Intracellular Metabolite Levels Research Articles

Suppression of brain cholesterol synthesis in male Mecp2-deficient mice is age dependent and not accompanied by a concurrent change in the rate of fatty acid synthesis

Mutations in the X-linked gene methyl-CpG-binding protein 2 (MECP2) are the principal cause of Rett syndrome, a progressive neurodevelopmental disorder afflicting 1 in 10,000 to 15,000 females. Studies using hemizygous Mecp2 mouse models have revealed disruptions to some aspects of their lipid metabolism including a partial suppression of cholesterol synthesis in the brains of mature Mecp2 mutants. The present studies investigated whether this suppression is evident from early neonatal life, or becomes manifest at a later stage of development. We measured the rate of cholesterol synthesis, in vivo, in the brains of male Mecp2−/y and their Mecp2+/y littermates at 7, 14, 21, 28, 42 and 56 days of age. Brain weight was consistently lower in the Mecp2−/y mice than in their Mecp2+/y controls except at 7 days of age. In the 7- and 14-day-old mice there was no genotypic difference in the rate of brain cholesterol synthesis but, from 21 days and later, it was always marginally lower in the Mecp2−/y mice than in age-matched Mecp2+/y littermates. At no age was a genotypic difference detected in either the rate of fatty acid synthesis or cholesterol concentration in the brain. Cholesterol synthesis rates in the liver and lungs of 56-day-old Mecp2−/y mice were normal. The onset of lower rates of brain cholesterol synthesis at about the time closure of the blood brain barrier purportedly occurs might signify a disruption to mechanism(s) that dictate intracellular levels of cholesterol metabolites including oxysterols known to exert a regulatory influence on the cholesterol biosynthetic pathway.

Src Family Kinases and p38 Mitogen-Activated Protein Kinases Regulate Pluripotent Cell Differentiation in Culture.

Multiple pluripotent cell populations, which together comprise the pluripotent cell lineage, have been identified. The mechanisms that control the progression between these populations are still poorly understood. The formation of early primitive ectoderm-like (EPL) cells from mouse embryonic stem (mES) cells provides a model to understand how one such transition is regulated. EPL cells form from mES cells in response to l-proline uptake through the transporter Slc38a2. Using inhibitors of cell signaling we have shown that Src family kinases, p38 MAPK, ERK1/2 and GSK3β are required for the transition between mES and EPL cells. ERK1/2, c-Src and GSK3β are likely to be enforcing a receptive, primed state in mES cells, while Src family kinases and p38 MAPK are involved in the establishment of EPL cells. Inhibition of these pathways prevented the acquisition of most, but not all, features of EPL cells, suggesting that other pathways are required. L-proline activation of differentiation is mediated through metabolism and changes to intracellular metabolite levels, specifically reactive oxygen species. The implication of multiple signaling pathways in the process suggests a model in which the context of Src family kinase activation determines the outcomes of pluripotent cell differentiation.

Usefulness of mean corpuscular volume as a surrogate marker for monitoring thiopurine treatment in inflammatory bowel disease.

Thiopurines are commonly used drugs in inflammatory bowel disease. Intracellular levels of thiopurine metabolites [i.e. 6-thioguaninenucleotides (6-TGN)] are associated with efficacy and toxicity. Because 6-TGN measurement is not globally available, the mean corpuscular volume (MCV) has been proposed as a surrogate marker for monitoring thiopurine therapy. To analyze the relationship between MCV and efficacy of thiopurines, defined as either response to therapy or 6-TGN levels. A systematic search on PubMed was performed. Fifteen studies were included. In six studies, a positive association was found between ΔMCV and 6-TGN. In four studies, it was suggested that ΔMCV can be used to predict clinical remission. In five articles, no association was found. In the majority of articles, it was reported that ΔMCV is useful in guiding intracellular metabolite levels. However, there is insufficient evidence showing that ΔMCV can predict clinical remission.

Fast Filtration of Bacterial or Mammalian Suspension Cell Cultures for Optimal Metabolomics Results.

