-xenografted mice. Higher levels of miR-143 were found in both blood and tumor tissues after the systemic administration with miR-143#12/PIC compared to those with lipoplexes in the xenografted mice. These findings indicated that this synthetic miR-143#12 induced a marked growth inhibition by impairing K-RAS-signaling networks in vitro and in vivo.
Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.

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Gut microbiome-derived phenyl sulfate contributes to albuminuria in diabetic kidney disease

Kikuchi K., Saigusa D., Kanemitsu Y., Matsumoto Y., Thanai P., Suzuki N., Mise K., Yamaguchi H., Nakamura T., Asaji K., Mukawa C., Tsukamoto H., Sato T., Oikawa Y., Iwasaki T., Oe Y., Tsukimi T., Fukuda N., Ho H., Nanto-Hara F., Ogura J., Saito R., Nagao S., Ohsaki Y., Shimada S., Suzuki T., Toyohara T., Mishima E., Shima H., Akiyama Y., Akiyama Y., Ichijo M., Matsuhashi T., Matsuo A., Ogata Y., Yang C., Suzuki C., Breeggemann M., Heymann J., Shimizu M., Ogawa S., Takahashi N., Suzuki T., Owada Y., Kure S., Mano N., Soga T., Wada T., Kopp J., Fukuda S., Hozawa A., Yamamoto M., Ito S., Wada J., Tomioka Y., Abe T.

Nat. Commun. 10 ( 1 ) 1835 - - 2019.04

Research paper (scientific journal), Joint Work, Accepted

Diabetic kidney disease is a major cause of renal failure that urgently necessitates a breakthrough in disease management. Here we show using untargeted metabolomics that levels of phenyl sulfate, a gut microbiota-derived metabolite, increase with the progression of diabetes in rats overexpressing human uremic toxin transporter SLCO4C1 in the kidney, and are decreased in rats with limited proteinuria. In experimental models of diabetes, phenyl sulfate administration induces albuminuria and podocyte damage. In a diabetic patient cohort, phenyl sulfate levels significantly correlate with basal and predicted 2-year progression of albuminuria in patients with microalbuminuria. Inhibition of tyrosine phenol-lyase, a bacterial enzyme responsible for the synthesis of phenol from dietary tyrosine before it is metabolized into phenyl sulfate in the liver, reduces albuminuria in diabetic mice. Together, our results suggest that phenyl sulfate contributes to albuminuria and could be used as a disease marker and future therapeutic target in diabetic kidney disease.

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Autophagy regulates lipid metabolism through selective turnover of NCoR1

Saito, T., Kuma, A., Sugiura, Y., Ichimura, Y., Obata, M., Kitamura, H., Okuda, S., Lee, HC., Ikeda, K., Kanegae, Y., Saito, I., Auwerx, J., Motohashi, H., Suematsu, M., Soga, T., Yokomizo, T., Waguri, S., Mizushima, N., Komatsu M.

Nat. Commun. 10 ( 1 ) 1567 - - 2019.04

Research paper (scientific journal), Joint Work, Accepted

Selective autophagy ensures the removal of specific soluble proteins, protein aggregates, damaged mitochondria, and invasive bacteria from cells. Defective autophagy has been directly linked to metabolic disorders. However how selective autophagy regulates metabolism remains largely uncharacterized. Here we show that a deficiency in selective autophagy is associated with suppression of lipid oxidation. Hepatic loss of Atg7 or Atg5 significantly impairs the production of ketone bodies upon fasting, due to decreased expression of enzymes involved in β-oxidation following suppression of transactivation by PPARα. Mechanistically, nuclear receptor co-repressor 1 (NCoR1), which interacts with PPARα to suppress its transactivation, binds to the autophagosomal GABARAP family proteins and is degraded by autophagy. Consequently, loss of autophagy causes accumulation of NCoR1, suppressing PPARα activity and resulting in impaired lipid oxidation. These results suggest that autophagy contributes to PPARα activation upon fasting by promoting degradation of NCoR1 and thus regulates β-oxidation and ketone bodies production.

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Cancer stem-like properties and gefitinib resistance are dependent on purine synthetic metabolism mediated by the mitochondrial enzyme MTHFD2

Nishimura T., Nakata A., Chen X., Nishi K., Meguro-Horike M., Sasaki S., Kita K., Horike S., Saitoh K., Kato K., Igarashi K., Murayama T., Kohno S., Takahashi C., Mukaida N., Yano S., Soga T., Tojo A., Gotoh N.

Oncogene 38 ( 14 ) 2464 - 2481 2019.04

Research paper (scientific journal), Joint Work, Accepted, ISSN 09509232

Tumor recurrence is attributable to cancer stem-like cells (CSCs), the metabolic mechanisms of which currently remain obscure. Here, we uncovered the critical role of folate-mediated one-carbon (1C) metabolism involving mitochondrial methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) and its downstream purine synthesis pathway. MTHFD2 knockdown greatly reduced tumorigenesis and stem-like properties, which were associated with purine nucleotide deficiency, and caused marked accumulation of 5-aminoimidazole carboxamide ribonucleotide (AICAR)-the final intermediate of the purine synthesis pathway. Lung cancer cells with acquired resistance to the targeted drug gefitinib, caused by elevated expression of components of the β-catenin pathway, exhibited increased stem-like properties and enhanced expression of MTHFD2. MTHFD2 knockdown or treatment with AICAR reduced the stem-like properties and restored gefitinib sensitivity in these gefitinib-resistant cancer cells. Moreover, overexpression of MTHFD2 in gefitinib-sensitive lung cancer cells conferred resistance to gefitinib. Thus, MTHFD2-mediated mitochondrial 1C metabolism appears critical for cancer stem-like properties and resistance to drugs including gefitinib through consumption of AICAR, leading to depletion of the intracellular pool of AICAR. Because CSCs are dependent on MTHFD2, therapies targeting MTHFD2 may eradicate tumors and prevent recurrence.

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Axis elongation during Xenopus tail-bud stage is regulated by GABA expressed in the anterior-to-mid neural tube

Furukawa, T., Yamasaki, Y., Hara, Y., Otsuki, C., Maki, H., Soga, T., Moriyama, Y., Kuroda, H.

Int. J. Dev. Biol. 63 ( 1-2 ) 37 - 43 2019.04

Research paper (scientific journal), Joint Work, Accepted, ISSN 02146282

The receptors of gamma-aminobutyric acid (GABA), which is a well-known neurotransmitter, are expressed in the anterior-to-mid neural tube at an early stage of Xenopus development, but there has been no report on the role of GABA in the presumptive central nervous system. Therefore, we tried to reveal the function of GABA for Xenopus early embryogenesis. We first confirmed that the region expressing a gene encoding glutamate decarboxylase 1 (gad1), which is an enzyme that catalyzes the decarboxylation of L-glutamate to GABA, overlapped with that of several genes encoding GABA receptors (gabr) in the neural tube. Metabolome analysis of culture medium of dorsal tail-bud explants containing the neural tube region of tail-bud stage embryos also revealed that GABA was expressed at this stage. Then, we examined the treatment of pentylenetetrazole (PTZ) and picrotoxin (PTX), which are known as inhibitors of GABA receptors (GABA-R), on the early stages of Xenopus embryogenesis, and found that axis elongation in the tail-bud was inhibited by both treatments, and these phenotypic effects were rescued by co-treatment with GABA. Moreover, our spatial- and temporal-specific inhibitor treatments revealed that the gabr- and gad1-overlapped region, which presents at the anterior-to-mid neural tube during the tail-bud stages, was much more sensitive to PTZ and thus caused severe inhibition of axis elongation. Taken together, our results indicate that the small ligand molecule GABA functions as a regulator to induce the axis elongation event in the tail-bud during early embryogenesis via direct stimulation of the neural tube and indirect stimulation of the surrounding area.

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Pyruvate dehydrogenase activation precedes the down-regulation of fatty acid oxidation in monocrotaline-induced myocardial toxicity in mice

Nakai G., Shimura D., Uesugi K., Kajimura I., Jiao Q., Kusakari Y., Soga T., Goda N., Minamisawa S.

Heart Vessels. 34 ( 3 ) 545 - 555 2019.03

Research paper (scientific journal), Joint Work, ISSN 09108327

© 2018, Springer Japan KK, part of Springer Nature. Fatty acid (FA) oxidation is impaired and glycolysis is promoted in the damaged heart. However, the factor(s) in the early stages of myocardial metabolic impairment remain(s) unclear. C57B6 mice were subcutaneously administered monocrotaline (MCT) in doses of 0.3 mg/g body weight twice a week for 3 or 6 weeks. Right and left ventricles at 3 and 6 weeks after administration were subjected to capillary electrophoresis–mass spectrometry metabolomic analysis. We also examined mRNA and protein levels of key metabolic molecules. Although no evidence of PH and right ventricular failure was found in the MCT-administered mice by echocardiographic and histological analyzes, the expression levels of stress markers such as TNFα and IL-6 were increased in right and left ventricles even at 3 weeks, suggesting that there was myocardial damage. Metabolites in the tricarboxylic acid (TCA) cycle were decreased and those in glycolysis were increased at 6 weeks. The expression levels of FA oxidation-related factors were decreased at 6 weeks. The phosphorylation level of pyruvate dehydrogenase (PDH) was significantly decreased at 3 weeks. FA oxidation and the TCA cycle were down-regulated, whereas glycolysis was partially up-regulated by MCT-induced myocardial damage. PDH activation preceded these alterations, suggesting that PDH activation is one of the earliest events to compensate for a subtle metabolic impairment from myocardial damage.

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Role of smooth muscle cell p53 in pulmonary arterial hypertension

Wakasugi, T., Shimizu, I., Yoshida, Y., Hayashi, Y., Ikegami, R., Suda, M., Katsuumi, G., Nakao, M., Hoyano, M., Kashimura, T., Nakamura, K., Ito, H., Nojiri, T., Soga, T., Minamino, T.

PLoS One 14 ( 2 ) e0212889 2019.02

Research paper (scientific journal), Joint Work, Accepted

© 2019 Wakasugi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Pulmonary arterial hypertension (PAH) is characterized by remodeling and narrowing of the pulmonary arteries, which lead to elevation of right ventricular pressure, heart failure, and death. Proliferation of pulmonary artery smooth muscle cells (PASMCs) is thought to be central to the pathogenesis of PAH, although the underlying mechanisms are still being explored. The protein p53 is involved in cell cycle coordination, DNA repair, apoptosis, and cellular senescence, but its role in pulmonary hypertension (PH) is not fully known. We developed a mouse model of hypoxia-induced pulmonary hypertension (PH) and found significant reduction of p53 expression in the lungs. Our in vitro experiments with metabolomic analyses and the Seahorse XF extracellular flux analyzer indicated that suppression of p53 expression in PASMCs led to upregulation of glycolysis and downregulation of mitochondrial respiration, suggesting a proliferative phenotype resembling that of cancer cells. It was previously shown that systemic genetic depletion of p53 in a murine PH model led to more severe lung manifestations. Lack of information about the role of cell-specific p53 signaling promoted us to investigate it in our mouse PH model with the inducible Cre-loxP system. We generated a mouse model with SMC-specific gain or loss of p53 function by crossing Myh11-Cre/ERT2 mice with floxed Mdm4 mice or floxed Trp53 mice. After these animals were exposed to hypoxia for 4 weeks, we conducted hemodynamic and echocardiographic studies. Surprisingly, the severity of PH was similar in both groups of mice and there were no differences between the genotypes. Our findings in these mice indicate that activation or suppression of p53 signaling in SMCs has a minor role in the pathogenesis of PH and suggest that p53 signaling in other cells (endothelial cells, immune cells, or fibroblasts) may be involved in the progression of this condition.

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Perioperative serum and urine metabolome analyses in patients with hepatocellular carcinoma undergoing partial hepatectomy

Kajiura, D., Yamanaka-Okumura, H., Hirayama, A., Tatano, H., Endo, K., Honma, M., Igarashi, K., Syoji, F., Ikeda, S., Yamaguchi, N., Katayama, T., Morine, Y., Imura, S., Utsunomiya, T., Soga, T., Tomita, M., Shimada, M.

Nutrition 58 110 - 119 2019.02

Research paper (scientific journal), Joint Work, Accepted, ISSN 08999007

© 2018 Elsevier Ltd Objectives: Perioperative nutritional management is essential for early recovery after liver surgery. The aim of this study was to assess changes in amino acid levels in serum and urine after hepatectomy. Methods: Serum samples were collected from 16 patients with hepatocellular carcinoma before and 1, 3, and 14 d after hepatectomy (S0, S1, S3, and S14, respectively). Spot urine samples were collected before and 3 d after the hepatectomy (U0 and U3). Metabolites in the serum and urine were analyzed. Results: Compared with S0, insulin levels significantly increased in the S1 and S3 samples. Valine levels significantly decreased in S1 and S14, and leucine levels significantly decreased in S14. Phenylalanine levels significantly increased in S1 and S3, and tyrosine levels significantly increased in S1. The Fischer ratio (branched-chain/aromatic amino acids) significantly decreased in S1 and S3. In multiple regression analysis, changes in serum taurine levels were related to the white blood cell count in S1 and S3, and inversely related to alanine aminotransferase levels in S14. Changes in serum glutamine levels were negatively related to C-reactive protein levels in S3. Serum glutamine levels decreased in S3 and S14, and tended to increase in U3, suggesting a deficiency of glutamate resulting from the invasive surgical procedure. Conclusions: These findings highlight the usefulness of metabolome analysis for characterizing perioperative patterns after liver resection. The observed amino acid pattern, including the reduction in Fischer ratio, underscores the need for specialized nutritional support.

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Effects of inter-day and intra-day variation on salivary metabolomic profiles

Kawanishi N., Hoshi N., Masahiro S., Enomoto A., Ota S., Kaneko M., Soga T., Tomita M., Kimoto K.

Clin. Chim. Acta. 489 ( - ) 41 - 48 2019.02

Research paper (scientific journal), Joint Work, ISSN 00098981

© 2018 Elsevier B.V. Background: Salivary secretion is an important parameter reflecting the health status of an individual and has been used clinically for the diagnosis of various oral diseases, such as xerostomia. Salivary metabolomic profiling is considered an emerging potential tool for the detection of various systemic diseases. To our knowledge, this is the first study to investigate the quantitative relationship between salivary secretion volume and salivary metabolomic profile. Methods: To evaluate inter- and intra-day variations in salivary secretion, 234 saliva samples were collected three times per day for three days from 13 subjects and analyzed. Capillary electrophoresis-mass spectrometry was used for non-targeted quantification of water-soluble metabolites. Results: No significant inter- or intra-day variations were observed in salivary secretion volume. No significant inter-day variations were observed in metabolomic patterns. In contrast, significant intra-day variations were observed in salivary metabolomic profiles. The difference was more obvious for stimulated saliva than for unstimulated saliva. These profile changes were independent of salivary secretion volume. Conclusions: Our results indicated that diurnal change had a greater effect on salivary metabolomic profiles than the other factors. Hence, sampling time should be tightly controlled to minimize unexpected bias in the clinical use of salivary metabolomics.

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GLUT6 is a lysosomal transporter that is regulated by inflammatory stimuli and modulates glycolysis in macrophages

Maedera S., Mizuno T., Ishiguro H., Ito T., Soga T., Kusuhara H.

FEBS Lett. 593 ( 2 ) 195 - 208 2019.01

Research paper (scientific journal), Joint Work, ISSN 00145793

© 2018 Federation of European Biochemical Societies The solute carrier family is an important protein class governing compound transport across membranes. However, some of its members remain functionally unidentified. We analyzed ChIP-seq data for the NF-κB family transcription factor RelA and identified GLUT6 as a functionally uncharacterized transporter that putatively works in inflammatory responses. Inflammatory stimuli increase GLUT6 expression level, although GLUT6-knockout mice exhibit a subtle phenotype to lipopolysaccharide administration. Metabolomics and in vitro analyses show that GLUT6 functions as a glycolysis modulator in inflammatory macrophages. GLUT6 does not mediate glucose uptake and is localized on lysosomal membranes. We conclude that GLUT6 is a lysosomal transporter that is regulated by inflammatory stimuli and modulates inflammatory responses by affecting the metabolic shift in macrophages.

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Metabolomics Platform with Capillary Electrophoresis Coupled with High-Resolution Mass Spectrometry for Plasma Analysis

Sasaki, K., Sagawa, H., Suzuki, M., Yamamoto, H., Tomita, M., Soga, T., Ohashi, Y.

Anal. Chem. (Analytical Chemistry) 91 ( 2 ) 1295 - 1301 2019.01

Research paper (scientific journal), Joint Work, ISSN 00032700

© 2018 American Chemical Society. Metabolome analysis using capillary electrophoresis (CE) coupled with high-resolution mass spectrometry (HRMS) has the potential to improve coverage of metabolite detection because of its high selectivity and sensitivity. Configuration of the interface between CE and HRMS to meet the ground connection is essential for enabling independent regulation of the electrical currents in the CE and electrospray field. In the present study, we applied an electrospray-ionization adapter equipped with a grounded nebulizer to CE-HRMS and tested the analytical performance for 34 charged compounds. The extracted-ion electropherograms, consisting of seven sets of isomers, showed reasonable peak shapes and separation for the annotation of each metabolite. The levels of 34 target analytes in a standard mixture were determined with a dynamic range of at least 10 2 , maintaining linearity with r 2 > 0.9. The repeatability and intermediate precision above the lower limit of quantification showed the relative standard deviation to be lower than 20%. In the spike-recovery experiment, 27 of the 34 metabolites in plasma extract were recovered at a rate of 80 to 120%, suggesting high accuracy. Furthermore, we assessed the feasibility of our platform in metabolome analysis using human-plasma extract. The results showed successful detection of 270 metabolites, indicating the potential of our platform to yield higher coverage of the metabolome. In addition, analysis of dilution integrity demonstrated the quantitative ability of metabolome analysis with CE-HRMS, although the existence of saturation or matrix effects were seen in the case of 33 of the metabolites. This study indicates that our platform has great potential for large-scale metabolome analysis of plasma for biological studies and clinical biomarker screening.

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Differences in peritoneal solute transport rates in peritoneal dialysis

Asano, M., Ishii, T., Hirayama, A., Mizuno, M., Suzuki, Y., Sakata, F., Akiyama, S., Maruyama, S., Soga, T., Kinashi, H., Katsuno, T., Ito, Y.

Clin. Exp. Nephrol. 23 ( 1 ) 122 - 134 2019.01

Research paper (scientific journal), Joint Work, Accepted, ISSN 13421751

© 2018, Japanese Society of Nephrology. Background: Ultrafiltration failure associated with peritoneal membrane dysfunction is one of the main complications for patients on long-term peritoneal dialysis (PD). The dialysate-to-plasma concentration ratio (D/P) of creatinine is widely used to assess peritoneal membrane function. However, other small-sized solutes have not been studied in detail as potential indicators of peritoneal permeability. Methods: We studied the D/Ps of small, middle-sized and large molecules in peritoneal equilibration tests in 50 PD patients. We applied metabolomic analysis of comprehensive small molecular metabolites using capillary electrophoresis time-of-flight mass spectrometry. Results: D/Ps of middle-sized and large molecules correlated positively with D/P creatinine. Most D/Ps of small molecules correlated positively with D/P creatinine. Among 38 small molecules contained in the dialysate, urea, citrulline and choline showed significantly lower ability to permeate than creatinine. In the relationship between D/Ps of creatinine and small molecules, regression coefficients of three molecules were less than 0.3, representing no correlation to D/P creatinine. Five molecules showed negative regression coefficients. Among these molecules, hippurate and 3-indoxyl sulfate showed relatively high teinpro binding rates, which may affect permeability. Serum concentrations of two molecules were higher in the Low Kt/V group, mainly due to high protein binding rates. Conclusions: D/Ps of some molecules did not correlate with D/P creatinine. Factors other than molecular weight, such as charge and protein binding rate, are involved in peritoneal transport rates. Metabolomic analysis appears useful to analyze small molecular uremic toxins, which could accumulate in PD patients, and the status of peritoneal membrane transport for each molecule.

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A Metabologenomic Approach Reveals Changes in the Intestinal Environment of Mice Fed on American Diet

Ishii, C., Nakanishi, Y., Murakami, S., Nozu, R., Ueno, M., Hioki, K., Aw, W., Hirayama, A., Soga, T., Ito, M., Tomita, M., Fukuda, S.

Int J Mol Sci. 19 ( 12 ) pii: E4079 2018.12

Research paper (scientific journal), Joint Work, ISSN 16616596

Intestinal microbiota and their metabolites are strongly associated with host physiology. Developments in DNA sequencing and mass spectrometry technologies have allowed us to obtain additional data that enhance our understanding of the interactions among microbiota, metabolites, and the host. However, the strategies used to analyze these datasets are not yet well developed. Here, we describe an original analytical strategy, metabologenomics, consisting of an integrated analysis of mass spectrometry-based metabolome data and high-throughput-sequencing-based microbiome data. Using this approach, we compared data obtained from C57BL/6J mice fed an American diet (AD), which contained higher amounts of fat and fiber, to those from mice fed control rodent diet. The feces of the AD mice contained higher amounts of butyrate and propionate, and higher relative abundances of Oscillospira and Ruminococcus. The amount of butyrate positively correlated with the abundance of these bacterial genera. Furthermore, integrated analysis of the metabolome data and the predicted metagenomic data from Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) indicated that the abundance of genes associated with butyrate metabolism positively correlated with butyrate amounts. Thus, our metabologenomic approach is expected to provide new insights and understanding of intestinal metabolic dynamics in complex microbial ecosystems.

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Drying and extraction effects on three edible brown seaweeds for metabolomics

Hamid, S.S., Wakayama, M., Soga, T., Tomita, M.

J. Appl. Phycol. (Journal of Applied Phycology) 30 ( 6 ) 3335 - 3350 2018.12

Research paper (scientific journal), Joint Work, Accepted, ISSN 09218971

© 2018, Springer Nature B.V. Metabolomics is often used to comprehensively elucidate the metabolites in organisms like seaweed. Amino acids hydrolysed from proteins and certain targeted metabolites in seaweed have been investigated. However, water-soluble metabolites like free amino acids, organic acids, and sugars have seldom been comprehensively analysed. Metabolomics are valuable tools for these studies, but they require optimisation of pre-treatment methodology. Here, we evaluated various pre-treatment drying and extraction methods for brown seaweed metabolomics. Three edible brown seaweeds (Cladosiphon okamuranus [Mozuku], Saccharina japonica [Kombu], and Undaria pinnatifida [Wakame]) were used. Freeze-drying and oven-drying at both 40 and 80 °C were investigated. Methanol-water extracts with and without chloroform were compared. Metabolites were evaluated and quantified using liquid chromatography-mass spectrometry and capillary electrophoresis-mass spectrometry. The results showed that metabolite profiling was determined mainly by seaweed species identity rather than pre-treatment method. Freeze-drying yielded higher metabolite concentrations than oven-drying at either 40 or 80 °C. The effects of extraction with and without chloroform on metabolite concentration varied with seaweed species.

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Metabolomics-based profiles predictive of low bone mass in menopausal women

Miyamoto, T., Hirayama, A., Sato, Y., Koboyashi, T., Katsuyama, E., Kanagawa, H., Fujie, A., Morita, M., Watanabe, R., Tando, T., Miyamoto, K., Tsuji, T., Funayama, A., Soga, T., Tomita, M., Nakamura, M., Matsumoto, M.

Bone Rep. 9 11 - 18 2018.12

Research paper (scientific journal), Joint Work, Accepted

© 2018 The Authors Osteoporosis is a skeletal disorder characterized by compromised bone strength and increased risk of fracture. Low bone mass and/or pre-existing bone fragility fractures serve as diagnostic criteria in deciding when to start medication for osteoporosis. Although osteoporosis is a metabolic disorder, metabolic markers to predict reduced bone mass are unknown. Here, we show serum metabolomics profiles of women grouped as pre-menopausal with normal bone mineral density (BMD) (normal estrogen and normal BMD; NN), post-menopausal with normal BMD (low estrogen and normal BMD; LN) or post-menopausal with low BMD (low estrogen and low BMD; LL) using comprehensive metabolomics analysis. To do so, we enrolled healthy volunteer and osteoporosis patient female subjects, surveyed them with a questionnaire, measured their BMD, and then undertook a comprehensive metabolomics analysis of sera of the three groups named above. We identified 24 metabolites whose levels differed significantly between NN/LN and NN/LL groups, as well as 18 or 10 metabolites whose levels differed significantly between NN/LN and LN/LL, or LN/LL and NN/LN groups, respectively. Our data shows metabolomics changes represent useful markers to predict estrogen deficiency and/or bone loss.

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Folliculin regulates osteoclastogenesis through metabolic regulation

Baba, M., Endoh, M., Ma, W., Toyama, H., Hirayama, A., Nishikawa, K., Takubo, K., Hano, H., Hasumi, H., Umemoto, T., Hashimoto, M., Irie, N., Esumi, C., Kataoka, M., Nakagata, N., Soga, T., Yao, M., Kamba, T., Minami, T., Ishii, M., Suda, T.

J. Bone Miner. Res. (Journal of Bone and Mineral Research) 33 ( 10 ) 1785 - 1798 2018.10

Research paper (scientific journal), Joint Work, Accepted, ISSN 08840431

© 2018 The Authors Journal of Bone and Mineral Research published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research (ASBMR) Osteoclast differentiation is a dynamic differentiation process, which is accompanied by dramatic changes in metabolic status as well as in gene expression. Recent findings have revealed an essential connection between metabolic reprogramming and dynamic gene expression changes during osteoclast differentiation. However, the upstream regulatory mechanisms that drive these metabolic changes in osteoclastogenesis remain to be elucidated. Here, we demonstrate that induced deletion of a tumor suppressor gene, Folliculin (Flcn), in mouse osteoclast precursors causes severe osteoporosis in 3 weeks through excess osteoclastogenesis. Flcn-deficient osteoclast precursors reveal cell autonomous accelerated osteoclastogenesis with increased sensitivity to receptor activator of NF-κB ligand (RANKL). We demonstrate that Flcn regulates oxidative phosphorylation and purine metabolism through suppression of nuclear localization of the transcription factor Tfe3, thereby inhibiting expression of its target gene Pgc1. Metabolome studies revealed that Flcn-deficient osteoclast precursors exhibit significant augmentation of oxidative phosphorylation and nucleotide production, resulting in an enhanced purinergic signaling loop that is composed of controlled ATP release and autocrine/paracrine purinergic receptor stimulation. Inhibition of this purinergic signaling loop efficiently blocks accelerated osteoclastogenesis in Flcn-deficient osteoclast precursors. Here, we demonstrate an essential and novel role of the Flcn-Tfe3-Pgc1 axis in osteoclastogenesis through the metabolic reprogramming of oxidative phosphorylation and purine metabolism. © 2018 The Authors Journal of Bone and Mineral Research published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research (ASBMR).

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Beta-galactosidase-responsive synthetic biomarker for targeted tumor detection.

Nishihara, T. Kuno, S., Nonaka, H., Tabata, S., Saito, N., Fukuda, S., Tomita, M., Sando, S., *Soga, T.

Chem.Comm.(Camb) 4 ( 83 ) 11745 - 11745 2018.10

Research paper (scientific journal), Joint Work, Accepted, ISSN 13597345

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Metabolic characterization of antifolate responsiveness and nonresponsiveness in malignant pleural mesothelioma cells

Sato, Y., Matsuda, S., Maruyama, A., Nakayama, J., Miyashita, T., Udagawa, H., Umemura, S., Yanagihara, K., Ochiai, A., Tomita, M., Soga, T., Tsuchihara, K., Makinoshima, H.

Front. Pharmacol. 9 1129 2018.10

Research paper (scientific journal), Joint Work, Accepted

© 2007 - 2018 Frontiers Media S.A. All Rights Reserved. Antifolates are a class of drugs effective for treating malignant pleural mesothelioma (MPM). The majority of antifolates inhibit enzymes involved in purine and pyrimidine synthesis such as dihydrofolate reductase (DHFR), thymidylate synthase (TYMS), and glycinamide ribonucleotide formyltransferase (GART). In order to select the most suitable patients for effective therapy with drugs targeting specific metabolic pathways, there is a need for better predictive metabolic biomarkers. Antifolates can alter global metabolic pathways in MPM cells, yet the metabolic profile of treated cells has not yet been clearly elucidated. Here we found that MPM cell lines could be categorized into two groups according to their sensitivity or resistance to pemetrexed treatment. We show that pemetrexed susceptibility could be reversed and DNA synthesis rescued in drug-treated cells by the exogenous addition of the nucleotide precursors hypoxanthine and thymidine (HT). We observed that the expression of pemetrexed-targeted enzymes in resistant MPM cells was quantitatively lower than that seen in pemetrexed-sensitive cells. Metabolomic analysis revealed that glycine and choline, which are involved in one-carbon metabolism, were altered after drug treatment in pemetrexed-sensitive but not resistant MPM cells. The addition of HT upregulated the concentration of inosine monophosphate (IMP) in pemetrexed-sensitive MPM cells, indicating that the nucleic acid biosynthesis pathway is important for predicting the efficacy of pemetrexed in MPM cells. Our data provide evidence that may link therapeutic response to the regulation of metabolism, and points to potential biomarkers for informing clinical decisions regarding the most effective therapies for patients with MPM.

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SRSF3, a Splicer of the PKM Gene, Regulates Cell Growth and Maintenance of Cancer-Specific Energy Metabolism in Colon Cancer Cells

Kuranaga, Y., Sugito, N., Shinohara, H., Tsujino, T., Taniguchi, K., Komura, K., Ito, Y., Soga, T., Akao, Y.

Int. J. Mol. Sci. (International Journal of Molecular Sciences) 19 ( 10 ) E3012 2018.10

Research paper (scientific journal), Joint Work, Accepted, ISSN 16616596

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. Serine and arginine rich splicing factor 3 (SRSF3), an SR-rich family protein, has an oncogenic function in various kinds of cancer. However, the detailed mechanism of the function had not been previously clarified. Here, we showed that the SRSF3 splicer regulated the expression profile of the pyruvate kinase, which is one of the rate-limiting enzymes in glycolysis. Most cancer cells express pyruvate kinase muscle 2 (PKM2) dominantly to maintain a glycolysis-dominant energy metabolism. Overexpression of SRSF3, as well as that of another splicer, polypyrimidine tract binding protein 1 (PTBP1) and heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), in clinical cancer samples supported the notion that these proteins decreased the Pyruvate kinase muscle 1 (PKM1)/PKM2 ratio, which positively contributed to a glycolysis-dominant metabolism. The silencing of SRSF3 in human colon cancer cells induced a marked growth inhibition in both in vitro and in vivo experiments and caused an increase in the PKM1/PKM2 ratio, thus resulting in a metabolic shift from glycolysis to oxidative phosphorylation. At the same time, the silenced cells were induced to undergo autophagy. SRSF3 contributed to PKM mRNA splicing by co-operating with PTBP1 and hnRNPA1, which was validated by the results of RNP immunoprecipitation (RIP) and immunoprecipitation (IP) experiments. These findings altogether indicated that SRSF3 as a PKM splicer played a positive role in cancer-specific energy metabolism.

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Canagliflozin reduces plasma uremic toxins and alters the intestinal microbiota composition in a chronic kidney disease mouse model

Mishima E., Fukuda S., Kanemitsu Y., Saigusa D., Mukawa C., Asaji K., Matsumoto Y., Tsukamoto H., Tachikawa T., Tsukimi T., Fukuda N., Ho H., Kikuchi K., Suzuki C., Nanto F., Suzuki T., Ito S., Soga T., Tomioka Y., Abe T.

Am. J. Physiol. Renal. Physiol. (American Journal of Physiology - Renal Physiology) 315 ( 4 ) F824 - F833 2018.10

Research paper (scientific journal), Joint Work, Accepted, ISSN 03636127

© 2018 the American Physiological Society. Mishima E, Fukuda S, Kanemitsu Y, Saigusa D, Mukawa C, Asaji K, Matsumoto Y, Tsukamoto H, Tachikawa T, Tsukimi T, Fukuda NN, Ho HJ, Kikuchi K, Suzuki C, Nanto F, Suzuki T, Ito S, Soga T, Tomioka Y, Abe T. Canagliflozin reduces plasma uremic toxins and alters the intestinal microbiota composition in a chronic kidney disease mouse model. Am J Physiol Renal Physiol 315: F824–F833, 2018. First published November 22, 2017; doi:10.1152/ajprenal.00314.2017.—Accumulation of uremic toxins, which exert deleterious effects in chronic kidney disease, is influenced by the intestinal environment; the microbiota contributes to the production of representative uremic toxins, including p-cresyl sulfate and indoxyl sulfate. Canagliflozin is a sodium-glucose cotransporter (SGLT) 2 inhibitor, and it also exerts a modest inhibitory effect on SGLT1. The inhibition of intestinal SGLT1 can influence the gastrointestinal environment. We examined the effect of canagliflozin on the accumulation of uremic toxins in chronic kidney disease using adenine-induced renal failure mice. Two-week canagliflozin (10 mg/kg po) treatment did not influence the impaired renal function; however, it significantly reduced the plasma levels of p-cresyl sulfate and indoxyl sulfate in renal failure mice (a 75% and 26% reduction, respectively, compared with the vehicle group). Additionally, canagliflozin significantly increased cecal short-chain fatty acids in the mice, suggesting the promotion of bacterial carbohydrate fermentation in the intestine. Analysis of the cecal microbiota showed that canagliflozin significantly altered microbiota composition in the renal failure mice. These results indicate that canagliflozin exerts intestinal effects that reduce the accumulation of uremic toxins including p-cresyl sulfate. Reduction of accumulated uremic toxins by canagliflozin could provide a potential therapeutic option in chronic kidney disease.

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A Metabolomic-Based Evaluation of the Role of Commensal Microbiota throughout the Gastrointestinal Tract in Mice

Yamamoto, Y., Nakanishi, Y., Murakami, S., Aw, W., Tsukimi, T., Nozu, R., Ueno, M., Hioki, K., Nakahigashi, K., Hirayama, A., Sugimoto, M., Soga, T., Ito, M., Tomita, M., Fukuda, S.

Microorganisms 6 ( 4 ) 101 2018.09

Research paper (scientific journal), Joint Work, Accepted

Commensal microbiota colonize the surface of our bodies. The inside of the gastrointestinal tract is one such surface that provides a habitat for them. The gastrointestinal tract is a long organ system comprising of various parts, and each part possesses various functions. It has been reported that the composition of intestinal luminal metabolites between the small and large intestine are different; however, comprehensive metabolomic and commensal microbiota profiles specific to each part of the gastrointestinal lumen remain obscure. In this study, by using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS)-based metabolome and 16S rRNA gene-based microbiome analyses of specific pathogen-free (SPF) and germ-free (GF) murine gastrointestinal luminal profiles, we observed the different roles of commensal microbiota in each part of the gastrointestinal tract involved in carbohydrate metabolism and nutrient production. We found that the concentrations of most amino acids in the SPF small intestine were higher than those in the GF small intestine. Furthermore, sugar alcohols such as mannitol and sorbitol accumulated only in the GF large intestine, but not in the SPF large intestine. On the other hand, pentoses, such as arabinose and xylose, gradually accumulated from the cecum to the colon only in SPF mice, but were undetected in GF mice. Correlation network analysis between the gastrointestinal microbes and metabolites showed that niacin metabolism might be correlated to Methylobacteriaceae. Collectively, commensal microbiota partially affects the gastrointestinal luminal metabolite composition based on their metabolic dynamics, in cooperation with host digestion and absorption.

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Trans-omic Analysis Reveals Selective Responses to Induced and Basal Insulin across Signaling, Transcriptional, and Metabolic Networks

Kawata K., Hatano A., Yugi K., Kubota H., Sano T., Fujii M., Tomizawa Y., Kokaji T., Tanaka K., Uda S., Suzuki Y., Matsumoto M., Nakayama K., Saitoh K., Kato K., Ueno A., Ohishi M., Hirayama A., Soga T., Kuroda S.

iScience 7 ( - ) 212 - 229 2018.09

Research paper (scientific journal), Joint Work, Accepted

The concentrations of insulin selectively regulate multiple cellular functions. To understand how insulin concentrations are interpreted by cells, we constructed a trans-omic network of insulin action in FAO hepatoma cells using transcriptomic data, western blotting analysis of signaling proteins, and metabolomic data. By integrating sensitivity into the trans-omic network, we identified the selective trans-omic networks stimulated by high and low doses of insulin, denoted as induced and basal insulin signals, respectively. The induced insulin signal was selectively transmitted through the pathway involving Erk to an increase in the expression of immediate-early and upregulated genes, whereas the basal insulin signal was selectively transmitted through a pathway involving Akt and an increase of Foxo phosphorylation and a reduction of downregulated gene expression. We validated the selective trans-omic network in vivo by analysis of the insulin-clamped rat liver. This integrated analysis enabled molecular insight into how liver cells interpret physiological insulin signals to regulate cellular functions.

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Gamma-aminobutyric acid signaling in brown adipose tissue promotes systemic metabolic derangement in obesity

Ikegami, R., Shimizu, I., Sato, T., Yoshida, Y., Hayashi, Y., Suda, M., Katsuumi, G., Li, J., Wakasugi, T., Minokoshi, Y., Okamoto, S., Hinoi, E., Nielsen, S., Jespersen, N. Z., Scheele, C., Soga, T., Minamino, T.

Cell Rep. 24 ( 11 ) 2827 - 2837 2018.09

Research paper (scientific journal), Joint Work, Accepted

Brown adipose tissue (BAT) is a metabolically active organ that contributes to the maintenance of systemic metabolism. The sympathetic nervous system plays important roles in the homeostasis of BAT and promotes its browning and activation. However, the role of other neurotransmitters in BAT homeostasis remains largely unknown. Our metabolomic analyses reveal that gamma-aminobutyric acid (GABA) levels are increased in the interscapular BAT of mice with dietary obesity. We also found a significant increase in GABA-type B receptor subunit 1 (GABA-BR1) in the cell membranes of brown adipocytes of dietary obese mice. When administered to obese mice, GABA induces BAT dysfunction together with systemic metabolic disorder. Conversely, the genetic inactivation or inhibition of GABA-BR1 leads to the re-browning of BAT under conditions of metabolic stress and ameliorated systemic glucose intolerance. These results indicate that the constitutive activation of GABA/GABA-BR1 signaling in obesity promotes BAT dysfunction and systemic metabolic derangement.

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Effect of storage conditions on salivary polyamines quantified via liquid chromatography-mass spectrometry

Tomita, A., Mori, M., Hiwatari, K., Yamaguchi, E., Itoi, T., Sunamura, M., Soga, T., Tomita, M., Sugimoto, M.

Sci. Rep. 8 ( 1 ) 12075 2018.08

Research paper (scientific journal), Joint Work, Accepted

© 2018, The Author(s). Salivary polyamines are potential non-invasive tools for screening various types of cancers. For clinical use, the reproducibility of these metabolites should be evaluated under various storage conditions, including duration and temperature, to establish standard operating protocols. Polyamines and amino acids in unstimulated whole saliva were quantified via liquid chromatography-mass spectrometry. Concentrations of time course samples were analysed after short-term storage for up to 240 min and long-term storage for up to 8 days under various storage conditions. As expected, storage at the lowest temperature (−18 °C) exerted the least pronounced effects on the quantified values in both tests. At a higher temperature, polyamines were more stable than amino acids, as evident from polyamine profiling. Addition of ethanol significantly stabilized polyamine profiles even at a higher temperature. Comparative processing of saliva revealed a minor effect of the solvent, whereas drying had a more prominent effect on polyamine profiles. Computational analyses evaluated the ability of polyamines to discriminate pancreatic cancer from controls. Repeated noise added tests were designed on the basis of the results of the storage tests; these analyses confirmed that the discriminative abilities were robust. These data contribute to the standardization of salivary storage conditions, thereby highlighting the clinical utility of saliva.

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Lacking ketohexokinase-A exacerbates renal injury in streptozotocin-induced diabetic mice

Doke, T., Ishimoto, T., Hayasaki, T., Ikeda, S., Hasebe, M., Hirayama, A., Soga, T., Kato, N., Kosugi, T., Tsuboi, N., Lanaspa, MA., Johnson, RJ., Kadomatsu, K., Maruyama, S.

Metabolism (Metabolism: Clinical and Experimental) 85 161 - 170 2018.08

Research paper (scientific journal), Joint Work, ISSN 00260495

© 2018 Elsevier Inc. Objective: Ketohexokinase (KHK), a primary enzyme in fructose metabolism, has two isoforms, namely, KHK-A and KHK-C. Previously, we reported that renal injury was reduced in streptozotocin-induced diabetic mice which lacked both isoforms. Although both isoforms express in kidney, it has not been elucidated whether each isoform plays distinct roles in the development of diabetic kidney disease (DKD). The aim of the study is to elucidate the role of KHK-A for DKD progression. Materials and Methods: Diabetes was induced by five consecutive daily intraperitoneal injections of streptozotocin (50 mg/kg) in C57BL/6J wild-type mice, mice lacking KHK-A alone (KHK-A KO), and mice lacking both KHK-A and KHK-C (KHK-A/C KO). At 35 weeks, renal injury, inflammation, hypoxia, and oxidative stress were examined. Metabolomic analysis including polyol pathway, fructose metabolism, glycolysis, TCA (tricarboxylic acid) cycle, and NAD (nicotinamide adenine dinucleotide) metabolism in kidney and urine was done. Results: Diabetic KHK-A KO mice developed severe renal injury compared to diabetic wild-type mice, and this was associated with further increases of intrarenal fructose, dihydroxyacetone phosphate (DHAP), TCA cycle intermediate levels, and severe inflammation. In contrast, renal injury was prevented in diabetic KHK-A/C KO mice compared to both wild-type and KHK-A KO diabetic mice. Further, diabetic KHK-A KO mice contained decreased renal NAD + level with the increase of renal hypoxia-inducible factor 1-alpha expression despite having increased renal nicotinamide (NAM) level. Conclusion: These results suggest that KHK-C might play a deleterious role in DKD progression through endogenous fructose metabolism, and that KHK-A plays a unique protective role against the development of DKD.

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Low tumor glutathione level as a sensitivity marker for glutamate-cysteine ligase inhibitors

Nishizawa S., Araki H., Ishikawa Y., Kitazawa S., Hata A., Soga T., Hara T.

Oncol. Lett. 15 ( 6 ) 8735 - 8743 2018.06

Research paper (scientific journal), Joint Work, ISSN 17921074

© 2018, Spandidos Publications. All rights reserved. Previous metabolomic analyses of cancer have revealed elevated glutathione levels in tumors. An inhibitor of cystine uptake was identified to suppress glutathione biosynthesis, leading to ferroptosis, a novel iron-dependent form of cell death that differs from apoptosis and necrosis. Glutamate-cysteine ligase (GCL) is the rate-limiting enzyme in the glutathione biosynthesis pathway. Buthionine sulfoximine (BSO), a GCL inhibitor, has previously demonstrated limited clinical benefits. Therefore, selecting patients who respond well to the inhibitor is a key approach for successful future drug development. Ferroptosis induction by BSO has not been fully examined in prior studies. Therefore, the present study investigated the pharmacological effects of BSO and the association between basal intracellular glutathione levels and sensitivity to BSO in cultured cell lines derived from various types of cancer, including those of the kidney [769P, 786-O, A-498, A704, ACHN, Caki-1, Caki-2, G401, G402, RCC4 VHL(-/-), RCC4 VHL(+/+), SK-NEP-1 and SW156] and ovaries (A2780 and A2780/CDDP). BSO was demonstrated to suppress glutathione levels and induce lipid peroxidation, thereby inhibiting cell viability. The viability-reducing effects of BSO were attenuated by ferroptosis inhibition and enhanced by iron, indicating that BSO induced ferroptosis in cancer cells. The cell lines sensitive to BSO, including G402, tended to exhibit non-significantly lower levels of glutathione compared with the BSO-insensitive cell lines, including Caki-2 (P=0.08). Patient sample data indicated the existence of a population of colorectal tumors with lower glutathione levels compared with those of matched normal tissues that might be suitable for the clinical testing of sensitivity to GCLC inhibitors. Collectively, these data suggest that GCL inhibition leads to ferroptosis in cancer cells, and that low glutathione tumor levels may be a patient selection marker for the use of GCL inhibitors in the treatment of tumors.

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Development of a sheathless CE‐ESI‐MS interface

Hirayama, A., Abe, H., Yamaguchi, N., Tabata, S., Tomita, T., Soga, T.

Electrophoresis 39 ( 11 ) 1382 - 1389 2018.06

Research paper (scientific journal), Joint Work, Accepted, ISSN 01730835

© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
A sheath-flow interface is the most common ionization technique in CE-ESI-MS. However, this interface dilutes the analytes with the sheath liquid and decreases the sensitivity. In this study, we developed a sheathless CE-MS interface to improve sensitivity. The interface was fabricated by making a small crack approximately 2 cm from the end of a capillary column fixed on a plastic plate, and then covering the crack with a dialysis membrane to prevent metabolite loss during separation. A voltage for CE separation was applied between the capillary inlet and the buffer reservoir. Under optimum conditions, 52 cationic metabolite standards were separated and selectively detected using MS. With a pressure injection of 5 kPa for 15 s (ca. 1.4 nL), the detection limits for the tested compounds were between 0.06 and 1.7 μmol/L (S/N = 3). The method was applied to analysis of cationic metabolites extracted from a small number (12 000) of cancer cells, and the number of peaks detected was about 2.5 times higher than when using conventional sheath-flow CE-MS. Because the interface is easy to construct, it is cost-effective and can be adapted to any commercially available capillaries. This method is a powerful new tool for highly sensitive CE-MS-based metabolomic analysis.

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Changes of Liver Metabolites following Hepatectomy with Ischemia Reperfusion towards Liver Regeneration

Saito, Y., Morine Y., Iwahashi, S., Ikemoto, T., Imura, S., Yamanaka-Okumura, H., Hirayama, A., Soga, T., Tomita, M., Shimada, M.

Ann. Gastroenterol. Surg. 2 ( 3 ) 204 - 211 2018.05

Research paper (scientific journal), Joint Work, Accepted

Background: Metabolome analysis is one of the omics which investigates the final product of a central dogma. Changes of liver metabolites during liver regeneration following hepatectomy (Hx) continue to remain unclear. The aim of the present study was to investigate the changes of liver metabolites following Hx with ischemia reperfusion (I/R) towards liver regeneration. Methods: Twenty-three patients who underwent Hx were enrolled in this study. Non-tumor tissues were sampled immediately before and after Hx and a comparison was made between the liver samples taken before and after Hx using capillary electrophoresis (CE)-time-of-flight mass spectrometry (TOFMS) as metabolome analysis. Results: The metabolic pathway showed that there was a significant increase in "lactate" following Hx. There was a significant decrease in metabolites only in the first half of the tricarboxylic acid cycle (TCA) cycle, and adenosine triphosphate (ATP) by anaerobiotic glycolysis did not occur in time for energy consumption of the Hx. Principal component analysis revealed remarkably different component profiles between the samples taken before and after Hx. One hundred and three metabolites were selected as critical metabolites for separating components. Valine and tryptophan increased significantly after Hx and they were regulated by resected liver volume, ischemic time and liver function. Conclusion: The liver metabolites changed remarkably between before and after Hx. Especially, liver valine and tryptophan were increased.

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Thymidine catabolism promotes NADPH oxidase-derived reactive oxygen species (ROS) signalling in KB and yumoto cells

Tabata, S., Yamamoto, M., Goto, H., Hirayama, A., Ohishi, M., Kuramoto, T., Mitsuhashi, A., Ikeda, R., Haraguchi, M., Kawahara, K., Shinsato, Y., Minami, K., Saijo, A., Toyoda, Y., Hanibuchi, M., Nishioka, Y., Sone, S., Esumi, H., Tomita, M., Soga, T., Furukawa, T., Akiyama, S.

Sci. Rep. 8 ( 1 ) 6760 2018.04

Research paper (scientific journal), Joint Work, Accepted

Thymidine phosphorylase (TP) is a rate-limiting enzyme in the thymidine catabolic pathway. TP is identical to platelet-derived endothelial cell growth factor and contributes to tumour angiogenesis. TP induces the generation of reactive oxygen species (ROS) and enhances the expression of oxidative stress-responsive genes, such as interleukin (IL)-8. However, the mechanism underlying ROS induction by TP remains unclear. In the present study, we demonstrated that TP promotes NADPH oxidase-derived ROS signalling in cancer cells. NADPH oxidase inhibition using apocynin or small interfering RNAs (siRNAs) abrogated the induction of IL-8 and ROS in TP-expressing cancer cells. Meanwhile, thymidine catabolism induced by TP increased the levels of NADPH and intermediates of the pentose phosphate pathway (PPP). Both siRNA knockdown of glucose 6-phosphate dehydrogenase (G6PD), a rate-limiting enzyme in PPP, and a G6PD inhibitor, dihydroepiandrosterone, reduced TP-induced ROS production. siRNA downregulation of 2-deoxy-D-ribose 5-phosphate (DR5P) aldolase, which is needed for DR5P to enter glycolysis, also suppressed the induction of NADPH and IL-8 in TP-expressing cells. These results suggested that TP-mediated thymidine catabolism increases the intracellular NADPH level via the PPP, which enhances the production of ROS by NADPH oxidase and activates its downstream signalling.

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Purine nucleotide metabolism regulates expression of the human immune ligand MICA

McCarthy, MT., Moncayo, G., Hiron, TK., Jakobsen, NA., Valli, A., Soga, T., Adam, J., O'Callaghan, CA.

