Details of a Researcher - Soga, Tomoyoshi

Papers - Soga, Tomoyoshi

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Disruption of HIF-1α in Hepatocytes Impairs Glucose Metabolism in Diet-induced Obesity Mice

Ochiai, D., Goda, N., Hishiki, T., Kanai, M., Senoo-Matsuda, N., Soga, T., Johnson, R.S., Yoshimura, Y., Suematsu, M.

Biochem. Biophys. Res. Commun 415 ( 3 ) 445 - 449 2011.11

Research paper (scientific journal), Joint Work, Accepted

The liver plays a central role in glucose homeostasis in the whole-body by responding to environmental factors including nutrients, hormones, and oxygen. In conditions of metabolic overload such as diabetes mellitus and obesity, coordinated regulation between oxygen supply and consumption has been reported to be disrupted and subsequently cause tissue hypoxia, although pathological significance of the disease-related hypoxia remains elusive. To investigate the role of tissue hypoxia in the liver on systemic glucose homeostasis, mice lacking HIF-1α gene, a critical component of a master regulator of hypoxic response, in hepatocytes were exposed to high fat/sucrose diet (HFSD). Exposure to HFSD for 5 weeks elicited liver hypoxia with a transient increase in HIF-1α protein expression in the liver of control mice. Glucose disposal was marginally impaired in control mice when challenged oral glucose tolerance test, but such impairment was enhanced in the mutant mice. This alteration was accompanied by a complete inhibition of glucokinase induction with a significant reduction of hepatic glucose uptake. Mice fed HFSD for 20 weeks exhibited fasting hyperglycemia and glucose intolerance, whereas these metabolic phenotypes deteriorated considerably with severe insulin resistance in skeletal muscles and adipose tissues in the mutant mice. These findings suggest that HIF-1 in hepatocytes plays protective roles against the progression of diabetes mellitus.

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Renal Cyst Formation in Fh1-Deficient Mice Is Independent of the Hif/Phd Pathway: Roles for Fumarate in KEAP1 Succination and Nrf2 Signaling

Adam, J., Hatipoglu, E., O’Flaherty, L., Ternette, N., Sahgal, N., Lockstone, H., Baban D., Nye, E., Stamp, G.W., Wolhuter, K., Stevens, M., Fischer, R., Carmeliet, P., Maxwell, P.H., Pugh, C.W., Frizzell, N., Soga, T., Kessler, B.M., El-Bahrawy, M., Ratcliffe, P.J., Pollard, P.J.

Cancer Cell 20 ( 4 ) 524 - 537 2011.10

Research paper (scientific journal), Joint Work, Accepted

The Krebs cycle enzyme fumarate hydratase (FH) is a human tumor suppressor whose inactivation is associated with the development of leiomyomata, renal cysts, and tumors. It has been proposed that activation of hypoxia inducible factor (HIF) by fumarate-mediated inhibition of HIF prolyl hydroxylases drives oncogenesis. Using a mouse model, we provide genetic evidence that Fh1-associated cyst formation is Hif independent, as is striking upregulation of antioxidant signaling pathways revealed by gene expression profiling. Mechanistic analysis revealed that fumarate modifies cysteine residues within the Kelch-like ECH-associated protein 1 (KEAP1), abrogating its ability to repress the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-mediated antioxidant response pathway, suggesting a role for Nrf2 dysregulation in FH-associated cysts and tumors.

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Sex-dependent Hepatic Transcripts and Metabolites in the Development of Glucose Intolerance and Insulin Resistance in Zucker Diabetic Fatty Rats

Gustavsson, C., Soga, T., Wahlström, E, Vesterlund, M, Azimi, A., Norstedt, G., Tollet-Egnell, P.

