Details of a Researcher - Soga, Tomoyoshi

Papers - Soga, Tomoyoshi

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Dynamic Simulation and Metabolome Analysis of Long-Term Erythrocyte Storage in Adenine–Guanosine Solution

Nishino, T., *Yachie-Kinoshita, A., Hirayama, A., Soga, T., Suematsu, M., Tomita, M.

PLoS ONE 8 ( 8 ) e71060 2013.08

Joint Work, Accepted

Although intraerythrocytic ATP and 2,3-bisphophoglycerate (2,3-BPG) are known as direct indicators of the viability of preserved red blood cells and the efficiency of post-transfusion oxygen delivery, no current blood storage method in practical use has succeeded in maintaining both these metabolites at high levels for long periods. In this study, we constructed a mathematical kinetic model of comprehensive metabolism in red blood cells stored in a recently developed blood storage solution containing adenine and guanosine, which can maintain both ATP and 2,3-BPG. The predicted dynamics of metabolic intermediates in glycolysis, the pentose phosphate pathway, and purine salvage pathway were consistent with time-series metabolome data measured with capillary electrophoresis time-of-flight mass spectrometry over 5 weeks of storage. From the analysis of the simulation model, the metabolic roles and fates of the 2 major additives were illustrated: (1) adenine could enlarge the adenylate pool, which maintains constant ATP levels throughout the storage period and leads to production of metabolic waste, including hypoxanthine; (2) adenine also induces the consumption of ribose phosphates, which results in 2,3-BPG reduction, while (3) guanosine is converted to ribose phosphates, which can boost the activity of upper glycolysis and result in the efficient production of ATP and 2,3-BPG. This is the first attempt to clarify the underlying metabolic mechanism for maintaining levels of both ATP and 2,3-BPG in stored red blood cells with in silico analysis, as well as to analyze the trade-off and the interlock phenomena between the benefits and possible side effects of the storage-solution additives.

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Metabolomic profiling analysis reveals chamber-dependent metabolite patterns in the mouse heart

Shimura, D., Nakai, G., Jiao, Q., Osanai, K., Kashikura, K., Endo, K., Soga, T., Goda, N., Minamisawa, S.

Am J Physiol Heart Circ Physiol. 305 ( 4 ) H494 - 505 2013.08

Research paper (scientific journal), Joint Work, Accepted

Energy of the cardiac muscle largely depends on fatty acid oxidation. It is known that the atrium and ventricle have chamber-specific functions, structures, gene expressions, and pathologies. The left ventricle works as a high-pressure chamber to pump blood toward the body, and its muscle wall is thicker than those of the other chambers, suggesting that energy utilization in each of the chambers should be different. However, a chamber-specific pattern of metabolism remains incompletely understood. Recently, innovative techniques have enabled the comprehensive analysis of metabolites. Therefore, we aimed to clarify differences in metabolic patterns among the chambers. Male C57BL6 mice at 6 wk old were subject to a comprehensive measurement of metabolites in the atria and ventricles by capillary electrophoresis and mass spectrometry. We found that overall metabolic profiles, including nucleotides and amino acids, were similar between the right and left ventricles. On the other hand, the atria exhibited a distinct metabolic pattern from those of the ventricles. Importantly, the high-energy phosphate pool (the total concentration of ATP, ADP, and AMP) was higher in both ventricles. In addition, the levels of lactate, acetyl CoA, and tricarboxylic acid cycle contents were higher in the ventricles. Accordingly, the activities and/or expression levels of key enzymes were higher in the ventricles to produce more energy. The present study provides a basis for understanding the chamber-specific metabolism underlining pathophysiology in the heart.

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Persistent Overexpression of Phosphoglycerate Mutase, a Glycolytic Enzyme, Modifies Energy Metabolism and Reduces Stress Resistance of Heart in Mice

Okuda, J., Niizuma, S., Shioi, T., Kato, T., Inuzuka, Y., Kawashima, T., Tamaki, Y., Kawamoto, A., Tanada, Y., Iwanaga, Y., Narazaki, M., Matsuda, T., Adachi, S., Soga, T., Takemura, G., Kondoh, H., Kita, T., Kimura, T.