The metabolome offers real time detection of the adaptive, multi-parametric response of the organisms to environmental changes, pathophysiological stimuli or genetic modifications and thus rationalizes the optimization of cell cultures in bioprocessing. In bioprocessing the measurement of physiological intracellular metabolite levels is imperative for successful applications. However, a sampling method applicable to all cell types with little to no validation effort which simultaneously offers high recovery rates, high metabolite coverage and sufficient removal of extracellular contaminations is still missing. Here, quenching, centrifugation and fast filtration were compared and fast filtration in combination with a stabilizing washing solution was identified as the most promising sampling method. Different influencing factors such as filter type, vacuum pressure, washing solutions were comprehensively tested. The improved fast filtration method (MxP® FastQuench) followed by routine lipid/polar extraction delivers a broad metabolite coverage and recovery reflecting well physiological intracellular metabolite levels for different cell types, such as bacteria (Escherichia coli) as well as mammalian cells chinese hamster ovary (CHO) and mouse myeloma cells (NS0).The proposed MxP® FastQuench allows sampling, i.e. separation of cells from medium with washing and quenching, in less than 30 seconds and is robustly designed to be applicable to all cell types. The washing solution contains the carbon source respectively the 13C-labeled carbon source to avoid nutritional stress during sampling. This method is also compatible with automation which would further reduce sampling times and the variability of metabolite profiling data.

Abstract B49: Bioluminescent assays for measuring glycolytic rate and glucose homeostasis

Abstract We describe a set of bioluminescence assays for systematic evaluation of metabolic alterations in response to different growth conditions or treatments. These newly developed cell-based bioluminescence assays offer new methods to detect different metabolites such as NAD(P)/NAD(P)H, lactate, glutamate, glucose-6-phosphate, glucose and 2-deoxyglucose-6-phosphate (for glucose uptake). These assays address the need for more rapid, sensitive and easier to use approaches to quantify key metabolites in cell lysates and tissues. Using the glucose uptake assay, compounds altering translocation of glucose transporters can be screened and characterized rapidly. The lactate detection assay offers a sensitive and rapid approach for glycolytic rate measurements. The glucose detection assay provides information on glucose consumption rates. When these glucose consumption rates are correlated with lactate secretion, the results serve as an indicator of the metabolic status of the cells (e.g. a shift from oxidative phosphorylation to glycolysis). The glucose assay also provides a convenient and robust approach for studying activation or inhibition of gluconeogenesis or glycogenolysis. These bioluminescent assays are robust and sensitive with assay windows significantly larger than analogous fluorescent or colorimetric methods. The sensitivity (1-5pmol/sample) and wide dynamic range (maximum signal-to-background > 100 fold) of the assays allow simultaneous detection of multiple metabolites from the same set of samples. Most importantly, the assays incorporate rapid inactivation of endogenous enzymes, which overcomes the limitations of the deprotonization step required by other methods. The improved workflow in combination with the high sensitivity of luminescence enables measurement of intracellular metabolite levels by simply adding detection reagents directly to treated cells. Alternatively, small samples (2-5ul) can be removed from the media and used for multiple metabolite (lactate, glucose, glutamate) measurements. By consolidating dose- and time-dependent measurements, these assays conserve cells and reagents, streamline workflow, and produce internally controlled data sets. The assays can be used in various formats (e.g. 96- and 384-well plates), applied to various sample types (e.g. mammalian cells, tissues, and 3D microtissues), and multiplexed with other assays, including our new real time cell viability assay. The bioluminescent real time cell viability assay is a homogeneous, non-lytic method to measure cell viability in real time. It overcomes the limitations of standard end-point lytic assays by providing a comprehensive representation of changes in cell viability occurring in live cells over the entire length of an experiment. Changes in cell viability can be conveniently correlated with changes in metabolite levels. Citation Format: Jolanta Vidugiriene, Donna Leippe, Mary Sobol, Mike Valley, Natasha Karassina, Sarah Duellman, James Cali. Bioluminescent assays for measuring glycolytic rate and glucose homeostasis. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B49.