J. Biol. Chem. 293 ( 11 ) 3913-3924 - 3924 2018.03

Research paper (scientific journal), Joint Work, Accepted, ISSN 00219258

© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.
Expression of the cell-surface glycoprotein MHC class I polypeptide- related sequence A (MICA) is induced in dangerous, abnormal, or "stressed" cells, including cancer cells, virus-infected cells, and rapidly proliferating cells. MICA is recognized by the activating immune cell receptor natural killer group 2D (NKG2D), providing a mechanism by which immune cells can identify and potentially eliminate pathological cells. Immune recognition through NKG2D is implicated in cancer, atherosclerosis, transplant rejection, and inflammatory diseases, such as rheumatoid arthritis. Despite the wide range of potential therapeutic applications ofMICAmanipulation, the factors that control MICA expression are unclear. Here we use metabolic interventions and metabolomic analyses to show that the transition from quiescent cellular metabolism to a "Warburg" or biosynthetic metabolic state induces MICA expression. Specifically, we show that glucose transport into the cell and active glycolytic metabolism are necessary to up-regulate MICA expression. Active purine synthesis is necessary to support this effect of glucose, and increases in purine nucleotide levels are sufficient to induce MICA expression. Metabolic induction of MICA expression directly influences NKG2D-dependent cytotoxicity by immune cells. These findings support a model of MICA regulation whereby the purine metabolic activity of individual cells is reflected by cell-surface MICA expression and is the subject of surveillance by NKG2D receptor-expressing immune cells.

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PKM1 Confers Metabolic Advantages and Promotes Cell-Autonomous Tumor Cell Growth

Morita, M., Sato, T., Nomura, M., Sakamoto, Y., Inoue, Y., Tanaka, R., Ito, S., Kurosawa, K., Yamaguchi, K., Sugiura, Y., Takizaki, H., Yamashita, Y., Katakura, R., Sato, I., Kawai, M., Okada, Y., Watanabe, H., Kondoh, G., Matsumoto, S., Kishimoto, A., Obata, M., Matsumoto, M., Fukuhara, T., Motohashi, H., Suematsu, M., Komatsu, M., Nakayama, KI., Watanabe, T., Soga, T., Shima, H., Maemondo, M., Tanuma. N.

Cancer Cell (Cancer Cell) 33 ( 3 ) 355 - 367.e7 2018.03

Research paper (scientific journal), Joint Work, Accepted, ISSN 15356108

Expression of PKM2, which diverts glucose-derived carbon from catabolic to biosynthetic pathways, is a hallmark of cancer. However, PKM2 function in tumorigenesis remains controversial. Here, we show that, when expressed rather than PKM2, the PKM isoform PKM1 exhibits a tumor-promoting function in KRASG12D-induced or carcinogen-initiated mouse models or in some human cancers. Analysis of Pkm mutant mouse lines expressing specific PKM isoforms established that PKM1 boosts tumor growth cell intrinsically. PKM1 activated glucose catabolism and stimulated autophagy/mitophagy, favoring malignancy. Importantly, we observed that pulmonary neuroendocrine tumors (NETs), including small-cell lung cancer (SCLC), express PKM1, and that PKM1 expression is required for SCLC cell proliferation. Our findings provide a rationale for targeting PKM1 therapeutically in certain cancer subtypes, including pulmonary NETs.

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Urinary Polyamine Biomarker Panels with Machine-Learning Differentiated Colorectal Cancers, Benign Disease, and Healthy Controls

Nakajima, T., Katsumata, K., Kuwabara, H., Soya, R., Enomoto, M., Ishizaki, T., Tsuchida, A., Mori, M., Hiwatari, K., Soga, T., Tomita, M., Sugimoto, M.

Int. J. Mol. Sci. (Int. J. Mol. Sci.) 19 ( 3 ) 756 2018.03

Research paper (scientific journal), Joint Work, Accepted, ISSN 16616596

Colorectal cancer (CRC) is one of the most daunting diseases due to its increasing worldwide prevalence, which requires imperative development of minimally or non-invasive screening tests. Urinary polyamines have been reported as potential markers to detect CRC, and an accurate pattern recognition to differentiate CRC with early stage cases from healthy controls are needed. Here, we utilized liquid chromatography triple quadrupole mass spectrometry to profile seven kinds of polyamines, such as spermine and spermidine with their acetylated forms. Urinary samples from 201 CRCs and 31 non-CRCs revealed the N₁,N12-diacetylspermine showing the highest area under the receiver operating characteristic curve (AUC), 0.794 (the 95% confidence interval (CI): 0.704-0.885, p < 0.0001), to differentiate CRC from the benign and healthy controls. Overall, 59 samples were analyzed to evaluate the reproducibility of quantified concentrations, acquired by collecting three times on three days each from each healthy control. We confirmed the stability of the observed quantified values. A machine learning method using combinations of polyamines showed a higher AUC value of 0.961 (95% CI: 0.937-0.984, p < 0.0001). Computational validations confirmed the generalization ability of the models. Taken together, polyamines and a machine-learning method showed potential as a screening tool of CRC.

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Microhomology-assisted scarless genome editing in human iPSCs

Kim, S., Matsumoto, T., Kagawa, H., Nakamura, M., Hirohata, R., Ueno, A., Ohishi, M., Sakuma, T., Soga, T., Yamamoto, T., Woltjen, K.

Nat. Commun. 9 ( 1 ) 939 2018.03

Research paper (scientific journal), Joint Work, Accepted

© 2018 The Author(s). Gene-edited induced pluripotent stem cells (iPSCs) provide relevant isogenic human disease models in patient-specific or healthy genetic backgrounds. Towards this end, gene targeting using antibiotic selection along with engineered point mutations remains a reliable method to enrich edited cells. Nevertheless, integrated selection markers obstruct scarless transgene-free gene editing. Here, we present a method for scarless selection marker excision using engineered microhomology-mediated end joining (MMEJ). By overlapping the homology arms of standard donor vectors, short tandem microhomologies are generated flanking the selection marker. Unique CRISPR-Cas9 protospacer sequences nested between the selection marker and engineered microhomologies are cleaved after gene targeting, engaging MMEJ and scarless excision. Moreover, when point mutations are positioned unilaterally within engineered microhomologies, both mutant and normal isogenic clones are derived simultaneously. The utility and fidelity of our method is demonstrated in human iPSCs by editing the X-linked HPRT1 locus and biallelic modification of the autosomal APRT locus, eliciting disease-relevant metabolic phenotypes.

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Human AK2 links intracellular bioenergetic redistribution to the fate of hematopoietic progenitors

Oshima, K., Saiki, N., Tanaka, M., Imamura, H. Niwa, A., Tanimura, A., Nagahashi;, A., Hirayama, A., Okita, K., Hotta, A., Kitayama, S., Osawa, M., Kaneko, S., Watanabe, A., Asaka, I., Fujibuchi W., Imai, K., Yabe, H., Kamachi, Y., Hara J., Kojima, S., Tomita, M., Soga, T., Noma, T., Nonoyama, S., Nakahata, T., Saito, M.K.

Biochem. Biophys. Res. Commun. 497 ( 2 ) 719-725 - 725 2018.03

Research paper (scientific journal), Joint Work, Accepted, ISSN 0006291X

AK2 is an adenylate phosphotransferase that localizes at the intermembrane spaces of the mitochondria, and its mutations cause a severe combined immunodeficiency with neutrophil maturation arrest named reticular dysgenesis (RD). Although the dysfunction of hematopoietic stem cells (HSCs) has been implicated, earlier developmental events that affect the fate of HSCs and/or hematopoietic progenitors have not been reported. Here, we used RD-patient-derived induced pluripotent stem cells (iPSCs) as a model of AK2-deficient human cells. Hematopoietic differentiation from RD-iPSCs was profoundly impaired. RD-iPSC-derived hemoangiogenic progenitor cells (HAPCs) showed decreased ATP distribution in the nucleus and altered global transcriptional profiles. Thus, AK2 has a stage-specific role in maintaining the ATP supply to the nucleus during hematopoietic differentiation, which affects the transcriptional profiles necessary for controlling the fate of multipotential HAPCs. Our data suggest that maintaining the appropriate energy level of each organelle by the intracellular redistribution of ATP is important for controlling the fate of progenitor cells.

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Elevated Polyamines in Saliva of Pancreatic Cancer

Asai, Y., Itoi, T., Sugimoto, M., Sofuni, A., Tsuchiya, T, Tanaka, R., Tonozuka, R., Honjo, M., Mukai, S., Fujita, M., Yamamoto, K., Matsunami, Y., Kurosawa, T., Nagakawa, Y., Kaneko, M., Ota, S., Kawachi, S., Shimazu, M., Soga, T., Tomita, M., Sunamura., M.

Cancers 10 ( 2 ) pii: E43 2018.02

Research paper (scientific journal), Joint Work, Accepted

Detection of pancreatic cancer (PC) at a resectable stage is still difficult because of the lack of accurate detection tests. The development of accurate biomarkers in low or non-invasive biofluids is essential to enable frequent tests, which would help increase the opportunity of PC detection in early stages. Polyamines have been reported as possible biomarkers in urine and saliva samples in various cancers. Here, we analyzed salivary metabolites, including polyamines, using capillary electrophoresis-mass spectrometry. Salivary samples were collected from patients with PC (n = 39), those with chronic pancreatitis (CP, n = 14), and controls (C, n = 26). Polyamines, such as spermine, N₁-acetylspermidine, and N₁-acetylspermine, showed a significant difference between patients with PC and those with C, and the combination of four metabolites including N₁-acetylspermidine showed high accuracy in discriminating PC from the other two groups. These data show the potential of saliva as a source for tests screening for PC.

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A Transient Rise in Free Mg2+ Ions Released from ATP-Mg Hydrolysis Contributes to Mitotic Chromosome Condensation

Maeshima, K., Matsuda, T., Shindo, Y., Imamura, H., Tamura, S., Imai, R., Kawakami, S., Nagashima, R., Soga, T., Noji, H., Oka, K., Nagai, T.

Curr. Biol. 28 ( 3 ) 444 - 451 2018.02

Research paper (scientific journal), Joint Work, Accepted

For cell division, negatively charged chromatin, in which nucleosome fibers (10 nm fibers) are irregularly folded [1-5], must be condensed into chromosomes and segregated. While condensin and other proteins are critical for organizing chromatin into the appropriate chromosome shape [6-17], free divalent cations such as Mg2+ and Ca2+, which condense chromatin or chromosomes in vitro [18-28], have long been considered important, especially for local condensation, because the nucleosome fiber has a net negative charge and is by itself stretched like "beads on a string" by electrostatic repulsion. For further folding, other positively charged factors are required to decrease the charge and repulsion [29]. However, technical limitations to measure intracellular free divalent cations, but not total cations [30], especially Mg2+, have prevented us from elucidating their function. Here, we developed a Förster resonance energy transfer (FRET)-based Mg2+ indicator that monitors free Mg2+ dynamics throughout the cell cycle. By combining this indicator with Ca2+ [31] and adenosine triphosphate (ATP) [32] indicators, we demonstrate that the levels of free Mg2+, but not Ca2+, increase during mitosis. The Mg2+ increase is coupled with a decrease in ATP, which is normally bound to Mg2+ in the cell [33]. ATP inhibited Mg2+-dependent chromatin condensation in vitro. Chelating Mg2+ induced mitotic cell arrest and chromosome decondensation, while ATP reduction had the opposite effect. Our results suggest that ATP-bound Mg2+ is released by ATP hydrolysis and contributes to mitotic chromosome condensation with increased rigidity, suggesting a novel regulatory mechanism for higher-order chromatin organization by the intracellular Mg2+-ATP balance.

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Ketone body 3-hydroxybutyrate mimics calorie restriction via the Nrf2 activator, fumarate, in the retina

Izuta, Y., Imada, T., Hisamura, R., Oonishi, E., Nakamura. S., Inagaki, E., Ito, M., Soga, T., Tsubota, K.

Aging Cell 17 ( 1 ) e12699 2018.02

Research paper (scientific journal), Joint Work, Accepted, ISSN 14749718

Calorie restriction (CR) being the most robust dietary intervention provides various health benefits. D-3-hydroxybutyrate (3HB), a major physiological ketone, has been proposed as an important endogenous molecule for CR. To investigate the role of 3HB in CR, we investigated potential shared mechanisms underlying increased retinal 3HB induced by CR and exogenously applied 3HB without CR to protect against ischemic retinal degeneration. The repeated elevation of retinal 3HB, with or without CR, suppressed retinal degeneration. Metabolomic analysis showed that the antioxidant pentose phosphate pathway and its limiting enzyme, glucose-6-phosphate dehydrogenase (G6PD), were concomitantly preserved. Importantly, the upregulation of nuclear factor erythroid 2 p45-related factor 2 (Nrf2), a regulator of G6PD, and elevation of the tricarboxylic acid cycle's Nrf2 activator, fumarate, were also shared. Together, our findings suggest that CR provides retinal antioxidative defense by 3HB through the antioxidant Nrf2 pathway via modification of a tricarboxylic acid cycle intermediate during 3HB metabolism.

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Comparative analysis of cerebrospinal fluid metabolites in Alzheimer’s disease and idiopathic normal pressure hydrocephalus in a Japanese cohort

Nagata, Y., Hirayama, A., Ikeda, S., Shirahata, A., Shoji, F., Maruyama, M., Kayano, M., Bundo, M., Hattori, K., Yoshida, S., Goto, Y., Urakami, K., Soga, T., Ozaki, K., Niida, S.

Biomark. Res. 6 ( 1 ) 5 2018.01

Research paper (scientific journal), Joint Work, Accepted

© 2018 The Author(s).
Background: Alzheimer's disease (AD) is a most common dementia in elderly people. Since AD symptoms resemble those of other neurodegenerative diseases, including idiopathic normal pressure hydrocephalus (iNPH), it is difficult to distinguish AD from iNPH for a precise and early diagnosis. iNPH is caused by the accumulation of cerebrospinal fluid (CSF) and involves gait disturbance, urinary incontinence, and dementia. iNPH is treatable with shunt operation which removes accumulated CSF from the brain ventricles. Methods: We performed metabolomic analysis in the CSF of patients with AD and iNPH with capillary electrophoresis-mass spectrometry. We assessed metabolites to discriminate between AD and iNPH with Welch's t-test, receiver operating characteristic (ROC) curve analysis, and multiple logistic regression analysis. Results: We found significant increased levels of glycerate and N-acetylneuraminate and significant decreased levels of serine and 2-hydroxybutyrate in the CSF of patients with AD compared to the CSF of patients with iNPH. The ROC curve analysis with these four metabolites showed that the area under the ROC curve was 0.90, indicating good discrimination between AD and iNPH. Conclusions: This study identified four metabolites that could possibly discriminate between AD and iNPH, which previous research has shown are closely related to the risk factors, pathogenesis, and symptoms of AD. Analyzing pathway-specific metabolites in the CSF of patients with AD may further elucidate the mechanism and pathogenesis of AD.

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Reliability of plasma polar metabolite concentrations in a large-scale cohort study using capillary electrophoresis-mass spectrometry

Harada, S., Hirayama, A., Chan, Q., Kurihara, A., Fukai, K., Iida, M., Kato, S., Sugiyama, D., Kuwabara, K., Takeuchi, A., Akiyama, M., Okamura, T., Ebbels, TMD., Elliott, P., Tomita, M., Sato, A., Suzuki, C., Sugimoto, M., Soga, T., Takebayashi, T.

PLoS One 13 ( 1 ) e0191230 2018.01

Research paper (scientific journal), Single Work, Accepted

Background: Cohort studies with metabolomics data are becoming more widespread, however, large-scale studies involving 10,000s of participants are still limited, especially in Asian populations. Therefore, we started the Tsuruoka Metabolomics Cohort Study enrolling 11,002 community-dwelling adults in Japan, and using capillary electrophoresis-mass spectrometry (CE-MS) and liquid chromatography-mass spectrometry. The CE-MS method is highly amenable to absolute quantification of polar metabolites, however, its reliability for large-scale measurement is unclear. The aim of this study is to examine reproducibility and validity of large-scale CE-MS measurements. In addition, the study presents absolute concentrations of polar metabolites in human plasma, which can be used in future as reference ranges in a Japanese population.

Methods: Metabolomic profiling of 8,413 fasting plasma samples were completed using CE-MS, and 94 polar metabolites were structurally identified and quantified. Quality control (QC) samples were injected every ten samples and assessed throughout the analysis. Inter- and intra-batch coefficients of variation of QC and participant samples, and technical intraclass correlation coefficients were estimated. Passing-Bablok regression of plasma concentrations by CE-MS on serum concentrations by standard clinical chemistry assays was conducted for creatinine and uric acid.

Results and conclusions: In QC samples, coefficient of variation was less than 20% for 64 metabolites, and less than 30% for 80 metabolites out of the 94 metabolites. Inter-batch coefficient of variation was less than 20% for 81 metabolites. Estimated technical intraclass correlation coefficient was above 0.75 for 67 metabolites. The slope of Passing-Bablok regression was estimated as 0.97 (95% confidence interval: 0.95, 0.98) for creatinine and 0.95 (0.92, 0.96) for uric acid. Compared to published data from other large cohort measurement platforms, reproducibility of metabolites common to the platforms was similar to or better than in the other studies. These results show that our CE-MS platform is suitable for conducting large-scale epidemiological studies.

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Antioxidant role of autophagy in maintaining the integrity of glomerular capillaries

Matsuda, J., Namba, T., Takabatake, Y., Kimura, T., Takahashi, A., Yamamoto, T., Minami, S., Sakai, S., Fujimura, R., Kaimori, J.Y., Matsui, I., Hamano, T., Fukushima, Y., Matsui, K., Soga, T., Isaka, Y.

Autophagy 14 ( 1 ) 53 - 65 2018.01

Research paper (scientific journal), Joint Work, Accepted, ISSN 15548627

Autophagy is a lysosomal degradation system by which cytosolic materials and damaged organelles are broken down into basic components. To explore the physiological role of autophagy in glomerular endothelial cells (GEnCs), we compared the autophagic flux among cells in the kidney under starvation. Inhibition of autophagy by chloroquine administration significantly increased the number of autophagosomes or autolysosomes in GEnCs and proximal tubular cells, but not in podocytes, suggesting that the GEnCs exhibit substantial autophagic activity. Next, we analyzed endothelial and hematopoietic cell-specific atg5-deficient mice (atg5-conditional KO [cKO] mice). Glomeruli of 4-wk-old atg5-cKO mice exhibited slightly distended capillary loops accompanied by an accumulation of reactive oxygen species (ROS). Glomeruli of 8-wk-old atg5-cKO mice showed a lobular pattern with thickening of the capillary loops and mesangial matrix expansion; however, the vasculature of other organs was preserved. The atg5-cKO mice died by 12 wk of age, presumably due to pancytopenia resulting from the defect in their hematopoietic lineages. Therefore, we subjected 4-wk atg5-cKO mice to irradiation followed by bone marrow transplantation from normal littermates. Transplanted mice recapitulated the glomerular phenotypes of the atg5-cKO mice with no obvious histological changes in other organs. Twelve-mo-old transplanted mice developed mesangiolysis and glomerulosclerosis with significant deterioration of kidney function. Administration of N-acetyl-l-cysteine, a ROS scavenger, to atg5-cKO mice rescued the glomerular phenotypes. These data suggest that endothelial autophagy protects glomeruli from oxidative stress and maintains the integrity of glomerular capillaries. Enhancing endothelial autophagy may provide a novel therapeutic approach to minimizing glomerular diseases.

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Dynamic metabolomics reveals that insulin primes the adipocyte for glucose metabolism

Krycer, JR., Yugi, K., Hirayama, A., Fazakerley, DJ., Quek, LE., Scalzo, R., Ohno, S., Hodson, MP. Ikeda, S., Shoji, F., Suzuki, K., Domanova, W., Parker, BL.,. Nelson, ME ., Humphrey, SJ., Turner, N., Hoehn, KL., Cooney, GJ., Soga, T., Kuroda, S., James, DE.

Cell Rep. 21 ( 12 ) 3536 - 3547 2017.12

Research paper (scientific journal), Joint Work, Accepted

Insulin triggers an extensive signaling cascade to coordinate adipocyte glucose metabolism. It is considered that the major role of insulin is to provide anabolic substrates by activating GLUT4-dependent glucose uptake. However, insulin stimulates phosphorylation of many metabolic proteins. To examine the implications of this on glucose metabolism, we performed dynamic tracer metabolomics in cultured adipocytes treated with insulin. Temporal analysis of metabolite concentrations and tracer labeling revealed rapid and distinct changes in glucose metabolism, favoring specific glycolytic branch points and pyruvate anaplerosis. Integrating dynamic metabolomics and phosphoproteomics data revealed that insulin-dependent phosphorylation of anabolic enzymes occurred prior to substrate accumulation. Indeed, glycogen synthesis was activated independently of glucose supply. We refer to this phenomenon as metabolic priming, whereby insulin signaling creates a demand-driven system to "pull" glucose into specific anabolic pathways. This complements the supply-driven regulation of anabolism by substrate accumulation and highlights an additional role for insulin action in adipocyte glucose metabolism.

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Cancer-Specific Energy Metabolism in Rhabdomyosarcoma Cells Is Regulated by MicroRNA

Sugito, N., Taniguchi, K., Kuranaga, Y., Ohishi, M., Soga, T., Ito, Y., Miyachi, M., Kikuchi, K., Hosoi, H., Akao, Y.

Nucleic Acid Ther. 27 ( 6 ) 365 - 377 2017.12

Research paper (scientific journal), Accepted

Rhabdomyosarcoma (RMS) is a soft tissue sarcoma and is most frequently found in children. In RMS, there are two major subtypes, that is, embryonal RMS and alveolar RMS (ARMS). ARMS has exclusively the worse prognosis and is caused by formation of the chimeric PAX3-FOXO1 gene. Regarding cancer, the Warburg effect is known as a feature of cancer-specific metabolism. Polypyrimidine tract-binding protein 1 (PTBP1), a splicer of pyruvate kinase muscle (PKM) mRNA, is a positive regulator of cancer-specific energy metabolism. We investigated the expression and effects of muscle-specific miR-1 and miR-133b on RMS cells (RD, KYM-1, Rh30, and Rh41) from the view of energy metabolism and regulation of the chimeric gene. As a result, downregulated miR-1 and miR-133b/upregulated PTBP1 were found in RMS cell lines as well as in RMS clinical cases. Ectopic expression of either miR in both types of RMS cells induced autophagic cell death through silencing of PTBP1. Interestingly, we validated that miR-133b also knock downed PAX3-FOXO1. Moreover, we found that PAX3-FOXO1 positively regulated the PKM2-dominant expression through enhanced expression of PTBP1. These findings suggest that the miR-1 and miR-133b/PTBP1 axis and miR-133b/PAX3-FOXO1/PTBP1 axis contributed to the maintenance of cancer-specific energy metabolism.

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UCP1-independent signaling involving SERCA2b-mediated calcium cycling regulates beige fat thermogenesis and systemic glucose homestasis

Ikeda, K., Kang, Q., Yoneshiro, T., Camporez, J.P., Maki, H., Homma, M., Shinoda, K., Chen, Y., Lu, X., Maretich, P., Tajima, K., Ajuwon, K.M., Soga, T., Kajimura, S.

Nat. Med. 23 ( 12 ) 1454 - 1465 2017.12

Research paper (scientific journal), Joint Work, Accepted

Uncoupling protein 1 (UCP1) plays a central role in nonshivering thermogenesis in brown fat; however, its role in beige fat remains unclear. Here we report a robust UCP1-independent thermogenic mechanism in beige fat that involves enhanced ATP-dependent Ca2+ cycling by sarco/endoplasmic reticulum Ca2+-ATPase 2b (SERCA2b) and ryanodine receptor 2 (RyR2). Inhibition of SERCA2b impairs UCP1-independent beige fat thermogenesis in humans and mice as well as in pigs, a species that lacks a functional UCP1 protein. Conversely, enhanced Ca2+ cycling by activation of α1- and/or β3-adrenergic receptors or the SERCA2b-RyR2 pathway stimulates UCP1-independent thermogenesis in beige adipocytes. In the absence of UCP1, beige fat dynamically expends glucose through enhanced glycolysis, tricarboxylic acid metabolism and pyruvate dehydrogenase activity for ATP-dependent thermogenesis through the SERCA2b pathway; beige fat thereby functions as a 'glucose sink' and improves glucose tolerance independently of body weight loss. Our study uncovers a noncanonical thermogenic mechanism through which beige fat controls whole-body energy homeostasis via Ca2+ cycling.

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Genetic, metabolomic and transcriptomic analyses of the de novo L-cysteine biosynthetic pathway in the enteric protozoan parasite Entamoeba histolytica

Jeelani, G., Sato, D., Soga, T., Nozaki, T.

Sci. Rep. 7 ( 1 ) 15649 2017.11

Research paper (scientific journal), Joint Work, Accepted

The de novo L-cysteine biosynthetic pathway is critical for the growth, antioxidative stress defenses, and pathogenesis of bacterial and protozoan pathogens, such as Salmonella typhimurium and Entamoeba histolytica. This pathway involves two key enzymes, serine acetyltransferase (SAT) and cysteine synthase (CS), which are absent in mammals and therefore represent rational drug targets. The human parasite E. histolytica possesses three SAT and CS isozymes; however, the specific roles of individual isoforms and significance of such apparent redundancy remains unclear. In the present study, we generated E. histolytica cell lines in which CS and SAT expression was knocked down by transcriptional gene silencing. The strain in which CS1, 2 and 3 were simultaneously silenced and the SAT3 gene-silenced strain showed impaired growth when cultured in a cysteine lacking BI-S-33 medium, whereas silencing of SAT1 and SAT2 had no effects on growth. Combined transcriptomic and metabolomic analyses revealed that, CS and SAT3 are involved in S-methylcysteine/cysteine synthesis. Furthermore, silencing of the CS1-3 or SAT3 caused upregulation of various iron-sulfur flavoprotein genes. Taken together, these results provide the first direct evidence of the biological importance of SAT3 and CS isoforms in E. histolytica and justify the exploitation of these enzymes as potential drug targets.

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ACSL3 promotes intratumoral steroidogenesis in prostate cancer cells

Migita, T., Takayama, K., Urano, T., Obinata, D., Ikeda, K., Soga, T., Takahashi, S., Inoue, S.

Cancer Sci. 108 ( 10 ) 2011 - 2021 2017.10

Research paper (scientific journal), Joint Work, Accepted

Long-chain acyl-coenzyme A (CoA) synthetase 3 (ACSL3) is an androgen-responsive gene involved in the generation of fatty acyl-CoA esters. ACSL3 is expressed in both androgen-sensitive and castration-resistant prostate cancer (CRPC). However, its role in prostate cancer remains elusive. We overexpressed ACSL3 in androgen-dependent LNCaP cells and examined the downstream effectors of ACSL3. Furthermore, we examined the role of ACSL3 in the androgen metabolism of prostate cancer. ACSL3 overexpression led to upregulation of several genes such as aldo-keto reductase 1C3 (AKR1C3) involved in steroidogenesis, which utilizes adrenal androgen dehydroepiandrosterone sulfate (DHEAS) as substrate, and downregulated androgen-inactivating enzyme UDP-glucuronosyltransferase 2 (UGT2B). Exposure to DHEAS significantly increased testosterone levels and cell proliferative response in ACSL3-overexpressing cells when compared to that in control cells. A public database showed that ACSL3 level was higher in CRPC than in hormone-sensitive prostate cancer. CRPC cells showed an increased expression of ACSL3 and an expression pattern of AKR1C3 and UGT2B similar to ACSL3-overexpressing cells. DHEAS stimulation significantly promoted the proliferation of CRPC cells when compared to that of LNCaP cells. These findings suggest that ACSL3 contributes to the growth of CRPC through intratumoral steroidogenesis (i.e. promoting androgen synthesis from DHEAS and preventing the catabolism of active androgens).

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Fumarate hydratase deletion in pancreatic β cells leads to progressive diabetes

Adam, J., Ramracheya, R., Chibalina, M.V., Ternette, N., Hamilton, A., Tarasov, A.I., Zhang, Q., Rebelato, E., Rorsman, N.J.G., Martin-del-Rio, R., Lewis, A., Ozkan, G., Do, H.W., Spegal, P., Saitoh, K., Kato, K., Igarashi, K., Kessler, B.M., Pugh, C.W., Tamarit-Rodriques, J., Mulder, H., Clark, A., Fizzell, N., Soga, T., Ashcroft, F.M., Silver, A., Pollard, P.J., Rorsman, P.

Cell Rep. 20 ( 13 ) 3135 - 3148 2017.09

Research paper (scientific journal), Joint Work, Accepted

We explored the role of the Krebs cycle enzyme fumarate hydratase (FH) in glucose-stimulated insulin secretion (GSIS). Mice lacking Fh1 in pancreatic β cells (Fh1βKO mice) appear normal for 6-8 weeks but then develop progressive glucose intolerance and diabetes. Glucose tolerance is rescued by expression of mitochondrial or cytosolic FH but not by deletion of Hif1α or Nrf2. Progressive hyperglycemia in Fh1βKO mice led to dysregulated metabolism in β cells, a decrease in glucose-induced ATP production, electrical activity, cytoplasmic [Ca2+]i elevation, and GSIS. Fh1 loss resulted in elevated intracellular fumarate, promoting succination of critical cysteines in GAPDH, GMPR, and PARK 7/DJ-1 and cytoplasmic acidification. Intracellular fumarate levels were increased in islets exposed to high glucose and in islets from human donors with type 2 diabetes (T2D). The impaired GSIS in islets from diabetic Fh1βKO mice was ameliorated after culture under normoglycemic conditions. These studies highlight the role of FH and dysregulated mitochondrial metabolism in T2D.

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Global metabolic reprogramming of colorectal cancer occurs at adenoma stage and is induced by MYC

Satoh, K., Yachida, S., Sugimoto, M., Oshima, M., Nakagawa, T., Akamoto, S., Tabata, S., Saitoh, K., Kato, K., Sato, S., Igarashi, K., Aizawa, Y., Kajino-Sakamoto, R., Kojima, Y., Fujishita, T., Enomoto, A., Hirayama, A., Ishikawa, T., Taketo, M.M., Kushida, Y., Haba, R., Okano, K., Tomita, M., Suzuki, Y., Fukuda, S., Aoki, M., Soga, T.

Proc. Natl. Acad. Sci. USA 114 ( 37 ) E7697 - E7706 2017.09

Research paper (scientific journal), Joint Work, Accepted

Cancer cells alter their metabolism for the production of precursors of macromolecules. However, the control mechanisms underlying this reprogramming are poorly understood. Here we show that metabolic reprogramming of colorectal cancer is caused chiefly by aberrant MYC expression. Multiomics-based analyses of paired normal and tumor tissues from 275 patients with colorectal cancer revealed that metabolic alterations occur at the adenoma stage of carcinogenesis, in a manner not associated with specific gene mutations involved in colorectal carcinogenesis. MYC expression induced at least 215 metabolic reactions by changing the expression levels of 121 metabolic genes and 39 transporter genes. Further, MYC negatively regulated the expression of genes involved in mitochondrial biogenesis and maintenance but positively regulated genes involved in DNA and histone methylation. Knockdown of MYC in colorectal cancer cells reset the altered metabolism and suppressed cell growth. Moreover, inhibition of MYC target pyrimidine synthesis genes such as CAD, UMPS, and CTPS blocked cell growth, and thus are potential targets for colorectal cancer therapy.

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Evaluation of the impact of gut microbiota on uremic solute accumulation by CE-TOFMS-based metabolomics approach

Mishima, E., Fukuda, S. (equally contributed), Mukawa, C., Yuri, A., Kanemitsu, Y., Matsumoto, Y., Akiyama, Y., Fukuda, NN., Tsukamoto, H., Asaji, K., Shima, H., Kikuchi, K., Suzuki, T., Tomioka, Y., Soga, T., Ito, S., Abe, T.

Kidney Int. 92 ( 3 ) 634 - 645 2017.09

Research paper (scientific journal), Joint Work

Gut microbiota is involved in the metabolism of uremic solutes. However, the precise influence of microbiota to the retention of uremic solutes in CKD is obscure. To clarify this, we compared adenine-induced renal failure and control mice under germ-free or specific pathogen-free (SPF) conditions, examining the metabolite profiles of plasma, feces, and urine using a capillary electrophoresis time-of-flight mass spectrometry-based approach. Mice with renal failure under germ-free conditions demonstrated significant changes in plasma metabolites. Among 183 detected solutes, plasma levels of 11 solutes, including major uremic toxins, were significantly lower in germ-free mice than in SPF mice with renal failure. These 11 solutes were considered microbiota-derived uremic solutes and included indoxyl sulfate, p-cresyl sulfate, phenyl sulfate, cholate, hippurate, dimethylglycine, γ-guanidinobutyrate, glutarate, 2-hydroxypentanoate, trimethylamine N-oxide, and phenaceturate. Metabolome profiling showed that these solutes were classified into three groups depending on their origins: completely derived from microbiota (indoxyl sulfate, p-cresyl sulfate), derived from both host and microbiota (dimethylglycine), and derived from both microbiota and dietary components (trimethylamine N-oxide). Additionally, germ-free renal failure conditions resulted in the disappearance of colonic short-chain fatty acids, decreased utilization of intestinal amino acids, and more severe renal damage compared with SPF mice with renal failure. Microbiota-derived short-chain fatty acids and efficient amino acid utilization may have a renoprotective effect, and loss of these factors may exacerbate renal damage in germ-free mice with renal failure. Thus, microbiota contributes substantially to the production of harmful uremic solutes, but conversely, growth without microbiota has harmful effects on CKD progression.

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Effect of masticatory stimulation on the quantity and quality of saliva and the salivary metabolomic profile

Okuma, N., Saita, M., Hoshi, N., Soga, T., Tomita, M., Sugimoto, M., Kimoto, K.

PLoS One 12 ( 8 ) e0183109 2017.08

Research paper (scientific journal), Joint Work, Accepted

Background: This study characterized the changes in quality and quantity of saliva, and changes in the salivary metabolomic profile, to understand the effects of masticatory stimulation.
Methods: Stimulated and unstimulated saliva samples were collected from 55 subjects and salivary hydrophilic metabolites were comprehensively quantified using capillary electrophoresis-time-of-flight mass spectrometry.
Results: In total, 137 metabolites were identified and quantified. The concentrations of 44 metabolites in stimulated saliva were significantly higher than those in unstimulated saliva. Pathway analysis identified the upregulation of the urea cycle and synthesis and degradation pathways of glycine, serine, cysteine and threonine in stimulated saliva. A principal component analysis revealed that the effect of masticatory stimulation on salivary metabolomic profiles was less dependent on sample population sex, age, and smoking. The concentrations of only 1 metabolite in unstimulated saliva, and of 3 metabolites stimulated saliva, showed significant correlation with salivary secretion volume, indicating that the salivary metabolomic profile and salivary secretion volume were independent factors.
Conclusions: Masticatory stimulation affected not only salivary secretion volume, but also metabolite concentration patterns. A low correlation between the secretion volume and these patterns supports the conclusion that the salivary metabolomic profile may be a new indicator to characterize masticatory stimulation.

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Inhibition of dipeptidyl peptidase-4 ameliorates cardiac ischemia and systolic dysfunction by up-regulating the FGF-2/EGR-1 pathway

Suda, M., Shimizu, I. , Yoshida, Y., Hayashi, Y., Ikegami, R., Katsuumi, G., Wakasugi, T., Yoshida, Y., Okuda, S., Soga, T., Minamino, T.

PLoS One 12 ( 8 ) e0182422 2017.08

Research paper (scientific journal), Joint Work

Dipeptidyl peptidase 4 inhibitors are used worldwide in the management of diabetes, but their role in the prevention or treatment of cardiovascular disorders has yet to be defined. We found that linagliptin, a DPP-4 inhibitor, suppressed capillary rarefaction in the hearts of mice with dietary obesity. Metabolomic analysis performed with capillary electrophoresis/mass spectrometry (LC-MS/MS) showed that linagliptin promoted favorable metabolic remodeling in cardiac tissue, which was characterized by high levels of citrulline and creatine. DNA microarray analysis revealed that the cardiac tissue level of early growth response protein 1 (EGR-1), which activates angiogenesis, was significantly reduced in untreated mice with dietary obesity, while this decrease was inhibited by administration of linagliptin. Mature fibroblast growth factor 2 (FGF-2) has a putative truncation site for DPP-4 at the NH2-terminal, and LC-MS/MS showed that recombinant DPP-4 protein cleaved the NH2-terminal dipeptides of mature FGF-2. Incubation of cultured neonatal rat cardiomyocytes with FGF-2 increased Egr1 expression, while it was suppressed by recombinant DPP-4 protein. Furthermore, vascular endothelial growth factor-A had a critical role in mediating FGF-2/EGR-1 signaling. In conclusion, pharmacological inhibition of DPP-4 suppressed capillary rarefaction and contributed to favorable remodeling of cardiac metabolism in mice with dietary obesity.

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Distinct requirements for energy metabolism in mouse primordial germ cells and their reprogramming to embryonic germ cells

Hayashi, Y., Otsuka, K., Ebina, M., Igarashi, K., Takehara, A., Matsumoto, M., Kanai, A., Igarashi, K., Soga, T., Matsui, Y.

Proc. Natl. Acad. Sci. USA 114 ( 31 ) 8289 - 8294 2017.08

Research paper (scientific journal), Joint Work, Accepted

Primordial germ cells (PGCs), undifferentiated embryonic germ cells, are the only cells that have the ability to become gametes and to reacquire totipotency upon fertilization. It is generally understood that the development of PGCs proceeds through the expression of germ cell-specific transcription factors and characteristic epigenomic changes. However, little is known about the properties of PGCs at the metabolite and protein levels, which are directly responsible for the control of cell function. Here, we report the distinct energy metabolism of PGCs compared with that of embryonic stem cells. Specifically, we observed remarkably enhanced oxidative phosphorylation (OXPHOS) and decreased glycolysis in embryonic day 13.5 (E13.5) PGCs, a pattern that was gradually established during PGC differentiation. We also demonstrate that glycolysis and OXPHOS are important for the control of PGC reprogramming and specification of pluripotent stem cells (PSCs) into PGCs in culture. Our findings about the unique metabolic property of PGCs provide insights into our understanding of the importance of distinct facets of energy metabolism for switching PGC and PSC status.

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Metabolomics of an in vitro liver model containing primary hepatocytes assembling around an endothelial cell network: comparative study on the metabolic stability and the effect of acetaminophen treatment

Toyoda, Y., Kashikura, K., Soga, T., Tagawa, Y.

J. Toxicol. Sci. 42 ( 4 ) 445 - 454 2017.08

Research paper (scientific journal), Joint Work, Accepted

Recently, a novel culture system consisting of primary hepatocytes structured over a network of endothelial cells on the Engelbreth-Holm-Swarm (EHS) gel has been reported. This in vitro liver model on the EHS gel (IVLEHS) has been shown to maintain the expression of hepatic genes and their functional activity. Moreover, the IVLEHS was more sensitive to xenobiotics than hepatocyte monocultures, suggesting the potential utility of this culture system for compound hepatotoxicity screening. However, the effect of this three-dimensional structure formation on the cellular metabolic profile of hepatocytes in the IVLEHS is not well understood. To address this concern, we performed metabolome analysis using capillary electrophoresis-time of flight mass spectrometry. Between the IVLEHS and mono-cultured hepatocytes on the EHS gel, there was no significant difference in the levels of metabolites of the urea cycle and the tricarboxylic acid cycle, essential amino acids, and adenylate energy charge (AEC) which is an important indicator of cellular energy status. On the other hand, acetaminophen-dependent decrease of the AEC in the IVLEHS was greater than that in the monoculture, suggesting the higher sensitivity of IVLEHS to acetaminophen-induced hepatotoxicity which is caused by metabolic activation of this drug. Further analysis showed that the levels of taurocholate, one of the major conjugated bile acids, were higher in the IVLEHS than in the monoculture. Considering that the construction of the IVLEHS did not seem to disturb the major cellular metabolism, our findings would strengthen the concept that IVLEHS would have beneficial effects on the maintenance of hepatic functions.

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Cancer with low cathepsin D levels is susceptible to vacuolar (H+)-ATPase inhibition

Kitazawa, S., Nishizawa, S., Nakagawa, H., Funata, M., Nishimura, K., Soga, T., Hara, T.

Cancer Sci. 108 ( 6 ) 1185 - 1193 2017.06

Research paper (scientific journal), Joint Work, Accepted

Vacuolar (H+ )-ATPases (V-ATPases) have important roles in the supply of nutrients to tumors by mediating autophagy and the endocytic uptake of extracellular fluids. Accordingly, V-ATPases are attractive therapeutic targets for cancer. However, the clinical use of V-ATPase inhibitors as anticancer drugs has not been realized, possibly owing to their high toxicity in humans. Inhibition of V-ATPase may be an appropriate strategy in highly susceptible cancers. In this study, we explored markers of V-ATPase inhibitor sensitivity. V-ATPase inhibitors led to pH impairment in acidic intracellular compartments, suppression of macropinocytosis, and decreased intracellular amino acid levels. The sensitivity of cells to V-ATPase inhibitors was correlated with low cathepsin D expression, and cancer cells showed increased sensitivity to V-ATPase inhibitors after pretreatment with a cathepsin D inhibitor and siRNA targeting the cathepsin D gene (CTSD). In addition, V-ATPase inhibitor treatment led to the induction of the amino acid starvation response, upregulation of endoplasmic reticulum stress markers, and suppression of mammalian target of rapamycin (mTOR) signaling in cells expressing low levels of cathepsin D. Some colorectal cancer patients showed the downregulation of cathepsin D in tumor tissues compared with matched normal tissues. These findings indicate that V-ATPase inhibitors are promising therapeutic options for cancers with downregulated cathepsin D.

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The metabolic profile of a rat model of chronic kidney disease

Tanada, Y., Okuda, J., Kato, T., Minamino-Muta, E., Murata, I., Soga, T., Shioi, T., Kimura, T.

Peer J. 5 e3352 2017.05

Research paper (scientific journal), Joint Work, Accepted

Background: The kidney is always subjected to high metabolic demand. The aim of this study was to characterize metabolic profiles of a rat model of chronic kidney disease (CKD) with cardiorenal syndrome (CRS) induced by prolonged hypertension.
Methods: We used inbred male Dahl salt-sensitive (DS) rats fed an 8% NaCl diet from six weeks of age (high-salt; HS group) or a 0.3% NaCl diet as controls (low-salt; LS group). We analyzed function, pathology, metabolome, and the gene expression related to energy metabolism of the kidney.
Results: DS rats with a high-salt diet showed hypertension at 11 weeks of age and elevated serum levels of creatinine and blood urea nitrogen with heart failure at 21 weeks of age. The fibrotic area in the kidneys increased at 21 weeks of age. In addition, gene expression related to mitochondrial function was largely decreased. The levels of citrate and isocitrate increased and the gene expression of alpha-ketoglutaratedehydrogenase and succinyl-CoA synthetase decreased; these are enzymes that metabolize citrate and isocitrate, respectively. In addition, the levels of succinate and acetyl Co-A, both of which are metabolites of the tricarboxylic acid (TCA) cycle, decreased.
Conclusions: DS rats fed a high-salt diet were deemed a suitable model of CKD with CRS. Gene expression and metabolites related to energy metabolism and mitochondria in the kidney significantly changed in DS rats with hypertension in accordance with the progression of renal injury.

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Synthetic Biomarker Design by Using Analyte-Responsive Acetaminophen

Nishihara, T., Inoue, J., Tabata, S., Murakami, S., Ishikawa, T., Saito, N., Fukuda, S., Tomita, M., Soga, T.

Chembiochem 18 ( 10 ) 910 - 913 2017.05

Research paper (scientific journal), Joint Work, Accepted

The use of synthetic biomarkers is an emerging technique to improve disease diagnosis. Here, we report a novel design strategy that uses analyte-responsive acetaminophen (APAP) to expand the catalogue of analytes available for synthetic biomarker development. As proof-of-concept, we designed hydrogen peroxide (H2 O2 )-responsive APAP (HR-APAP) and succeeded in H2 O2 detection with cellular and animal experiments. In fact, for blood samples following HR-APAP injection, we demonstrated that the plasma concentration ratio [APAP+APAP conjugates]/[HR-APAP] accurately reflects in vivo differences in H2 O2 levels. We anticipate that our practical methodology will be broadly useful for the preparation of various synthetic biomarkers.

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Thymidine Catabolism as a Metabolic Strategy for Cancer Survival

Tabata, S., Yamamoto, M., Goto, H., Hirayama, A., Ohishi, M., Kuramoto, T., Mitsuhashi, A., Ikeda, R., Haraguchi, M., Kawahara, K., Shinsato, Y., Minami, K., Saijo, A., Hanibuchi, M., Nishioka, Y., Sone, S., Esumi, H., Tomita, M., Soga, T., Furukawa, T., Akiyama, S.

Cell Rep. 19 ( 7 ) 1313 - 1321 2017.05

Research paper (scientific journal), Joint Work, Accepted

Thymidine phosphorylase (TP), a rate-limiting enzyme in thymidine catabolism, plays a pivotal role in tumor progression; however, the mechanisms underlying this role are not fully understood. Here, we found that TP-mediated thymidine catabolism could supply the carbon source in the glycolytic pathway and thus contribute to cell survival under conditions of nutrient deprivation. In TP-expressing cells, thymidine was converted to metabolites, including glucose 6-phosphate, lactate, 5-phospho-α-D-ribose 1-diphosphate, and serine, via the glycolytic pathway both in vitro and in vivo. These thymidine-derived metabolites were required for the survival of cells under low-glucose conditions. Furthermore, activation of thymidine catabolism was observed in human gastric cancer. These findings demonstrate that thymidine can serve as a glycolytic pathway substrate in human cancer cells.

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Modelling urea-cycle disorder citrullinemia type 1 with disease-specific iPSCs

Yoshitoshi-Uebayashi, E.Y., Toyoda, T., Yasuda, K., Kotaka, M., Nomoto, K., Okita, K., Yasuchika, K., Okamoto, S., Takubo, N., Nishikubo, T., Soga, T., Uemoto, S., Osafune, K.

Biochem Biophys Res Commun. 486 ( 3 ) 613 - 619 2017.05

Research paper (scientific journal), Joint Work, Accepted

Citrullinemia type 1 (CTLN1) is a urea cycle disorder (UCD) caused by mutations of the ASS1 gene, which is responsible for production of the enzyme argininosuccinate synthetase (ASS), and classically presented as life-threatening hyperammonemia in newborns. Therapeutic options are limited, and neurological sequelae may persist. To understand the pathophysiology and find novel treatments, induced pluripotent stem cells (iPSCs) were generated from a CTLN1 patient and differentiated into hepatocyte-like cells (HLCs). CTLN1-HLCs have lower ureagenesis, recapitulating part of the patient's phenotype. l-arginine, an amino acid clinically used for UCD treatment, improved this phenotype in vitro. Metabolome analysis revealed an increase in tricarboxylic acid (TCA) cycle metabolites in CTLN1, suggesting a connection between CTLN1 and the TCA cycle. This CTLN1-iPSC model improves the understanding of CTLN1 pathophysiology and can be used to pursue new therapeutic approaches.

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Cell competition with normal epithelial cells promotes apical extrusion of transformed cells through metabolic changes

Kon, S., Ishibashi, K., Katoh, H., Kitamoto, S., Shirai, T., Tanaka, S., Kajita, M., Ishikawa, S., Yamauchi, H., Yako, Y., Kamasaki, T., Matsumoto, T., Watanabe, H., Egami, R., Sasaki, A., Nishikawa, A., Kameda, I., Maruyama, T., Narumi, R., Morita, T., Sasaki, Y., Enoki, R., Honma, S., Imamura, H., Oshima, M., Soga, T., Miyazaki, J., Duchen, MR.., Nam, J., Onodera, Y., Yoshioka, S., Kikuta, J., Ishii, M., Imajo, M., Nishida, E., Fujioka, Y., Ohba, Y., Sato, T., Fujita, Y.

Nat. Cell Biol. 19 ( 5 ) 530 - 541 2017.05

Research paper (scientific journal), Joint Work, Accepted

Recent studies have revealed that newly emerging transformed cells are often apically extruded from epithelial tissues. During this process, normal epithelial cells can recognize and actively eliminate transformed cells, a process called epithelial defence against cancer (EDAC). Here, we show that mitochondrial membrane potential is diminished in RasV12-transformed cells when they are surrounded by normal cells. In addition, glucose uptake is elevated, leading to higher lactate production. The mitochondrial dysfunction is driven by upregulation of pyruvate dehydrogenase kinase 4 (PDK4), which positively regulates elimination of RasV12-transformed cells. Furthermore, EDAC from the surrounding normal cells, involving filamin, drives the Warburg-effect-like metabolic alteration. Moreover, using a cell-competition mouse model, we demonstrate that PDK-mediated metabolic changes promote the elimination of RasV12-transformed cells from intestinal epithelia. These data indicate that non-cell-autonomous metabolic modulation is a crucial regulator for cell competition, shedding light on the unexplored events at the initial stage of carcinogenesis.

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Succinate dehydrogenase B-deficient cancer cells are highly sensitive to bromodomain and extra-terminal inhibitors

Kitazawa, S., Ebara, S., Ando, A., Baba, Y., Satomi, Y., Soga, T., Hara, T.

Oncotarget 8 ( 17 ) 28922 - 28938 2017.04

Research paper (scientific journal), Joint Work, Accepted

Mutations in succinate dehydrogenase B (SDHB) gene are frequently observed in several tumors and associated with poor prognosis in these tumors. Therefore, drugs effective for SDHB-deficient tumors could fulfill an unmet medical need. In addition, such drugs would have an advantage in that selection of patients with SDHB-mutant cancer could increase the probability of success in clinical trials. Currently, however, the characteristics of SDHB-deficient cancers are not completely understood. Here, we established SDHB knockout cancer cell lines from human colon cancer HCT116 cells using the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 knockout system, and clarified its metabolic characteristics.In the SDHB knockout cells, succinate was accumulated and fumarate was decreased. The oxygen consumption rate was decreased while the extracellular acidification rate was increased in the SDHB knockout cells. Accordingly, an enhanced glycolysis pathway in the SDHB knockout cells was demonstrated by metabolomics analysis. Tracer experiments showed bidirectional metabolic flow in the tricarboxylic acid (TCA) cycle, possibly to maintain the necessary amounts of metabolites in the SDHB knockout cells. The proliferation of SDHB knockout cells was suppressed by a glycolysis inhibitor but not by a mitochondrial inhibitor. Additionally, partial dependence on glutaminolysis was observed in the SDHB knockout cells. Compound screening revealed that a bromodomain and extra-terminal (BET) inhibitor, which downregulated c-Myc, suppressed the growth of the SDHB knockout cells more potently than that of control cells. These findings provide an understanding of the metabolic characteristics of SDHB-deficient cancer and its vulnerabilities, which may lead to new therapeutic options.

