WorldWideScience

Sample records for metabolic flux phenotypes

  1. Linking high-resolution metabolic flux phenotypes and transcriptional regulation in yeast modulated by the global regulator Gcn4p.

    Science.gov (United States)

    Moxley, Joel F; Jewett, Michael C; Antoniewicz, Maciek R; Villas-Boas, Silas G; Alper, Hal; Wheeler, Robert T; Tong, Lily; Hinnebusch, Alan G; Ideker, Trey; Nielsen, Jens; Stephanopoulos, Gregory

    2009-04-21

    Genome sequencing dramatically increased our ability to understand cellular response to perturbation. Integrating system-wide measurements such as gene expression with networks of protein-protein interactions and transcription factor binding revealed critical insights into cellular behavior. However, the potential of systems biology approaches is limited by difficulties in integrating metabolic measurements across the functional levels of the cell despite their being most closely linked to cellular phenotype. To address this limitation, we developed a model-based approach to correlate mRNA and metabolic flux data that combines information from both interaction network models and flux determination models. We started by quantifying 5,764 mRNAs, 54 metabolites, and 83 experimental (13)C-based reaction fluxes in continuous cultures of yeast under stress in the absence or presence of global regulator Gcn4p. Although mRNA expression alone did not directly predict metabolic response, this correlation improved through incorporating a network-based model of amino acid biosynthesis (from r = 0.07 to 0.80 for mRNA-flux agreement). The model provides evidence of general biological principles: rewiring of metabolic flux (i.e., use of different reaction pathways) by transcriptional regulation and metabolite interaction density (i.e., level of pairwise metabolite-protein interactions) as a key biosynthetic control determinant. Furthermore, this model predicted flux rewiring in studies of follow-on transcriptional regulators that were experimentally validated with additional (13)C-based flux measurements. As a first step in linking metabolic control and genetic regulatory networks, this model underscores the importance of integrating diverse data types in large-scale cellular models. We anticipate that an integrated approach focusing on metabolic measurements will facilitate construction of more realistic models of cellular regulation for understanding diseases and constructing

  2. DNA methylation dynamics, metabolic fluxes, gene splicing, and alternative phenotypes in honey bees.

    Science.gov (United States)

    Foret, Sylvain; Kucharski, Robert; Pellegrini, Matteo; Feng, Suhua; Jacobsen, Steven E; Robinson, Gene E; Maleszka, Ryszard

    2012-03-27

    In honey bees (Apis mellifera), the development of a larva into either a queen or worker depends on differential feeding with royal jelly and involves epigenomic modifications by DNA methyltransferases. To understand the role of DNA methylation in this process we sequenced the larval methylomes in both queens and workers. We show that the number of differentially methylated genes (DMGs) in larval head is significantly increased relative to adult brain (2,399 vs. 560) with more than 80% of DMGs up-methylated in worker larvae. Several highly conserved metabolic and signaling pathways are enriched in methylated genes, underscoring the connection between dietary intake and metabolic flux. This includes genes related to juvenile hormone and insulin, two hormones shown previously to regulate caste determination. We also tie methylation data to expressional profiling and describe a distinct role for one of the DMGs encoding anaplastic lymphoma kinase (ALK), an important regulator of metabolism. We show that alk is not only differentially methylated and alternatively spliced in Apis, but also seems to be regulated by a cis-acting, anti-sense non-protein-coding transcript. The unusually complex regulation of ALK in Apis suggests that this protein could represent a previously unknown node in a process that activates downstream signaling according to a nutritional context. The correlation between methylation and alternative splicing of alk is consistent with the recently described mechanism involving RNA polymerase II pausing. Our study offers insights into diet-controlled development in Apis.

  3. Linking high-resolution metabolic flux phenotypes and transcriptional regulation in yeast modulated by the global regulator Gcn4p

    DEFF Research Database (Denmark)

    Moxley, Joel F.; Jewett, Michael Christopher; Antoniewicz, Maciek R.

    2009-01-01

    Gcn4p. Although mRNA expression alone did not directly predict metabolic response, this correlation improved through incorporating a network-based model of amino acid biosynthesis (from r = 0.07 to 0.80 for mRNA-flux agreement). The model provides evidence of general biological principles: rewiring...

  4. Analysis of metabolic flux phenotypes for two Arabidopsis mutants with severe impairment in seed storage lipid synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Lonien, J.; Schwender, J.

    2009-11-01

    Major storage reserves of Arabidopsis (Arabidopsis thaliana) seeds are triacylglycerols (seed oils) and proteins. Seed oil content is severely reduced for the regulatory mutant wrinkled1 (wri1-1; At3g54320) and for a double mutant in two isoforms of plastidic pyruvate kinase (pkp{beta}{sub 1}pkp{alpha}; At5g52920 and At3g22960). Both already biochemically well-characterized mutants were now studied by {sup 13}C metabolic flux analysis of cultured developing embryos based on comparison with their respective genetic wild-type backgrounds. For both mutations, in seeds as well as in cultured embryos, the oil fraction was strongly reduced while the fractions of proteins and free metabolites increased. Flux analysis in cultured embryos revealed changes in nutrient uptakes and fluxes into biomass as well as an increase in tricarboxylic acid cycle activity for both mutations. While in both wild types plastidic pyruvate kinase (PK{sub p}) provides most of the pyruvate for plastidic fatty acid synthesis, the flux through PK{sub p} is reduced in pkp{beta}{sub 1}pkp{alpha} by 43% of the wild-type value. In wri1-1, PK{sub p} flux is even more reduced (by 82%), although the genes PKp{beta}{sub 1} and PKp{alpha} are still expressed. Along a common paradigm of metabolic control theory, it is hypothesized that a large reduction in PK{sub p} enzyme activity in pkp{beta}{sub 1}pkp{alpha} has less effect on PK{sub p} flux than multiple smaller reductions in glycolytic enzymes in wri1-1. In addition, only in the wri1-1 mutant is the large reduction in PK{sub p} flux compensated in part by an increased import of cytosolic pyruvate and by plastidic malic enzyme. No such limited compensatory bypass could be observed in pkp{beta}{sub 1}pkp{alpha}.

  5. Principal Metabolic Flux Mode Analysis.

    Science.gov (United States)

    Bhadra, Sahely; Blomberg, Peter; Castillo, Sandra; Rousu, Juho; Wren, Jonathan

    2018-02-06

    In the analysis of metabolism, two distinct and complementary approaches are frequently used: Principal component analysis (PCA) and stoichiometric flux analysis. PCA is able to capture the main modes of variability in a set of experiments and does not make many prior assumptions about the data, but does not inherently take into account the flux mode structure of metabolism. Stoichiometric flux analysis methods, such as Flux Balance Analysis (FBA) and Elementary Mode Analysis, on the other hand, are able to capture the metabolic flux modes, however, they are primarily designed for the analysis of single samples at a time, and not best suited for exploratory analysis on a large sets of samples. We propose a new methodology for the analysis of metabolism, called Principal Metabolic Flux Mode Analysis (PMFA), which marries the PCA and stoichiometric flux analysis approaches in an elegant regularized optimization framework. In short, the method incorporates a variance maximization objective form PCA coupled with a stoichiometric regularizer, which penalizes projections that are far from any flux modes of the network. For interpretability, we also introduce a sparse variant of PMFA that favours flux modes that contain a small number of reactions. Our experiments demonstrate the versatility and capabilities of our methodology. The proposed method can be applied to genome-scale metabolic network in efficient way as PMFA does not enumerate elementary modes. In addition, the method is more robust on out-of-steady steady-state experimental data than competing flux mode analysis approaches. Matlab software for PMFA and SPMFA and data set used for experiments are available in https://github.com/aalto-ics-kepaco/PMFA. sahely@iitpkd.ac.in, juho.rousu@aalto.fi, Peter.Blomberg@vtt.fi, Sandra.Castillo@vtt.fi. Detailed results are in Supplementary files. Supplementary data are available at https://github.com/aalto-ics-kepaco/PMFA/blob/master/Results.zip.

  6. Derivative processes for modelling metabolic fluxes

    Science.gov (United States)

    Žurauskienė, Justina; Kirk, Paul; Thorne, Thomas; Pinney, John; Stumpf, Michael

    2014-01-01

    Motivation: One of the challenging questions in modelling biological systems is to characterize the functional forms of the processes that control and orchestrate molecular and cellular phenotypes. Recently proposed methods for the analysis of metabolic pathways, for example, dynamic flux estimation, can only provide estimates of the underlying fluxes at discrete time points but fail to capture the complete temporal behaviour. To describe the dynamic variation of the fluxes, we additionally require the assumption of specific functional forms that can capture the temporal behaviour. However, it also remains unclear how to address the noise which might be present in experimentally measured metabolite concentrations. Results: Here we propose a novel approach to modelling metabolic fluxes: derivative processes that are based on multiple-output Gaussian processes (MGPs), which are a flexible non-parametric Bayesian modelling technique. The main advantages that follow from MGPs approach include the natural non-parametric representation of the fluxes and ability to impute the missing data in between the measurements. Our derivative process approach allows us to model changes in metabolite derivative concentrations and to characterize the temporal behaviour of metabolic fluxes from time course data. Because the derivative of a Gaussian process is itself a Gaussian process, we can readily link metabolite concentrations to metabolic fluxes and vice versa. Here we discuss how this can be implemented in an MGP framework and illustrate its application to simple models, including nitrogen metabolism in Escherichia coli. Availability and implementation: R code is available from the authors upon request. Contact: j.norkunaite@imperial.ac.uk; m.stumpf@imperial.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online. PMID:24578401

  7. Population FBA predicts metabolic phenotypes in yeast.

    Directory of Open Access Journals (Sweden)

    Piyush Labhsetwar

    2017-09-01

    Full Text Available Using protein counts sampled from single cell proteomics distributions to constrain fluxes through a genome-scale model of metabolism, Population flux balance analysis (Population FBA successfully described metabolic heterogeneity in a population of independent Escherichia coli cells growing in a defined medium. We extend the methodology to account for correlations in protein expression arising from the co-regulation of genes and apply it to study the growth of independent Saccharomyces cerevisiae cells in two different growth media. We find the partitioning of flux between fermentation and respiration predicted by our model agrees with recent 13C fluxomics experiments, and that our model largely recovers the Crabtree effect (the experimentally known bias among certain yeast species toward fermentation with the production of ethanol even in the presence of oxygen, while FBA without proteomics constraints predicts respirative metabolism almost exclusively. The comparisons to the 13C study showed improvement upon inclusion of the correlations and motivated a technique to systematically identify inconsistent kinetic parameters in the literature. The minor secretion fluxes for glycerol and acetate are underestimated by our method, which indicate a need for further refinements to the metabolic model. For yeast cells grown in synthetic defined (SD medium, the calculated broad distribution of growth rates matches experimental observations from single cell studies, and we characterize several metabolic phenotypes within our modeled populations that make use of diverse pathways. Fast growing yeast cells are predicted to perform significant amount of respiration, use serine-glycine cycle and produce ethanol in mitochondria as opposed to slow growing cells. We use a genetic algorithm to determine the proteomics constraints necessary to reproduce the growth rate distributions seen experimentally. We find that a core set of 51 constraints are essential but

  8. Primary Metabolic Pathways and Metabolic Flux Analysis

    DEFF Research Database (Denmark)

    Villadsen, John

    2015-01-01

    his chapter introduces the metabolic flux analysis (MFA) or stoichiometry-based MFA, and describes the quantitative basis for MFA. It discusses the catabolic pathways in which free energy is produced to drive the cell-building anabolic pathways. An overview of these primary pathways provides...... the reader who is primarily trained in the engineering sciences with atleast a preliminary introduction to biochemistry and also shows how carbon is drained off the catabolic pathways to provide precursors for cell mass building and sometimes for important industrial products. The primary pathways...... to be examined in the following are: glycolysis, primarily by the EMP pathway, but other glycolytic pathways is also mentioned; fermentative pathways in which the redox generated in the glycolytic reactions are consumed; reactions in the tricarboxylic acid (TCA) cycle, which produce biomass precursors and redox...

  9. Impact of stoichiometry representation on simulation of genotype-phenotype relationships in metabolic networks

    DEFF Research Database (Denmark)

    Brochado, Ana Rita; Andrejev, Sergej; Maranas, Costas D.

    2012-01-01

    Genome-scale metabolic networks provide a comprehensive structural framework for modeling genotype-phenotype relationships through flux simulations. The solution space for the metabolic flux state of the cell is typically very large and optimization-based approaches are often necessary for predic......Genome-scale metabolic networks provide a comprehensive structural framework for modeling genotype-phenotype relationships through flux simulations. The solution space for the metabolic flux state of the cell is typically very large and optimization-based approaches are often necessary...

  10. Controlling fluxes for microbial metabolic engineering

    OpenAIRE

    Sachdeva, Gairik

    2014-01-01

    This thesis presents novel synthetic biology tools and design principles usable for microbial metabolic engineering. Controlling metabolic fluxes is essential for biological manufacturing of fuels, materials, and high value chemicals. Insulating the flow of metabolites is a successful natural strategy for metabolic flux regulation. Recently, approaches using scaffolds, both in vitro and in vivo, to spatially co-localize enzymes have reported significant gains in product yields. RNA is suitabl...

  11. Nutrient-gene interaction: metabolic genotype-phenotype relationship.

    Science.gov (United States)

    Go, Vay Liang W; Nguyen, Christine T H; Harris, Diane M; Lee, Wai-Nang Paul

    2005-12-01

    The U.S. Department of Health and Human Services (DHHS)/USDA Dietary Guidelines for Americans is a science and population evidence-based guide on diet and physical activity, providing advice and recommendations to promote a healthier lifestyle and reduce the risk of chronic diseases, including cancer. These recommendations are supported by the comprehensive evidence-based review on diet and cancer prevention conducted by the American Institute for Cancer Research, National Cancer Institute, World Health Organization/International Agency for Research on Cancer, and others. However, influencing dietary effects are the individual genetic predispositions that are the basis for considerable interindividual variations in cancer risk within the population and in nutrient homeostasis, which is maintained by genomic-nutrient and metabolic-phenotype interactions. Although genetics is an important component, it accounts for only a portion of this variation. An individual's overall phenotype, including health status, is achieved and maintained by the sum of metabolic activities functioning under differing circumstances within the life cycle and the complex interactions among genotype, metabolic phenotype, and the environment. In this postgenomic era, high-throughput groups of technologies in genomics, proteomics, and metabolomics measure and analyze DNA sequences, RNA transcripts, proteins, and nutrient-metabolic fluxes in a single experiment. These advances have transformed biomarker studies on nutrient-gene interactions from a reductionist concept into a holistic practice in which many regulated genes involved in metabolism, along with its metabolic phenotypes, can be measured through functional genomics and metabolic profiling. The overall integration of data and information from the building blocks of metabolism-based nutrient-gene interaction can lead to future individualized dietary recommendations to diminish cancer risk.

  12. Optimal flux patterns in cellular metabolic networks

    Energy Technology Data Exchange (ETDEWEB)

    Almaas, E

    2007-01-20

    The availability of whole-cell level metabolic networks of high quality has made it possible to develop a predictive understanding of bacterial metabolism. Using the optimization framework of flux balance analysis, I investigate metabolic response and activity patterns to variations in the availability of nutrient and chemical factors such as oxygen and ammonia by simulating 30,000 random cellular environments. The distribution of reaction fluxes is heavy-tailed for the bacteria H. pylori and E. coli, and the eukaryote S. cerevisiae. While the majority of flux balance investigations have relied on implementations of the simplex method, it is necessary to use interior-point optimization algorithms to adequately characterize the full range of activity patterns on metabolic networks. The interior-point activity pattern is bimodal for E. coli and S. cerevisiae, suggesting that most metabolic reaction are either in frequent use or are rarely active. The trimodal activity pattern of H. pylori indicates that a group of its metabolic reactions (20%) are active in approximately half of the simulated environments. Constructing the high-flux backbone of the network for every environment, there is a clear trend that the more frequently a reaction is active, the more likely it is a part of the backbone. Finally, I briefly discuss the predicted activity patterns of the central-carbon metabolic pathways for the sample of random environments.

