研究者詳細 - 曽我　朋義

論文 - 曽我　朋義

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Metabolic Profiling to Identify Potential Serum Biomarkers for gastric ulceration induced by non-steroid anti-inflammatory drugs

Takeuchi, K., Ohishi, M., Ota, S., Suzumura, K., Naraoka, H., Ohata, T., Seki, J., Miyamae, Y., Honma, M., Soga, T.

J. Proteome Res. 12 （ 3 ） 1399 - 1407 2013年03月

研究論文（学術雑誌）, 共著, 査読有り

Nonsteroid anti-inflammatory drugs (NSAIDs) are among the most frequently prescribed drugs currently available. The most frequently reported serious side effects associated with NSAIDs are gastric mucosal ulceration and gastric hemorrhage. Presently, these side effects are only detectable by endoscopy, however, and no biomarkers have yet been identified. The ability to identify serum biomarkers would likely improve the safety of NSAID use. In this study we performed capillary electrophoresis-mass spectrometry (CE-MS)-based metabolomic profiling in stomach extract and serum from rats administered NSAIDs. Results showed drug-induced decreases in levels of citrate, cis-aconitate, succinate, 3-hydroxy butanoic acid, o-acetyl carnitine, proline, and hydroxyproline. We consider that these changes are due to NSAID-induced depression of mitochondrial function and activation of collagenase by lesions in the stomach. In addition, four of these changes in metabolite levels in the stomach were significantly correlated with changes in the serum. While further study is needed to clarify the mechanism of change in the level of these biomarkers, limitation of indications, and extrapolation to humans, these new serum biomarker candidates of gastric injury may be useful in the monitoring of NSAID-induced tissue damage.

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Changes of Metabolic Profiles in an Oral Squamous Cell Carcinoma Cell Line Induced by Eugenol

Koh, T., Murakami, Y., Tanaka, S., Machino, M., Onuma, H., Kaneko, M., Sugimoto, M., Soga, T., Tomita, M., Sakagami, H.

In Vivo 27 （ 2 ） 233 - 243 2013年03月

研究論文（学術雑誌）, 共著, 査読有り

Background: We have recently reported that eugenol exerted indiscriminate cytotoxicity towards normal oral cells and oral squamous cell carcinoma (OSCC) cell lines without induction of apoptosis markers. In order to investigate the underlying mechanisms of cytotoxicity induction, we investigated the effect of short-term treatment with eugenol on the metabolic profiles of a human OSCC cell line (HSC-2).
Materials and methods: The viable cell number was determined by direct cell counting with a hemocytometer after trypsinization. After washing with 5% D-mannitol solution (found to retain the highest amounts of intracellular metabolites among several washing conditions), cellular metabolites were extracted with methanol with internal markers and then subjected to metabolomic analysis.
Results: Cytotoxic concentrations of eugenol induced the reduction of ATP utilization (assessed by a significant reduction of the AMP/ATP and ADP/ATP ratio), of oxidative stress (assessed by the increase in oxidized form of glutathione, cysteine-glutathione disulfide and methionine sulfoxide), and an increase in the polyamines and glycolytic metabolites.
Conclusion: The metabolic changes observed in this study suggest the induction of non-apoptotic cell death by eugenol.

Biochemical and functional characterization of novel NADH kinase in the enteric protozoan parasite Entamoeba histolytica

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

Biochimie 95 （ 2 ） 309 - 319 2013年03月

研究論文（学術雑誌）, 査読有り

NAD(H) kinase catalyzes the phosphorylation of NAD(H) to form NADP(H) using ATP or inorganic polyphosphate as a phosphoryl donor. While the enzyme is conserved throughout prokaryotes and eukaryotes, remarkable differences in kinetic parameters including substrate preference, cation dependence, and physiological roles exist among the organisms. In the present study, we biochemically characterized NAD(H) kinase from the anaerobic/microaerophilic fermentative protozoan parasite Entamoeba histolytica, which lacks the conventional mitochondria capable of oxidative phosphorylation, leading to ATP. The kinetic properties of E. histolytica NAD(H) kinase recombinantly produced in Escherichia coli showed remarkable differences from those in bacteria and higher eukaryotes. Entamoeba NAD(H) kinase preferred NADH to NAD+ as the phosphoryl acceptor, utilized nucleoside triphosphates including ATP, GTP and deoxyATP, but not nucleoside di-, mono-phosphates, or inorganic polyphosphates, as the phosphoryl donor. To further understand the physiological roles in E. histolytica, we generated a stable transformant overexpressing NAD(H) kinase. Overexpression of NAD(H) kinase resulted in a 1.6-2 fold increase in the NADPH and NADP+ concentrations, a 40% reduction of the intracellular concentration of reactive oxygen species, and also led to increased tolerance toward hydrogen peroxide. These data, together with the essentially of NAD(H) kinase gene, underscore its significance as an NADP(H)-producing enzyme in this organism, and should help in designing of drugs targeting this enzyme.