J. Mol. Endcrinol. 47 ( 2 ) 129 - 143 2011.10

Research paper (scientific journal), Joint Work, Accepted

Male Zucker diabetic fatty (mZDF) rats spontaneously develop type 2 diabetes, whereas females only become diabetic when fed a diabetogenic high-fat diet (high-fat-fed female ZDF rat, HF-fZDF). The aim of this study was to investigate if differences in liver functions could provide clues to this sex difference. Non-diabetic obese fZDF rats were compared with either mZDF or HF-fZDF regarding hepatic molecular profiles, to single out those components that might be protective in the females. High-fat feeding in fZDF led to enhanced weight gain, increased blood glucose and insulin levels, reduced insulin sensitivity and a trend towards reduced glucose tolerance, indicative of a prediabetic state. mZDF rats were diabetic, with low levels of insulin, high levels of glucose, reduced insulin sensitivity and impaired glucose tolerance. Transcript profiling and capillary electrophoresis time-of-flight mass spectrometry were used to indentify hepatic transcripts and metabolites that might be related to this. Many diet-induced alterations in transcript and metabolite levels in female rats were towards a 'male-like' phenotype, including reduced lipogenesis, increased fatty acid (FA) oxidation and increased oxidative stress responses. Alterations detected at the level of hepatic metabolites, indicated lower capacity for glutathione (GSH) production in male rats, and higher GSH turnover in females. Taken together, this could be interpreted as if anabolic pathways involving lipogenesis and lipid output might limit the degree of FA oxidation and oxidative stress in female rats. Together with a greater capacity to produce GSH, these hepatic sex differences might contribute to the sex-different development of diabetes in ZDF rats.

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Metabolomic profiling of the autosomal dominant polycystic kidney disease rat model

Toyohara, T., Suzuki, T., Akiyama, Y., Yoshihara, D., Takeuchi, Y., Mishima, E., Kikuchi, K., Suzuki, C., Tanemoto, M., Ito, S., Nagao, S., Soga T., Abe, T.

Clin Exp Nephrol 15 ( 5 ) 676 - 687 2011.10

Research paper (scientific journal), Accepted

Background: Autosomal dominant polycystic kidney disease (ADPKD) is an inherited systemic disease characterized by renal cyst expansion, resulting in renal failure. With the progression of renal damage, the accumulation of uremic compounds is recently reported to subsequently cause further renal damage and hypertension. Finding uremic toxins and sensitive markers for detecting the early stage of ADPKD is necessary to clarify its pathophysiological process and to prevent its progression. The aim of this study was to analyze the profile of uremic retention solutes of ADPKD by capillary electrophoresis-mass spectrometry (CE-MS) using the Han:SPRD rat model.
Methods: Two hundred and ninety-seven cations and 190 anions were comprehensively analyzed by CE-MS in Han:SPRD rats and control rats.
Results: We found 21 cations and 19 anions that accumulated significantly in the heterozygous (Cy/+) ADPKD rat model compared with control rats. Among the compounds, increases in 5-methyl-2'-deoxycytidine, glucosamine, ectoine, allantoate, α-hydroxybenzoate, phenaceturate and 3-phenylpropionate and decreases in 2-deoxycytidine, decanoate and 10-hydroxydecanoate were newly identified in the ADPKD Cy/+ rats.
Conclusion: We identified uremic retention solutes in ADPKD Cy/+ rats. Compounds related to ADPKD could be useful markers for detecting the early stage of ADPKD.

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Serum metabolomics reveals γ-glutamyl dipeptides as biomarkers for discrimination among different forms of liver disease

Soga, T., Sugimoto, M., Honma, M., Mori, M., Igarashi, K., Kashikura, K., Ikeda, S., Hirayama, A., Yamamoto, T., Yoshida, H., Otsuka, M., Tsuji, S., Yatomi, Y., Sakuragawa, T., Watanabe, H., Nihei, K., Saito, T., Kawata, S., Suzuki, H., Tomita, M., Suematsu, M.

