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Branched-chain amino acid (BCAA; valine, leucine and isoleucine) supplementation is often beneficial to energy expenditure; however, increased circulatinglevels of BCAA are linked to obesity and diabetes. The mechanisms of this paradox remain unclear. Here we report that, on cold exposure, brown adipose tissue (BAT) actively utilizes BCAA in the mitochondria for thermogenesis and promotes systemic BCAA clearance in mice and humans. In turn, a BAT-specific defect in BCAA catabolism attenuates systemic BCAA clearance, BAT fuel oxidation and thermogenesis, leading to diet-induced obesity and glucose intolerance. Mechanistically, active BCAA catabolism in BAT is mediated by SLC25A44, which transports BCAAs into mitochondria. Our results suggest that BAT serves as a key metabolic filter that controls BCAA clearance via SLC25A44, thereby contributing to the improvement of metabolic health.

Regulatory mechanisms of germline differentiation have generally been explained via the function of signaling pathways, transcription factors, and epigenetic regulation; however, little is known regarding proteomic and metabolomic regulation and their contribution to germ cell development. Here, we conducted integrated proteomic and metabolomic analyses of fetal germ cells in mice on embryonic day (E)13.5 and E18.5 and demonstrate sex- and developmental stage-dependent changes in these processes. In male germ cells, RNA processing, translation, oxidative phosphorylation, and nucleotide synthesis are dominant in E13.5 and then decline until E18.5, which corresponds to the prolonged cell division and more enhanced hyper-transcription/translation in male primordial germ cells and their subsequent repression. Tricarboxylic acid cycle and one-carbon pathway are consistently upregulated in fetal male germ cells, suggesting their involvement in epigenetic changes preceding in males. Increased protein stability and oxidative phosphorylation during female germ cell differentiation suggests an upregulation of aerobic energy metabolism, which likely contributes to the proteostasis required for oocyte maturation in subsequent stages. The features elucidated in this study shed light on the unrevealed mechanisms of germ cell development.

Benzoxazaborine derivatives were synthesized by the dehydration condensation reaction of 2-aminobenzyl alcohols with arylboronic acids. The insensitivity of the benzoxazaborines to hydrolysis allowed these compounds to be isolated by silica gel column chromatography. The single crystal X-ray structural analysis demonstrated the highly planar structure of the benzoxazaborine unit, and the B N bond length indicated an extended π-conjugated system.

Summary: Tolerance to severe tumor microenvironments, including hypoxia and nutrient starvation, is a common feature of aggressive cancer cells and can be targeted. However, metabolic alterations that support cancer cells upon nutrient starvation are not well understood. Here, by comprehensive metabolome analyses, we show that glutamine deprivation leads to phosphoethanolamine (PEtn) accumulation in cancer cells via the downregulation of PEtn cytidylyltransferase (PCYT2), a rate-limiting enzyme of phosphatidylethanolamine biosynthesis. PEtn accumulation correlated with tumor growth under nutrient starvation. PCYT2 suppression was partially mediated by downregulation of the transcription factor ELF3. Furthermore, PCYT2 overexpression reduced PEtn levels and tumor growth. In addition, PEtn accumulation and PCYT2 downregulation in human breast tumors correlated with poor prognosis. Thus, we show that glutamine deprivation leads to tumor progression by regulating PE biosynthesis via the ELF3-PCYT2 axis. Furthermore, manipulating glutamine-responsive genes could be a therapeutic approach to limit cancer progression. : Osawa et al. find that accumulation of phosphoethanolamine (PEtn) protects cancer cells under glutamine starvation through the downregulation of PCYT2. Glutamine regulates PE biosynthesis through PCYT2, resulting in pro-tumorigenic metabolite PEtn accumulation. PEtn stimulates the tolerance of cancer cells to starvation, and lowered PCYT2 expression correlates with decreased survival in patients. Keywords: cancer metabolism, tumor microenvironments, hypoxia, nutrient starvation, glutamine deprivation, amino acids, phosphoethanolamine, PCYT2, PE biosynthesis