Metabolomic study of Chilean biomining bacteria Acidithiobacillus ferrooxidans strain Wenelen and Acidithiobacillus thiooxidans strain Licanantay

Martı´nez, P., Ga´lvez, S., Ohtsuka, N., Budinich, M., Corte´s, M.P., Serpell, C., Nakahigashi, K., Hirayama, A., Tomita, M., Soga, T., Martı´nez, S., Maass, A., Parada, P.

Metabolomics 9 （ 1 ） 247 - 257 2013年02月

研究論文（学術雑誌）, 共著, 査読有り

In this study, we present the first metabolic profiles for two bioleaching bacteria using capillary electrophoresis coupled with mass spectrometry. The bacteria, Acidithiobacillus ferrooxidans strain Wenelen (DSM 16786) and Acidithiobacillus thiooxidans strain Licanantay (DSM 17318), were sampled at different growth phases and on different substrates: the former was grown with iron and sulfur, and the latter with sulfur and chalcopyrite. Metabolic profiles were scored from planktonic and sessile states. Spermidine was detected in intra- and extracellular samples for both strains, suggesting it has an important role in biofilm formation in the presence of solid substrate. The canonical pathway for spermidine synthesis seems absent as its upstream precursor, putrescine, was not present in samples. Glutathione, a catalytic activator of elemental sulfur, was identified as one of the most abundant metabolites in the intracellular space in A. thiooxidans strain Licanantay, confirming its participation in the sulfur oxidation pathway. Amino acid profiles varied according to the growth conditions and bioleaching species. Glutamic and aspartic acid were highly abundant in intra- and extracellular extracts. Both are constituents of the extracellular matrix, and have a probable role in cell detoxification. This novel metabolomic information validates previous knowledge from in silico metabolic reconstructions based on genomic sequences, and reveals important biomining functions such as biofilm formation, energy management and stress responses.

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Regulation of Glycolysis by Pdk Functions as a Metabolic Checkpoint for Cell Cycle Quiescence in Hematopoietic Stem Cells

Takubo, K., Nagamatsu, G., Kobayashi, C. I, Nakamura-Ishizu, A., Kobayashi, H., Ikeda, E., Goda, N., Rahimi, Y., Johnson, R. S, Soga, T., Hirao, A., Suematsu, M., Suda, T.

Cell Stem Cell. 12 （ 1 ） 49 - 61 2013年01月

研究論文（学術雑誌）, 共著, 査読有り

Defining the metabolic programs that underlie stem cell maintenance will be essential for developing strategies to manipulate stem cell capacity. Mammalian hematopoietic stem cells (HSCs) maintain cell cycle quiescence in a hypoxic microenvironment. It has been proposed that HSCs exhibit a distinct metabolic phenotype under these conditions. Here we directly investigated this idea using metabolomic analysis and found that HSCs generate adenosine-5'-triphosphate by anaerobic glycolysis through a pyruvate dehydrogenase kinase (Pdk)-dependent mechanism. Elevated Pdk expression leads to active suppression of the influx of glycolytic metabolites into mitochondria. Pdk overexpression in glycolysis-defective HSCs restored glycolysis, cell cycle quiescence, and stem cell capacity, while loss of both Pdk2 and Pdk4 attenuated HSC quiescence, glycolysis, and transplantation capacity. Moreover, treatment of HSCs with a Pdk mimetic promoted their survival and transplantation capacity. Thus, glycolytic metabolic status governed by Pdk acts as a cell cycle checkpoint that modulates HSC quiescence and function.