J. Hepatol. 55 ( 4 ) 896 - 905 2011.10

Research paper (scientific journal), Joint Work, Accepted

Background & aims: We applied a metabolome profiling approach to serum samples obtained from patients with different liver diseases, to discover noninvasive and reliable biomarkers for rapid-screening diagnosis of liver diseases.
Methods: Using capillary electrophoresis and liquid chromatography mass spectrometry, we analyzed low molecular weight metabolites in a total of 248 serum samples obtained from patients with nine types of liver disease and healthy controls.
Results: We found that γ-glutamyl dipeptides, which were biosynthesized through a reaction with γ-glutamylcysteine synthetase, were indicative of the production of reduced glutathione, and that measurement of their levels could distinguish among different liver diseases. Multiple logistic regression models facilitated the discrimination between specific and other liver diseases and yielded high areas under receiver-operating characteristic curves. The area under the curve values in training and independent validation data were 0.952 and 0.967 in healthy controls, 0.817 and 0.849 in drug-induced liver injury, 0.754 and 0.763 in asymptomatic hepatitis B virus infection, 0.820 and 0.762 in chronic hepatitis B, 0.972 and 0.895 in hepatitis C with persistently normal alanine transaminase, 0.917 and 0.707 in chronic hepatitis C, 0.803 and 0.993 in cirrhosis type C, and 0.762 and 0.803 in hepatocellular carcinoma, respectively. Several γ-glutamyl dipeptides also manifested potential for differentiating between nonalcoholic steatohepatitis and simple steatosis.
Conclusions: γ-Glutamyl dipeptides are novel biomarkers for liver diseases, and varying levels of individual or groups of these peptides have the power to discriminate among different forms of hepatic disease.

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Hepatitis C Virus Infection Promotes Hepatic Gluconeogenesis through an NS5A-Mediated, FoxO1-Dependent Pathway

Deng, L., Shoji, I., Ogawa, W., Kaneda, S., Soga, T., Jiang, D., Ide, Y., Hotta, H.

J. Virol. 85 ( 17 ) 8556 - 8568 2011.09

Research paper (scientific journal), Joint Work, Accepted

Chronic hepatitis C virus (HCV) infection is often associated with type 2 diabetes. However, the precise mechanism underlying this association is still unclear. Here, using Huh-7.5 cells either harboring HCV-1b RNA replicons or infected with HCV-2a, we showed that HCV transcriptionally upregulated the genes for phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase), the rate-limiting enzymes for hepatic gluconeogenesis. In this way, HCV enhanced the cellular production of glucose 6-phosphate (G6P) and glucose. PEPCK and G6Pase gene expressions are controlled by the transcription factor forkhead box O1 (FoxO1). We observed that although neither the mRNA levels nor the protein levels of FoxO1 expression were affected by HCV, the level of phosphorylation of FoxO1 at Ser319 was markedly diminished in HCV-infected cells compared to the control cells, resulting in an increased nuclear accumulation of FoxO1, which is essential for sustaining its transcriptional activity. It was unlikely that the decreased level of FoxO1 phosphorylation was mediated through Akt inactivation, as we observed an increased phosphorylation of Akt at Ser473 in HCV-infected cells compared to control cells. By using specific inhibitors of c-Jun N-terminal kinase (JNK) and reactive oxygen species (ROS), we demonstrated that HCV infection induced JNK activation via increased mitochondrial ROS production, resulting in decreased FoxO1 phosphorylation, FoxO1 nuclear accumulation, and, eventually, increased glucose production. We also found that HCV NS5A mediated increased ROS production and JNK activation, which is directly linked with the FoxO1-dependent increased gluconeogenesis. Taken together, these observations suggest that HCV promotes hepatic gluconeogenesis through an NS5A-mediated, FoxO1-dependent pathway.

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Transcriptional regulation of Organic anion transporting polypeptide SLCO4C1 as a new therapeutic modality to prevent chronic kidney disease

Suzuki, T., Toyohara, T., Akiyama, Y., Takeuchi, Y., Mishima, E., Suzuki, C., Ito, S., Soga, T., Abe, T.