Cancer cells alter their metabolism for the production of precursors of macromolecules. However, the control mechanisms underlying this reprogramming are poorly understood. Here we show that metabolic reprogramming of colorectal cancer is caused chiefly by aberrant MYC expression. Multiomics-based analyses of paired normal and tumor tissues from 275 patients with colorectal cancer revealed that metabolic alterations occur at the adenoma stage of carcinogenesis, in a manner not associated with specific gene mutations involved in colorectal carcinogenesis. MYC expression induced at least 215 metabolic reactions by changing the expression levels of 121 metabolic genes and 39 transporter genes. Further, MYC negatively regulated the expression of genes involved in mitochondrial biogenesis and maintenance but positively regulated genes involved in DNA and histone methylation. Knockdown of MYC in colorectal cancer cells reset the altered metabolism and suppressed cell growth. Moreover, inhibition of MYC target pyrimidine synthesis genes such as CAD, UMPS, and CTPS blocked cell growth, and thus are potential targets for colorectal cancer therapy.

Objectives Perioperative nutritional management is essential for early recovery after liver surgery. The aim of this study was to assess changes in amino acid levels in serum and urine after hepatectomy. Methods Serum samples were collected from 16 patients with hepatocellular carcinoma before and 1, 3, and 14 d after hepatectomy (S0, S1, S3, and S14, respectively). Spot urine samples were collected before and 3 d after the hepatectomy (U0 and U3). Metabolites in the serum and urine were analyzed. Results Compared with S0, insulin levels significantly increased in the S1 and S3 samples. Valine levels significantly decreased in S1 and S14, and leucine levels significantly decreased in S14. Phenylalanine levels significantly increased in S1 and S3, and tyrosine levels significantly increased in S1. The Fischer ratio (branched-chain/aromatic amino acids) significantly decreased in S1 and S3. In multiple regression analysis, changes in serum taurine levels were related to the white blood cell count in S1 and S3, and inversely related to alanine aminotransferase levels in S14. Changes in serum glutamine levels were negatively related to C-reactive protein levels in S3. Serum glutamine levels decreased in S3 and S14, and tended to increase in U3, suggesting a deficiency of glutamate resulting from the invasive surgical procedure. Conclusions These findings highlight the usefulness of metabolome analysis for characterizing perioperative patterns after liver resection. The observed amino acid pattern, including the reduction in Fischer ratio, underscores the need for specialized nutritional support.

Living bodies make use of endogenous electric energies for biological processes and can be affected by exogenous energies as well. Certain exogenous energies, such as the electric field of a polarized bioceramic surface, have been reported to have potency as part of an adjunct therapy for malignant tumors. Here, we investigated the effects of an electrostatic field on a leiomyosarcoma cell line cultured on polarized hydroxyapatite. We found that growth on the polarized surface suppressed cell proliferation, but the occurrence of apoptosis was low. In addition, our examination of cell cyclerelated genes revealed that the expression levels of two markers of myogenic differentiation were increased and tumor suppressor genes were up-regulated. These results indicated that inhibition of DNA replication led to the suppression of proliferation and acceleration of differentiation. Further studies on the electric fields produced around polarized bioceramic surfaces and their effects on cell cycle regulation might enable the design of an effective differentiation-inducing therapy for leiomyosarcoma and other cancers.

To clarify the characteristics of metabolite profiles in virus-related hepatocellular carcinoma (HCC) patients using serum metabolome analysis.The serum levels of low-molecular-weight metabolites in 68 patients with HCC were quantified using capillary electrophoresis chromatography and mass spectrometry. Thirty and 38 of the patients suffered from hepatitis B virus-related HCC (HCC-B) and hepatitis C virus-related HCC (HCC-C), respectively.The main metabolites characteristic of HCC were those associated with glutathione metabolism, notably 13 γ-glutamyl peptides, which are by-products of glutathione induction. Two major profiles, i.e., concentration patterns, of metabolites were identified in HCC patients, and these were classified into two groups: an HCC-B group and an HCC-C group including some of the HCC-B cases. The receiver operating characteristic curve for the multiple logistic regression model discriminating HCC-B from HCC-C incorporating the concentrations of glutamic acid, methionine and γ-glutamyl-glycine-glycine showed a highly significant area under the curve value of 0.94 (95%CI: 0.89-1.0, P0.0001).The serum levels of γ-glutamyl peptides, as well as their concentration patterns, contribute to the development of potential biomarkers for virus-related HCC. The difference in metabolite profiles between HCC-B and HCC-C may reflect the respective metabolic reactions that underlie the different pathogeneses of these two types of HCC.

