Details of a Researcher - Soga, Tomoyoshi

Papers - Soga, Tomoyoshi

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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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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 2012.12

Research paper (scientific journal), Joint Work, Accepted

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 2012.12

Research paper (scientific journal), Joint Work, Accepted

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 2012.12

Research paper (scientific journal), Joint Work, Accepted

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 2012.11

Research paper (scientific journal), Joint Work, Accepted

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 2012.11

Research paper (scientific journal), Joint Work, Accepted

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 2012.11

Research paper (scientific journal), Joint Work, Accepted

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 2012.11

Research paper (scientific journal), Joint Work, Accepted

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 2012.11

Research paper (scientific journal), Joint Work, Accepted

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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