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A medium-chain fatty acid as an alternative energy source in mouse preimplantation development

Yamada, M., Takanashi, K., *Hamatani, T., Hirayama, A., Akutsu, H., Fukunaga, T. Ogawa, S., Sugawara, K., Shinoda, K., Soga, T., Umezawa, A., Kuji, N., Yoshimura ,Y. and Tomita, M.

Sci. Rep. 2 930 2012年12月

研究論文（学術雑誌）, 共著, 査読有り

To further optimize the culturing of preimplantation embryos, we undertook metabolomic analysis of relevant culture media using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). We detected 28 metabolites: 23 embryo-excreted metabolites including 16 amino acids and 5 media-derived metabolites (e.g., octanoate, a medium-chain fatty acid (MCFA)). Due to the lack of information on MCFAs in mammalian preimplantation development, this study examined octanoate as a potential alternative energy source for preimplantation embryo cultures. No embryos survived in culture media lacking FAs, pyruvate, and glucose, but supplementation of octanoate rescued the embryonic development. Immunoblotting showed significant expression of acyl-CoA dehydrogenase and hydroxyacyl-CoA dehydrogenase, important enzymes for ß-oxidation of MCFAs, in preimplantation embryo. Furthermore, CE-TOFMS traced [1-(13)C(8)] octanoate added to the culture media into intermediate metabolites of the TCA cycle via ß-oxidation in mitochondria. These results are the first demonstration that octanoate could provide an efficient alternative energy source throughout preimplantation development.

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Metabolic profiling reveals new serum biomarkers for differentiating diabetic nephropathy

Hirayama, A., Nakashima, E., Sugimoto, M., Akiyama, S., Sato, W., Maruayama, S., Matsuo, S., Tomita, M., Yuzawa, Y., Soga, T.

Anal Bioanal Chem. 404 （ 10 ） 3101 - 3109 2012年12月

研究論文（学術雑誌）, 共著, 査読有り

Capillary electrophoresis coupled with time-of-flight mass spectrometry was used to explore new serum biomarkers with high sensitivity and specificity for diabetic nephropathy (DN) diagnosis, through comprehensive analysis of serum metabolites with 78 diabetic patients. Multivariate analyses were used for identification of marker candidates and development of discriminative models. Of the 289 profiled metabolites, orthogonal partial least-squares discriminant analysis identified 19 metabolites that could distinguish between DN with macroalbuminuria and diabetic patients without albuminuria. These identified metabolites included creatinine, aspartic acid, γ-butyrobetaine, citrulline, symmetric dimethylarginine (SDMA), kynurenine, azelaic acid, and galactaric acid. Significant correlations between all these metabolites and urinary albumin-to-creatinine ratios (p < 0.009, Spearman’s rank test) were observed. When five metabolites (including γ-butyrobetaine, SDMA, azelaic acid and two unknowns) were selected from 19 metabolites and applied for multiple logistic regression model, AUC value for diagnosing DN was 0.927 using the whole dataset, and 0.880 in a cross-validation test. In addition, when four known metabolites (aspartic acid, SDMA, azelaic acid and galactaric acid) were applied, the resulting AUC was still high at 0.844 with the whole dataset and 0.792 with cross-validation. Combination of serum metabolomics with multivariate analyses enabled accurate discrimination of DN patients. The results suggest that capillary electrophoresis-mass spectrometry based metabolome analysis could be used for DN diagnosis.

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Glycogen is the primary source of glucose during the lag phase of E. coli proliferation

Yamamotoya, T., Dose, H., Tian, Z., Fauré, A., Toya, Y., Honma, M., Igarashi, K., Nakahigashi, K., Soga, T., Mori, H., Matsuno, H.