  13. From elementary flux modes to elementary flux vectors: Metabolic pathway analysis with arbitrary linear flux constraints

    Science.gov (United States)

    Klamt, Steffen; Gerstl, Matthias P.; Jungreuthmayer, Christian; Mahadevan, Radhakrishnan; Müller, Stefan

    2017-01-01

    Elementary flux modes (EFMs) emerged as a formal concept to describe metabolic pathways and have become an established tool for constraint-based modeling and metabolic network analysis. EFMs are characteristic (support-minimal) vectors of the flux cone that contains all feasible steady-state flux vectors of a given metabolic network. EFMs account for (homogeneous) linear constraints arising from reaction irreversibilities and the assumption of steady state; however, other (inhomogeneous) linear constraints, such as minimal and maximal reaction rates frequently used by other constraint-based techniques (such as flux balance analysis [FBA]), cannot be directly integrated. These additional constraints further restrict the space of feasible flux vectors and turn the flux cone into a general flux polyhedron in which the concept of EFMs is not directly applicable anymore. For this reason, there has been a conceptual gap between EFM-based (pathway) analysis methods and linear optimization (FBA) techniques, as they operate on different geometric objects. One approach to overcome these limitations was proposed ten years ago and is based on the concept of elementary flux vectors (EFVs). Only recently has the community started to recognize the potential of EFVs for metabolic network analysis. In fact, EFVs exactly represent the conceptual development required to generalize the idea of EFMs from flux cones to flux polyhedra. This work aims to present a concise theoretical and practical introduction to EFVs that is accessible to a broad audience. We highlight the close relationship between EFMs and EFVs and demonstrate that almost all applications of EFMs (in flux cones) are possible for EFVs (in flux polyhedra) as well. In fact, certain properties can only be studied with EFVs. Thus, we conclude that EFVs provide a powerful and unifying framework for constraint-based modeling of metabolic networks. PMID:28406903

  14. The Metabolic Phenotype of Prostate Cancer

    Directory of Open Access Journals (Sweden)

    Eric Eidelman

    2017-06-01

    Full Text Available Prostate cancer is the most common non-cutaneous cancer in men in the United States. Cancer metabolism has emerged as a contemporary topic of great interest for improved mechanistic understanding of tumorigenesis. Prostate cancer is a disease model of great interest from a metabolic perspective. Prostatic tissue exhibits unique metabolic activity under baseline conditions. Benign prostate cells accumulate zinc, and this excess zinc inhibits citrate oxidation and metabolism within the citric acid cycle, effectively resulting in citrate production. Malignant cells, however, actively oxidize citrate and resume more typical citric acid cycle function. Of further interest, prostate cancer does not exhibit the Warburg effect, an increase in glucose uptake, seen in many other cancers. These cellular metabolic differences and others are of clinical interest as they present a variety of potential therapeutic targets. Furthermore, understanding of the metabolic profile differences between benign prostate versus low- and high-grade prostate cancers also represents an avenue to better understand cancer progression and potentially develop new diagnostic testing. In this paper, we review the current state of knowledge on the metabolic phenotypes of prostate cancer.

  15. Diversity of flux distribution in central carbon metabolism of S. cerevisiae strains from diverse environments.

    Science.gov (United States)

    Nidelet, Thibault; Brial, Pascale; Camarasa, Carole; Dequin, Sylvie

    2016-04-05

    S. cerevisiae has attracted considerable interest in recent years as a model for ecology and evolutionary biology, revealing a substantial genetic and phenotypic diversity. However, there is a lack of knowledge on the diversity of metabolic networks within this species. To identify the metabolic and evolutionary constraints that shape metabolic fluxes in S. cerevisiae, we used a dedicated constraint-based model to predict the central carbon metabolism flux distribution of 43 strains from different ecological origins, grown in wine fermentation conditions. In analyzing these distributions, we observed a highly contrasted situation in flux variability, with quasi-constancy of the glycolysis and ethanol synthesis yield yet high flexibility of other fluxes, such as the pentose phosphate pathway and acetaldehyde production. Furthermore, these fluxes with large variability showed multimodal distributions that could be linked to strain origin, indicating a convergence between genetic origin and flux phenotype. Flux variability is pathway-dependent and, for some flux, a strain origin effect can be found. These data highlight the constraints shaping the yeast operative central carbon network and provide clues for the design of strategies for strain improvement.

  16. Regulation of flux through metabolic cycles

    International Nuclear Information System (INIS)

    Walsh, K.

    1984-01-01

    The branchpoint of the tricarboxylic acid and glyoxylate shunt was characterized in the intact organism by a multidimensional approach. Theory and methodology were developed to determine velocities for the net flow of carbon through the major steps of acetate metabolism in E. coli. Rates were assigned based on the 13 C-NMR spectrum of intracellular glutamate, measured rates of substrate incorporation into end products, the constituent composition of E. coli and a series of conservation equations which described the system at steady state. The in vivo fluxes through the branchpoint of the tricarboxylic acid and glyoxylate cycles were compared to rates calculated from the kinetic constants of the branchpoint enzymes and the intracellular concentrations of their substrates. These studies elucidated the role of isocitrate dehydrogenase phosphorylation in the Krebs cycle and led to the development of a generalized mathematical description of the sensitivity of branchpoints to regulatory control. This theoretical analysis was termed the branchpoint effect and it describes conditions which result in large changes in the flux through an enzyme even though that enzyme is not subject to direct regulatory control. The theoretical and experimental characterization of this system provided a framework to study the effects of enzyme overproduction and underproduction on metabolic processes in the cell. An in vivo method was developed to determine the extent to which an enzyme catalyzes a rate-controlling reaction. The enzyme chosen for this study was citrate synthase

  17. Maximum entropy modeling of metabolic networks by constraining growth-rate moments predicts coexistence of phenotypes

    Science.gov (United States)

    De Martino, Daniele

    2017-12-01

    In this work maximum entropy distributions in the space of steady states of metabolic networks are considered upon constraining the first and second moments of the growth rate. Coexistence of fast and slow phenotypes, with bimodal flux distributions, emerges upon considering control on the average growth (optimization) and its fluctuations (heterogeneity). This is applied to the carbon catabolic core of Escherichia coli where it quantifies the metabolic activity of slow growing phenotypes and it provides a quantitative map with metabolic fluxes, opening the possibility to detect coexistence from flux data. A preliminary analysis on data for E. coli cultures in standard conditions shows degeneracy for the inferred parameters that extend in the coexistence region.

  18. Metabolic fuels: regulating fluxes to select mix.

    Science.gov (United States)

    Weber, Jean-Michel

    2011-01-15

    Animals must regulate the fluxes of multiple fuels to support changing metabolic rates that result from variation in physiological circumstances. The aim of fuel selection strategies is to exploit the advantages of individual substrates while minimizing the impact of disadvantages. All exercising mammals share a general pattern of fuel selection: at the same %V(O(2,max)) they oxidize the same ratio of lipids to carbohydrates. However, highly aerobic species rely more on intramuscular fuels because energy supply from the circulation is constrained by trans-sarcolemmal transfer. Fuel selection is performed by recruiting different muscles, different fibers within the same muscles or different pathways within the same fibers. Electromyographic analyses show that shivering humans can modulate carbohydrate oxidation either through the selective recruitment of type II fibers within the same muscles or by regulating pathway recruitment within type I fibers. The selection patterns of shivering and exercise are different: at the same %V(O(2,max)), a muscle producing only heat (shivering) or significant movement (exercise) strikes a different balance between lipid and carbohydrate oxidation. Long-distance migrants provide an excellent model to characterize how to increase maximal substrate fluxes. High lipid fluxes are achieved through the coordinated upregulation of mobilization, transport and oxidation by activating enzymes, lipid-solubilizing proteins and membrane transporters. These endurance athletes support record lipolytic rates in adipocytes, use lipoprotein shuttles to accelerate transport and show increased capacity for lipid oxidation in muscle mitochondria. Some migrant birds use dietary omega-3 fatty acids as performance-enhancing agents to boost their ability to process lipids. These dietary fatty acids become incorporated in membrane phospholipids and bind to peroxisome proliferator-activated receptors to activate membrane proteins and modify gene expression.

  19. Phototransduction Influences Metabolic Flux and Nucleotide Metabolism in Mouse Retina.

    Science.gov (United States)

    Du, Jianhai; Rountree, Austin; Cleghorn, Whitney M; Contreras, Laura; Lindsay, Ken J; Sadilek, Martin; Gu, Haiwei; Djukovic, Danijel; Raftery, Dan; Satrústegui, Jorgina; Kanow, Mark; Chan, Lawrence; Tsang, Stephen H; Sweet, Ian R; Hurley, James B

    2016-02-26

    Production of energy in a cell must keep pace with demand. Photoreceptors use ATP to maintain ion gradients in darkness, whereas in light they use it to support phototransduction. Matching production with consumption can be accomplished by coupling production directly to consumption. Alternatively, production can be set by a signal that anticipates demand. In this report we investigate the hypothesis that signaling through phototransduction controls production of energy in mouse retinas. We found that respiration in mouse retinas is not coupled tightly to ATP consumption. By analyzing metabolic flux in mouse retinas, we also found that phototransduction slows metabolic flux through glycolysis and through intermediates of the citric acid cycle. We also evaluated the relative contributions of regulation of the activities of α-ketoglutarate dehydrogenase and the aspartate-glutamate carrier 1. In addition, a comprehensive analysis of the retinal metabolome showed that phototransduction also influences steady-state concentrations of 5'-GMP, ribose-5-phosphate, ketone bodies, and purines. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. OpenFLUX: efficient modelling software for 13C-based metabolic flux analysis

    Directory of Open Access Journals (Sweden)

    Nielsen Lars K

    2009-05-01

    Full Text Available Abstract Background The quantitative analysis of metabolic fluxes, i.e., in vivo activities of intracellular enzymes and pathways, provides key information on biological systems in systems biology and metabolic engineering. It is based on a comprehensive approach combining (i tracer cultivation on 13C substrates, (ii 13C labelling analysis by mass spectrometry and (iii mathematical modelling for experimental design, data processing, flux calculation and statistics. Whereas the cultivation and the analytical part is fairly advanced, a lack of appropriate modelling software solutions for all modelling aspects in flux studies is limiting the application of metabolic flux analysis. Results We have developed OpenFLUX as a user friendly, yet flexible software application for small and large scale 13C metabolic flux analysis. The application is based on the new Elementary Metabolite Unit (EMU framework, significantly enhancing computation speed for flux calculation. From simple notation of metabolic reaction networks defined in a spreadsheet, the OpenFLUX parser automatically generates MATLAB-readable metabolite and isotopomer balances, thus strongly facilitating model creation. The model can be used to perform experimental design, parameter estimation and sensitivity analysis either using the built-in gradient-based search or Monte Carlo algorithms or in user-defined algorithms. Exemplified for a microbial flux study with 71 reactions, 8 free flux parameters and mass isotopomer distribution of 10 metabolites, OpenFLUX allowed to automatically compile the EMU-based model from an Excel file containing metabolic reactions and carbon transfer mechanisms, showing it's user-friendliness. It reliably reproduced the published data and optimum flux distributions for the network under study were found quickly ( Conclusion We have developed a fast, accurate application to perform steady-state 13C metabolic flux analysis. OpenFLUX will strongly facilitate and

  1. Metabolic flux distribution and mathematical models for dynamic ...

    African Journals Online (AJOL)

    A simple model was build for the metabolic flux determination based on published articles. A method for metabolic flux determination by carbon labeling experiments was described and developed here in the first part of this study that allows mathematical description relating the measured quantities and the intracellular ...

  2. Metabolic flux distribution and mathematical models for dynamic ...

    African Journals Online (AJOL)

    user

    2011-03-21

    Mar 21, 2011 ... for metabolic flux determination by carbon labeling experiments was described and developed here in the first part of this study that allows mathematical description relating the measured quantities and the intracellular fluxes. The described method was used to investigate the central carbon metabolism of.

  3. Inferring metabolic phenotypes from the exometabolome through a thermodynamic variational principle

    International Nuclear Information System (INIS)

    Martino, Daniele De; Martino, Andrea De; Capuani, Fabrizio

    2014-01-01

    Networks of biochemical reactions, like cellular metabolic networks, are kept in non-equilibrium steady states by the exchange fluxes connecting them to the environment. In most cases, feasible flux configurations can be derived from minimal mass-balance assumptions upon prescribing in- and outtake fluxes. Here we consider the problem of inferring intracellular flux patterns from extracellular metabolite levels. Resorting to a thermodynamic out of equilibrium variational principle to describe the network at steady state, we show that the switch from fermentative to oxidative phenotypes in cells can be characterized in terms of the glucose, lactate, oxygen and carbon dioxide concentrations. Results obtained for an exactly solvable toy model are fully recovered for a large scale reconstruction of human catabolism. Finally we argue that, in spite of the many approximations involved in the theory, available data for several human cell types are well described by the predicted phenotypic map of the problem. (paper)

  4. Improving the phenotype predictions of a yeast genome-scale metabolic model by incorporating enzymatic constraints

    DEFF Research Database (Denmark)

    Sanchez, Benjamin J.; Zhang, Xi-Cheng; Nilsson, Avlant

    2017-01-01

    , which act as limitations on metabolic fluxes, are not taken into account. Here, we present GECKO, a method that enhances a GEM to account for enzymes as part of reactions, thereby ensuring that each metabolic flux does not exceed its maximum capacity, equal to the product of the enzyme's abundance...... and turnover number. We applied GECKO to a Saccharomyces cerevisiae GEM and demonstrated that the new model could correctly describe phenotypes that the previous model could not, particularly under high enzymatic pressure conditions, such as yeast growing on different carbon sources in excess, coping...... with stress, or overexpressing a specific pathway. GECKO also allows to directly integrate quantitative proteomics data; by doing so, we significantly reduced flux variability of the model, in over 60% of metabolic reactions. Additionally, the model gives insight into the distribution of enzyme usage between...

  5. Metabolic cartography: experimental quantification of metabolic fluxes from isotopic labelling studies

    Energy Technology Data Exchange (ETDEWEB)

    O' Grady J.; Schwender J.; Shachar-Hill, Y.; Morgan, J. A.

    2012-03-01

    For the past decade, flux maps have provided researchers with an in-depth perspective on plant metabolism. As a rapidly developing field, significant headway has been made recently in computation, experimentation, and overall understanding of metabolic flux analysis. These advances are particularly applicable to the study of plant metabolism. New dynamic computational methods such as non-stationary metabolic flux analysis are finding their place in the toolbox of metabolic engineering, allowing more organisms to be studied and decreasing the time necessary for experimentation, thereby opening new avenues by which to explore the vast diversity of plant metabolism. Also, improved methods of metabolite detection and measurement have been developed, enabling increasingly greater resolution of flux measurements and the analysis of a greater number of the multitude of plant metabolic pathways. Methods to deconvolute organelle-specific metabolism are employed with increasing effectiveness, elucidating the compartmental specificity inherent in plant metabolism. Advances in metabolite measurements have also enabled new types of experiments, such as the calculation of metabolic fluxes based on {sup 13}CO{sub 2} dynamic labelling data, and will continue to direct plant metabolic engineering. Newly calculated metabolic flux maps reveal surprising and useful information about plant metabolism, guiding future genetic engineering of crops to higher yields. Due to the significant level of complexity in plants, these methods in combination with other systems biology measurements are necessary to guide plant metabolic engineering in the future.