PLoS ONE 8 ( 8 ) e72173 2013.08

Research paper (scientific journal), Joint Work, Accepted

Background: Heart failure is associated with changes in cardiac energy metabolism. Glucose metabolism in particular is thought to be important in the pathogenesis of heart failure. We examined the effects of persistent overexpression of phosphoglycerate mutase 2 (Pgam2), a glycolytic enzyme, on cardiac energy metabolism and function.
Methods and results: Transgenic mice constitutively overexpressing Pgam2 in a heart-specific manner were generated, and cardiac energy metabolism and function were analyzed. Cardiac function at rest was normal. The uptake of analogs of glucose or fatty acids and the phosphocreatine/βATP ratio at rest were normal. A comprehensive metabolomic analysis revealed an increase in the levels of a few metabolites immediately upstream and downstream of Pgam2 in the glycolytic pathway, whereas the levels of metabolites in the initial few steps of glycolysis and lactate remained unchanged. The levels of metabolites in the tricarboxylic acid (TCA) cycle were altered. The capacity for respiration by isolated mitochondria in vitro was decreased, and that for the generation of reactive oxygen species (ROS) in vitro was increased. Impaired cardiac function was observed in response to dobutamine. Mice developed systolic dysfunction upon pressure overload.
Conclusions: Constitutive overexpression of Pgam2 modified energy metabolism and reduced stress resistance of heart in mice.

Abundant Nucleostemin Expression Supports the Undifferentiated Properties of Germ Cell Tumors

Uema, N., Ooshio, T., Harada, K., Naito, M., Naka, K., Hoshi T., Tadokoro, Y., Ohta, K., Ali, M. A.E., Katanno, M., Soga, T., Nakanuma, Y., Okuda, A., Hirao, A.

Am J Pathol. 183 ( 2 ) 592 - 603 2013.08

Research paper (scientific journal), Joint Work

Nucleostemin (NS) is a nucleolar GTP-binding protein that is involved in ribosomal biogenesis and protection of telomeres. We investigated the expression of NS in human germ cell tumors and its function in a mouse germ cell tumor model. NS was abundantly expressed in undifferentiated, but not differentiated, types of human testicular germ cell tumors. NS was expressed concomitantly with OCT3/4, a critical regulator of the undifferentiated status of pluripotent stem cells in primordial germ cells and embryonal carcinomas. To investigate the roles of NS in tumor growth in vivo, we used a mouse teratoma model. Analysis of teratomas derived from embryonic stem cells in which the NS promoter drives GFP expression showed that cells highly expressing NS were actively proliferating and exhibited the characteristics of tumor-initiating cells, including the ability to initiate and propagate tumor cells in vivo. NS-expressing cells exhibited higher levels of GTP than non-NS-expressing cells. Because NS protein is stabilized by intracellular GTP, metabolic changes may contribute to abundant NS expression in the undifferentiated cells. OCT3/4 deficiency in teratomas led to loss of NS expression, resulting in growth retardation. Finally, we found that teratomas deficient in NS lost their undifferentiated characteristics, resulting in defective tumor proliferation. These data indicate that abundant expression of NS supports the undifferentiated properties of germ cell tumors.

Global metabolomic analysis of heart tissue in a hamster model for dilated cardiomyopathy

Maekawa, K., Hirayama, A., Iwata Y., Tajima Y., Nishimaki-Mogami T., Sugawara S., Ueno, N., Abe, H., Ishikawa M., Murayama M., Matsuzawa Y., Nakanishi H., Ikeda K., Arita M., Taguchi, R., Minamino N., Wakabayashi, S., Soga T. *, Saito, Y.*