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Remodelling of microRNAs in colorectal cancer by hypoxia alters metabolism profiles and 5-fluorouracil resistance

Nijhuis, A., Thompson, H., Adam, J., Paker A., Gammmon, L., Lewis, A., Bundy, J.G., Soga, T., Jalaly, A., Propper, D., Jeffery, R., Suraweera, N., McDonald, S., Thaha, M.A., Feakins, R., Lowe, R., Bishop, C., Silver, A.

Hum Mol Genet. 26 ( 8 ) 1552 - 1564 2017.04

Research paper (scientific journal), Joint Work, Accepted

Solid tumours have oxygen gradients and areas of near and almost total anoxia. Hypoxia reduces sensitivity to 5-fluorouracil (5-FU)-chemotherapy for colorectal cancer (CRC). MicroRNAs (miRNAs) are hypoxia sensors and were altered consistently in six CRC cell lines (colon cancer: DLD-1, HCT116 and HT29; rectal cancer: HT55, SW837 and VACO4S) maintained in hypoxia (1 and 0.2% oxygen) compared with normoxia (20.9%). CRC cell lines also showed altered amino acid metabolism in hypoxia and hypoxia-responsive miRNAs were predicted to target genes in four metabolism pathways: beta-alanine; valine, leucine, iso-leucine; aminoacyl-tRNA; and alanine, aspartate, glutamate. MiR-210 was increased in hypoxic areas of CRC tissues and hypoxia-responsive miR-21 and miR-30d, but not miR-210, were significantly increased in 5-FU resistant CRCs. Treatment with miR-21 and miR-30d antagonists sensitized hypoxic CRC cells to 5-FU. Our data highlight the complexity and tumour heterogeneity caused by hypoxia. MiR-210 as a hypoxic biomarker, and the targeting of miR-21 and miR-30d and/or the amino acid metabolism pathways may offer translational opportunities.

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Serum Metabolomic Profiles for Human Pancreatic Cancer Discrimination

Itoi, T., Sugimoto, M., Umeda, J., Sofuni, A., Tsuchiya, T., Tsuji, S., Tanaka, R., Tonozuka, R., Honjo, M., Moriyasu, F., Kasuya, K., Nagakawa, Y., Abe, Y., Takano, K., Kawachi, S., Shimazu, M., Soga, T., Tomita, M., Sunamura, M.

Int. J. Mol. Sci. 18 ( 4 ) 767 2017.04

Research paper (scientific journal), Joint Work, Accepted

This study evaluated the clinical use of serum metabolomics to discriminate malignant cancers including pancreatic cancer (PC) from malignant diseases, such as biliary tract cancer (BTC), intraductal papillary mucinous carcinoma (IPMC), and various benign pancreaticobiliary diseases. Capillary electrophoresismass spectrometry was used to analyze charged metabolites. We repeatedly analyzed serum samples (n = 41) of different storage durations to identify metabolites showing high quantitative reproducibility, and subsequently analyzed all samples (n = 140). Overall, 189 metabolites were quantified and 66 metabolites had a 20% coefficient of variation and, of these, 24 metabolites showed significant differences among control, benign, and malignant groups (p < 0.05; Steel-Dwass test). Four multiple logistic regression models (MLR) were developed and one MLR model clearly discriminated all disease patients from healthy controls with an area under receiver operating characteristic curve (AUC) of 0.970 (95% confidential interval (CI), 0.946-0.994, p < 0.0001). Another model to discriminate PC from BTC and IPMC yielded AUC = 0.831 (95% CI, 0.650-1.01, p = 0.0020) with higher accuracy compared with tumor markers including carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA19-9), pancreatic cancer-associated antigen (DUPAN2) and s-pancreas-1 antigen (SPAN1). Changes in metabolomic profiles might be used to screen for malignant cancers as well as to differentiate between PC and other malignant diseases.

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Sodium chloride promotes tissue inflammation via osmotic stimuli in subtotal-nephrectomized mice

Sakata, F., Ito, Y., Mizuno, M., Sawai, A., Suzuki, Y., Tomita, T., Tawada, M., Tanaka, A., Hirayama, A., Sagara, A., Wada, T., Maruyama, S., Soga, T., Matsuo, S., Imai, E., Takei, Y.

Lab. Invest. 97 ( 4 ) 432 - 446 2017.04

Research paper (scientific journal), Joint Work, Accepted

Chronic inflammation, which is often associated with high all-cause and cardiovascular mortality, is prevalent in patients with renal failure; however, the precise mechanisms remain unclear. High-salt intake was reported to induce lymphangiogenesis and autoimmune diseases via osmotic stimuli with accumulation of sodium or chloride. In addition, sodium was recently reported to be stored in the extremities of dialysis patients. We studied the effects and mechanisms of high salt loading on tissue and systemic inflammation in subtotal-nephrectomized mice (5/6Nx) and in cultured cells. Macrophage infiltration in the peritoneal wall (P<0.001), heart (P<0.05) and para-aortic tissues (P<0.001) was significantly higher in 5/6Nx with salt loading (5/6Nx/NaCl) than in 5/6Nx without salt loading (5/6Nx/Water); however, there were no significant differences in blood pressure and renal function between the groups. Tissue interleukin-6, monocyte chemotactic protein-1 (MCP-1), serum- and glucocorticoid-inducible kinase 1 (Sgk1) and tonicity-responsive enhancer binding protein (TonEBP) mRNA were significantly elevated in the peritoneal wall and heart with 5/6Nx/NaCl when compared with 5/6Nx/Water. Sodium was stored in the abdominal wall, exerting high-osmotic conditions. Reversal of salt loading reduced macrophage infiltration associated with decreased TonEBP in 5/6Nx/NaCl. Macrophage infiltration associated with fibrosis induced by salt loading was decreased in the 5/6Nx/NaCl/CC chemokine receptor 2 (CCR2, receptor of MCP-1)-deficient mice when compared with 5/6Nx/NaCl/Wild mice, suggesting that CCR2 is required for macrophage infiltration in 5/6Nx with NaCl loading. In cultured mesothelial cells and cardiomyocytes, culture media with high NaCl concentration induced MCP-1, Sgk1 and TonEBP mRNA, all of which were suppressed by TonEBP siRNA, indicating that both MCP-1 and Sgk1 are downstream of TonEBP. Our study indicates that high NaCl intake induces MCP-1 expression leading to macrophage infiltration via the TonEBP-MCP-1 pathway in 5/6Nx/NaCl mice, and that TonEBP has a central role in inflammation in patients with renal failure taking high salt.

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A serum metabolomics-based profile in low bone mineral density postmenopausal women

Miyamoto, T., Hirayama, A., Sato, Y., Koboyashi, T., Katsuyama, E., Kanagawa, H., Miyamoto, H., Mori, T., Yoshida, S., Fujie, A., Morita, M., Watanabe, R., Tando, T., Miyamoto, K., Tsuji, T., Funayama, A., Nakamura, M., Matsumoto, M., Soga, T., Tomita, M., Toyama, Y.

Bone 95 1 - 4 2017.02

Research paper (scientific journal), Accepted

Osteoporosis is characterized as a metabolic disorder of bone tissue, and various metabolic markers are now available to support its diagnosis and evaluate treatment effects. Substances produced as end products of metabolomic activities are the correlated factors to the biological or metabolic status, and thus, metabolites are considered highly sensitive markers of particular pathological states, including osteoporosis. Here we undertook comprehensive serum metabolomics analysis in postmenopausal women with or without low bone mineral density (low BMD vs controls) for the first time using capillary electrophoresis/mass spectrometry. Among the metabolites tested, 57 were detected in sera. Levels of hydroxyproline, Gly-Gly and cystine, differed significantly between groups, with Gly-Gly and cystine significantly lower in the low BMD group and hydroxyproline, a reported marker of osteoporosis, significantly higher. Levels of TRACP5b, a bone resorption marker, were significantly higher in the low BMD group, supporting the study's validity. Taken together, our findings represent novel metabolomic profiling in low BMD in postmenopausal women.

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Metabolomic profiling as a possible reverse engineering tool for estimating processing conditions of dry-cured hams

Sugimoto, M., Obiya, S., Kaneko, M., Enomoto, A., Honma, M., Wakayama, M., Soga, T., Tomita, M.

J. Agric. Food Chem 65 ( 2 ) 402 - 410 2017.01

Research paper (scientific journal), Joint Work, Accepted

Dry-cured hams are popular among consumers. To increase the attractiveness of the product, objective analytical methods and algorithms to evaluate the relationship between observable properties and consumer acceptability are required. In this study, metabolomics, which is used for quantitative profiling of hundreds of small molecules, was applied to 12 kinds of dry-cured hams from Japan and Europe. In total, 203 charged metabolites, including amino acids, organic acids, nucleotides, and peptides, were successfully identified and quantified. Metabolite profiles were compared for the samples with different countries of origin and processing methods (e.g., smoking or use of a starter culture). Principal component analysis of the metabolite profiles with sensory properties revealed significant correlations for redness, homogeneity, and fat whiteness. This approach could be used to design new ham products by objective evaluation of various features.

Stimulating S-adenosyl-L-methionine synthesis extends lifespan via activation of AMPK

Ogawa, T., Tsubakiyama, R., Kanai, M., Koyama , T., Fujii, T., Iefuji, H., Soga, T., Kume, K., Miyakawa, T., Hirata, D., Mizunuma, M.

Proc. Natl. Acad. Sci. USA 113 ( 42 ) 11913 - 11918 2016.10

Research paper (scientific journal), Joint Work, Accepted

Dietary restriction (DR), such as calorie restriction (CR) or methionine (Met) restriction, extends the lifespan of diverse model organisms. Although studies have identified several metabolites that contribute to the beneficial effects of DR, the molecular mechanism underlying the key metabolites responsible for DR regimens is not fully understood. Here we show that stimulating S-adenosyl-l-methionine (AdoMet) synthesis extended the lifespan of the budding yeast Saccharomyces cerevisiae The AdoMet synthesis-mediated beneficial metabolic effects, which resulted from consuming both Met and ATP, mimicked CR. Indeed, stimulating AdoMet synthesis activated the universal energy-sensing regulator Snf1, which is the S. cerevisiae ortholog of AMP-activated protein kinase (AMPK), resulting in lifespan extension. Furthermore, our findings revealed that S-adenosyl-l-homocysteine contributed to longevity with a higher accumulation of AdoMet only under the severe CR (0.05% glucose) conditions. Thus, our data uncovered molecular links between Met metabolites and lifespan, suggesting a unique function of AdoMet as a reservoir of Met and ATP for cell survival.

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Metabolic Profiling of Total Physical Activity and Sedentary Behavior in Community-Dwelling Men

Fukai, K., Harada, S., Iida, M., Kuriara, A., Takeuchi, A., Kuwabara, K., Sugiyama, D., Okamura, T., Akiyama, M., Nishiwaki, Y., Oguma, Y., Suzuki, A., Suzuki, C, Hirayama, A., Sugimoto, M., Soga, T.,c Tomita, M., Takebayashi, T.

PLoS One 11 ( 10 ) e0164877 2016.10

Research paper (scientific journal), Joint Work, Accepted

Objective: Physical activity is known to be preventive against various non-communicable diseases. We investigated the relationship between daily physical activity level and plasma metabolites using a targeted metabolomics approach in a population-based study.
Methods: A total of 1,193 participants (male, aged 35 to 74 years) with fasting blood samples were selected from the baseline survey of a cohort study. Information on daily total physical activity, classified into four levels by quartile of metabolic equivalent scores, and sedentary behavior, defined as hours of sitting per day, was collected through a self-administered questionnaire. Plasma metabolite concentrations were quantified by capillary electrophoresis mass spectrometry method. We performed linear regression analysis models with multivariable adjustment and corrected p-values for multiple testing in the original population (n = 808). The robustness of the results was confirmed by replication analysis in a separate population (n = 385) created by random allocation.
Results: Higher levels of total physical activity were associated with various metabolite concentrations, including lower concentrations of amino acids and their derivatives, and higher concentrations of pipecolate (FDR p <0.05 in original population). The findings persisted after adjustment for age, body mass index, smoking, alcohol intake, and energy intake. Isoleucine, leucine, valine, 4-methyl-2-oxoisopentanoate, 2-oxoisopentanoate, alanine, and proline concentrations were lower with a shorter sitting time.
Conclusions: Physical activity is related to various plasma metabolites, including known biomarkers for future insulin resistance or type 2 diabetes. These metabolites might potentially play a key role in the protective effects of higher physical activity and/or less sedentary behavior on non-communicable diseases.

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Index markers of chronic fatigue syndrome with dysfunction of TCA and urea cycles

Yamano, E., Sugimoto, M., Hirayama, A., Kume, S., Yamato, M., Jin, G., Tajima, S., Goda, N., Iwai, K., Fukuda, S., Yamaguti, K, Kuratsune, H., Soga, T., Watanabe, Y., Kataoka, Y.

Sci Rep. 6 34990 2016.10

Research paper (scientific journal), Joint Work, Accepted

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Expression of Idh1R132H in the murine subventricular zone stem cell niche recapitulates features of early gliomagenesis

Bardella C, Al-Dalahmah O, Krell D, Brazauskas P, Al-Qahtani K, Tomkova M, Adam J, Serres S, Lockstone H, Freeman-Mills L, Pfeffer I, Sibson N, Goldin R, Schuster-Böeckler B, Pollard, PJ, Soga T, McCullagh JS, Schofield CJ, Mulholland P, Ansorge O, Kriaucionis S, Ratcliffe PJ, Szele, FG, Tomlinson I

Cancer Cell 30 ( 4 ) 578 - 594 2016.10

Research paper (scientific journal), Joint Work

Isocitrate dehydrogenase 1 mutations drive human gliomagenesis, probably through neomorphic enzyme activity that produces D-2-hydroxyglutarate. To model this disease, we conditionally expressed Idh1R132H in the subventricular zone (SVZ) of the adult mouse brain. The mice developed hydrocephalus and grossly dilated lateral ventricles, with accumulation of 2-hydroxyglutarate and reduced α-ketoglutarate. Stem and transit amplifying/progenitor cell populations were expanded, and proliferation increased. Cells expressing SVZ markers infiltrated surrounding brain regions. SVZ cells also gave rise to proliferative subventricular nodules. DNA methylation was globally increased, while hydroxymethylation was decreased. Mutant SVZ cells overexpressed Wnt, cell-cycle and stem cell genes, and shared an expression signature with human gliomas. Idh1R132H mutation in the major adult neurogenic stem cell niche causes a phenotype resembling gliomagenesis.

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Therapeutic Strategy for Targeting Aggressive Malignant Gliomas by Disrupting Their Energy Balance

Hegazy, A.M., Yamada, D., Kobayashi, M., Kohno, S., Ueno, M., Ali, MA., Ohta, K., Tadokoro, Y., Ino, Y., Todo, T., Soga, T., Takahashi, C., Hirao, A.

J. Biol. Chem. 291 ( 41 ) 21496 - 21509 2016.10

Research paper (scientific journal), Joint Work, Accepted

Although abnormal metabolic regulation is a critical determinant of cancer cell behavior, it is still unclear how an altered balance between ATP production and consumption contributes to malignancy. Here we show that disruption of this energy balance efficiently suppresses aggressive malignant gliomas driven by mammalian target of rapamycin complex 1 (mTORC1) hyperactivation. In a mouse glioma model, mTORC1 hyperactivation induced by conditional Tsc1 deletion increased numbers of glioma-initiating cells (GICs) in vitro and in vivo Metabolic analysis revealed that mTORC1 hyperactivation enhanced mitochondrial biogenesis, as evidenced by elevations in oxygen consumption rate and ATP production. Inhibition of mitochondrial ATP synthetase was more effective in repressing sphere formation by Tsc1-deficient glioma cells than that by Tsc1-competent glioma cells, indicating a crucial function for mitochondrial bioenergetic capacity in GIC expansion. To translate this observation into the development of novel therapeutics targeting malignant gliomas, we screened drug libraries for small molecule compounds showing greater efficacy in inhibiting the proliferation/survival of Tsc1-deficient cells compared with controls. We identified several compounds able to preferentially inhibit mitochondrial activity, dramatically reducing ATP levels and blocking glioma sphere formation. In human patient-derived glioma cells, nigericin, which reportedly suppresses cancer stem cell properties, induced AMPK phosphorylation that was associated with mTORC1 inactivation and induction of autophagy and led to a marked decrease in sphere formation with loss of GIC marker expression. Furthermore, malignant characteristics of human glioma cells were markedly suppressed by nigericin treatment in vivo Thus, targeting mTORC1-driven processes, particularly those involved in maintaining a cancer cell's energy balance, may be an effective therapeutic strategy for glioma patients.

Identification of salivary metabolomic biomarkers for oral cancer screening

Ishikawa, S., Sugimoto, M., Kitabatake, K., Sugano, A., Nakamura, M., Kaneko, M., Ota, S., Hiwatari, K., Enomoto, A., Soga, T., Tomita, M., Iino, M.

Sci. Rep. 6 ( - ) 31520 - - 2016.08

Research paper (scientific journal), Joint Work, Accepted

The objective of this study was to explore salivary metabolite biomarkers by profiling both saliva and tumor tissue samples for oral cancer screening. Paired tumor and control tissues were obtained from oral cancer patients and whole unstimulated saliva samples were collected from patients and healthy controls. The comprehensive metabolomic analysis for profiling hydrophilic metabolites was conducted using capillary electrophoresis time-of-flight mass spectrometry. In total, 85 and 45 metabolites showed significant differences between tumor and matched control samples, and between salivary samples from oral cancer and controls, respectively (P < 0.05 correlated by false discovery rate); 17 metabolites showed consistent differences in both saliva and tissue-based comparisons. Of these, a combination of only two biomarkers yielded a high area under receiver operating characteristic curves (0.827; 95% confidence interval, 0.726-0.928, P < 0.0001) for discriminating oral cancers from controls. Various validation tests confirmed its high generalization ability. The demonstrated approach, integrating both saliva and tumor tissue metabolomics, helps eliminate pseudo-molecules that are coincidentally different between oral cancers and controls. These combined salivary metabolites could be the basis of a clinically feasible method of non-invasive oral cancer screening.

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p38α activates purine metabolism to initiate hematopoietic stem/progenitor cell cycling in response to stress

Karigane, D., Kobayashi, H., Morikawa, T., Ootomo, Y., Sakai, M., Nagamatsu, G., Kubota, Y., Goda, N., Matsumoto, M., Nishimura, E. K., Soga, T., Otsu, K., Suematsu, M., Okamoto, S., Suda, T., Takubo, K.

Cell Stem Cell 19 ( 2 ) 192 - 204 2016.08

Research paper (scientific journal), Joint Work

Hematopoietic stem cells (HSCs) maintain quiescence by activating specific metabolic pathways, including glycolysis. We do not yet have a clear understanding of how this metabolic activity changes during stress hematopoiesis, such as bone marrow transplantation. Here, we report a critical role for the p38MAPK family isoform p38α in initiating hematopoietic stem and progenitor cell (HSPC) proliferation during stress hematopoiesis in mice. We found that p38MAPK is immediately phosphorylated in HSPCs after a hematological stress, preceding increased HSPC cycling. Conditional deletion of p38α led to defective recovery from hematological stress and a delay in initiation of HSPC proliferation. Mechanistically, p38α signaling increases expression of inosine-5'-monophosphate dehydrogenase 2 in HSPCs, leading to altered levels of amino acids and purine-related metabolites and changes in cell-cycle progression in vitro and in vivo. Our studies have therefore uncovered a p38α-mediated pathway that alters HSPC metabolism to respond to stress and promote recovery.

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Mitochondrial Mg2+ homeostasis decides cellular energy metabolism and vulnerability to stress

Yamanaka, R., Tabata, S., Shindo, Y., Hotta, K., Suzuki, K., Soga, T., Oka, K.

Sci. Rep. 6 30027 2016.07

Research paper (scientific journal), Joint Work, Accepted

Cellular energy production processes are composed of many Mg(2+) dependent enzymatic reactions. In fact, dysregulation of Mg(2+) homeostasis is involved in various cellular malfunctions and diseases. Recently, mitochondria, energy-producing organelles, have been known as major intracellular Mg(2+) stores. Several biological stimuli alter mitochondrial Mg(2+) concentration by intracellular redistribution. However, in living cells, whether mitochondrial Mg(2+) alteration affect cellular energy metabolism remains unclear. Mg(2+) transporter of mitochondrial inner membrane MRS2 is an essential component of mitochondrial Mg(2+) uptake system. Here, we comprehensively analyzed intracellular Mg(2+) levels and energy metabolism in Mrs2 knockdown (KD) cells using fluorescence imaging and metabolome analysis. Dysregulation of mitochondrial Mg(2+) homeostasis disrupted ATP production via shift of mitochondrial energy metabolism and morphology. Moreover, Mrs2 KD sensitized cellular tolerance against cellular stress. These results indicate regulation of mitochondrial Mg(2+) via MRS2 critically decides cellular energy status and cell vulnerability via regulation of mitochondrial Mg(2+) level in response to physiological stimuli.

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Serum metabolome profiles characterized by patients with hepatocellular carcinoma associated with hepatitis B and C

Saito, T., Sugimoto, M., Okumoto, K., Haga, H., Katsumi, T., Mizuno, K., Nishina, T., Sato, S., Igarashi, K., Maki, H., Tomita, M., Ueno, Y., Soga, T.

World J. Gastroenterol. 22 ( 27 ) 6224 - 6234 2016.07

Research paper (scientific journal), Joint Work

Aim: To clarify the characteristics of metabolite profiles in virus-related hepatocellular carcinoma (HCC) patients using serum metabolome analysis.
Methods: The serum levels of low-molecular-weight metabolites in 68 patients with HCC were quantified using capillary electrophoresis chromatography and mass spectrometry. Thirty and 38 of the patients suffered from hepatitis B virus-related HCC (HCC-B) and hepatitis C virus-related HCC (HCC-C), respectively.
Results: The main metabolites characteristic of HCC were those associated with glutathione metabolism, notably 13 γ-glutamyl peptides, which are by-products of glutathione induction. Two major profiles, i.e., concentration patterns, of metabolites were identified in HCC patients, and these were classified into two groups: an HCC-B group and an HCC-C group including some of the HCC-B cases. The receiver operating characteristic curve for the multiple logistic regression model discriminating HCC-B from HCC-C incorporating the concentrations of glutamic acid, methionine and γ-glutamyl-glycine-glycine showed a highly significant area under the curve value of 0.94 (95%CI: 0.89-1.0, P < 0.0001).

Conclusion: The serum levels of γ-glutamyl peptides, as well as their concentration patterns, contribute to the development of potential biomarkers for virus-related HCC. The difference in metabolite profiles between HCC-B and HCC-C may reflect the respective metabolic reactions that underlie the different pathogeneses of these two types of HCC.

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Profiling of plasma metabolites in postmenopausal women with metabolic syndrome

Iida, M., Harada, S., Kurihara, A., Fukai, K., Kuwabara, K., Sugiyama, D., Takeuchi, A., Okamura, T., Akiyama, M., Nishiwaki, Y., Suzuki, A., Hirayama, A., Sugimoto, M., Soga, T., Tomita, M., Banno, K., Aoki, D., Takebayashi, T.

Menopause 23 ( 7 ) 749 - 758 2016.07

Research paper (scientific journal), Joint Work, Accepted

Objective: The aim of the study was to investigate the associations of amino acids and other polar metabolites with metabolic syndrome (MetS) in postmenopausal women in a lean Asian population.
Methods: The participants were 1,422 female residents enrolled in a cohort study from April to August 2012. MetS was defined according to the National Cholesterol Education Program Adult Treatment Panel III modified for Japanese women. Associations were examined between MetS and 78 metabolites assayed in fasting plasma samples using capillary electrophoresis-mass spectrometry. Replication analysis was performed to confirm the robustness of the results in a separate population created by random allocation.
Results: Analysis was performed for 877 naturally postmenopausal women, including 594 in the original population and 283 in the replication population. The average age, body mass index, and levels of high- and low-density lipoprotein cholesterol of the entire population were 64.6 years, 23.0 kg/m, 72.1 mg/dL, and 126.1 mg/dL, respectively. There was no significant difference in low-density lipoprotein cholesterol levels between women with and without MetS. Thirteen metabolites were significantly related to MetS: multiple plasma amino acids were elevated in women with MetS, including branched-chain amino acids, alanine, glutamate, and proline; and alpha-aminoadipate, which is generated by lysine degradation, was also significantly increased.
Conclusions: Our large-scale metabolomic profiling indicates that Japanese postmenopausal women with MetS have abnormal polar metabolites, suggesting altered catabolic pathways. These results may help to understand metabolic disturbance, including in persons with normal body mass index and relatively high levels of high-density lipoprotein cholesterol, and may have clinical utility based on further studies.

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p62/Sqstm1 promotes malignancy of HCV-positive hepatocellular carcinoma through Nrf2-dependent metabolic reprogramming

Saito, T., Ichimura, Y., Taguchi, K., Suzuki, T., Mizushima, T., Takagi, K., Hirose, Y., Nagahashi, M., Iso, T., Fukutomi, T., Ohishi, M., Endo, K., Uemura, T., Nishito, Y., Okuda, S., Obata, M., Kouno, T., Imamura, R., Tada, Y., Obata, R., Yasuda, D., Takahashi, K., Fujimura, T., Pi, J., Lee, MS., Ueno, T., Ohe, T., Mashino, T., Wakai, T., Kojima, H., Okabe, T., Nagano, T., Motohashi, H., Waguri, S., Soga, T., Yamamoto, M., Tanaka, K., Komatsu, M.

Nat. Commun. 7 12030 2016.06

Research paper (scientific journal), Joint Work

p62/Sqstm1 is a multifunctional protein involved in cell survival, growth and death, that is degraded by autophagy. Amplification of the p62/Sqstm1 gene, and aberrant accumulation and phosphorylation of p62/Sqstm1, have been implicated in tumour development. Herein, we reveal the molecular mechanism of p62/Sqstm1-dependent malignant progression, and suggest that molecular targeting of p62/Sqstm1 represents a potential chemotherapeutic approach against hepatocellular carcinoma (HCC). Phosphorylation of p62/Sqstm1 at Ser349 directs glucose to the glucuronate pathway, and glutamine towards glutathione synthesis through activation of the transcription factor Nrf2. These changes provide HCC cells with tolerance to anti-cancer drugs and proliferation potency. Phosphorylated p62/Sqstm1 accumulates in tumour regions positive for hepatitis C virus (HCV). An inhibitor of phosphorylated p62-dependent Nrf2 activation suppresses the proliferation and anticancer agent tolerance of HCC. Our data indicate that this Nrf2 inhibitor could be used to make cancer cells less resistant to anticancer drugs, especially in HCV-positive HCC patients.

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Smad2/3 are required for immobilization-induced skeletal muscle atrophy

Tando, T., Hirayama, A., Furukawa, M., Sato, Y., Kobayashi, T., Funayama1, A., Kanaji, A., Hao, W., Watanabe, R., Morita, M., Oike, T., Miyamoto, K., Soga, T., Nomura, M., Yoshimura, A., Tomita, M., Matsumoto, M., Nakamura, M., Toyama, Y., Miyamoto, T.

J. Biol. Chem. 291 ( 23 ) 12184 - 12194 2016.06

Research paper (scientific journal), Joint Work, Accepted

Skeletal muscle atrophy promotes muscle weakness, limiting activities of daily living. However, mechanisms underlying atrophy remain unclear. Here, we show that skeletal muscle immobilization elevates Smad2/3 protein but not mRNA levels in muscle, promoting atrophy. Furthermore, we demonstrate that myostatin, which negatively regulates muscle hypertrophy, is dispensable for denervation-induced muscle atrophy and Smad2/3 protein accumulation. Moreover, muscle-specific Smad2/3-deficient mice exhibited significant resistance to denervation-induced muscle atrophy. In addition, expression of the atrogenes Atrogin-1 and MuRF1, which underlie muscle atrophy, did not increase in muscles of Smad2/3-deficient mice following denervation. We also demonstrate that serum starvation promotes Smad2/3 protein accumulation in C2C12 myogenic cells, an in vitro muscle atrophy model, an effect inhibited by IGF1 treatment. In vivo, we observed IGF1 receptor deactivation in immobilized muscle, even in the presence of normal levels of circulating IGF1. Denervation-induced muscle atrophy was accompanied by reduced glucose intake and elevated levels of branched-chain amino acids, effects that were Smad2/3-dependent. Thus, muscle immobilization attenuates IGF1 signals at the receptor rather than the ligand level, leading to Smad2/3 protein accumulation, muscle atrophy, and accompanying metabolic changes.

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Effects of fractionated radiation on murine glioma stem cell metabolism

Fujita, S., Osuka, S. Shibao, S., Igarashi, K., Soga, T., Sampetrean, O.

Toho J. Med. 2 ( 2 ) 45 - 52 2016.06

Research paper (scientific journal), Joint Work, Accepted

Background: Glioma stem cells (GSCs) have an important role in tumor recurrence after treatment. We recently showed that, in a mouse model of glioma, GSCs adapt to repeated radiation exposures by changing
their secretory profile and modulating survival signaling. Irradiated GSCs also reduce their rate of proliferation. In the present study we examined whether these changes require a specific adaptation of GSC energy
metabolism.
Methods: Initial tumors were established in mice by orthotopic implantation of Ink4a/Arf-null neural stem
cells transduced with human HRASV12. GSCs isolated from the tumors were exposed (or not) to fractionated
radiation (12 fractions of 5 Gy), and five clones in each group of cells were assessed for glucose consumption,
lactate production, and content of intracellular metabolites.
Results: Rates of glucose consumption and lactate production were lower in clones exposed to radiation
than in nonirradiated clones, which suggests a reduction in glycolysis. Among intracellular metabolites, the
high concentrations of intermediates of glycolysis and nucleoside metabolism were reduced in irradiated
clones, whereas levels of essential amino acids were largely unchanged.
Conclusions: Repeated radiation changes the metabolic preferences of GSCs, in conjunction with their
slowed proliferation. Future studies should thus investigate intra- and extracellular metabolites, to identify
potential diagnostic and therapeutic targets for glioma recurrence.

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Identification of biomarkers for development of end-stage kidney disease in chronic kidney disease by metabolomic profiling

Kimura, T., Yasuda, K., Yamamoto, R., Soga, T., Rakugi, H., Hayashi, T., Isaka, Y.

Sci. Rep. 6 ( - ) 26138 - - 2016.05

Research paper (scientific journal), Joint Work

A critical issue in the management of chronic kidney disease (CKD) is to prevent patients from the progression to end-stage kidney disease (ESKD), however, there is only limited number of biomarkers for the discrimination of the high-risk CKD patients. We aimed to identify the metabolites which possess the ability to predict the earlier kidney deterioration. We performed capillary electrophoresis and liquid chromatography mass spectrometry (CE-MS)-based metabolic profiling in a prospective cohort, which consisted of referred 112 CKD patients with median follow-up period of 4.4 years. The association between the levels of candidate metabolites and the outcomes (progression to ESKD alone or in combination with death before ESKD) were assessed by multivariate Cox proportional hazard models after adjusting for the baseline covariates. A total of 218 metabolites were detected in the plasma of CKD patients. We identified 16 metabolites which have predictive values for the composite outcome: The risk for composite outcome was elevated from 2.0- to 8.0-fold in those with higher levels of 16 plasma metabolites. Our results suggest that the measurement of these metabolites may facilitate CKD management by predicting the risk of progression to ESKD.

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Metabolomic profiling reveals novel biomarkers of alcohol intake and alcohol-induced liver injury in community-dwelling men

Harada S., Takebayashi T., Kurihara A., Akiyama M., Suzuki A., Hatakeyama Y., Sugiyama D., Kuwabara K., Takeuchi A., Okamura T., Nishiwaki Y., Tanaka T., Hirayama A., Sugimoto M., Soga T., Tomita M.

Environ. Health Prev. Med. 21 ( 1 ) 18(283) - 26(284) 2016.01

Research paper (scientific journal), Joint Work, Accepted

Objective: Metabolomics is a promising approach to the identification of biomarkers in plasma. Here, we performed a population-based, cross-sectional study to identify potential biomarkers of alcohol intake and alcohol-induced liver injury by metabolomic profiling using capillary electrophoresis-mass spectrometry (CE-MS).
Methods: Fasting plasma samples were collected from 896 Japanese men who participated in the baseline survey of the Tsuruoka Metabolomics Cohort Study, and 115 polar metabolites were identified and absolutely quantified by CE-MS. Information on daily ethanol intake was collected through a standardized, self-administered questionnaire. The associations between ethanol intake and plasma concentration of metabolites were examined. Relationships between metabolite concentrations or their ratios and serum liver enzyme levels in the highest ethanol intake group (>46.0 g/day) were then examined by linear regression analysis. Replication analysis was conducted in 193 samples collected from independent population of this cohort.
Results: Nineteen metabolites were identified to have an association with daily alcohol consumption both in the original and replication population. Three of these metabolites (threonine, glutamine, and guanidinosuccinate) were found to associate well with elevated levels of serum liver enzymes in the highest ethanol intake group, but not in the non-drinker group. We also found that the glutamate/glutamine ratio had a much stronger relation to serum γ-glutamyltransferase, aspartate transaminase, and alanine transaminase than glutamate or glutamine alone (standardized beta = 0.678, 0.558, 0.498, respectively).
Conclusions: We found 19 metabolites associated with alcohol intake, and three biomarker candidates (threonine, guanidinosuccinate and glutamine) of alcohol-induced liver injury. Glutamate/glutamine ratio might also be good biomarker.

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Effects of 3-styrylchromones on metabolic profiles and cell death in oral squamous cell carcinoma cells

Sakagami, H., Shimada, C., Kanda, Y., Amano, O., Sugimoto, M., Ota S., Soga, T., Tomita, M., Sato, A., Tanuma, S., Takao, K., Sugita, Y.

Toxicol. Rep. 2 1281 - 1290 2015.12

Research paper (scientific journal), Joint Work, Accepted

4H-1-benzopyran-4-ones (chromones) are important naturally-distributing compounds. As compared with flavones, isoflavones and 2-styrylchromones, there are only few papers of 3-styrylchromones that have been published. We have previously reported that among fifteen 3-styrylchromone derivatives, three new synthetic compounds that have OCH3 group at the C-6 position of chromone ring, (E)-3-(4-hydroxystyryl)-6-methoxy-4H-chromen-4-one (compound 11), (E)-6-methoxy-3-(4-methoxystyryl)-4H-chromen-4-one (compound 4), (E)-6-methoxy-3-(3,4,5-trimethoxystyryl)-4H-chromen-4-one (compound 6) showed much higher cytotoxicities against four epithelial human oral squamous cell carcinoma (OSCC) lines than human normal oral mesenchymal cells. In order to further confirm the tumor specificities of these compounds, we compared their cytotoxicities against both human epithelial malignant and non-malignant cells, and then investigated their effects on fine cell structures and metabolic profiles and cell death in human OSCC cell line HSC-2. Cytotoxicities of compounds 4, 6, 11 were assayed with MTT method. Fine cell structures were observed under transmission electron microscope. Cellular metabolites were extracted with methanol and subjected to CE-TOFMS analysis. Compounds 4, 6, 11 showed much weaker cytotoxicity against human oral keratinocyte and primary human gingival epithelial cells, as compared with HSC-2, confirming their tumor-specificity, whereas doxorubicin and 5-FU were highly cytotoxic to these normal epithelial cells, giving unexpectedly lower tumor-specificity. The most cytotoxic compound 11, induced the mitochondrial vacuolization, autophagy suppression followed by apoptosis induction, and changes in the metabolites involved in amino acid and glycerophospholipid metabolisms. Chemical modification of lead compound 11 may be a potential choice for designing new type of anticancer drugs.

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Effects of processing and storage conditions on charged metabolomic profiles in blood

Hirayama, A., Sugimoto, M., Suzuki, A., Hatakeyama, Y., Enomoto, A., Harada, S., Soga, T., Tomita, M., Takebayashi, T.

Electrophoresis 36 ( 18 ) 2148 - 2155 2015.09

Research paper (scientific journal), Joint Work, Accepted

The development of high-throughput metabolite measurement technologies has enabled the use of metabolomics for epidemiologic studies by profiling metabolite concentrations in large cohorts of human blood samples. Standard protocols are necessary to obtain unbiased profiles through multiple runs over long periods of time and to allow reliable statistical analyses. This study assessed the effects of sampling procedures and storage conditions on the stability of metabolomic profiles in plasma and serum. Charged metabolomic profiles were determined by capillary electrophoresis-mass spectrometry (CE-MS) and compared by multivariate analyses. The effects of pre-analytical procedures, including times for clotting and incubation of serum and plasma, respectively; incubation temperatures; and number of freeze-thaw cycles, were assessed. Overall, inter-individual differences in profiles were larger than intra-individual differences, and profiles in plasma showed better stability than those in serum. These quantified datasets of metabolites, along with their stability and variation, may help in interpreting data from long-term cohort studies.

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Metabolomic alterations in human cancer cells by vitamin C-induced oxidative stress

Uetaki, M., Tabata, S., Nakasuka, F., Soga, T., Tomita, M.

Sci. Rep. 5 ( - ) 13896 - - 2015.09

Research paper (scientific journal), Joint Work, Accepted

Intravenous administration of high-dose vitamin C has recently attracted attention as a cancer therapy. High-dose vitamin C induces pro-oxidant effects and selectively kills cancer cells. However, the anticancer mechanisms of vitamin C are not fully understood. Here, we analyzed metabolic changes induced by vitamin C in MCF7 human breast adenocarcinoma and HT29 human colon cancer cells using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). The metabolomic profiles of both cell lines were dramatically altered after exposure to cytotoxic concentrations of vitamin C. Levels of upstream metabolites in the glycolysis pathway and tricarboxylic acid (TCA) cycle were increased in both cell lines following treatment with vitamin C, while adenosine triphosphate (ATP) levels and adenylate energy charges were decreased concentration-dependently. Treatment with N-acetyl cysteine (NAC) and reduced glutathione (GSH) significantly inhibited vitamin C-induced cytotoxicity in MCF7 cells. NAC also suppressed vitamin C-dependent metabolic changes, and NAD treatment prevented vitamin C-induced cell death. Collectively, our data suggests that vitamin C inhibited energy metabolism through NAD depletion, thereby inducing cancer cell death.

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Acquisition of chemoresistance to gemcitabine is induced by a loss-of-function missense mutation of DCK

Nakano, T., Saiki, Y., Kudo, C., Hirayama, A., Mizuguchi, Y., Fujiwara, S., Soga, T., Sunamura, M., Matsumura, N., Motoi, F., Unno, M., Horii, A.

Biochem. Biophys. Res. Commun. 464 ( 4 ) 1084 - 1089 2015.09

Research paper (scientific journal), Joint Work, Accepted

The anti-tumor activity of gemcitabine (GEM) has been clinically proven in several solid tumors, including pancreatic cancer, biliary tract cancer, urinary bladder cancer, and non-small cell lung cancer. However, problems remain with issues such as acquisition of chemoresistance against GEM. GEM is activated after phosphorylation by deoxycytidine kinase (DCK) inside of the cell; thus, DCK inactivation is one of the important mechanisms for acquisition of GEM resistance. We previously investigated the DCK gene in multiple GEM resistant cancer cell lines and identified frequent inactivating mutations. In this study, we identified two crucial genetic alteration in DCK. (1) A total deletion of DCK in RTGBC1-TKB, an acquired GEM resistant cell line derived from a gall bladder cancer cell line TGBC1-TKB. (2) An E197K missense alteration of DCK in MKN28, a gastric cancer cell line; its acquired GEM resistant cancer cell line, RMKN28, showed a loss of the normal E197 allele. We introduced either normal DCK or altered DCK_E197K into RMKN28 and proved that only the introduction of normal DCK restored GEM sensitivity. Furthermore, we analyzed 104 healthy volunteers and found that none of them carried the same base substitution observed in MKN28. These results strongly suggest that (1) the E197K alteration in DCK causes inactivation of DCK, and that (2) loss of the normal E197 allele is the crucial mechanism in acquisition of GEM resistance in RMKN28.

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Alteration of the Intestinal Environment by Lubiprostone Is Associated with Amelioration of Adenine-Induced CKD

Mishima, E., Fukuda, S., Shima, H., Hirayama, A., Akiyama, Y., Takeuchi, Y., Fukuda, N. N., Suzuki, T., Suzuki, C., Yuri, A., Kikuchi, K., Tomioka, Y., Ito, S., Soga, T., Abe, T.

J. Am. Soc. Nephrol. 26 ( 8 ) 1787 - 1794 2015.08

Research paper (scientific journal), Joint Work, Accepted

The accumulation of uremic toxins is involved in the progression of CKD. Various uremic toxins are derived from gut microbiota, and an imbalance of gut microbiota or dysbiosis is related to renal failure. However, the pathophysiologic mechanisms underlying the relationship between the gut microbiota and renal failure are still obscure. Using an adenine-induced renal failure mouse model, we evaluated the effects of the ClC-2 chloride channel activator lubiprostone (commonly used for the treatment of constipation) on CKD. Oral administration of lubiprostone (500 µg/kg per day) changed the fecal and intestinal properties in mice with renal failure. Additionally, lubiprostone treatment reduced the elevated BUN and protected against tubulointerstitial damage, renal fibrosis, and inflammation. Gut microbiome analysis of 16S rRNA genes in the renal failure mice showed that lubiprostone treatment altered their microbial composition, especially the recovery of the levels of the Lactobacillaceae family and Prevotella genus, which were significantly reduced in the renal failure mice. Furthermore, capillary electrophoresis-mass spectrometry-based metabolome analysis showed that lubiprostone treatment decreased the plasma level of uremic toxins, such as indoxyl sulfate and hippurate, which are derived from gut microbiota, and a more recently discovered uremic toxin, trans-aconitate. These results suggest that lubiprostone ameliorates the progression of CKD and the accumulation of uremic toxins by improving the gut microbiota and intestinal environment.

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Alteration of metabolomic profiles by titanium dioxide nanoparticles in human gingivitis model

Garcia-Contreras, R., Sugimoto, M., Umemura, N., Kaneko, M., Hatakeyama, Y., Soga, T., Tomita, M., Scougall-Vilchis, R.J., Contreras-Bulnes, R., Nakajima, H., Sakagami, H.

Biomaterials 57 33 - 40 2015.07

Research paper (scientific journal), Joint Work, Accepted

Although nanoparticles (NPs) has afforded considerable benefits in various fields of sciences, several reports have shown their harmful effects, suggesting the necessity of adequate risk assessment. To clarify the mechanism of titanium dioxide nanoparticles (TiO2 NPs)-enhanced gingival inflammation, we conducted the full-scale metabolomic analyses of human gingival fibroblast cells treated with IL-1β alone or in combination with TiO2 NPs. Observation with transmission electron microscope demonstrated the incorporation of TiO2 NPs into vacuoles of the cells. TiO2 NPs significantly enhanced the IL-1β-induced prostaglandin E2 production and COX-1 and COX-2 protein expression. IL-1β reduced the intracellular concentrations of overall primary metabolites especially those of amino acid, urea cycle, polyamine, S-adenosylmethione and glutathione synthetic pathways. The addition of TiO2 NPs further augmented these IL-1β-induced metabolic changes, recommending careful use of dental materials containing TiO2 NPs towards patients with gingivitis or periodontitis. The impact of the present study is to identify the molecular targets of TiO2 NPs for the future establishment of new metabolic markers and therapeutic strategy of gingival inflammation.

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IDH2 and NPM1 mutations cooperate to activate Hoxa9/Meis1 and hypoxia pathways in acute myeloid leukemia

Ogawara, Y., Katsumoto, T., Aikawa, Y., Shima, Y., Kagiyama, Y., Soga, T., Matsunaga, H., Seki, T., Araki, K., Kitabayashi, I.

Cancer Res. 75 ( 10 ) 2005 - 2016 2015.05

Research paper (scientific journal), Joint Work, Accepted

IDH1 and IDH2 mutations occur frequently in acute myeloid leukemia (AML) and other cancers. The mutant isocitrate dehydrogenase (IDH) enzymes convert α-ketoglutarate (α-KG) to the oncometabolite 2-hydroxyglutarate (2-HG), which dysregulates a set of α-KG-dependent dioxygenases. To determine whether mutant IDH enzymes are valid targets for cancer therapy, we created a mouse model of AML in which mice were transplanted with nucleophosmin1 (NPM)(+/-) hematopoietic stem/progenitor cells cotransduced with four mutant genes (NPMc, IDH2/R140Q, DNMT3A/R882H, and FLT3/ITD), which often occur simultaneously in human AML patients. Conditional deletion of IDH2/R140Q blocked 2-HG production and maintenance of leukemia stem cells, resulting in survival of the AML mice. IDH2/R140Q was necessary for the engraftment or survival of NPMc(+) cells in vivo. Gene expression analysis indicated that NPMc increased expression of Hoxa9. IDH2/R140Q also increased the level of Meis1 and activated the hypoxia pathway in AML cells. IDH2/R140Q decreased the 5hmC modification and expression of some differentiation-inducing genes (Ebf1 and Spib). Taken together, our results indicated that IDH2 mutation is critical for the development and maintenance of AML stem-like cells, and they provided a preclinical justification for targeting mutant IDH enzymes as a strategy for anticancer therapy.
©2015 American Association for Cancer Research.

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Decreased miR122 in hepatocellular carcinoma leads to chemoresistance with increased arginine

Kishikawa, T., *Otsuka, M., Tan, P., Ohno, M., Sun, X., Yoshikawa, T., Shibata, C., Takata, A., Kojima, K., Takehana, K., Ohishi, M., Ota, S., Noyama, T., Kondo, Y., Sato, M., Soga, T., Hoshida, Y., Koike, K.

Oncotarget 6 ( 10 ) 8339 - 8352 2015.04

Research paper (scientific journal), Joint Work

Reduced expression of microRNA122 (miR122), a liver-specific microRNA, is frequent in hepatocellular carcinoma (HCC). However, its biological significances remain poorly understood. Because deregulated amino acid levels in cancers can affect their biological behavior, we determined the amino acid levels in miR122-silenced mouse liver tissues, in which intracellular arginine levels were significantly increased. The increased intracellular arginine levels were through upregulation of the solute carrier family 7 (SLC7A1), a transporter of arginine and a direct target of miR122. Arginine is the substrate for nitric oxide (NO) synthetase, and intracellular NO levels were increased in miR122-silenced HCC cells, with increased resistance to sorafenib, a multikinase inhibitor. Conversely, maintenance of the miR122-silenced HCC cells in arginine-depleted culture media, as well as overexpression of miR122 in miR122-low-expressing HCC cells, reversed these effects and rendered the cells more sensitive to sorafenib. Using a reporter knock-in construct, chemical compounds were screened, and Wee1 kinase inhibitor was identified as upregulators of miR122 transcription, which increased the sensitivity of the cells to sorafenib. These results provide an insight into sorafenib resistance in miR122-low HCC, and suggest that arginine depletion or a combination of sorafenib with the identified compound may provide promising approaches to managing this HCC subset.

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Cystathionine is a novel substrate of cystine/glutamate transporter: implications for immune function

Kobayashi, S., Sato, M., Kasakoshi, T., Tsutsui, T., Sugimoto, M., Osaki, M., Okada, F., Igarashi, K., Hiratake, J., Homma, T., Conrad, M., Fujii, J., Soga, T., Bannai, S., *Sato, H.

J. Biol. Chem. 290 ( 14 ) 8778 - 8788 2015.04

Research paper (scientific journal), Joint Work

The cystine/glutamate transporter, designated as system xc(-), is important for maintaining intracellular glutathione levels and extracellular redox balance. The substrate-specific component of system xc(-), xCT, is strongly induced by various stimuli, including oxidative stress, whereas it is constitutively expressed only in specific brain regions and immune tissues, such as the thymus and spleen. Although cystine and glutamate are the well established substrates of system xc(-) and the knockout of xCT leads to alterations of extracellular redox balance, nothing is known about other potential substrates. We thus performed a comparative metabolite analysis of tissues from xCT-deficient and wild-type mice using capillary electrophoresis time-of-flight mass spectrometry. Although most of the analyzed metabolites did not show significant alterations between xCT-deficient and wild-type mice, cystathionine emerged as being absent specifically in the thymus and spleen of xCT-deficient mice. No expression of either cystathionine β-synthase or cystathionine γ-lyase was observed in the thymus and spleen of mice. In embryonic fibroblasts derived from wild-type embryos, cystine uptake was significantly inhibited by cystathionine in a concentration-dependent manner. Wild-type cells showed an intracellular accumulation of cystathionine when incubated in cystathionine-containing buffer, which concomitantly stimulated an increased release of glutamate into the extracellular space. By contrast, none of these effects could be observed in xCT-deficient cells. Remarkably, unlike knock-out cells, wild-type cells could be rescued from cystine deprivation-induced cell death by cystathionine supplementation. We thus conclude that cystathionine is a novel physiological substrate of system xc(-) and that the accumulation of cystathionine in immune tissues is exclusively mediated by system xc(-).

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Potential Biomarkers of Fatigue Identified by Plasma Metabolome Analysis in Rats

Kume, S., Yamato, M., Tamura, Y., Jin, G., Nakano, M., Miyashige, Y., Eguchi, A., Ogata, Y., Goda, N., Iwai, K., Yamano, E., Watanabe, Y., Soga, T., *Kataoka, Y.