J. Pharm. Sci. 100 ( 9 ) 3696 - 3707 2011.09

Research paper (scientific journal), Joint Work, Accepted

Uremic toxins accumulate in patients with chronic kidney diseases (CKDs) and cause further progression of renal damage and cardiovascular diseases. Recently, it was reported that some of the organic anion transporting polypeptides (OATPs) and the organic anion transporters (OATs) are involved in the renal elimination of uremic toxins. SLCO4C1 is the only OATP expressed at the basolateral side of proximal tubular cells in human kidney, and it mediates the excretion of uremic toxins. The overexpression of human SLCO4C1 in rat kidney promotes the renal excretion of uremic toxins and reduces hypertension, cardiomegaly, and renal inflammation in renal failure. Statins induce SLCO4C1 expression thorough transcriptional factor Aryl hydrocarbon receptor through binding of the xenobiotic responsive element at its promoter region. The administration of statin in a rat renal failure model facilitated the elimination of uremic toxins and mitigated organ damage. In addition, metabolomic analysis of rat renal failure models and patients with CKD by capillary electrophoresis-mass spectrometry is a useful method for identifying new uremic solutes and explores surrogate biomarkers for detecting the progression of early stage CKD.

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Use of Serum and Urine Metabolome Analysis for the Detection of Metabolic Changes in Patients with Stage 1-2 Chronic Kidney Disease

Hayashi, K., Sasamura, H., Hishiki, T., Suematsu, M., Ikeda, S., Soga, T., Itoh, H.

Nephro-Urol. Mon. 3 ( 3 ) 164 - 171 2011.07

Research paper (scientific journal), Joint Work, Accepted

Background: Chronic kidney disease (CKD) is a major health problem throughout the world, and understanding the pathological condition of CKD has become increasingly important. The recent development of advanced metabolomic assay techniques now allows the human metabolic condition to be evaluated sensitively and comprehensively.
Objectives: The aim of this study was to use metabolomic analysis to perform a preliminary survey of metabolic changes occurring in patients with stage 1-2 CKD.
Patients and Methods: Serum and urine metabolomic profiles of 15 patients with stage 1-2 CKD were analyzed using our previously reported capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) systems, and compared to 7 healthy volunteers.
Results: The CE-TOFMS systems in three different modes for cation, anion, and nucleotide analyses detected multiple metabolites in serum and urine samples. In cation analysis mode, several increases in nonessential amino acids were identified in patients with stage 1-2 CKD, similar to those reported for end-stage renal disease (ESRD). Free-radical scavengers carnosine and hypotaurine were decreased in the urine, whereas serum hypotaurine and taurine were increased, consistent with changes in renal and/or systemic oxidative stress. Moreover, the cardiotoxin hypoxanthine was markedly increased in the serum, whereas serum and urine adenosine and urine guanine were decreased, suggesting changes in purine nucleotide metabolism which could affect cardiovascular prognosis. Changes in other unidentified metabolites were also detected.
Conclusions: These results suggest that multiple changes in the metabolism are already detectable in stage 1-2 CKD using metabolome analysis. Further studies on these metabolic changes may result in new strategies to prevent cardiovascular events and progression to ESRD in patients with CKD.

Autophagy Protects the Proximal Tubule from Degeneration and Acute Ischemic Injury

Kimura, T., Takabatake, Y., Takahashi, A., Kaimori, J., Matsui, I., Namba, T., Kitamura, H., Niimura, F., Matsusaka, T., Soga, T., Rakugi, H., Isaka, Y.