NT5C2 As a Major Contributor to Thiopurine Resistance at ALL Relapse Via Multiple Mechanisms

Background Relapse remains as a formidable challenge for acute lymphoblastic leukemia (ALL), particularly because it is associated with dramatic drug resistance and thus dismal prognosis. It is crucial to understand the genetic basis of relapsed ALL to develop new therapeutic strategies. Recently, somatic mutations in NT5C2 were identified as one of the most common genomic lesions specific to relapsed ALL and were directly linked to thiopurine resistance (Nat Genet 2013, Nat Med 2013, Nat Commun 2015). A nucleotidase, NT5C2 dephosphorylates purine monophosphate nucleotides and is important for cellular nucleic acid homeostasis. However, the exact mechanisms by which NT5C2 mutations influence thiopurine resistance in ALL are not completely understood.Methods Wildtype and mutant human NT5C2 (p.R39Q, p.R238W, and p.R367Q) were expressed in E. Coli and purified by affinity chromatography followed by gel filtration. Nucleotidase activity was measured by quantifying the release of free phosphate with the Malachite Green assay. NT5C2 enzyme kinetics (Vmax and Km) were determined for endogenous purine nucleotide substrates (inosine monophosphate [IMP] and guanosine monophosphate [GMP]) and thiopurine drug metabolite substrates (thioinosine monophosphate [TIMP] and thioguanosine monophosphate [TGMP]). Human ALL cell lines (Nalm6 and REH) stably overexpressing wildtype or mutant NT5C2 were established by lentiviral transduction. Thiopurine transport was characterized in vitro by quantifying the influx and efflux of 14 C labeled mercaptopurine (MP) and its metabolites.Results To characterize NT5C2 substrate specificity, we first determined the nucleotidase activity of wildtype and mutant proteins with endogenous purine nucleotides (IMP and GMP) and thiopurine metabolites (TIMP and TGMP). Wildtype NT5C2 exhibited nucleotidase activity against all 4 nucleotide metabolites, but IMP and TIMP were clearly the preferred substrates with up to 4-fold higher reaction velocities compared to GMP and TGMP, respectively. All leukemia-derived NT5C2 mutations resulted in substantial increase in nucleotidase activity across substrates, with the p.R367Q mutant consistently exhibiting the greatest gain of activity. By comparison, mutant NT5C2 proteins showed similar substrate preference of IMP over GMP (e.g., reaction rate of 26.6 pmol/min and 10.4 pmol/min for IMP and GMP, respectively, with the p.R367Q NT5C2). However, this inosine over guanosine bias in substrate specificity was reversed with thiopurine metabolites: leukemia-derived mutant NT5C2s were substantially more efficient to dephosphorylate TGMP than TIMP (e.g., reaction rate of 27.8 pmol/min and 41.5 pmol/min for TIMP and TGMP, respectively, with the p.R367Q NT5C2). The "mutant to wildtype" ratio of NT5C2 nucleotidase activity (e.g., the relative increase of Vmax) was significantly greater for thiopurine metabolites than what was observed when endogenous purine nucleotides were used as substrates (e.g., 15.2- and 1.6-fold increase in Vmax for TIMP and IMP with the p.R367Q NT5C2). These results suggest that ALL relapse-associated NT5C2 mutations likely confer not only increase in endogenous nucleotidase activity but also novel enzymatic functions that specially affect thiopurine metabolism.In addition to the increase in thiopurine resistance, human ALL cell lines expressing mutant NT5C2 also showed significant reduction in intracellular levels of thiopurine metabolites after drug treatment, pointing to influence of NT5C2 mutations on thiopurine drug transport. Using 14 C labeled MP, we traced both the influx and efflux of drug metabolites in isogenic cell lines expressing wildtype or mutant NT5C2 s. In both Nalm6 and REH cells, intracellular uptake of MP was reduced by 51% in mutant cells compared to cells with wildtype protein. In contrast, the level of MP metabolites in the extracellular medium was 3 times higher with mutant cells, reflecting increased drug efflux. Therefore, expression of mutant NT5C2 resulted in a net reduction of intracellular exposure to thiopurine, suggesting disturbance of the drug transport pathway as a distinct mechanism for NT5C2-mediated thiopurine resistance.ConclusionsNT5C2 mutations specifically influence thiopurine metabolism and transport, and therefore are critical determinants for drug resistance in relapsed ALL. DisclosuresNo relevant conflicts of interest to declare.