PLoS ONE 10 ( 3 ) e0120106 2015.03

Research paper (scientific journal), Joint Work

In the present study, prior to the establishment of a method for the clinical diagnosis of chronic fatigue in humans, we validated the utility of plasma metabolomic analysis in a rat model of fatigue using capillary electrophoresis-mass spectrometry (CE-MS). In order to obtain a fatigued animal group, rats were placed in a cage filled with water to a height of 2.2 cm for 5 days. A food-restricted group, in which rats were limited to 10 g/d of food (around 50% of the control group), was also assessed. The food-restricted group exhibited weight reduction similar to that of the fatigued group. CE-MS measurements were performed to evaluate the profile of food intake-dependent metabolic changes, as well as the profile in fatigue loading, resulting in the identification of 48 metabolites in plasma. Multivariate analyses using hierarchical clustering and principal component analysis revealed that the plasma metabolome in the fatigued group showed clear differences from those in the control and food-restricted groups. In the fatigued group, we found distinctive changes in metabolites related to branched-chain amino acid metabolism, urea cycle, and proline metabolism. Specifically, the fatigued group exhibited significant increases in valine, leucine, isoleucine, and 2-oxoisopentanoate, and significant decreases in citrulline and hydroxyproline compared with the control and food-restricted groups. Plasma levels of total nitric oxide were increased in the fatigued group, indicating systemic oxidative stress. Further, plasma metabolites involved in the citrate cycle, such as cis-aconitate and isocitrate, were reduced in the fatigued group. The levels of ATP were significantly decreased in the liver and skeletal muscle, indicative of a deterioration in energy metabolism in these organs. Thus, this comprehensive metabolic analysis furthered our understanding of the pathophysiology of fatigue, and identified potential diagnostic biomarkers based on fatigue pathophysiology.

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Hypoxia Induces a Lipogenic Cancer Cell Phenotype via HIF1α-Dependent and -Independent Pathways

*Valli, A., Rodriguez, M., Moutsianas, L., Fischer, R., Fedele, V., Huang, H.L., Van Stiphout, R., Jones, D., Mccarthy, M., Vinaxia, M., Igarashi, K., Sato, M., Soga, T., Buffa, F., Mccullagh, J., Yanes, O., Harris, A., Kessler. B.

Oncotarget 6 ( 4 ) 1920 - 1941 2015.02

Research paper (scientific journal), Joint Work, Accepted

The biochemistry of cancer cells diverges significantly from normal cells as a result of a comprehensive reprogramming of metabolic pathways. A major factor influencing cancer metabolism is hypoxia, which is mediated by HIF1α and HIF2α. HIF1α represents one of the principal regulators of metabolism and energetic balance in cancer cells through its regulation of glycolysis, glycogen synthesis, Krebs cycle and the pentose phosphate shunt. However, less is known about the role of HIF1α in modulating lipid metabolism. Lipids serve cancer cells to provide molecules acting as oncogenic signals, energetic reserve, precursors for new membrane synthesis and to balance redox biological reactions. To study the role of HIF1α in these processes, we used HCT116 colorectal cancer cells expressing endogenous HIF1α and cells in which the hif1α gene was deleted to characterize HIF1α-dependent and independent effects on hypoxia regulated lipid metabolites. Untargeted metabolomics integrated with proteomics revealed that hypoxia induced many changes in lipids metabolites. Enzymatic steps in fatty acid synthesis and the Kennedy pathway were modified in a HIF1α-dependent fashion. Palmitate, stearate, PLD3 and PAFC16 were regulated in a HIF-independent manner. Our results demonstrate the impact of hypoxia on lipid metabolites, of which a distinct subset is regulated by HIF1α.

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Development of quantitative method for determination of γ-glutamyl peptides by capillary electrophoresis tandem mass spectrometry: An efficient approach avoiding matrix effect

Hirayama, A., Igarashi, K., Tomita, M., *Soga, T.

J Chromatogr A. 1369 161 - 169 2014.11

Research paper (scientific journal), Joint Work, Accepted

Serum γ-glutamyl di- and tripeptides have proven to be useful biomarkers to accurately predict nine different forms of liver disease. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with multiple reaction monitoring (MRM), serum and liver samples spiked with γ-glutamyl peptide standards were analyzed to estimate accuracy. Unexpectedly, the recovery rates for several γ-glutamyl peptides in the serum samples were quite low, whereas values for some γ-glutamyl peptides in the liver samples were highly elevated. Most of these peptides were barely retained on the reverse-phase column, resulting in significant ion suppression or enhancement. In contrast, a capillary electrophoresis tandem mass spectrometry (CE-MS/MS) method with MRM was minimally affected by matrix effects. Of the 39 tested compounds, most of γ-glutamyl peptides that did not contain a thiol substituent in its structure gave acceptable recoveries (70-120%), and limits of detection for the analytes were between 3.6 and 800 nmol/l with pressure injection at 5 kPa for 10 s (ca. 10 nl). The CE-MS/MS method provided high resolution and proved to be highly selective and sensitive, its utility being demonstrated by the determination of γ-glutamyl di- and tripeptides in serum and liver samples.

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Thymidine phosphorylase activates NFκB and stimulates the expression of angiogenic and metastatic factors in human cancer cells

Tabata, S., Ikeda, R., Yamamoto, M., Shimaoka, S., Mukaida, N., Takeda, Y., Yamada, K., Soga, T., Furukawa, T., Akiyama, S.

Oncotarget 5 ( 21 ) 10473 - 10485 2014.11

Research paper (scientific journal), Joint Work, Accepted

Thymidine phosphorylase (TP) promotes angiogenesis and metastasis, and confers resistance to anticancer agents in some cancer cell types. We previously reported that TP stimulates the expression of interleukin (IL)-8 in human KB cancer cells by an unknown mechanism. A mutation in the nuclear factor (NF)κB binding site of the IL-8 promoter suppressed promoter activity in KB/TP cells that overexpress TP. Specifically inhibiting NFκB by using BY11-7082 also suppressed TP-induced IL-8 promoter activity and IL-8 expression. Moreover, TP overexpression led to the activation of NFκB and an upregulation in the expression of its target genes, and increased phosphorylated IKKα/β protein levels, while promoting IκBα degradation as well as p65 phosphorylation and nuclear localization. The activation of NFκB in KB/TP cells was suppressed by the antioxidants N-acetylcysteine and EUK-8. In addition, in gastric cancer tissue samples, the expression of the NFκB-regulated genes, including IL-8, IL-6, and fibronectin-1 was positively correlated with TP expression. These findings indicate that reactive oxygen species mediated NFκB activation by TP increases the expression of genes that promote angiogenesis and metastasis in gastric cancer.

Mass spectrometric analysis of L-cysteine metabolism: physiological role and fate of L-cysteine in the enteric protozoan parasite Entamoeba histolytica

Jeelani, G., Sato, D., Soga, T., Watanabe, H., *Nozaki, T.

mBio 5 ( 6 ) e01995-14 2014.11

Research paper (scientific journal), Joint Work

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Disturbed biopterin and folate metabolism in the Qdpr-deficient mouse

Xu, F., Sudo, Y., Sanechika, S., Yamashita, J., Shimaguchi, S., Honda, S., Sumi-Ichinose, C., Mori-Kojima, M., Nakata, R., Furuta, T., Sakurai, M., Sugimoto, M., Soga, T., Kondo, K., Ichinose, H.

FEBS Lett. 588 3924-3931 2014.11

Joint Work

Quinonoid dihydropteridine reductase (QDPR) catalyzes the regeneration of tetrahydrobiopterin (BH4), a cofactor for monoamine synthesis, phenylalanine hydroxylation and nitric oxide production. Here, we produced and analyzed a transgenic Qdpr(-/-) mouse model. Unexpectedly, the BH4 contents in the Qdpr(-/-) mice were not decreased and even increased in some tissues, whereas those of the oxidized form dihydrobiopterin (BH2) were significantly increased. We demonstrated that unlike the wild-type mice, dihydrofolate reductase regenerated BH4 from BH2 in the mutants. Furthermore, we revealed wide alterations in folate-associated metabolism in the Qdpr(-/-) mice, which suggests an interconnection between folate and biopterin metabolism in the transgenic mouse model.

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Conformational Change in Transfer RNA Is an Early Indicator of Acute Cellular Damage

Mishima, E., Inoue, C., Saigusa, D., Inoue, R., Ito, K., Suzuki, Y., Jinno, D., Tsukui, Y., Akamatsu, Y., Araki, M., Araki, K., Shimizu, R., Shinke, H., Suzuki, T., Takeuchi, Y., Shima, H., Akiyama, Y., Toyohara, T., Suzuki, C., Saiki, Y., Tominaga, T., Miyagi, S., Kawagisihi, N., Soga, T., Ohkubo, T., Yamamura, K., Imai, Y., Masuda, S., Sabbisetti, V., Ichimura, T., Mount, D.B., Bonventre, J.V., Ito, S., Tomioka, Y., Itoh, K., *Abe, T.

J. Am. Soc. Nephrol. 25 ( 10 ) 2316 - 2326 2014.10

Research paper (scientific journal), Joint Work

Tissue damage by oxidative stress is a key pathogenic mechanism in various diseases, including AKI and CKD. Thus, early detection of oxidative tissue damage is important. Using a tRNA-specific modified nucleoside 1-methyladenosine (m1A) antibody, we show that oxidative stress induces a direct conformational change in tRNA structure that promotes subsequent tRNA fragmentation and occurs much earlier than DNA damage. In various models of tissue damage (ischemic reperfusion, toxic injury, and irradiation), the levels of circulating tRNA derivatives increased rapidly. In humans, the levels of circulating tRNA derivatives also increased under conditions of acute renal ischemia, even before levels of other known tissue damage markers increased. Notably, the level of circulating free m1A correlated with mortality in the general population (n=1033) over a mean follow-up of 6.7 years. Compared with healthy controls, patients with CKD had higher levels of circulating free m1A, which were reduced by treatment with pitavastatin (2 mg/d; n=29). Therefore, tRNA damage reflects early oxidative stress damage, and detection of tRNA damage may be a useful tool for identifying organ damage and forming a clinical prognosis.

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Metabolomic Analysis of the Effects of Omeprazole and Famotidine on Aspirin-induced Gastric Injury

Takeuchi, K., Ohishi, M., Endo, K., Suzumura, K., Naraoka, H., Ohata, T., Seki, J., Miyamae, Y., Honma, M., Soga, T.

Metabolomics 10 ( 5 ) 995 - 1004 2014.10

Research paper (scientific journal), Joint Work, Accepted

Gastric mucosal ulceration and gastric hemorrhage are frequently associated with treatment by non-steroid anti-inflammatory drugs (NSAIDs); however, no convenient biomarker-based diagnostic methods for these adverse reactions are currently available, requiring the use of endoscopic evaluation. We recently reported five biomarker candidates in serum which predict gastric injury induced by NSAIDs in rats, but were unable to clarify the mechanism of change in the levels of these biomarker candidates. In this study, we performed capillary electrophoresis–mass spectrometry-based metabolomic profiling in stomach and serum from rats in which gastric ulcer was induced by aspirin and prevented by co-administration of omeprazole and famotidine. Results showed drug-induced decreases in the levels of citrate, cis-aconitate, succinate, 3-hydroxy butanoic acid, and O-acetyl carnitine in all animals administered aspirin. In contrast, aspirin-induced decreases in the level of 4-hydroxyproline were suppressed by co-administration of omeprazole and famotidine. We consider that these changes were due to the prevention of gastric ulcer and decrease in the amount of collagen in stomach tissue by omeprazole and famotidine, without prevention of the NSAID-induced depression of mitochondrial function. In addition, the decreases in 4-hydroxyproline in the stomach was also detectable as changes in the serum. While further study is needed to clarify limitations of indications and extrapolation to humans, this new serum biomarker candidate of gastric injury may be useful in the monitoring of NSAID-induced tissue damage.

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Metabolome analysis based on capillary electrophoresis-mass spectrometry

Hirayama, A., Wakayama, M., Soga, T.

Trends Anal. Chem. 61 215 - 222 2014.10

Research paper (scientific journal), Joint Work, Accepted

Capillary electrophoresis-mass spectrometry (CE-MS) has emerged as a powerful new tool for comprehensive analysis of charged compounds. In this review, we provide a general description of the application of CE-MS in metabolome analysis, including the separation modes of CE, various interfaces and the mass spectrometers used. We also discuss strategies for sample pretreatment, data processing and peak identification, which are important processes in metabolome analysis. In addition, we highlight a number of new techniques to improve metabolite extraction, peak resolution and sensitivity. Finally, we provide some general conclusions and future perspectives.

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Prolyl hydroxylase domain enzymes: important regulators of cancer metabolism

Yang, M., Su, H., Soga, T., Kranc, K.R., Pollard, P.J.

Hypoxia 2 127 - 142 2014.08

Research paper (scientific journal), Joint Work, Accepted

The hypoxia-inducible factor (HIF) prolyl hydroxylase domain enzymes (PHDs) regulate the stability of HIF protein by post-translational hydroxylation of two conserved prolyl residues in its α subunit in an oxygen-dependent manner. Trans-4-prolyl hydroxylation of HIFα under normal oxygen (O2) availability enables its association with the von Hippel-Lindau (VHL) tumor suppressor pVHL E3 ligase complex, leading to the degradation of HIFα via the ubiquitin-proteasome pathway. Due to the obligatory requirement of molecular O2 as a co-substrate, the activity of PHDs is inhibited under hypoxic conditions, resulting in stabilized HIFα, which dimerizes with HIFβ and, together with transcriptional co-activators CBP/p300, activates the transcription of its target genes. As a key molecular regulator of adaptive response to hypoxia, HIF plays important roles in multiple cellular processes and its overexpression has been detected in various cancers. The HIF1α isoform in particular has a strong impact on cellular metabolism, most notably by promoting anaerobic, whilst inhibiting O2-dependent, metabolism of glucose. The PHD enzymes also seem to have HIF-independent functions and are subject to regulation by factors other than O2, such as by metabolic status, oxidative stress, and abnormal levels of endogenous metabolites (oncometabolites) that have been observed in some types of cancers. In this review, we aim to summarize current understandings of the function and regulation of PHDs in cancer with an emphasis on their roles in metabolism.

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Fetal Growth Retardation and Lack of Hypotaurine in Ezrin Knockout Mice

Nishimura, T., Higuchi, K., Sai, Y., Sugita, Y., Yoshida, Y., Tomi, M., Wada, M., Wakayama, T., Tamura, A., Tsukita, S., Soga, T., and Nakashima, E.

PLoS ONE 9 ( 8 ) e105423 2014.08

Research paper (scientific journal), Joint Work, Accepted

Ezrin is a membrane-associated cytoplasmic protein that serves to link cell-membrane proteins with the actin-based cytoskeleton, and also plays a role in regulation of the functional activities of some transmembrane proteins. It is expressed in placental trophoblasts. We hypothesized that placental ezrin is involved in the supply of nutrients from mother to fetus, thereby influencing fetal growth. The aim of this study was firstly to clarify the effect of ezrin on fetal growth and secondly to determine whether knockout of ezrin is associated with decreased concentrations of serum and placental nutrients. Ezrin knockout mice (Ez(-/-)) were confirmed to exhibit fetal growth retardation. Metabolome analysis of fetal serum and placental extract of ezrin knockout mice by means of capillary electrophoresis-time-of-flight mass spectrometry revealed a markedly decreased concentration of hypotaurine, a precursor of taurine. However, placental levels of cysteine and cysteine sulfinic acid (precursors of hypotaurine) and taurine were not affected. Lack of hypotaurine in Ez(-/-) mice was confirmed by liquid chromatography with tandem mass spectrometry. Administration of hypotaurine to heterogenous dams significantly decreased the placenta-to-maternal plasma ratio of hypotaurine in wild-type fetuses but only slightly decreased it in ezrin knockout fetuses, indicating that the uptake of hypotaurine from mother to placenta is saturable and that disruption of ezrin impairs the uptake of hypotaurine by placental trophoblasts. These results indicate that ezrin is required for uptake of hypotaurine from maternal serum by placental trophoblasts, and plays an important role in fetal growth.

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Reconstruction of insulin signal flow from phosphoproteome and metabolome data

Yugi, K., Kubota, H., Toyoshima, Y., Noguchi, R., Kawata, K., Komori, Y., Uda, S., Kunida, K., Tomizawa, Y., Funato, Y., Miki, H., Matsumoto, M., Nakayama, K.I., Kashikura, K., Endo, K., Ikeda, K., Soga, T., Kuroda, S.

Cell Rep. 8 ( 4 ) 1171 - 1183 2014.08

Research paper (scientific journal), Joint Work, Accepted

Cellular homeostasis is regulated by signals through multiple molecular networks that include protein phosphorylation and metabolites. However, where and when the signal flows through a network and regulates homeostasis has not been explored. We have developed a reconstruction method for the signal flow based on time-course phosphoproteome and metabolome data, using multiple databases, and have applied it to acute action of insulin, an important hormone for metabolic homeostasis. An insulin signal flows through a network, through signaling pathways that involve 13 protein kinases, 26 phosphorylated metabolic enzymes, and 35 allosteric effectors, resulting in quantitative changes in 44 metabolites. Analysis of the network reveals that insulin induces phosphorylation and activation of liver-type phosphofructokinase 1, thereby controlling a key reaction in glycolysis. We thus provide a versatile method of reconstruction of signal flow through the network using phosphoproteome and metabolome data.

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Hydroxyproline, a Serum Biomarker Candidate for Gastric Ulcer in Rats: A Comparison Study of Metabolic Analysis of Gastric Ulcer Models Induced by Ethanol, Stress, and Aspirin

Takeuchi, K., Ohishi, M., Endo, K., Suzumura, K., Naraoka, H., Ohata, T., Seki, J., Miyamae, Y., Honma, M., and Soga, T.

Biomark. Insights. 9 61 - 66 2014.07

Research paper (scientific journal), Joint Work, Accepted

Gastrointestinal symptoms are a common manifestation of adverse drug effects. Non-steroid anti-inflammatory drugs (NSAIDs) are widely prescribed drugs that induce the serious side effect of gastric mucosal ulceration. Biomarkers for these side effects have not been identified and ulcers are now only detectable by endoscopy. We previously identified five metabolites as biomarker candidates for NSAID-induced gastric ulcer using capillary electrophoresis-mass spectrometry (CE-MS)-based metabolomic analysis of serum and stomach from rats. Here, to clarify mechanism of changes and limitations of indications of biomarker candidates, we performed CE-MS-based metabolomic profiling in stomach and serum from rats with gastric ulcers induced by ethanol, stress, and aspirin. The results suggest that a decrease in hydroxyproline reflects the induction of gastric injury and may be useful in identifying gastric ulcer induced by multiple causes. While extrapolation to humans requires further study, hydroxyproline can be a new serum biomarker of gastric injury regardless of cause.

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Inhibition of ATP citrate lyase induces triglyceride accumulation with altered fatty acid composition in cancer cells

Migita, T., Okabe, S., Ikeda, K., Igarashi, S., Sugawara, S., Tomida, A., Soga, T., Taguchi, R., Seimiya, H.

Int J Cancer 135 ( 1 ) 37 - 47 2014.07

Joint Work, Accepted

De novo lipogenesis is activated in most cancers and several lipogenic enzymes have been implicated as therapeutic targets. Here, we demonstrate a novel function of the lipogenic enzyme, ATP citrate lyase (ACLY), in lipid metabolism in cancer cells. ACLY depletion by small interfering RNAs caused growth suppression and/or apoptosis in a subset of cancer cell lines. To investigate the effect of ACLY inhibition on lipid metabolism, metabolome and transcriptome analysis was performed. ACLY depletion blocks the fatty acid chain elongation from C16 to C18 in triglyceride (TG), but not in other lipid classes. Meanwhile, wild-type ACLY overexpression enhanced fatty acid elongation of TG, whereas an inactive mutant ACLY did not change it. ACLY depletion-mediated blockade of fatty acid elongation was coincident with downregulation of long-chain fatty acid elongase ELOVL6, which resides in endoplasmic reticulum (ER). Paradoxically, ACLY depletion-mediated growth suppression was associated with TG accumulation. ACLY depletion downregulated the expression of carnitine palmitoyltransferase 1A, which is a mitochondrial fatty acid transporter. Consistent with this finding, metabolome analysis revealed that ACLY positively regulates the carnitine system, which plays as an essential cofactor for fatty acid transport across mitochondrial membrane. AICAR, an activator of mitochondrial fatty acid oxidation (FAO), significantly reduced ACLY depletion-mediated TG accumulation. These data indicate that inhibition of ACLY might affect both fatty acid elongation in ER and FAO in mitochondria, thereby explaining the TG accumulation with altered fatty acid composition. This phenotype may be a hallmark of growth suppression mediated by ACLY inhibition.

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Quantitative metabolome analysis profiles activation of glutaminolysis in glioma with IDH1 mutation

Ohka, F., Ito, M., Ranjit, M., Senga, T., Motomura, A., Motomura, K., Saito, K., Kato, K., Kato, Y., Wakabayashi, T., Soga, T., Natsume, A.

Tumor Biol. 35 ( 6 ) 5911 - 5920 2014.06

Research paper (scientific journal), Joint Work, Accepted

Isocitrate dehydrogenase 1 (IDH1), which localizes to the cytosol and peroxisomes, catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG) and in parallel converts NADP(+) to NADPH. IDH1 mutations are frequently detected in grades 2-4 gliomas and in acute myeloid leukemias (AML). Mutations of IDH1 have been identified at codon 132, with arginine being replaced with histidine in most cases. Mutant IDH1 gains novel enzyme activity converting α-KG to D-2-hydroxyglutarate (2-HG) which acts as a competitive inhibitor of α-KG. As a result, the activity of α-KG-dependent enzyme is reduced. Based on these findings, 2-HG has been proposed to be an oncometabolite. In this study, we established HEK293 and U87 cells that stably expressed IDH1-WT and IDH1-R132H and investigated the effect of glutaminase inhibition on cell proliferation with 6-diazo-5-oxo-L-norleucine (DON). We found that cell proliferation was suppressed in IDH1-R132H cells. The addition of α-KG restored cell proliferation. The metabolic features of 33 gliomas with wild type IDH1 (IDH1-WT) and with IDH1-R132H mutation were examined by global metabolome analysis using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). We showed that the 2-HG levels were highly elevated in gliomas with IDH1-R132H mutation. Intriguingly, in gliomas with IDH1-R132H, glutamine and glutamate levels were significantly reduced which implies replenishment of α-KG by glutaminolysis. Based on these results, we concluded that glutaminolysis is activated in gliomas with IDH1-R132H mutation and that development of novel therapeutic approaches targeting activated glutaminolysis is warranted.

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Rare insights into cancer biology

Adam, J., Yang, M., Soga, T., and Pollard, P.J.

Oncogene 33 ( 20 ) 2547 - 2556 2014.05

Research paper (scientific journal), Joint Work, Accepted

Cancer-associated mutations have been identified in the metabolic genes succinate dehydrogenase (SDH), fumarate hydratase (FH) and isocitrate dehydrogenase (IDH), advancing and challenging our understanding of cellular function and disease mechanisms and providing direct links between dysregulated metabolism and cancer. Some striking parallels exist in the cellular consequences of the genetic mutations within this triad of cancer syndromes, including accumulation of oncometabolites and competitive inhibition of 2-oxoglutarate-dependent dioxygenases, particularly, hypoxia-inducible factor (HIF) prolyl hydroxylases, JmjC domain-containing histone demethylases (part of the JMJD family) and the ten-eleven translocation (TET) family of 5methyl cytosine (5mC) DNA hydroxylases. These lead to activation of HIF-dependent oncogenic pathways and inhibition of histone and DNA demethylation. Mutations in FH, resulting in loss of enzyme activity, predispose affected individuals to a rare cancer, hereditary leiomyomatosis and renal cell cancer (HLRCC), characterised by benign smooth muscle cutaneous and uterine tumours (leiomyomata) and an aggressive form of collecting duct and type 2 papillary renal cancer. Interestingly, loss of FH activity results in the accumulation of high levels of fumarate that can lead to the non-enzymatic modification of cysteine residues in multiple proteins (succination) and in some cases to their disrupted function. Here we consider that the study of rare diseases such as HLRCC, combining analyses of human tumours and cell lines with in vitro and in vivo murine models has provided novel insights into cancer biology associated with dysregulated metabolism and represents a useful paradigm for cancer research.

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Necrosis-driven systemic immune response alters SAM metabolism through the FOXO-GNMT axis

Obata, F., Kuranaga, E., Tomioka, K., Ming M., Takeishi, A., Chen, C., Soga, T., Miura, M.

Cell Rep. 7 ( 8 ) 821 - 833 2014.05

Research paper (scientific journal), Joint Work, Accepted

Sterile inflammation triggered by endogenous factors is thought to contribute to the pathogenesis of acute and chronic inflammatory diseases. Here, we demonstrate that apoptosis-deficient mutants spontaneously develop a necrosis-driven systemic immune response in Drosophila and provide an in vivo model for studying the organismal response to sterile inflammation. Metabolomic analysis of hemolymph from apoptosis-deficient mutants revealed increased sarcosine and reduced S-adenosyl-methionine (SAM) levels due to glycine N-methyltransferase (Gnmt) upregulation. We showed that Gnmt was elevated in response to Toll activation induced by the local necrosis of wing epidermal cells. Necrosis-driven inflammatory conditions induced dFoxO hyperactivation, leading to an energy-wasting phenotype. Gnmt was cell-autonomously upregulated by dFoxO in the fat body as a possible rheostat for controlling energy loss, which functioned during fasting as well as inflammatory conditions. We propose that the dFoxO-Gnmt axis is essential for the maintenance of organismal SAM metabolism and energy homeostasis.

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Comparative metabolite profiling of foxglove aphids (Aulacorthum solani Kaltenbach) on leaves from resistant and susceptible soybean strains

Sato, D., Sugimoto, M., Akashi, H., Tomita, M., Soga, T.

Mol. BioSyst. 10 ( 4 ) 909 - 915 2014.04

Research paper (scientific journal), Joint Work, Accepted

Aphid infestations can cause severe decreases in soybean (Glycine max [L.] Merr.) yield. Since planting aphid-resistant soybean strains is a promising approach for pest control, understanding the resistance mechanisms employed by aphids is of considerable importance. We compared aphid resistance in seven soybean strains and found that strain Tohoku149 was the most resistant to the foxglove aphid, Aulacorthum solani Kaltenbach. We subsequently analyzed the metabolite profiles of aphids cultured on the leaves of resistant and susceptible soybean strains using capillary electrophoresis-time-of-flight mass spectrometry. Our findings showed that the metabolite profiles of several amino acids, glucose 6-phosphate, and components of the tricarboxylic acid cycle were similar in aphids reared on Tohoku149 leaves and in aphids maintained under conditions of starvation, suggesting that Tohoku149 is more resistant to aphid feeding. Compared to susceptible strains, we also found that two methylated metabolites, S-methylmethionine and trigonelline, were either not detected or decreased in aphids reared on Tohoku149 plants. Since these metabolites function as important sulfur transporters in phloem sap and osmoprotectants involved in salt and drought stress, respectively, aphid-resistance is considered to be related to sulfur metabolism and methylation. These results contribute to an increase in our understanding of soybean aphid resistance mechanisms at the molecular level.

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Metabolomic profiling of sodium fluoride-induced cytotoxicity in an oral squamous cell carcinoma cell line

Sakagami, H., Sugimoto, M., Tanaka, S., Onuma, H., Ota, S., Kaneko, M., Soga, T., Tomita, M.

Metabolomics 10 ( 2 ) 270 - 279 2014.04

Research paper (scientific journal), Joint Work, Accepted

Sodium fluoride (NaF) is used in dentistry as a preventive agent for dental caries because of its ability to remineralize the tooth surface and its antibacterial effect. Although one of its target molecules in bacteria is enolase, its site of action in human cells has not been identified. The aim of this study was to identify target metabolites that are coupled to NaF-induced cytotoxicity in the HSC-2 human oral squamous cell carcinoma cell line. Cell viability, membrane integrity and apoptosis induction were analyzed by MTT assay, trypan blue exclusion and caspase-3 activation, respectively. Cells were treated with a minimal cytotoxic concentration of NaF for various times and subjected to comprehensive metabolomics analysis using capillary electrophoresis-mass spectrometry. In the early stages, inhibition of the enolase reaction in glycolysis pathway was observed. This was coupled with rapid inhibition of the progression of TCA cycle. In the later stages, gradual increases in the AMP/ATP ratio (a putative marker of apoptosis) and oxidized products (e.g. GSSH, and methionine sulfoxide), and marginal changes in polyamine levels (putative marker of necrosis) were observed. This manuscript provides the new insight into the global impact of NaF on metabolic pathways in human oral squamous cell carcinoma cells.

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Differences in the fatty acid metabolism of visceral adipose tissue in postmenopausal women

Yamatani, H., Takahashi, K., Yoshida, T., Soga, T., Kurachi, H.

Menopause 21 ( 2 ) 170 - 176 2014.02

Research paper (scientific journal), Joint Work, Accepted

Objective: Visceral fat accumulation and metabolic syndrome incidence among women increase after menopause; therefore, fat metabolic changes and fat redistribution may occur according to menstrual status. The aim of our study was to clarify differences in subcutaneous and visceral adipose tissue metabolism between premenopausal and postmenopausal women, using metabolomics.
Methods: Thirty-nine (16 premenopausal and 23 postmenopausal) women were recruited through elective gynecologic surgery, and both subcutaneous and visceral adipose tissues were collected during surgical operation. Metabolite profiling of adipose tissue was performed by capillary electrophoresis with electrospray ionization time-of-flight mass spectrometry.
Results: Sedoheptulose 7-phosphate, a midproduct of the pentose phosphate pathway, was significantly higher (P < 0.05) in visceral adipose tissues of premenopausal women. Dihydroxyacetone phosphate and fructose-1,6-biphosphate, midproducts of glycolysis, were significantly higher (P < 0.05) in subcutaneous adipose tissues of postmenopausal women. The concentrations of fatty acid metabolites-heptanoate (C7:0; premenopausal vs postmenopausal, 4.07 [0.72] vs 2.64 [0.28] nmol/g), octanoate (C8:0; 3.52 [0.29] vs 5.20 [0.29] nmol/g), and pelargonate (C9:0; 8.03 [0.49] vs 10.66 [0.44] nmol/g)-in the visceral fat (but not in subcutaneous fat) of postmenopausal women were significantly higher (P < 0.05) than those in the visceral fat of premenopausal women.
Conclusions: Fatty acid metabolites increase in visceral fat (but not in subcutaneous fat) after menopause. The change in fatty acid metabolism in visceral adipose tissues might be related to metabolic syndrome in postmenopausal women.

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Global metabolic network reorganization by adaptive mutations allows fast growth of Escherichia coli on glycerol

Cheng, K., Lee, B., Masuda, T., Ito, T., Ikeda, K., Hirayama, A., Deng, L., Dong, J., Shimizu, K., Soga, T., Tomita, M., Palsson, B. O. & Robert, M.

Nat. Commun. 5 3233 2014.01

Research paper (scientific journal), Joint Work

Comparative whole-genome sequencing enables the identification of specific mutations during adaptation of bacteria to new environments and allelic replacement can establish their causality. However, the mechanisms of action are hard to decipher and little has been achieved for epistatic mutations, especially at the metabolic level. Here we show that a strain of Escherichia coli carrying mutations in the rpoC and glpK genes, derived from adaptation in glycerol, uses two distinct metabolic strategies to gain growth advantage. A 27-bp deletion in the rpoC gene first increases metabolic efficiency. Then, a point mutation in the glpK gene promotes growth by improving glycerol utilization but results in increased carbon wasting as overflow metabolism. In a strain carrying both mutations, these contrasting carbon/energy saving and wasting mechanisms work together to give an 89% increase in growth rate. This study provides insight into metabolic reprogramming during adaptive laboratory evolution for fast cellular growth.

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Autophagy protects kidney proximal tubule epithelial cells from mitochondrial metabolic stress

Kimura, T., Takahashi, A., Takabatake, Y., Namba, T, Yamamoto, T., Kaimori, J.Y., Matsui, I., Kitamura, H., Niimura, F., Matsusaka T., Soga, T., Rakugi, H., *Isaka, Y.

Autophagy 9 ( 11 ) 1876 - 1886 2013.11

Joint Work, Accepted

Chronic metabolic stress is related to diseases, whereas autophagy supplies nutrients by recycling the degradative products. Cyclosporin A (CsA), a frequently used immunosuppressant, induces metabolic stress via effects on mitochondrial respiration, and thereby, its chronic usage is often limited. Here we show that autophagy plays a protective role against CsA-induced metabolic stress in kidney proximal tubule epithelial cells. Autophagy deficiency leads to decreased mitochondrial membrane potential, which coincides with metabolic abnormalities as characterized by decreased levels of amino acids, increased tricarboxylic acid (TCA) ratio (the levels of intermediates of the latter part of the TCA cycle, over levels of intermediates in the earlier part), and decreased products of oxidative phosphorylation (ATP). In addition to the altered profile of amino acids, CsA decreased the hyperpolarization of mitochondria with the disturbance of mitochondrial energy metabolism in autophagy-competent cells, i.e., increased TCA ratio and worsening of the NAD(+)/NADH ratio, coupled with decreased energy status, which suggests that adaptation to CsA employs autophagy to supply electron donors from amino acids via intermediates of the latter part of the TCA cycle. The TCA ratio of autophagy-deficient cells was further worsened with decreased levels of amino acids in response to CsA, and, as a result, the deficiency of autophagy failed to adapt to the CsA-induced metabolic stress. Deterioration of the TCA ratio further worsened energy status. The CsA-induced metabolic stress also activated regulatory genes of metabolism and apoptotic signals, whose expressions were accelerated in autophagy-deficient cells. These data provide new perspectives on autophagy in conditions of chronic metabolic stress in disease.

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Dynamics of serum metabolites in patients with chronic hepatitis C receiving pegylated interferon plus ribavirin: A metabolomics analysis

Saito, T., Sugimoto, M., Igarashi, K., Saito, K., Shao, L., Katsumi, T., Tomita, K., Sato, C., Okumoto, K., Nishise, Y., Watanabe, H., Tomita, M., Ueno, Y., Soga, T.

Metabolism 62 ( 11 ) 1577 - 1586 2013.11

Joint Work, Accepted

Objectives: Serum samples from patients with chronic hepatitis C were subjected to metabolomics analysis to clarify the pretreatment characteristics of their metabolites and also changes in specific metabolites resulting from antiviral therapy with pegylated interferon plus ribavirin (PegIFN/RBV).
Materials/methods: The serum levels of low-molecular-weight metabolites in the twenty patients before and 24weeks after completion of PegIFN/RBV therapy were analyzed using capillary electrophoresis and liquid chromatography-mass spectrometry.
Results: Ten patients showed a non-virological response (NVR) and 10 achieved a sustained virological response (SVR) with eradication of viremia. The pretreatment levels of tryptophan were significantly higher in the patients of SVR than in those of NVR (p=0.010). The area under the curve (AUC) value of tryptophan calculated from the receiver operating characteristic (ROC) curve for discriminating SVR from NVR was 0.84 (95% confidential interval, 0.66-1.02, p=0.010). The ROC curve of multiple logistic regression model incorporating the pretreatment levels of tryptophan and γ-glutamate-arginine showed that the AUC value was highly significant (AUC=0.92, 95% confidential interval, 0.79-1.05, p=0.002). Twenty four weeks after completion of treatment, the levels of γ-glutamyl dipeptides, glutamic acid, 5-oxoproline, glucosamine and methionine sulfoxide were decreased, whereas those of 5-methoxy-3-indoleacetate, glutamine, kynurenine and lysine were increased significantly (p<0.05) in both the NVR and SVR patients.
Conclusions: The pretreatment serum levels of certain metabolites including tryptophan are associated with the response to PegIFN/RBV therapy. PegIFN/RBV therapy can ameliorate the oxidative stress responsible for glutathione metabolism.

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Capillary electrophoresis-mass spectrometry-based metabolome analysis of serum and saliva from neurodegenerative dementia patients

Tsuruoka, M., Hara, J., Hirayama, A., Sugimoto, M., Soga, T., Shankle, W.R., Tomita , M.

Electrophoresis 34 ( 19 ) 2865 - 2872 2013.10

Research paper (scientific journal), Joint Work, Accepted

Despite increasing global prevalence, the precise pathogenesis and terms for objective diagnosis of neurodegenerative dementias remain controversial, and comprehensive understanding of the disease remains lacking. Here, we conducted metabolomic analysis of serum and saliva obtained from patients with neurodegenerative dementias (n = 10), including Alzheimer's disease, frontotemporal lobe dementia, and Lewy body disease, as well as from age-matched healthy controls (n = 9). Using CE-TOF-MS, six metabolites in serum (β-alanine, creatinine, hydroxyproline, glutamine, iso-citrate, and cytidine) and two in saliva (arginine and tyrosine) were significantly different between dementias and controls. Using multivariate analysis, serum was confirmed as a more efficient biological fluid for diagnosis compared to saliva; additionally, 45 metabolites in total were identified as candidate markers that could discriminate at least one pair of diagnostic groups from the healthy control group. These metabolites possibly provide an objective method for diagnosing dementia-type by multiphase screening. Moreover, diagnostic-type-dependent differences were observed in several tricarboxylic acid cycle compounds detected in serum, indicating that some pathways in glucose metabolism may be altered in dementia patients. This pilot study revealed novel alterations in metabolomic profiles between various neurodegenerative dementias, which would contribute to etiological investigations.

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Metabolome analysis of erythrocytes from patients with chronic hepatitis C reveals the etiology of ribavirin-induced hemolysis

Karasawa, T., *Saito, T., Ueno, Y., Sugimoto, M., Soga, T.

Int J Med Sci. 10 ( 11 ) 1575 - 1577 2013.09

Research paper (scientific journal), Joint Work, Accepted

Ribavirin is one of the major agents used in combination therapy with interferon for chronic hepatitis C, but is often associated with hemolytic anemia as a serious adverse event. Employing metabolome analysis, we demonstrated that the concentrations of intermediate metabolites produced by glycolysis and the pentose phosphate cycle in patients' erythrocytes were significantly decreased after administration of ribavirin. Our findings suggest that hemolysis associated with ribavirin is triggered by an energy crisis and consequent oxidative stress, thus having implications for the prevention of such hemolysis.

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Oncometabolites: linking altered metabolism with cancer

Yang, M., Soga, T., *Pollard, P. J.

J. Clin. Invest. 123 ( 9 ) 3652 - 3658 2013.09

Research paper (scientific journal), Joint Work, Accepted

The discovery of cancer-associated mutations in genes encoding key metabolic enzymes has provided a direct link between altered metabolism and cancer. Advances in mass spectrometry and nuclear magnetic resonance technologies have facilitated high-resolution metabolite profiling of cells and tumors and identified the accumulation of metabolites associated with specific gene defects. Here we review the potential roles of such "oncometabolites" in tumor evolution and as clinical biomarkers for the detection of cancers characterized by metabolic dysregulation.

Time-resolved metabolomics of a novel trebouxiophycean alga using 13CO2 feeding

Ito, T., Sugimoto, M., Toya, Y., Ano, Y., Kurano, N., Soga, T., Tomita, M.

J Biosci Bioeng. 116 ( 3 ) 408 - 415 2013.09

Research paper (scientific journal), Joint Work, Accepted

Oil-rich algae are potentially promising as next-generation biofuel feedstock. However, the productivity of oil needs to be improved for industrial use. The biosynthesis of oil and its control mechanism have not been characterized in any algae, and understanding the metabolic network is vital to achieve the precise engineering of algae metabolic pathways. "Pseudochoricystis ellipsoidea" MBIC 11204, a novel microalgal strain, accumulates a large amount of lipids in nitrogen-deficient conditions. In this study, "P. ellipsoidea" was grown in flat flasks with continuous illumination and aeration with 1% CO2 at 25°C. During the exponential growth phase, CO2 was switched to (13)C-labeled CO2 and samples were collected for time-course experiments. Seventy-eight pairs of unlabeled and uniformly (13)C-labeled metabolites were quantified using a capillary electrophoresis- and liquid chromatography-mass spectrometry for ionic primary metabolites and lipids, respectively. The (13)C-exchange indices of the metabolites were calculated from a concentration of unlabeled and uniformly-labeled metabolites. A hierarchical clustering analysis of the dynamics of the indices revealed 4 characteristic clusters, two of which represented rapidly-labeled metabolites, mainly composed of primary metabolites, while the two other clusters represented slowly-labeled metabolites, mainly composed of lipids. Moreover, the labeling order of these clusters was mainly matched to the metabolic process of Chlamydomonas reinhardtii, a model organism of green algae. In TCA cycle, anomalistically different of the labeling order was found. To the author's knowledge, this study for the first time in literature, characterize the features of global metabolism in "P. ellipsoidea."

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Dynamic Simulation and Metabolome Analysis of Long-Term Erythrocyte Storage in Adenine–Guanosine Solution

Nishino, T., *Yachie-Kinoshita, A., Hirayama, A., Soga, T., Suematsu, M., Tomita, M.

PLoS ONE 8 ( 8 ) e71060 2013.08

Joint Work, Accepted

Although intraerythrocytic ATP and 2,3-bisphophoglycerate (2,3-BPG) are known as direct indicators of the viability of preserved red blood cells and the efficiency of post-transfusion oxygen delivery, no current blood storage method in practical use has succeeded in maintaining both these metabolites at high levels for long periods. In this study, we constructed a mathematical kinetic model of comprehensive metabolism in red blood cells stored in a recently developed blood storage solution containing adenine and guanosine, which can maintain both ATP and 2,3-BPG. The predicted dynamics of metabolic intermediates in glycolysis, the pentose phosphate pathway, and purine salvage pathway were consistent with time-series metabolome data measured with capillary electrophoresis time-of-flight mass spectrometry over 5 weeks of storage. From the analysis of the simulation model, the metabolic roles and fates of the 2 major additives were illustrated: (1) adenine could enlarge the adenylate pool, which maintains constant ATP levels throughout the storage period and leads to production of metabolic waste, including hypoxanthine; (2) adenine also induces the consumption of ribose phosphates, which results in 2,3-BPG reduction, while (3) guanosine is converted to ribose phosphates, which can boost the activity of upper glycolysis and result in the efficient production of ATP and 2,3-BPG. This is the first attempt to clarify the underlying metabolic mechanism for maintaining levels of both ATP and 2,3-BPG in stored red blood cells with in silico analysis, as well as to analyze the trade-off and the interlock phenomena between the benefits and possible side effects of the storage-solution additives.

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Metabolomic profiling analysis reveals chamber-dependent metabolite patterns in the mouse heart

Shimura, D., Nakai, G., Jiao, Q., Osanai, K., Kashikura, K., Endo, K., Soga, T., Goda, N., Minamisawa, S.

Am J Physiol Heart Circ Physiol. 305 ( 4 ) H494 - 505 2013.08

Research paper (scientific journal), Joint Work, Accepted

Energy of the cardiac muscle largely depends on fatty acid oxidation. It is known that the atrium and ventricle have chamber-specific functions, structures, gene expressions, and pathologies. The left ventricle works as a high-pressure chamber to pump blood toward the body, and its muscle wall is thicker than those of the other chambers, suggesting that energy utilization in each of the chambers should be different. However, a chamber-specific pattern of metabolism remains incompletely understood. Recently, innovative techniques have enabled the comprehensive analysis of metabolites. Therefore, we aimed to clarify differences in metabolic patterns among the chambers. Male C57BL6 mice at 6 wk old were subject to a comprehensive measurement of metabolites in the atria and ventricles by capillary electrophoresis and mass spectrometry. We found that overall metabolic profiles, including nucleotides and amino acids, were similar between the right and left ventricles. On the other hand, the atria exhibited a distinct metabolic pattern from those of the ventricles. Importantly, the high-energy phosphate pool (the total concentration of ATP, ADP, and AMP) was higher in both ventricles. In addition, the levels of lactate, acetyl CoA, and tricarboxylic acid cycle contents were higher in the ventricles. Accordingly, the activities and/or expression levels of key enzymes were higher in the ventricles to produce more energy. The present study provides a basis for understanding the chamber-specific metabolism underlining pathophysiology in the heart.

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Persistent Overexpression of Phosphoglycerate Mutase, a Glycolytic Enzyme, Modifies Energy Metabolism and Reduces Stress Resistance of Heart in Mice

Okuda, J., Niizuma, S., Shioi, T., Kato, T., Inuzuka, Y., Kawashima, T., Tamaki, Y., Kawamoto, A., Tanada, Y., Iwanaga, Y., Narazaki, M., Matsuda, T., Adachi, S., Soga, T., Takemura, G., Kondoh, H., Kita, T., Kimura, T.

PLoS ONE 8 ( 8 ) e72173 2013.08

Research paper (scientific journal), Joint Work, Accepted

Background: Heart failure is associated with changes in cardiac energy metabolism. Glucose metabolism in particular is thought to be important in the pathogenesis of heart failure. We examined the effects of persistent overexpression of phosphoglycerate mutase 2 (Pgam2), a glycolytic enzyme, on cardiac energy metabolism and function.
Methods and results: Transgenic mice constitutively overexpressing Pgam2 in a heart-specific manner were generated, and cardiac energy metabolism and function were analyzed. Cardiac function at rest was normal. The uptake of analogs of glucose or fatty acids and the phosphocreatine/βATP ratio at rest were normal. A comprehensive metabolomic analysis revealed an increase in the levels of a few metabolites immediately upstream and downstream of Pgam2 in the glycolytic pathway, whereas the levels of metabolites in the initial few steps of glycolysis and lactate remained unchanged. The levels of metabolites in the tricarboxylic acid (TCA) cycle were altered. The capacity for respiration by isolated mitochondria in vitro was decreased, and that for the generation of reactive oxygen species (ROS) in vitro was increased. Impaired cardiac function was observed in response to dobutamine. Mice developed systolic dysfunction upon pressure overload.
Conclusions: Constitutive overexpression of Pgam2 modified energy metabolism and reduced stress resistance of heart in mice.

Abundant Nucleostemin Expression Supports the Undifferentiated Properties of Germ Cell Tumors

Uema, N., Ooshio, T., Harada, K., Naito, M., Naka, K., Hoshi T., Tadokoro, Y., Ohta, K., Ali, M. A.E., Katanno, M., Soga, T., Nakanuma, Y., Okuda, A., Hirao, A.

Am J Pathol. 183 ( 2 ) 592 - 603 2013.08

Research paper (scientific journal), Joint Work

Nucleostemin (NS) is a nucleolar GTP-binding protein that is involved in ribosomal biogenesis and protection of telomeres. We investigated the expression of NS in human germ cell tumors and its function in a mouse germ cell tumor model. NS was abundantly expressed in undifferentiated, but not differentiated, types of human testicular germ cell tumors. NS was expressed concomitantly with OCT3/4, a critical regulator of the undifferentiated status of pluripotent stem cells in primordial germ cells and embryonal carcinomas. To investigate the roles of NS in tumor growth in vivo, we used a mouse teratoma model. Analysis of teratomas derived from embryonic stem cells in which the NS promoter drives GFP expression showed that cells highly expressing NS were actively proliferating and exhibited the characteristics of tumor-initiating cells, including the ability to initiate and propagate tumor cells in vivo. NS-expressing cells exhibited higher levels of GTP than non-NS-expressing cells. Because NS protein is stabilized by intracellular GTP, metabolic changes may contribute to abundant NS expression in the undifferentiated cells. OCT3/4 deficiency in teratomas led to loss of NS expression, resulting in growth retardation. Finally, we found that teratomas deficient in NS lost their undifferentiated characteristics, resulting in defective tumor proliferation. These data indicate that abundant expression of NS supports the undifferentiated properties of germ cell tumors.

Global metabolomic analysis of heart tissue in a hamster model for dilated cardiomyopathy

Maekawa, K., Hirayama, A., Iwata Y., Tajima Y., Nishimaki-Mogami T., Sugawara S., Ueno, N., Abe, H., Ishikawa M., Murayama M., Matsuzawa Y., Nakanishi H., Ikeda K., Arita M., Taguchi, R., Minamino N., Wakabayashi, S., Soga T. *, Saito, Y.*

J. Mol. Cell. Cardiol. 59 76 - 85 2013.06

Research paper (scientific journal), Joint Work, Accepted

Dilated cardiomyopathy (DCM), a common cause of heart failure, is characterized by cardiac dilation and reduced left ventricular ejection fraction, but the underlying mechanisms remain unclear. To investigate the mechanistic basis, we performed global metabolomic analysis of myocardial tissues from the left ventricles of J2N-k cardiomyopathic hamsters. This model exhibits symptoms similar to those of human DCM, owing to the deletion of the δ-sarcoglycan gene. Charged and lipid metabolites were measured by capillary electrophoresis mass spectrometry (MS) and liquid chromatography MS(/MS), respectively, and J2N-k hamsters were compared with J2N-n healthy controls at 4 (presymptomatic phase) and 16weeks (symptomatic) of age. Disturbances in membrane phospholipid homeostasis were initiated during the presymptomatic phase. Significantly different levels of charged metabolites, occurring mainly in the symptomatic phase, were mapped to primary metabolic pathways. Reduced levels of metabolites in glycolysis, the pentose phosphate pathway, and the tricarboxylic acid cycle, together with large decreases in major triacylglycerol levels, suggested that decreased energy production leads to cardiac contractile dysfunction in the symptomatic phase. A mild reduction in glutathione and a compensatory increase in ophthalmate levels suggest increased oxidative stress in diseased tissues, which was confirmed by histochemical staining. Increased levels of 4 eicosanoids, including prostaglandin (PG) E2 and 6-keto-PGF1α, in the symptomatic phase suggested activation of the protective response pathways. These results provide mechanistic insights into DCM pathogenesis and may help identify new targets for therapeutic intervention and diagnosis.

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Metabolome profiling of floral scent production in Petunia axillaris

Oyama-Okubo, N., Sakai , T., Ando, T., Nakayama, M., Soga, T.