Heme oxygenase (HO) is the rate-limiting enzyme that catalyzes the degradation of heme into iron, carbon monoxide, and biliverdin. This enzyme has important functions in cellular homeostasis, including the regulation of oxidative load, apoptosis, and inflammation. Two isoforms of HO, the inducible HO-1 and the constitutive HO-2, are expressed and are known to play a role in the normal human endometrium throughout the menstrual cycle, but there is little evidence for HO expression and behavior in adenomyosis, which is the occurrence of intramural ectopic endometrial tissue. The aim of this study was to investigate the presence and localization of the two HO isoforms in both eutopic and ectopic endometrium of women with adenomyosis during the menstrual cycle. The oxidative stress and apoptosis related to HO-1 expression were also assessed. The expression of HO-1 and HO-2 in both eutopic and ectopic endometrium was confirmed, and their levels in the ectopic endometrium were lower than those in the eutopic endometrium. The cyclic variability of HO expression was lost in the ectopic endometrium during the menstrual cycle, whereas this variability was apparent in the eutopic endometrium. Moreover, HO-1 expression corresponded to apoptotic events in the eutopic endometrium. Constitutive HO-2 expression corresponded to endometrial proliferation and degradation. These results reveal that both HO-1 and HO-2 contribute little in the pathophysiology of adenomyosis.

The effects of ionizing radiation (IR) on tumor neovascularization are still unclear. We previously reported that vascular endothelial cells (ECs) expressing the IR-induced senescence-like (IRSL) phenotype exhibit a significant decrease in angiogenic activity in vitro. In this study, we examined the effects of the IRSL phenotype on adhesion to early endothelial progenitor cells (early EPCs). Adhesion of human peripheral blood-derived early EPCs to human umbilical vein endothelial cells (HUVECs) expressing the IRSL phenotype was evaluated by an adhesion assay under static conditions. It was revealed that the IRSL HUVECs supported significantly more adhesion of early EPCs than normal HUVECs. Expressions of ICAM-1, VCAM-1 and E-selectin were up-regulated in IRSL HUVECs. Pre-treatment of IRSL HUVECs with adhesion-blocking monoclonal antibodies against E-selectin and VCAM-1 significantly reduced early EPC adhesion to IRSL HUVECs, suggesting a potential role for the E-selectin and VCAM-1 in the adhesion between IRSL ECs and early EPCs. Therefore, the IRSL phenotype expressed in ECs may enhance neovascularization via increased homing of early EPCs. Our findings are first to implicate the complex effects of this phenotype on tumor neovascularization following irradiation.

The feasibility of using a Compton camera for multitracer imaging has been demonstrated with the results of two biological sample imaging experiments. The distribution of the multitracer administered to a soybean sample and a tumor-bearing mouse has been visualized for each nuclide simultaneously. Three-dimensional images of the multitracer have been obtained even though the samples were measured from a fixed direction.

Iron deficiency is one of the major nutritional problems in developing countries. Food fortification is assumed to be an effective method for enhancing iron absorption. Sodium iron ethylenediaminetetraacetic acid (NaFeEDTA), a metal chelate, is one of the iron fortificants. It is a flavorless and inert compound. However, it is possible that EDTA may bind to metals other than iron and influence their absorption because of its strong chelating property. In this study, we determined the effect of NaFeEDTA on the absorption of various trace elements in the presence and absence of inhibitor using a multitracer technique. The multitracer is a carrier-free and salt-free solution containing a large number of radioactive nuclides. This method allows the simultaneous evaluation of behavior for a number of trace elements under strictly identical experimental conditions. As a result, incorporating NaFeEDTA into the rat diet did not lead to a deficiency in essential metals such as Zn, Mn, and Se in the presence of tannic acid. Furthermore, the uptake of various elements in the NaFeEDTA-fed rats is not inhibited by tannic acid. Thus, NaFeEDTA may be a suitable fortificant for improving iron-deficiency status in developing countries. Our findings in this study demonstrated that the multitracer technique provides us with many findings in nutritional research.

