論文 - 曽我　朋義

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

　概要を見る

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

　概要を見る

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

　概要を見る

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

　概要を見る

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

　概要を見る

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

　概要を見る

L-cysteine is ubiquitous in all living organisms and is involved in a variety of functions, including the synthesis of iron-sulfur clusters and glutathione and the regulation of the structure, stability, and catalysis of proteins. In the protozoan parasite Entamoeba histolytica, the causative agent of amebiasis, L-cysteine plays an essential role in proliferation, adherence, and defense against oxidative stress; however, the essentiality of this amino acid in the pathways it regulates is not well understood. In the present study, we applied capillary electrophoresis time-of-flight mass spectrometry to quantitate charged metabolites modulated in response to L-cysteine deprivation in E. histolytica, which was selected as a model for examining the biological roles of L-cysteine. L-cysteine deprivation had profound effects on glycolysis, amino acid, and phospholipid metabolism, with sharp decreases in the levels of L-cysteine, L-cystine, and S-adenosylmethionine and a dramatic accumulation of O-acetylserine and S-methylcysteine. We further demonstrated that S-methylcysteine is synthesized from methanethiol and O-acetylserine by cysteine synthase, which was previously considered to be involved in sulfur-assimilatory L-cysteine biosynthesis. In addition, L-cysteine depletion repressed glycolysis and energy generation, as it reduced acetyl-CoA, ethanol, and the major nucleotide di- and triphosphates, and led to the accumulation of glycolytic intermediates. Interestingly, L-cysteine depletion increased the synthesis of isopropanolamine and phosphatidylisopropanolamine, and it was confirmed that their increment was not a result of oxidative stress but was a specific response to L-cysteine depletion. We also identified a pathway in which isopropanolamine is synthesized from methylglyoxal via aminoacetone. To date, this study represents the first case where L-cysteine deprivation leads to drastic changes in core metabolic pathways, including energy, amino acid, and phospholipid metabolism.

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Functional Expression of Carnitine/Organic Cation Transporter OCTN1/SLC22A4 in Mouse Small Intestine and Liver

Sugiura, T., Kato, S., Shimizu, T., Wakayama, T., Nakamichi, N., Kubo, Y., Iwata, D., Suzuki, K., Soga, T., Asano, M., Iseki, S., Tamai, I., Tsuji, A., *Kato, Y

Drug Metab Dispos 38 （ 10 ） 1665 - 1672 2010年10月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0090-9556

　概要を見る

Carnitine/organic cation transporter (OCTN1/SLC22A4) accepts various therapeutic agents as substrates in vitro and is expressed ubiquitously, although its function in most organs has not yet been examined. The purpose of the present study was to evaluate functional expression of OCTN1 in small intestine and liver, using octn1 gene knockout [octn1(−/−)] mice. After oral administration of [3H]ergothioneine ([3H]ERGO), a typical substrate of OCTN1, the amount of [3H]ERGO remaining in the small intestinal lumen was much higher in octn1(−/−) mice than in wild-type mice. In addition, uptake of [3H]ERGO by human embryonic kidney 293 cells heterologously expressing OCTN1 gene product and uptake of [3H]ERGO at the apical surface of intestinal everted sacs from wild-type mice were inhibited by OCTN1 substrates, tetraethylammonium and verapamil. Immunohistochemical analysis revealed that OCTN1 is localized on the apical surface of small intestine in mice and humans. These results suggest that OCTN1 is responsible for small intestinal absorption of [3H]ERGO. However, the plasma concentration of [3H]ERGO after oral administration was higher in octn1(−/−) mice than in wild-type mice, despite the lower absorption in octn1(−/−) mice. This was probably because of efficient hepatic uptake of [3H]ERGO, as revealed by integration plot analysis; the uptake clearance was close to the hepatic plasma flow rate. The uptake of [3H]ERGO by isolated hepatocytes was minimal, whereas [3H]ERGO uptake was observed in isolated nonparenchymal cells. This finding is consistent with immunostaining of OCTN1 in liver sinusoids. Thus, our results indicate that OCTN1 is functionally expressed in nonparenchymal liver cells.