Phytochemistry 90 37 - 42 2013.06

Research paper (scientific journal), Joint Work, Accepted

Emission of floral scent benzenoid/phenylpropanoid compounds in Petunia axillaris increases significantly at night, a change that is primarily determined by the endogenous concentration of these compounds in the corolla. Among wild type P. axillaris plants, there are lines that emit different amounts of scent. To understand how the nocturnal rhythm of floral scent concentrations is controlled, the concentration profiles of metabolites in the scent biosynthetic pathway in two lines of P. axillaris, a strongly scented line and a weakly scented line, are reported. In the strongly scented line, the concentration of a series of compounds from glucose-6-phosphate (G6P) to the scent compounds changed synchronously. In the weakly scented lines, the concentrations of some metabolites including 6-phosphogluconate (6PG) and downstream metabolites of shikimic acid were remarkably lower, suggesting a reduction in metabolism of G6P to 6PG and the metabolism of shikimic acid in the weakly scented line. Nocturnal increases in the concentrations of sucrose, fructose, and glucose were not found in strongly scented line. Nocturnal increases in concentrations of S-adenosylhomocysteine (SAH) and methionine and reductions in the concentrations of S-adenosylmethionine (SAM), a methylation donor to benzenoid-skeletons, were observed only in strongly scented line. It is concluded that the biosynthetic regulation of each step from G6P to the volatile scent benzenoids is performed by, at least in part, concentrations of substrates, and the regulation also affects concentrations of SAM cycle compounds.

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A Role for Cytosolic Fumarate Hydratase in Urea Cycle Metabolism and Renal Neoplasia

Adam, J., Yang, M., Bauerschmidt, C., Kitagawa, M., O’Flaherty, L., Maheswaran, P., O¨ zkan, G., Sahgal, N., Baban, D., Kato, K., Saito, K., Iino, K., Igarashi, K., Stratford, M., Pugh, C., Tenant, D.A., Ludwig, C., Davies, B., Ratcliffe, P. J., El-Bahrawy, M., Ashrafian, H., Soga, T., *Pollard, P. J.

Cell Rep. 3 ( 5 ) 1440 - 1448 2013.05

Research paper (scientific journal), Joint Work, Accepted

The identification of mutated metabolic enzymes in hereditary cancer syndromes has established a direct link between metabolic dysregulation and cancer. Mutations in the Krebs cycle enzyme, fumarate hydratase (FH), predispose affected individuals to leiomyomas, renal cysts, and cancers, though the respective pathogenic roles of mitochondrial and cytosolic FH isoforms remain undefined. On the basis of comprehensive metabolomic analyses, we demonstrate that FH1-deficient cells and tissues exhibit defects in the urea cycle/arginine metabolism. Remarkably, transgenic re-expression of cytosolic FH ameliorated both renal cyst development and urea cycle defects associated with renal-specific FH1 deletion in mice. Furthermore, acute arginine depletion significantly reduced the viability of FH1-deficient cells in comparison to controls. Our findings highlight the importance of extramitochondrial metabolic pathways in FH-associated oncogenesis and the urea cycle/arginine metabolism as a potential therapeutic target.

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The selective control of glycolysis, gluconeogenesis and glycogenesis by temporal insulin patterns

Noguchi, R., Kubota, H., Yugi, K., Toyoshima, Y., Komori, Y., Soga, T., Kuroda, S.

Mol Syst Biol. 9 664 2013.05

Research paper (scientific journal), Joint Work, Accepted

Insulin governs systemic glucose metabolism, including glycolysis, gluconeogenesis and glycogenesis, through temporal change and absolute concentration. However, how insulin-signalling pathway selectively regulates glycolysis, gluconeogenesis and glycogenesis remains to be elucidated. To address this issue, we experimentally measured metabolites in glucose metabolism in response to insulin. Step stimulation of insulin induced transient response of glycolysis and glycogenesis, and sustained response of gluconeogenesis and extracellular glucose concentration (GLC(ex)). Based on the experimental results, we constructed a simple computational model that characterises response of insulin-signalling-dependent glucose metabolism. The model revealed that the network motifs of glycolysis and glycogenesis pathways constitute a feedforward (FF) with substrate depletion and incoherent feedforward loop (iFFL), respectively, enabling glycolysis and glycogenesis responsive to temporal changes of insulin rather than its absolute concentration. In contrast, the network motifs of gluconeogenesis pathway constituted a FF inhibition, enabling gluconeogenesis responsive to absolute concentration of insulin regardless of its temporal patterns. GLC(ex) was regulated by gluconeogenesis and glycolysis. These results demonstrate the selective control mechanism of glucose metabolism by temporal patterns of insulin.

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Inhibition of ATP citrate lyase induces an anticancer effect via reactive oxygen species: AMPK as a predictive biomarker for therapeutic impact

Migita, T., Okabe, S., Ikeda, K., Igarashi, S., Sugawara, S., Tomida, A., Taguchi, R., Soga, T., Seimiya, H.

Am J Pathol. 182 ( 5 ) 1800 - 1810 2013.05

Research paper (scientific journal), Joint Work, Accepted

De novo lipogenesis is activated in most cancers. Inhibition of ATP citrate lyase (ACLY), the enzyme that catalyzes the first step of de novo lipogenesis, leads to growth suppression and apoptosis in a subset of human cancer cells. Herein, we found that ACLY depletion increases the level of intracellular reactive oxygen species (ROS), whereas addition of an antioxidant reduced ROS and attenuated the anticancer effect. ACLY depletion or exogenous hydrogen peroxide induces phosphorylation of AMP-activated protein kinase (p-AMPK), a crucial regulator of lipid metabolism, independently of energy status. Analysis of various cancer cell lines revealed that cancer cells with a higher susceptibility to ACLY depletion have lower levels of basal ROS and p-AMPK. Mitochondrial-deficient ρ(0) cells retained high levels of ROS and p-AMPK and were resistant to ACLY depletion, whereas the replenishment of normal mitochondrial DNA reduced the levels of ROS and p-AMPK and restored the sensitivity to ACLY depletion, indicating that low basal levels of mitochondrial ROS are critical for the anticancer effect of ACLY depletion. Finally, p-AMPK levels were significantly correlated to the levels of oxidative DNA damage in colon cancer tissues, suggesting that p-AMPK reflects cellular ROS levels in vitro and in vivo. Together, these data suggest that ACLY inhibition exerts an anticancer effect via increased ROS, and p-AMPK could be a predictive biomarker for its therapeutic outcome.

Metabolomic profiling of the response of susceptible and resistant soybean strains to foxglove aphid, Aulacorthum solani Kaltenbach

Sato, D., Akashi, H., Sugimoto, M., Tomita, M., Soga, T.

J. Chromatogr. B. 925 95 - 103 2013.04

Research paper (scientific journal), Joint Work, Accepted

Aphid infection reduces soybean (Glycine max [L.] Merr.) yield. Consequently, cultivation of aphid-resistant strains is a promising approach to pest control, and understanding the resistance mechanism is of importance. Here, we characterized the resistance of soybeans to foxglove aphid, Aulacorthum solani Kaltenbach, at the metabolite level. First, we evaluated aphid mortality and settlement rates on the leaves of two soybean strains, ‘Tohoku149’ and ‘Suzuyutaka’, and found that the former had strong resistance soon after introduction of the aphids. The metabolomic response to aphid introduction was analyzed using capillary electrophoresis–time-of-flight mass spectrometry. We found the following three features in the profiles: (1) concentrations of citrate, amino acids, and their intermediates were intrinsically higher for Tohoku149 than Suzuyutaka, (2) concentrations of several metabolites producing secondary metabolites, such as flavonoids and alkaloids, drastically changed 6 h after aphid introduction, and (3) concentrations of TCA cycle metabolites increased in Tohoku149 48 h after aphid introduction. We also profiled free amino acids in aphids reared on both soybean strains and under starvation, and found that the profile of the aphids on Tohoku149 was similar to that of the starved aphids, but different to that of aphids on Suzuyutaka. These tests confirmed that aphids suck phloem sap even from Tohoku149. This study demonstrates the metabolomic profiles of both soybean strains and aphids, which will contribute to the molecular level understanding of mechanisms of soybean resistance to aphids.

Physiological and environmental parameters associated with mass spectrometry-based salivary metabolomic profiles

Sugimoto, M., Saruta, J., Matsuki, C., To, M., Onuma, H., Kaneko, M., Soga, T., Tomita, M., and Tsukinoki, K.

Metabolomics 9 ( 2 ) 454 - 463 2013.04

Research paper (scientific journal), Joint Work, Accepted

Mass spectrometry (MS)-based metabolomic methods enable simultaneous profiling of hundreds of salivary metabolites, and may be useful to diagnose a wide range of diseases using saliva. However, few studies have evaluated the effects of physiological or environmental factors on salivary metabolomic profiles. Therefore, we used capillary electrophoresis-MS to analyze saliva metabolite profiles in 155 subjects with reasonable oral hygiene, and examined the effects of physiological and environmental factors on the metabolite profiles. Overall, 257 metabolites were identified and quantified. The global profiles and individual metabolites were evaluated by principle component analysis and univariate tests, respectively. Collection method, collection time, sex, body mass index, and smoking affected the global metabolite profiles. However, age also might contribute to the bias in sex and collection time. The profiles were relatively unaffected by other parameters, such as alcohol consumption and smoking, tooth brushing, or the use of medications or nutritional supplements. Temporomandibular joint disorders had relatively greater effects on salivary metabolites than other dental abnormalities (e.g., stomatitis, tooth alignment, and dental caries). These findings provide further insight into the diversity and stability of salivary metabolomic profiles, as well as the generalizability of disease-specific biomarkers.

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Metabolomic Profiling of Lung and Prostate Tumor Tissues by Capillary Electrophoresis Time-of-flight Mass Spectrometry

Kami, K., Fujimori, T., Sato, H., Sato, M., Yamamoto, H., Ohashi, Y., Sugiyama, N., Ishihama, Y., Onozuka, H., Ochiai, A., Esumi, H., Soga, T., Tomita, M.

Metabolomics 9 ( 2 ) 444 - 453 2013.04

Research paper (scientific journal), Joint Work, Accepted

Metabolic microenvironment of tumor cells is influenced by oncogenic signaling and tissue-specific metabolic demands, blood supply, and enzyme expression. To elucidate tumor-specific metabolism, we compared the metabolomics of normal and tumor tissues surgically resected pairwise from nine lung and seven prostate cancer patients, using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). Phosphorylation levels of enzymes involved in central carbon metabolism were also quantified. Metabolomic profiles of lung and prostate tissues comprised 114 and 86 metabolites, respectively, and the profiles not only well distinguished tumor from normal tissues, but also squamous cell carcinoma from the other tumor types in lung cancer and poorly differentiated tumors from moderately differentiated tumors in prostate cancer. Concentrations of most amino acids, especially branched-chain amino acids, were significantly higher in tumor tissues, independent of organ type, but of essential amino acids were particularly higher in poorly differentiated than moderately differentiated prostate cancers. Organ-dependent differences were prominent at the levels of glycolytic and tricarboxylic acid cycle intermediates and associated energy status. Significantly high lactate concentrations and elevated activating phosphorylation levels of phosphofructokinase and pyruvate kinase in lung tumors confirmed hyperactive glycolysis. We highlighted the potential of CE-TOFMS-based metabolomics combined with phosphorylated enzyme analysis for understanding tissue-specific tumor microenvironments, which may lead to the development of more effective and specific anticancer therapeutics.

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Inhibition of Mitochondrial Aconitase by Succination in Fumarate Hydratase Deficiency

Ternette N., Yang M., Laroyia M., Kitagawa M., O'Flaherty L., Wolhulter K., Igarashi K., Saito K., Kato K., Fischer R., Berquand A., Kessler B.M., Lappin T., Frizzell N., Soga T., Adam J, Pollard P.J.

Cell Rep. 3 ( 3 ) 689 - 700 2013.03

Research paper (scientific journal), Joint Work, Accepted

The gene encoding the Krebs cycle enzyme fumarate hydratase (FH) is mutated in hereditary leiomyomatosis and renal cell cancer (HLRCC). Loss of FH activity causes accumulation of intracellular fumarate, which can directly modify cysteine residues to form 2-succinocysteine through succination. We undertook a proteomic-based screen in cells and renal cysts from Fh1 (murine FH)-deficient mice and identified 94 protein succination targets. Notably, we identified the succination of three cysteine residues in mitochondrial Aconitase2 (ACO2) crucial for iron-sulfur cluster binding. We show that fumarate exerts a dose-dependent inhibition of ACO2 activity, which correlates with increased succination as determined by mass spectrometry, possibly by interfering with iron chelation. Importantly, we show that aconitase activity is impaired in FH-deficient cells. Our data provide evidence that succination, resulting from FH deficiency, targets and potentially alters the function of multiple proteins and may contribute to the dysregulated metabolism observed in HLRCC.

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Cancer metabolism: key players in metabolic reprogramming

Soga, T.

Cancer Sci. 104 ( 3 ) 275 - 281 2013.03

Research paper (scientific journal), Single Work, Accepted

Over 80 years ago, Warburg discovered that cancer cells generate ATP through the glycolytic pathway, even in the presence of oxygen. The finding of this phenomenon, termed the "Warburg effect," stimulated much research on tumorigenesis, but few explanations were forthcoming to explain the observation. Recently, advanced developments in molecular biology and high-throughput molecular analyses have revealed that many of the signaling pathways altered by gene mutations regulate cell metabolism in cancer. Furthermore, mutations in isocitrate dehydrogenase 1 and 2 were shown to elevate 2-hydroxyglutarate, which led to changes in α-ketoglutarate-dependent dioxygenase enzyme activity, resulting in an increased risk of malignant tumors. Although these findings led to a renewed interest in cancer metabolism, our knowledge on the specifics of tumor metabolism is still fragmented. This paper reviews recent findings related to key transcription factors and enzymes that play an important role in the regulation of cancer metabolism.

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Metabolic and morphological changes of an oil accumulating trebouxiophycean alga in nitrogen-deficient conditions

Ito, T., Tanaka, M., Shinkawa, H., Nakada, T., Ano, Y., Kurano N., Soga T., Tomita, M.

Metabolomics 9 ( 1Supplement ) 178 - 187 2013.03

Research paper (scientific journal), Joint Work, Accepted

Oil-rich algae have promising potential for a next-generation biofuel feedstock. Pseudochoricystis ellipsoidea MBIC 11204, a novel unicellular green algal strain, accumulates a large amount of oil (lipids) in nitrogen-deficient (-N) conditions. Although the oil bodies are easily visualized by lipophilic staining in the cells, little is known about how oil bodies are metabolically synthesized. Clarifying the metabolic profiles in -N conditions is important to understand the physiological mechanisms of lipid accumulations and will be useful to optimize culture conditions efficiently produce industrial oil. Metabolome and lipidome profiles were obtained, respectively, using capillary electrophoresis- and liquid chromatography-mass spectrometry from P. ellipsoidea in both nitrogen-rich (+N; rapid growth) and -N conditions. Relative quantities of more than 300 metabolites were systematically compared between these two conditions. Amino acids in nitrogen assimilation and N-transporting metabolisms were decreased to 1/20 the amount, or less, in -N conditions. In lipid metabolism, the quantities of neutral lipids increased greatly in -N conditions; however, quantities of nearly all the other lipids either decreased or only changed slightly. The morphological changes in +N and -N conditions were also provided by microscopy, and we discuss their relationship to the metabolic changes. This is the first approach to understand the novel algal strain's metabolism using a combination of wide-scale metabolome analysis and morphological analysis.

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Metabolic Profiling to Identify Potential Serum Biomarkers for gastric ulceration induced by non-steroid anti-inflammatory drugs

Takeuchi, K., Ohishi, M., Ota, S., Suzumura, K., Naraoka, H., Ohata, T., Seki, J., Miyamae, Y., Honma, M., Soga, T.

J. Proteome Res. 12 ( 3 ) 1399 - 1407 2013.03

Research paper (scientific journal), Joint Work, Accepted

Nonsteroid anti-inflammatory drugs (NSAIDs) are among the most frequently prescribed drugs currently available. The most frequently reported serious side effects associated with NSAIDs are gastric mucosal ulceration and gastric hemorrhage. Presently, these side effects are only detectable by endoscopy, however, and no biomarkers have yet been identified. The ability to identify serum biomarkers would likely improve the safety of NSAID use. In this study we performed capillary electrophoresis-mass spectrometry (CE-MS)-based metabolomic profiling in stomach extract and serum from rats administered NSAIDs. Results showed drug-induced decreases in levels of citrate, cis-aconitate, succinate, 3-hydroxy butanoic acid, o-acetyl carnitine, proline, and hydroxyproline. We consider that these changes are due to NSAID-induced depression of mitochondrial function and activation of collagenase by lesions in the stomach. In addition, four of these changes in metabolite levels in the stomach were significantly correlated with changes in the serum. While further study is needed to clarify the mechanism of change in the level of these biomarkers, limitation of indications, and extrapolation to humans, these new serum biomarker candidates of gastric injury may be useful in the monitoring of NSAID-induced tissue damage.

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Changes of Metabolic Profiles in an Oral Squamous Cell Carcinoma Cell Line Induced by Eugenol

Koh, T., Murakami, Y., Tanaka, S., Machino, M., Onuma, H., Kaneko, M., Sugimoto, M., Soga, T., Tomita, M., Sakagami, H.

In Vivo 27 ( 2 ) 233 - 243 2013.03

Research paper (scientific journal), Joint Work, Accepted

Background: We have recently reported that eugenol exerted indiscriminate cytotoxicity towards normal oral cells and oral squamous cell carcinoma (OSCC) cell lines without induction of apoptosis markers. In order to investigate the underlying mechanisms of cytotoxicity induction, we investigated the effect of short-term treatment with eugenol on the metabolic profiles of a human OSCC cell line (HSC-2).
Materials and methods: The viable cell number was determined by direct cell counting with a hemocytometer after trypsinization. After washing with 5% D-mannitol solution (found to retain the highest amounts of intracellular metabolites among several washing conditions), cellular metabolites were extracted with methanol with internal markers and then subjected to metabolomic analysis.
Results: Cytotoxic concentrations of eugenol induced the reduction of ATP utilization (assessed by a significant reduction of the AMP/ATP and ADP/ATP ratio), of oxidative stress (assessed by the increase in oxidized form of glutathione, cysteine-glutathione disulfide and methionine sulfoxide), and an increase in the polyamines and glycolytic metabolites.
Conclusion: The metabolic changes observed in this study suggest the induction of non-apoptotic cell death by eugenol.

Biochemical and functional characterization of novel NADH kinase in the enteric protozoan parasite Entamoeba histolytica

Jeelani, G., Husain, A., Sato, D., Soga, T., Suematsu, M., Nozaki, T.

Biochimie 95 ( 2 ) 309 - 319 2013.03

Research paper (scientific journal), Accepted

NAD(H) kinase catalyzes the phosphorylation of NAD(H) to form NADP(H) using ATP or inorganic polyphosphate as a phosphoryl donor. While the enzyme is conserved throughout prokaryotes and eukaryotes, remarkable differences in kinetic parameters including substrate preference, cation dependence, and physiological roles exist among the organisms. In the present study, we biochemically characterized NAD(H) kinase from the anaerobic/microaerophilic fermentative protozoan parasite Entamoeba histolytica, which lacks the conventional mitochondria capable of oxidative phosphorylation, leading to ATP. The kinetic properties of E. histolytica NAD(H) kinase recombinantly produced in Escherichia coli showed remarkable differences from those in bacteria and higher eukaryotes. Entamoeba NAD(H) kinase preferred NADH to NAD+ as the phosphoryl acceptor, utilized nucleoside triphosphates including ATP, GTP and deoxyATP, but not nucleoside di-, mono-phosphates, or inorganic polyphosphates, as the phosphoryl donor. To further understand the physiological roles in E. histolytica, we generated a stable transformant overexpressing NAD(H) kinase. Overexpression of NAD(H) kinase resulted in a 1.6-2 fold increase in the NADPH and NADP+ concentrations, a 40% reduction of the intracellular concentration of reactive oxygen species, and also led to increased tolerance toward hydrogen peroxide. These data, together with the essentially of NAD(H) kinase gene, underscore its significance as an NADP(H)-producing enzyme in this organism, and should help in designing of drugs targeting this enzyme.

Metabolomic study of Chilean biomining bacteria Acidithiobacillus ferrooxidans strain Wenelen and Acidithiobacillus thiooxidans strain Licanantay

Martı´nez, P., Ga´lvez, S., Ohtsuka, N., Budinich, M., Corte´s, M.P., Serpell, C., Nakahigashi, K., Hirayama, A., Tomita, M., Soga, T., Martı´nez, S., Maass, A., Parada, P.

Metabolomics 9 ( 1 ) 247 - 257 2013.02

Research paper (scientific journal), Joint Work, Accepted

In this study, we present the first metabolic profiles for two bioleaching bacteria using capillary electrophoresis coupled with mass spectrometry. The bacteria, Acidithiobacillus ferrooxidans strain Wenelen (DSM 16786) and Acidithiobacillus thiooxidans strain Licanantay (DSM 17318), were sampled at different growth phases and on different substrates: the former was grown with iron and sulfur, and the latter with sulfur and chalcopyrite. Metabolic profiles were scored from planktonic and sessile states. Spermidine was detected in intra- and extracellular samples for both strains, suggesting it has an important role in biofilm formation in the presence of solid substrate. The canonical pathway for spermidine synthesis seems absent as its upstream precursor, putrescine, was not present in samples. Glutathione, a catalytic activator of elemental sulfur, was identified as one of the most abundant metabolites in the intracellular space in A. thiooxidans strain Licanantay, confirming its participation in the sulfur oxidation pathway. Amino acid profiles varied according to the growth conditions and bioleaching species. Glutamic and aspartic acid were highly abundant in intra- and extracellular extracts. Both are constituents of the extracellular matrix, and have a probable role in cell detoxification. This novel metabolomic information validates previous knowledge from in silico metabolic reconstructions based on genomic sequences, and reveals important biomining functions such as biofilm formation, energy management and stress responses.

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Regulation of Glycolysis by Pdk Functions as a Metabolic Checkpoint for Cell Cycle Quiescence in Hematopoietic Stem Cells

Takubo, K., Nagamatsu, G., Kobayashi, C. I, Nakamura-Ishizu, A., Kobayashi, H., Ikeda, E., Goda, N., Rahimi, Y., Johnson, R. S, Soga, T., Hirao, A., Suematsu, M., Suda, T.

Cell Stem Cell. 12 ( 1 ) 49 - 61 2013.01

Research paper (scientific journal), Joint Work, Accepted

Defining the metabolic programs that underlie stem cell maintenance will be essential for developing strategies to manipulate stem cell capacity. Mammalian hematopoietic stem cells (HSCs) maintain cell cycle quiescence in a hypoxic microenvironment. It has been proposed that HSCs exhibit a distinct metabolic phenotype under these conditions. Here we directly investigated this idea using metabolomic analysis and found that HSCs generate adenosine-5'-triphosphate by anaerobic glycolysis through a pyruvate dehydrogenase kinase (Pdk)-dependent mechanism. Elevated Pdk expression leads to active suppression of the influx of glycolytic metabolites into mitochondria. Pdk overexpression in glycolysis-defective HSCs restored glycolysis, cell cycle quiescence, and stem cell capacity, while loss of both Pdk2 and Pdk4 attenuated HSC quiescence, glycolysis, and transplantation capacity. Moreover, treatment of HSCs with a Pdk mimetic promoted their survival and transplantation capacity. Thus, glycolytic metabolic status governed by Pdk acts as a cell cycle checkpoint that modulates HSC quiescence and function.

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A medium-chain fatty acid as an alternative energy source in mouse preimplantation development

Yamada, M., Takanashi, K., *Hamatani, T., Hirayama, A., Akutsu, H., Fukunaga, T. Ogawa, S., Sugawara, K., Shinoda, K., Soga, T., Umezawa, A., Kuji, N., Yoshimura ,Y. and Tomita, M.

Sci. Rep. 2 930 2012.12

Research paper (scientific journal), Joint Work, Accepted

To further optimize the culturing of preimplantation embryos, we undertook metabolomic analysis of relevant culture media using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). We detected 28 metabolites: 23 embryo-excreted metabolites including 16 amino acids and 5 media-derived metabolites (e.g., octanoate, a medium-chain fatty acid (MCFA)). Due to the lack of information on MCFAs in mammalian preimplantation development, this study examined octanoate as a potential alternative energy source for preimplantation embryo cultures. No embryos survived in culture media lacking FAs, pyruvate, and glucose, but supplementation of octanoate rescued the embryonic development. Immunoblotting showed significant expression of acyl-CoA dehydrogenase and hydroxyacyl-CoA dehydrogenase, important enzymes for ß-oxidation of MCFAs, in preimplantation embryo. Furthermore, CE-TOFMS traced [1-(13)C(8)] octanoate added to the culture media into intermediate metabolites of the TCA cycle via ß-oxidation in mitochondria. These results are the first demonstration that octanoate could provide an efficient alternative energy source throughout preimplantation development.

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Metabolic profiling reveals new serum biomarkers for differentiating diabetic nephropathy

Hirayama, A., Nakashima, E., Sugimoto, M., Akiyama, S., Sato, W., Maruayama, S., Matsuo, S., Tomita, M., Yuzawa, Y., Soga, T.

Anal Bioanal Chem. 404 ( 10 ) 3101 - 3109 2012.12

Research paper (scientific journal), Joint Work, Accepted

Capillary electrophoresis coupled with time-of-flight mass spectrometry was used to explore new serum biomarkers with high sensitivity and specificity for diabetic nephropathy (DN) diagnosis, through comprehensive analysis of serum metabolites with 78 diabetic patients. Multivariate analyses were used for identification of marker candidates and development of discriminative models. Of the 289 profiled metabolites, orthogonal partial least-squares discriminant analysis identified 19 metabolites that could distinguish between DN with macroalbuminuria and diabetic patients without albuminuria. These identified metabolites included creatinine, aspartic acid, γ-butyrobetaine, citrulline, symmetric dimethylarginine (SDMA), kynurenine, azelaic acid, and galactaric acid. Significant correlations between all these metabolites and urinary albumin-to-creatinine ratios (p < 0.009, Spearman’s rank test) were observed. When five metabolites (including γ-butyrobetaine, SDMA, azelaic acid and two unknowns) were selected from 19 metabolites and applied for multiple logistic regression model, AUC value for diagnosing DN was 0.927 using the whole dataset, and 0.880 in a cross-validation test. In addition, when four known metabolites (aspartic acid, SDMA, azelaic acid and galactaric acid) were applied, the resulting AUC was still high at 0.844 with the whole dataset and 0.792 with cross-validation. Combination of serum metabolomics with multivariate analyses enabled accurate discrimination of DN patients. The results suggest that capillary electrophoresis-mass spectrometry based metabolome analysis could be used for DN diagnosis.

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Glycogen is the primary source of glucose during the lag phase of E. coli proliferation

Yamamotoya, T., Dose, H., Tian, Z., Fauré, A., Toya, Y., Honma, M., Igarashi, K., Nakahigashi, K., Soga, T., Mori, H., Matsuno, H.

Biochim Biophys Acta 1824 ( 12 ) 1142 - 1148 2012.12

Research paper (scientific journal), Joint Work, Accepted

In the studies of Escherichia coli (E. coli), metabolomics analyses have mainly been performed using steady state culture. However, to analyze the dynamic changes in cellular metabolism, we performed a profiling of concentration of metabolites by using batch culture. As a first step, we focused on glucose uptake and the behavior of the first metabolite, G6P (glucose-6-phosphate). A computational formula was derived to express the glucose uptake rate by a single cell from two kinds of experimental data, extracellular glucose concentration and cell growth, being simulated by Cell Illustrator. In addition, average concentration of G6P has been measured by CE-MS. The existence of another carbon source was suggested from the computational result. After careful comparison between cell growth, G6P concentration, and the computationally obtained curve of glucose uptake rate, we predicted the consumption of glycogen in lag phase and its accumulation as an energy source in an E. coli cell for the next proliferation. We confirmed our prediction experimentally. This behavior indicates the importance of glycogen participation in the lag phase for the growth of E. coli. This article is part of a Special Issue entitled: Computational Methods for Protein Interaction and Structural Prediction

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Analysis of liver metabolism in a rat model of heart failure

Kato, T., Niizuma, S., Inuzuka, Y., Kawashima, T., Okuda, J., Kawamoto, A., Tamai, Y., Iwanaga, Y., Soga, T., Kita, T., Kimura, T., Shioi, T.

Int. J. Cardiol. 161 ( 3 ) 130 - 136 2012.11

Research paper (scientific journal), Joint Work, Accepted

Background: Cachexia, namely body wasting, is a common complication in cases of congestive heart failure (CHF). Although, neurohumoral and immune abnormalities are associated with the condition, precisely how the imbalance of catabolism and anabolism is responsible for the wasting process is not known.
Methods: We analyzed markers of cachexia in Dahl salt-sensitive rats which show marked hypertension with preserved systolic function at 11 weeks and CHF at 17-19 weeks of age. We also analyzed the change in hepatic metabolism associated with CHF since liver plays a central role in the systemic regulation of catabolism and anabolism.
Results: In CHF rats, a failure to grow was observed and blood hepatic protein levels were decreased associated with increased blood proinflammatory cytokine levels, indicating that Dahl rats serve as a model of cardiac cachexia. Food intake was reduced, and blood sugar and insulin levels were decreased. Despite the apparent fasting condition, blood fatty acid levels were decreased and triglycerides levels were increased. In CHF rats, liver incorporated more glucose, the gene expression related to gluconeogenesis was decreased, the gene expression related to lipogenesis was increased, and the triglyceride content of the liver was increased. The paradoxical production of triglycerides synthesis in fasting rats was associated with a proinflammatory response in liver.
Conclusions: The Dahl salt-sensitive rat can be used as a model of cardiac cachexia. The cachexia was associated with abnormal hepatic metabolism that might work as a maladaptive response during the progression of CHF.

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A Metabolomic Approach to Clarifying the Effect of AST-120 on 5/6 Nephrectomized Rats by Capillary Electrophoresis with Mass Spectrometry (CE-MS)

Akiyama, Y., Takeuchi, Y., Kikuchi, K., Mishima, E., Yamamoto, Y., Suzuki, C., Toyohara, T., Suzuki, T., Hozawa, A., Ito, S., Soga, T., Abe, T.

Toxins 4 ( 11 ) 1309-1322 2012.11

Research paper (scientific journal), Joint Work, Accepted

The oral adsorbent AST-120 is composed of spherical carbon particles and has an adsorption ability for certain small-molecular-weight compounds that accumulate in patients with chronic kidney disease (CKD). So far, very few compounds are known to be adsorbed by AST-120 in vivo. To examine the effect of AST-120 in vivo, we comprehensively evaluated the plasma concentrations of 146 compounds (61 anions and 85 cations) in CKD model rats, with or without four weeks of treatment with AST-120. By capillary electrophoresis with mass spectrometry, we identified 6 anions and 17 cations that were significantly decreased by AST-120 treatment. In contrast, we also identified 2 cations that were significantly increased by AST-120. Among them, 4 anions, apart from indoxyl sulfate and hippurate, and 19 cations were newly identified in this study. The plasma levels of N-acetyl-neuraminate, 4-pyridoxate, 4-oxopentanoate, glycine, γ-guanidinobutyrate, N-γ-ethylglutamine, allantoin, cytosine, 5-methylcytosine and imidazole-4-acetate were significantly increased in the CKD model compared with the sham-operated group, and were significantly decreased by AST-120 treatment. Therefore, these 10 compounds could be added as uremic compounds that indicate the effect of AST-120 treatment. This study provides useful information not only for identifying the indicators of AST-120, but also for clarifying changes in the metabolic profile by AST-120 treatment in the clinical setting. View

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Sheathless capillary electrophoresis-mass spectrometry with a high-sensitivity porous sprayer for cationic metabolome analysis

Hirayama, A., Tomita, M., Soga, T.

Analyst 137 ( 21 ) 5026 - 5033 2012.11

Research paper (scientific journal), Joint Work, Accepted

Sheath-flow capillary electrophoresis-mass spectrometry (CE-MS) has emerged as a new tool for comprehensive analysis of charged metabolites. However, it needs to be more sensitive. Here, we report a sheathless capillary electrophoresis-electrospray ionization-mass spectrometry method for cationic metabolome analysis. This system used a high-sensitivity porous sprayer interface and 10% (v/v) acetic acid as the background electrolyte (BGE). Under optimized conditions, 53 cationic metabolites, including amino acids and their derivatives, amines, nucleic acids and small peptides, were successfully separated and selectively detected with a time-of-flight mass spectrometer. At a signal-to-noise ratio of three, the concentration detection limits for these compounds were between 0.004 and 0.8 μmol L(-1) (amount detection limit, 0.01 to 2 fmol) with pressure injection at 20.7 kPa for 5 s (2.6 nL). Compared with conventional sheath-flow CE-MS, the detection limit of the present method was increased more than 5-fold for 21 (40%) of the compounds detected. When the method was applied to the analysis of cationic metabolites obtained from human urine, there was a 10-fold increase in the number of detected peaks compared with conventional methods. More than 180 successive runs could be conducted without any problems, and only the BGE needed to be changed.

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Pilot Study of Changes in Salivary Metabolic Profiles Induced by Template Therapy

Tanaka, S., Taga, H., Maehara, K., *Kaneshima, A., Machino, M., Onuma, H., Kaneko, M., Sakagami, H., Sugimoto, M., Soga, T. and Tomita, M.

In Vivo 26 ( 6 ) 1015 - 1020 2012.11

Research paper (scientific journal), Joint Work, Accepted

Background: Occlusal raising method (so-called 'Template therapy') has been reported to alleviate various diseases and symptoms, but the underlying mechanism is not clear. We searched the low-molecular weight metabolite(s) in the saliva, the concentration of which is significantly changed by the template therapy.
Materials and methods: One female patient with headache underwent the template therapy for 12 days, and her total saliva was subjected to non-targeted analysis using capillary electrophoresis time-of-flight mass spectrometry (CE-TOF-MS).
Results: One hundred and thirteen substances were identified in the saliva. Glycine was the most abundant amino acid in the saliva, followed by alanine, serine and proline. After the start of the template therapy, her headache was alleviated, accompanied by a significant (p=0.042) increase of salivary concentration of glycine, as compared with total amino acids whereas that of other amino acids was not significantly changed. In the metabolomics profile, salivary concentration of large number of metabolites as compared with total metabolite concentration decreased, including N-acetylneuraminate (p=0.025) and p-hydroxyphenylacetate (p=0.039).
Conclusion: This pilot study demonstrated, to our knowledge for the first time, that only glycine exhibited unique changes among total metabolites, suggesting its significant role in template therapy.

Metabolomic Profiling Rationalized Pyruvate Efficacy in Cybrid Cells Harboring MELAS Mitochondrial DNA Mutations

Kami, K., Fujita, Y., Igarashi, S., Koike, S., Sugawara, S., Ikeda, S., Sato, N., Ito, M., Tanaka, M., Tomita, M., Soga, T.

Mitochondrion 12 ( 6 ) 644 - 653 2012.11

Research paper (scientific journal), Joint Work, Accepted

Pyruvate treatment was found to alleviate clinical symptoms of mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome and is highly promising therapeutic. Using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS), we measured time-changes of 161 intracellular and 85 medium metabolites to elucidate metabolic effects of pyruvate treatment on cybrid human 143B osteosarcoma cells harboring normal (2SA) and MELAS mutant (2SD) mitochondria. The results demonstrated dramatic and sustainable effects of pyruvate administration on the energy metabolism of 2SD cells, corroborating pyruvate as a metabolically rational treatment regimen for improving symptoms associated with MELAS and possibly other mitochondrial diseases.

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In vivo role of aldehyde reductase

Takahashi, M., Miyata, S., Fujii, J., Inai, Y., Ueyama, S., Araki, M., Soga, T., Fujinawa, R., Nishitani C., Ariki S., Shimizu T., Abe T., Ihara Y., Nishikimi, M., Kozutsumi Y., Taniguchi N., and Kuroki, Y.

Biochim. biophys. Acta 1820 ( 11 ) 1787 - 1796 2012.11

Research paper (scientific journal), Joint Work, Accepted

Background: Aldehyde reductase (AKR1A; EC 1.1.1.2) catalyzes the reduction of various types of aldehydes. To ascertain the physiological role of AKR1A, we examined AKR1A knockout mice.
Methods: Ascorbic acid concentrations in AKR1A knockout mice tissues were examined, and the effects of human AKR1A transgene were analyzed. We purified AKR1A and studied the activities of glucuronate reductase and glucuronolactone reductase, which are involved in ascorbic acid biosynthesis. Metabolomic analysis and DNA microarray analysis were performed for a comprehensive study of AKR1A knockout mice.
Results: The levels of ascorbic acid in tissues of AKR1A knockout mice were significantly decreased which were completely restored by human AKR1A transgene. The activities of glucuronate reductase and glucuronolactone reductase, which are involved in ascorbic acid biosynthesis, were suppressed in AKR1A knockout mice. The accumulation of d-glucuronic acid and saccharate in knockout mice tissue and the expression of acute-phase proteins such as serum amyloid A2 are significantly increased in knockout mice liver.
Conclusions: AKR1A plays a predominant role in the reduction of both d-glucuronic acid and d-glucurono-γ-lactone in vivo. The knockout of AKR1A in mice results in accumulation of d-glucuronic acid and saccharate as well as a deficiency of ascorbic acid, and also leads to upregulation of acute phase proteins.
General significance: AKR1A is a major enzyme that catalyzes the reduction of d-glucuronic acid and d-glucurono-γ-lactone in vivo, besides acting as an aldehyde-detoxification enzyme. Suppression of AKR1A by inhibitors, which are used to prevent diabetic complications, may lead to the accumulation of d-glucuronic acid and saccharate.

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Mitochondrial dysfunction associated with increased oxidative stress and α-synuclein accumulation in PARK2 iPSC-derived neurons and postmortem brain tissue

Imaizumi, Y., Okada, Y., Akamatsu, W., Koike, M., Kuzumaki, N., Hayakawa, H., Nihira, T., Kobayashi, T., Ohyama, M., ,Sato, S., Takanashi, M., Funayama, M., Hirayama, A., Soga, T., Hishiki, T., Suematsu, M., Yagi, T., Ito, D., Kosakai, A., Hayashi, K., Shouji, M., Nakanishi, A., Suzuki, N., Mizuno, Y., Mizushima, N., Amagai, M., Uchiyama, Y., Mochizuki, H., Hattori, N., Okano H.

Mol Brain. 5 35 2012.10

Research paper (scientific journal), Joint Work, Accepted

Background: Parkinson's disease (PD) is a neurodegenerative disease characterized by selective degeneration of dopaminergic neurons in the substantia nigra (SN). The familial form of PD, PARK2, is caused by mutations in the parkin gene. parkin-knockout mouse models show some abnormalities, but they do not fully recapitulate the pathophysiology of human PARK2.
Results: Here, we generated induced pluripotent stem cells (iPSCs) from two PARK2 patients. PARK2 iPSC-derived neurons showed increased oxidative stress and enhanced activity of the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. iPSC-derived neurons, but not fibroblasts or iPSCs, exhibited abnormal mitochondrial morphology and impaired mitochondrial homeostasis. Although PARK2 patients rarely exhibit Lewy body (LB) formation with an accumulation of α-synuclein, α-synuclein accumulation was observed in the postmortem brain of one of the donor patients. This accumulation was also seen in the iPSC-derived neurons in the same patient.
Conclusions: Thus, pathogenic changes in the brain of a PARK2 patient were recapitulated using iPSC technology. These novel findings reveal mechanistic insights into the onset of PARK2 and identify novel targets for drug screening and potential modified therapies for PD.

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Dramatic Increase in Glycerol Biosynthesis upon Oxidative Stress in the Anaerobic Protozoan Parasite Entamoeba histolytica

Husain, A., Sato, D., Jeelani, G., Soga, T., Nozaki, T.

PLoS Negl Trop Dis. 6 ( 9 ) e1831 2012.09

Research paper (scientific journal), Joint Work, Accepted

Entamoeba histolytica, a microaerophilic enteric protozoan parasite, causes amebic colitis and extra intestinal abscesses in millions of inhabitants of endemic areas. Trophozoites of E. histolytica are exposed to a variety of reactive oxygen and nitrogen species during infection. Since E. histolytica lacks key components of canonical eukaryotic anti-oxidative defense systems, such as catalase and glutathione system, alternative not-yet-identified anti-oxidative defense strategies have been postulated to be operating in E. histolytica. In the present study, we investigated global metabolic responses in E. histolytica in response to H(2)O(2)- and paraquat-mediated oxidative stress by measuring charged metabolites on capillary electrophoresis and time-of-flight mass spectrometry. We found that oxidative stress caused drastic modulation of metabolites involved in glycolysis, chitin biosynthesis, and nucleotide and amino acid metabolism. Oxidative stress resulted in the inhibition of glycolysis as a result of inactivation of several key enzymes, leading to the redirection of metabolic flux towards glycerol production, chitin biosynthesis, and the non-oxidative branch of the pentose phosphate pathway. As a result of the repression of glycolysis as evidenced by the accumulation of glycolytic intermediates upstream of pyruvate, and reduced ethanol production, the levels of nucleoside triphosphates were decreased. We also showed for the first time the presence of functional glycerol biosynthetic pathway in E. histolytica as demonstrated by the increased production of glycerol 3-phosphate and glycerol upon oxidative stress. We proposed the significance of the glycerol biosynthetic pathway as a metabolic anti-oxidative defense system in E. histolytica.

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Human blood metabolite timetable indicates internal body time

Kasukawa, T., Sugimoto, M., Hida, A., Minami, Y., Mori, M., Honma, S., Honma, K., Mishima, K., Soga, T., Ueda, H.R.

Proc. Natl. Acad. Sci. USA 109 ( 37 ) 15036 - 15041 2012.09

Research paper (scientific journal), Joint Work, Accepted

A convenient way to estimate internal body time (BT) is essential for chronotherapy and time-restricted feeding, both of which use body-time information to maximize potency and minimize toxicity during drug administration and feeding, respectively. Previously, we proposed a molecular timetable based on circadian-oscillating substances in multiple mouse organs or blood to estimate internal body time from samples taken at only a few time points. Here we applied this molecular-timetable concept to estimate and evaluate internal body time in humans. We constructed a 1.5-d reference timetable of oscillating metabolites in human blood samples with 2-h sampling frequency while simultaneously controlling for the confounding effects of activity level, light, temperature, sleep, and food intake. By using this metabolite timetable as a reference, we accurately determined internal body time within 3 h from just two anti-phase blood samples. Our minimally invasive, molecular-timetable method with human blood enables highly optimized and personalized medicine.

L-Carnitine Prevents the Development of Ventricular Fibrosis and Heart Failure with Preserved Ejection Fraction in Hypertensive Heart Disease

Omori, Y., Ohtani, T., Sakata, Y., Mano, T., Takeda, Y., Tamaki, S., Tsukamoto. Y.,　Kamimura, D., Aizawa, Y., Miwa, T., Komuro, I., Soga, T., Yamamoto, K.

J. Hypertens. 30 ( 9 ) 1834 - 1844 2012.09

Research paper (scientific journal), Joint Work, Accepted

Objectives: Prognosis of heart failure with preserved ejection fraction (HFpEF) remains poor because of unknown pathophysiology and unestablished therapeutic strategy. This study aimed to identify a potential therapeutic intervention for HFpEF through metabolomics-based analysis.
Methods and results: Metabolomics with capillary electrophoresis time-of-flight mass spectrometry was performed using plasma of Dahl salt-sensitive rats fed high-salt diet, a model of hypertensive HFpEF, and showed decreased free-carnitine levels. Reassessment with enzymatic cycling method revealed the decreased plasma and left-ventricular free-carnitine levels in the HFpEF model. Urinary free-carnitine excretion was increased, and the expression of organic cation/carnitine transporter 2, which transports free-carnitine into cells, was down-regulated in the left ventricle (LV) and kidney in the HFpEF model. L-Carnitine was administered to the hypertensive HFpEF model. L-Carnitine treatment restored left-ventricular free-carnitine levels, attenuated left-ventricular fibrosis and stiffening, prevented pulmonary congestion, and improved survival in the HFpEF model independent of the antihypertensive effects, accompanied with increased expression of fatty acid desaturase (FADS) 1/2, rate-limiting enzymes in forming arachidonic acid, and enhanced production of arachidonic acid, a precursor of prostacyclin, and prostacyclin in the LV. In cultured cardiac fibroblasts, L-carnitine attenuated the angiotensin II-induced collagen production with increased FADS1/2 expression and enhanced production of arachidonic acid and prostacyclin. L-Carnitine-induced increase of arachidonic acid was canceled by knock-down of FADS1 or FADS2 in cultured cardiac fibroblasts. Serum free-carnitine levels were decreased in HFpEF patients.
Conclusions: L-carnitine supplementation attenuates cardiac fibrosis by increasing prostacyclin production through arachidonic acid pathway, and may be a promising therapeutic option for HFpEF

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A Yeast Metabolite Extraction Protocol Optimised for Time-Series Analyses

Sasidharan , K., Soga, T., Tomita, M., Murray, D.B.

PLoS One 7 ( 8 ) e44283 2012.08

Research paper (scientific journal), Joint Work, Accepted

There is an increasing call for the absolute quantification of time-resolved metabolite data. However, a number of technical issues exist, such as metabolites being modified/degraded either chemically or enzymatically during the extraction process. Additionally, capillary electrophoresis mass spectrometry (CE-MS) is incompatible with high salt concentrations often used in extraction protocols. In microbial systems, metabolite yield is influenced by the extraction protocol used and the cell disruption rate. Here we present a method that rapidly quenches metabolism using dry-ice ethanol bath and methanol N-ethylmaleimide solution (thus stabilising thiols), disrupts cells efficiently using bead-beating and avoids artefacts created by live-cell pelleting. Rapid sample processing minimised metabolite leaching. Cell weight, number and size distribution was used to calculate metabolites to an attomol/cell level. We apply this method to samples obtained from the respiratory oscillation that occurs when yeast are grown continuously.

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Self-enhancement of hepatitis C virus replication by promotion of specific sphingolipid biosynthesis

Hirata, Y., Ikeda, K., Sudoh, M., Tokunaga, Y., Suzuki, A., Weng, L., Ohta, M., Tobita, Y., Okano, K., Ozeki, K., Kawasaki, K., Tsukuda, T., Katsume, A., Aoki, Y., Umehara, T., Sekiguchi, S., Toyoda, T., Shimotohno, K., Soga, T., Nishijima, M., Taguchi, R., Kohara, M.

PLoS Pathog. 8 ( 8 ) e1002860 2012.08

Research paper (scientific journal), Joint Work, Accepted

Lipids are key components in the viral life cycle that affect host-pathogen interactions. In this study, we investigated the effect of HCV infection on sphingolipid metabolism, especially on endogenous SM levels, and the relationship between HCV replication and endogenous SM molecular species. We demonstrated that HCV induces the expression of the genes (SGMS1 and 2) encoding human SM synthases 1 and 2. We observed associated increases of both total and individual sphingolipid molecular species, as assessed in human hepatocytes and in the detergent-resistant membrane (DRM) fraction in which HCV replicates. SGMS1 expression had a correlation with HCV replication. Inhibition of sphingolipid biosynthesis with a hepatotropic serine palmitoyltransferase (SPT) inhibitor, NA808, suppressed HCV-RNA production while also interfering with sphingolipid metabolism. Further, we identified the SM molecular species that comprise the DRM fraction and demonstrated that these endogenous SM species interacted with HCV nonstructural 5B polymerase to enhance viral replication. Our results reveal that HCV alters sphingolipid metabolism to promote viral replication, providing new insights into the formation of the HCV replication complex and the involvement of host lipids in the HCV life cycle.

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Non-targeted metabolite profiling in activated macrophage secretion

Sugimoto, M., Sakagami, H., Yokote, Y., Onuma, H., Kaneko, M., Mori, M., Sakaguchi, Y., Soga, T., Tomita, M.

Metabolomics 8 ( 4 ) 624 - 633 2012.08

Research paper (scientific journal), Joint Work, Accepted

Periodontal diseases are inflammatory infectious diseases that affect the periodontal tissue. Macrophages play a central role in inflammatory conditions, leading to the destruction of tissues. Identifying the signaling molecules secreted by macrophages would be valuable to the study of these diseases. Here, we present non-targeted analysis using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) for the profiling of extracellular metabolites released during macrophage activation. Lipopolysaccharide (LPS)-induced activation of a mouse macrophage-like cell line RAW264.7 was used as a model system. Cells were treated without (control) or with LPS for 22 h and, after washing, were incubated for 1 h in phosphate-buffered saline. The accumulation of metabolites in the culture supernatant was monitored. LPS treatment significantly enhanced the accumulation of prostaglandins, tumor necrosis factor-α, nitric oxide and citrulline in the culture medium. RAW264.7 cells produced 46 metabolites and 66% of these showed significant changes (P < 0.05) following cell activation. In particular, the production of leucine, hypoxanthine, choline, putrecine, N 8-acetylspermidine, succinate, itaconate, and 4-methyl-2-oxopentanoate was significantly increased by cell activation (P < 0.001). Significantly elevated production of lactate and glycine was also observed. Here, we present the first catalog of the up and down-regulation of the various metabolites secreted by macrophages following inflammatory activation.

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The emerging role of fumarate as an oncometabolite

Yang, M., Soga, T., Pollard, P.J., Adam, J.

Front. Oncol. 2 85 2012.07

Research paper (scientific journal), Joint Work, Accepted

The drive to understand how altered cellular metabolism and cancer are linked has caused a paradigm shift in the focus of cancer research. The discovery of a mutated metabolic enzyme, isocitrate dehydrogenase 1, that leads to accumulation of the oncometabolite 2-hydroxyglutarate, provided significant direct evidence that dysfunctional metabolism plays an important role in oncogenesis. Striking parallels exist with the Krebs cycle enzyme fumarate hydratase (FH), a tumor suppressor, whose mutation is associated with the development of leiomyomata, renal cysts, and tumors. Loss of FH enzymatic activity results in accumulation of intracellular fumarate which has been proposed to act as a competitive inhibitor of 2-oxoglutarate-dependent oxygenases including the hypoxia-inducible factor (HIF) hydroxylases, thus activating oncogenic HIF pathways. Interestingly, our studies have questioned the role of HIF and have highlighted other candidate mechanisms, in particular the non-enzymatic modification of cysteine residues (succination) that could lead to disruption or loss of protein functions, dysfunctional cell metabolism and cell signaling. Here, we discuss the evidence for proposing fumarate as an onco-metabolite.

