研究者詳細 - 曽我　朋義

論文 - 曽我　朋義

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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年03月

研究論文（学術雑誌）, 共著

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年03月

研究論文（学術雑誌）, 共著, 査読有り

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年03月

共著, 査読有り

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年02月

研究論文（学術雑誌）, 共著, 査読有り

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年01月

研究論文（学術雑誌）, 共著, 査読有り

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年01月

研究論文（学術雑誌）, 共著, 査読有り

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年12月

研究論文（学術雑誌）, 共著, 査読有り

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年11月

研究論文（学術雑誌）, 共著, 査読有り

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年10月

研究論文（学術雑誌）, 共著, 査読有り

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. Endrinol. 47 （ 2 ） 129 - 143 2011年10月

研究論文（学術雑誌）, 共著, 査読有り

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年10月

研究論文（学術雑誌）, 査読有り

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年10月

研究論文（学術雑誌）, 共著, 査読有り

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年09月

研究論文（学術雑誌）, 共著, 査読有り

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年09月

研究論文（学術雑誌）, 共著, 査読有り

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年07月

研究論文（学術雑誌）, 共著, 査読有り

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年05月

研究論文（学術雑誌）, 共著, 査読有り

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年04月

研究論文（学術雑誌）, 共著, 査読有り

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年04月

研究論文（学術雑誌）, 共著, 査読有り

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年02月

研究論文（学術雑誌）, 共著, 査読有り, 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年12月

研究論文（学術雑誌）, 共著, 査読有り, 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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