Metabolomic, Proteomic and Genomic Profiling Identifies Biomarakers of Sensitivity to Glutaminase Inhibitor CB-839 in Multiple Myeloma

IntroductionGlutaminase is a mitochondrial enzyme that converts glutamine to glutamate to support several metabolic processes including amino acid and nucleotide synthesis, maintenance of cellular redox homeostasis, and the replacement of TCA cycle intermediates. Selective glutaminase inhibitors BPTES and CB-839 have anti-proliferative activity in several pre-clinical cancer models including breast, pancreatic, lung, renal, brain, leukemia, and lymphoma. Across a panel of twenty-nine multiple myeloma cell lines, we found that glutaminase inhibition with CB-839 caused tumor cell death or growth inhibition in only a subset of cell lines. To identify biomarkers that predict sensitivity to CB-839 in multiple myeloma cells, we profiled cellular metabolites, mRNA transcripts, and signaling pathways in eight multiple myeloma cell (four CB-839-sensitive and four CB-839-resistant).ResultsProteomic analysis showed that CB-839 treatment suppressed the activity of the amino-acid sensing kinase mTORC1 in CB-839-sensitive cells, leading to down regulation of protein synthesis and expression of metabolic genes. Analysis of steady-state levels of intra-cellular metabolites revealed that CB-839-sensitive cells had more profound decreases in nucleotide levels and less pronounced increases in essential amino acids upon CB-839 treatment compared to CB-839-resistant cells. This suggests that the metabolic response to glutaminase inhibition is fundamentally different in sensitive versus resistant multiple myeloma cell lines. Consistent with the in vitro data, in a xenograft model with the CB-839-sensitive cell line RPMI8226, CB-839 treatment produced a 71% reduction in tumor growth that was associated with reduced levels of intratumoral nucleotides and no changes in the levels of essential amino acids. We next explored protein biomarkers that predict resistance to CB-839 and found that pyruvate carboxylase (PC) expression strongly correlated with resistance. siRNA-mediated knockdown of PC reduced TCA cycle activity and sensitized cells to CB-839 treatment, suggesting that PC can rescue cells from glutaminase inhibition by supporting anapleurotic utilization of glucose. This hypothesis was further substantiated by the observation that treatment of CB-839-resistant cells with the AKT inhibitor MK2206 led to a decrease in glucose utilization, and when combined with CB-839, produced a significant decrease in TCA cycle activity and a profound synergistic anti-proliferative response.ConclusionMultiple myeloma cells show varying anti-proliferative responses to glutaminase inhibition by CB-839. CB-839 treatment inhibits mTORC1 pathway signaling and causes decreases in nucleotides in sensitive multiple myeloma cells. Multiple myeloma cells that are resistant to glutaminase inhibition have high expression of PC, which may allow these cells to utilize glucose instead of glutamine to resupply TCA cycle intermediates. Knockdown of PC or treatment with an AKT inhibitor causes cells to utilize less glucose and sensitizes resistant cells to glutaminase inhibition with CB-839. CB-839 is currently being evaluated in Phase 1 clinical trials for the treatment of various solid and hematological cancers including multiple myeloma. We are exploring the utility of PC and mTORC1 pathway signaling biomarkers to identify multiple myeloma patients that may respond to CB-839 treatment. DisclosuresMacKinnon:Calithera Biosciences: Employment, Equity Ownership. Bennett:Calithera Biosciences: Employment, Equity Ownership. Gross:Calithera Biosciences: Employment, Equity Ownership. Janes:Calithera Biosciences: Employment, Equity Ownership. Li:Calithera Biosciences: Employment, Equity Ownership. Rodriquez:Calithera Biosciences: Employment, Equity Ownership. Wang:Calithera Biosciences: Employment, Equity Ownership. Zhang:Calithera Biosciences: Employment, Equity Ownership. Parlati:Calithera Biosciences: Employment, Equity Ownership.

Long Noncoding RNA Ceruloplasmin Promotes Cancer Growth by Altering Glycolysis.

Long noncoding RNAs (lncRNAs) significantly influence the development and regulation of genome expression in cells. Here, we demonstrate the role of lncRNA ceruloplasmin (NRCP) in cancer metabolism and elucidate functional effects leading to increased tumor progression. NRCP was highly upregulated in ovarian tumors, and knockdown of NRCP resulted in significantly increased apoptosis, decreased cell proliferation, and decreased glycolysis compared with control cancer cells. In an orthotopic mouse model of ovarian cancer, siNRCP delivered via a liposomal carrier significantly reduced tumor growth compared with control treatment. We identified NRCP as an intermediate binding partner between STAT1 and RNA polymerase II, leading to increased expression of downstream target genes such as glucose-6-phosphate isomerase. Collectively, we report a previously unrecognized role of the lncRNA NRCP in modulating cancer metabolism. As demonstrated, DOPC nanoparticle-incorporated siRNA-mediated silencing of this lncRNA in vivo provides therapeutic avenue toward modulating lncRNAs in cancer.