J. Am. Soc. Nephrol. 22 ( 5 ) 902 - 913 2011.05

Research paper (scientific journal), Joint Work, Accepted

Autophagy is a bulk protein degradation system that likely plays an important role in normal proximal tubule function and recovery from acute ischemic kidney injury. Using conditional Atg5 gene deletion to eliminate autophagy in the proximal tubule, we determined whether autophagy prevents accumulation of damaged proteins and organelles with aging and ischemic renal injury. Autophagy-deficient cells accumulated deformed mitochondria and cytoplasmic inclusions, leading to cellular hypertrophy and eventual degeneration not observed in wildtype controls. In autophagy-deficient mice, I/R injury increased proximal tubule cell apoptosis with accumulation of p62 and ubiquitin positive cytoplasmic inclusions. Compared with control animals, autophagy-deficient mice exhibited significantly greater elevations in serum urea nitrogen and creatinine. These data suggest that autophagy maintains proximal tubule cell homeostasis and protects against ischemic injury. Enhancing autophagy may provide a novel therapeutic approach to minimize acute kidney injury and slow CKD progression.

Positive Autoregulation Delays the Expression Phase of Mammalian Clock Gene Per2

Ogawa, Y., Koike, N., Kurisawa. G., Soga, T., Tomita, M., Tei, H.

PLoS One 6 ( 4 ) e18663 2011.04

Research paper (scientific journal), Joint Work, Accepted

In mammals, cellular circadian rhythms are generated by a transcriptional-translational autoregulatory network that consists of clock genes that encode transcriptional regulators. Of these clock genes, Period1 (Per1) and Period2 (Per2) are essential for sustainable circadian rhythmicity and photic entrainment. Intriguingly, Per1 and Per2 mRNAs exhibit circadian oscillations with a 4-hour phase difference, but they are similarly transactivated by CLOCK-BMAL1. In this study, we investigated the mechanism underlying the phase difference between Per1 and Per2 through a combination of mathematical simulations and molecular experiments. Mathematical analyses of a model for the mammalian circadian oscillator demonstrated that the slow synthesis and fast degradation of mRNA tend to advance the oscillation phase of mRNA expression. However, the phase difference between Per1 and Per2 was not reproduced by the model, which implemented a 1.1-fold difference in degradation rates and a 3-fold difference in CLOCK-BMAL1 mediated inductions of Per1 and Per2 as estimated in cultured mammalian cells. Thus, we hypothesized the existence of a novel transcriptional activation of Per2 by PER1/2 such that the Per2 oscillation phase was delayed. Indeed, only the Per2 promoter, but not Per1, was strongly induced by both PER1 and PER2 in the presence of CLOCK-BMAL1 in a luciferase reporter assay. Moreover, a 3-hour advance was observed in the transcriptional oscillation of the delta-Per2 reporter gene lacking cis-elements required for the induction by PER1/2. These results indicate that the Per2 positive feedback regulation is a significant factor responsible for generating the phase difference between Per1 and Per2 gene expression.

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Metabolomic Anatomy of an Animal Model Revealing Homeostatic Imbalances in Dyslipidaemia

Ooga, T., Sato, H., Nagashima, A., Sasaki, K., Tomita, M., Soga, T., Ohashi, Y

Mol. Biosyst 7 ( 4 ) 1217 - 1223 2011.04

Research paper (scientific journal), Joint Work, Accepted

Metabolomics is an emerging technology that reveals homeostatic imbalances in biological systems. Global determination of metabolite concentrations in body fluid and tissues provides novel anatomical aspects of pathological conditions that cannot be obtained from target-specific measurements. Here, we characterised metabolic imbalance in Watanabe heritable hyperlipidaemic rabbits as a model of hypercholesterolaemia. Using a mass spectrometry-based system, we measured a total of 335 metabolites in plasma and tissues (liver, aorta, cardiac muscle, and brain) from WHHL and healthy control rabbits. From the comparison between two metabolomic profiles, pathophysiological features including glutathione and phosphatidylcholine metabolism indicated the occurrence of oxidative stress in several tissues. Especially for the liver, imbalanced purine catabolism shed light on the transcriptional activation of xanthine oxidase, which is thought to act in absorbing or possibly triggering oxidative stress. We also applied this system to assess the therapeutic effects of simvastatin administration. After the treatment, a portion of the metabolomic features in pathological conditions showed alterations suggesting restoration of metabolism to the healthy condition. These changes were considered to be due to the pleiotropic action of statin, including antioxidant effects, rather than its main inhibitory action on cholesterol biosynthesis.