Heat shock induces a variety of biological events including gene activation, cell cycle arrest, and apoptosis. Heat shock has recently been shown to be potentially useful when combined with radiation in cancer therapy, probably because, in mammalian cells, heat inhibits the repair of double-strand breaks (DSBs) induced by ionizing radiation. It remains unclear, however, whether heat shock by itself induces DSBs. In this communication, we present the first evidence that heat shock induces the phosphorylated form of histone H2AX, which is thought to be generated at the chromatin proximal to DSB sites. These results suggest that heat shock induces DSBs in mammalian cells and may provide direct evidence to explain previous reports on DSB-related events occurring after heat shock treatment.

Summary 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., Graphical Abstract, Highlights • FH-deficient cells accumulate fumarate and urea cycle metabolites • Cytosolic FH is critical for renal cyst development in FH1-deficient mice • Restoration of cytosolic FH activity restores urea cycle defects • Arginine depletion reduces viability of FH1-deficient cells, In this study, Pollard and colleagues demonstrate the importance of cytosolic fumarate hydratase (FH) in renal cyst development in mice. Metabolite analysis of FH-deficient cysts and tumors identified alterations in urea cycle metabolism. re-expression of extramitochondrial FH in mice with renal-specific FH1 deletion was sufficient to ameliorate cyst development and metabolic alterations in the urea cycle. Significantly, FH-deficient cells exhibit increased sensitivity to arginine depletion. Exploitation of this vulnerability may be a potential route for therapy for FH-mutant tumors.

Objectives Serum samples from patients with chronic hepatitis C were subjected to metabolomics analysis to clarify the pretreatment characteristics of their metabolites and also changes in specific metabolites resulting from antiviral therapy with pegylated interferon plus ribavirin (PegIFN/RBV). Materials/Methods The serum levels of low-molecular-weight metabolites in the twenty patients before and 24 weeks after completion of PegIFN/RBV therapy were analyzed using capillary electrophoresis and liquid chromatography–mass spectrometry. Results Ten patients showed a non-virological response (NVR) and 10 achieved a sustained virological response (SVR) with eradication of viremia. The pretreatment levels of tryptophan were significantly higher in the patients of SVR than in those of NVR (p = 0.010). The area under the curve (AUC) value of tryptophan calculated from the receiver operating characteristic (ROC) curve for discriminating SVR from NVR was 0.84 (95% confidential interval, 0.66–1.02, p = 0.010). The ROC curve of multiple logistic regression model incorporating the pretreatment levels of tryptophan and γ-glutamate-arginine showed that the AUC value was highly significant (AUC = 0.92, 95% confidential interval, 0.79–1.05, p = 0.002). Twenty four weeks after completion of treatment, the levels of γ-glutamyl dipeptides, glutamic acid, 5-oxoproline, glucosamine and methionine sulfoxide were decreased, whereas those of 5-methoxy-3-indoleacetate, glutamine, kynurenine and lysine were increased significantly (p Conclusions The pretreatment serum levels of certain metabolites including tryptophan are associated with the response to PegIFN/RBV therapy. PegIFN/RBV therapy can ameliorate the oxidative stress responsible for glutathione metabolism.

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.

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.

Igarashi, K. and Miura, M. Inhibition of a Radiation-Induced Senescence-Like Phenotype: A Possible Mechanism for Potentially Lethal Damage Repair in Vascular Endothelial Cells. Radiat. Res. 170, 534–539 (2008). The well-established process of potentially lethal damage (PLD) repair enhances plateau-phase cell survival after exposure to ionizing radiation. PLD repair requires that confluent cells be incubated prior to plating for a colony-forming assay rather than being plated immediately. Enhanced double-strand break (DSB) repair during this incubation period has been implicated in the enhanced survival, but the precise molecular mechanism and its biological significance remain largely unclear. Radiation has been recently reported to induce premature senescence, and increasing evidence suggests that DSBs commonly mediate cellular senescence. Here we successfully related these two biological phenomena using bovine aortic endothelial cells, and propose that enhanced DSB repair during the plateau-pha...