Comprehensive metabolomic analyses of anode-respiring Geobacter sulfurreducens cells: The impact of anode-respiration activity on intracellular metabolite levels

Comprehensive metabolomic analysis of anode-respiring Geobacter sulfurreducens cells revealed that intracellular levels of metabolites related to the tricarboxylic acid (TCA) cycle, gluconeogenesis, consumptions of nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH), and adenosine triphosphate (ATP) generation correlated well with the activity of microbial anode-respiration detected as microbial electric current. Use of a glassy carbon electrode as the anode material in a three-electrode system resulted in a higher microbial current at +0.2V (vs. Ag/AgCl) than at −0.2V. A larger current flow resulted in higher concentrations of TCA cycle metabolites and lower concentrations of gluconeogenesis metabolites. Metabolomic analysis also revealed that higher anode-respiration activity resulted in a higher ATP/adenosine diphosphate (ADP) ratio and lower ratios of NADH/NAD+ and NADPH/NADP+. These findings provide direct experimental evidence that microbial anode-respiration activity controlled by the anode potential influences both the flux of central metabolic pathways and the redox balance in microbial fuel cells.

Nitrate and amino acid availability affects glycine betaine and mycosporine-2-glycine in response to changes of salinity in a halotolerant cyanobacteriumAphanothece halophytica

A halotolerant cyanobacterium Aphanothece halophytica thrives in extreme salinity with accumulation of a potent osmoprotectant glycine betaine. Recently, this cyanobacterium was shown to accumulate sunscreen molecule mycosporine-2-glycine significantly at high salinity. In this study, we investigated effects of nitrate and amino acid provision on the accumulation of glycine betaine and mycosporine-2-glycine. With elevated nitrate concentrations at high salinity, intracellular levels of both metabolites were enhanced. Six-fold high nitrate concentration increased the relative amounts of glycine betaine and mycosporine-2-glycine to be 1.5 and 2.0 folds compared with control condition : Increased levels were time- and dose-dependent manner. Exogenous supply of glycine/serine at high salinity resulted in the similar trends as observed in excess nitrate experiment. Intracellular level of glycine betaine increased ∼1.6 folds with glycine/serine supplementation. These supplementations also caused the increased level of mycosporine-2-glycine, namely 1.4 and 2 folds by glycine and serine, respectively. The transcription of glycine betaine and mycosporine-2-glycine biosynthetic genes was strongly induced under high-nitrate-salt condition. These results suggest the dependence of glycine betaine and mycosporine-2-glycine productions on substrate availability, and the effect of nitrate was possibly associated with stimulation of osmoprotectant increment in this extremophile.

Bypasses in intracellular glucose metabolism in iron-limited Pseudomonas putida.

Decreased biomass growth in iron (Fe)-limited Pseudomonas is generally attributed to downregulated expression of Fe-requiring proteins accompanied by an increase in siderophore biosynthesis. Here, we applied a stable isotope-assisted metabolomics approach to explore the underlying carbon metabolism in glucose-grown Pseudomonas putida KT2440. Compared to Fe-replete cells, Fe-limited cells exhibited a sixfold reduction in growth rate but the glucose uptake rate was only halved, implying an imbalance between glucose uptake and biomass growth. This imbalance could not be explained by carbon loss via siderophore production, which accounted for only 10% of the carbon-equivalent glucose uptake. In lieu of the classic glycolytic pathway, the Entner-Doudoroff (ED) pathway in Pseudomonas is the principal route for glucose catabolism following glucose oxidation to gluconate. Remarkably, gluconate secretion represented 44% of the glucose uptake in Fe-limited cells but only 2% in Fe-replete cells. Metabolic (13) C flux analysis and intracellular metabolite levels under Fe limitation indicated a decrease in carbon fluxes through the ED pathway and through Fe-containing metabolic enzymes. The secreted siderophore was found to promote dissolution of Fe-bearing minerals to a greater extent than the high extracellular gluconate. In sum, bypasses in the Fe-limited glucose metabolism were achieved to promote Fe availability via siderophore secretion and to reroute excess carbon influx via enhanced gluconate secretion.