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Clarification as to why alcoholic beverages have the ability to induce superconductivity in Fe1+dTe1−xSx

Deguchi, K., Sato, D., Sugimoto, M., Hara, H., Kawasaki, Y., Demura, S., Watanabe, T., Denholme, S. J., Okazaki, H., Ozaki, T., Yamaguchi, T., Takeya, H., Soga, T., Tomita, M., Takano, Y.

Supercond. Sci. Technol. 25 ( 8 ) 084025 2012.07

Research paper (scientific journal), Joint Work, Accepted

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mTORC1 is Essential for Leukemia propagation but Not Stem Cell Self- renewl

Hoshi, T., Tadokoro, Y., Naka, K., Ooshio, T., Muraguchi, T., Sugiyama, N., Soga, T., Araki, K., Yamamura, K., Hirao, A.

J. Clin. Invest. 122 ( 6 ) 2114 - 2129 2012.06

Research paper (scientific journal), Joint Work, Accepted

Although dysregulation of mTOR complex 1 (mTORC1) promotes leukemogenesis, how mTORC1 affects established leukemia is unclear. We investigated the role of mTORC1 in mouse hematopoiesis using a mouse model of conditional deletion of Raptor, an essential component of mTORC1. Raptor deficiency impaired granulocyte and B cell development but did not alter survival or proliferation of hematopoietic progenitor cells. In a mouse model of acute myeloid leukemia (AML), Raptor deficiency significantly suppressed leukemia progression by causing apoptosis of differentiated, but not undifferentiated, leukemia cells. mTORC1 did not control cell cycle or cell growth in undifferentiated AML cells in vivo. Transplantation of Raptor-deficient undifferentiated AML cells in a limiting dilution revealed that mTORC1 is essential for leukemia initiation. Strikingly, a subset of AML cells with undifferentiated phenotypes survived long-term in the absence of mTORC1 activity. We further demonstrated that the reactivation of mTORC1 in those cells restored their leukemia-initiating capacity. Thus, AML cells lacking mTORC1 activity can self-renew as AML stem cells. Our findings provide mechanistic insight into how residual tumor cells circumvent anticancer therapies and drive tumor recurrence.

Metabolic Profiling of the Protozoan Parasite Entamoeba invadens Revealed Activation of Unpredicted Pathway during Encystation

Jeelani, G., Sato, D., Husain, A., Cadiz, A.E., Sugimoto, M., Soga, T., Suematsu, M., Nozaki, T.

PLoS One 7 ( 5 ) e37740 2012.05

Research paper (scientific journal), Joint Work, Accepted

Encystation, which is cellular differentiation from the motile, proliferative, labile trophozoite form to the dormant, resistant cyst form, is a crucial process found in parasitic and free-living protozoa such as Entamoeba, Giardia, Acanthamoeba, and Balamuthia. Since encystation is an essential process to deal with the adverse external environmental changes during the life cycle, and often integral to the transmission of the diseases, biochemical understanding of the process potentially provides useful measures against the infections caused by this group of protozoa. In this study, we investigated metabolic and transcriptomic changes that occur during encystation in Entamoeba invadens, the reptilian sibling of mammal-infecting E. histolytica, using capillary electrophoresis-tandem mass spectrometry-based metabolite profiling and DNA microarray-based expression profiling. As the encystation progressed, the levels of majority of metabolites involved in glycolysis and nucleotides drastically decreased, indicating energy generation is ceased. Furthermore, the flux of glycolysis was redirected toward chitin wall biosynthesis. We found remarkable temporal increases in biogenic amines such as isoamylamine, isobutylamine, and cadaverine, during the early period of encystation, when the trophozoites form large multicellular aggregates (precyst). We also found remarkable induction of γ-aminobutyric acid (GABA) during encystation. This study has unveiled for the first time the dynamics of the transcriptional and metabolic regulatory networks during encystation, and should help in better understanding of the process in pathogenic eukaryotes, and further development of measures controlling infections they cause.

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Antihypertensive Effects of Unripe Persimmon (Diospyros kaki L.cv. Hiratanenashi) Fruit and Its Component in Spontaneously Hypertensive Rats

Liu, C., Kurakane, S., Takita, J., Itano R., Soga, T., Oikawa A., and Igarashi, K.

Food Sci. Technol. Res. 18 ( 3 ) 391 - 398 2012.05

Research paper (scientific journal), Joint Work, Accepted

The feeding of unripe persimmon (PP) (‘Hiratanenashi’, pollination variant) and soluble tannin (PST), as its component, on blood pressure was examined in spontaneously hypertensive rats (SHR). Systolic blood pressure (SBP) was decreased by PP and PST administration for 2-3 days and increased after switching to the basal diet alone. Increases in serum nitric oxide level and liver NOS3 gene expression in SHR fed PP suggests that the induction of nitric oxide formation by PP administration might be involved in decreasing SBP. The decrease in ACE activity by PST administration suggests that, as a component of PP, PST mainly lowers SBP through its angiotensin I-converting enzyme (ACE) inhibitory effect. Scopo-letin, contained as a minor component in PP and PST, decreased SBP by single oral administration, sug-gesting that it also might be involved in lowering SBP.

Serum Glycerophosphate Levels are Increased in Japanese Men with Type 2 Diabetes

Dimon, M., Soga, T., Hozawa, A., Oizumi, T., Kaino, W., Takase, K., Karasawa, S., Jimbu, Y., Wada, K., Kameda, W., Susa, S., Kayama, T., Saito, K., Tomita, M., Kato, T.

Internal Medicine 51 ( 6 ) 545 - 551 2012.03

Research paper (scientific journal), Joint Work

Objective To identify metabolites showing changes in serum levels among Japanese male with diabetes.
Methods We performed metabolite profiling by coupling capillary electrophoresis with electrospray ionization time-of-flight mass spectrometry using fasting serum samples from Japanese male subjects with diabetes (n=17), impaired glucose tolerance (IGT; n=5) and normal glucose tolerance (NGT; n=14).
Results Other than the expected differences in characteristics related to abnormal glucose metabolism, the percent body fat was significantly different among subjects with diabetes, IGT and NGT (27.3±6.2, 22.2±4.5 and 19.2±6.0%, respectively, p=0.0022). Therefore, percent body fat was considered as a possible confounding factor in subsequent analyses. Of 560 metabolites detected using our platform, the levels of 74 metabolites were quantified in all of the serum samples. Significant differences between diabetes and NGT were observed for 24 metabolites. The top-ranked metabolite was glycerol-3-phophate (glycerophosphate), which was significantly higher in subjects with diabetes than in those with NGT, even after Bonferroni correction for multiple testing (11.7±3.6 vs. 6.4±1.9 μM, respectively; corrected p=0.0222). Stepwise multiple regression analyses revealed that serum glycerophosphate levels were significantly correlated with 2-h plasma glucose after a 75-g oral glucose tolerance test (r=0.553, p=0.0005), independently of other characteristics, including FPG and HbA1c.
Conclusion Serum glycerophosphate levels were found to be elevated in Japanese men with diabetes, and correlated with 2-h PG, independent of FPG and HbA1c. Namely, serum glycerophosphate level at fasting condition can be a marker for predicting glucose intolerance. These results warrant further studies to evaluate the relevance of glycerophosphate in the pathophysiology of diabetes.

Changes in the Charged Metabolite and Sugar Profiles of Pasteurized and Unpasteurized Japanese Sake with Storage

Sugimoto, M., Kaneko, M., Onuma, H., Sakaguchi, Y., Mori, M., Abe, S., Soga, T., Tomita, M.

J. Agric. Food Chem. 60 ( 10 ) 2586 - 2593 2012.03

Research paper (scientific journal), Joint Work, Accepted

Japanese sake (rice wine) is commonly heat treated (pasteurized) to maintain its quality. In this study, temporal changes in the metabolite profiles of pasteurized and unpasteurized sake were investigated during storage. Metabolomic analyses were conducted for eight sets of pasteurized and unpasteurized sake obtained from single process batches stored at 8 or 20 °C for 0, 1, 2, or 4 months. Capillary electrophoresis time-of-flight mass spectrometry and liquid chromatography tandem mass spectrometry were used to obtain charged metabolite and sugar profiles, respectively. The total amino acid concentration decreased with storage, and the decrease was faster in pasteurized sake than in unpasteurized. The organic acid concentrations were relatively constant in both types of sake. Peptide and glucose concentrations increased and polysaccharide concentrations decreased in unpasteurized sake, while they were relatively constant in pasteurized sake. Rather than stabilizing the sake metabolite profile during storage, pasteurization results in characteristic changes compared to unpasteurized sake.

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Bioinfomatics Tools for Mass Spectroscopy-Based Metabolomic Data Processing and Analysis

Sugimoto, M., Kawakami, M., Robert, M., Soga, T., Tomita, M.

Curr. Bioinf. 7 ( 1 ) 96 - 108 2012.03

Joint Work, Accepted

Biological systems are increasingly being studied in a holistic manner, using omics approaches, to provide quantitative and qualitative descriptions of the diverse collection of cellular components. Among the omics approaches, metabolomics, which deals with the quantitative global profiling of small molecules or metabolites, is being used extensively to explore the dynamic response of living systems, such as organelles, cells, tissues, organs and whole organisms, under diverse physiological and pathological conditions. This technology is now used routinely in a number of applications, including basic and clinical research, agriculture, microbiology, food science, nutrition, pharmaceutical research, environmental science and the development of biofuels. Of the multiple analytical platforms available to perform such analyses, nuclear magnetic resonance and mass spectrometry have come to dominate, owing to the high resolution and large datasets that can be generated with these techniques. The large multidimensional datasets that result from such studies must be processed and analyzed to render this data meaningful. Thus, bioinformatics tools are essential for the efficient processing of huge datasets, the characterization of the detected signals, and to align multiple datasets and their features. This paper provides a state-of-the-art overview of the data processing tools available, and reviews a collection of recent reports on the topic. Data conversion, pre-processing, alignment, normalization and statistical analysis are introduced, with their advantages and disadvantages, and comparisons are made to guide the reader.

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Profiling of the Charged Metabolites of Traditional Herbal Medicines Using Capillary Electrophoresis time-of-flight Mass Spectrometry

Iino, K., Sugimoto, M., Soga, T., Tomita, M.

Metabolomics 8 ( 1 ) 99 - 108 2012.02

Research paper (scientific journal), Joint Work, Accepted

The quantification of a small number of bioactive components in herbal medicines is often inadequate when attempting to elucidate a medicine’s biological effects. Despite rapid advances in analytical technologies, obtaining comprehensive metabolomic profiles of herbal medicines remains difficult, due to the complexity of natural product mixtures. Toki-Shakuyaku-San is a Chinese medicine used widely to treat gynecological and obstetric disorders, such as infertility, dysmenorrhea, toxemia during pregnancy and neural dysfunction. It consists of Angelica acutiloba Radix (Toki), Cnidium officinale Rhizoma (Senkyu), Paeonia lactiflora Radix (Shakuyaku), Atractylodes lancea Rhizoma (Sojutsu), Alisma orientale Rhizoma (Takusha) and Poria cocos Hoelen (Bukuryo). To elucidate the composition of these herbal medicines individually, we conducted non-targeted profiling analyses of extracts of these herbs using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS), which allows the simultaneous quantification of hundreds of charged metabolites. In total, 737 ± 183.1 (average ± SD) metabolite-derived features were observed, and of these, 119 metabolites were identified. Score plots of principal component analysis (PCA) showed a clear cluster including Shakuyaku, Bukuryo, and Sojutsu, while the other three herbs were distributed over PCA spaces. Loading plots revealed that amino acids and shikimate-derived alkaloids were the predominant metabolite constituents. Hierarchical clustering analysis revealed that few clusters overlapped in the herbal medicines tested. This report is the first demonstration of the characterization of a herbal medicine using large-scale metabolomic analysis, which is complementary to traditional quality control methods.

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MMMDB: Mouse Multiple Tissue Metabolome Database

Sugimoto, M., Ikeda, S., Niigata, K., Tomita, M., Sato, H., Soga, T.

Nucl. Acids Res. 40 ( D1 ) D809 - D814 2012.01

Research paper (scientific journal), Joint Work, Accepted

The Mouse Multiple Tissue Metabolome Database (MMMDB) provides comprehensive and quantitative metabolomic information for multiple tissues from single mice. Manually curated databases that integrate literature-based individual metabolite information have been available so far. However, data sets on the absolute concentration of a single metabolite integrated from multiple resources are often difficult to be used when different metabolomic studies are compared because the relative balance of the multiple metabolite concentrations in the metabolic pathways as a snapshot of a dynamic system is more important than the absolute concentration of a single metabolite. We developed MMMDB by performing non-targeted analyses of cerebra, cerebella, thymus, spleen, lung, liver, kidney, heart, pancreas, testis and plasma using capillary electrophoresis time-of-flight mass spectrometry and detected 428 non-redundant features from which 219 metabolites were successfully identified. Quantified concentrations of the individual metabolites and the corresponding processed raw data; for example, the electropherograms and mass spectra with their annotations, such as isotope and fragment information, are stored in the database. MMMDB is designed to normalize users' data, which can be submitted online and used to visualize overlaid electropherograms. Thus, MMMDB allows newly measured data to be compared with the other data in the database. MMMDB is available at: http://mmmdb.iab.keio.ac.jp.

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Acetaminophen-induced Hepatotoxicity in a Liver Tissue Model Consisting of Primary Hepatocytes Assembling Around an Endothelial Cell Network

Toyoda, Y., Tamai, M., Kashikura, K., Kobayashi, S., Fujiyama, Y., Soga, T., Tagawa, Y.

Drug Metab. Dispos. 40 ( 1 ) 169 - 177 2012.01

Research paper (scientific journal), Joint Work, Accepted

Primary hepatocytes have been used in drug development for the evaluation of hepatotoxicity of candidate compounds. However, the rapid depression of their hepatic characters in vitro must be improved to predict toxicity with higher accuracy. We have hypothesized that a well organized tissue construct that includes nonparenchymal cells and appropriate scaffold material(s) could overcome this difficulty by remediating the viability and physiological function of primary hepatocytes. In this study, we constructed an in vitro liver tissue model, consisting of mouse primary hepatocytes assembling around an endothelial cell network on Engelbreth-Holm-Swarm gel, and examined its response to acetaminophen treatment. The increase in lactate dehydrogenase release after the exposure to acetaminophen was induced earlier in the liver tissue model than in monolayer hepatocytes alone, suggesting that the tissue model was more sensitive to an acetaminophen-induced toxicity. On the basis of our results, we conclude that liver tissue models of this kind may enhance the responses of hepatocytes against xenobiotics via the maintenance of hepatic genes and functions such as cytochrome P450s. These findings will contribute to the development of more accurate systems for evaluating hepatotoxicity.

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GTP-dependent RNA 3′-terminal phosphate cyclase from the hyperthermophilic archaeon Pyrococcus furiosus

Sato, A., Soga, T., Igarashi, K., Takesue, K., Tomita, M., Kanai, A.

Genes Cell 16 ( 12 ) 1190 - 1199 2011.12

Research paper (scientific journal), Joint Work, Accepted

We discovered that the PF1549 gene in Pyrococcus furiosus encodes a very heat-stable RNA 3'-terminal phosphate cyclase (Pf-Rtc). Although all previously reported Rtc proteins are ATP-dependent enzymes, we found that Pf-Rtc requires GTP for its cyclase activity at 95 °C. Low-level activation of the enzyme was also observed in the presence of dGTP but not other dNTPs, indicating that the guanine base is very important for Pf-Rtc activity. We analyzed a series of GTP analogues and found that the conversion from GTP to GMP is important for Pf-Rtc activity and that an excess of GMP inhibits this activity. Gel-shift analysis clearly showed that the RNA-binding activity of Pf-Rtc is totally dependent on the linear form of the 3'-terminal phosphate, with an apparent K(d) value of 20 nm at 95°C. Furthermore, we found that Pf-Rtc may contribute to GTP-dependent RNA ligation activity through the PF0027 protein (a 2'-5' RNA ligase-like protein in P. furiosus). The possible roles of Pf-Rtc and the importance of terminal phosphate structures in RNA are discussed.

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Disruption of HIF-1α in Hepatocytes Impairs Glucose Metabolism in Diet-induced Obesity Mice

Ochiai, D., Goda, N., Hishiki, T., Kanai, M., Senoo-Matsuda, N., Soga, T., Johnson, R.S., Yoshimura, Y., Suematsu, M.

Biochem. Biophys. Res. Commun 415 ( 3 ) 445 - 449 2011.11

Research paper (scientific journal), Joint Work, Accepted

The liver plays a central role in glucose homeostasis in the whole-body by responding to environmental factors including nutrients, hormones, and oxygen. In conditions of metabolic overload such as diabetes mellitus and obesity, coordinated regulation between oxygen supply and consumption has been reported to be disrupted and subsequently cause tissue hypoxia, although pathological significance of the disease-related hypoxia remains elusive. To investigate the role of tissue hypoxia in the liver on systemic glucose homeostasis, mice lacking HIF-1α gene, a critical component of a master regulator of hypoxic response, in hepatocytes were exposed to high fat/sucrose diet (HFSD). Exposure to HFSD for 5 weeks elicited liver hypoxia with a transient increase in HIF-1α protein expression in the liver of control mice. Glucose disposal was marginally impaired in control mice when challenged oral glucose tolerance test, but such impairment was enhanced in the mutant mice. This alteration was accompanied by a complete inhibition of glucokinase induction with a significant reduction of hepatic glucose uptake. Mice fed HFSD for 20 weeks exhibited fasting hyperglycemia and glucose intolerance, whereas these metabolic phenotypes deteriorated considerably with severe insulin resistance in skeletal muscles and adipose tissues in the mutant mice. These findings suggest that HIF-1 in hepatocytes plays protective roles against the progression of diabetes mellitus.

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Renal Cyst Formation in Fh1-Deficient Mice Is Independent of the Hif/Phd Pathway: Roles for Fumarate in KEAP1 Succination and Nrf2 Signaling

Adam, J., Hatipoglu, E., O’Flaherty, L., Ternette, N., Sahgal, N., Lockstone, H., Baban D., Nye, E., Stamp, G.W., Wolhuter, K., Stevens, M., Fischer, R., Carmeliet, P., Maxwell, P.H., Pugh, C.W., Frizzell, N., Soga, T., Kessler, B.M., El-Bahrawy, M., Ratcliffe, P.J., Pollard, P.J.

Cancer Cell 20 ( 4 ) 524 - 537 2011.10

Research paper (scientific journal), Joint Work, Accepted

The Krebs cycle enzyme fumarate hydratase (FH) is a human tumor suppressor whose inactivation is associated with the development of leiomyomata, renal cysts, and tumors. It has been proposed that activation of hypoxia inducible factor (HIF) by fumarate-mediated inhibition of HIF prolyl hydroxylases drives oncogenesis. Using a mouse model, we provide genetic evidence that Fh1-associated cyst formation is Hif independent, as is striking upregulation of antioxidant signaling pathways revealed by gene expression profiling. Mechanistic analysis revealed that fumarate modifies cysteine residues within the Kelch-like ECH-associated protein 1 (KEAP1), abrogating its ability to repress the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-mediated antioxidant response pathway, suggesting a role for Nrf2 dysregulation in FH-associated cysts and tumors.

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Sex-dependent Hepatic Transcripts and Metabolites in the Development of Glucose Intolerance and Insulin Resistance in Zucker Diabetic Fatty Rats

Gustavsson, C., Soga, T., Wahlström, E, Vesterlund, M, Azimi, A., Norstedt, G., Tollet-Egnell, P.

J. Mol. Endcrinol. 47 ( 2 ) 129 - 143 2011.10

Research paper (scientific journal), Joint Work, Accepted

Male Zucker diabetic fatty (mZDF) rats spontaneously develop type 2 diabetes, whereas females only become diabetic when fed a diabetogenic high-fat diet (high-fat-fed female ZDF rat, HF-fZDF). The aim of this study was to investigate if differences in liver functions could provide clues to this sex difference. Non-diabetic obese fZDF rats were compared with either mZDF or HF-fZDF regarding hepatic molecular profiles, to single out those components that might be protective in the females. High-fat feeding in fZDF led to enhanced weight gain, increased blood glucose and insulin levels, reduced insulin sensitivity and a trend towards reduced glucose tolerance, indicative of a prediabetic state. mZDF rats were diabetic, with low levels of insulin, high levels of glucose, reduced insulin sensitivity and impaired glucose tolerance. Transcript profiling and capillary electrophoresis time-of-flight mass spectrometry were used to indentify hepatic transcripts and metabolites that might be related to this. Many diet-induced alterations in transcript and metabolite levels in female rats were towards a 'male-like' phenotype, including reduced lipogenesis, increased fatty acid (FA) oxidation and increased oxidative stress responses. Alterations detected at the level of hepatic metabolites, indicated lower capacity for glutathione (GSH) production in male rats, and higher GSH turnover in females. Taken together, this could be interpreted as if anabolic pathways involving lipogenesis and lipid output might limit the degree of FA oxidation and oxidative stress in female rats. Together with a greater capacity to produce GSH, these hepatic sex differences might contribute to the sex-different development of diabetes in ZDF rats.

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Metabolomic profiling of the autosomal dominant polycystic kidney disease rat model

Toyohara, T., Suzuki, T., Akiyama, Y., Yoshihara, D., Takeuchi, Y., Mishima, E., Kikuchi, K., Suzuki, C., Tanemoto, M., Ito, S., Nagao, S., Soga T., Abe, T.

Clin Exp Nephrol 15 ( 5 ) 676 - 687 2011.10

Research paper (scientific journal), Accepted

Background: Autosomal dominant polycystic kidney disease (ADPKD) is an inherited systemic disease characterized by renal cyst expansion, resulting in renal failure. With the progression of renal damage, the accumulation of uremic compounds is recently reported to subsequently cause further renal damage and hypertension. Finding uremic toxins and sensitive markers for detecting the early stage of ADPKD is necessary to clarify its pathophysiological process and to prevent its progression. The aim of this study was to analyze the profile of uremic retention solutes of ADPKD by capillary electrophoresis-mass spectrometry (CE-MS) using the Han:SPRD rat model.
Methods: Two hundred and ninety-seven cations and 190 anions were comprehensively analyzed by CE-MS in Han:SPRD rats and control rats.
Results: We found 21 cations and 19 anions that accumulated significantly in the heterozygous (Cy/+) ADPKD rat model compared with control rats. Among the compounds, increases in 5-methyl-2'-deoxycytidine, glucosamine, ectoine, allantoate, α-hydroxybenzoate, phenaceturate and 3-phenylpropionate and decreases in 2-deoxycytidine, decanoate and 10-hydroxydecanoate were newly identified in the ADPKD Cy/+ rats.
Conclusion: We identified uremic retention solutes in ADPKD Cy/+ rats. Compounds related to ADPKD could be useful markers for detecting the early stage of ADPKD.

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Serum metabolomics reveals γ-glutamyl dipeptides as biomarkers for discrimination among different forms of liver disease

Soga, T., Sugimoto, M., Honma, M., Mori, M., Igarashi, K., Kashikura, K., Ikeda, S., Hirayama, A., Yamamoto, T., Yoshida, H., Otsuka, M., Tsuji, S., Yatomi, Y., Sakuragawa, T., Watanabe, H., Nihei, K., Saito, T., Kawata, S., Suzuki, H., Tomita, M., Suematsu, M.

J. Hepatol. 55 ( 4 ) 896 - 905 2011.10

Research paper (scientific journal), Joint Work, Accepted

Background & aims: We applied a metabolome profiling approach to serum samples obtained from patients with different liver diseases, to discover noninvasive and reliable biomarkers for rapid-screening diagnosis of liver diseases.
Methods: Using capillary electrophoresis and liquid chromatography mass spectrometry, we analyzed low molecular weight metabolites in a total of 248 serum samples obtained from patients with nine types of liver disease and healthy controls.
Results: We found that γ-glutamyl dipeptides, which were biosynthesized through a reaction with γ-glutamylcysteine synthetase, were indicative of the production of reduced glutathione, and that measurement of their levels could distinguish among different liver diseases. Multiple logistic regression models facilitated the discrimination between specific and other liver diseases and yielded high areas under receiver-operating characteristic curves. The area under the curve values in training and independent validation data were 0.952 and 0.967 in healthy controls, 0.817 and 0.849 in drug-induced liver injury, 0.754 and 0.763 in asymptomatic hepatitis B virus infection, 0.820 and 0.762 in chronic hepatitis B, 0.972 and 0.895 in hepatitis C with persistently normal alanine transaminase, 0.917 and 0.707 in chronic hepatitis C, 0.803 and 0.993 in cirrhosis type C, and 0.762 and 0.803 in hepatocellular carcinoma, respectively. Several γ-glutamyl dipeptides also manifested potential for differentiating between nonalcoholic steatohepatitis and simple steatosis.
Conclusions: γ-Glutamyl dipeptides are novel biomarkers for liver diseases, and varying levels of individual or groups of these peptides have the power to discriminate among different forms of hepatic disease.

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Hepatitis C Virus Infection Promotes Hepatic Gluconeogenesis through an NS5A-Mediated, FoxO1-Dependent Pathway

Deng, L., Shoji, I., Ogawa, W., Kaneda, S., Soga, T., Jiang, D., Ide, Y., Hotta, H.

J. Virol. 85 ( 17 ) 8556 - 8568 2011.09

Research paper (scientific journal), Joint Work, Accepted

Chronic hepatitis C virus (HCV) infection is often associated with type 2 diabetes. However, the precise mechanism underlying this association is still unclear. Here, using Huh-7.5 cells either harboring HCV-1b RNA replicons or infected with HCV-2a, we showed that HCV transcriptionally upregulated the genes for phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase), the rate-limiting enzymes for hepatic gluconeogenesis. In this way, HCV enhanced the cellular production of glucose 6-phosphate (G6P) and glucose. PEPCK and G6Pase gene expressions are controlled by the transcription factor forkhead box O1 (FoxO1). We observed that although neither the mRNA levels nor the protein levels of FoxO1 expression were affected by HCV, the level of phosphorylation of FoxO1 at Ser319 was markedly diminished in HCV-infected cells compared to the control cells, resulting in an increased nuclear accumulation of FoxO1, which is essential for sustaining its transcriptional activity. It was unlikely that the decreased level of FoxO1 phosphorylation was mediated through Akt inactivation, as we observed an increased phosphorylation of Akt at Ser473 in HCV-infected cells compared to control cells. By using specific inhibitors of c-Jun N-terminal kinase (JNK) and reactive oxygen species (ROS), we demonstrated that HCV infection induced JNK activation via increased mitochondrial ROS production, resulting in decreased FoxO1 phosphorylation, FoxO1 nuclear accumulation, and, eventually, increased glucose production. We also found that HCV NS5A mediated increased ROS production and JNK activation, which is directly linked with the FoxO1-dependent increased gluconeogenesis. Taken together, these observations suggest that HCV promotes hepatic gluconeogenesis through an NS5A-mediated, FoxO1-dependent pathway.

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Transcriptional regulation of Organic anion transporting polypeptide SLCO4C1 as a new therapeutic modality to prevent chronic kidney disease

Suzuki, T., Toyohara, T., Akiyama, Y., Takeuchi, Y., Mishima, E., Suzuki, C., Ito, S., Soga, T., Abe, T.

J. Pharm. Sci. 100 ( 9 ) 3696 - 3707 2011.09

Research paper (scientific journal), Joint Work, Accepted

Uremic toxins accumulate in patients with chronic kidney diseases (CKDs) and cause further progression of renal damage and cardiovascular diseases. Recently, it was reported that some of the organic anion transporting polypeptides (OATPs) and the organic anion transporters (OATs) are involved in the renal elimination of uremic toxins. SLCO4C1 is the only OATP expressed at the basolateral side of proximal tubular cells in human kidney, and it mediates the excretion of uremic toxins. The overexpression of human SLCO4C1 in rat kidney promotes the renal excretion of uremic toxins and reduces hypertension, cardiomegaly, and renal inflammation in renal failure. Statins induce SLCO4C1 expression thorough transcriptional factor Aryl hydrocarbon receptor through binding of the xenobiotic responsive element at its promoter region. The administration of statin in a rat renal failure model facilitated the elimination of uremic toxins and mitigated organ damage. In addition, metabolomic analysis of rat renal failure models and patients with CKD by capillary electrophoresis-mass spectrometry is a useful method for identifying new uremic solutes and explores surrogate biomarkers for detecting the progression of early stage CKD.

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Use of Serum and Urine Metabolome Analysis for the Detection of Metabolic Changes in Patients with Stage 1-2 Chronic Kidney Disease

Hayashi, K., Sasamura, H., Hishiki, T., Suematsu, M., Ikeda, S., Soga, T., Itoh, H.

Nephro-Urol. Mon. 3 ( 3 ) 164 - 171 2011.07

Research paper (scientific journal), Joint Work, Accepted

Background: Chronic kidney disease (CKD) is a major health problem throughout the world, and understanding the pathological condition of CKD has become increasingly important. The recent development of advanced metabolomic assay techniques now allows the human metabolic condition to be evaluated sensitively and comprehensively.
Objectives: The aim of this study was to use metabolomic analysis to perform a preliminary survey of metabolic changes occurring in patients with stage 1-2 CKD.
Patients and Methods: Serum and urine metabolomic profiles of 15 patients with stage 1-2 CKD were analyzed using our previously reported capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) systems, and compared to 7 healthy volunteers.
Results: The CE-TOFMS systems in three different modes for cation, anion, and nucleotide analyses detected multiple metabolites in serum and urine samples. In cation analysis mode, several increases in nonessential amino acids were identified in patients with stage 1-2 CKD, similar to those reported for end-stage renal disease (ESRD). Free-radical scavengers carnosine and hypotaurine were decreased in the urine, whereas serum hypotaurine and taurine were increased, consistent with changes in renal and/or systemic oxidative stress. Moreover, the cardiotoxin hypoxanthine was markedly increased in the serum, whereas serum and urine adenosine and urine guanine were decreased, suggesting changes in purine nucleotide metabolism which could affect cardiovascular prognosis. Changes in other unidentified metabolites were also detected.
Conclusions: These results suggest that multiple changes in the metabolism are already detectable in stage 1-2 CKD using metabolome analysis. Further studies on these metabolic changes may result in new strategies to prevent cardiovascular events and progression to ESRD in patients with CKD.

Autophagy Protects the Proximal Tubule from Degeneration and Acute Ischemic Injury

Kimura, T., Takabatake, Y., Takahashi, A., Kaimori, J., Matsui, I., Namba, T., Kitamura, H., Niimura, F., Matsusaka, T., Soga, T., Rakugi, H., Isaka, Y.

J. Am. Soc. Nephrol. 22 ( 5 ) 902 - 913 2011.05

Research paper (scientific journal), Joint Work, Accepted

Autophagy is a bulk protein degradation system that likely plays an important role in normal proximal tubule function and recovery from acute ischemic kidney injury. Using conditional Atg5 gene deletion to eliminate autophagy in the proximal tubule, we determined whether autophagy prevents accumulation of damaged proteins and organelles with aging and ischemic renal injury. Autophagy-deficient cells accumulated deformed mitochondria and cytoplasmic inclusions, leading to cellular hypertrophy and eventual degeneration not observed in wildtype controls. In autophagy-deficient mice, I/R injury increased proximal tubule cell apoptosis with accumulation of p62 and ubiquitin positive cytoplasmic inclusions. Compared with control animals, autophagy-deficient mice exhibited significantly greater elevations in serum urea nitrogen and creatinine. These data suggest that autophagy maintains proximal tubule cell homeostasis and protects against ischemic injury. Enhancing autophagy may provide a novel therapeutic approach to minimize acute kidney injury and slow CKD progression.

Positive Autoregulation Delays the Expression Phase of Mammalian Clock Gene Per2

Ogawa, Y., Koike, N., Kurisawa. G., Soga, T., Tomita, M., Tei, H.

PLoS One 6 ( 4 ) e18663 2011.04

Research paper (scientific journal), Joint Work, Accepted

In mammals, cellular circadian rhythms are generated by a transcriptional-translational autoregulatory network that consists of clock genes that encode transcriptional regulators. Of these clock genes, Period1 (Per1) and Period2 (Per2) are essential for sustainable circadian rhythmicity and photic entrainment. Intriguingly, Per1 and Per2 mRNAs exhibit circadian oscillations with a 4-hour phase difference, but they are similarly transactivated by CLOCK-BMAL1. In this study, we investigated the mechanism underlying the phase difference between Per1 and Per2 through a combination of mathematical simulations and molecular experiments. Mathematical analyses of a model for the mammalian circadian oscillator demonstrated that the slow synthesis and fast degradation of mRNA tend to advance the oscillation phase of mRNA expression. However, the phase difference between Per1 and Per2 was not reproduced by the model, which implemented a 1.1-fold difference in degradation rates and a 3-fold difference in CLOCK-BMAL1 mediated inductions of Per1 and Per2 as estimated in cultured mammalian cells. Thus, we hypothesized the existence of a novel transcriptional activation of Per2 by PER1/2 such that the Per2 oscillation phase was delayed. Indeed, only the Per2 promoter, but not Per1, was strongly induced by both PER1 and PER2 in the presence of CLOCK-BMAL1 in a luciferase reporter assay. Moreover, a 3-hour advance was observed in the transcriptional oscillation of the delta-Per2 reporter gene lacking cis-elements required for the induction by PER1/2. These results indicate that the Per2 positive feedback regulation is a significant factor responsible for generating the phase difference between Per1 and Per2 gene expression.

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Metabolomic Anatomy of an Animal Model Revealing Homeostatic Imbalances in Dyslipidaemia

Ooga, T., Sato, H., Nagashima, A., Sasaki, K., Tomita, M., Soga, T., Ohashi, Y

Mol. Biosyst 7 ( 4 ) 1217 - 1223 2011.04

Research paper (scientific journal), Joint Work, Accepted

Metabolomics is an emerging technology that reveals homeostatic imbalances in biological systems. Global determination of metabolite concentrations in body fluid and tissues provides novel anatomical aspects of pathological conditions that cannot be obtained from target-specific measurements. Here, we characterised metabolic imbalance in Watanabe heritable hyperlipidaemic rabbits as a model of hypercholesterolaemia. Using a mass spectrometry-based system, we measured a total of 335 metabolites in plasma and tissues (liver, aorta, cardiac muscle, and brain) from WHHL and healthy control rabbits. From the comparison between two metabolomic profiles, pathophysiological features including glutathione and phosphatidylcholine metabolism indicated the occurrence of oxidative stress in several tissues. Especially for the liver, imbalanced purine catabolism shed light on the transcriptional activation of xanthine oxidase, which is thought to act in absorbing or possibly triggering oxidative stress. We also applied this system to assess the therapeutic effects of simvastatin administration. After the treatment, a portion of the metabolomic features in pathological conditions showed alterations suggesting restoration of metabolism to the healthy condition. These changes were considered to be due to the pleiotropic action of statin, including antioxidant effects, rather than its main inhibitory action on cholesterol biosynthesis.

A novel mechanism regulates H2S and SO2 production in Saccharomyces cerevisiae

Yoshida, S., Imoto, J., Minato, T., Oouchi, R., Kamada, Y., Tomita, M., Soga, T., Yoshimoto, H.

Yeast 28 ( 2 ) 109 - 121 2011.02

Research paper (scientific journal), Joint Work, Accepted, ISSN 0749-503X

Sulfite (SO2) plays an important role in flavour stability in alcoholic beverages, whereas hydrogen sulfide (H2S) has an undesirable aroma. To discover the cellular processes that control SO2 and H2S production, we screened a library of Saccharomyces cerevisiae deletion mutants. Deletion of 12 genes led to increased H2S productivity. Ten of these genes are known to be involved in sulfur‐containing amino acid metabolism, whereas UBI4 functions in the ubiquitin‐proteasome system and SKP2 encodes an F‐box‐containing protein whose function is unknown. We found that the skp2 mutant accumulated H2S and SO2, because the adenosylphophosulfate kinase Met14p is a substrate of SCFSkp2 and more stable in the skp2 mutant than in the wild‐type strain. Furthermore, the skp2 mutant grew more slowly than the wild‐type strain under nutrient‐limited conditions. Metabolome analysis showed that the concentration of intracellular cysteine is lower in the skp2 mutant than in the wild‐type strain. The slow growth of the skp2 mutant was due to a lower concentration of intracellular cysteine, because the addition of cysteine suppressed the slow growth. In the skp2 mutant, the cysteine biosynthesis proteins Str2p, Str3p and Str4p are more stable than in the wild‐type strain. Moreover, supplementation with methionine, S‐adenosylmethionine, S‐adenosylhomocysteine and homocysteine also suppressed the slow growth. Overexpression of STR1 or STR4 caused a more severe defect in the skp2 mutant. These results suggest that the balance of methionine and cysteine biosynthesis is important for yeast cell growth. Thus, Skp2p is one of the key components regulating this balance and H2S/SO2 production. Copyright © 2010 John Wiley & Sons, Ltd.

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Metabolome Analysis Revealed Increase in S-Methylcysteine and Phosphatidylisopropanolamine Synthesis upon L-Cysteine Deprivation in the Anaerobic Protozoan Parasite Entamoeba histolytica

Husain, A., Sato, D., Jeelani, G., Mi-ichi F., Ali V., Suematsu, M., Soga, T., Nozaki, T.

J Biol Chem 285 ( 50 ) 39160 - 39170 2010.12

Research paper (scientific journal), Joint Work, Accepted, ISSN 0021-9258

L-cysteine is ubiquitous in all living organisms and is involved in a variety of functions, including the synthesis of iron-sulfur clusters and glutathione and the regulation of the structure, stability, and catalysis of proteins. In the protozoan parasite Entamoeba histolytica, the causative agent of amebiasis, L-cysteine plays an essential role in proliferation, adherence, and defense against oxidative stress; however, the essentiality of this amino acid in the pathways it regulates is not well understood. In the present study, we applied capillary electrophoresis time-of-flight mass spectrometry to quantitate charged metabolites modulated in response to L-cysteine deprivation in E. histolytica, which was selected as a model for examining the biological roles of L-cysteine. L-cysteine deprivation had profound effects on glycolysis, amino acid, and phospholipid metabolism, with sharp decreases in the levels of L-cysteine, L-cystine, and S-adenosylmethionine and a dramatic accumulation of O-acetylserine and S-methylcysteine. We further demonstrated that S-methylcysteine is synthesized from methanethiol and O-acetylserine by cysteine synthase, which was previously considered to be involved in sulfur-assimilatory L-cysteine biosynthesis. In addition, L-cysteine depletion repressed glycolysis and energy generation, as it reduced acetyl-CoA, ethanol, and the major nucleotide di- and triphosphates, and led to the accumulation of glycolytic intermediates. Interestingly, L-cysteine depletion increased the synthesis of isopropanolamine and phosphatidylisopropanolamine, and it was confirmed that their increment was not a result of oxidative stress but was a specific response to L-cysteine depletion. We also identified a pathway in which isopropanolamine is synthesized from methylglyoxal via aminoacetone. To date, this study represents the first case where L-cysteine deprivation leads to drastic changes in core metabolic pathways, including energy, amino acid, and phospholipid metabolism.

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Functional Expression of Carnitine/Organic Cation Transporter OCTN1/SLC22A4 in Mouse Small Intestine and Liver

Sugiura, T., Kato, S., Shimizu, T., Wakayama, T., Nakamichi, N., Kubo, Y., Iwata, D., Suzuki, K., Soga, T., Asano, M., Iseki, S., Tamai, I., Tsuji, A., *Kato, Y

Drug Metab Dispos 38 ( 10 ) 1665 - 1672 2010.10

Research paper (scientific journal), Joint Work, Accepted, ISSN 0090-9556

Carnitine/organic cation transporter (OCTN1/SLC22A4) accepts various therapeutic agents as substrates in vitro and is expressed ubiquitously, although its function in most organs has not yet been examined. The purpose of the present study was to evaluate functional expression of OCTN1 in small intestine and liver, using octn1 gene knockout [octn1(−/−)] mice. After oral administration of [3H]ergothioneine ([3H]ERGO), a typical substrate of OCTN1, the amount of [3H]ERGO remaining in the small intestinal lumen was much higher in octn1(−/−) mice than in wild-type mice. In addition, uptake of [3H]ERGO by human embryonic kidney 293 cells heterologously expressing OCTN1 gene product and uptake of [3H]ERGO at the apical surface of intestinal everted sacs from wild-type mice were inhibited by OCTN1 substrates, tetraethylammonium and verapamil. Immunohistochemical analysis revealed that OCTN1 is localized on the apical surface of small intestine in mice and humans. These results suggest that OCTN1 is responsible for small intestinal absorption of [3H]ERGO. However, the plasma concentration of [3H]ERGO after oral administration was higher in octn1(−/−) mice than in wild-type mice, despite the lower absorption in octn1(−/−) mice. This was probably because of efficient hepatic uptake of [3H]ERGO, as revealed by integration plot analysis; the uptake clearance was close to the hepatic plasma flow rate. The uptake of [3H]ERGO by isolated hepatocytes was minimal, whereas [3H]ERGO uptake was observed in isolated nonparenchymal cells. This finding is consistent with immunostaining of OCTN1 in liver sinusoids. Thus, our results indicate that OCTN1 is functionally expressed in nonparenchymal liver cells.

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Metabolomic Identification of the Target of the Filopodia Protrusion Inhibitor Glucopiericidin A

Kitagawa, M., Ikeda, S., Tashiro, E., Soga, T., Imoto, M.

Chem. Biol. 17 ( 9 ) 989 - 998 2010.09

Research paper (scientific journal), Joint Work, Accepted

Identifying the targets of bioactive compounds is a major challenge in chemical biological research. Here, we identified the functional target of the natural bioactive compound glucopiericidin A (GPA) through metabolomic analysis. We isolated GPA while screening microbial samples for a filopodia protrusion inhibitor. Interestingly, GPA alone did not inhibit filopodia protrusion, but synergistically inhibit protrusion with the mitochondrial respiration inhibitor, piericidin A (PA). These results suggested that GPA might inhibit glycolysis. Capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) provided strong evidence that GPA suppresses glycolysis by functionally targeting the glucose transporter. GPA may therefore serve as a glucose transporter chemical probe. Simultaneous inhibition of both glycolysis and mitochondrial respiration dramatically decreased intracellular ATP levels, indicating that GPA inhibits ATP-dependent filopodia protrusion with PA. Our results represent a challenge of molecular target identification using metabolomic analysis.

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Metabolomic profiling of uremic solutes in CKD patients

Toyohara, T., Akiyama, Y., Suzuki, T., Takeuchi, Y., Mishima, E., Tanemoto, M., Momose, A., Toki, N., Sato, H., Nakayama, M., Hozawa, A., Tsuji, I., Ito, S., Soga, T. and Abe. T.

Hypertens. Res. 33 ( 9 ) 944 - 952 2010.09

Research paper (scientific journal), Joint Work, Accepted

Early detection and accurate monitoring of patients with chronic kidney disease (CKD) is likely to improve care and decrease the risk of cardiovascular and cerebrovascular diseases. As a new diagnostic tool, we examined the retention of uremic solutes as a simpler, more accurate method to assess renal function. To achieve this, we comprehensively evaluated these solutes in CKD patients. By capillary electrophoresis with mass spectrometry, we found 22 cations and 30 anions that accumulated significantly as the estimated glomerular filtration rate (eGFR) decreased. These compounds included 9 cations and 27 anions that were newly identified in this study. In contrast, we also found 7 cations (2 new) and 5 anions (all new) that decrease significantly as eGFR declines. We evaluated each substance for its suitability to detect early CKD stage. Compounds that are highly correlated with eGFR and whose plasma concentration changed in a manner approximated by the first-degree equation are excellent candidates for detecting CKD and identifying uremic toxins that might aggravate kidney function in the early stage of CKD. These results identify a number of uremic compounds, many of which are novel and which predict worsening renal function. These compounds provide diagnostic information and may be targets for therapies designed to treat the complications of CKD patients.

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Two Atypical L-cysteine-regulated NADPH-dependent Oxidoreductases Involved in Redox Maintenance, L-cystine and Iron Reduction, and Metronidazole Activation in the Enteric Protozoan Entamoeba Histolytica

Jeelani, G., Husain, A., Sato, D., Ali, V., Suematsu, M., Soga, T., Nozaki, T.

J Biol Chem 285 ( 35 ) 26889 - 26899 2010.08

Research paper (scientific journal), Joint Work, Accepted, ISSN 0021-9258

We discovered novel catalytic activities of two atypical NADPH-dependent oxidoreductases (EhNO1/2) from the enteric protozoan parasite Entamoeba histolytica. EhNO1/2 were previously annotated as the small subunit of glutamate synthase (glutamine:2-oxoglutarate amidotransferase) based on similarity to authentic bacterial homologs. As E. histolytica lacks the large subunit of glutamate synthase, EhNO1/2 were presumed to play an unknown role other than glutamine/glutamate conversion. Transcriptomic and quantitative reverse PCR analyses revealed that supplementation or deprivation of extracellular L-cysteine caused dramatic up- or down-regulation, respectively, of EhNO2, but not EhNO1 expression. Biochemical analysis showed that these FAD- and 2[4Fe-4S]-containing enzymes do not act as glutamate synthases, a conclusion which was supported by phylogenetic analyses. Rather, they catalyze the NADPH-dependent reduction of oxygen to hydrogen peroxide and L-cystine to L-cysteine and also function as ferric and ferredoxin-NADP(+) reductases. EhNO1/2 showed notable differences in substrate specificity and catalytic efficiency; EhNO1 had lower K(m) and higher k(cat)/K(m) values for ferric ion and ferredoxin than EhNO2, whereas EhNO2 preferred L-cystine as a substrate. In accordance with these properties, only EhNO1 was observed to physically interact with intrinsic ferredoxin. Interestingly, EhNO1/2 also reduced metronidazole, and E. histolytica transformants overexpressing either of these proteins were more sensitive to metronidazole, suggesting that EhNO1/2 are targets of this anti-amebic drug. To date, this is the first report to demonstrate that small subunit-like proteins of glutamate synthase could play an important role in redox maintenance, L-cysteine/L-cystine homeostasis, iron reduction, and the activation of metronidazole.

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13C-Metabolic Flux Analysis for Batch Culture of Escherichia coli and Its pyk and pgi Gene Knockout Mutants based on Mass Isotopomer Distribution of Intracellular Metabolites

Toya, Y., Ishii, N., Nakahigashi, K., Hirasawa, T., Soga, T., Tomita, M., *Shimizu, K

Biotechnol Prog 26 ( 4 ) 975 - 992 2010.08

Research paper (scientific journal), Joint Work, Accepted, ISSN 8756-7938

Since most bio‐production processes are conducted in a batch or fed‐batch manner, the evaluation of metabolism with respect to time is highly desirable. Toward this aim, we applied 13C‐metabolic flux analysis to nonstationary conditions by measuring the mass isotopomer distribution of intracellular metabolites. We performed our analysis on batch cultures of wild‐type Escherichia coli, as well as on Pyk and Pgi mutants, obtained the fluxes and metabolite concentrations as a function of time. Our results for the wild‐type indicated that the TCA cycle flux tended to increase during growth on glucose. Following glucose exhaustion, cells controlled the branch ratio between the glyoxylate pathway and the TCA cycle, depending on the availability of acetate. In the Pyk mutant, the concentrations of glycolytic intermediates changed drastically over time due to the dumping and feedback inhibition caused by PEP accumulation. Nevertheless, the flux distribution and free amino acid concentrations changed little. The growth rate and the fluxes remained constant in the Pgi mutant and the glucose‐6‐phosphate dehydrogenase reaction was the rate‐limiting step. The measured fluxes were compared with those predicted by flux balance analysis using maximization of biomass yield or ATP production. Our findings indicate that the objective function of biosynthesis became less important as time proceeds on glucose in the wild‐type, while it remained highly important in the Pyk mutant. Furthermore, ATP production was the primary objective function in the Pgi mutant. This study demonstrates how cells adjust their metabolism in response to environmental changes and/or genetic perturbations in the batch cultivation. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010

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Metabolomic Profiles and Sensory Attributes of Edamame under Various Storage Duration and Temperature Conditions

Sugimoto, M., Goto, H., Otomo, K., Ito, M., Onuma, H., Suzuki, A., Sugawara, M., Abe, S., Tomita, M., Soga, T.

J Agric Food Chem 58 ( 14 ) 8418 - 8425 2010.07

Research paper (scientific journal), Joint Work, Accepted, ISSN 0021-8561

Its high nutritional content and sensory characteristics make edamame a popular vegetable bean. However, because of its short shelf-life, it is important to optimize the storage conditions to maintain its quality during distribution to consumers. We focused on storage conditions to investigate the temporal changes in the metabolic profiles and sensory characteristics of edamame during transportation from the site of harvest to the site of purchase/consumption. We conducted metabolomic analysis and sensory evaluation tests of edamame stored for different lengths and at different temperatures. Charged metabolites were profiled by capillary electrophoresis-mass spectrometry, and free sugars were quantified by liquid chromatography-tandem mass spectrometry. In comparison to the gradual decrease in its sensory characteristics over time, the changes in metabolite profiles manifested four different patterns. In particular, changes in amino acid levels were related to sensory attributes. The downstream metabolites of shikimate as well as phospholipids and gamma-aminobutyric acid increased with increasing storage temperatures.