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Metabolomic Identification of the Target of the Filopodia Protrusion Inhibitor Glucopiericidin A

Kitagawa, M., Ikeda, S., Tashiro, E., Soga, T., Imoto, M.

Chem. Biol. 17 （ 9 ） 989 - 998 2010年09月

研究論文（学術雑誌）, 共著, 査読有り

　概要を見る

Identifying the targets of bioactive compounds is a major challenge in chemical biological research. Here, we identified the functional target of the natural bioactive compound glucopiericidin A (GPA) through metabolomic analysis. We isolated GPA while screening microbial samples for a filopodia protrusion inhibitor. Interestingly, GPA alone did not inhibit filopodia protrusion, but synergistically inhibit protrusion with the mitochondrial respiration inhibitor, piericidin A (PA). These results suggested that GPA might inhibit glycolysis. Capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) provided strong evidence that GPA suppresses glycolysis by functionally targeting the glucose transporter. GPA may therefore serve as a glucose transporter chemical probe. Simultaneous inhibition of both glycolysis and mitochondrial respiration dramatically decreased intracellular ATP levels, indicating that GPA inhibits ATP-dependent filopodia protrusion with PA. Our results represent a challenge of molecular target identification using metabolomic analysis.

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Metabolomic profiling of uremic solutes in CKD patients

Toyohara, T., Akiyama, Y., Suzuki, T., Takeuchi, Y., Mishima, E., Tanemoto, M., Momose, A., Toki, N., Sato, H., Nakayama, M., Hozawa, A., Tsuji, I., Ito, S., Soga, T. and Abe. T.

Hypertens. Res. 33 （ 9 ） 944 - 952 2010年09月

研究論文（学術雑誌）, 共著, 査読有り

　概要を見る

Early detection and accurate monitoring of patients with chronic kidney disease (CKD) is likely to improve care and decrease the risk of cardiovascular and cerebrovascular diseases. As a new diagnostic tool, we examined the retention of uremic solutes as a simpler, more accurate method to assess renal function. To achieve this, we comprehensively evaluated these solutes in CKD patients. By capillary electrophoresis with mass spectrometry, we found 22 cations and 30 anions that accumulated significantly as the estimated glomerular filtration rate (eGFR) decreased. These compounds included 9 cations and 27 anions that were newly identified in this study. In contrast, we also found 7 cations (2 new) and 5 anions (all new) that decrease significantly as eGFR declines. We evaluated each substance for its suitability to detect early CKD stage. Compounds that are highly correlated with eGFR and whose plasma concentration changed in a manner approximated by the first-degree equation are excellent candidates for detecting CKD and identifying uremic toxins that might aggravate kidney function in the early stage of CKD. These results identify a number of uremic compounds, many of which are novel and which predict worsening renal function. These compounds provide diagnostic information and may be targets for therapies designed to treat the complications of CKD patients.

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Two Atypical L-cysteine-regulated NADPH-dependent Oxidoreductases Involved in Redox Maintenance, L-cystine and Iron Reduction, and Metronidazole Activation in the Enteric Protozoan Entamoeba Histolytica

Jeelani, G., Husain, A., Sato, D., Ali, V., Suematsu, M., Soga, T., Nozaki, T.

J Biol Chem 285 （ 35 ） 26889 - 26899 2010年08月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0021-9258