Biochim Biophys Acta 1824 （ 12 ） 1142 - 1148 2012年12月

研究論文（学術雑誌）, 共著, 査読有り

In the studies of Escherichia coli (E. coli), metabolomics analyses have mainly been performed using steady state culture. However, to analyze the dynamic changes in cellular metabolism, we performed a profiling of concentration of metabolites by using batch culture. As a first step, we focused on glucose uptake and the behavior of the first metabolite, G6P (glucose-6-phosphate). A computational formula was derived to express the glucose uptake rate by a single cell from two kinds of experimental data, extracellular glucose concentration and cell growth, being simulated by Cell Illustrator. In addition, average concentration of G6P has been measured by CE-MS. The existence of another carbon source was suggested from the computational result. After careful comparison between cell growth, G6P concentration, and the computationally obtained curve of glucose uptake rate, we predicted the consumption of glycogen in lag phase and its accumulation as an energy source in an E. coli cell for the next proliferation. We confirmed our prediction experimentally. This behavior indicates the importance of glycogen participation in the lag phase for the growth of E. coli. This article is part of a Special Issue entitled: Computational Methods for Protein Interaction and Structural Prediction

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Analysis of liver metabolism in a rat model of heart failure

Kato, T., Niizuma, S., Inuzuka, Y., Kawashima, T., Okuda, J., Kawamoto, A., Tamai, Y., Iwanaga, Y., Soga, T., Kita, T., Kimura, T., Shioi, T.

Int. J. Cardiol. 161 （ 3 ） 130 - 136 2012年11月

研究論文（学術雑誌）, 共著, 査読有り

Background: Cachexia, namely body wasting, is a common complication in cases of congestive heart failure (CHF). Although, neurohumoral and immune abnormalities are associated with the condition, precisely how the imbalance of catabolism and anabolism is responsible for the wasting process is not known.
Methods: We analyzed markers of cachexia in Dahl salt-sensitive rats which show marked hypertension with preserved systolic function at 11 weeks and CHF at 17-19 weeks of age. We also analyzed the change in hepatic metabolism associated with CHF since liver plays a central role in the systemic regulation of catabolism and anabolism.
Results: In CHF rats, a failure to grow was observed and blood hepatic protein levels were decreased associated with increased blood proinflammatory cytokine levels, indicating that Dahl rats serve as a model of cardiac cachexia. Food intake was reduced, and blood sugar and insulin levels were decreased. Despite the apparent fasting condition, blood fatty acid levels were decreased and triglycerides levels were increased. In CHF rats, liver incorporated more glucose, the gene expression related to gluconeogenesis was decreased, the gene expression related to lipogenesis was increased, and the triglyceride content of the liver was increased. The paradoxical production of triglycerides synthesis in fasting rats was associated with a proinflammatory response in liver.
Conclusions: The Dahl salt-sensitive rat can be used as a model of cardiac cachexia. The cachexia was associated with abnormal hepatic metabolism that might work as a maladaptive response during the progression of CHF.

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A Metabolomic Approach to Clarifying the Effect of AST-120 on 5/6 Nephrectomized Rats by Capillary Electrophoresis with Mass Spectrometry (CE-MS)

Akiyama, Y., Takeuchi, Y., Kikuchi, K., Mishima, E., Yamamoto, Y., Suzuki, C., Toyohara, T., Suzuki, T., Hozawa, A., Ito, S., Soga, T., Abe, T.

Toxins 4 （ 11 ） 1309-1322 2012年11月

研究論文（学術雑誌）, 共著, 査読有り

The oral adsorbent AST-120 is composed of spherical carbon particles and has an adsorption ability for certain small-molecular-weight compounds that accumulate in patients with chronic kidney disease (CKD). So far, very few compounds are known to be adsorbed by AST-120 in vivo. To examine the effect of AST-120 in vivo, we comprehensively evaluated the plasma concentrations of 146 compounds (61 anions and 85 cations) in CKD model rats, with or without four weeks of treatment with AST-120. By capillary electrophoresis with mass spectrometry, we identified 6 anions and 17 cations that were significantly decreased by AST-120 treatment. In contrast, we also identified 2 cations that were significantly increased by AST-120. Among them, 4 anions, apart from indoxyl sulfate and hippurate, and 19 cations were newly identified in this study. The plasma levels of N-acetyl-neuraminate, 4-pyridoxate, 4-oxopentanoate, glycine, γ-guanidinobutyrate, N-γ-ethylglutamine, allantoin, cytosine, 5-methylcytosine and imidazole-4-acetate were significantly increased in the CKD model compared with the sham-operated group, and were significantly decreased by AST-120 treatment. Therefore, these 10 compounds could be added as uremic compounds that indicate the effect of AST-120 treatment. This study provides useful information not only for identifying the indicators of AST-120, but also for clarifying changes in the metabolic profile by AST-120 treatment in the clinical setting. View

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Sheathless capillary electrophoresis-mass spectrometry with a high-sensitivity porous sprayer for cationic metabolome analysis

Hirayama, A., Tomita, M., Soga, T.