  6. Metabolic cartography: experimental quantification of metabolic fluxes from isotopic labelling studies

    Energy Technology Data Exchange (ETDEWEB)

    O' Grady, J; Schwender, J; Shachar-Hill, Y; Morgan, JA

    2012-03-26

    For the past decade, flux maps have provided researchers with an in-depth perspective on plant metabolism. As a rapidly developing field, significant headway has been made recently in computation, experimentation, and overall understanding of metabolic flux analysis. These advances are particularly applicable to the study of plant metabolism. New dynamic computational methods such as non-stationary metabolic flux analysis are finding their place in the toolbox of metabolic engineering, allowing more organisms to be studied and decreasing the time necessary for experimentation, thereby opening new avenues by which to explore the vast diversity of plant metabolism. Also, improved methods of metabolite detection and measurement have been developed, enabling increasingly greater resolution of flux measurements and the analysis of a greater number of the multitude of plant metabolic pathways. Methods to deconvolute organelle-specific metabolism are employed with increasing effectiveness, elucidating the compartmental specificity inherent in plant metabolism. Advances in metabolite measurements have also enabled new types of experiments, such as the calculation of metabolic fluxes based on (CO2)-C-13 dynamic labelling data, and will continue to direct plant metabolic engineering. Newly calculated metabolic flux maps reveal surprising and useful information about plant metabolism, guiding future genetic engineering of crops to higher yields. Due to the significant level of complexity in plants, these methods in combination with other systems biology measurements are necessary to guide plant metabolic engineering in the future.

  7. Metabolic cartography: experimental quantification of metabolic fluxes from isotopic labelling studies.

    Science.gov (United States)

    O'Grady, John; Schwender, Jörg; Shachar-Hill, Yair; Morgan, John A

    2012-03-01

    For the past decade, flux maps have provided researchers with an in-depth perspective on plant metabolism. As a rapidly developing field, significant headway has been made recently in computation, experimentation, and overall understanding of metabolic flux analysis. These advances are particularly applicable to the study of plant metabolism. New dynamic computational methods such as non-stationary metabolic flux analysis are finding their place in the toolbox of metabolic engineering, allowing more organisms to be studied and decreasing the time necessary for experimentation, thereby opening new avenues by which to explore the vast diversity of plant metabolism. Also, improved methods of metabolite detection and measurement have been developed, enabling increasingly greater resolution of flux measurements and the analysis of a greater number of the multitude of plant metabolic pathways. Methods to deconvolute organelle-specific metabolism are employed with increasing effectiveness, elucidating the compartmental specificity inherent in plant metabolism. Advances in metabolite measurements have also enabled new types of experiments, such as the calculation of metabolic fluxes based on (13)CO(2) dynamic labelling data, and will continue to direct plant metabolic engineering. Newly calculated metabolic flux maps reveal surprising and useful information about plant metabolism, guiding future genetic engineering of crops to higher yields. Due to the significant level of complexity in plants, these methods in combination with other systems biology measurements are necessary to guide plant metabolic engineering in the future.

  8. OptFlux: an open-source software platform for in silico metabolic engineering

    Directory of Open Access Journals (Sweden)

    Pinto José P

    2010-04-01

    Full Text Available Abstract Background Over the last few years a number of methods have been proposed for the phenotype simulation of microorganisms under different environmental and genetic conditions. These have been used as the basis to support the discovery of successful genetic modifications of the microbial metabolism to address industrial goals. However, the use of these methods has been restricted to bioinformaticians or other expert researchers. The main aim of this work is, therefore, to provide a user-friendly computational tool for Metabolic Engineering applications. Results OptFlux is an open-source and modular software aimed at being the reference computational application in the field. It is the first tool to incorporate strain optimization tasks, i.e., the identification of Metabolic Engineering targets, using Evolutionary Algorithms/Simulated Annealing metaheuristics or the previously proposed OptKnock algorithm. It also allows the use of stoichiometric metabolic models for (i phenotype simulation of both wild-type and mutant organisms, using the methods of Flux Balance Analysis, Minimization of Metabolic Adjustment or Regulatory on/off Minimization of Metabolic flux changes, (ii Metabolic Flux Analysis, computing the admissible flux space given a set of measured fluxes, and (iii pathway analysis through the calculation of Elementary Flux Modes. OptFlux also contemplates several methods for model simplification and other pre-processing operations aimed at reducing the search space for optimization algorithms. The software supports importing/exporting to several flat file formats and it is compatible with the SBML standard. OptFlux has a visualization module that allows the analysis of the model structure that is compatible with the layout information of Cell Designer, allowing the superimposition of simulation results with the model graph. Conclusions The OptFlux software is freely available, together with documentation and other resources, thus

  9. OptFlux: an open-source software platform for in silico metabolic engineering.

    Science.gov (United States)

    Rocha, Isabel; Maia, Paulo; Evangelista, Pedro; Vilaça, Paulo; Soares, Simão; Pinto, José P; Nielsen, Jens; Patil, Kiran R; Ferreira, Eugénio C; Rocha, Miguel

    2010-04-19

    Over the last few years a number of methods have been proposed for the phenotype simulation of microorganisms under different environmental and genetic conditions. These have been used as the basis to support the discovery of successful genetic modifications of the microbial metabolism to address industrial goals. However, the use of these methods has been restricted to bioinformaticians or other expert researchers. The main aim of this work is, therefore, to provide a user-friendly computational tool for Metabolic Engineering applications. OptFlux is an open-source and modular software aimed at being the reference computational application in the field. It is the first tool to incorporate strain optimization tasks, i.e., the identification of Metabolic Engineering targets, using Evolutionary Algorithms/Simulated Annealing metaheuristics or the previously proposed OptKnock algorithm. It also allows the use of stoichiometric metabolic models for (i) phenotype simulation of both wild-type and mutant organisms, using the methods of Flux Balance Analysis, Minimization of Metabolic Adjustment or Regulatory on/off Minimization of Metabolic flux changes, (ii) Metabolic Flux Analysis, computing the admissible flux space given a set of measured fluxes, and (iii) pathway analysis through the calculation of Elementary Flux Modes. OptFlux also contemplates several methods for model simplification and other pre-processing operations aimed at reducing the search space for optimization algorithms. The software supports importing/exporting to several flat file formats and it is compatible with the SBML standard. OptFlux has a visualization module that allows the analysis of the model structure that is compatible with the layout information of Cell Designer, allowing the superimposition of simulation results with the model graph. The OptFlux software is freely available, together with documentation and other resources, thus bridging the gap from research in strain optimization

  10. Metabolic flux analysis of heterotrophic growth in Chlamydomonas reinhardtii.

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    Nanette R Boyle

    Full Text Available Despite the wealth of knowledge available for C. reinhardtii, the central metabolic fluxes of growth on acetate have not yet been determined. In this study, 13C-metabolic flux analysis (13C-MFA was used to determine and quantify the metabolic pathways of primary metabolism in C. reinhardtii cells grown under heterotrophic conditions with acetate as the sole carbon source. Isotopic labeling patterns of compartment specific biomass derived metabolites were used to calculate the fluxes. It was found that acetate is ligated with coenzyme A in the three subcellular compartments (cytosol, mitochondria and plastid included in the model. Two citrate synthases were found to potentially be involved in acetyl-coA metabolism; one localized in the mitochondria and the other acting outside the mitochondria. Labeling patterns demonstrate that Acetyl-coA synthesized in the plastid is directly incorporated in synthesis of fatty acids. Despite having a complete TCA cycle in the mitochondria, it was also found that a majority of the malate flux is shuttled to the cytosol and plastid where it is converted to oxaloacetate providing reducing equivalents to these compartments. When compared to predictions by flux balance analysis, fluxes measured with 13C-MFA were found to be suboptimal with respect to biomass yield; C. reinhardtii sacrifices biomass yield to produce ATP and reducing equivalents.

  11. Genome-scale metabolic flux analysis of Streptomyces lividans growing on a complex medium.

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    D'Huys, Pieter-Jan; Lule, Ivan; Vercammen, Dominique; Anné, Jozef; Van Impe, Jan F; Bernaerts, Kristel

    2012-09-15

    Constraint-based metabolic modeling comprises various excellent tools to assess experimentally observed phenotypic behavior of micro-organisms in terms of intracellular metabolic fluxes. In combination with genome-scale metabolic networks, micro-organisms can be investigated in much more detail and under more complex environmental conditions. Although complex media are ubiquitously applied in industrial fermentations and are often a prerequisite for high protein secretion yields, such multi-component conditions are seldom investigated using genome-scale flux analysis. In this paper, a systematic and integrative approach is presented to determine metabolic fluxes in Streptomyces lividans TK24 grown on a nutritious and complex medium. Genome-scale flux balance analysis and randomized sampling of the solution space are combined to extract maximum information from exometabolome profiles. It is shown that biomass maximization cannot predict the observed metabolite production pattern as such. Although this cellular objective commonly applies to batch fermentation data, both input and output constraints are required to reproduce the measured biomass production rate. Rich media hence not necessarily lead to maximum biomass growth. To eventually identify a unique intracellular flux vector, a hierarchical optimization of cellular objectives is adopted. Out of various tested secondary objectives, maximization of the ATP yield per flux unit returns the closest agreement with the maximum frequency in flux histograms. This unique flux estimation is hence considered as a reasonable approximation for the biological fluxes. Flux maps for different growth phases show no active oxidative part of the pentose phosphate pathway, but NADPH generation in the TCA cycle and NADPH transdehydrogenase activity are most important in fulfilling the NADPH balance. Amino acids contribute to biomass growth by augmenting the pool of available amino acids and by boosting the TCA cycle, particularly

  12. Metabolic flux prediction in cancer cells with altered substrate uptake.

    Science.gov (United States)

    Schwartz, Jean-Marc; Barber, Michael; Soons, Zita

    2015-12-01

    Proliferating cells, such as cancer cells, are known to have an unusual metabolism, characterized by an increased rate of glycolysis and amino acid metabolism. Our understanding of this phenomenon is limited but could potentially be used in order to develop new therapies. Computational modelling techniques, such as flux balance analysis (FBA), have been used to predict fluxes in various cell types, but remain of limited use to explain the unusual metabolic shifts and altered substrate uptake in human cancer cells. We implemented a new flux prediction method based on elementary modes (EMs) and structural flux (StruF) analysis and tested them against experimentally measured flux data obtained from (13)C-labelling in a cancer cell line. We assessed the quality of predictions using different objective functions along with different techniques in normalizing a metabolic network with more than one substrate input. Results show a good correlation between predicted and experimental values and indicate that the choice of cellular objective critically affects the quality of predictions. In particular, lactate gives an excellent correlation and correctly predicts the high flux through glycolysis, matching the observed characteristics of cancer cells. In contrast with FBA, which requires a priori definition of all uptake rates, often hard to measure, atomic StruFs (aStruFs) are able to predict uptake rates of multiple substrates. © 2015 Authors; published by Portland Press Limited.

  13. Constraining genome-scale models to represent the bow tie structure of metabolism for 13C metabolic flux analysis

    DEFF Research Database (Denmark)

    Backman, Tyler W.H.; Ando, David; Singh, Jahnavi

    2018-01-01

    Determination of internal metabolic fluxes is crucial for fundamental and applied biology because they map how carbon and electrons flow through metabolism to enable cell function. 13C Metabolic Flux Analysis (13C MFA) and Two-Scale 13C Metabolic Flux Analysis (2S-13C MFA) are two techniques used...

  14. Body Temperature Measurements for Metabolic Phenotyping in Mice

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    Carola W. Meyer

    2017-07-01

    Full Text Available Endothermic organisms rely on tightly balanced energy budgets to maintain a regulated body temperature and body mass. Metabolic phenotyping of mice, therefore, often includes the recording of body temperature. Thermometry in mice is conducted at various sites, using various devices and measurement practices, ranging from single-time probing to continuous temperature imaging. Whilst there is broad agreement that body temperature data is of value, procedural considerations of body temperature measurements in the context of metabolic phenotyping are missing. Here, we provide an overview of the various methods currently available for gathering body temperature data from mice. We explore the scope and limitations of thermometry in mice, with the hope of assisting researchers in the selection of appropriate approaches, and conditions, for comprehensive mouse phenotypic analyses.

  15. Body Temperature Measurements for Metabolic Phenotyping in Mice

    Science.gov (United States)

    Meyer, Carola W.; Ootsuka, Youichirou; Romanovsky, Andrej A.

    2017-01-01

    Endothermic organisms rely on tightly balanced energy budgets to maintain a regulated body temperature and body mass. Metabolic phenotyping of mice, therefore, often includes the recording of body temperature. Thermometry in mice is conducted at various sites, using various devices and measurement practices, ranging from single-time probing to continuous temperature imaging. Whilst there is broad agreement that body temperature data is of value, procedural considerations of body temperature measurements in the context of metabolic phenotyping are missing. Here, we provide an overview of the various methods currently available for gathering body temperature data from mice. We explore the scope and limitations of thermometry in mice, with the hope of assisting researchers in the selection of appropriate approaches, and conditions, for comprehensive mouse phenotypic analyses. PMID:28824441

  16. 1H NMR spectroscopy-based interventional metabolic phenotyping

    DEFF Research Database (Denmark)

    Lauridsen, Michael B; Bliddal, Henning; Christensen, Robin

    2010-01-01

    -up with assessments of disease activity (DAS-28) and 1H NMR spectroscopy of plasma samples. Discriminant analysis provided evidence that the metabolic profiles predicted disease severity. Cholesterol, lactate, acetylated glycoprotein, and lipid signatures were found to be candidate biomarkers for disease severity.......0007). However, after 31 days of optimized therapy, the two patient groups were not significantly different (P=0.91). The metabolic profiles of both groups of RA patients were different from the healthy subjects. 1H NMR-based metabolic phenotyping of plasma samples in patients with RA is well suited...

  17. Synergy between 13C-metabolic flux analysis and flux balance analysis for understanding metabolic adaption to anaerobiosis in e. coli

    Science.gov (United States)

    Genome-based Flux Balance Analysis (FBA, constraints based flux analysis) and steady state isotopic-labeling-based Metabolic Flux Analysis (MFA) are complimentary approaches to predicting and measuring the operation and regulation of metabolic networks. Here a genome-derived model of E. coli metabol...

  18. Genetic and Environmental Regulation on Longitudinal Change of Metabolic Phenotypes in Danish and Chinese Adult Twins

    DEFF Research Database (Denmark)

    Li, Shuxia; Kyvik, Kirsten Ohm; Pang, Zengchang

    2016-01-01

    OBJECTIVE: The rate of change in metabolic phenotypes can be highly indicative of metabolic disorders and disorder-related modifications. We analyzed data from longitudinal twin studies on multiple metabolic phenotypes in Danish and Chinese twins representing two populations of distinct ethnic, c...... differential patterns of genetic and common environmental regulation on changes over time in metabolic phenotypes across the two samples....

  19. PFA toolbox: a MATLAB tool for Metabolic Flux Analysis.

    Science.gov (United States)

    Morales, Yeimy; Bosque, Gabriel; Vehí, Josep; Picó, Jesús; Llaneras, Francisco

    2016-07-11

    Metabolic Flux Analysis (MFA) is a methodology that has been successfully applied to estimate metabolic fluxes in living cells. However, traditional frameworks based on this approach have some limitations, particularly when measurements are scarce and imprecise. This is very common in industrial environments. The PFA Toolbox can be used to face those scenarios. Here we present the PFA (Possibilistic Flux Analysis) Toolbox for MATLAB, which simplifies the use of Interval and Possibilistic Metabolic Flux Analysis. The main features of the PFA Toolbox are the following: (a) It provides reliable MFA estimations in scenarios where only a few fluxes can be measured or those available are imprecise. (b) It provides tools to easily plot the results as interval estimates or flux distributions. (c) It is composed of simple functions that MATLAB users can apply in flexible ways. (d) It includes a Graphical User Interface (GUI), which provides a visual representation of the measurements and their uncertainty. (e) It can use stoichiometric models in COBRA format. In addition, the PFA Toolbox includes a User's Guide with a thorough description of its functions and several examples. The PFA Toolbox for MATLAB is a freely available Toolbox that is able to perform Interval and Possibilistic MFA estimations.