　概要を見る

We discovered novel catalytic activities of two atypical NADPH-dependent oxidoreductases (EhNO1/2) from the enteric protozoan parasite Entamoeba histolytica. EhNO1/2 were previously annotated as the small subunit of glutamate synthase (glutamine:2-oxoglutarate amidotransferase) based on similarity to authentic bacterial homologs. As E. histolytica lacks the large subunit of glutamate synthase, EhNO1/2 were presumed to play an unknown role other than glutamine/glutamate conversion. Transcriptomic and quantitative reverse PCR analyses revealed that supplementation or deprivation of extracellular L-cysteine caused dramatic up- or down-regulation, respectively, of EhNO2, but not EhNO1 expression. Biochemical analysis showed that these FAD- and 2[4Fe-4S]-containing enzymes do not act as glutamate synthases, a conclusion which was supported by phylogenetic analyses. Rather, they catalyze the NADPH-dependent reduction of oxygen to hydrogen peroxide and L-cystine to L-cysteine and also function as ferric and ferredoxin-NADP(+) reductases. EhNO1/2 showed notable differences in substrate specificity and catalytic efficiency; EhNO1 had lower K(m) and higher k(cat)/K(m) values for ferric ion and ferredoxin than EhNO2, whereas EhNO2 preferred L-cystine as a substrate. In accordance with these properties, only EhNO1 was observed to physically interact with intrinsic ferredoxin. Interestingly, EhNO1/2 also reduced metronidazole, and E. histolytica transformants overexpressing either of these proteins were more sensitive to metronidazole, suggesting that EhNO1/2 are targets of this anti-amebic drug. To date, this is the first report to demonstrate that small subunit-like proteins of glutamate synthase could play an important role in redox maintenance, L-cysteine/L-cystine homeostasis, iron reduction, and the activation of metronidazole.

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13C-Metabolic Flux Analysis for Batch Culture of Escherichia coli and Its pyk and pgi Gene Knockout Mutants based on Mass Isotopomer Distribution of Intracellular Metabolites

Toya, Y., Ishii, N., Nakahigashi, K., Hirasawa, T., Soga, T., Tomita, M., *Shimizu, K

Biotechno Prog 26 （ 4 ） 975 - 992 2010年08月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  8756-7938

　概要を見る

Since most bio‐production processes are conducted in a batch or fed‐batch manner, the evaluation of metabolism with respect to time is highly desirable. Toward this aim, we applied 13C‐metabolic flux analysis to nonstationary conditions by measuring the mass isotopomer distribution of intracellular metabolites. We performed our analysis on batch cultures of wild‐type Escherichia coli, as well as on Pyk and Pgi mutants, obtained the fluxes and metabolite concentrations as a function of time. Our results for the wild‐type indicated that the TCA cycle flux tended to increase during growth on glucose. Following glucose exhaustion, cells controlled the branch ratio between the glyoxylate pathway and the TCA cycle, depending on the availability of acetate. In the Pyk mutant, the concentrations of glycolytic intermediates changed drastically over time due to the dumping and feedback inhibition caused by PEP accumulation. Nevertheless, the flux distribution and free amino acid concentrations changed little. The growth rate and the fluxes remained constant in the Pgi mutant and the glucose‐6‐phosphate dehydrogenase reaction was the rate‐limiting step. The measured fluxes were compared with those predicted by flux balance analysis using maximization of biomass yield or ATP production. Our findings indicate that the objective function of biosynthesis became less important as time proceeds on glucose in the wild‐type, while it remained highly important in the Pyk mutant. Furthermore, ATP production was the primary objective function in the Pgi mutant. This study demonstrates how cells adjust their metabolism in response to environmental changes and/or genetic perturbations in the batch cultivation. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010

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Metabolomic Profiles and Sensory Attributes of Edamame under Various Storage Duration and Temperature Conditions

Sugimoto, M., Goto, H., Otomo, K., Ito, M., Onuma, H., Suzuki, A., Sugawara, M., Abe, S., Tomita, M., Soga, T.

J Agric Food Chem 58 （ 14 ） 8418 - 8425 2010年07月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0021-8561

　概要を見る

Its high nutritional content and sensory characteristics make edamame a popular vegetable bean. However, because of its short shelf-life, it is important to optimize the storage conditions to maintain its quality during distribution to consumers. We focused on storage conditions to investigate the temporal changes in the metabolic profiles and sensory characteristics of edamame during transportation from the site of harvest to the site of purchase/consumption. We conducted metabolomic analysis and sensory evaluation tests of edamame stored for different lengths and at different temperatures. Charged metabolites were profiled by capillary electrophoresis-mass spectrometry, and free sugars were quantified by liquid chromatography-tandem mass spectrometry. In comparison to the gradual decrease in its sensory characteristics over time, the changes in metabolite profiles manifested four different patterns. In particular, changes in amino acid levels were related to sensory attributes. The downstream metabolites of shikimate as well as phospholipids and gamma-aminobutyric acid increased with increasing storage temperatures.

