Details of a Researcher - Soga, Tomoyoshi

Papers - Soga, Tomoyoshi

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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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