  20. Tracking the metabolic pulse of plant lipid production with isotopic labeling and flux analyses: Past, present and future

    Science.gov (United States)

    Metabolic networks are comprised of chemical transformations that are the basis of cellular operation and function to sustain life. The molecular rate of transitioning through biochemical pathways (i.e. flux) establishes cellular phenotypes that can be studied in response to genetic or environmental...

  1. Predicting growth conditions from internal metabolic fluxes in an in-silico model of E. coli.

    Directory of Open Access Journals (Sweden)

    Viswanadham Sridhara

    Full Text Available A widely studied problem in systems biology is to predict bacterial phenotype from growth conditions, using mechanistic models such as flux balance analysis (FBA. However, the inverse prediction of growth conditions from phenotype is rarely considered. Here we develop a computational framework to carry out this inverse prediction on a computational model of bacterial metabolism. We use FBA to calculate bacterial phenotypes from growth conditions in E. coli, and then we assess how accurately we can predict the original growth conditions from the phenotypes. Prediction is carried out via regularized multinomial regression. Our analysis provides several important physiological and statistical insights. First, we show that by analyzing metabolic end products we can consistently predict growth conditions. Second, prediction is reliable even in the presence of small amounts of impurities. Third, flux through a relatively small number of reactions per growth source (∼10 is sufficient for accurate prediction. Fourth, combining the predictions from two separate models, one trained only on carbon sources and one only on nitrogen sources, performs better than models trained to perform joint prediction. Finally, that separate predictions perform better than a more sophisticated joint prediction scheme suggests that carbon and nitrogen utilization pathways, despite jointly affecting cellular growth, may be fairly decoupled in terms of their dependence on specific assortments of molecular precursors.

  2. Finding elementary flux modes in metabolic networks based on flux balance analysis and flux coupling analysis: application to the analysis of Escherichia coli metabolism.

    Science.gov (United States)

    Tabe-Bordbar, Shayan; Marashi, Sayed-Amir

    2013-12-01

    Elementary modes (EMs) are steady-state metabolic flux vectors with minimal set of active reactions. Each EM corresponds to a metabolic pathway. Therefore, studying EMs is helpful for analyzing the production of biotechnologically important metabolites. However, memory requirements for computing EMs may hamper their applicability as, in most genome-scale metabolic models, no EM can be computed due to running out of memory. In this study, we present a method for computing randomly sampled EMs. In this approach, a network reduction algorithm is used for EM computation, which is based on flux balance-based methods. We show that this approach can be used to recover the EMs in the medium- and genome-scale metabolic network models, while the EMs are sampled in an unbiased way. The applicability of such results is shown by computing “estimated” control-effective flux values in Escherichia coli metabolic network.

  3. Comparison between elementary flux modes analysis and 13C-metabolic fluxes measured in bacterial and plant cells.

    Science.gov (United States)

    Beurton-Aimar, Marie; Beauvoit, Bertrand; Monier, Antoine; Vallée, François; Dieuaide-Noubhani, Martine; Colombié, Sophie

    2011-06-20

    (13)C metabolic flux analysis is one of the pertinent ways to compare two or more physiological states. From a more theoretical standpoint, the structural properties of metabolic networks can be analysed to explore feasible metabolic behaviours and to define the boundaries of steady state flux distributions. Elementary flux mode analysis is one of the most efficient methods for performing this analysis. In this context, recent approaches have tended to compare experimental flux measurements with topological network analysis. Metabolic networks describing the main pathways of central carbon metabolism were set up for a bacteria species (Corynebacterium glutamicum) and a plant species (Brassica napus) for which experimental flux maps were available. The structural properties of each network were then studied using the concept of elementary flux modes. To do this, coefficients of flux efficiency were calculated for each reaction within the networks by using selected sets of elementary flux modes. Then the relative differences - reflecting the change of substrate i.e. a sugar source for C. glutamicum and a nitrogen source for B. napus - of both flux efficiency and flux measured experimentally were compared. For both organisms, there is a clear relationship between these parameters, thus indicating that the network structure described by the elementary flux modes had captured a significant part of the metabolic activity in both biological systems. In B. napus, the extension of the elementary flux mode analysis to an enlarged metabolic network still resulted in a clear relationship between the change in the coefficients and that of the measured fluxes. Nevertheless, the limitations of the method to fit some particular fluxes are discussed. This consistency between EFM analysis and experimental flux measurements, validated on two metabolic systems allows us to conclude that elementary flux mode analysis could be a useful tool to complement (13)C metabolic flux analysis

  4. Reduced TCA flux in diabetic myotubes: A governing influence on the diabetic phenotype?

    DEFF Research Database (Denmark)

    Gaster, Michael

    2009-01-01

    The diabetic phenotype is complex, requiring elucidation of key initiating defects. It is unknown whether the reduced tricarboxylic acid cycle (TCA) flux in skeletal muscle of obese and obese type 2 diabetic (T2D) subjects is of primary origin. Acetate oxidation (measurement of TCA-flux) was sign......The diabetic phenotype is complex, requiring elucidation of key initiating defects. It is unknown whether the reduced tricarboxylic acid cycle (TCA) flux in skeletal muscle of obese and obese type 2 diabetic (T2D) subjects is of primary origin. Acetate oxidation (measurement of TCA...

  5. Tracking the metabolic pulse of plant lipid production with isotopic labeling and flux analyses: Past, present and future.

    Science.gov (United States)

    Allen, Doug K; Bates, Philip D; Tjellström, Henrik

    2015-04-01

    Metabolism is comprised of networks of chemical transformations, organized into integrated biochemical pathways that are the basis of cellular operation, and function to sustain life. Metabolism, and thus life, is not static. The rate of metabolites transitioning through biochemical pathways (i.e., flux) determines cellular phenotypes, and is constantly changing in response to genetic or environmental perturbations. Each change evokes a response in metabolic pathway flow, and the quantification of fluxes under varied conditions helps to elucidate major and minor routes, and regulatory aspects of metabolism. To measure fluxes requires experimental methods that assess the movements and transformations of metabolites without creating artifacts. Isotopic labeling fills this role and is a long-standing experimental approach to identify pathways and quantify their metabolic relevance in different tissues or under different conditions. The application of labeling techniques to plant science is however far from reaching it potential. In light of advances in genetics and molecular biology that provide a means to alter metabolism, and given recent improvements in instrumentation, computational tools and available isotopes, the use of isotopic labeling to probe metabolism is becoming more and more powerful. We review the principal analytical methods for isotopic labeling with a focus on seminal studies of pathways and fluxes in lipid metabolism and carbon partitioning through central metabolism. Central carbon metabolic steps are directly linked to lipid production by serving to generate the precursors for fatty acid biosynthesis and lipid assembly. Additionally some of the ideas for labeling techniques that may be most applicable for lipid metabolism in the future were originally developed to investigate other aspects of central metabolism. We conclude by describing recent advances that will play an important future role in quantifying flux and metabolic operation in plant

  6. Mediterranean diet and mortality risk in metabolically healthy obese and metabolically unhealthy obese phenotypes.

    Science.gov (United States)

    Park, Y-M; Steck, S E; Fung, T T; Zhang, J; Hazlett, L J; Han, K; Merchant, A T

    2016-10-01

    The Mediterranean diet has been consistently associated with reduced mortality risk. Few prospective studies have examined whether the benefits from a Mediterranean diet are equally shared by obese individuals with varying metabolic health. The objective of this study was to investigate the association between Mediterranean diet, metabolic phenotypes and mortality risk in a representative obese US population. Data from 1739 adults aged 20-88 years were analyzed from participants of the National Health and Nutrition Examination Survey III, 1988-1994 followed up for deaths until 31 December 2011 in a prospective cohort analysis. Mediterranean Diet Scores (MDS) were created to assess the adherence to Mediterranean diet. Participants were classified as metabolically healthy obese (MHO) phenotype (0 or 1 metabolic abnormality) or metabolically unhealthy obese (MUO) phenotype (two or more metabolic abnormalities), based on high glucose, insulin resistance, blood pressure, triglycerides, C-reactive protein and low high-density lipoprotein cholesterol. The MHO phenotype (n=598) was observed in 34.8% (s.e., 1.7%) of those who were obese (mean body mass index was 33.4 and 34.8 in MHO and MUO phenotypes, respectively). During a median follow-up of 18.5 years, there were 77 (12.9%) and 309 (27.1%) deaths in MHO and MUO individuals, respectively. In MHO individuals, the multivariable-adjusted hazard ratio (HR) of all-cause mortality in the highest tertile compared with the first tertile of MDS was 0.44 (95% confidence interval (CI), 0.26-0.75; P for trend Mediterranean dietary pattern appears to reduce mortality in the MHO phenotype, but not among the MUO phenotype in an obese population.

  7. Designing metabolic engineering strategies with genome-scale metabolic flux modeling

    Directory of Open Access Journals (Sweden)

    Yen JY

    2015-01-01

    Full Text Available Jiun Y Yen,1,2 Imen Tanniche,1 Amanda K Fisher,1–3 Glenda E Gillaspy,2 David R Bevan,2,3 Ryan S Senger1 1Department of Biological Systems Engineering, 2Department of Biochemistry, 3Genomics, Bioinformatics, and Computational Biology Interdisciplinary Program, Virginia Tech, Blacksburg, VA, USA Abstract: New in silico tools that make use of genome-scale metabolic flux modeling are improving the design of metabolic engineering strategies. This review highlights the latest developments in this area, explains the interface between these in silico tools and the experimental implementation tools of metabolic engineers, and provides a way forward so that in silico predictions can better mimic reality and more experimental methods can be considered in simulation studies. The several methodologies for solving genome-scale models (eg, flux balance analysis [FBA], parsimonious FBA, flux variability analysis, and minimization of metabolic adjustment all have unique advantages and applications. There are two basic approaches to designing metabolic engineering strategies in silico, and both have demonstrated success in the literature. The first involves: 1 making a genetic manipulation in a model; 2 testing for improved performance through simulation; and 3 iterating the process. The second approach has been used in more recently designed in silico tools and involves: 1 comparing metabolic flux profiles of a wild-type and ideally engineered state and 2 designing engineering strategies based on the differences in these flux profiles. Improvements in genome-scale modeling are anticipated in areas such as the inclusion of all relevant cellular machinery, the ability to understand and anticipate the results of combinatorial enrichment experiments, and constructing dynamic and flexible biomass equations that can respond to environmental and genetic manipulations. Keywords: genome-scale modeling, genome-scale modeling, flux balance analysis, flux variability

  8. Two-Scale 13C Metabolic Flux Analysis for Metabolic Engineering.

    Science.gov (United States)

    Ando, David; Garcia Martin, Hector

    2018-01-01

    Accelerating the Design-Build-Test-Learn (DBTL) cycle in synthetic biology is critical to achieving rapid and facile bioengineering of organisms for the production of, e.g., biofuels and other chemicals. The Learn phase involves using data obtained from the Test phase to inform the next Design phase. As part of the Learn phase, mathematical models of metabolic fluxes give a mechanistic level of comprehension to cellular metabolism, isolating the principle drivers of metabolic behavior from the peripheral ones, and directing future experimental designs and engineering methodologies. Furthermore, the measurement of intracellular metabolic fluxes is specifically noteworthy as providing a rapid and easy-to-understand picture of how carbon and energy flow throughout the cell. Here, we present a detailed guide to performing metabolic flux analysis in the Learn phase of the DBTL cycle, where we show how one can take the isotope labeling data from a 13 C labeling experiment and immediately turn it into a determination of cellular fluxes that points in the direction of genetic engineering strategies that will advance the metabolic engineering process.For our modeling purposes we use the Joint BioEnergy Institute (JBEI) Quantitative Metabolic Modeling (jQMM) library, which provides an open-source, python-based framework for modeling internal metabolic fluxes and making actionable predictions on how to modify cellular metabolism for specific bioengineering goals. It presents a complete toolbox for performing different types of flux analysis such as Flux Balance Analysis, 13 C Metabolic Flux Analysis, and it introduces the capability to use 13 C labeling experimental data to constrain comprehensive genome-scale models through a technique called two-scale 13 C Metabolic Flux Analysis (2S- 13 C MFA) [1]. In addition to several other capabilities, the jQMM is also able to predict the effects of knockouts using the MoMA and ROOM methodologies. The use of the jQMM library is

  9. Phenotypic and metabolic traits of commercial Saccharomyces cerevisiae yeasts

    Science.gov (United States)

    2014-01-01

    Currently, pursuing yeast strains that display both a high potential fitness for alcoholic fermentation and a favorable impact on quality is a major goal in the alcoholic beverage industry. This considerable industrial interest has led to many studies characterizing the phenotypic and metabolic traits of commercial yeast populations. In this study, 20 Saccharomyces cerevisiae strains from different geographical origins exhibited high phenotypic diversity when their response to nine biotechnologically relevant conditions was examined. Next, the fermentation fitness and metabolic traits of eight selected strains with a unique phenotypic profile were evaluated in a high-sugar synthetic medium under two nitrogen regimes. Although the strains exhibited significant differences in nitrogen requirements and utilization rates, a direct relationship between nitrogen consumption, specific growth rate, cell biomass, cell viability, acetic acid and glycerol formation was only observed under high-nitrogen conditions. In contrast, the strains produced more succinic acid under the low-nitrogen regime, and a direct relationship with the final cell biomass was established. Glucose and fructose utilization patterns depended on both yeast strain and nitrogen availability. For low-nitrogen fermentation, three strains did not fully degrade the fructose. This study validates phenotypic and metabolic diversity among commercial wine yeasts and contributes new findings on the relationship between nitrogen availability, yeast cell growth and sugar utilization. We suggest that measuring nitrogen during the stationary growth phase is important because yeast cells fermentative activity is not exclusively related to population size, as previously assumed, but it is also related to the quantity of nitrogen consumed during this growth phase. PMID:24949272

  10. Flux-Enabled Exploration of the Role of Sip1 in Galactose Yeast Metabolism

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    Christopher M. Shymansky

    2017-05-01

    Full Text Available 13C metabolic flux analysis (13C MFA is an important systems biology technique that has been used to investigate microbial metabolism for decades. The heterotrimer Snf1 kinase complex plays a key role in the preference Saccharomyces cerevisiae exhibits for glucose over galactose, a phenomenon known as glucose repression or carbon catabolite repression. The SIP1 gene, encoding a part of this complex, has received little attention, presumably, because its knockout lacks a growth phenotype. We present a fluxomic investigation of the relative effects of the presence of galactose in classically glucose-repressing media and/or knockout of SIP1 using a multi-scale variant of 13C MFA known as 2-Scale 13C metabolic flux analysis (2S-13C MFA. In this study, all strains have the galactose metabolism deactivated (gal1Δ background so as to be able to separate the metabolic effects purely related to glucose repression from those arising from galactose metabolism. The resulting flux profiles reveal that the presence of galactose in classically glucose-repressing conditions, for a CEN.PK113-7D gal1Δ background, results in a substantial decrease in pentose phosphate pathway (PPP flux and increased flow from cytosolic pyruvate and malate through the mitochondria toward cytosolic branched-chain amino acid biosynthesis. These fluxomic redistributions are accompanied by a higher maximum specific growth rate, both seemingly in violation of glucose repression. Deletion of SIP1 in the CEN.PK113-7D gal1Δ cells grown in mixed glucose/galactose medium results in a further increase. Knockout of this gene in cells grown in glucose-only medium results in no change in growth rate and a corresponding decrease in glucose and ethanol exchange fluxes and flux through pathways involved in aspartate/threonine biosynthesis. Glucose repression appears to be violated at a 1/10 ratio of galactose-to-glucose. Based on the scientific literature, we may have conducted our experiments

  11. Metabolic flux balance analysis and the in silico analysis of Escherichia coli K-12 gene deletions

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    Edwards Jeremy S

    2000-07-01

    Full Text Available Abstract Background Genome sequencing and bioinformatics are producing detailed lists of the molecular components contained in many prokaryotic organisms. From this 'parts catalogue' of a microbial cell, in silico representations of integrated metabolic functions can be constructed and analyzed using flux balance analysis (FBA. FBA is particularly well-suited to study metabolic networks based on genomic, biochemical, and strain specific information. Results Herein, we have utilized FBA to interpret and analyze the metabolic capabilities of Escherichia coli. We have computationally mapped the metabolic capabilities of E. coli using FBA and examined the optimal utilization of the E. coli metabolic pathways as a function of environmental variables. We have used an in silico analysis to identify seven gene products of central metabolism (glycolysis, pentose phosphate pathway, TCA cycle, electron transport system essential for aerobic growth of E. coli on glucose minimal media, and 15 gene products essential for anaerobic growth on glucose minimal media. The in silico tpi-, zwf, and pta- mutant strains were examined in more detail by mapping the capabilities of these in silico isogenic strains. Conclusions We found that computational models of E. coli metabolism based on physicochemical constraints can be used to interpret mutant behavior. These in silica results lead to a further understanding of the complex genotype-phenotype relation. Supplementary information: http://gcrg.ucsd.edu/supplementary_data/DeletionAnalysis/main.htm

  12. Metabolic Flux Analysis of Shewanella spp. Reveals Evolutionary Robustness in Central Carbon Metabolism

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Yinjie J.; Martin, Hector Garcia; Dehal, Paramvir S.; Deutschbauer, Adam; Llora, Xavier; Meadows, Adam; Arkin, Adam; Keasling, Jay D.