Analyst 137 （ 21 ） 5026 - 5033 2012年11月

研究論文（学術雑誌）, 共著, 査読有り

Sheath-flow capillary electrophoresis-mass spectrometry (CE-MS) has emerged as a new tool for comprehensive analysis of charged metabolites. However, it needs to be more sensitive. Here, we report a sheathless capillary electrophoresis-electrospray ionization-mass spectrometry method for cationic metabolome analysis. This system used a high-sensitivity porous sprayer interface and 10% (v/v) acetic acid as the background electrolyte (BGE). Under optimized conditions, 53 cationic metabolites, including amino acids and their derivatives, amines, nucleic acids and small peptides, were successfully separated and selectively detected with a time-of-flight mass spectrometer. At a signal-to-noise ratio of three, the concentration detection limits for these compounds were between 0.004 and 0.8 μmol L(-1) (amount detection limit, 0.01 to 2 fmol) with pressure injection at 20.7 kPa for 5 s (2.6 nL). Compared with conventional sheath-flow CE-MS, the detection limit of the present method was increased more than 5-fold for 21 (40%) of the compounds detected. When the method was applied to the analysis of cationic metabolites obtained from human urine, there was a 10-fold increase in the number of detected peaks compared with conventional methods. More than 180 successive runs could be conducted without any problems, and only the BGE needed to be changed.

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Pilot Study of Changes in Salivary Metabolic Profiles Induced by Template Therapy

Tanaka, S., Taga, H., Maehara, K., *Kaneshima, A., Machino, M., Onuma, H., Kaneko, M., Sakagami, H., Sugimoto, M., Soga, T. and Tomita, M.

In Vivo 26 （ 6 ） 1015 - 1020 2012年11月

研究論文（学術雑誌）, 共著, 査読有り

Background: Occlusal raising method (so-called 'Template therapy') has been reported to alleviate various diseases and symptoms, but the underlying mechanism is not clear. We searched the low-molecular weight metabolite(s) in the saliva, the concentration of which is significantly changed by the template therapy.
Materials and methods: One female patient with headache underwent the template therapy for 12 days, and her total saliva was subjected to non-targeted analysis using capillary electrophoresis time-of-flight mass spectrometry (CE-TOF-MS).
Results: One hundred and thirteen substances were identified in the saliva. Glycine was the most abundant amino acid in the saliva, followed by alanine, serine and proline. After the start of the template therapy, her headache was alleviated, accompanied by a significant (p=0.042) increase of salivary concentration of glycine, as compared with total amino acids whereas that of other amino acids was not significantly changed. In the metabolomics profile, salivary concentration of large number of metabolites as compared with total metabolite concentration decreased, including N-acetylneuraminate (p=0.025) and p-hydroxyphenylacetate (p=0.039).
Conclusion: This pilot study demonstrated, to our knowledge for the first time, that only glycine exhibited unique changes among total metabolites, suggesting its significant role in template therapy.

Metabolomic Profiling Rationalized Pyruvate Efficacy in Cybrid Cells Harboring MELAS Mitochondrial DNA Mutations

Kami, K., Fujita, Y., Igarashi, S., Koike, S., Sugawara, S., Ikeda, S., Sato, N., Ito, M., Tanaka, M., Tomita, M., Soga, T.

Mitochondrion 12 （ 6 ） 644 - 653 2012年11月

研究論文（学術雑誌）, 共著, 査読有り

Pyruvate treatment was found to alleviate clinical symptoms of mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome and is highly promising therapeutic. Using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS), we measured time-changes of 161 intracellular and 85 medium metabolites to elucidate metabolic effects of pyruvate treatment on cybrid human 143B osteosarcoma cells harboring normal (2SA) and MELAS mutant (2SD) mitochondria. The results demonstrated dramatic and sustainable effects of pyruvate administration on the energy metabolism of 2SD cells, corroborating pyruvate as a metabolically rational treatment regimen for improving symptoms associated with MELAS and possibly other mitochondrial diseases.