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MassBank: a public repository for sharing mass spectral data for life sciences

Horai, H., Arita, M., Kanaya, S., Nihei, Y., Ikeda, T., Suwa, K., Ojima, Y., Tanaka, K., Tanaka, S., Aoshima, K., Oda, Y., Kakazu, Y., Kusano, M., Tohge, T., Matsuda, F., Sawada, Y., Hirai, Y. M., Nakanishi, H., Ikeda, K., Akimoto, N., Maoka, T., Takahashi, H., Ara, T., Sakurai, N., Suzuki, H., Shibata, D., Neumann, S., Iida, T., Tanaka, K., Funatsu, K., Matsuura, F., Soga, T., Taguchi, R., Saito, K., *Nishioka, T.

J. Mass Spectro. 45 ( 7 ) 703 - 714 2010.07

Research paper (scientific journal), Joint Work, Accepted, ISSN 1076-5174

MassBank is the first public repository of mass spectra of small chemical compounds for life sciences (<3000 Da). The database contains 605 electron-ionization mass spectrometry (EI-MS), 137 fast atom bombardment MS and 9276 electrospray ionization (ESI)-MS(n) data of 2337 authentic compounds of metabolites, 11 545 EI-MS and 834 other-MS data of 10,286 volatile natural and synthetic compounds, and 3045 ESI-MS(2) data of 679 synthetic drugs contributed by 16 research groups (January 2010). ESI-MS(2) data were analyzed under nonstandardized, independent experimental conditions. MassBank is a distributed database. Each research group provides data from its own MassBank data servers distributed on the Internet. MassBank users can access either all of the MassBank data or a subset of the data by specifying one or more experimental conditions. In a spectral search to retrieve mass spectra similar to a query mass spectrum, the similarity score is calculated by a weighted cosine correlation in which weighting exponents on peak intensity and the mass-to-charge ratio are optimized to the ESI-MS(2) data. MassBank also provides a merged spectrum for each compound prepared by merging the analyzed ESI-MS(2) data on an identical compound under different collision-induced dissociation conditions. Data merging has significantly improved the precision of the identification of a chemical compound by 21-23% at a similarity score of 0.6. Thus, MassBank is useful for the identification of chemical compounds and the publication of experimental data.

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Prediction of metabolite identity from accurate mass, migration time prediction and isotopic pattern information in CE-TOFMS data

Sugimoto, M., Hirayama, A., Robert, M., Abe, S., Soga, T., Tomita, M.

Electrophoresis 31 ( 14 ) 2311 - 2318 2010.07

Research paper (scientific journal), Joint Work, Accepted, ISSN 0173-0835

CE-TOFMS is a powerful method for profiling charged metabolites. However, the limited availability of metabolite standards hinders the process of identifying compounds from detected features in CE-TOFMS data sets. To overcome this problem, we developed a method to identify unknown peaks based on the predicted migration time (t(m)) and accurate m/z values. We developed a predictive model using 375 standard cationic metabolites and support vector regression. The model yielded good correlations between the predicted and measured t(m) (R=0.952 and 0.905 using complete and cross-validation data sets, respectively). Using the trained model, we subsequently predicted the t(m) for 2938 metabolites available from the public databases and assigned tentative identities to noise-filtered features in human urine samples. While 38.9% of the peaks were assigned metabolite names by matching with the standard library alone, the proportion increased to 52.2%. The proposed methodology increases the value of metabolomic data sets obtained from CE-TOFMS profiling.

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Unveiling cellular biochemical reactions via metabolomics-driven approaches

Saito, N., Ohashi, Y., Soga, T., Tomita, M.

Curr. Opin. Microbiol. 13 ( 3 ) 358 - 362 2010.06

Research paper (scientific journal), Joint Work, Accepted

Despite the widespread availability of genome sequences and the systematic assignment of individual open reading frames, many of the activities of metabolic enzymes are still veiled. It is difficult to assign biochemical functions to enzymes and uncover the in vivo regulation of their enzymatic activities, but this is crucial to understand cellular functions. The development of metabolomics and metabolite profiling, which can globally analyze intracellular and extracellular metabolites, has provided some valuable tools for systems biology. This report reviews the recent metabolomics approaches to elucidate unidentified metabolic systems while focusing on their enzymatic activities, including their biochemical and biological functions, and their regulation in response to environmental perturbations in microorganisms.

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A systematic survey of in vivo obligate chaperonin‐dependent substrates

Fujiwara, K., Ishihama, Y., Nakahigashi, K., Soga, T., Taguchi, H.,

EMBO J. 29 ( 9 ) 1552 - 1564 2010.05

Research paper (scientific journal), Joint Work, Accepted, ISSN 14602075

Chaperonins are absolutely required for the folding of a subset of proteins in the cell. An earlier proteome-wide analysis of Escherichia coli chaperonin GroEL/GroES (GroE) interactors predicted obligate chaperonin substrates, which were termed Class III substrates. However, the requirement of chaperonins for in vivo folding has not been fully examined. Here, we comprehensively assessed the chaperonin requirement using a conditional GroE expression strain, and concluded that only approximately 60% of Class III substrates are bona fide obligate GroE substrates in vivo. The in vivo obligate substrates, combined with the newly identified obligate substrates, were termed Class IV substrates. Class IV substrates are restricted to proteins with molecular weights that could be encapsulated in the chaperonin cavity, are enriched in alanine/glycine residues, and have a strong structural preference for aggregation-prone folds. Notably, approximately 70% of the Class IV substrates appear to be metabolic enzymes, supporting a hypothetical role of GroE in enzyme evolution.

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Gene knockout and metabolome analysis of carnitine/organic cation transporter OCTN1

Kato, Y., Kubo, Y., Iwata, D., Kato, S., Sudo, T., Sugiura, T., Kagaya, T., Wakayama, T., Hirayama, A., Sugimoto, M., Sugihara, K., Kaneko, S., Soga, T., Asano, M., Tomita, M., Matsui, T., Wada, M., Tsuji, A.

Pharm. Res. 27 ( 5 ) 832 - 840 2010.05

Research paper (scientific journal), Joint Work, Accepted

Purpose: Solute carrier OCTN1 (SLC22A4) is an orphan transporter, the physiologically important substrate of which is still unidentified. The aim of the present study was to examine physiological roles of OCTN1.
Methods: We first constructed octn1 gene knockout (octn1 ( -/- )) mice. Metabolome analysis was then performed to identify substrates in vivo. The possible association of the substrate identified with diseased conditions was further examined.
Results: The metabolome analysis of blood and several organs indicated complete deficiency of a naturally occurring potent antioxidant ergothioneine in octn1 ( -/- ) mice among 112 metabolites examined. Pharmacokinetic analyses after oral administration revealed the highest distribution to small intestines and extensive renal reabsorption of [(3)H]ergothioneine, both of which were much reduced in octn1 ( -/- ) mice. The octn1 ( -/- ) mice exhibited greater susceptibility to intestinal inflammation under the ischemia and reperfusion model. The blood ergothioneine concentration was also much reduced in Japanese patients with Crohn's disease, compared with healthy volunteers and patients with another inflammatory bowel disease, ulcerative colitis.
Conclusions: These results indicate that OCTN1 plays a pivotal role for maintenance of systemic and intestinal exposure of ergothioneine, which could be important for protective effects against intestinal tissue injuries, providing a possible diagnostic tool to distinguish the inflammatory bowel diseases.

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Analysis of Metabolic Remodeling in Compensated Left Ventricular Hypertrophy and Heart Failure

Kato, T., Niizuma, S., Inuzuka, Y., Kawashima, T., Okuda, J., Tamaki, Y., Iwanaga, Y., Narazaki, M., Matsuda, T., Soga, T., Kita, T., Kimura, T., Shioi, T.

Circ. Heart Fail. 3 ( 3 ) 420 - 430 2010.05

Research paper (scientific journal), Joint Work, Accepted, ISSN 1941-3289

Background: Congestive heart failure (CHF) is associated with a change in cardiac energy metabolism. However, the mechanism by which this change is induced and causes the progression of CHF is unclear.
Methods and results: We analyzed the cardiac energy metabolism of Dahl salt-sensitive rats fed a high-salt diet, which showed a distinct transition from compensated left ventricular hypertrophy to CHF. Glucose uptake increased at the left ventricular hypertrophy stage, and glucose uptake further increased and fatty acid uptake decreased at the CHF stage. The gene expression related to glycolysis, fatty acid oxidation, and mitochondrial function was preserved at the left ventricular hypertrophy stage but decreased at the CHF stage and was associated with decreases in levels of transcriptional regulators. In a comprehensive metabolome analysis, the pentose phosphate pathway that regulates the cellular redox state was found to be activated at the CHF stage. Dichloroacetate (DCA), a compound known to enhance glucose oxidation, increased energy reserves and glucose uptake. DCA improved cardiac function and the survival of the animals. DCA activated the pentose phosphate pathway in the rat heart. DCA activated the pentose phosphate pathway, decreased oxidative stress, and prevented cell death of cultured cardiomyocytes.
Conclusions: Left ventricular hypertrophy or CHF is associated with a distinct change in the metabolic profile of the heart. DCA attenuated the transition associated with increased energy reserves, activation of the pentose phosphate pathway, and reduced oxidative stress.

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Chlorogonium complexum sp. nov. (Volvocales, Chlorophyceae), and morphological evolution of Chlorogonium

Nakada, T., Soga, T., Tomita, M., Nozaki, H.

Eur. J. Phycol. 45 ( 1 ) 97 - 107 2010.03

Research paper (scientific journal), Joint Work, Accepted

We isolated strains of a new species of Chlorogonium (Cg.), Cg. complexum Nakada sp. nov. The genus Chlorogonium was recently emended to have several, irregularly distributed contractile vacuoles, two-to-many pyrenoids associated with fragmented starch plates and tubular thylakoid lamellae penetrating their matrices, and stigma composed of a single layer of globules. Chlorogonium complexum has these diagnostic characters of the emended genus. It is distinguished from other species of Chlorogonium, Cg. euchlorum, Cg. elongatum and Cg. capillatum, on the combination of diagnostic characters, such as the number and stability of pyrenoids and the mode of encystment. Based on a dataset combining 18S rRNA, RuBisCO large subunit (rbcL) and P700 chl a-apoprotein A2 (psaB) gene sequences, the phylogenetic position of Chlorogonium was determined. Chlorogonium complexum represented an independent lineage, sister to Cg. euchlorum and Cg. capillatum. The evolution of diagnostic characters among Chlorogonium species is discussed based on the molecular phylogenetic analyses, and a key to species of Chlorogonium and fusiform chlamydomonads with irregularly distributed contractile vacuoles is presented.

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Hypotaurine is an energy-saving hepatoprotective compound against ischemia-reperfusion injury of the rat liver

Sakuragawa, T., Hishiki, T., Ueno, Y., Ikeda, S., Soga, T., Yachie-Kinoshita, A., Kajimura, M., Suematsu, M.

J. Clin. Biochem. Nutr. 46 ( 2 ) 126 - 134 2010.03

Research paper (scientific journal), Joint Work, Accepted, ISSN 0912-0009

Metabolome analyses assisted by capillary electrophoresis-mass spectrometry (CE-MS) have allowed us to systematically grasp changes in small molecular metabolites under disease conditions. We applied CE-MS to mine out biomarkers in hepatic ischemia-reperfusion. Rat livers were exposed to ischemia by clamping of the portal inlet followed by reperfusion. Metabolomic profiling revealed that l contents of taurine in liver and plasma were significantly increased. Of interest is an elevation of hypotaurine, collectively suggesting significance of hypotaurine/taurine in post-ischemic responses. Considering the anti-oxidative capacity of hypotaurine, we examined if supplementation of the compound or its precursor amino acids could affect hepatocellular viability and contents of taurine in liver and plasma. Administration of hypotaurine, N-acetylcysteine or methionine upon reperfusion comparablly attenuated the post-ischemic hepatocellular injury but with different metabolomic profiling among groups: rats treated with methionine or N-acetylcysteine but not those treated with hypotaurine, exhibited significant elevation of hepatic lactate generation without notable recovery of the energy charge. Furthermore, the group treated with hypotaurine exhibited elevation of the plasma taurine, suggesting that the exogenously administered compound was utilized as an antioxidant. These results suggest that taurine serves as a surrogate marker for ischemia-reperfusion indicating effectiveness of hypotaurine as an energy-saving hepatoprotective amino acid.

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Differential Metabolomics Software for Capillary Electrophoresis-Mass Spectrometry Data Analysis

Sugimoto, M., Hirayama, A., Ishikawa, T., Robert, M., Baran, R., Uehara, K., Kawai, K., Soga, T., Tomita, M.

Metabolomics 6 ( 1 ) 27 - 41 2010.03

Research paper (scientific journal), Joint Work, Accepted

In metabolomics, the rapid identification of quantitative differences between multiple biological samples remains a major challenge. While capillary electrophoresis–mass spectrometry (CE–MS) is a powerful tool to simultaneously quantify charged metabolites, reliable and easy-to-use software that is well suited to analyze CE–MS metabolic profiles is still lacking. Optimized software tools for CE–MS are needed because of the sometimes large variation in migration time between runs and the wider variety of peak shapes in CE–MS data compared with LC–MS or GC–MS. Therefore, we implemented a stand-alone application named JDAMP (Java application for Differential Analysis of Metabolite Profiles), which allows users to identify the metabolites that vary between two groups. The main features include fast calculation modules and a file converter using an original compact file format, baseline subtraction, dataset normalization and alignment, visualization on 2D plots (m/z and time axis) with matching metabolite standards, and the detection of significant differences between metabolite profiles. Moreover, it features an easy-to-use graphical user interface that requires only a few mouse-actions to complete the analysis. The interface also enables the analyst to evaluate the semiautomatic processes and interactively tune options and parameters depending on the input datasets. The confirmation of findings is available as a list of overlaid electropherograms, which is ranked using a novel difference-evaluation function that accounts for peak size and distortion as well as statistical criteria for accurate difference-detection. Overall, the JDAMP software complements other metabolomics data processing tools and permits easy and rapid detection of significant differences between multiple complex CE–MS profiles.

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Capillary Electrophoresis Mass Spectrometry-based Saliva Metabolomics Identifies Oral, Breast and Pancreatic Cancer-Specific Profiles

Sugimoto, M., Wong, D.T., Hirayama, A., Soga, T., Tomita, M.

Metabolomics 6 ( 1 ) 78 - 95 2010.03

Research paper (scientific journal), Joint Work, Accepted

Saliva is a readily accessible and informative biofluid, making it ideal for the early detection of a wide range of diseases including cardiovascular, renal, and autoimmune diseases, viral and bacterial infections and, importantly, cancers. Saliva-based diagnostics, particularly those based on metabolomics technology, are emerging and offer a promising clinical strategy, characterizing the association between salivary analytes and a particular disease. Here, we conducted a comprehensive metabolite analysis of saliva samples obtained from 215 individuals (69 oral, 18 pancreatic and 30 breast cancer patients, 11 periodontal disease patients and 87 healthy controls) using capillary electrophoresis time-of-flight mass spectrometry (CE-TOF-MS). We identified 57 principal metabolites that can be used to accurately predict the probability of being affected by each individual disease. Although small but significant correlations were found between the known patient characteristics and the quantified metabolites, the profiles manifested relatively higher concentrations of most of the metabolites detected in all three cancers in comparison with those in people with periodontal disease and control subjects. This suggests that cancer-specific signatures are embedded in saliva metabolites. Multiple logistic regression models yielded high area under the receiver-operating characteristic curves (AUCs) to discriminate healthy controls from each disease. The AUCs were 0.865 for oral cancer, 0.973 for breast cancer, 0.993 for pancreatic cancer, and 0.969 for periodontal diseases. The accuracy of the models was also high, with cross-validation AUCs of 0.810, 0.881, 0.994, and 0.954, respectively. Quantitative information for these 57 metabolites and their combinations enable us to predict disease susceptibility. These metabolites are promising biomarkers for medical screening. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-009-0178-y) contains supplementary material, which is available to authorized users.

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Correlation between Sensory Evaluation Scores of Japanese Sake and Metabolome Profiles

Sugimoto, M., Koseki, T., Hirayama, A., Abe, S., Sano, T., Tomita, M., Soga, T.

J. Agric. Food Chem. 58 ( 1 ) 374–383 2010.01

Research paper (scientific journal), Joint Work, Accepted

The aim of this study was to explore the association between taste and metabolite profiles of Japanese refined sake. Nontarget metabolome analysis was conducted using capillary electrophoresis mass spectrometry. Zatsumi, an unpleasant not clear flavor, and sweetness, bitterness, and sourness were graded by four experienced panelists. Regression models based on support vector regression (SVR) were used to estimate the relationships among sensory evaluation scores and quantified metabolites and visualized as a nonlinear relationship between sensory scores and metabolite components. The SVR model was highly accurate and versatile: the correlation coefficients for whole training data, cross-validation, and separated validation data were 0.86, 0.73, and 0.73, respectively, for zatsumi. Other sensory scores were also analyzed and modeled by SVR. The methodology demonstrated here carries great potential for predicting the relevant parameters and quantitative relationships between charged metabolites and sensory evaluation in Japanese refined sake.

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Phylogenetic position of a rare loricated green alga, Cephalomonas granulata N. L. Higinb (Volvocales, Chlorophyceae)

Nakada, T., Soga, T., Tomita, M.

Phycol. Res. 58 ( 1 ) 62 - 68 2010.01

Research paper (scientific journal), Joint Work, Accepted

Cephalomonas (Cp.) granulata N. L. Higinb. (Volvocales, Chlorophyceae), a rare volvocalean phytoflagellate, has recently been isolated from a paddy field in Japan and examined by light and fluorescence microscopy. The vegetative cells of Cp. granulata have granulate loricae and a characteristic mushroom‐like shape with a hemispherical to spherical anterior part and narrower posterior part. Cp. granulata has been classified among the Phacotaceae along with other loricated genera. However, its phylogenetic position has not been investigated using molecular phylogeny. To evaluate the phylogenetic position of Cephalomonas, the 18S rRNA gene sequence of the Japanese strain was determined. The phylogenetic analyses revealed that Cp. granulata was not closely related to other loricated taxa with known phylogenetic affinities, such as Phacotaceae sensu stricto (Phacotus, Pteromonas, and Wislouchiella) and Dysmorphococcus globosus. This indicates at least three independent origins of loricae within the Volvocales.

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Cytotoxic effect of amide derivatives of trifluoromethionine against the enteric protozoan parasite Entamoeba histolytica

Sato, D., Kobayashi, S., Yasui, H., Shibata, N., Toru, T., Yamamoto, M., Tokoro, G., Ali, V., Soga, T., Takeuchi, T., Suematsu, M., Nozaki, T.

Int. J. Antimicrob. Agents. 35 ( 1 ) 56-61 2010.01

Research paper (scientific journal), Joint Work, Accepted

Amoebiasis, caused by infection with the enteric protist Entamoeba histolytica, is one of the major parasitic diseases. Although metronidazole and its derivatives are currently employed in therapy, the paucity of effective drugs and potential clinical resistance necessitate the development of a novel drug. Trifluoromethionine (TFM) is a promising lead compound for antiamoebic drugs. To potentiate the antiamoebic effect of TFM, we synthesised various amide derivatives of TFM and evaluated their cytotoxicity. The amide derivatives of TFM were observed to have a superior cytotoxic effect compared with TFM and metronidazole against E. histolytica in vitro. Although TFM showed cytotoxicity following degradation by methionine gamma-lyase, the derivatives were degraded by the enzyme less efficiently compared with TFM. We further demonstrated that a representative derivative was hydrolysed by the amoebic cell lysate to first yield TFM, followed by degradation similar to TFM. Hydrolysis was partially inhibited by protease inhibitors. A single subcutaneous or oral administration of TFM and its amide derivatives also effectively prevented the formation of amoebic liver abscess in a rodent model. These data demonstrate the improved effectiveness of TFM derivatives against E. histolytica infection and elucidate the mechanisms underlining the mode of action of these compounds.

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SLCO4C1 Transporter Eliminates Uremic Toxins and Attenuates Hypertension and Renal Inflammation.

Toyohara, T., Suzuki, T., Morimoto, R., Akiyama, Y., Souma, T., Shiwaku, H.O., Takeuchi, Y., Mishima, E., Abe, M., Tanemoto, M., Masuda, S., Kawano, H., Maemura, K., Nakayama, M., Sato, H., Mikkaichi, T., Yamaguchi, H., Fukui, S., Fukumoto, Y., Shimokawa, H., Inui, K., Terasaki, T., Goto, J., Ito, S., Hishinuma, T., Rubera, I., Tauc, M., Fujii-Kuriyama, Y., Yabuuchi, H., Moriyama, Y., Soga, T., Abe, T.

J. Am. Soc. Nephrol. 20 ( 12 ) 2546 - 2555 2009.12

Research paper (scientific journal), Joint Work, Accepted

Hypertension in patients with chronic kidney disease (CKD) strongly associates with cardiovascular events. Among patients with CKD, reducing the accumulation of uremic toxins may protect against the development of hypertension and progression of renal damage, but there are no established therapies to accomplish this. Here, overexpression of human kidney-specific organic anion transporter SLCO4C1 in rat kidney reduced hypertension, cardiomegaly, and inflammation in the setting of renal failure. In addition, SLCO4C1 overexpression decreased plasma levels of the uremic toxins guanidino succinate, asymmetric dimethylarginine, and the newly identified trans-aconitate. We found that xenobiotic responsive element core motifs regulate SLCO4C1 transcription, and various statins, which act as inducers of nuclear aryl hydrocarbon receptors, upregulate SLCO4C1 transcription. Pravastatin, which is cardioprotective, increased the clearance of asymmetric dimethylarginine and trans-aconitate in renal failure. These data suggest that drugs that upregulate SLCO4C1 may have therapeutic potential for patients with CKD.

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In Silico Modeling and Metabolome Analysis of Long-stored Erythrocytes to Improve Blood Storage Methods

Nishino, T., Yachie-Kinoshita, A., Hirayama, A., Soga, T., Suematsu, M., Tomota, M.

J. Biotechnol. 144 212 - 223 2009.11

Research paper (scientific journal), Joint Work, Accepted

There is currently no effective method for preventing ATP and 2,3-bisphosphoglycerate (2,3-BPG) depletion during long-term erythrocyte storage in the cold, although these metabolites are strongly associated with cell viability and oxygen delivery after transfusion. Metabolite reduction is caused by whole metabolic networks in the cell, which are regulated by various physical or chemical factors. Mathematical modeling is a powerful tool for integrating such complex and dynamic systems. Here, we developed a mathematical model to predict metabolism in erythrocytes preserved with a mannitol-adenine-phosphate solution (MAP) at 4 degrees C, by modifying a published model of large-scale erythrocyte metabolism. Our model successfully reproduced the reported decreases in ATP and 2,3-BPG during storage. Analysis of our model identified several enzymatic reactions and factors related to ATP and 2,3-BPG depletions, which may serve as possible targets for improving blood storage methods. We also performed metabolome analysis of laboratory-made MAP-stored erythrocytes using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS), which provided a comprehensive view of the metabolism dynamics. Alterations in the metabolic intermediate concentrations after long storage were qualitatively predicted by the model. Finally, through further systematic analysis, we also discuss the usability of our model.

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HIF-1α is necessary to support gluconeogenesis during liver regeneration

Tajima, T., Goda, N., Fujiki, N., Hishiki, T., Nishiyama, Y., Senoo-Matsuda, N., Shimazu, M., Soga, T., Yoshimura, Y., Johnson, R.S., Suematsu, M.

Biochem. Bioph. Res. Commun. 387 789 - 794 2009.10

Research paper (scientific journal), Joint Work, Accepted

Coordinated recovery of hepatic glucose metabolism is prerequisite for normal liver regeneration. To examine roles of hypoxia inducible factor-1α (HIF-1α) for hepatic glucose homeostasis during the reparative process, we inactivated the gene in hepatocytes in vivo. Following partial hepatectomy (PH), recovery of residual liver weight was initially retarded in the mutant mice by down-regulation of hepatocyte proliferation, but occurred comparably between the mutant and control mice at 72 h after PH. At this time point, the mutant mice showed lowered blood glucose levels with enhanced accumulation of glycogen in the liver. The mutant mice exhibited impairment of hepatic gluconeogenesis as assessed by alanine tolerance test. This appeared to result from reduced expression of PGK-1 and PEPCK since 3-PG, PEP and malate were accumulated to greater extents in the regenerated liver. In conclusion, these findings provide evidence for roles of HIF-1α in the regulation of gluconeogenesis under liver regeneration.

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Systematic phenome analysis of Escherichia coli multiple-knockout mutants reveals hidden reactions in central carbon metabolism

Nakahigashi, K., Toya, Y., Ishii, N., Soga, T., Hasegawa, M., Watanabe, H., Takai, Y., Honma, M., Mori, H., Tomita, M.

Mol. Syst. Biol. 5 306 2009.09

Research paper (scientific journal), Joint Work, Accepted

Central carbon metabolism is a basic and exhaustively analyzed pathway. However, the intrinsic robustness of the pathway might still conceal uncharacterized reactions. To test this hypothesis, we constructed systematic multiple-knockout mutants involved in central carbon catabolism in Escherichia coli and tested their growth under 12 different nutrient conditions. Differences between in silico predictions and experimental growth indicated that unreported reactions existed within this extensively analyzed metabolic network. These putative reactions were then confirmed by metabolome analysis and in vitro enzymatic assays. Novel reactions regarding the breakdown of sedoheptulose-7-phosphate to erythrose-4-phosphate and dihydroxyacetone phosphate were observed in transaldolase-deficient mutants, without any noticeable changes in gene expression. These reactions, triggered by an accumulation of sedoheptulose-7-phosphate, were catalyzed by the universally conserved glycolytic enzymes ATP-dependent phosphofructokinase and aldolase. The emergence of an alternative pathway not requiring any changes in gene expression, but rather relying on the accumulation of an intermediate metabolite may be a novel mechanism mediating the robustness of these metabolic networks.

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Rescue of anaemia and autoimmune responses in SOD1-deficient mice by transgenic expression of human SOD1 in erythrocytes

Iuchi, Y., Okada, F., Takamiya, R., Kibe, N., Tsunoda, S., Nakajima, O., Toyoda, K., Nagae, R., Suematsu, M., Soga, T., Uchida, K., Fujii, J.

Biochem. J. 422 ( 2 ) 313 - 320 2009.08

Research paper (scientific journal), Joint Work, Accepted

Oxidative stress has been implicated as a cause of various diseases such as anaemia. We found that the SOD1 [Cu,Zn-SOD (superoxide dismutase)] gene deficiency causes anaemia, the production of autoantibodies against RBCs (red blood cells) and renal damage. In the present study, to further understand the role of oxidative stress in the autoimmune response triggered by SOD1 deficiency, we generated mice that had the hSOD1 (human SOD1) transgene under regulation of the GATA-1 promoter, and bred the transgene onto the SOD1(-/-) background (SOD1(-/-);hSOD1(tg/+)). The lifespan of RBCs, levels of intracellular reactive oxygen species, and RBC content in SOD1(-/-);hSOD1(tg/+) mice, were approximately equivalent to those of SOD1(+/+) mice. The production of antibodies against lipid peroxidation products, 4-hydroxy-2-nonenal and acrolein, as well as autoantibodies against RBCs and carbonic anhydrase II were elevated in the SOD1(-/-) mice, but were suppressed in the SOD1(-/-);hSOD1(tg/+) mice. Renal function, as judged by blood urea nitrogen, was improved in the transgenic mice. These results rule out the involvement of a defective immune system in the autoimmune response of SOD1-deficient mice, because SOD1(-/-);hSOD1(tg/+) mice carry the hSOD1 protein only in RBCs. Metabolomic analysis indicated a shift in glucose metabolism to the pentose phosphate pathway and a decrease in the energy charge potential of RBCs in SOD1-deficient mice. We conclude that the increase in reactive oxygen species due to SOD1 deficiency accelerates RBC destruction by affecting carbon metabolism and increasing oxidative modification of lipids and proteins. The resulting oxidation products are antigenic and, consequently, trigger autoantibody production, leading to autoimmune responses.

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Metabolomic Profiling of Anionic Metabolites by Capillary Electrophoresis Mass Spectrometry

Soga, T., Igarashi, K., Itoh, C., Mizobuchi, K., Zimmermann, H., Tomita, M.

Anal. Chem. 81 ( 15 ) 6165 - 6174 2009.08

Research paper (scientific journal), Joint Work, Accepted

We describe a sheath flow capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) method in the negative mode using a platinum electrospray ionization (ESI) spray needle, which allows the comprehensive analysis of anionic metabolites. The material of the spray needle had significant effect on the measurement of anions. A stainless steel spray needle was oxidized and corroded at the anodic electrode due to electrolysis. The precipitation of iron oxides (rust) plugged the capillary outlet, resulting in shortened capillary lifetime. Many anionic metabolites also formed complexes with the iron oxides or migrating nickel ion, which was also generated by electrolysis and moved toward the cathode (the capillary inlet). The metal-anion complex formation significantly reduced detection sensitivity of the anionic compounds. The use of a platinum ESI needle prevented both oxidation of the metals and needle corrosion. Sensitivity using the platinum needle increased from several- to 63-fold, with the largest improvements for anions exhibiting high metal chelating properties such as carboxylic acids, nucleotides, and coenzyme A compounds. The detection limits for most anions were between 0.03 and 0.87 micromol/L (0.8 and 24 fmol) at a signal-to-noise ratio of 3. This method is quantitative, sensitive, and robust, and its utility was demonstrated by the analysis of the metabolites in the central metabolic pathways extracted from mouse liver.

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Measurement of Internal Body Time by Blood Metabolomics

Minami, Y., Kasukawa, T., Kakazu, Y., Iigo, M., Sugimoto, M., Ikeda, S., Yasui, A., van der Horst, G.T., Soga, T., Ueda, H.

Proc. Natl. Acad. Sci. USA 106 ( 24 ) 9890 - 9895 2009.06

Research paper (scientific journal), Joint Work, Accepted

Detection of internal body time (BT) via a few-time-point assay has been a longstanding challenge in medicine, because BT information can be exploited to maximize potency and minimize toxicity during drug administration and thus will enable highly optimized medication. To address this challenge, we previously developed the concept, "molecular-timetable method," which was originally inspired by Linné's flower clock. In Linné's flower clock, one can estimate the time of the day by watching the opening and closing pattern of various flowers. Similarly, in the molecular-timetable method, one can measure the BT of the day by profiling the up and down patterns of substances in the molecular timetable. To make this method clinically feasible, we now performed blood metabolome analysis and here report the successful quantification of hundreds of clock-controlled metabolites in mouse plasma. Based on circadian blood metabolomics, we can detect individual BT under various conditions, demonstrating its robustness against genetic background, sex, age, and feeding differences. The power of this method is also demonstrated by the sensitive and accurate detection of circadian rhythm disorder in jet-lagged mice. These results suggest the potential for metabolomics-based detection of BT ("metabolite-timetable method"), which will lead to the realization of chronotherapy and personalized medicine.

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Metabolite profiling reveals YihU as a novel hydroxybutyrate dehydrogenase for alternative succinic semialdehyde metabolism in Escherichia coli

Saito, N., Robert, M., Kochi, H., Matsuo, G., Kakazu, Y., Soga, T., Tomita, M.

J. Biol. Chem 284 ( 24 ) 16442 - 16451 2009.06

Research paper (scientific journal), Joint Work, Accepted

The search for novel enzymes and enzymatic activities is important to map out all metabolic activities and reveal cellular metabolic processes in a more exhaustive manner. Here we present biochemical and physiological evidence for the function of the uncharacterized protein YihU in Escherichia coli using metabolite profiling by capillary electrophoresis time-of-flight mass spectrometry. To detect enzymatic activity and simultaneously identify possible substrates and products of the putative enzyme, we profiled a complex mixture of metabolites in the presence or absence of YihU. In this manner, succinic semialdehyde was identified as a substrate for YihU. The purified YihU protein catalyzed in vitro the NADH-dependent reduction of succinic semialdehyde to gamma-hydroxybutyrate. Moreover, a yihU deletion mutant displayed reduced tolerance to the cytotoxic effects of exogenous addition of succinic semialdehyde. Profiling of intracellular metabolites following treatment of E. coli with succinic semialdehyde supports the existence of a YihU-catalyzed reduction of succinic semialdehyde to gamma-hydroxybutyrate in addition to its known oxidation to succinate and through the tricarboxylic acid cycle. These findings suggest that YihU is a novel gamma-hydroxybutyrate dehydrogenase involved in the metabolism of succinic semialdehyde, and other potentially toxic intermediates that may accumulate under stress conditions in E. coli.

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Degradation of ppGpp by Nudix Pyrophosphatase Modulates the Transition of Growth Phase in the Bacterium Thermus thermophilus

Ooga, T., Ohashi, Y., Kuramitsu, S., Koyama, Y., Tomita, M., Soga, T., Masui, R.

J. Biol. Chem. 284 ( 23 ) 15549 - 15556 2009.06

Research paper (scientific journal), Joint Work, Accepted

A major bacterial alarmone, guanosine 3′,5′-bispyrophosphate (ppGpp), controls cellular growth under conditions of nutritional starvation. For most bacteria, intracellular ppGpp levels are tightly controlled by the synthesis/degradation cycle of RelA and SpoT activities. This study shows a novel ppGpp regulatory protein governing the cellular growth of Thermus thermophilus, Ndx8, a member of the Nudix pyrophosphatase family that degrades ppGpp to yield guanosine 3′,5′-bisphosphate. The ndx8-null mutant strain exhibited early stage growth arrest accompanied by the stationary phase-specific morphologies and global transcriptional modulation under nutritionally defined conditions. Several possible substrate compounds of Ndx8, which specifically accumulated in the ndx8 mutant cells, were identified by employing a capillary electrophoresis time-of-flight mass spectrometry-based metabolomics approach. Among them, the hydrolytic activity of Ndx8 for ppGpp was significant not only in vitro but also in vivo. Finally, the elimination of ppGpp synthetic activity suppressed the observed phenotype of the ndx8 mutation, suggesting that the function of Ndx8 as a growth regulator is involved in ppGpp accumulation, which is thought to act as a trigger of the growth phase transition. These results suggest a novel mechanism of ppGpp-mediated growth control by the functional relay between Ndx8 and SpoT activity as ppGpp scavengers.

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Quantitative Metabolome Profiling of Colon and Stomach Cancer Microenvironment by Capillary Electrophoresis Time-of-flight Mass Spectrometry

Hirayama, A., Kami, K., Sugimoto, M., Sugawara, M., Toki, N., Onozuka, H., Kinoshita, T., Saito, N., Ochiai, A., Tomita, M., Esumi, H., Soga, T.

Cancer Res. 69 ( 1 ) 4918 - 4925 2009.06

Research paper (scientific journal), Joint Work, Accepted

Most cancer cells predominantly produce energy by glycolysis rather than oxidative phosphorylation via the tricarboxylic acid (TCA) cycle, even in the presence of an adequate oxygen supply (Warburg effect). However, little has been reported regarding the direct measurements of global metabolites in clinical tumor tissues. Here, we applied capillary electrophoresis time-of-flight mass spectrometry, which enables comprehensive and quantitative analysis of charged metabolites, to simultaneously measure their levels in tumor and grossly normal tissues obtained from 16 colon and 12 stomach cancer patients. Quantification of 94 metabolites in colon and 95 metabolites in stomach involved in glycolysis, the pentose phosphate pathway, the TCA and urea cycles, and amino acid and nucleotide metabolisms resulted in the identification of several cancer-specific metabolic traits. Extremely low glucose and high lactate and glycolytic intermediate concentrations were found in both colon and stomach tumor tissues, which indicated enhanced glycolysis and thus confirmed the Warburg effect. Significant accumulation of all amino acids except glutamine in the tumors implied autophagic degradation of proteins and active glutamine breakdown for energy production, i.e., glutaminolysis. In addition, significant organ-specific differences were found in the levels of TCA cycle intermediates, which reflected the dependency of each tissue on aerobic respiration according to oxygen availability. The results uncovered unexpectedly poor nutritional conditions in the actual tumor microenvironment and showed that capillary electrophoresis coupled to mass spectrometry-based metabolomics, which is capable of quantifying the levels of energy metabolites in tissues, could be a powerful tool for the development of novel anticancer agents that target cancer-specific metabolism.

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Mono-(2-ethylhexyl) phthalate Targets Glycogen Debranching Enzyme and Affects Glycogen Metabolism in Rat Testis

Kuramori, C., Hase, Y., Hoshikawa, K., Watanabe, K., Nishi, T., Hishiki, T., Soga, T., Nashimoto, A., Kabe, Y., Yamaguchi, Y., Watanabe, H., Kataoka, K., Suematsu, M., Handa, H.

Toxicol. Sci. 109 ( 1 ) 143 - 151 2009.05

Research paper (scientific journal), Joint Work, Accepted

Phthalate esters are commonly used plasticizers; however, some are suspected to cause reproductive toxicity. Administration of high doses of di-(2-ethylhexyl) phthalate (DEHP) induces germ cell death in male rodents. Mono-(2-ethylhexyl) phthalate (MEHP), a hydrolyzed metabolite of DEHP, appears to be responsible for this testicular toxicity; however, the underlying mechanism of this chemical's action remains unknown. Here, using a one-step affinity purification procedure, we identified glycogen debranching enzyme (GDE) as a phthalate-binding protein. GDE has oligo-1,4-1,4-glucanotransferase and amylo-1,6-glucosidase activities, which are responsible for the complete degradation of glycogen to glucose. Our findings demonstrate that MEHP inhibits the activity of oligo-1,4-1,4-glucanotransferase, but not of amylo-1,6-glucosidase. Among various phthalate esters tested, MEHP specifically binds to and inhibits GDE. We also show that DEHP administration affects glycogen metabolism in rat testis. Thus, inhibition of GDE by MEHP may play a role in germ cell apoptosis in the testis.

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Cystathionine β-synthase as a Carbon Monoxide-sensitive Regulator of Bile Excretion

Shintani, T., Iwabuchi, T., Soga, T., Kato,Y., Yamamoto, T., Takano, N., Hishiki, T., Ueno, Y., Ikeda, S., Sakurakawa, T., Ishikawa, K., Goda, N., Yitagawa, Y., Kajiyama, M., Matsumoto, K., *Suematsu, M.

Hepatology 49 ( 1 ) 141 - 150 2009.01

Research paper (scientific journal), Joint Work, Accepted

Carbon monoxide (CO) is a stress-inducible gas generated by heme oxygenase (HO) eliciting adaptive responses against toxicants; however, mechanisms for its reception remain unknown. Serendipitous observation in metabolome analysis in CO-overproducing livers suggested roles of cystathionine beta-synthase (CBS) that rate-limits transsulfuration pathway and H(2)S generation, for the gas-responsive receptor. Studies using recombinant CBS indicated that CO binds to the prosthetic heme, stabilizing 6-coordinated CO-Fe(II)-histidine complex to block the activity, whereas nitric oxide (NO) forms 5-coordinated structure without inhibiting it. The CO-overproducing livers down-regulated H(2)S to stimulate HCO(3) (-)-dependent choleresis: these responses were attenuated by blocking HO or by donating H(2)S. Livers of heterozygous CBS knockout mice neither down-regulated H(2)S nor exhibited the choleresis while overproducing CO. In the mouse model of estradiol-induced cholestasis, CO overproduction by inducing HO-1 significantly improved the bile output through stimulating HCO(3) (-) excretion; such a choleretic response did not occur in the knockout mice.
Conclusion: Results collected from metabolome analyses suggested that CBS serves as a CO-sensitive modulator of H(2)S to support biliary excretion, shedding light on a putative role of the enzyme for stress-elicited adaptive response against bile-dependent detoxification processes.

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Time-resolved Metabolomics Reveals Metabolic Modulation in Rice Foliage

Sato, S., Arita, M., Soga, T., Nishioka, T., Tomita, M.

BMC Syst Biol. 2 51 2008.06

Research paper (scientific journal), Joint Work, Accepted

Background: To elucidate the interaction of dynamics among modules that constitute biological systems, comprehensive datasets obtained from "omics" technologies have been used. In recent plant metabolomics approaches, the reconstruction of metabolic correlation networks has been attempted using statistical techniques. However, the results were unsatisfactory and effective data-mining techniques that apply appropriate comprehensive datasets are needed.
Results: Using capillary electrophoresis mass spectrometry (CE-MS) and capillary electrophoresis diode-array detection (CE-DAD), we analyzed the dynamic changes in the level of 56 basic metabolites in plant foliage (Oryza sativa L. ssp. japonica) at hourly intervals over a 24-hr period. Unsupervised clustering of comprehensive metabolic profiles using Kohonen's self-organizing map (SOM) allowed classification of the biochemical pathways activated by the light and dark cycle. The carbon and nitrogen (C/N) metabolism in both periods was also visualized as a phenotypic linkage map that connects network modules on the basis of traditional metabolic pathways rather than pairwise correlations among metabolites. The regulatory networks of C/N assimilation/dissimilation at each time point were consistent with previous works on plant metabolism. In response to environmental stress, glutathione and spermidine fluctuated synchronously with their regulatory targets. Adenine nucleosides and nicotinamide coenzymes were regulated by phosphorylation and dephosphorylation. We also demonstrated that SOM analysis was applicable to the estimation of unidentifiable metabolites in metabolome analysis. Hierarchical clustering of a correlation coefficient matrix could help identify the bottleneck enzymes that regulate metabolic networks.
Conclusion: Our results showed that our SOM analysis with appropriate metabolic time-courses effectively revealed the synchronous dynamics among metabolic modules and elucidated the underlying biochemical functions. The application of discrimination of unidentified metabolites and the identification of bottleneck enzymatic steps even to non-targeted comprehensive analysis promise to facilitate an understanding of large-scale interactions among components in biological systems.

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Development of bottom-fermenting Saccharomyces strains that produce high SO2 levels, using integrated metabolome and transcriptome analysis

Yoshida, S., Imoto, J., Minato, T., Oouchi, R., Sugihara, M., Imai, T., Ishiguro, T., Mizutani, S., Tomita, M., Soga, T., Yoshimoto, H.

Appl. Environ. Microbiol. 74 ( 9 ) 2787 - 2796 2008.05

Research paper (scientific journal), Joint Work, Accepted

Sulfite plays an important role in beer flavor stability. Although breeding of bottom-fermenting Saccharomyces strains that produce high levels of SO2 is desirable, it is complicated by the fact that undesirable H2S is produced as an intermediate in the same pathway. Here, we report the development of a high-level SO2-producing bottom-fermenting yeast strain by integrated metabolome and transcriptome analysis. This analysis revealed that O-acetylhomoserine (OAH) is the rate-limiting factor for the production of SO2 and H2S. Appropriate genetic modifications were then introduced into a prototype strain to increase metabolic fluxes from aspartate to OAH and from sulfate to SO2, resulting in high SO2 and low H2S production. Spontaneous mutants of an industrial strain that were resistant to both methionine and threonine analogs were then analyzed for similar metabolic fluxes. One promising mutant produced much higher levels of SO2 than the parent but produced parental levels of H2S.

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Depiction of metabolome changes in histidine-starved Escherichia coli by CE-TOFMS

Ohashi, Y., Hirayama, A., Ishikawa, T., Nakamura, S., Shimizu, K., Ueno, Y., Tomita, M., *Soga, T.

Mol. BioSyst 4 ( 2 ) 135 - 147 2008.02

Research paper (scientific journal), Joint Work, Accepted

Metabolic changes in response to histidine starvation were observed in histidine-auxotrophic Escherichia coli using a capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS)-based metabolomics technique. Prior to the analysis, we prepared an E. coli metabolome list of 727 metabolites reported in the literature. An improved method for metabolite extraction was developed, which resulted in higher extraction efficiency in phosphate-rich metabolites, e.g., ATP and GTP. Based on the results, 375 charged, hydrophilic intermediates in primary metabolisms were analysed simultaneously, providing quantitative data of 198 metabolites. We confirmed that the intracellular levels of intermediates in histidine biosynthesis are rapidly accumulated in response to a drop in histidine level under histidine-starved conditions. Simultaneously, disciplined responses were observed in the glycolysis, tricarboxylic acid cycle, and amino acid and nucleotide biosynthesis pathways as regulated by amino acid starvation.

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Microelectrospray interface with coaxial sheath flow for high-resolution capillary electrophoresis/mass spectrometry separation.

Hashimoto, M., *Ishihama, Y., Tomita, M., Soga, T.

Rapid Commun. Mass Spectrom. 21 ( 22 ) 3579 - 3584 2007.11

Research paper (scientific journal), Joint Work, Accepted

We have fabricated a coaxial sheath liquid flow microelectrospray ionization (microESI) interface for capillary electrophoresis coupled with mass spectrometry (CE/MS). The ESI interface, which features a reduced probe diameter (130 microm i.d. x 174 microm o.d.) with a nebulizer-free format, can relatively easily electrospray a large amount of make-up sheath liquid (5-10 microL/min) over the long term (more than 80 runs) with a high degree of stability. The interface also provides higher separation qualities and improved detection sensitivities compared with a conventional ion spray (IS) interface.

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Metabolome Analysis by Capillary Electrophoresis – Mass Spectrometry

Monton M.R.N., Soga, T.

J. Chromatogr. A 1168 ( 1-2 ) 237 - 246 2007.10

Research paper (scientific journal), Joint Work, Accepted

Capillary electrophoresis (CE)-mass spectrometry (MS), as an analytical platform, has made significant contributions in advancing metabolomics research, if still limited up to this time. This review, covering reports published between 1998 and 2006, describes how CE-MS has been used thus far in this field, with the majority of the works dealing with targeted metabolite analyses and only a small fraction using it in the comprehensive context. It also discusses how some of the key features of CE-MS were exploited in selected metabolomic applications.

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Polymer Entrapment in Polymerized Silicate for Preparing Highly Stable Capillary Coatings in CE and CE-MS

Monton, R.M.N., Tomita, M., *Soga, T., *Ishihama, Y.

Anal. Chem. 79 ( 20 ) 7838 - 7844 2007.10

Research paper (scientific journal), Joint Work, Accepted

An easy-to-implement capillary coating strategy based on polymer entrapment in the network of polymerized silicate is described. In this manner, cationic polymers are tightly fixed onto the inner wall of the capillary for electroosmotic flow control without necessitating complex surface modification chemistries. The resulting coated capillary exhibited good stability over a wide range of pH, good reproducibility, strong endurance in more than 300 electrophoretic runs, and tolerance of commonly employed organic solvent additives in CE. Applications in CE-MS analysis of biologically important anions as well as sample enrichment are shown. Additionally, it was used as a durable base for attachment of multiple layers of charged polymers on the wall, via electrostatic interaction with the preceding layer. Thus, two novel types of highly stable coated capillaries, one with anodic EOF and the other cathodic, were developed.

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Direct measurement of isotopomer of intracellular metabolites using capillary electrophoresis time-of-flight mass spectrometry for efficient metabolic flux analysis

Toya, Y., Ishii, N., Hirasawa, T., Naba, M., Hirai, K., Sugawara, K., Igarashi, S., Shimizu, K., Tomita, M., *Soga, T.

J. Chromatogr. A 1159 ( 1-2 ) 134 - 141 2007.08

Research paper (scientific journal), Joint Work, Accepted

We have developed a metabolic flux analysis method that is based on 13C-labeling patterns of the intracellular metabolites directly measured by capillary electrophoresis time-of-flight mass spectrometry (CE–TOFMS). The flux distribution of the central carbon metabolism in Escherichia coli was determined by this new approach and the results were compared with findings obtained by conventional GC–MS analysis based on isotopomer of the proteinogenic amino acids. There were some differences in estimation results between new approach using CE–TOFMS and conventional approach using GC–MS. These were thought to be attributable to variations in measured mass distributions between amino acids and the corresponding precursors and to differences in the sensitivity of the exchange coefficients to mass distributions. However, our CE–TOFMS method facilitates high-throughput flux analysis without requiring complicated sample preparation such as hydrolysis of proteins and derivatization of amino acids.

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P-BOSS: A New Filtering Method for Treasure Hunting in Metabolomics

Morohashi, M., Shimizu, K., Ohashi, Y., Abe, J., Mori, H., Tomita, M., Soga, T.

J. Chromatogr. A 1159 ( 1-2 ) 142 - 148 2007.08

Research paper (scientific journal), Joint Work, Accepted

Metabolomics is expected to boost data driven research. In biomarker discovery, powerful filtering methods to remove noise and outliers are essential for screening significant candidates from the huge volume of omic data. Here we propose a post-measurement peak filtering method (named P-BOSS) for CE electrospray ionization–time-of-flight MS (CE–TOFMS) data. Combining outlier detection method functions in parallel, we applied P-BOSS to the data using Escherichia coli knockout mutants of the tryptophan and purine biosynthesis pathways. As the result, P-BOSS showed remarkably superior performance, reducing 65% of all peaks, while leaving significant peaks.

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Analysis of Nucleotides by Pressure-Assisted Capillary Electrophoresis Mass Spectrometry Using Silanol Mask Technique

Soga, T.*, Ishikawa, T., Igarashi, S., Sugawara, K., Kakazu Y., Tomita, M.

J. Chromatogr. A 1159 ( 1-2 ) 125 - 133 2007.08

Research paper (scientific journal), Joint Work, Accepted

A method for the determination of nucleotides based on pressure-assisted capillary electrophoresis–electrospray ionization mass spectrometry (PACE–MS) is described. To prevent multi-phosphorylated species from adsorbing onto the fused-silica capillary, silanol groups were masked with phosphate ions by preconditioning the capillary with the background electrolyte containing phosphate. During preconditioning, nebulizer gas was turned off to avoid contamination of MS detector with phosphate ions. To detect nucleotides using the CE positive mode at a pH 7.5, it was necessary to apply air pressure to the inlet capillary during electrophoresis to supplement the electroosmotic flow (EOF) toward the cathode. Moreover, we exchanged the running electrolyte every analysis using the buffer replenishment system to obtain the required reproducibility. Under the optimized conditions, 14 phosphorylated species such as nucleotides, nicotinamide–adenine dinucleotides and coenzyme A (CoA) compounds were well determined in less than 20 min. The relative standard deviations (n = 6) of the method were better than 0.9% for migration times and between 1.7% and 8.1% for peak areas. The detection limits for these species were between 0.5 and 1.7 μmol/L with pressure injection of 50 mbar for 30 s (30 nL) at a signal-to-noise ratio of 3. This approach is robust and quantitative compared to the previous method, and its utility is demonstrated by the analysis of intracellular nucleotides and CoA compounds extracted from Escherichia coli wild type, pfkA and pfkB knockout mutants. The methodology was used to suggest that pfkA is the main functional enzyme.