    2009-08-19

    Shewanella spp. are a group of facultative anaerobic bacteria widely distributed in marine and fresh-water environments. In this study, we profiled the central metabolic fluxes of eight recently sequenced Shewanella species grown under the same condition in minimal med-ium with [3-13C] lactate. Although the tested Shewanella species had slightly different growth rates (0.23-0.29 h31) and produced different amounts of acetate and pyruvate during early exponential growth (pseudo-steady state), the relative intracellular metabolic flux distributions were remarkably similar. This result indicates that Shewanella species share similar regulation in regard to central carbon metabolic fluxes under steady growth conditions: the maintenance of metabolic robustness is not only evident in a single species under genetic perturbations (Fischer and Sauer, 2005; Nat Genet 37(6):636-640), but also observed through evolutionary related microbial species. This remarkable conservation of relative flux profiles through phylogenetic differences prompts us to introduce the concept of metabotype as an alternative scheme to classify microbial fluxomics. On the other hand, Shewanella spp. display flexibility in the relative flux profiles when switching their metabolism from consuming lactate to consuming pyruvate and acetate.

  13. Maintenance metabolism and carbon fluxes in Bacillus species

    Directory of Open Access Journals (Sweden)

    Decasper Seraina

    2008-06-01

    Full Text Available Abstract Background Selection of an appropriate host organism is crucial for the economic success of biotechnological processes. A generally important selection criterion is a low maintenance energy metabolism to reduce non-productive consumption of substrate. We here investigated, whether various bacilli that are closely related to Bacillus subtilis are potential riboflavin production hosts with low maintenance metabolism. Results While B. subtilis exhibited indeed the highest maintenance energy coefficient, B. licheniformis and B. amyloliquefaciens exhibited only statistically insignificantly reduced maintenance metabolism. Both B. pumilus and B. subtilis (natto exhibited irregular growth patterns under glucose limitation such that the maintenance metabolism could not be determined. The sole exception with significantly reduced maintenance energy requirements was the B. licheniformis strain T380B. The frequently used spo0A mutation significantly increased the maintenance metabolism of B. subtilis. At the level of 13C-detected intracellular fluxes, all investigated bacilli exhibited a significant flux through the pentose phosphate pathway, a prerequisite for efficient riboflavin production. Different from all other species, B. subtilis featured high respiratory tricarboxylic acid cycle fluxes in batch and chemostat cultures. In particular under glucose-limited conditions, this led to significant excess formation of NADPH of B. subtilis, while anabolic consumption was rather balanced with catabolic NADPH formation in the other bacilli. Conclusion Despite its successful commercial production of riboflavin, B. subtilis does not seem to be the optimal cell factory from a bioenergetic point of view. The best choice of the investigated strains is the sporulation-deficient B. licheniformis T380B strain. Beside a low maintenance energy coefficient, this strain grows robustly under different conditions and exhibits only moderate acetate overflow, hence

  14. WUFlux: an open-source platform for 13C metabolic flux analysis of bacterial metabolism.

    Science.gov (United States)

    He, Lian; Wu, Stephen G; Zhang, Muhan; Chen, Yixin; Tang, Yinjie J

    2016-11-04

    Flux analyses, including flux balance analysis (FBA) and 13 C-metabolic flux analysis ( 13 C-MFA), offer direct insights into cell metabolism, and have been widely used to characterize model and non-model microbial species. Nonetheless, constructing the 13 C-MFA model and performing flux calculation are demanding for new learners, because they require knowledge of metabolic networks, carbon transitions, and computer programming. To facilitate and standardize the 13 C-MFA modeling work, we set out to publish a user-friendly and programming-free platform (WUFlux) for flux calculations in MATLAB ® . We constructed an open-source platform for steady-state 13 C-MFA. Using GUIDE (graphical user interface design environment) in MATLAB, we built a user interface that allows users to modify models based on their own experimental conditions. WUFlux is capable of directly correcting mass spectrum data of TBDMS (N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide)-derivatized proteinogenic amino acids by removing background noise. To simplify 13 C-MFA of different prokaryotic species, the software provides several metabolic network templates, including those for chemoheterotrophic bacteria and mixotrophic cyanobacteria. Users can modify the network and constraints, and then analyze the microbial carbon and energy metabolisms of various carbon substrates (e.g., glucose, pyruvate/lactate, acetate, xylose, and glycerol). WUFlux also offers several ways of visualizing the flux results with respect to the constructed network. To validate our model's applicability, we have compared and discussed the flux results obtained from WUFlux and other MFA software. We have also illustrated how model constraints of cofactor and ATP balances influence fluxome results. Open-source software for 13 C-MFA, WUFlux, with a user-friendly interface and easy-to-modify templates, is now available at http://www.13cmfa.org /or ( http://tang.eece.wustl.edu/ToolDevelopment.htm ). We will continue

  15. Metabolic phenotype in the mouse model of osteogenesis imperfecta.

    Science.gov (United States)

    Boraschi-Diaz, Iris; Tauer, Josephine T; El-Rifai, Omar; Guillemette, Delphine; Lefebvre, Geneviève; Rauch, Frank; Ferron, Mathieu; Komarova, Svetlana V

    2017-09-01

    Osteogenesis imperfecta (OI) is the most common heritable bone fragility disorder, usually caused by dominant mutations in genes coding for collagen type I alpha chains, COL1A1 or COL1A2 Osteocalcin (OCN) is now recognized as a bone-derived regulator of insulin secretion and sensitivity and glucose homeostasis. Since OI is associated with increased rates of bone formation and resorption, we hypothesized that the levels of undercarboxylated OCN are increased in OI. The objective of this study was to determine changes in OCN and to elucidate the metabolic phenotype in the Col1a1 Jrt/+ mouse, a model of dominant OI caused by a Col1a1 mutation. Circulating levels of undercarboxylated OCN were higher in 4-week-old OI mice and normal by 8 weeks of age. Young OI animals exhibited a sex-dependent metabolic phenotype, including increased insulin levels in males, improved glucose tolerance in females, lower levels of random glucose and low adiposity in both sexes. The rates of O 2 consumption and CO 2 production, as well as energy expenditure assessed using indirect calorimetry were significantly increased in OI animals of both sexes, whereas respiratory exchange ratio was significantly higher in OI males only. Although OI mice have significant physical impairment that may contribute to metabolic differences, we specifically accounted for movement and compared OI and WT animals during the periods of similar activity levels. Taken together, our data strongly suggest that OI animals have alterations in whole body energy metabolism that are consistent with the action of undercarboxylated osteocalcin. © 2017 Society for Endocrinology.

  16. FluxFix: automatic isotopologue normalization for metabolic tracer analysis.

    Science.gov (United States)

    Trefely, Sophie; Ashwell, Peter; Snyder, Nathaniel W

    2016-11-25

    Isotopic tracer analysis by mass spectrometry is a core technique for the study of metabolism. Isotopically labeled atoms from substrates, such as [ 13 C]-labeled glucose, can be traced by their incorporation over time into specific metabolic products. Mass spectrometry is often used for the detection and differentiation of the isotopologues of each metabolite of interest. For meaningful interpretation, mass spectrometry data from metabolic tracer experiments must be corrected to account for the naturally occurring isotopologue distribution. The calculations required for this correction are time consuming and error prone and existing programs are often platform specific, non-intuitive, commercially licensed and/or limited in accuracy by using theoretical isotopologue distributions, which are prone to artifacts from noise or unresolved interfering signals. Here we present FluxFix ( http://fluxfix.science ), an application freely available on the internet that quickly and reliably transforms signal intensity values into percent mole enrichment for each isotopologue measured. 'Unlabeled' data, representing the measured natural isotopologue distribution for a chosen analyte, is entered by the user. This data is used to generate a correction matrix according to a well-established algorithm. The correction matrix is applied to labeled data, also entered by the user, thus generating the corrected output data. FluxFix is compatible with direct copy and paste from spreadsheet applications including Excel (Microsoft) and Google sheets and automatically adjusts to account for input data dimensions. The program is simple, easy to use, agnostic to the mass spectrometry platform, generalizable to known or unknown metabolites, and can take input data from either a theoretical natural isotopologue distribution or an experimentally measured one. Our freely available web-based calculator, FluxFix ( http://fluxfix.science ), quickly and reliably corrects metabolic tracer data for

  17. Physiology and genetics of metabolic flux control in Zymomonas mobilis

    Energy Technology Data Exchange (ETDEWEB)

    Conway, T.

    1992-01-01

    This work seeks to understand the role of gene expression in regulating glycolytic enzyme synthesis in a balance that allows proper glycoltic flux control. The seven genes targeted for study in this laboratory have been cloned and sequenced, and molecular details of regulation have been investigated. Clear that glycolytic enzyme synthesis is coordinated to prevent the build up of toxic metabolic intermediates. The genetic mechanisms responsible for regulating balanced expression of the EntnerDoudoroff and glycolytic genes in Z. mobilis are beginning to be understood. Several layers of genetic control, perhaps in a hierarchal arrangement act in concert to determine the relative abundance of the glycolytic enzymes. These genetic controls involve differential translational efficiency, highly conserved promoter sequences, transcription factors, differential mRNA stabilities, and nucleolytic mRNA processing. The serendipitous cloning of the glucose facilitator, glf, as a result of linkage to several other genes of interest will have a significant impact on the study of Z. mobilis metabolism. The glucose facilitator is being characterized in a genetically reconstituted system in E. coli. Molecular genetic studies indicate that the ratio of glf expression to that of glk, zmf, and edd is carefully regulated, and suggests a critical role in metabolic control. Regulation of glycolytic gene expression is now sufficiently well understood to allow use of the glycolytic genes as tools to manipulate specified enzyme levels for the purpose of analyzing metabolic flux control. The critical genes have been subcloned for stable expression in Z. mobilis and placed under control of a regulated promoter system involving the tac promoter, the lacI repressor, and gene induction in by IPTG. HPLC methods have been developed that allow quantitation of virtually all of the metabolic intermediates in the cell pool.

  18. The Composition and Metabolic Phenotype of Neisseria gonorrhoeae Biofilms

    Directory of Open Access Journals (Sweden)

    Michael A Apicella

    2011-04-01

    Full Text Available N. gonorrhoeae has been shown to form biofilms during cervical infection. Thus, biofilm formation may play an important role in the infection of women. The ability of N. gonorrhoeae to form membrane blebs is crucial to biofilm formation. Blebs contain DNA and outer membrane structures, which have been shown to be major constituents of the biofilm matrix. The organism expresses a DNA thermonuclease that is involved in remodeling of the biofilm matrix. Comparison of the transcriptional profiles of gonococcal biofilms and planktonic runoff indicate that genes involved in anaerobic metabolism and oxidative stress tolerance are more highly expressed in biofilm. The expression of aniA, ccp, and norB, which encode nitrite reductase, cytochrome c peroxidase, and nitric oxide reductase respectively, is required for mature biofilm formation over glass and human cervical cells. In addition, anaerobic respiration occurs in the substratum of gonococcal biofilms and disruption of the norB gene required for anaerobic respiration, results in a severe biofilm attenuation phenotype. It has been demonstrated that accumulation of nitric oxide (NO contributes to the phenotype of a norB mutant and can retard biofilm formation. However, NO can also enhance biofilm formation, and this is largely dependent on the concentration and donation rate or steady state kinetics of NO. The majority of the genes involved in gonococcal oxidative stress tolerance are also required for normal biofilm formation, as mutations in the following genes result in attenuated biofilm formation over cervical cells and/or glass: oxyR, gor, prx, mntABC, trxB, and estD. Overall, biofilm formation appears to be an adaptation for coping with the environmental stresses present in the female genitourinary tract. Therefore, this review will discuss the studies, which describe the composition and metabolic phenotype of gonococcal biofilms.

  19. Constraining Genome-Scale Models to Represent the Bow Tie Structure of Metabolism for 13C Metabolic Flux Analysis

    Directory of Open Access Journals (Sweden)

    Tyler W. H. Backman

    2018-01-01

    Full Text Available Determination of internal metabolic fluxes is crucial for fundamental and applied biology because they map how carbon and electrons flow through metabolism to enable cell function. 13 C Metabolic Flux Analysis ( 13 C MFA and Two-Scale 13 C Metabolic Flux Analysis (2S- 13 C MFA are two techniques used to determine such fluxes. Both operate on the simplifying approximation that metabolic flux from peripheral metabolism into central “core” carbon metabolism is minimal, and can be omitted when modeling isotopic labeling in core metabolism. The validity of this “two-scale” or “bow tie” approximation is supported both by the ability to accurately model experimental isotopic labeling data, and by experimentally verified metabolic engineering predictions using these methods. However, the boundaries of core metabolism that satisfy this approximation can vary across species, and across cell culture conditions. Here, we present a set of algorithms that (1 systematically calculate flux bounds for any specified “core” of a genome-scale model so as to satisfy the bow tie approximation and (2 automatically identify an updated set of core reactions that can satisfy this approximation more efficiently. First, we leverage linear programming to simultaneously identify the lowest fluxes from peripheral metabolism into core metabolism compatible with the observed growth rate and extracellular metabolite exchange fluxes. Second, we use Simulated Annealing to identify an updated set of core reactions that allow for a minimum of fluxes into core metabolism to satisfy these experimental constraints. Together, these methods accelerate and automate the identification of a biologically reasonable set of core reactions for use with 13 C MFA or 2S- 13 C MFA, as well as provide for a substantially lower set of flux bounds for fluxes into the core as compared with previous methods. We provide an open source Python implementation of these algorithms at https://github.com/JBEI/limitfluxtocore.

  20. Reliable Metabolic Flux Estimation in Escherichia coli Central Carbon Metabolism Using Intracellular Free Amino Acids

    Directory of Open Access Journals (Sweden)

    Nobuyuki Okahashi

    2014-05-01

    Full Text Available 13C metabolic flux analysis (MFA is a tool of metabolic engineering for investigation of in vivo flux distribution. A direct 13C enrichment analysis of intracellular free amino acids (FAAs is expected to reduce time for labeling experiments of the MFA. Measurable FAAs should, however, vary among the MFA experiments since the pool sizes of intracellular free metabolites depend on cellular metabolic conditions. In this study, minimal 13C enrichment data of FAAs was investigated to perform the FAAs-based MFA. An examination of a continuous culture of Escherichia coli using 13C-labeled glucose showed that the time required to reach an isotopically steady state for FAAs is rather faster than that for conventional method using proteinogenic amino acids (PAAs. Considering 95% confidence intervals, it was found that the metabolic flux distribution estimated using FAAs has a similar reliability to that of the PAAs-based method. The comparative analysis identified glutamate, aspartate, alanine and phenylalanine as the common amino acids observed in E. coli under different culture conditions. The results of MFA also demonstrated that the 13C enrichment data of the four amino acids is required for a reliable analysis of the flux distribution.