A novel mechanism regulates H2S and SO2 production in Saccharomyces cerevisiae

Yoshida, S., Imoto, J., Minato, T., Oouchi, R., Kamada, Y., Tomita, M., Soga, T., Yoshimoto, H.

Yeast 28 ( 2 ) 109 - 121 2011.02

Research paper (scientific journal), Joint Work, Accepted, ISSN 0749-503X

Sulfite (SO2) plays an important role in flavour stability in alcoholic beverages, whereas hydrogen sulfide (H2S) has an undesirable aroma. To discover the cellular processes that control SO2 and H2S production, we screened a library of Saccharomyces cerevisiae deletion mutants. Deletion of 12 genes led to increased H2S productivity. Ten of these genes are known to be involved in sulfur‐containing amino acid metabolism, whereas UBI4 functions in the ubiquitin‐proteasome system and SKP2 encodes an F‐box‐containing protein whose function is unknown. We found that the skp2 mutant accumulated H2S and SO2, because the adenosylphophosulfate kinase Met14p is a substrate of SCFSkp2 and more stable in the skp2 mutant than in the wild‐type strain. Furthermore, the skp2 mutant grew more slowly than the wild‐type strain under nutrient‐limited conditions. Metabolome analysis showed that the concentration of intracellular cysteine is lower in the skp2 mutant than in the wild‐type strain. The slow growth of the skp2 mutant was due to a lower concentration of intracellular cysteine, because the addition of cysteine suppressed the slow growth. In the skp2 mutant, the cysteine biosynthesis proteins Str2p, Str3p and Str4p are more stable than in the wild‐type strain. Moreover, supplementation with methionine, S‐adenosylmethionine, S‐adenosylhomocysteine and homocysteine also suppressed the slow growth. Overexpression of STR1 or STR4 caused a more severe defect in the skp2 mutant. These results suggest that the balance of methionine and cysteine biosynthesis is important for yeast cell growth. Thus, Skp2p is one of the key components regulating this balance and H2S/SO2 production. Copyright © 2010 John Wiley & Sons, Ltd.

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Metabolome Analysis Revealed Increase in S-Methylcysteine and Phosphatidylisopropanolamine Synthesis upon L-Cysteine Deprivation in the Anaerobic Protozoan Parasite Entamoeba histolytica

Husain, A., Sato, D., Jeelani, G., Mi-ichi F., Ali V., Suematsu, M., Soga, T., Nozaki, T.

J Biol Chem 285 ( 50 ) 39160 - 39170 2010.12

Research paper (scientific journal), Joint Work, Accepted, ISSN 0021-9258

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

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

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

Drug Metab Dispos 38 ( 10 ) 1665 - 1672 2010.10

Research paper (scientific journal), Joint Work, Accepted, ISSN 0090-9556

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

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

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

Chem. Biol. 17 ( 9 ) 989 - 998 2010.09

Research paper (scientific journal), Joint Work, Accepted

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

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Metabolomic profiling of uremic solutes in CKD patients

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

Hypertens. Res. 33 ( 9 ) 944 - 952 2010.09

Research paper (scientific journal), Joint Work, Accepted

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

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

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

J Biol Chem 285 ( 35 ) 26889 - 26899 2010.08

Research paper (scientific journal), Joint Work, Accepted, ISSN 0021-9258

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

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

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

Biotechnol Prog 26 ( 4 ) 975 - 992 2010.08

Research paper (scientific journal), Joint Work, Accepted, ISSN 8756-7938

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

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

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

J Agric Food Chem 58 ( 14 ) 8418 - 8425 2010.07

Research paper (scientific journal), Joint Work, Accepted, ISSN 0021-8561

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

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

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

J. Mass Spectro. 45 ( 7 ) 703 - 714 2010.07

Research paper (scientific journal), Joint Work, Accepted, ISSN 1076-5174

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

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