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MassBank: a public repository for sharing mass spectral data for life sciences

Horai, H., Arita, M., Kanaya, S., Nihei, Y., Ikeda, T., Suwa, K., Ojima, Y., Tanaka, K., Tanaka, S., Aoshima, K., Oda, Y., Kakazu, Y., Kusano, M., Tohge, T., Matsuda, F., Sawada, Y., Hirai, Y. M., Nakanishi, H., Ikeda, K., Akimoto, N., Maoka, T., Takahashi, H., Ara, T., Sakurai, N., Suzuki, H., Shibata, D., Neumann, S., Iida, T., Tanaka, K., Funatsu, K., Matsuura, F., Soga, T., Taguchi, R., Saito, K., *Nishioka, T.

J. Mass Spectro. 45 （ 7 ） 703 - 714 2010年07月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  1076-5174

　概要を見る

MassBank is the first public repository of mass spectra of small chemical compounds for life sciences (<3000 Da). The database contains 605 electron-ionization mass spectrometry (EI-MS), 137 fast atom bombardment MS and 9276 electrospray ionization (ESI)-MS(n) data of 2337 authentic compounds of metabolites, 11 545 EI-MS and 834 other-MS data of 10,286 volatile natural and synthetic compounds, and 3045 ESI-MS(2) data of 679 synthetic drugs contributed by 16 research groups (January 2010). ESI-MS(2) data were analyzed under nonstandardized, independent experimental conditions. MassBank is a distributed database. Each research group provides data from its own MassBank data servers distributed on the Internet. MassBank users can access either all of the MassBank data or a subset of the data by specifying one or more experimental conditions. In a spectral search to retrieve mass spectra similar to a query mass spectrum, the similarity score is calculated by a weighted cosine correlation in which weighting exponents on peak intensity and the mass-to-charge ratio are optimized to the ESI-MS(2) data. MassBank also provides a merged spectrum for each compound prepared by merging the analyzed ESI-MS(2) data on an identical compound under different collision-induced dissociation conditions. Data merging has significantly improved the precision of the identification of a chemical compound by 21-23% at a similarity score of 0.6. Thus, MassBank is useful for the identification of chemical compounds and the publication of experimental data.

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Prediction of metabolite identity from accurate mass, migration time prediction and isotopic pattern information in CE-TOFMS data

Sugimoto, M., Hirayama, A., Robert, M., Abe, S., Soga, T., Tomita, M.

Electrophoresis 31 （ 14 ） 2311 - 2318 2010年07月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0173-0835

　概要を見る

CE-TOFMS is a powerful method for profiling charged metabolites. However, the limited availability of metabolite standards hinders the process of identifying compounds from detected features in CE-TOFMS data sets. To overcome this problem, we developed a method to identify unknown peaks based on the predicted migration time (t(m)) and accurate m/z values. We developed a predictive model using 375 standard cationic metabolites and support vector regression. The model yielded good correlations between the predicted and measured t(m) (R=0.952 and 0.905 using complete and cross-validation data sets, respectively). Using the trained model, we subsequently predicted the t(m) for 2938 metabolites available from the public databases and assigned tentative identities to noise-filtered features in human urine samples. While 38.9% of the peaks were assigned metabolite names by matching with the standard library alone, the proportion increased to 52.2%. The proposed methodology increases the value of metabolomic data sets obtained from CE-TOFMS profiling.

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Unveiling cellular biochemical reactions via metabolomics-driven approaches

Saito, N., Ohashi, Y., Soga, T., Tomita, M.

Curr. Opin. Microbiol. 13 （ 3 ） 358 - 362 2010年06月

研究論文（学術雑誌）, 共著, 査読有り

　概要を見る

Despite the widespread availability of genome sequences and the systematic assignment of individual open reading frames, many of the activities of metabolic enzymes are still veiled. It is difficult to assign biochemical functions to enzymes and uncover the in vivo regulation of their enzymatic activities, but this is crucial to understand cellular functions. The development of metabolomics and metabolite profiling, which can globally analyze intracellular and extracellular metabolites, has provided some valuable tools for systems biology. This report reviews the recent metabolomics approaches to elucidate unidentified metabolic systems while focusing on their enzymatic activities, including their biochemical and biological functions, and their regulation in response to environmental perturbations in microorganisms.