  1. Bacterial persistence is an active σS stress response to metabolic flux limitation.

    Science.gov (United States)

    Radzikowski, Jakub Leszek; Vedelaar, Silke; Siegel, David; Ortega, Álvaro Dario; Schmidt, Alexander; Heinemann, Matthias

    2016-09-21

    While persisters are a health threat due to their transient antibiotic tolerance, little is known about their phenotype and what actually causes persistence. Using a new method for persister generation and high-throughput methods, we comprehensively mapped the molecular phenotype of Escherichia coli during the entry and in the state of persistence in nutrient-rich conditions. The persister proteome is characterized by σ(S)-mediated stress response and a shift to catabolism, a proteome that starved cells tried to but could not reach due to absence of a carbon and energy source. Metabolism of persisters is geared toward energy production, with depleted metabolite pools. We developed and experimentally verified a model, in which persistence is established through a system-level feedback: Strong perturbations of metabolic homeostasis cause metabolic fluxes to collapse, prohibiting adjustments toward restoring homeostasis. This vicious cycle is stabilized and modulated by high ppGpp levels, toxin/anti-toxin systems, and the σ(S)-mediated stress response. Our system-level model consistently integrates past findings with our new data, thereby providing an important basis for future research on persisters. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

  2. Reduced TCA flux in diabetic myotubes: A governing influence on the diabetic phenotype?

    Science.gov (United States)

    Gaster, Michael

    2009-10-02

    The diabetic phenotype is complex, requiring elucidation of key initiating defects. It is unknown whether the reduced tricarboxylic acid cycle (TCA) flux in skeletal muscle of obese and obese type 2 diabetic (T2D) subjects is of primary origin. Acetate oxidation (measurement of TCA-flux) was significantly reduced in primary myotube cultures established from T2D versus lean subjects. Acetate oxidation was acutely stimulated by insulin and respiratory uncoupling. Inhibition of TCA flux in lean myotubes by malonate was followed by a measured decline in; acetate oxidation, complete palmitate oxidation, lipid uptake, glycogen synthesis, ATP content and increased glucose uptake, while glucose oxidation was unaffected. Acute TCA inhibition did not induce insulin resistance. Thus the reduced TCA cycle flux in T2D skeletal muscle may be of primary origin. The diabetic phenotype of increased basal glucose uptake and glucose oxidation, the reduced complete lipid oxidation and increased respiratory quotient, are likely to be adaptive responses to the reduced TCA cycle flux.

  3. Rumen microbial communities influence metabolic phenotypes in lambs

    Directory of Open Access Journals (Sweden)

    Diego P. Morgavi

    2015-10-01

    Full Text Available The rumen microbiota is an essential part of ruminants forging their nutrition and health. Despite its importance, it is not fully understood how various groups of rumen microbes affect host-microbe relationships and functions. The aim of the study was to simultaneously explore the rumen microbiota and the metabolic phenotype of lambs for identifying host-microbe associations and potential biomarkers of digestive functions. Twin lambs, separated in two groups after birth were exposed to practices (isolation and gavage with rumen fluid with protozoa or protozoa-depleted that differentially restricted the acquisition of microbes. Rumen microbiota, fermentation parameters, digestibility and growth were monitored for up to 31 weeks of age. Microbiota assembled in isolation from other ruminants lacked protozoa and had low bacterial and archaeal diversity whereas digestibility was not affected. Exposure to adult sheep microbiota increased bacterial and archaeal diversity independently of protozoa presence. For archaea, Methanomassiliicoccales displaced Methanosphaera. Notwithstanding, protozoa induced differences in functional traits such as digestibility and significantly shaped bacterial community structure, notably Ruminococcaceae and Lachnospiraceae lower up to 6 folds, Prevotellaceae lower by ~40%, and Clostridiaceae and Veillonellaceae higher up to 10 folds compared to microbiota without protozoa. An orthogonal partial least squares-discriminant analysis of urinary metabolome matched differences in microbiota structure. Discriminant metabolites were mainly involved in amino acids and protein metabolic pathways while a negative interaction was observed between methylotrophic methanogens Methanomassiliicoccales and trimethylamine N-oxide. These results stress the influence of gut microbes on animal phenotype and show the potential of metabolomics for monitoring rumen microbial functions.

  4. Estimation of dynamic flux profiles from metabolic time series data

    Directory of Open Access Journals (Sweden)

    Chou I-Chun

    2012-07-01

    Full Text Available Abstract Background Advances in modern high-throughput techniques of molecular biology have enabled top-down approaches for the estimation of parameter values in metabolic systems, based on time series data. Special among them is the recent method of dynamic flux estimation (DFE, which uses such data not only for parameter estimation but also for the identification of functional forms of the processes governing a metabolic system. DFE furthermore provides diagnostic tools for the evaluation of model validity and of the quality of a model fit beyond residual errors. Unfortunately, DFE works only when the data are more or less complete and the system contains as many independent fluxes as metabolites. These drawbacks may be ameliorated with other types of estimation and information. However, such supplementations incur their own limitations. In particular, assumptions must be made regarding the functional forms of some processes and detailed kinetic information must be available, in addition to the time series data. Results The authors propose here a systematic approach that supplements DFE and overcomes some of its shortcomings. Like DFE, the approach is model-free and requires only minimal assumptions. If sufficient time series data are available, the approach allows the determination of a subset of fluxes that enables the subsequent applicability of DFE to the rest of the flux system. The authors demonstrate the procedure with three artificial pathway systems exhibiting distinct characteristics and with actual data of the trehalose pathway in Saccharomyces cerevisiae. Conclusions The results demonstrate that the proposed method successfully complements DFE under various situations and without a priori assumptions regarding the model representation. The proposed method also permits an examination of whether at all, to what degree, or within what range the available time series data can be validly represented in a particular functional format of

  5. High throughput phenotypic analysis of Mycobacterium tuberculosis and Mycobacterium bovis strains' metabolism using biolog phenotype microarrays.

    Directory of Open Access Journals (Sweden)

    Bhagwati Khatri

    Full Text Available Tuberculosis is a major human and animal disease of major importance worldwide. Genetically, the closely related strains within the Mycobacterium tuberculosis complex which cause disease are well-characterized but there is an urgent need better to understand their phenotypes. To search rapidly for metabolic differences, a working method using Biolog Phenotype MicroArray analysis was developed. Of 380 substrates surveyed, 71 permitted tetrazolium dye reduction, the readout over 7 days in the method. By looking for ≥5-fold differences in dye reduction, 12 substrates differentiated M. tuberculosis H37Rv and Mycobacterium bovis AF2122/97. H37Rv and a Beijing strain of M. tuberculosis could also be distinguished in this way, as could field strains of M. bovis; even pairs of strains within one spoligotype could be distinguished by 2 to 3 substrates. Cluster analysis gave three clear groups: H37Rv, Beijing, and all the M. bovis strains. The substrates used agreed well with prior knowledge, though an unexpected finding that AF2122/97 gave greater dye reduction than H37Rv with hexoses was investigated further, in culture flasks, revealing that hexoses and Tween 80 were synergistic for growth and used simultaneously rather than in a diauxic fashion. Potential new substrates for growth media were revealed, too, most promisingly N-acetyl glucosamine. Osmotic and pH arrays divided the mycobacteria into two groups with different salt tolerance, though in contrast to the substrate arrays the groups did not entirely correlate with taxonomic differences. More interestingly, these arrays suggested differences between the amines used by the M. tuberculosis complex and enteric bacteria in acid tolerance, with some hydrophobic amino acids being highly effective. In contrast, γ-aminobutyrate, used in the enteric bacteria, had no effect in the mycobacteria. This study proved principle that Phenotype MicroArrays can be used with slow-growing pathogenic mycobacteria

  6. Identification of genetic elements in metabolism by high-throughput mouse phenotyping

    DEFF Research Database (Denmark)

    Rozman, Jan; Rathkolb, Birgit; Oestereicher, Manuela A.

    2018-01-01

    Metabolic diseases are a worldwide problem but the underlying genetic factors and their relevance to metabolic disease remain incompletely understood. Genome-wide research is needed to characterize so-far unannotated mammalian metabolic genes. Here, we generate and analyze metabolic phenotypic da...

  7. Metabolic Flux Analysis of the Synechocystis sp. PCC 6803 ΔnrtABCD Mutant Reveals a Mechanism for Metabolic Adaptation to Nitrogen-Limited Conditions.

    Science.gov (United States)

    Nakajima, Tsubasa; Yoshikawa, Katsunori; Toya, Yoshihiro; Matsuda, Fumio; Shimizu, Hiroshi

    2017-03-01

    Metabolic flux redirection during nitrogen-limited growth was investigated in the Synechocystis sp. PCC 6803 glucose-tolerant (GT) strain under photoautotrophic conditions by isotopically non-stationary metabolic flux analysis (INST-MFA). A ΔnrtABCD mutant of Synechocystis sp. PCC 6803 was constructed to reproduce phenotypes arising during nitrogen starvation. The ΔnrtABCD mutant and the wild-type GT strain were cultured under photoautotrophic conditions by a photobioreactor. Intracellular metabolites were labeled over a time course using NaH13CO3 as a carbon source. Based on these data, the metabolic flux distributions in the wild-type and ΔnrtABCD cells were estimated by INST-MFA. The wild-type GT and ΔnrtABCD strains displayed similar distribution patterns, although the absolute levels of metabolic flux were lower in ΔnrtABCD. Furthermore, the relative flux levels for glycogen metabolism, anaplerotic reactions and the oxidative pentose phosphate pathway were increased in ΔnrtABCD. This was probably due to the increased expression of enzyme genes that respond to nitrogen depletion. Additionally, we found that the ratio of ATP/NADPH demand increased slightly in the ΔnrtABCD mutant. These results indicated that futile ATP consumption increases under nitrogen-limited conditions because the Calvin-Benson cycle and the oxidative pentose phosphate pathway form a metabolic futile cycle that consumes ATP without CO2 fixation and NADPH regeneration. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  8. Transcript abundance on its own cannot be used to infer fluxes in central metabolism

    Directory of Open Access Journals (Sweden)

    Jörg eSchwender

    2014-11-01

    Full Text Available An attempt has been made to define the extent to which metabolic flux in central plant metabolism is reflected by changes in the transcriptome and metabolome, based on an analysis of in vitro cultured immature embryos of two oilseed rape (Brassica napus accessions which contrast for seed lipid accumulation. Metabolic flux analysis was used to constrain a flux balance metabolic model which included 671 biochemical and transport reactions within the central metabolism. This highly confident flux information was eventually used for comparative analysis of flux versus transcript (metabolite. Metabolite profiling succeeded in identifying 79 intermediates within the central metabolism, some of which differed quantitatively between the two accessions and displayed a significant shift corresponding to flux. An RNA-Seq based transcriptome analysis revealed a large number of genes which were differentially transcribed in the two accessions, including some enzymes/proteins active in major metabolic pathways. With a few exceptions, differential activity in the major pathways (glycolysis, TCA cycle, amino acid and fatty acid synthesis was not reflected in contrasting abundances of the relevant transcripts. The conclusion was that transcript abundance on its own cannot be used to infer metabolic activity/fluxes in central plant metabolism. This limitation needs to be borne in mind in evaluating transcriptome data and designing metabolic engineering experiments.

  9. Comprehensive metabolomic study of platelets reveals the expression of discrete metabolic phenotypes during storage

    DEFF Research Database (Denmark)

    Paglia, Giuseppe; Sigurjónsson, Ólafur E; Rolfsson, Óttar

    2014-01-01

    not undergo a monotonic decay, but experienced systematic changes in metabolism reflected in three discrete metabolic phenotypes: The first (Days 0-3) was associated with active glycolysis, pentose phosphate pathway, and glutathione metabolism and down regulation of tricarboxylic acid (TCA) cycle. The second...

  10. Metabolic flux analysis of the halophilic archaeon Haladaptatus paucihalophilus

    International Nuclear Information System (INIS)

    Liu, Guangxiu; Zhang, Manxiao; Mo, Tianlu; He, Lian; Zhang, Wei; Yu, Yi; Zhang, Qi; Ding, Wei

    2015-01-01

    This work reports the 13 C-assisted metabolic flux analysis of Haladaptatus paucihalophilus, a halophilic archaeon possessing an intriguing osmoadaption mechanism. We showed that the carbon flow is through the oxidative tricarboxylic acid (TCA) cycle whereas the reductive TCA cycle is not operative in H. paucihalophilus. In addition, both threonine and the citramalate pathways contribute to isoleucine biosynthesis, whereas lysine is synthesized through the diaminopimelate pathway and not through the α-aminoadipate pathway. Unexpected, the labeling patterns of glycine from the cells grown on [1- 13 C]pyruvate and [2- 13 C]pyruvate suggest that, unlike all the organisms investigated so far, in which glycine is produced exclusively from the serine hydroxymethyltransferase (SHMT) pathway, glycine biosynthesis in H. paucihalophilus involves different pathways including SHMT, threonine aldolase (TA) and the reverse reaction of glycine cleavage system (GCS), demonstrating for the first time that other pathways instead of SHMT can also make a significant contribution to the cellular glycine pool. Transcriptional analysis confirmed that both TA and GCS genes were transcribed in H. paucihalophilus, and the transcriptional level is independent of salt concentrations in the culture media. This study expands our understanding of amino acid biosynthesis and provides valuable insights into the metabolism of halophilic archaea. - Highlights: • Serine hydroxymethyltransferase, threonine aldolase, and glycine cleavage system all contribute to the glycine pool of H. paucihalophilus. • Threonine and the citramalate pathways contribute equally to the isoleucine biosynthesis in H. paucihalophilus. • Lysine in H. paucihalophilus is synthesized through the diaminopimelate pathway and not through the α-aminoadipate pathway. • Glycine biosynthesis is likely unrelated to the cell osmoadaption mechanism.

  11. Metabolic flux analysis of the halophilic archaeon Haladaptatus paucihalophilus

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Guangxiu; Zhang, Manxiao [Key Laboratory of Desert and Desertification, Cold and Arid Regions Environmental and Engineering Research Institute, Chinese Academy of Sciences, Lanzhou, 730000 (China); Key Laboratory of Extreme Environmental Microbial Resources and Engineering, Gansu Province, Lanzhou, 730000 (China); Mo, Tianlu [Department of Chemistry, Fudan University, Shanghai, 200433 (China); He, Lian [Key Laboratory of Combinatory Biosynthesis and Drug Discovery (Ministry of Education), School of Pharmaceutical Sciences, Wuhan University, Wuhan, 430071 (China); Zhang, Wei [Key Laboratory of Desert and Desertification, Cold and Arid Regions Environmental and Engineering Research Institute, Chinese Academy of Sciences, Lanzhou, 730000 (China); Key Laboratory of Extreme Environmental Microbial Resources and Engineering, Gansu Province, Lanzhou, 730000 (China); Yu, Yi, E-mail: yu_yi@whu.edu.cn [Key Laboratory of Combinatory Biosynthesis and Drug Discovery (Ministry of Education), School of Pharmaceutical Sciences, Wuhan University, Wuhan, 430071 (China); Zhang, Qi, E-mail: qizhang@sioc.ac.cn [Department of Chemistry, Fudan University, Shanghai, 200433 (China); Ding, Wei, E-mail: dingw@lzu.edu.cn [Key Laboratory of Desert and Desertification, Cold and Arid Regions Environmental and Engineering Research Institute, Chinese Academy of Sciences, Lanzhou, 730000 (China); Key Laboratory of Extreme Environmental Microbial Resources and Engineering, Gansu Province, Lanzhou, 730000 (China); Department of Chemistry, Fudan University, Shanghai, 200433 (China)

    2015-11-27

    This work reports the {sup 13}C-assisted metabolic flux analysis of Haladaptatus paucihalophilus, a halophilic archaeon possessing an intriguing osmoadaption mechanism. We showed that the carbon flow is through the oxidative tricarboxylic acid (TCA) cycle whereas the reductive TCA cycle is not operative in H. paucihalophilus. In addition, both threonine and the citramalate pathways contribute to isoleucine biosynthesis, whereas lysine is synthesized through the diaminopimelate pathway and not through the α-aminoadipate pathway. Unexpected, the labeling patterns of glycine from the cells grown on [1-{sup 13}C]pyruvate and [2-{sup 13}C]pyruvate suggest that, unlike all the organisms investigated so far, in which glycine is produced exclusively from the serine hydroxymethyltransferase (SHMT) pathway, glycine biosynthesis in H. paucihalophilus involves different pathways including SHMT, threonine aldolase (TA) and the reverse reaction of glycine cleavage system (GCS), demonstrating for the first time that other pathways instead of SHMT can also make a significant contribution to the cellular glycine pool. Transcriptional analysis confirmed that both TA and GCS genes were transcribed in H. paucihalophilus, and the transcriptional level is independent of salt concentrations in the culture media. This study expands our understanding of amino acid biosynthesis and provides valuable insights into the metabolism of halophilic archaea. - Highlights: • Serine hydroxymethyltransferase, threonine aldolase, and glycine cleavage system all contribute to the glycine pool of H. paucihalophilus. • Threonine and the citramalate pathways contribute equally to the isoleucine biosynthesis in H. paucihalophilus. • Lysine in H. paucihalophilus is synthesized through the diaminopimelate pathway and not through the α-aminoadipate pathway. • Glycine biosynthesis is likely unrelated to the cell osmoadaption mechanism.