J. Mol. Cell. Cardiol. 59 76 - 85 2013.06

Research paper (scientific journal), Joint Work, Accepted

Dilated cardiomyopathy (DCM), a common cause of heart failure, is characterized by cardiac dilation and reduced left ventricular ejection fraction, but the underlying mechanisms remain unclear. To investigate the mechanistic basis, we performed global metabolomic analysis of myocardial tissues from the left ventricles of J2N-k cardiomyopathic hamsters. This model exhibits symptoms similar to those of human DCM, owing to the deletion of the δ-sarcoglycan gene. Charged and lipid metabolites were measured by capillary electrophoresis mass spectrometry (MS) and liquid chromatography MS(/MS), respectively, and J2N-k hamsters were compared with J2N-n healthy controls at 4 (presymptomatic phase) and 16weeks (symptomatic) of age. Disturbances in membrane phospholipid homeostasis were initiated during the presymptomatic phase. Significantly different levels of charged metabolites, occurring mainly in the symptomatic phase, were mapped to primary metabolic pathways. Reduced levels of metabolites in glycolysis, the pentose phosphate pathway, and the tricarboxylic acid cycle, together with large decreases in major triacylglycerol levels, suggested that decreased energy production leads to cardiac contractile dysfunction in the symptomatic phase. A mild reduction in glutathione and a compensatory increase in ophthalmate levels suggest increased oxidative stress in diseased tissues, which was confirmed by histochemical staining. Increased levels of 4 eicosanoids, including prostaglandin (PG) E2 and 6-keto-PGF1α, in the symptomatic phase suggested activation of the protective response pathways. These results provide mechanistic insights into DCM pathogenesis and may help identify new targets for therapeutic intervention and diagnosis.

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Metabolome profiling of floral scent production in Petunia axillaris

Oyama-Okubo, N., Sakai , T., Ando, T., Nakayama, M., Soga, T.

Phytochemistry 90 37 - 42 2013.06

Research paper (scientific journal), Joint Work, Accepted

Emission of floral scent benzenoid/phenylpropanoid compounds in Petunia axillaris increases significantly at night, a change that is primarily determined by the endogenous concentration of these compounds in the corolla. Among wild type P. axillaris plants, there are lines that emit different amounts of scent. To understand how the nocturnal rhythm of floral scent concentrations is controlled, the concentration profiles of metabolites in the scent biosynthetic pathway in two lines of P. axillaris, a strongly scented line and a weakly scented line, are reported. In the strongly scented line, the concentration of a series of compounds from glucose-6-phosphate (G6P) to the scent compounds changed synchronously. In the weakly scented lines, the concentrations of some metabolites including 6-phosphogluconate (6PG) and downstream metabolites of shikimic acid were remarkably lower, suggesting a reduction in metabolism of G6P to 6PG and the metabolism of shikimic acid in the weakly scented line. Nocturnal increases in the concentrations of sucrose, fructose, and glucose were not found in strongly scented line. Nocturnal increases in concentrations of S-adenosylhomocysteine (SAH) and methionine and reductions in the concentrations of S-adenosylmethionine (SAM), a methylation donor to benzenoid-skeletons, were observed only in strongly scented line. It is concluded that the biosynthetic regulation of each step from G6P to the volatile scent benzenoids is performed by, at least in part, concentrations of substrates, and the regulation also affects concentrations of SAM cycle compounds.

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A Role for Cytosolic Fumarate Hydratase in Urea Cycle Metabolism and Renal Neoplasia

Adam, J., Yang, M., Bauerschmidt, C., Kitagawa, M., O’Flaherty, L., Maheswaran, P., O¨ zkan, G., Sahgal, N., Baban, D., Kato, K., Saito, K., Iino, K., Igarashi, K., Stratford, M., Pugh, C., Tenant, D.A., Ludwig, C., Davies, B., Ratcliffe, P. J., El-Bahrawy, M., Ashrafian, H., Soga, T., *Pollard, P. J.

Cell Rep. 3 ( 5 ) 1440 - 1448 2013.05

Research paper (scientific journal), Joint Work, Accepted

The identification of mutated metabolic enzymes in hereditary cancer syndromes has established a direct link between metabolic dysregulation and cancer. Mutations in the Krebs cycle enzyme, fumarate hydratase (FH), predispose affected individuals to leiomyomas, renal cysts, and cancers, though the respective pathogenic roles of mitochondrial and cytosolic FH isoforms remain undefined. On the basis of comprehensive metabolomic analyses, we demonstrate that FH1-deficient cells and tissues exhibit defects in the urea cycle/arginine metabolism. Remarkably, transgenic re-expression of cytosolic FH ameliorated both renal cyst development and urea cycle defects associated with renal-specific FH1 deletion in mice. Furthermore, acute arginine depletion significantly reduced the viability of FH1-deficient cells in comparison to controls. Our findings highlight the importance of extramitochondrial metabolic pathways in FH-associated oncogenesis and the urea cycle/arginine metabolism as a potential therapeutic target.

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The selective control of glycolysis, gluconeogenesis and glycogenesis by temporal insulin patterns

Noguchi, R., Kubota, H., Yugi, K., Toyoshima, Y., Komori, Y., Soga, T., Kuroda, S.