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In vivo role of aldehyde reductase

Takahashi, M., Miyata, S., Fujii, J., Inai, Y., Ueyama, S., Araki, M., Soga, T., Fujinawa, R., Nishitani C., Ariki S., Shimizu T., Abe T., Ihara Y., Nishikimi, M., Kozutsumi Y., Taniguchi N., and Kuroki, Y.

Biochim. biophys. Acta 1820 （ 11 ） 1787 - 1796 2012年11月

研究論文（学術雑誌）, 共著, 査読有り

Background: Aldehyde reductase (AKR1A; EC 1.1.1.2) catalyzes the reduction of various types of aldehydes. To ascertain the physiological role of AKR1A, we examined AKR1A knockout mice.
Methods: Ascorbic acid concentrations in AKR1A knockout mice tissues were examined, and the effects of human AKR1A transgene were analyzed. We purified AKR1A and studied the activities of glucuronate reductase and glucuronolactone reductase, which are involved in ascorbic acid biosynthesis. Metabolomic analysis and DNA microarray analysis were performed for a comprehensive study of AKR1A knockout mice.
Results: The levels of ascorbic acid in tissues of AKR1A knockout mice were significantly decreased which were completely restored by human AKR1A transgene. The activities of glucuronate reductase and glucuronolactone reductase, which are involved in ascorbic acid biosynthesis, were suppressed in AKR1A knockout mice. The accumulation of d-glucuronic acid and saccharate in knockout mice tissue and the expression of acute-phase proteins such as serum amyloid A2 are significantly increased in knockout mice liver.
Conclusions: AKR1A plays a predominant role in the reduction of both d-glucuronic acid and d-glucurono-γ-lactone in vivo. The knockout of AKR1A in mice results in accumulation of d-glucuronic acid and saccharate as well as a deficiency of ascorbic acid, and also leads to upregulation of acute phase proteins.
General significance: AKR1A is a major enzyme that catalyzes the reduction of d-glucuronic acid and d-glucurono-γ-lactone in vivo, besides acting as an aldehyde-detoxification enzyme. Suppression of AKR1A by inhibitors, which are used to prevent diabetic complications, may lead to the accumulation of d-glucuronic acid and saccharate.

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Mitochondrial dysfunction associated with increased oxidative stress and α-synuclein accumulation in PARK2 iPSC-derived neurons and postmortem brain tissue

Imaizumi, Y., Okada, Y., Akamatsu, W., Koike, M., Kuzumaki, N., Hayakawa, H., Nihira, T., Kobayashi, T., Ohyama, M., ,Sato, S., Takanashi, M., Funayama, M., Hirayama, A., Soga, T., Hishiki, T., Suematsu, M., Yagi, T., Ito, D., Kosakai, A., Hayashi, K., Shouji, M., Nakanishi, A., Suzuki, N., Mizuno, Y., Mizushima, N., Amagai, M., Uchiyama, Y., Mochizuki, H., Hattori, N., Okano H.

Mol Brain. 5 35 2012年10月

研究論文（学術雑誌）, 共著, 査読有り

Background: Parkinson's disease (PD) is a neurodegenerative disease characterized by selective degeneration of dopaminergic neurons in the substantia nigra (SN). The familial form of PD, PARK2, is caused by mutations in the parkin gene. parkin-knockout mouse models show some abnormalities, but they do not fully recapitulate the pathophysiology of human PARK2.
Results: Here, we generated induced pluripotent stem cells (iPSCs) from two PARK2 patients. PARK2 iPSC-derived neurons showed increased oxidative stress and enhanced activity of the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. iPSC-derived neurons, but not fibroblasts or iPSCs, exhibited abnormal mitochondrial morphology and impaired mitochondrial homeostasis. Although PARK2 patients rarely exhibit Lewy body (LB) formation with an accumulation of α-synuclein, α-synuclein accumulation was observed in the postmortem brain of one of the donor patients. This accumulation was also seen in the iPSC-derived neurons in the same patient.
Conclusions: Thus, pathogenic changes in the brain of a PARK2 patient were recapitulated using iPSC technology. These novel findings reveal mechanistic insights into the onset of PARK2 and identify novel targets for drug screening and potential modified therapies for PD.