  12. NIBBS-Search for Fast and Accurate Prediction of Phenotype-Biased Metabolic Systems

    Science.gov (United States)

    Padmanabhan, Kanchana; Shpanskaya, Yekaterina; Banfield, Jill; Scott, Kathleen; Mihelcic, James R.; Samatova, Nagiza F.

    2012-01-01

    Understanding of genotype-phenotype associations is important not only for furthering our knowledge on internal cellular processes, but also essential for providing the foundation necessary for genetic engineering of microorganisms for industrial use (e.g., production of bioenergy or biofuels). However, genotype-phenotype associations alone do not provide enough information to alter an organism's genome to either suppress or exhibit a phenotype. It is important to look at the phenotype-related genes in the context of the genome-scale network to understand how the genes interact with other genes in the organism. Identification of metabolic subsystems involved in the expression of the phenotype is one way of placing the phenotype-related genes in the context of the entire network. A metabolic system refers to a metabolic network subgraph; nodes are compounds and edges labels are the enzymes that catalyze the reaction. The metabolic subsystem could be part of a single metabolic pathway or span parts of multiple pathways. Arguably, comparative genome-scale metabolic network analysis is a promising strategy to identify these phenotype-related metabolic subsystems. Network Instance-Based Biased Subgraph Search (NIBBS) is a graph-theoretic method for genome-scale metabolic network comparative analysis that can identify metabolic systems that are statistically biased toward phenotype-expressing organismal networks. We set up experiments with target phenotypes like hydrogen production, TCA expression, and acid-tolerance. We show via extensive literature search that some of the resulting metabolic subsystems are indeed phenotype-related and formulate hypotheses for other systems in terms of their role in phenotype expression. NIBBS is also orders of magnitude faster than MULE, one of the most efficient maximal frequent subgraph mining algorithms that could be adjusted for this problem. Also, the set of phenotype-biased metabolic systems output by NIBBS comes very close to

  13. Common phenotype of resting mouse extensor digitorum longus and soleus muscles: equal ATPase and glycolytic flux during transient anoxia.

    Science.gov (United States)

    Vinnakota, Kalyan C; Rusk, Joshua; Palmer, Lauren; Shankland, Eric; Kushmerick, Martin J

    2010-06-01

    Rates of ATPase and glycolysis are several times faster in actively contracting mouse extensor digitorum longus muscle (EDL) than soleus (SOL), but we find these rates are not distinguishable at rest. We used a transient anoxic perturbation of steady state energy balance to decrease phosphocreatine (PCr) reversibly and to measure the rates of ATPase and of lactate production without muscle activation or contraction. The rate of glycolytic ATP synthesis is less than the ATPase rate, accounting for the continual PCr decrease during anoxia in both muscles. We fitted a mathematical model validated with properties of enzymes and solutes measured in vitro and appropriate for the transient perturbation of these muscles to experimental data to test whether the model accounts for the results. Simulations showed equal rates of ATPase and lactate production in both muscles. ATPase controls glycolytic flux by feedback from its products. Adenylate kinase function is critical because a rise in [AMP] is necessary to activate glycogen phosphorylase. ATPase is the primary source of H+ production. The sum of contributions of the 13 reactions of the glycogenolytic and glycolytic network to total proton load is negligible. The stoichiometry of lactate and H+ production is near unity. These results identify a default state of energy metabolism for resting muscle in which there is no difference in the metabolic phenotype of EDL and SOL. Therefore, additional control mechanisms, involving higher ATPase flux and [Ca2+], must exist to explain the well-known difference in glycolytic rates in fast-twitch and slow-twitch muscles in actively contracting muscle.

  14. Role of hormones in cartilage and joint metabolism: understanding an unhealthy metabolic phenotype in osteoarthritis.

    Science.gov (United States)

    Bay-Jensen, Anne C; Slagboom, Eline; Chen-An, Pingping; Alexandersen, Peter; Qvist, Per; Christiansen, Claus; Meulenbelt, Ingrid; Karsdal, Morten A

    2013-05-01

    Joint health is affected by local and systemic hormones. It is well accepted that systemic factors regulate the metabolism of joint tissues, and that substantial cross-talk between tissues actively contributes to homeostasis. In the current review, we try to define a subtype of osteoarthritis (OA), metabolic OA, which is dependent on an unhealthy phenotype. Peer-reviewed research articles and reviews were reviewed and summarized. Only literature readily available online, either by download or by purchase order, was included. OA is the most common joint disease and is more common in women after menopause. OA is a disease that affects the whole joint, including cartilage, subchondral bone, synovium, tendons, and muscles. The clinical endpoints of OA are pain and joint space narrowing, which is characterized by cartilage erosion and subchondral sclerosis, suggesting that cartilage is a central tissue of joint health. Thus, the joint, more specifically the cartilage, may be considered a target of endocrine function in addition to the well-described traditional risk factors of disease initiation and progression such as long-term loading of the joint due to obesity. Metabolic syndrome affects a range of tissues and may in part be molecularly described as a dysregulation of cytokines, adipokines, and hormones (e.g., estrogen and thyroid hormone). Consequently, metabolic imbalance may both directly and indirectly influence joint health and cartilage turnover, altering the progression of diseases such as OA. There is substantial evidence for a connection between metabolic health and development of OA. We propose that more focus be directed to understanding this connection to improve the management of menopausal health and associated comorbidities.

  15. MicrobesFlux: a web platform for drafting metabolic models from the KEGG database

    Directory of Open Access Journals (Sweden)

    Feng Xueyang

    2012-08-01

    Full Text Available Abstract Background Concurrent with the efforts currently underway in mapping microbial genomes using high-throughput sequencing methods, systems biologists are building metabolic models to characterize and predict cell metabolisms. One of the key steps in building a metabolic model is using multiple databases to collect and assemble essential information about genome-annotations and the architecture of the metabolic network for a specific organism. To speed up metabolic model development for a large number of microorganisms, we need a user-friendly platform to construct metabolic networks and to perform constraint-based flux balance analysis based on genome databases and experimental results. Results We have developed a semi-automatic, web-based platform (MicrobesFlux for generating and reconstructing metabolic models for annotated microorganisms. MicrobesFlux is able to automatically download the metabolic network (including enzymatic reactions and metabolites of ~1,200 species from the KEGG database (Kyoto Encyclopedia of Genes and Genomes and then convert it to a metabolic model draft. The platform also provides diverse customized tools, such as gene knockouts and the introduction of heterologous pathways, for users to reconstruct the model network. The reconstructed metabolic network can be formulated to a constraint-based flux model to predict and analyze the carbon fluxes in microbial metabolisms. The simulation results can be exported in the SBML format (The Systems Biology Markup Language. Furthermore, we also demonstrated the platform functionalities by developing an FBA model (including 229 reactions for a recent annotated bioethanol producer, Thermoanaerobacter sp. strain X514, to predict its biomass growth and ethanol production. Conclusion MicrobesFlux is an installation-free and open-source platform that enables biologists without prior programming knowledge to develop metabolic models for annotated microorganisms in the KEGG

  16. Flux balance analysis of genome-scale metabolic model of rice ...

    Indian Academy of Sciences (India)

    Here, we analyse a genome-scale metabolic model of rice leaf using Flux Balance Analysis to investigate whether it has potential metabolic flexibility to increase the biosynthesis of any of the biomass components. We initially simulate the metabolic responses under an objective to maximize the biomass components.

  17. Secular change in 13 metabolic phenotypes: A Chinese longitudinal twin study

    DEFF Research Database (Denmark)

    Li, Shuxia; Pang, Zengchang; Zhang, Dongfeng

    Aims: The genetic and environmental influences on metabolic phenotypes have been intensively studied by twin modeling in different populations. However, twin studies on secular change in metabolic phenotypes have been rare due to high expenses, losses of follow up, and long waiting time in prospe......Aims: The genetic and environmental influences on metabolic phenotypes have been intensively studied by twin modeling in different populations. However, twin studies on secular change in metabolic phenotypes have been rare due to high expenses, losses of follow up, and long waiting time......; LDL), weight and blood pressure (SBP, DBP) have AE model as the best fitting model with low to moderate (a2: 0.25 - 0.46) genetic control. Variations in secular change in the rest of the phenotypes have CE model as the best fitting model with low to moderate (c2: 0.21 - 0.55) control by shared...... environmental factors. Secular changes in all phenotypes are under moderate to high (e2: 0.45 - 0.79) control by unique environmental factors. Conclusions: Variations in secular change in the 13 metabolic phenotypes show limited genetic control. Our results emphasize the special importance of unique environment...

  18. Longitudinal Investigation into Genetics in the Conservation of Metabolic Phenotypes in Danish and Chinese Twins

    DEFF Research Database (Denmark)

    Li, Shuxia; Kyvik, Kirsten Ohm; Duan, Haiping

    2016-01-01

    twin study on long-term stability of metabolic phenotypes in Danish and Chinese twins identified a common pattern of high genetic control over phenotype conservation, and at the same time revealed population-specific patterns of genetic and common environmental regulation on the variance as well...

  19. [Clinical, hormonal and metabolic characteristics of different phenotypes of polycystic ovary syndrome, in Bulgarian population].

    Science.gov (United States)

    Kavardzhikova, S; Pechlivanov, B

    2010-01-01

    Our aim was to investigate the percentage occurrence of different phenotypes of polycystic ovary syndrome (PCOS) in a Bulgarian population, and their clinical, metabolic and hormonal characteristics. The study included 110 women with PCOS, diagnosed according to the Europian Society of Human Reproduction & Embriology/American Society for Reproductive Medicine criteria. The women were divided into four phenotypes: hyperandrogenism (HA) + oligo-/ anovulation (OA) + polycystic ovaries at ultrasound (PCO) ( full-blown syndrome, phenotype A); HA + OA (former Institute of Health definition, phenotype B); OA + PCO (phenotype C); and HA + PCO (phenotype D). Serum levels of testosteron, immune-reactive insulin, sex hormone-binding globulin, dehydroepiandrosterone sulfate and lipid metabolism parameters were measured. Free androgen index and homeostasis model assessment of insulin resistance were calculated. Body mass index and waist- to--hip ratio were assessed. The percentage of phenotypes A, B, C and D in a Bulgarian Population are 53.6%, 12.8%, 11%, 22.6% respectively. The women with the classical form of PCOS (phenotypes A and B) were more obese, had more strongly expressed hyperandrogenemia, and were more insulin--resistant compared with the women of phenotypes C and D. There is a significant difference in anthropometric, hormonal and metabolic indices between the classical form and the clinical variants of PCOS in the studied Bulgarian population.

  20. Exome sequencing-driven discovery of coding polymorphisms associated with common metabolic phenotypes

    NARCIS (Netherlands)

    Albrechtsen, A.; Grarup, N.; Li, Y.; Sparso, T.; Tian, G.; Cao, H.; Jiang, T.; Kim, S.Y.; Korneliussen, T.; Li, Q.; Nie, C.; Wu, R.; Skotte, L.; Morris, A.P.; Ladenvall, C.; Cauchi, S.; Stancakova, A.; Andersen, G.; Astrup, A.; Banasik, K.; Bennett, A.J.; Bolund, L.; Charpentier, G.; Chen, Y.; Dekker, J.M.; Doney, A.S.F.; Dorkhan, M.; Forsen, T.; Frayling, T.M.; Groves, C.J.; Gui, Y; Hallmans, G.; Hattersley, A.T.; He, K.; Hitman, G.A.; Holmkvist, J.; Huang, S.; Jiang, H.; Jin, X.; Justesen, J.M.; Kristiansen, K.; Kuusisto, J.; Lajer, M.; Lantieri, O.; Li, W.; Liang, H.; Liao, Q.; Liu, X.; Ma, T.; Ma, X.; Manijak, M.P.; Marre, M.; Mokrosinski, J.; Morris, A.D.; Mu, B.; Nielsen, A.A.; Nijpels, G.; Nilsson, P.; Palmer, C.N.A.; Rayner, N.W.; Renstrom, F.; Ribel-Madsen, R.; Robertson, N.; Rolandsson, O.; Rossing, P.; Schwartz, T.W.; Slagboom, P.E.; Sterner, M.; Tang, M.; Tarnow, L.; Tuomi, T.; van 't Riet, E.; van Leeuwen, N.; Varga, T.V.; Vestmar, M.A.; Walker, M.; Wang, B.; Wang, Y.; Wu, H.; Xi, F.; Yengo, L.; Yu, C.; Zhang, X.; Zhang, J.; Zhang, Q.; Zhang, W.; Zheng, H.; Zhou, Y.; Altshuler, D.; 't Hart, L.M.; Franks, P.W.; Balkau, B.; Froguel, P.; McCarthy, M.I.; Laakso, M.; Groop, L.; Christensen, C.; Brandslund, I.; Lauritzen, T.; Witte, D.R.; Linneberg, A.; Jorgensen, T.; Hansen, T.; Wang, J.; Nielsen, R.; Pedersen, O.

    2013-01-01

    Aims/hypothesis: Human complex metabolic traits are in part regulated by genetic determinants. Here we applied exome sequencing to identify novel associations of coding polymorphisms at minor allele frequencies (MAFs) >1% with common metabolic phenotypes. Methods: The study comprised three stages.

  1. Exome sequencing-driven discovery of coding polymorphisms associated with common metabolic phenotypes

    DEFF Research Database (Denmark)

    Albrechtsen, Anders; Grarup, Niels; Li, Y

    2013-01-01

    AIMS/HYPOTHESIS: Human complex metabolic traits are in part regulated by genetic determinants. Here we applied exome sequencing to identify novel associations of coding polymorphisms at minor allele frequencies (MAFs) >1% with common metabolic phenotypes. METHODS: The study comprised three stages...

  2. Whole genome sequencing of Saccharomyces cerevisiae: from genotype to phenotype for improved metabolic engineering applications

    DEFF Research Database (Denmark)

    Otero, José Manuel; Vongsangnak, Wanwipa; Asadollahi, Mohammadali

    2010-01-01

    selective pressure is applied to a partially genetically engineered strain to confer a desirable phenotype. The exact genetic modification or resulting genotype that leads to the improved phenotype is often not identified or understood to enable further metabolic engineering. RESULTS: In this work we...... and CEN.PK113-7D in both glucose and galactose batch cultures did not provide a clear hypothesis for major phenotypes observed, suggesting that genotype to phenotype correlations are manifested post-transcriptionally or post-translationally either through protein concentration and/or function. CONCLUSIONS...