Growth-rate dependency of de novo resveratrol production in chemostat cultures of an engineered Saccharomyces cerevisiae strain.

IntroductionSaccharomyces cerevisiae has become a popular host for production of non-native compounds. The metabolic pathways involved generally require a net input of energy. To maximize the ATP yield on sugar in S. cerevisiae, industrial cultivation is typically performed in aerobic, sugar-limited fed-batch reactors which, due to constraints in oxygen transfer and cooling capacities, have to be operated at low specific growth rates. Because intracellular levels of key metabolites are growth-rate dependent, slow growth can significantly affect biomass-specific productivity. Using an engineered Saccharomyces cerevisiae strain expressing a heterologous pathway for resveratrol production as a model energy-requiring product, the impact of specific growth rate on yeast physiology and productivity was investigated in aerobic, glucose-limited chemostat cultures.ResultsStoichiometric analysis revealed that de novo resveratrol production from glucose requires 13 moles of ATP per mole of produced resveratrol. The biomass-specific production rate of resveratrol showed a strong positive correlation with the specific growth rate. At low growth rates a substantial fraction of the carbon source was invested in cellular maintenance-energy requirements (e.g. 27 % at 0.03 h−1). This distribution of resources was unaffected by resveratrol production. Formation of the by-products coumaric, phloretic and cinnamic acid had no detectable effect on maintenance energy requirement and yeast physiology in chemostat. Expression of the heterologous pathway led to marked differences in transcript levels in the resveratrol-producing strain, including increased expression levels of genes involved in pathways for precursor supply (e.g. ARO7 and ARO9 involved in phenylalanine biosynthesis). The observed strong differential expression of many glucose-responsive genes in the resveratrol producer as compared to a congenic reference strain could be explained from higher residual glucose concentrations and higher relative growth rates in cultures of the resveratrol producer.ConclusionsDe novo resveratrol production by engineered S. cerevisiae is an energy demanding process. Resveratrol production by an engineered strain exhibited a strong correlation with specific growth rate. Since industrial production in fed-batch reactors typically involves low specific growth rates, this study emphasizes the need for uncoupling growth and product formation via energy-requiring pathways.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0321-6) contains supplementary material, which is available to authorized users.

Abstract 4450: CB839, an orally bioavailable glutaminase inhibitor, shows potent antitumor activity in vitro against models of soft tissue sarcoma and chondrosarcoma

Abstract CB839 (Glutaminase inhibitor) is a potent, orally bioavailable and selective inhibitor of the first enzyme in glutamine utilization in Krebs tricarboxylic acid (TCA) cycle. CB839 has been shown to have significant anti proliferative activity in triple negative breast cancer cell lines in vitro as well as in mouse xenograft models. We tested the in vitro anti proliferative activity of CB839 in chondrosarcoma cell lines carrying a mutation in isocitrate dehydrogenase (IDH), a key enzyme in TCA cycle. These cell lines included JJ012 (IDH1 mutant) and SW1353 (IDH2 mutant). CH2879 cell line carrying wild-type IDH was used as a control. In addition to IDH mutant chondrosarcoma cell lines, we tested in vitro efficacy of CB839 in a panel of soft tissue sarcoma cell lines including malignant peripheral nerve sheath tumor (MPNST), Ewing sarcoma (CHP100) and Liposarcoma (LS141). Results from our in vitro proliferation assay showed that compared to wild-type IDH cell line CH2879, mutant IDH cell lines JJ012 and SW1353 were more sensitive to inhibition of proliferation by CB839. Among the soft tissue sarcoma cell lines tested, MPNST and ST8814, which carry an NF1 mutation (neurofibromatosis 1) or NF1 loss respectively, showed greater sensitivity to inhibition. NF1 negatively regulates Ras activity and NF1 mutation or loss in these cells is known to up regulate Ras/MAP kinase pathway. The extent of sensitivity observed in soft-tissue and chondrosarcoma cell lines correlated with their extracellular glutamine dependency. This was confirmed by cell proliferation assay in glutamine containing versus glutamine depleted media. Target inhibition by CB839 was confirmed by gas chromatography mass spectrometry (GC/MS) analysis, which showed reduced levels of intracellular intermediary metabolites including Glutamate, α-ketoglutarate, succinate, fumarate and 2-hydroxy-glutarate. Previous reports have suggested that α-ketoglutarate is a rate limiting factor in the activation of mTORC1. To explore whether CB839 affects mTORC1 signaling, we carried out western blot analysis to determine the phosphorylation levels of S6 (pS6), a direct substrate of mTORC1 activity. We observed down regulation of pS6 by CB839 only in IDH1 mutant cell line, JJ012, but not in wild-type IDH cell line, CH2879. This data strongly supports cross-talk between glutamine metabolism and mTORC activity in IDH mutant cell line. Some reports have suggested that under conditions of glutamine deprivation, cells use pyruvate carboxylase for anapleurosis. Treatment of chondrosarcoma cell lines by CB839 showed dose dependent suppression of pyruvate carboxylase only in IDH mutant but not wild type cell line. Taken together, our data strongly supports further pre-clinical research in order to explore the role played by IDH and other mutations such as NF1 in determining sensitivity to Glutaminase inhibition by CB839. Citation Format: Tahir N. Sheikh, Parag P. Patwardhan, Serge Cremers, Gary K. Schwartz. CB839, an orally bioavailable glutaminase inhibitor, shows potent antitumor activity in vitro against models of soft tissue sarcoma and chondrosarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4450. doi:10.1158/1538-7445.AM2015-4450