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A systematic survey of in vivo obligate chaperonin‐dependent substrates

Fujiwara, K., Ishihama, Y., Nakahigashi, K., Soga, T., Taguchi, H.,

EMBO J. 29 （ 9 ） 1552 - 1564 2010年05月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  14602075

　概要を見る

Chaperonins are absolutely required for the folding of a subset of proteins in the cell. An earlier proteome-wide analysis of Escherichia coli chaperonin GroEL/GroES (GroE) interactors predicted obligate chaperonin substrates, which were termed Class III substrates. However, the requirement of chaperonins for in vivo folding has not been fully examined. Here, we comprehensively assessed the chaperonin requirement using a conditional GroE expression strain, and concluded that only approximately 60% of Class III substrates are bona fide obligate GroE substrates in vivo. The in vivo obligate substrates, combined with the newly identified obligate substrates, were termed Class IV substrates. Class IV substrates are restricted to proteins with molecular weights that could be encapsulated in the chaperonin cavity, are enriched in alanine/glycine residues, and have a strong structural preference for aggregation-prone folds. Notably, approximately 70% of the Class IV substrates appear to be metabolic enzymes, supporting a hypothetical role of GroE in enzyme evolution.

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Gene knockout and metabolome analysis of carnitine/organic cation transporter OCTN1

Kato, Y., Kubo, Y., Iwata, D., Kato, S., Sudo, T., Sugiura, T., Kagaya, T., Wakayama, T., Hirayama, A., Sugimoto, M., Sugihara, K., Kaneko, S., Soga, T., Asano, M., Tomita, M., Matsui, T., Wada, M., Tsuji, A.

Pharm. Res. 27 （ 5 ） 832 - 840 2010年05月

研究論文（学術雑誌）, 共著, 査読有り

　概要を見る

Purpose: Solute carrier OCTN1 (SLC22A4) is an orphan transporter, the physiologically important substrate of which is still unidentified. The aim of the present study was to examine physiological roles of OCTN1.
Methods: We first constructed octn1 gene knockout (octn1 ( -/- )) mice. Metabolome analysis was then performed to identify substrates in vivo. The possible association of the substrate identified with diseased conditions was further examined.
Results: The metabolome analysis of blood and several organs indicated complete deficiency of a naturally occurring potent antioxidant ergothioneine in octn1 ( -/- ) mice among 112 metabolites examined. Pharmacokinetic analyses after oral administration revealed the highest distribution to small intestines and extensive renal reabsorption of [(3)H]ergothioneine, both of which were much reduced in octn1 ( -/- ) mice. The octn1 ( -/- ) mice exhibited greater susceptibility to intestinal inflammation under the ischemia and reperfusion model. The blood ergothioneine concentration was also much reduced in Japanese patients with Crohn's disease, compared with healthy volunteers and patients with another inflammatory bowel disease, ulcerative colitis.
Conclusions: These results indicate that OCTN1 plays a pivotal role for maintenance of systemic and intestinal exposure of ergothioneine, which could be important for protective effects against intestinal tissue injuries, providing a possible diagnostic tool to distinguish the inflammatory bowel diseases.


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Analysis of Metabolic Remodeling in Compensated Left Ventricular Hypertrophy and Heart Failure

Kato, T., Niizuma, S., Inuzuka, Y., Kawashima, T., Okuda, J., Tamaki, Y., Iwanaga, Y., Narazaki, M., Matsuda, T., Soga, T., Kita, T., Kimura, T., Shioi, T.