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Model based definition of population heterogeneity and its effects on metabolism in sporulating Bacillus subtilis

Morohashi, M., *Ohashi, Y., Tani S., Ishii, K., Itaya, M., Nanamiya, H., Kawamura, F., Tomita, M., Soga, T.

J. Biochem. 142 ( 2 ) 183 - 191 2007.08

Research paper (scientific journal), Joint Work, Accepted

The soil bacterium Bacillus subtilis forms dormant, robust spores as a tactic to ensure survival under conditions of starvation. However, the sporulating culture includes sporulating and non-sporulating cells, because a portion of the cell population initiates sporulation in wild-type strain. We anticipated that the population effect must be considered carefully to analyse samples yielding population heterogeneity. We first built a mathematical model and simulated for signal transduction of the sporulation cue to see what mechanisms are responsible for generating the heterogeneity. The simulated results were confirmed experimentally, where heterogeneity is primarily modulated by negative feedback circuits, resulting in generation of a bistable response within the sporulating culture. We also confirmed that mutants relevant to negative feedback yield either sporulating or non-sporulating subpopulations. To see the effect of molecular mechanism between sporulating and non-sporulating cells in distinct manner, metabolome analysis was conducted using the above mutants. The metabolic profiles exhibited distinct characteristics with time regardless of whether sporulation was initiated or not. In addition, several distinct characteristics of metabolites were observed between strains, which was inconsistent with previously reported data. The results imply that careful consideration must be made in the interpretation of data obtained from cells yielding population heterogeneity.

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Multiple High-throughput Analyses Monitor the Response of E. coli to Perturbations

Ishii, N., Nakahigashi, K., Baba, T., Robert, M., Soga, T., Kanai, A., Hirasawa, T., Naba, M., Hirai, K., Hoque, A., Ho, P.Y., Kakazu, Y., Sugawara, K., Igarashi, S.,Harada, S., Masuda, T., Sugiyama, N., Togashi, T., Hasegawa, M., Takai, Y., Yugi, K., Arakawa, K., Iwata, N., Toya, Y., Nakayama, Y., Nishioka, T., Shimizu, K., Mori, H., *Tomita, M

Science 316 ( 5824 ) 593 - 597 2007.04

Research paper (scientific journal), Joint Work, Accepted

Analysis of cellular components at multiple levels of biological information can provide valuable functional insights. We performed multiple high-throughput measurements to study the response of Escherichia coli cells to genetic and environmental perturbations. Analysis of metabolic enzyme gene disruptants revealed unexpectedly small changes in messenger RNA and proteins for most disruptants. Overall, metabolite levels were also stable, reflecting the rerouting of fluxes in the metabolic network. In contrast, E. coli actively regulated enzyme levels to maintain a stable metabolic state in response to changes in growth rate. E. coli thus seems to use complementary strategies that result in a metabolic network robust against perturbations.

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Roles of Hemoglobin Allostery in Hypoxia-induced Metabolic Alterations in Erythrocytes: Simulation and Its Demonstration by Metabolome Analysis

Kinoshita, A., Tsukada, K., Soga, T., Hishiki, T., Ueno, Y., Nakayama, Y., Tomita, M., Suematsu, M

J. Biol. Chem. 282 ( 14 ) 10731 - 10741 2007.04

Research paper (scientific journal), Joint Work, Accepted

When erythrocytes are exposed to hypoxia, hemoglobin (Hb) stabilizes in the T-state by capturing 2,3-bisphosphoglycerate. This process could reduce the intracellular pool of glycolytic substrates, jeopardizing cellular energetics. Recent observations suggest that hypoxia-induced activation of glycolytic enzymes is correlated with their release from Band III (BIII) on the cell membrane. Based on these data, we developed a mathematical model of erythrocyte metabolism and compared hypoxia-induced differences in predicted activities of the enzymes, their products, and cellular energetics between models with and without the interaction of Hb with BIII. The models predicted that the allostery-dependent Hb interaction with BIII accelerates consumption of upstream glycolytic substrates such as glucose 6-phosphate and increases downstream products such as phosphoenolpyruvate. This prediction was consistent with metabolomic data from capillary electrophoresis mass spectrometry. The hypoxia-induced alterations in the metabolites resulted from acceleration of glycolysis, as judged by increased conversion of [13C]glucose to [13C]lactate. The allostery-dependent interaction of Hb with BIII appeared to contribute not only to maintenance of energy charge but also to further synthesis of 2,3-bisphosphoglycerate, which could help sustain stabilization of T-state Hb during hypoxia. Furthermore, such an activation of glycolysis was not observed when Hb was stabilized in R-state by treating the cells with CO. These results suggest that Hb allostery in erythrocytes serves as an O2-sensing trigger that drives glycolytic acceleration to stabilize intracellular energetics and promote the ability to release O2 from the cells.

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Visualization of Three-way Comparisons of Omics Data

Baran, R., Robert, M.*, Suematsu, M., Soga, T., Tomita, M.

BMC Bioinformatics 8 72 2007.03

Research paper (scientific journal), Joint Work, Accepted

Background: Density plot visualizations (also referred to as heat maps or color maps) are widely used in different fields including large-scale omics studies in biological sciences. However, the current color-codings limit the visualizations to single datasets or pairwise comparisons.
Results: We propose a color-coding approach for the representation of three-way comparisons. The approach is based on the HSB (hue, saturation, brightness) color model. The three compared values are assigned specific hue values from the circular hue range (e.g. red, green, and blue). The hue value representing the three-way comparison is calculated according to the distribution of three compared values. If two of the values are identical and one is different, the resulting hue is set to the characteristic hue of the differing value. If all three compared values are different, the resulting hue is selected from a color gradient running between the hues of the two most distant values (as measured by the absolute value of their difference) according to the relative position of the third value between the two. The saturation of the color representing the three-way comparison reflects the amplitude (or extent) of the numerical difference between the two most distant values according to a scale of interest. The brightness is set to a maximum value by default but can be used to encode additional information about the three-way comparison.
Conclusion: We propose a novel color-coding approach for intuitive visualization of three-way comparisons of omics data.

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MathDAMP: a package for differential analysis of metabolite profiles

Baran, R., Kochi, H., Saito, N., Suematsu, M., Soga, T., Nishioka, T., Robert, M., Tomita, M.

BMC Bioinformatics 7 530 2006.12

Research paper (scientific journal), Joint Work, Accepted

Background: With the advent of metabolomics as a powerful tool for both functional and biomarker discovery, the identification of specific differences between complex metabolite profiles is becoming a major challenge in the data analysis pipeline. The task remains difficult, given the datasets' size, complexity, and common shifts in migration (elution/retention) times between samples analyzed by hyphenated mass spectrometry methods.
Results: We present a Mathematica (Wolfram Research, Inc.) package MathDAMP (Mathematica package for Differential Analysis of Metabolite Profiles), which highlights differences between raw datasets acquired by hyphenated mass spectrometry methods by applying arithmetic operations to all corresponding signal intensities on a datapoint-by-datapoint basis. Peak identification and integration is thus bypassed and the results are displayed graphically. To facilitate direct comparisons, the raw datasets are automatically preprocessed and normalized in terms of both migration times and signal intensities. A combination of dynamic programming and global optimization is used for the alignment of the datasets along the migration time dimension. The processed datasets and the results of direct comparisons between them are visualized using density plots (axes represent migration time and m/z values while peaks appear as color-coded spots) providing an intuitive overall view. Various forms of comparisons and statistical tests can be applied to highlight subtle differences. Overlaid electropherograms (chromatograms) corresponding to the vicinities of the candidate differences from any result may be generated in a descending order of significance for visual confirmation. Additionally, a standard library table (a list of m/z values and migration times for known compounds) may be aligned and overlaid on the plots to allow easier identification of metabolites.
Conclusion: Our tool facilitates the visualization and identification of differences between complex metabolite profiles according to various criteria in an automated fashion and is useful for data-driven discovery of biomarkers and functional genomics.

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Prediction of liquid chromatographic retention times of peptides generated by protease digestion of the Escherichia coli proteome using artificial neural networks

Shinoda, K., Sugimoto, M., Yachie, N., Sugiyama, N., Masuda, T., Robert, M., Soga, T., Tomita, M.,

J. Proteome Res 5 ( 12 ) 3312 - 3317 2006.12

Research paper (scientific journal), Joint Work, Accepted

We developed a computational method to predict the retention times of peptides in HPLC using artificial neural networks (ANN). We performed stepwise multiple linear regressions and selected for ANN input amino acids that significantly affected the LC retention time. Unlike conventional linear models, the trained ANN accurately predicted the retention time of peptides containing up to 50 amino acid residues. In 834 peptides, there was a strong correlation (R2 = 0.928) between measured and predicted retention times. We demonstrated the utility of our method by the prediction of the retention time of 121,273 peptides resulting from LysC-digestion of the Escherichia coli proteome. Our approach is useful for the proteome-wide characterization of peptides and the identification of unknown peptide peaks obtained in proteome analysis.

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HybGFS: a hybrid method for genome-fingerprint scanning

Shinoda, K., Yachie, N., Masuda, T., Sugiyama, N., Sugimoto, M., Soga, T., Tomita, M.,

BMC Bioinformatics 7 479 2006.10

Research paper (scientific journal), Joint Work, Accepted

Background: Protein identification based on mass spectrometry (MS) has previously been performed using peptide mass fingerprinting (PMF) or tandem MS (MS/MS) database searching. However, these methods cannot identify proteins that are not already listed in existing databases. Moreover, the alternative approach of de novo sequencing requires costly equipment and the interpretation of complex MS/MS spectra. Thus, there is a need for novel high-throughput protein-identification methods that are independent of existing predefined protein databases.
Results: Here, we present a hybrid method for genome-fingerprint scanning, known as HybGFS. This technique combines genome sequence-based peptide MS/MS ion searching with liquid-chromatography elution-time (LC-ET) prediction, to improve the reliability of identification. The hybrid method allows the simultaneous identification and mapping of proteins without a priori information about their coding sequences. The current study used standard LC-MS/MS data to query an in silico-generated six-reading-frame translation and the enzymatic digest of an entire genome. Used in conjunction with precursor/product ion-mass searching, the LC-ETs increased confidence in the peptide-identification process and reduced the number of false-positive matches. The power of this method was demonstrated using recombinant proteins from the Escherichia coli K12 strain.
Conclusion: The novel hybrid method described in this study will be useful for the large-scale experimental confirmation of genome coding sequences, without the need for transcriptome-level expression analysis or costly MS database searching.

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Evaluation of Metabolic Alteration in Transgenic Rice Overexpressing Dihydroflavonol-4-reductase

Takahashi, H., Hayashi, M., Sato, S., Soga, T., Nishioka, T., Tomita, M., Kawai-Yamada, M., Uchimiya, H.,

Ann. Bot. 98 ( 4 ) 819 - 825 2006.10

Research paper (scientific journal), Joint Work, Accepted

Background and aims: Previous studies have shown that transgenic rice plants overexpressing YK1, which possesses dihydroflavonol-4-reductase (DFR) activity, showed biotic and abiotic stress tolerance. High throughput profiles of metabolites have also been shown in such transgenic plants by Fourier transform ion cyclotron mass spectrometry. In this study, capillary electrophoresis mass spectrometry analysis (CE/MS) was employed to identify precise metabolites such as organic acids, amino acids and sugars.
Methods: Using CE/MS, we analysed several metabolites of glycolysis, the tricarboxylic acid (TCA) cycle and the pentose phosphate pathway. In addition, the concentrations of sugars and ion were quantified.
Key results: In YK1 (DFR)-overexpressing plants, the concentrations of cis-aconitate, isocitrate and 2-oxoglutarate were higher in leaves, whereas those of fructose-1,6-bisphosphate and glyceraldehyde-3-phosphate were lower in roots. In seeds, the amounts of free amino acids and metals were altered, whereas sugars in seeds were kept constant. In YK1 calli, an approx. 3-fold increase in glutathione was observed, whereas the activities of glutathione peroxidase and glutathione reductase were concomitantly increased.
Conclusions: The overexpression of YK1 (DFR) was associated with slight changes in the amounts of several metabolites analysed in whole plants, whilst glutathione derivatives were substantially increased in suspension-cultured cells.

Metabolomics Approach for Enzyme Discovery

Saito, N., Robert, M., Kitamura, S., Baran, R., Soga, T., Mori, H., Nishioka, T., Tomita, M.

J. Proteome Res. 5 ( 8 ) 1979 - 1987 2006.08

Research paper (scientific journal), Joint Work, Accepted

The search for novel enzymes is an important but difficult task in functional genomics. Here, we present a systematic method based on in vitro assays in combination with metabolite profiling to discover novel enzymatic activities. A complex mixture of metabolites is incubated with purified candidate proteins and the reaction mixture is subsequently profiled by capillary electrophoresis electrospray ionization mass spectrometry (CE-MS). Specific changes in the metabolite composition can directly suggest the presence of an enzymatic activity while subsequent identification of the compounds whose level changed specifically can pinpoint the actual substrate(s) and product(s) of the reaction. We first evaluated the method using several Escherichia coli metabolic enzymes and then applied it to the functional screening of uncharacterized proteins. In this manner, YbhA and YbiV proteins were found to display both phosphotransferase and phosphatase activity toward different sugars/sugar phosphates. Our approach should be broadly applicable and useful for enzyme discovery in any system.

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Differential Metabolomics Reveals Ophthalmic Acid As An Oxidative Stress Biomarker Indicating Hepatic Glutathione Consumption

Soga, T., Baran, R., Suematsu M., Ueno, Y., Ikeda, S., Sakurakawa T., Kakazu, Y., Ishikawa, T., Robert, M., Nishioka, T., Tomita, M.

J. Biol. Chem. 281 ( 24 ) 16768 - 16776 2006.06

Research paper (scientific journal), Joint Work, Accepted

Metabolomics is an emerging tool that can be used to gain insights into cellular and physiological responses. Here we present a metabolome differential display method based on capillary electrophoresis time-of-flight mass spectrometry to profile liver metabolites following acetaminophen-induced hepatotoxicity. We globally detected 1,859 peaks in mouse liver extracts and highlighted multiple changes in metabolite levels, including an activation of the ophthalmate biosynthesis pathway. We confirmed that ophthalmate was synthesized from 2-aminobutyrate through consecutive reactions with gamma-glutamylcysteine and glutathione synthetase. Changes in ophthalmate level in mouse serum and liver extracts were closely correlated and ophthalmate levels increased significantly in conjunction with glutathione consumption. Overall, our results provide a broad picture of hepatic metabolite changes following acetaminophen treatment. In addition, we specifically found that serum ophthalmate is a sensitive indicator of hepatic GSH depletion, and may be a new biomarker for oxidative stress. Our method can thus pinpoint specific metabolite changes and provide insights into the perturbation of metabolic pathways on a large scale and serve as a powerful new tool for discovering low molecular weight biomarkers.

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Conductivity detection in capillary zone electrophoresis: Inspection by PeakMaster

Jaros, M., Soga, T., Van de Goor, T., *Gas, B.

Electrophoresis 26 ( 10 ) 1948 - 1953 2005.05

Research paper (scientific journal), Joint Work, Accepted, ISSN 01730835

A simple rule stating that the signal in conductivity detection in capillary zone electrophoresis is proportional to the difference between the analyte mobility and mobility of the background electrolyte (BGE) co‐ion is valid only for systems with fully ionized electrolytes. In zone electrophoresis systems with weak electrolytes both conductivity signal and electromigration dispersion of analyte peaks depend on the conductivity and pH effects. This allows optimization of the composition of BGEs to give a good conductivity signal of analytes while still keeping electromigration dispersion near zero, regardless of the injected amount of sample. The demands to achieve minimum electromigration dispersion and high sensitivity in conductivity detection can be accomplished at the same time. PeakMaster software is used for inspection of BGEs commonly used for separation of sugars (carbohydrates, saccharides) at highly alkaline pH. It is shown that the terms direct and indirect conductivity detection are misleading and should not be used.

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Metabolome analysis and metabolic simulation

Ishii, N., Soga, T., Nishioka, T., Tomita, M.

Metabolomics 1 ( 1 ) 29 - 37 2005.03

Research paper (scientific journal), Joint Work, Accepted

For many decades microorganisms have been used for industrial purposes; traditional fermentations such as brewing and production of food additives, aroma molecules, organic acids and pharmaceutical-like antibiotics or recombinant proteins are instances of the industrial microorganism utilization. Therefore, microorganism modeling and simulation have been required for engineering purposes, because of demands for design, optimization and quality control of large-scale fermentation plants. Modeling has recently become more highly developed, aided by the deciphering of microorganism genomes, the completion of metabolic databases, the development of analytical methodologies and improvements in the performance of computers. This paper reviews past and recent metabolic simulation of microorganisms, and also discusses the metabolome analytical techniques and the construction of large-scale microorganism models which are now being developed in our group.

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Large-Scale Prediction of Cationic Metabolite Identity and Migration Time in Capillary Electrophoresis Mass Spectrometry Using Artificial Neural Networks

Sugimoto, M., Kikuchi, S., Arita, M., Soga, T., Nishioka, T., Tomita, M.

Anal. Chem. 77 ( 1 ) 78 - 84 2005.01

Research paper (scientific journal), Joint Work, Accepted

We developed a computational technique to assist in the large-scale identification of charged metabolites. The electrophoretic mobility of metabolites in capillary electrophoresis-mass spectrometry (CE-MS) was predicted from their structure, using an ensemble of artificial neural networks (ANNs). Comparison between relative migration times of 241 various cations measured by CE-MS and predicted by a trained ANN ensemble produced a correlation coefficient of 0.931. When we used our technique to characterize all metabolites listed in the KEGG ligand database, the correct compounds among the top three candidates were predicted in 78.0% of cases. We suggest that this approach can be used for the prediction of the migration time of any cation and that it represents a powerful method for the identification of uncharacterized CE-MS peaks in metabolome analysis.

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Simultaneous Determination of Main Metabolites in Rice Leaves using Capillary Electrophoresis Mass Spectrometry and Capillary Electrophoresis Diode Array Detection

Sato, S., Soga, T., Nishioka, T., Tomita, M.

Plant J. 40 ( 1 ) 151 - 163 2004.10

Research paper (scientific journal), Joint Work, Accepted

The study of the metabolomics of primary metabolites using conventional chemical analyses requires a high‐throughput method. Chemical derivatizations are a prerequisite for gas‐chromatographic separation, and a large sample quantity is needed for liquid‐chromatographic separation and nuclear magnetic resonance detection systems. Recently, we have developed a capillary electrophoresis–mass spectrometry (CE–MS) technology that can simultaneously quantify a large number of primary metabolites, using only a small quantity of samples, and without any chemical derivatizations. Parallel use of a capillary electrophoresis–diode array detector (CE–DAD) system further enables almost all water‐soluble intracellular metabolites to be analyzed. We demonstrate, with rice leaves, a simple and rapid method of sample preparation for CE analysis; using this method, we have successfully measured the levels of 88 main metabolites involved in glycolysis, the tricarboxylic acid cycle, the pentose phosphate pathway, photorespiration, and amino acid biosynthesis.

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Application of capillary electrophoresis‐mass spectrometry to synthetic in vitro glycolysis studies

Itoh, A., Ohashi, Y., Soga, T., Mori, H., Nishioka, T., Tomita, M.

Electrophoresis 25 ( 13 ) 1996 - 2002 2004.07

Research paper (scientific journal), Joint Work, Accepted

We propose an approach designed to reconstitute a metabolic pathway composed of multistep biochemical reactions, rather than to dissect the individual reactions that make up the pathway. A synthetic in vitro glycolysis was reconstructed from ten purified Escherichia coli (E. coli) enzymes to obtain a better understanding of the regulation of sequential enzymatic reactions. The key to the success of this approach is the ability to perform direct and simultaneous determination of the diverse metabolic intermediates in the pathway by capillary electrophoresis-mass spectrometry. We observed that the pathway is regulated by a delicate balance between the changing metabolite concentrations and behaves like a natural biological oscillating network that has hitherto not been reported for E. coli glycolysis. The end-product, pyruvate, was periodically synthesized from glucose at an overall efficiency of 30%, corresponding to an average of 90% conversion efficiency for each of the ten steps involved. This approach is likely useful for the synthesis of natural products requiring complex sequential biocatalytic reactions.

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Qualitative and quantitative analysis of amino acids by capillary electrophoresis‐electrospray ionization‐tandem mass spectrometry

Soga, T., Kakazu, Y., Robert.M., Tomita, M., Nishioka, T.

Electrophoresis 25 ( 13 ) 1964 - 1972 2004.07

Research paper (scientific journal), Joint Work, Accepted

We describe a method to identify and quantify amino acids using capillary electrophoresis-electrospray ionization-triple-quadrupole tandem mass spectrometry (CE-ESI-MS/MS). Amino acids, including physiological amino acids, were first separated by CE under acidic pH conditions and then detected by MS/MS. To efficiently introduce the whole sample into the capillary, no electrical potential was applied to the electrospray probe until running electrophoresis. The position of the electrosprayer with respect to the MS capillary entrance drastically affected sensitivity and generation of cluster ions. MS/MS with multiple reaction monitoring (MRM) detection was performed to obtain sufficient selectivity and sensitivity. Under optimized CE-MS/MS conditions, the minimum detectable levels for 32 free amino acids normally found in proteins and other physiological amino acids were between 0.1 and 14 micromol/L with pressure injection of 50 mbar for 3 s (3 nL) at a signal-to-noise ratio of 3. For most amino acids, this constitutes a severalfold increase in sensitivity compared to CE-MS. The relative standard deviations (% RSD) for all amino acids were better than 0.4% for migration times and between 1.4% and 8.6% for peak areas (n = 10). Since amino acids exhibited characteristic MS/MS spectra, this approach is useful for the simultaneous, selective, quantitative, and reproducible analysis of amino acids in physiological and biological samples that contain various kinds of matrices. The power of the method was demonstrated by analyzing amino acids in human urine.

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Quantitative Metabolome Analysis Using Capillary ElectrophoresisMass Spectrometry

Soga T, Ohashi Y, Ueno Y, Naraoka H, Tomita M, Nishioka T

J Proteome Res. 2 ( 5 ) 488 - 494 2003.10

Research paper (scientific journal), Joint Work, Accepted

A new approach for the comprehensive and quantitative analysis of charged metabolites by capillary electrophoresis mass spectrometry (CE-MS) is proposed. Metabolites are first separated by CE based on charge and size and then selectively detected using MS by monitoring over a large range of m/z values. This method enabled the determination of 352 metabolic standards and its utility was demonstrated in the analysis of 1692 metabolites from Bacillus subtilis extracts, revealing significant changes in metabolites during B. subtilis sporulation.

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Pressure-Assisted Capillary Electrophoresis Electrospray Ionization Mass Spectrometry for Analysis of Multivalent Anions

Soga, T., Ueno, Y., Naraoka, H., Matsuda, K., Tomita, M., and Nishioka, T.

Anal. Chem. 74 ( 24 ) 6224 - 6229 2002.12

Research paper (scientific journal), Joint Work, Accepted

We describe a method, based on pressure-assisted capillary electrophoresis coupled to electrospray ionization mass spectrometry (PACE/ESI-MS), that allows the simultaneous and quantitative analysis of multivalent anions, such as citrate isomers, nucleotides, nicotinamide-adenine dinucleotides, and flavin adenine dinucleotide, and coenzyme A (CoA) compounds. Key to the analysis was using a noncharged polymer, poly(dimethylsiloxane), coated to the inner surface of the capillary to prevent anionic species from adsorbing onto the capillary wall. It was also necessary to drive a constant liquid flow toward the MS by applying air pressure to the inlet capillary during electrophoresis to maintain a conductive liquid junction between the capillary and the electrospray needle. Although theoretical plates were inferior to those obtained by CE/ESI-MS using a cationic polymer-coated capillary, the PACE/ESI-MS method improved reproducibility and sensitivity of these anions. Eighteen anions were separated by PACE and selectively detected by a quadrupole mass spectrometer with a sheath-flow electrospray ionization interface. The relative standard deviations (n = 6) of the method were better than 0.6% for migration times and between 1.4% and 6.2% for peak areas. The detection limits for these species were between 0.4 and 3.7 μmol/L with pressure injection of 50 mbar for 30 s (30 nL), that is, mass detection limits calculated in the range from 12 to 110 fmol at a signal-to-noise ratio of 3. The utility of the method was demonstrated by analysis of citrate isomers, nucleotides, dinucleotides, and CoA compounds extracted from Bacillus subtilis cells.

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Combined Metabolome and Transcriptome Analysis of Bacillus subtilis Cells Cultured on Different Carbon Sources

Matsuda, K., Yamaguchi, H., Ueno, Y., Soga, T., Fujita, Y., and Nishioka, T.

Genome Informatics 13 593 - 594 2002.12

Research paper (scientific journal), Joint Work, Accepted

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Simultaneous Determination of Anionic Intermediates for Bacillus subtilis Metabolic Pathways by Capillary Electrophoresis Electrospray Ionization Mass Spectrometry

Soga, T., Ueno, Y., Naraoka, H., Ohashi, Y., Tomita, M., and Nishioka, T.

Anal. Chem. 74 ( 10 ) 2233 - 2239 2002.05

Research paper (scientific journal), Joint Work, Accepted

A method for simultaneous determination of anionic metabolites based on capillary electrophoresis (CE) coupled to electrospray ionization mass spectrometry is described. To prevent current drop by the system, electroosmotic flow (EOF) reversal by using a cationic polymer-coated capillary was indispensable. A mixture containing 32 standards including carboxylic acids, phosphorylated carboxylic acids, phosphorylated saccharides, nucleotides, and nicotinamide and flavin adenine coenzymes of glycolysis and the tricarboxylic acid cycle pathways were separated by CE and selectively detected by a quadrupole mass spectrometer with a sheath-flow electrospray ionization interface. Key to the analysis was EOF reversal using a cationic polymer-coated capillary and an electrolyte system consisting of 50 mM ammonium acetate, pH 9.0. The relative standard deviations of the method were better than 0.4% for migration times and between 0.9% and 5.4% for peak areas. The concentration detection limits for these metabolites were between 0.3 and 6.7 μmol/L with pressure injection of 50 mbar for 30 s (30 nL); i.e., mass detection limits ranged from 9 to 200 fmol, at a signal-to-noise ratio of 3. This method was applied to the comprehensive analysis of metabolic intermediates extracted from Bacillus subtilis, and 27 anionic metabolites could be directly detected and quantified.

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Capillary electrophoresis method for the analysis of inorganic anions, organic acids, amino acids, nucleotides, carbohydrates and other anionic compounds

Soga, T., Imaizumi, M.

Electrophoresis 22 ( 16 ) 3418 - 3425 2001.10

Research paper (scientific journal), Joint Work, Accepted

A previously developed capillary zone electrophoresis (CZE) method with indirect UV detection for the simultaneous determination of inorganic and organic anions, amino acids and carbohydrates using 20 mM 2,6‐pyridinedicarboxylic acid (PDC) as the background electrolyte was extended to allow determination of 206 anions including those above – mentioned and physiological amino acids, nucleotides, aromatic acids, haloacetic acids, alcohols, phosphorylated saccharides, oxyhalides, metal oxoacids, metal‐ethylenediaminetetraacetic acid (EDTA) complexes, forensic anions, Good's buffers and herbicides. Every compound could be analyzed and their electrophoretic mobility determined simply by selecting detection wavelength. This method is simple and universal for anion analysis, and could be readily applied to the simultaneous determination of anionic compounds. In this work, it was used to identify and quantify important anions in sea urchin and sake.

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Direct chiral resolution of malic acid in apple juice by ligand‐exchange capillary electrophoresis using copper(II)‐L‐tartaric acid as a chiral selector

Kodama, S., Yamamoto, A., Matsunaga, A., Soga, T., Hayakawa, K.

Electrophoresis 22 ( 15 ) 3286 - 3290 2001.09

Research paper (scientific journal), Joint Work, Accepted

Chiral resolution of native DL-malic acid was achieved by ligand-exchange capillary electrophoresis using copper(II)-L-tartrate as a chiral selector. Factors affecting chiral resolution, migration time, and peak area of malic acid were studied. The running conditions for optimum separation of malic acid were found to be 1 mM copper(II) sulfate-1 mM L-tartrate (pH 5.1) with an effective voltage of -20 kV at 30 degrees C, using direct detection at 280 nm, and resolution (Rs) of racemic malic acid was approximately 4. With this system, D- and L-malic acids in apple juice were analyzed successfully.

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Determination of carbohydrates in food samples by capillary electrophoresis with indirect UV detection

Soga, T., Serwe, M

Food Chem. 69 ( 3 ) 339 - 344 2000.05

Research paper (scientific journal), Joint Work, Accepted

An easy and reproducible capillary electrophoretic method with indirect UV detection for the determination of carbohydrates in food samples was described. A highly alkaline pH condition was used in order to charge every carbohydrate negatively. Electroosmotic flow (EOF) was reversed to detect sugar alcohols and to improve the separation of neutral and amino sugars. Under optimized conditions 28 carbohydrates including mono- and disaccharides, uronic acids, sialic acids, amino sugars and sugar alcohols were separated in less than 25 min. The detection limits for fructose, glucose and sucrose were in the range from 12 to 16 mg l−1 with pressure injection of 50 mbar for 6 s (ca. 6 nl) at a signal to noise ratio of 3. The calibration curves were linear over the range from 50 to 10 000 mg l−1 with correlation coefficients better than 0.9999. This method could be readily applied to the analysis of carbohydrates in several foods and beverages. Sample preparation was minimal and consisted either of dilution or dilution with filtration.

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Direct chiral resolution of lactic acid in food products by capillary electrophoresis

Kodama, S., Yamamoto, A., Matsunaga, A., Soga, T., and Minoura, K.

J. Chromatogr. A. 875 ( 1-2 ) 371 - 377 2000.04

Research paper (scientific journal), Joint Work, Accepted

Chiral resolution of native dl-lactic acid was performed by capillary electrophoresis using 2-hydroxypropyl-β-cyclodextrin as a chiral selector. Various factors affecting chiral resolution, migration time, and peak area of lactic acid were studied. The running conditions for optimum separation of lactic acid were found to be 90 mM phosphate buffer (pH 6.0) containing 240 mM 2-hydroxypropyl-β-cyclodextrin with an effective voltage of −30 kV at 16°C, using direct detection at 200 nm. In order to enhance the sensitivity, sample injection was done under a pressure of 50 mbar for 200 s. On-line sample concentration was accomplished by sample stacking. With this system, d- and l-lactic acids in food products were analyzed successfully.

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Amino Acid Analysis by Capillary Electrophoresis Electrospray Ionization Mass Spectrometry

Soga, T., Heiger, D.N.

Anal. Chem. 72 ( 6 ) 1236 - 1241 2000.03

Research paper (scientific journal), Joint Work, Accepted

A method for the determination of underivatized amino acids based on capillary electrophoresis coupled to electrospray ionization mass spectrometry (CE−ESI-MS) is described. To analyze free amino acids simultaneously a low acidic pH condition was used to confer positive charge on whole amino acids. The choice of the electrolyte and its concentration influenced resolution and peak shape of the amino acids, and 1 M formic acid was selected as the optimal electrolyte. Meanwhile, the sheath liquid composition had a significant effect on sensitivity and the highest sensitivity was obtained when 5 mM ammonium acetate in 50% (v/v) methanol−water was used. Protonated amino acids were roughly separated by CE and selectively detected by a quadrupole mass spectrometer with a sheath flow electrospray ionization interface. Under the optimized conditions, 19 free amino acids normally found in proteins and several physiological amino acids were well determined in less than 17 min. The detection limits for basic amino acids were between 0.3 and 1.1 μmol/L and for acidic and low molecular weight amino acids were less than 6.0 μmol/L with pressure injection of 50 mbar for 3 s (3 nL) at a signal-to-noise ratio of 3. This method is simple, rapid, and selective compared with conventional techniques and could be readily applied to the analysis of free amino acids in soy sauce.

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Capillary Electrophoresis for the Determination of Forensic Anions in Adulterated Foods and Beverages

Soga, T., Tajima, I., Heiger, D.N.

Am. Lab. 32 ( 3 ) 124 - 128 2000.02

Research paper (scientific journal), Joint Work, Accepted

Determination of Aspartic Acid Enantiomers in Bio-samples by Capillary Electrophoresis

Tsunoda, M., Kato, M., Fukushima, T., Santa, T., Homma, H., Yani, H., Soga, T., and Imai, K.

Biomed. Chromatogr 13 ( 5 ) 335 - 339 1999.08

Research paper (scientific journal), Joint Work, Accepted

Enantiomeric separation and detection of D,L-aspartic acid (Asp) derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) by capillary electrophoresis (CE) using modified cyclodextrins as chiral selectors was studied. Heptakis(2,3, 6-tri-O-methyl)-beta-cyclodextrin(TM-beta-CD) was most effective for enantiomeric separation of NBD-D,L-Asp with optimum conditions of 30 mM TM-beta-CD in 50 mM phosphate buffer (pH 4.0) and the limit of detection (LOD) attained was 100 nM for each enantiomer. The method proposed in the present study was convenient for both D- and L-Asp determination since the other amino compounds migrated differently and D-Asp in bio-samples such as rat pineal gland and foods was determined with a simple sample pretreatment and a short analysis run time.

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Simultaneous determination of inorganic anions, organic acids, amino acids and carbohydrates by capillary electrophoresis

Soga, T., Ross, G.A.

J. Chromatogr. A 837 ( 1-2 ) 231 - 239 1999.04

Research paper (scientific journal), Joint Work, Accepted

A capillary zone electrophoretic method for the simultaneous analysis of inorganic anions, organic acids, amino acids and carbohydrates was developed with indirect UV detection using 2,6-pyridine dicarboxylic acid as the background electrolyte. This is the first paper to report their simultaneous analysis. Highly alkaline conditions were used in order to confer a negative charge not only on inorganic and organic anions but also on amino acids and carbohydrates, and to promote their migration towards the anode. Electroosmotic flow was reversed in the direction of the anode by adding cetyltrimethylammonium hydroxide to the electrolyte. Outstanding separations were obtained and electrophoretic mobilities of 82 compounds including nine inorganic anions, 23 organic acids, 18 amino acids and 32 carbohydrates were determined by the method. Under the optimized conditions 43 compounds were well separated in a single run. The relative standard deviations (n=5) of the method were better than 0.5% for migration times and between 0.8% and 4.9% for peak areas except for a few compounds. The detection limits for anions and amino acids were in the range from 6 to 12 mg/l and for carbohydrates from 23 to 35 mg/l with 300 mbar·s pressure injection (6 nl) at a signal-to-noise ratio of three. This method could be readily applied to the simultaneous determination of inorganic anions, organic acids, amino acids and carbohydrates in soy sauce, nutrient tonic and pineapple.

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Simultaneous determination of inorganic anions, organic acids and metal cations by capillary electrophoresis

Soga, T., Ross, G.A.

J. Chromatogr. A 834 ( 1-2 ) 65 - 71 1999.02

Research paper (scientific journal), Joint Work, Accepted

A capillary electrophoresis method for the simultaneous analysis of inorganic anions, organic acids and metal cations was developed. 2,6-Pyridinedicarboxylic acid (PDC) was used as the background electrolyte to permit indirect UV detection of anions. The PDC also worked to confer a negative charge on metal cations via PDC-complex formation. In order to detect both anions and metal–PDC complexes simultaneously, the electroosmotic flow was reversed by adding cetyltrimethylammonium hydroxide. In the method detection of anions was carried out with indirect UV detection, whereas metal cations were detected directly. Thirteen anions and three metal cations were completely separated at the operating buffer, pH 5.7. The R.S.D. (n=6) values of the method were better than 0.2% for migration time and between 1.1% and 3.9% for peak area. Calibration graphs were linear with correlation coefficients better than 0.9992. The minimum detectable levels ranged from 0.2 mg l−1 to 1.9 mg l−1 with 300 mbar pressure injection (6 nl) at a signal-to-noise ratio of 3. This method is simple, rapid and reproducible and could be readily applied to the simultaneous determination of anions and metal cations in a plating bath sample.

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Simultaneous Determination of Monosaccharides in Glycoproteins by Capillary Electrophoresis

Soga, T., Heiger, D.N.

Anal. Biochem. 261 ( 1 ) 73 - 78 1998.07

Research paper (scientific journal), Joint Work, Accepted

A rapid, easy, and reproducible capillary electrophoretic method for the simultaneous determination of acidic, neutral, and amino sugars and sugar alcohols was developed. Underivatized mannuronic acid, glucuronic acid, galacturonic acid, N-glycolylneuraminic acid, N-acetylneuraminic acid, glucosamine, galactosamine, mannose, xylose, glucose, galactose, fucose, ribose, mannitol, sorbitol, xylitol, and inositol were simultaneously determined with indirect UV detection using 2,6-pyridinedicarboxylic acid as a background electrolyte. A highly alkaline pH condition was used in order to charge carbohydrates negatively and to promote migration toward the anode. Electroosmotic flow was reversed to the direction of the anode by adding cetyltrimethylammonium bromide to the electrolyte. The separation of the carbohydrates was investigated by optimizing the operating pH value and satisfactory resolution was obtained at pH 12.1. The relative standard deviations of the method for carbohydrates were between 0.02 and 0.33% for migration times and were greater than 2.7% for peak areas (n = 6). The minimum detectable level ranged from 23 to 71 microM with a 6-nl injection at a signal-to-noise ratio of 3. This method was applied to the composition analysis of monosaccharides in glycoprotein. After acid hydrolysis of fetuin under each optimum condition, sialo, neutral, and amino sugars were quantified under the same electrophoretic condition.

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Kapillarelektrophorese-eine Prädestinierte Technik für die Analyse von Kationen und Anionen in Elektrolyten für die Galvanische und Chemische Metallisierung

Jegle, U., Serwe, M., Soga, T.

Galvanotechnik 89 1478 - 1485 1998

Research paper (scientific journal), Joint Work, Accepted

Capillary electrophoretic determination of inorganic and organic anions using 2,6-pyridinedicarboxylic acid: effect of electrolyte's complexing ability

Soga, T., Ross, G.A.

J. Chromatogr. A 767 ( 1-2 ) 223 - 230 1997.04

Research paper (scientific journal), Joint Work, Accepted

A capillary zone electrophoresis method was developed with indirect UV detection for the analysis of inorganic and organic anions using a background electrolyte (BGE), 2,6-pyridinedicarboxylic acid (PDC). The parameters which influence the separation of these anions, such as electrolyte pH, concentration of PDC and applied voltage, were investigated. Three inorganic anions and nine organic acids were determined simulataneously in less than 7 min. The R.S.D. (n66) values of the method were around 0.1% for migration time and better than 2.6% for peak area. This method is rapid, sensitive and quantitative and could be readily applied to analysis of inorganic and organic anions in beer. In the present paper, the effect of the complexing ability of the BGE on the analysis of organic acids is also discussed.

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Determination of Inorganic and Organic Anions in Beer and Wort by Capillary Electrophoresis

Soga, T., Wakaura, M

J. Am. Soc. Brew. Chem 55 ( 2 ) 44 - 46 1997.04

Research paper (scientific journal), Joint Work, Accepted

A rapid, easy, and reproducible method for the determination of inorganic and organic anions in beer and wort by capillary electrophoresis was developed. Three inorganic anions (chloride, sulfate, and phosphate) and nine organic acids (oxalate, formate, malate, citrate, succinate, pyruvate, acetate, lactate, and pyroglutamate) were separated and quantified in less than 7 min. Sample preparation was simple and consisted only of degassing the beer by sonication and 1:5 dilution with Milli Q water prior to injection. Satisfactory reproducibility was obtained, and the concentrations of anions were in good agreement with those found by conventional techniques.

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Poly(vinyl alcohol)-Coated Capillaries – A Solution for Many Application Areas in Capillary Electrophoresis

Jegle, U., Grimm, R., Godel, H., Schuster, R., Ross, G. A., Soga, T.

LC GC International 10 ( 3 ) 176 - 188 1997.03

Research paper (scientific journal), Joint Work, Accepted

Analysis of halides, oxyhalides and metal oxoacids by capillary electrophoresis with suppressed electroosmotic flow

Soga, T., Inoue, Y., Ross, G.A.

J. Chromatogr. A 718 ( 2 ) 421 - 428 1995.12

Research paper (scientific journal), Joint Work, Accepted

A rapid and easy method for the determination of halides, oxyhalides and metal oxoacids by capillary electrophoresis was developed. This is the first paper to report their simultaneous analysis. Electroosmotic flow was suppressed more than 40-fold by using a poly(ethyleneglycol)-coated capillary, compared with that in a bare fused-silica capillary under similar conditions. Using this capillary, the separation of eleven anions was accomplished. Anion migration was dependent only on their mobility and detection was carried out directly with a diode-array detector. The R.S.D.s were better than 0.6% for migration time and between 1.0% and 3.4% for peak area. Calibration graphs for all the anions were linear, with correlation coefficients better than 0.9995. Using stacking sample preconcentration, the achievable lower detection limits were found to be in the range 14–260 μg 1−1. This represents a 33–43-fold increase in sensitivity over the results obtained by using 200 mbar·s pressure injection.

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Determination of catecholamines in urine and plasma by on-line sample pretreatment using an internal surface boronic acid gel

Soga, T., Inoue, Y.

J. Chromatogr. Biomed. 620 ( 2 ) 175 - 181 1993.10

Research paper (scientific journal), Joint Work, Accepted

An automated method of analysis of catecholamines using a new packing material, internal surface boronic acid gel, was developed. The new support is designed with a carboxymethylcellulose-bonded external surface in order to be non-adsorptive to proteins and with a phenylboronic acid-bonded internal surface to retain only catecholamines. This packing support displayed an affinity for basic or neutral catecholic compounds with no protein adsorption and enabled on-line sample pretreatment of catecholamines in urine and deproteinized plasma. The catecholamines were selectively adsorbed on the new material and separated on a reversed-phase or a cation-exchange column. These compounds were then detected electrochemically. The limits of quantitation were 1.5–3.0 ng/ml in urine and 10–15 pg/ml in plasma, at a signal-to-noise ratio of 5.

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Determination of carbohydrates by hydrophilic interaction chromatography with pulsed amperometric detection using postcolumn pH adjustment

Soga, T., Inoue, Y., Yamaguchi, K.

J. Chromatogr. 625 ( 2 ) 151 - 155 1992.11

Research paper (scientific journal), Joint Work, Accepted

An HPLC method for the determination of carbohydrates using a postcolumn pH adjustment technique with lithium hydroxide solution followed by pulsed amperometric detection was developed. In conjunction with hydrophilic interaction chromatography, the technique was used to determine reducing sugars, non-reducing sugars and sugar alcohols. Detection limits from 0.7 to 2.7 ng with a signal-to-noise ratio of 3 were achieved. The relative standard deviation for peak was better than 2.7%. The postcolumn pH adjustment technique enabled gradient elution to be used.

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Comprehensive Measurement and Comparison of Ionic Small Molecules Contained in Citrus Unshiu Marc. Using Metabolomic Analysis

Hara, Y., Sugawara, A., Yamada, KC, Endo, K., Ashitani, S., Igarashi, K., Soga, T., Shinjo, A., Kuroda, H.

JOURNAL OF THE JAPANESE SOCIETY FOR FOOD SCIENCE AND TECHNOLOGY (THE JAPANESE SOCIETY FOR FOOD SCIENCE AND TECHNOLOGY) 67 ( 12 ) 499 - 513 2020.12

Research paper (scientific journal), Joint Work, Accepted

Trigonelline Attenuates Lipopolysaccharide-Induced Nitric Oxide Production and Downregulates Inflammation Associated Gene Expressions

Aizawa, Y., Suzuki, T., Saito, A., Satoh, K., Ogura, T., Wakayama, M., Soga, T., Tsuji, Y., Honmma, K., Inoue, H.

Food preserv. Sci. 46 ( 6 ) 275 - 280 2020.11

Research paper (scientific journal), Joint Work, Accepted

Trigonelline, an N-methylnicotinic acid, is found in fenugreek (Trigonella foenum-graecum L.) and coffee beans. Trigonelline has been reported to prevent cardiovascular and Alzheimer’s diseases.However, the potential pharmacological application of trigonelline in in fl ammatory responses is poorly understood, particularly in macrophages. In the present study, we investigated the effects and underlying molecular mechanisms of trigonelline on 100 ng /mL lipopolysaccharide (LPS) -induced inflammatory responses in RAW 264.7 mouse macrophages. 50 uM Trigonelline elicited a marked reduction in LPS - mediated production of nitric oxide (NO) compared with LPS - treated cells. Subsequently, the mRNA expression levels of inducible nitric oxide (iNOS), cyclooxygenase-2 (COX2),and pro-inflammatory cytokines, including tumor necrosis factor a (TNF-a), interleukin (IL) -6, and IL-1beta in LPS-treated cells were significantly decreased by trigonelline co-treatment. Furthermore, we found that treatment of cells with LPS elevated the gene expression of reactive oxidative species (ROS)-associated molecules, such as p47phox and p67phox, whereas trigonelline treatment significantly decreased them. Our data suggested that trigonelline inhibited inflammation and oxidative stress, resulting in the potential application of this compound in the prevention of inflammatory diseases.

Establishment of Basic Lipidomics Platforms for Discovery of Lipid Biomarkers

Ikeda, K., Taguchi, R., Soga, T.

Bunseki Kagaku (The Japan Society for Analytical Chemistry) 61 ( 6 ) 501 - 512 2012.06

Research paper (scientific journal), Joint Work

Lipidomics is one of the major approaches in metabolomics, and often uses a liquid chromatography-mass spectrometer (LC-MS) to obtain profiling of lipid metabolites and to detect changing molecules statistically. However, previously reported methods have been technically insufficient in the simultaneous analysis of amphiphilic glycosphingolipids, such as gangliosides, and in the global detection of various polar metabolites from complex lipid mixtures. Here, we report on newly targeted and non-targeted methods to measure these lipid metabolites sensitively. In the targeted approach, we were able to detect individual ganglioside molecular species using the combination of reversed-phase or hydrophilic interaction liquid chromatography and ESI-MS with multiple reaction monitoring (MRM).
Meanwhile, in a non-targeted approach, global analysis of various polar metabolites was possible by reverse-phased high-resolution (LC) coupled quadrapole time-of-flight MS and sensitive twodimensional (2D) profiling. Our methods will be useful for the elucidation of cancers, metabolic syndrome and nervous-system diseases, etc. ; these diagnostic markers may be detectable.

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Metabolome Analysis of Colon Tumor Tissues by Capillary Electrophoresis Time-of-Flight Mass Spectrometer

Hirayama, A., Kami, K., Sugimoto, M., Tomita, M., Soga, T.

J. Mass Spectrom. Soc. Jpn. 57 ( 3 ) 183 - 186 2009.06

Research paper (scientific journal), Joint Work

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CE-MSによるメタボローム解析

Soga, T.

生物物理化学 52 ( 3 ) 91 - 94 2008.09

Research paper (scientific journal), Single Work

Metabolomics, which can be defined as the measurement of the level of all intracellular metabolites, has become a powerful new tool for gaining insight into cellular and physiological responses. Here we have developed a comprehensive and quantitative analysis method based on capillary electrophoresis mass spectrometry (CE-MS). In this marriage of techniques, CE confers rapid analysis and efficient resolution, and MS provides excellent selectivity and sensitivity. Previous work in our laboratory demonstrated that CE-MS techniques were quite useful for the metabolome analysis. We applied this approach to profile liver metabolites following acetaminophen-induced hepatotoxicity and revealed ophthalmate as a sensitive indicator of reduced glutathione (GSH) depletion. Our results specifically indicate that serum ophthalmate is a sensitive indicator of hepatic GSH depletion, and may be a new biomarker for oxidative stress.

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Development of CE-MS for Meabolomics.

Soga, T.

J Mass Spectrom Soc Jpn 51 ( 3 ) 407 - 411 2003.06

Research paper (scientific journal), Single Work, ISSN 1340-8097

Metabolomics, which can be defined as the measurement of the level of all intracellular metabolites, has become a powerful new tool for gaining insight into functional biology. Intercellular metabolites not only provide metabolic phenotypes but also inducers to gene expression. Thus, metabolome analysis will be as important as genome and proteome research. However, very few methods for a large-scale metabolite analysis have been reported. This paper reports a method for the direct and quantitative analysis of charged metabolite using capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS). Its utility is demonstrated in the determination of basal metabolic intermediates of glycolysis and the TCA cycle pathways in Bacillus subtilis cells, yielding new information about how changes in metabolites are related to sporulation events.

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Simultaneous Determination of 8 Kinds of Organic Acids in Formula Feed by Capillary Electrophoresis.

Ishikuro, E., Hibino, H., Soga, T., Yanai, H., Sawada, H.

食品衛生学雑誌 41 ( 4 ) 261 - 267 2000.08

Research paper (scientific journal), Joint Work, ISSN 0015-6426

配合飼料中の8種類の有機酸 (ギ酸, フマル酸, 酒石酸, リンゴ酸, クエン酸, 酢酸, 乳酸及びプロピオン酸) のキャピラリー電気泳動 (CE) による分析法を開発した. 各有機酸は水で同時抽出し, ろ過した後, ろ液を0.02mol/Lの水酸化ナトリウムで希釈し, 限外ろ過フィルターユニット (分画分子量30,000) で除タンパクを行いCEに供した. 3種類の配合飼料に8種類の有機酸を0.05～2.0%の範囲で添加して回収試験を行ったところ, 平均回収率は85.1～107.3%で繰り返し精度 (RSDr) は13.6%以内であった. また, 3試験室において共同試験を行った結果, 平均回収率は87.3～105.0%, 室間再現精度 (RSDR) は16.3%以内であった.

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