e15568 Background: Recently, omics study such as metabolome analysis and whole genome sequencing has been applied to various disorders. We predicted the dysfunction of isocitrate dehydrogenase 1 (IDH1) in renal cell carcinoma (RCC) using metabolome analysis and identified a novel mutation of IDH1. In addition, we examined its function. Methods: We determined the global-scale metabolome profiling of human RCC by capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS), and compared the metabolite levels of tumors and paired normal tissues in 10 patients with RCC. We performed the genotyping of IDH1 and von Hippel-Lindau (VHL) gene. We also performed transfection experiments by using Lipofectamine and measured the enzymatic activity of IDH1 in RCC cell lines. All protocols were performed by the Institutional Review Board (IRB) of Hamamatsu University School of Medicine and Keio University. Informed consent was obtained from the patient in accordance with the IRB of Hamamatsu University School of Medicine. Results: The acceleration of glycolysis with low level of NADPH and citrate in one patient led to IDH1 dysfunction followed by identifying a novel missense mutation, V178I. In transfection experiment, the reductive activity of IDH1 variant (V178I) increased 2.5 times than that of IDH1 wild type while the oxidative activity of IDH1 variant oxidative activity was as well as that of IDH1 wild type. Furthermore, in the metabolome analysis using labeling glutamine, RCC cell lines deficient in the von Hippel-Lindau (VHL) protein preferentially use reductive glutamine metabolism. On the other hand, this patient with V178I mutation of IDH1 had no VHL mutation. Conclusions: Metabolome analysis led to the identification a novel IDH1 missense mutation in RCC. This mutation might be involved with acceleration of reductive reaction in tricarboxylic acid cycle. Metabolome analysis using CE-TOFMS could be a powerful tool for the global-scale exploration of disease-specific metabolism.

// Alessandro Valli 1,2,7 , Miguel Rodriguez 3,4 , Loukas Moutsianas 5 , Roman Fischer 2 , Vita Fedele 2 , Hong-Lei Huang 2 , Ruud Van Stiphout 1 , Dylan Jones 1 , Michael Mccarthy 2 , Maria Vinaxia 3,4 , Kaori Igarashi 6 , Maya Sato 6 , Tomoyoshi Soga 6 , Francesca Buffa 1 , James Mccullagh 7 , Oscar Yanes 3,4 , Adrian Harris 1,* and Benedikt Kessler 2,* 1 Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK 2 Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Oxford, UK 3 Centre for Omic Sciences, Rovira i Virgili University, Reus, Spain 4 Biomedical Research Centre in Diabetes and Associated Metabolic Disorders, Madrid, Spain 5 The Wellcome Trust Centre for Human Genetics, Nuffield Department of Medicine, Oxford, UK 6 Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, Japan 7 Mass Spectrometry Research Facility CRL, Department of Chemistry, University of Oxford, Oxford, UK * equal senior authors Correspondence: Alessandro Valli, email: // Keywords : cancer metabolism, fatty acids, HIF1α, HIF2α, hypoxia, Kennedy pathway, lipidomics, PAF Received : June 01, 2014 Accepted : December 10, 2014 Published : December 11, 2014 Abstract The biochemistry of cancer cells diverges significantly from normal cells as a result of a comprehensive reprogramming of metabolic pathways. A major factor influencing cancer metabolism is hypoxia, which is mediated by HIF1α and HIF2α. HIF1α represents one of the principal regulators of metabolism and energetic balance in cancer cells through its regulation of glycolysis, glycogen synthesis, Krebs cycle and the pentose phosphate shunt. However, less is known about the role of HIF1α in modulating lipid metabolism. Lipids serve cancer cells to provide molecules acting as oncogenic signals, energetic reserve, precursors for new membrane synthesis and to balance redox biological reactions. To study the role of HIF1α in these processes, we used HCT116 colorectal cancer cells expressing endogenous HIF1α and cells in which the hif1α gene was deleted to characterize HIF1α-dependent and independent effects on hypoxia regulated lipid metabolites. Untargeted metabolomics integrated with proteomics revealed that hypoxia induced many changes in lipids metabolites. Enzymatic steps in fatty acid synthesis and the Kennedy pathway were modified in a HIF1α-dependent fashion. Palmitate, stearate, PLD3 and PAFC16 were regulated in a HIF-independent manner. Our results demonstrate the impact of hypoxia on lipid metabolites, of which a distinct subset is regulated by HIF1α.