Mol Syst Biol. 9 664 2013.05

Research paper (scientific journal), Joint Work, Accepted

Insulin governs systemic glucose metabolism, including glycolysis, gluconeogenesis and glycogenesis, through temporal change and absolute concentration. However, how insulin-signalling pathway selectively regulates glycolysis, gluconeogenesis and glycogenesis remains to be elucidated. To address this issue, we experimentally measured metabolites in glucose metabolism in response to insulin. Step stimulation of insulin induced transient response of glycolysis and glycogenesis, and sustained response of gluconeogenesis and extracellular glucose concentration (GLC(ex)). Based on the experimental results, we constructed a simple computational model that characterises response of insulin-signalling-dependent glucose metabolism. The model revealed that the network motifs of glycolysis and glycogenesis pathways constitute a feedforward (FF) with substrate depletion and incoherent feedforward loop (iFFL), respectively, enabling glycolysis and glycogenesis responsive to temporal changes of insulin rather than its absolute concentration. In contrast, the network motifs of gluconeogenesis pathway constituted a FF inhibition, enabling gluconeogenesis responsive to absolute concentration of insulin regardless of its temporal patterns. GLC(ex) was regulated by gluconeogenesis and glycolysis. These results demonstrate the selective control mechanism of glucose metabolism by temporal patterns of insulin.

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Inhibition of ATP citrate lyase induces an anticancer effect via reactive oxygen species: AMPK as a predictive biomarker for therapeutic impact

Migita, T., Okabe, S., Ikeda, K., Igarashi, S., Sugawara, S., Tomida, A., Taguchi, R., Soga, T., Seimiya, H.

Am J Pathol. 182 ( 5 ) 1800 - 1810 2013.05

Research paper (scientific journal), Joint Work, Accepted

De novo lipogenesis is activated in most cancers. Inhibition of ATP citrate lyase (ACLY), the enzyme that catalyzes the first step of de novo lipogenesis, leads to growth suppression and apoptosis in a subset of human cancer cells. Herein, we found that ACLY depletion increases the level of intracellular reactive oxygen species (ROS), whereas addition of an antioxidant reduced ROS and attenuated the anticancer effect. ACLY depletion or exogenous hydrogen peroxide induces phosphorylation of AMP-activated protein kinase (p-AMPK), a crucial regulator of lipid metabolism, independently of energy status. Analysis of various cancer cell lines revealed that cancer cells with a higher susceptibility to ACLY depletion have lower levels of basal ROS and p-AMPK. Mitochondrial-deficient ρ(0) cells retained high levels of ROS and p-AMPK and were resistant to ACLY depletion, whereas the replenishment of normal mitochondrial DNA reduced the levels of ROS and p-AMPK and restored the sensitivity to ACLY depletion, indicating that low basal levels of mitochondrial ROS are critical for the anticancer effect of ACLY depletion. Finally, p-AMPK levels were significantly correlated to the levels of oxidative DNA damage in colon cancer tissues, suggesting that p-AMPK reflects cellular ROS levels in vitro and in vivo. Together, these data suggest that ACLY inhibition exerts an anticancer effect via increased ROS, and p-AMPK could be a predictive biomarker for its therapeutic outcome.

Metabolomic profiling of the response of susceptible and resistant soybean strains to foxglove aphid, Aulacorthum solani Kaltenbach

Sato, D., Akashi, H., Sugimoto, M., Tomita, M., Soga, T.

J. Chromatogr. B. 925 95 - 103 2013.04

Research paper (scientific journal), Joint Work, Accepted

Aphid infection reduces soybean (Glycine max [L.] Merr.) yield. Consequently, cultivation of aphid-resistant strains is a promising approach to pest control, and understanding the resistance mechanism is of importance. Here, we characterized the resistance of soybeans to foxglove aphid, Aulacorthum solani Kaltenbach, at the metabolite level. First, we evaluated aphid mortality and settlement rates on the leaves of two soybean strains, ‘Tohoku149’ and ‘Suzuyutaka’, and found that the former had strong resistance soon after introduction of the aphids. The metabolomic response to aphid introduction was analyzed using capillary electrophoresis–time-of-flight mass spectrometry. We found the following three features in the profiles: (1) concentrations of citrate, amino acids, and their intermediates were intrinsically higher for Tohoku149 than Suzuyutaka, (2) concentrations of several metabolites producing secondary metabolites, such as flavonoids and alkaloids, drastically changed 6 h after aphid introduction, and (3) concentrations of TCA cycle metabolites increased in Tohoku149 48 h after aphid introduction. We also profiled free amino acids in aphids reared on both soybean strains and under starvation, and found that the profile of the aphids on Tohoku149 was similar to that of the starved aphids, but different to that of aphids on Suzuyutaka. These tests confirmed that aphids suck phloem sap even from Tohoku149. This study demonstrates the metabolomic profiles of both soybean strains and aphids, which will contribute to the molecular level understanding of mechanisms of soybean resistance to aphids.