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Dramatic Increase in Glycerol Biosynthesis upon Oxidative Stress in the Anaerobic Protozoan Parasite Entamoeba histolytica

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

PLoS Negl Trop Dis. 6 （ 9 ） e1831 2012年09月

研究論文（学術雑誌）, 共著, 査読有り

Entamoeba histolytica, a microaerophilic enteric protozoan parasite, causes amebic colitis and extra intestinal abscesses in millions of inhabitants of endemic areas. Trophozoites of E. histolytica are exposed to a variety of reactive oxygen and nitrogen species during infection. Since E. histolytica lacks key components of canonical eukaryotic anti-oxidative defense systems, such as catalase and glutathione system, alternative not-yet-identified anti-oxidative defense strategies have been postulated to be operating in E. histolytica. In the present study, we investigated global metabolic responses in E. histolytica in response to H(2)O(2)- and paraquat-mediated oxidative stress by measuring charged metabolites on capillary electrophoresis and time-of-flight mass spectrometry. We found that oxidative stress caused drastic modulation of metabolites involved in glycolysis, chitin biosynthesis, and nucleotide and amino acid metabolism. Oxidative stress resulted in the inhibition of glycolysis as a result of inactivation of several key enzymes, leading to the redirection of metabolic flux towards glycerol production, chitin biosynthesis, and the non-oxidative branch of the pentose phosphate pathway. As a result of the repression of glycolysis as evidenced by the accumulation of glycolytic intermediates upstream of pyruvate, and reduced ethanol production, the levels of nucleoside triphosphates were decreased. We also showed for the first time the presence of functional glycerol biosynthetic pathway in E. histolytica as demonstrated by the increased production of glycerol 3-phosphate and glycerol upon oxidative stress. We proposed the significance of the glycerol biosynthetic pathway as a metabolic anti-oxidative defense system in E. histolytica.

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Human blood metabolite timetable indicates internal body time

Kasukawa, T., Sugimoto, M., Hida, A., Minami, Y., Mori, M., Honma, S., Honma, K., Mishima, K., Soga, T., Ueda, H.R.

Proc. Natl. Acad. Sci. USA 109 （ 37 ） 15036 - 15041 2012年09月

研究論文（学術雑誌）, 共著, 査読有り

A convenient way to estimate internal body time (BT) is essential for chronotherapy and time-restricted feeding, both of which use body-time information to maximize potency and minimize toxicity during drug administration and feeding, respectively. Previously, we proposed a molecular timetable based on circadian-oscillating substances in multiple mouse organs or blood to estimate internal body time from samples taken at only a few time points. Here we applied this molecular-timetable concept to estimate and evaluate internal body time in humans. We constructed a 1.5-d reference timetable of oscillating metabolites in human blood samples with 2-h sampling frequency while simultaneously controlling for the confounding effects of activity level, light, temperature, sleep, and food intake. By using this metabolite timetable as a reference, we accurately determined internal body time within 3 h from just two anti-phase blood samples. Our minimally invasive, molecular-timetable method with human blood enables highly optimized and personalized medicine.

L-Carnitine Prevents the Development of Ventricular Fibrosis and Heart Failure with Preserved Ejection Fraction in Hypertensive Heart Disease

Omori, Y., Ohtani, T., Sakata, Y., Mano, T., Takeda, Y., Tamaki, S., Tsukamoto. Y.,　Kamimura, D., Aizawa, Y., Miwa, T., Komuro, I., Soga, T., Yamamoto, K.