Amino Acids: Sensing and Implication into Aging

An ability to sense and respond to nutrient availability is an important requisite for life.Nutrient limitation is among main factors to influence the evolution of most cellular processes.Different pathways that sense intracellular and extracellular levels of carbohydtrates, amino acids,lipids, and intermediate metabolites are integrated and coordinated at the organismal levelthrough neuronal and humoral signals. During food abundance, nutrient-sensing pathwaysengage anabolism and storage, whereas limitation triggers the mechanisms, such as themobilization of internal stores including through autophagy. These processes are affected duringaging and are themselves important regulators of longevity, stress resistance, and age-relatedcomplications

Lysine-specific demethylase 2 suppresses lipid influx and metabolism in hepatic cells.

Cells link environmental fluctuations, such as nutrition, to metabolic remodeling. Epigenetic factors are thought to be involved in such cellular processes, but the molecular basis remains unclear. Here we report that the lysine-specific demethylase 2 (LSD2) suppresses the flux and metabolism of lipids to maintain the energy balance in hepatic cells. Using transcriptome and chromatin immunoprecipitation-sequencing analyses, we revealed that LSD2 represses the genes involved in lipid influx and metabolism through demethylation of histone H3K4. Selective recruitment of LSD2 at lipid metabolism gene loci was mediated in part by a stress-responsive transcription factor, c-Jun. Intriguingly, LSD2 depletion increased the intracellular levels of many lipid metabolites, which was accompanied by an increased susceptibility to toxic cell damage in response to fatty acid exposure. Our data demonstrate that LSD2 maintains metabolic plasticity under fluctuating environment in hepatocytes by mediating the cross talk between the epigenome and metabolism.

CarbonQuest: Unfolding the Map of Seed Metabolism

In view of our rapid growing demands on vegetable oil, enhancing fatty acid synthesis in maize (Zea mays) represents tremendous nutritional and economic benefits. In corn kernels, the endosperm and the embryo are the main site for synthesis and accumulation of starch and oil, respectively. So far, breeding efforts to achieve elevated oil content in maize resulted in smaller endosperm and therefore lower yield. We hypothesize that the key to increasing oil content in maize kernels without affecting the yield lies on directly changing their carbon metabolism. To test our hypothesis, we compared the intracellular metabolite levels in maize embryos from two different maize lines, Alex synthetic (Alex) and LH59, which accumulate 48 and 34% of oil, respectively. The comparative metabolomics highlighted the metabolites and pathways that were active in maize embryos and important for oil production. The contribution of each pathway to fatty acid synthesis in terms of carbon, reductant and energy provision was assessed by measuring the carbon flow through the metabolic network (13C‐Metabolic Flux Analysis) in developing Alex and LH59 embryos to build maps of carbon flow through central metabolism. Our approach combined mathematical modeling with biochemical quantification to identify metabolic bottlenecks in fatty acid synthesis in maize embryos. Transgenic maize plants that we generated to overexpress a lead gene increased embryo fatty acid content by 35% without affecting the size of the kernel. This study hence describes the combination of innovative tools that will pave the way for controlling seed composition in important food crops.