Physiological and environmental parameters associated with mass spectrometry-based salivary metabolomic profiles

Sugimoto, M., Saruta, J., Matsuki, C., To, M., Onuma, H., Kaneko, M., Soga, T., Tomita, M., and Tsukinoki, K.

Metabolomics 9 ( 2 ) 454 - 463 2013.04

Research paper (scientific journal), Joint Work, Accepted

Mass spectrometry (MS)-based metabolomic methods enable simultaneous profiling of hundreds of salivary metabolites, and may be useful to diagnose a wide range of diseases using saliva. However, few studies have evaluated the effects of physiological or environmental factors on salivary metabolomic profiles. Therefore, we used capillary electrophoresis-MS to analyze saliva metabolite profiles in 155 subjects with reasonable oral hygiene, and examined the effects of physiological and environmental factors on the metabolite profiles. Overall, 257 metabolites were identified and quantified. The global profiles and individual metabolites were evaluated by principle component analysis and univariate tests, respectively. Collection method, collection time, sex, body mass index, and smoking affected the global metabolite profiles. However, age also might contribute to the bias in sex and collection time. The profiles were relatively unaffected by other parameters, such as alcohol consumption and smoking, tooth brushing, or the use of medications or nutritional supplements. Temporomandibular joint disorders had relatively greater effects on salivary metabolites than other dental abnormalities (e.g., stomatitis, tooth alignment, and dental caries). These findings provide further insight into the diversity and stability of salivary metabolomic profiles, as well as the generalizability of disease-specific biomarkers.

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Metabolomic Profiling of Lung and Prostate Tumor Tissues by Capillary Electrophoresis Time-of-flight Mass Spectrometry

Kami, K., Fujimori, T., Sato, H., Sato, M., Yamamoto, H., Ohashi, Y., Sugiyama, N., Ishihama, Y., Onozuka, H., Ochiai, A., Esumi, H., Soga, T., Tomita, M.

Metabolomics 9 ( 2 ) 444 - 453 2013.04

Research paper (scientific journal), Joint Work, Accepted

Metabolic microenvironment of tumor cells is influenced by oncogenic signaling and tissue-specific metabolic demands, blood supply, and enzyme expression. To elucidate tumor-specific metabolism, we compared the metabolomics of normal and tumor tissues surgically resected pairwise from nine lung and seven prostate cancer patients, using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). Phosphorylation levels of enzymes involved in central carbon metabolism were also quantified. Metabolomic profiles of lung and prostate tissues comprised 114 and 86 metabolites, respectively, and the profiles not only well distinguished tumor from normal tissues, but also squamous cell carcinoma from the other tumor types in lung cancer and poorly differentiated tumors from moderately differentiated tumors in prostate cancer. Concentrations of most amino acids, especially branched-chain amino acids, were significantly higher in tumor tissues, independent of organ type, but of essential amino acids were particularly higher in poorly differentiated than moderately differentiated prostate cancers. Organ-dependent differences were prominent at the levels of glycolytic and tricarboxylic acid cycle intermediates and associated energy status. Significantly high lactate concentrations and elevated activating phosphorylation levels of phosphofructokinase and pyruvate kinase in lung tumors confirmed hyperactive glycolysis. We highlighted the potential of CE-TOFMS-based metabolomics combined with phosphorylated enzyme analysis for understanding tissue-specific tumor microenvironments, which may lead to the development of more effective and specific anticancer therapeutics.

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Inhibition of Mitochondrial Aconitase by Succination in Fumarate Hydratase Deficiency

Ternette N., Yang M., Laroyia M., Kitagawa M., O'Flaherty L., Wolhulter K., Igarashi K., Saito K., Kato K., Fischer R., Berquand A., Kessler B.M., Lappin T., Frizzell N., Soga T., Adam J, Pollard P.J.