J. Hypertens. 30 （ 9 ） 1834 - 1844 2012年09月

研究論文（学術雑誌）, 共著, 査読有り

Objectives: Prognosis of heart failure with preserved ejection fraction (HFpEF) remains poor because of unknown pathophysiology and unestablished therapeutic strategy. This study aimed to identify a potential therapeutic intervention for HFpEF through metabolomics-based analysis.
Methods and results: Metabolomics with capillary electrophoresis time-of-flight mass spectrometry was performed using plasma of Dahl salt-sensitive rats fed high-salt diet, a model of hypertensive HFpEF, and showed decreased free-carnitine levels. Reassessment with enzymatic cycling method revealed the decreased plasma and left-ventricular free-carnitine levels in the HFpEF model. Urinary free-carnitine excretion was increased, and the expression of organic cation/carnitine transporter 2, which transports free-carnitine into cells, was down-regulated in the left ventricle (LV) and kidney in the HFpEF model. L-Carnitine was administered to the hypertensive HFpEF model. L-Carnitine treatment restored left-ventricular free-carnitine levels, attenuated left-ventricular fibrosis and stiffening, prevented pulmonary congestion, and improved survival in the HFpEF model independent of the antihypertensive effects, accompanied with increased expression of fatty acid desaturase (FADS) 1/2, rate-limiting enzymes in forming arachidonic acid, and enhanced production of arachidonic acid, a precursor of prostacyclin, and prostacyclin in the LV. In cultured cardiac fibroblasts, L-carnitine attenuated the angiotensin II-induced collagen production with increased FADS1/2 expression and enhanced production of arachidonic acid and prostacyclin. L-Carnitine-induced increase of arachidonic acid was canceled by knock-down of FADS1 or FADS2 in cultured cardiac fibroblasts. Serum free-carnitine levels were decreased in HFpEF patients.
Conclusions: L-carnitine supplementation attenuates cardiac fibrosis by increasing prostacyclin production through arachidonic acid pathway, and may be a promising therapeutic option for HFpEF

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A Yeast Metabolite Extraction Protocol Optimised for Time-Series Analyses

Sasidharan, K., Soga, T., Tomita, M., Murray, D.B.

PLoS One 7 （ 8 ） e44283 2012年08月

研究論文（学術雑誌）, 共著, 査読有り

There is an increasing call for the absolute quantification of time-resolved metabolite data. However, a number of technical issues exist, such as metabolites being modified/degraded either chemically or enzymatically during the extraction process. Additionally, capillary electrophoresis mass spectrometry (CE-MS) is incompatible with high salt concentrations often used in extraction protocols. In microbial systems, metabolite yield is influenced by the extraction protocol used and the cell disruption rate. Here we present a method that rapidly quenches metabolism using dry-ice ethanol bath and methanol N-ethylmaleimide solution (thus stabilising thiols), disrupts cells efficiently using bead-beating and avoids artefacts created by live-cell pelleting. Rapid sample processing minimised metabolite leaching. Cell weight, number and size distribution was used to calculate metabolites to an attomol/cell level. We apply this method to samples obtained from the respiratory oscillation that occurs when yeast are grown continuously.

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Self-enhancement of hepatitis C virus replication by promotion of specific sphingolipid biosynthesis

Hirata, Y., Ikeda, K., Sudoh, M., Tokunaga, Y., Suzuki, A., Weng, L., Ohta, M., Tobita, Y., Okano, K., Ozeki, K., Kawasaki, K., Tsukuda, T., Katsume, A., Aoki, Y., Umehara, T., Sekiguchi, S., Toyoda, T., Shimotohno, K., Soga, T., Nishijima, M., Taguchi, R., Kohara, M.

PLoS Pathog. 8 （ 8 ） e1002860 2012年08月

研究論文（学術雑誌）, 共著, 査読有り

Lipids are key components in the viral life cycle that affect host-pathogen interactions. In this study, we investigated the effect of HCV infection on sphingolipid metabolism, especially on endogenous SM levels, and the relationship between HCV replication and endogenous SM molecular species. We demonstrated that HCV induces the expression of the genes (SGMS1 and 2) encoding human SM synthases 1 and 2. We observed associated increases of both total and individual sphingolipid molecular species, as assessed in human hepatocytes and in the detergent-resistant membrane (DRM) fraction in which HCV replicates. SGMS1 expression had a correlation with HCV replication. Inhibition of sphingolipid biosynthesis with a hepatotropic serine palmitoyltransferase (SPT) inhibitor, NA808, suppressed HCV-RNA production while also interfering with sphingolipid metabolism. Further, we identified the SM molecular species that comprise the DRM fraction and demonstrated that these endogenous SM species interacted with HCV nonstructural 5B polymerase to enhance viral replication. Our results reveal that HCV alters sphingolipid metabolism to promote viral replication, providing new insights into the formation of the HCV replication complex and the involvement of host lipids in the HCV life cycle.

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曽我　朋義

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