Circ. Heart Fail. 3 （ 3 ） 420 - 430 2010年05月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  1941-3289

　概要を見る

Background: Congestive heart failure (CHF) is associated with a change in cardiac energy metabolism. However, the mechanism by which this change is induced and causes the progression of CHF is unclear.
Methods and results: We analyzed the cardiac energy metabolism of Dahl salt-sensitive rats fed a high-salt diet, which showed a distinct transition from compensated left ventricular hypertrophy to CHF. Glucose uptake increased at the left ventricular hypertrophy stage, and glucose uptake further increased and fatty acid uptake decreased at the CHF stage. The gene expression related to glycolysis, fatty acid oxidation, and mitochondrial function was preserved at the left ventricular hypertrophy stage but decreased at the CHF stage and was associated with decreases in levels of transcriptional regulators. In a comprehensive metabolome analysis, the pentose phosphate pathway that regulates the cellular redox state was found to be activated at the CHF stage. Dichloroacetate (DCA), a compound known to enhance glucose oxidation, increased energy reserves and glucose uptake. DCA improved cardiac function and the survival of the animals. DCA activated the pentose phosphate pathway in the rat heart. DCA activated the pentose phosphate pathway, decreased oxidative stress, and prevented cell death of cultured cardiomyocytes.
Conclusions: Left ventricular hypertrophy or CHF is associated with a distinct change in the metabolic profile of the heart. DCA attenuated the transition associated with increased energy reserves, activation of the pentose phosphate pathway, and reduced oxidative stress.


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Chlorogonium complexum sp. nov. (Volvocales, Chlorophyceae), and morphological evolution of Chlorogonium

Nakada, T., Soga, T., Tomita, M., Nozaki, H.

Eur. J. Phycol. 45 （ 1 ） 97 - 107 2010年03月

研究論文（学術雑誌）, 共著, 査読有り

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We isolated strains of a new species of Chlorogonium (Cg.), Cg. complexum Nakada sp. nov. The genus Chlorogonium was recently emended to have several, irregularly distributed contractile vacuoles, two-to-many pyrenoids associated with fragmented starch plates and tubular thylakoid lamellae penetrating their matrices, and stigma composed of a single layer of globules. Chlorogonium complexum has these diagnostic characters of the emended genus. It is distinguished from other species of Chlorogonium, Cg. euchlorum, Cg. elongatum and Cg. capillatum, on the combination of diagnostic characters, such as the number and stability of pyrenoids and the mode of encystment. Based on a dataset combining 18S rRNA, RuBisCO large subunit (rbcL) and P700 chl a-apoprotein A2 (psaB) gene sequences, the phylogenetic position of Chlorogonium was determined. Chlorogonium complexum represented an independent lineage, sister to Cg. euchlorum and Cg. capillatum. The evolution of diagnostic characters among Chlorogonium species is discussed based on the molecular phylogenetic analyses, and a key to species of Chlorogonium and fusiform chlamydomonads with irregularly distributed contractile vacuoles is presented.

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Hypotaurine is an energy-saving hepatoprotective compound against ischemia-reperfusion injury of the rat liver

Sakuragawa, T., Hishiki, T., Ueno, Y., Ikeda, S., Soga, T., Yachie-Kinoshita, A., Kajimura, M., Suematsu, M.

J. Clin. Biochem. Nutr. 46 （ 2 ） 126 - 134 2010年03月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0912-0009

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Metabolome analyses assisted by capillary electrophoresis-mass spectrometry (CE-MS) have allowed us to systematically grasp changes in small molecular metabolites under disease conditions. We applied CE-MS to mine out biomarkers in hepatic ischemia-reperfusion. Rat livers were exposed to ischemia by clamping of the portal inlet followed by reperfusion. Metabolomic profiling revealed that l contents of taurine in liver and plasma were significantly increased. Of interest is an elevation of hypotaurine, collectively suggesting significance of hypotaurine/taurine in post-ischemic responses. Considering the anti-oxidative capacity of hypotaurine, we examined if supplementation of the compound or its precursor amino acids could affect hepatocellular viability and contents of taurine in liver and plasma. Administration of hypotaurine, N-acetylcysteine or methionine upon reperfusion comparablly attenuated the post-ischemic hepatocellular injury but with different metabolomic profiling among groups: rats treated with methionine or N-acetylcysteine but not those treated with hypotaurine, exhibited significant elevation of hepatic lactate generation without notable recovery of the energy charge. Furthermore, the group treated with hypotaurine exhibited elevation of the plasma taurine, suggesting that the exogenously administered compound was utilized as an antioxidant. These results suggest that taurine serves as a surrogate marker for ischemia-reperfusion indicating effectiveness of hypotaurine as an energy-saving hepatoprotective amino acid.

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