Cell Rep. 3 ( 3 ) 689 - 700 2013.03

Research paper (scientific journal), Joint Work, Accepted

The gene encoding the Krebs cycle enzyme fumarate hydratase (FH) is mutated in hereditary leiomyomatosis and renal cell cancer (HLRCC). Loss of FH activity causes accumulation of intracellular fumarate, which can directly modify cysteine residues to form 2-succinocysteine through succination. We undertook a proteomic-based screen in cells and renal cysts from Fh1 (murine FH)-deficient mice and identified 94 protein succination targets. Notably, we identified the succination of three cysteine residues in mitochondrial Aconitase2 (ACO2) crucial for iron-sulfur cluster binding. We show that fumarate exerts a dose-dependent inhibition of ACO2 activity, which correlates with increased succination as determined by mass spectrometry, possibly by interfering with iron chelation. Importantly, we show that aconitase activity is impaired in FH-deficient cells. Our data provide evidence that succination, resulting from FH deficiency, targets and potentially alters the function of multiple proteins and may contribute to the dysregulated metabolism observed in HLRCC.

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Cancer metabolism: key players in metabolic reprogramming

Soga, T.

Cancer Sci. 104 ( 3 ) 275 - 281 2013.03

Research paper (scientific journal), Single Work, Accepted

Over 80 years ago, Warburg discovered that cancer cells generate ATP through the glycolytic pathway, even in the presence of oxygen. The finding of this phenomenon, termed the "Warburg effect," stimulated much research on tumorigenesis, but few explanations were forthcoming to explain the observation. Recently, advanced developments in molecular biology and high-throughput molecular analyses have revealed that many of the signaling pathways altered by gene mutations regulate cell metabolism in cancer. Furthermore, mutations in isocitrate dehydrogenase 1 and 2 were shown to elevate 2-hydroxyglutarate, which led to changes in α-ketoglutarate-dependent dioxygenase enzyme activity, resulting in an increased risk of malignant tumors. Although these findings led to a renewed interest in cancer metabolism, our knowledge on the specifics of tumor metabolism is still fragmented. This paper reviews recent findings related to key transcription factors and enzymes that play an important role in the regulation of cancer metabolism.

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Metabolic and morphological changes of an oil accumulating trebouxiophycean alga in nitrogen-deficient conditions

Ito, T., Tanaka, M., Shinkawa, H., Nakada, T., Ano, Y., Kurano N., Soga T., Tomita, M.

Metabolomics 9 ( 1Supplement ) 178 - 187 2013.03

Research paper (scientific journal), Joint Work, Accepted

Oil-rich algae have promising potential for a next-generation biofuel feedstock. Pseudochoricystis ellipsoidea MBIC 11204, a novel unicellular green algal strain, accumulates a large amount of oil (lipids) in nitrogen-deficient (-N) conditions. Although the oil bodies are easily visualized by lipophilic staining in the cells, little is known about how oil bodies are metabolically synthesized. Clarifying the metabolic profiles in -N conditions is important to understand the physiological mechanisms of lipid accumulations and will be useful to optimize culture conditions efficiently produce industrial oil. Metabolome and lipidome profiles were obtained, respectively, using capillary electrophoresis- and liquid chromatography-mass spectrometry from P. ellipsoidea in both nitrogen-rich (+N; rapid growth) and -N conditions. Relative quantities of more than 300 metabolites were systematically compared between these two conditions. Amino acids in nitrogen assimilation and N-transporting metabolisms were decreased to 1/20 the amount, or less, in -N conditions. In lipid metabolism, the quantities of neutral lipids increased greatly in -N conditions; however, quantities of nearly all the other lipids either decreased or only changed slightly. The morphological changes in +N and -N conditions were also provided by microscopy, and we discuss their relationship to the metabolic changes. This is the first approach to understand the novel algal strain's metabolism using a combination of wide-scale metabolome analysis and morphological analysis.

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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 2013.03

Research paper (scientific journal), Joint Work, Accepted

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 2013.03

Research paper (scientific journal), Joint Work, Accepted

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 2013.03

Research paper (scientific journal), Accepted

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 2013.02

Research paper (scientific journal), Joint Work, Accepted

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 2013.01

Research paper (scientific journal), Joint Work, Accepted

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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