Substrate-dependent activation of the Vibrio cholerae vexAB RND efflux system requires vexR.

Vibrio cholerae encodes six resistance-nodulation-division (RND) efflux systems which function in antimicrobial resistance, virulence factor production, and intestinal colonization. Among the six RND efflux systems, VexAB exhibited broad substrate specificity and played a predominant role in intrinsic antimicrobial resistance. The VexAB system was encoded in an apparent three gene operon that included vexR; which encodes an uncharacterized TetR family regulator. In this work we examined the role of vexR in vexRAB expression. We found that VexR bound to the vexRAB promoter and vexR deletion resulted in decreased vexRAB expression and increased susceptibility to VexAB antimicrobial substrates. Substrate-dependent induction of vexRAB was dependent on vexR and episomal vexR expression provided a growth advantage in the presence of the VexAB substrate deoxycholate. The expression of vexRAB increased, in a vexR-dependent manner, in response to the loss of RND efflux activity. This suggested that VexAB may function to export intracellular metabolites. Support for this hypothesis was provided by data showing that vexRAB was upregulated in several metabolic mutants including tryptophan biosynthetic mutants that were predicted to accumulate indole. In addition, vexRAB was found to be upregulated in response to exogenous indole and to contribute to indole resistance. The collective results indicate that vexR is required for vexRAB expression in response to VexAB substrates and that the VexAB RND efflux system modulates the intracellular levels of metabolites that could otherwise accumulate to toxic levels.

Nutrient-sensing mechanisms and pathways.

The ability to sense and respond to fluctuations in environmental nutrient levels is a requisite for life. Nutrient scarcity is a selective pressure that has shaped the evolution of most cellular processes. Different pathways that detect intracellular and extracellular levels of sugars, amino acids, lipids and surrogate metabolites are integrated and coordinated at the organismal level through hormonal signals. During food abundance, nutrient-sensing pathways engage anabolism and storage, whereas scarcity triggers homeostatic mechanisms, such as the mobilization of internal stores through autophagy. Nutrient-sensing pathways are commonly deregulated in human metabolic diseases.

Metabolomics-based approach for assessing the toxicity mechanisms of dibutyl phthalate to abalone (Haliotis diversicolor supertexta).

Dibutyl phthalate (DBP) is a ubiquitous contaminant in the marine environment, and relatively little is known about the toxicological mechanisms of this compound at the metabolite level. In this study, marine gastropods (abalone) were exposed to DBP at environmentally relevant concentrations (2, 10, and 50μg/L) for 30days. The plasma metabolite profiles were determined at the 5th, 15th, and 30th. The major metabolite changes corresponding to DBP exposure were related to osmotic regulation, energy metabolism, and environmental stress, and the effects displayed a dose-dependent pattern. The most obvious change was the increase in the levels of intracellular metabolites (betaine, dimethylglycine, homarine, glutamine, and lactate) and tricarboxylic acid cycle intermediates. The results revealed that DBP may lead to abalone oxidative stress, lipid metabolism dysfunction, energy metabolism disturbance, and osmoregulation imbalance. These results would be helpful in better understanding the mechanisms of abalone response to DBP stress at the system level.

Intracellular metabolite levels shape sulfur isotope fractionation during microbial sulfate respiration

We present a quantitative model for sulfur isotope fractionation accompanying bacterial and archaeal dissimilatory sulfate respiration. By incorporating independently available biochemical data, the model can reproduce a large number of recent experimental fractionation measurements with only three free parameters: (i) the sulfur isotope selectivity of sulfate uptake into the cytoplasm, (ii) the ratio of reduced to oxidized electron carriers supporting the respiration pathway, and (iii) the ratio of in vitro to in vivo levels of respiratory enzyme activity. Fractionation is influenced by all steps in the dissimilatory pathway, which means that environmental sulfate and sulfide levels control sulfur isotope fractionation through the proximate influence of intracellular metabolites. Although sulfur isotope fractionation is a phenotypic trait that appears to be strain specific, we show that it converges on near-thermodynamic behavior, even at micromolar sulfate levels, as long as intracellular sulfate reduction rates are low enough (<<1 fmol H2S⋅cell(-1)⋅d(-1)).