研究者詳細 - 曽我　朋義

論文 - 曽我　朋義

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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年08月

研究論文（学術雑誌）, 共著, 査読有り, 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年07月

研究論文（学術雑誌）, 共著, 査読有り, 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年06月

研究論文（学術雑誌）, 共著, 査読有り

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年06月

研究論文（学術雑誌）, 共著, 査読有り, 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年05月

研究論文（学術雑誌）, 共著, 査読有り, 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年04月

研究論文（学術雑誌）, 共著, 査読有り

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年04月

研究論文（学術雑誌）, 共著, 査読有り

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年04月

研究論文（学術雑誌）, 共著, 査読有り, 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年04月

研究論文（学術雑誌）, 共著, 査読有り, 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年03月

研究論文（学術雑誌）, 共著, ISSN 09108327

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年02月

研究論文（学術雑誌）, 共著, 査読有り

© 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., Shoji F., Ikeda S., Yamaguchi N., Katayama T., Morine Y., Imura S., Utsunomiya T., Soga T., Tomita M., Shimada M.

Nutrition 58 110 - 119 2019年02月

研究論文（学術雑誌）, 共著, 査読有り, 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年02月

研究論文（学術雑誌）, 共著, 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年01月

研究論文（学術雑誌）, 共著, 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.

Analytical Chemistry （Analytical Chemistry） 91 （ 2 ） 1295 - 1301 2019年01月

研究論文（学術雑誌）, 共著, 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年01月

研究論文（学術雑誌）, 共著, 査読有り, 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年12月

研究論文（学術雑誌）, 共著, ISSN 16616596

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. 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., Wakayama M., Soga T., Tomita M.

J. Appl. Phycol. （Journal of Applied Phycology） 30 （ 6 ） 3335 - 3350 2018年12月

研究論文（学術雑誌）, 共著, 査読有り, 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年12月

研究論文（学術雑誌）, 共著, 査読有り

© 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年10月

研究論文（学術雑誌）, 共著, 査読有り, 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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