  3. Flux balance analysis of genome-scale metabolic model of rice ...

    Indian Academy of Sciences (India)

    2015-09-28

    Sep 28, 2015 ... genome-scale metabolic model of rice leaf using Flux Balance Analysis to investigate whether it has potential .... is number of reactions. In steady state. S.v = 0. (2) where v is the flux vector of reactions (Kauffman et al. 2003). Objective function is. Z = w.v. (3). 820 .... All the reactions are not included.

  4. Invariability of Central Metabolic Flux Distribution in Shewanella oneidensis MR-1 Under Environmental or Genetic Perturbations

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Yinjie; Martin, Hector Garcia; Deutschbauer, Adam; Feng, Xueyang; Huang, Rick; Llora, Xavier; Arkin, Adam; Keasling, Jay D.

    2009-04-21

    An environmentally important bacterium with versatile respiration, Shewanella oneidensis MR-1, displayed significantly different growth rates under three culture conditions: minimal medium (doubling time {approx} 3 hrs), salt stressed minimal medium (doubling time {approx} 6 hrs), and minimal medium with amino acid supplementation (doubling time {approx}1.5 hrs). {sup 13}C-based metabolic flux analysis indicated that fluxes of central metabolic reactions remained relatively constant under the three growth conditions, which is in stark contrast to the reported significant changes in the transcript and metabolite profiles under various growth conditions. Furthermore, ten transposon mutants of S. oneidensis MR-1 were randomly chosen from a transposon library and their flux distributions through central metabolic pathways were revealed to be identical, even though such mutational processes altered the secondary metabolism, for example, glycine and C1 (5,10-Me-THF) metabolism.

  5. Detecting the Significant Flux Backbone of Escherichia coli metabolism.

    Science.gov (United States)

    Güell, Oriol; Sagués, Francesc; Serrano, M Ángeles

    2017-05-01

    The heterogeneity of computationally predicted reaction fluxes in metabolic networks within a single flux state can be exploited to detect their significant flux backbone. Here, we disclose the backbone of Escherichia coli, and compare it with the backbones of other bacteria. We find that, in general, the core of the backbones is mainly composed of reactions in energy metabolism corresponding to ancient pathways. In E. coli, the synthesis of nucleotides and the metabolism of lipids form smaller cores which rely critically on energy metabolism. Moreover, the consideration of different media leads to the identification of pathways sensitive to environmental changes. The metabolic backbone of an organism is thus useful to trace simultaneously both its evolution and adaptation fingerprints. © 2017 The Authors FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

  6. Heritability of eleven metabolic phenotypes in Danish and Chinese twins

    DEFF Research Database (Denmark)

    Li, Shuxia; Duan, Hongmei; Pang, Zengchang

    2013-01-01

    modeling was performed on full and nested models with the best fitting models selected. Results: Heritability estimates were compared between Danish and Chinese samples to identify differential genetic influences on each of the phenotypes. Except for hip circumference, all other body measures exhibited...

  7. Rumen microbial communities influence metabolic phenotypes in lambs

    DEFF Research Database (Denmark)

    Morgavi, Diego P.; Rahahao-Paris, Estelle; Popova, Milka

    2015-01-01

    in microbiota structure. Discriminant metabolites were mainly involved in amino acids and protein metabolic pathways while a negative interaction was observed between methylotrophic methanogens Methanomassiliicoccales and trimethylamine N-oxide. These results stress the influence of gut microbes on animal...

  8. COMPLETE-MFA: complementary parallel labeling experiments technique for metabolic flux analysis.

    Science.gov (United States)

    Leighty, Robert W; Antoniewicz, Maciek R

    2013-11-01

    We have developed a novel approach for measuring highly accurate and precise metabolic fluxes in living cells, termed COMPLETE-MFA, short for complementary parallel labeling experiments technique for metabolic flux analysis. The COMPLETE-MFA method is based on combined analysis of multiple isotopic labeling experiments, where the synergy of using complementary tracers greatly improves the precision of estimated fluxes. In this work, we demonstrate the COMPLETE-MFA approach using all singly labeled glucose tracers, [1-(13)C], [2-(13)C], [3-(13)C], [4-(13)C], [5-(13)C], and [6-(13)C]glucose to determine precise metabolic fluxes for wild-type Escherichia coli. Cells were grown in six parallel cultures on defined medium with glucose as the only carbon source. Mass isotopomers of biomass amino acids were measured by gas chromatography-mass spectrometry (GC-MS). The data from all six experiments were then fitted simultaneously to a single flux model to determine accurate intracellular fluxes. We obtained a statistically acceptable fit with more than 300 redundant measurements. The estimated flux map is the most precise flux result obtained thus far for E. coli cells. To our knowledge, this is the first time that six isotopic labeling experiments have been successfully integrated for high-resolution (13)C-flux analysis. Copyright © 2013 Elsevier Inc. All rights reserved.

  9. Evaluation of 13C isotopic tracers for metabolic flux analysis in mammalian cells.

    Science.gov (United States)

    Metallo, Christian M; Walther, Jason L; Stephanopoulos, Gregory

    2009-11-01

    (13)C metabolic flux analysis (MFA) is the most comprehensive means of characterizing cellular metabolic states. Uniquely labeled isotopic tracers enable more focused analyses to probe specific reactions within the network. As a result, the choice of tracer largely determines the precision with which one can estimate metabolic fluxes, especially in complex mammalian systems that require multiple substrates. Here we have experimentally determined metabolic fluxes in a tumor cell line, successfully recapitulating the hallmarks of cancer cell metabolism. Using these data, we computationally evaluated specifically labeled (13)C glucose and glutamine tracers for their ability to precisely and accurately estimate fluxes in central carbon metabolism. These methods enabled us to identify the optimal tracer for analyzing individual fluxes, specific pathways, and central carbon metabolism as a whole. [1,2-(13)C(2)]glucose provided the most precise estimates for glycolysis, the pentose phosphate pathway, and the overall network. Tracers such as [2-(13)C]glucose and [3-(13)C]glucose also outperformed the more commonly used [1-(13)C]glucose. [U-(13)C(5)]glutamine emerged as the preferred isotopic tracer for the analysis of the tricarboxylic acid (TCA) cycle. These results provide valuable, quantitative information on the performance of (13)C-labeled substrates and can aid in the design of more informative MFA experiments in mammalian cell culture.

  10. Flux analysis uncovers key role of functional redundancy in formaldehyde metabolism.

    Directory of Open Access Journals (Sweden)

    Christopher J Marx

    2005-02-01

    Full Text Available Genome-scale analysis of predicted metabolic pathways has revealed the common occurrence of apparent redundancy for specific functional units, or metabolic modules. In many cases, mutation analysis does not resolve function, and instead, direct experimental analysis of metabolic flux under changing conditions is necessary. In order to use genome sequences to build models of cellular function, it is important to define function for such apparently redundant systems. Here we describe direct flux measurements to determine the role of redundancy in three modules involved in formaldehyde assimilation and dissimilation in a bacterium growing on methanol. A combination of deuterium and (14C labeling was used to measure the flux through each of the branches of metabolism for growth on methanol during transitions into and out of methylotrophy. The cells were found to differentially partition formaldehyde among the three modules depending on the flux of methanol into the cell. A dynamic mathematical model demonstrated that the kinetic constants of the enzymes involved are sufficient to account for this phenomenon. We demonstrate the role of redundancy in formaldehyde metabolism and have uncovered a new paradigm for coping with toxic, high-flux metabolic intermediates: a dynamic, interconnected metabolic loop.

  11. Flux-Enabled Exploration of the Role of Sip1 in galactose yeast metabolism

    DEFF Research Database (Denmark)

    Shymansky, Christopher M.; Wang, George; Baidoo, Edward E. K.

    2017-01-01

    13C metabolic flux analysis (13C MFA) is an important systems biology technique that has been used to investigate microbial metabolism for decades. The heterotrimer Snf1 kinase complex plays a key role in the preference Saccharomyces cerevisiae exhibits for glucose over galactose, a phenomenon kn...

  12. Isotopically nonstationary metabolic flux analysis (INST-MFA) of photosynthesis and photorespiration in plants

    Science.gov (United States)

    Photorespiration is a central component of photosynthesis; however to better understand its role it should be viewed in the context of an integrated metabolic network rather than a series of individual reactions that operate independently. Isotopically nonstationary 13C metabolic flux analysis (INST...

  13. Modelling central metabolic fluxes by constraint-based optimization reveals metabolic reprogramming of developing Solanum lycopersicum (tomato) fruit.

    Science.gov (United States)

    Colombié, Sophie; Nazaret, Christine; Bénard, Camille; Biais, Benoît; Mengin, Virginie; Solé, Marion; Fouillen, Laëtitia; Dieuaide-Noubhani, Martine; Mazat, Jean-Pierre; Beauvoit, Bertrand; Gibon, Yves

    2015-01-01

    Modelling of metabolic networks is a powerful tool to analyse the behaviour of developing plant organs, including fruits. Guided by our current understanding of heterotrophic metabolism of plant cells, a medium-scale stoichiometric model, including the balance of co-factors and energy, was constructed in order to describe metabolic shifts that occur through the nine sequential stages of Solanum lycopersicum (tomato) fruit development. The measured concentrations of the main biomass components and the accumulated metabolites in the pericarp, determined at each stage, were fitted in order to calculate, by derivation, the corresponding external fluxes. They were used as constraints to solve the model by minimizing the internal fluxes. The distribution of the calculated fluxes of central metabolism were then analysed and compared with known metabolic behaviours. For instance, the partition of the main metabolic pathways (glycolysis, pentose phosphate pathway, etc.) was relevant throughout fruit development. We also predicted a valid import of carbon and nitrogen by the fruit, as well as a consistent CO2 release. Interestingly, the energetic balance indicates that excess ATP is dissipated just before the onset of ripening, supporting the concept of the climacteric crisis. Finally, the apparent contradiction between calculated fluxes with low values compared with measured enzyme capacities suggest a complex reprogramming of the metabolic machinery during fruit development. With a powerful set of experimental data and an accurate definition of the metabolic system, this work provides important insight into the metabolic and physiological requirements of the developing tomato fruits. © 2014 The Authors The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.

  14. Synergizing metabolic flux analysis and nucleotide sugar metabolism to understand the control of glycosylation of recombinant protein in CHO cells

    LENUS (Irish Health Repository)

    Burleigh, Susan C

    2011-10-18

    Abstract Background The glycosylation of recombinant proteins can be altered by a range of parameters including cellular metabolism, metabolic flux and the efficiency of the glycosylation process. We present an experimental set-up that allows determination of these key processes associated with the control of N-linked glycosylation of recombinant proteins. Results Chinese hamster ovary cells (CHO) were cultivated in shake flasks at 0 mM glutamine and displayed a reduced growth rate, glucose metabolism and a slower decrease in pH, when compared to other glutamine-supplemented cultures. The N-linked glycosylation of recombinant human chorionic gonadotrophin (HCG) was also altered under these conditions; the sialylation, fucosylation and antennarity decreased, while the proportion of neutral structures increased. A continuous culture set-up was subsequently used to understand the control of HCG glycosylation in the presence of varied glutamine concentrations; when glycolytic flux was reduced in the absence of glutamine, the glycosylation changes that were observed in shake flask culture were similarly detected. The intracellular content of UDP-GlcNAc was also reduced, which correlated with a decrease in sialylation and antennarity of the N-linked glycans attached to HCG. Conclusions The use of metabolic flux analysis illustrated a case of steady state multiplicity, where use of the same operating conditions at each steady state resulted in altered flux through glycolysis and the TCA cycle. This study clearly demonstrated that the control of glycoprotein microheterogeneity may be examined by use of a continuous culture system, metabolic flux analysis and assay of intracellular nucleotides. This system advances our knowledge of the relationship between metabolic flux and the glycosylation of biotherapeutics in CHO cells and will be of benefit to the bioprocessing industry.

  15. SUMOFLUX: A Generalized Method for Targeted 13C Metabolic Flux Ratio Analysis.

    Directory of Open Access Journals (Sweden)

    Maria Kogadeeva

    2016-09-01

    Full Text Available Metabolic fluxes are a cornerstone of cellular physiology that emerge from a complex interplay of enzymes, carriers, and nutrients. The experimental assessment of in vivo intracellular fluxes using stable isotopic tracers is essential if we are to understand metabolic function and regulation. Flux estimation based on 13C or 2H labeling relies on complex simulation and iterative fitting; processes that necessitate a level of expertise that ordinarily preclude the non-expert user. To overcome this, we have developed SUMOFLUX, a methodology that is broadly applicable to the targeted analysis of 13C-metabolic fluxes. By combining surrogate modeling and machine learning, we trained a predictor to specialize in estimating flux ratios from measurable 13C-data. SUMOFLUX targets specific flux features individually, which makes it fast, user-friendly, applicable to experimental design and robust in terms of experimental noise and exchange flux magnitude. Collectively, we predict that SUMOFLUX's properties realistically pave the way to high-throughput flux analyses.

  16. Co-regulation of metabolic genes is better explained by flux coupling than by network distance.

    Directory of Open Access Journals (Sweden)

    Richard A Notebaart

    2008-01-01

    Full Text Available To what extent can modes of gene regulation be explained by systems-level properties of metabolic networks? Prior studies on co-regulation of metabolic genes have mainly focused on graph-theoretical features of metabolic networks and demonstrated a decreasing level of co-expression with increasing network distance, a naïve, but widely used, topological index. Others have suggested that static graph representations can poorly capture dynamic functional associations, e.g., in the form of dependence of metabolic fluxes across genes in the network. Here, we systematically tested the relative importance of metabolic flux coupling and network position on gene co-regulation, using a genome-scale metabolic model of Escherichia coli. After validating the computational method with empirical data on flux correlations, we confirm that genes coupled by their enzymatic fluxes not only show similar expression patterns, but also share transcriptional regulators and frequently reside in the same operon. In contrast, we demonstrate that network distance per se has relatively minor influence on gene co-regulation. Moreover, the type of flux coupling can explain refined properties of the regulatory network that are ignored by simple graph-theoretical indices. Our results underline the importance of studying functional states of cellular networks to define physiologically relevant associations between genes and should stimulate future developments of novel functional genomic tools.

  17. Objective assessment of dietary patterns by use of metabolic phenotyping

    DEFF Research Database (Denmark)

    Garcia-Perez, Isabel; Posma, Joram M; Gibson, Rachel

    2017-01-01

    BACKGROUND: Accurate monitoring of changes in dietary patterns in response to food policy implementation is challenging. Metabolic profiling allows simultaneous measurement of hundreds of metabolites in urine, the concentrations of which can be affected by food intake. We hypothesised that metabo...

  18. Methionine metabolism and phenotypic variability in X-linked adrenoleukodystrophy

    NARCIS (Netherlands)

    Linnebank, M.; Kemp, S.; Wanders, R. J. A.; Kleijer, W. J.; van der Sterre, M. L. T.; Gärtner, J.; Fliessbach, K.; Semmler, A.; Sokolowski, P.; Köhler, W.; Schlegel, U.; Schmidt, S.; Klockgether, T.; Wüllner, U.

    2006-01-01

    A combined genotype of polymorphisms of methionine metabolism has been associated with CNS demyelination in methotrexate-treated patients. Within a sample of 86 patients with X-linked adrenoleukodystrophy, this genotype was overrepresented in a subgroup of 15 patients with adrenomyeloneuropathy

  19. In vivo dynamics of galactose metabolism in Saccharomyces cerevisiae: Metabolic fluxes and metabolite levels

    DEFF Research Database (Denmark)

    Østergaard, Simon; Olsson, Lisbeth; Nielsen, Jens

    2001-01-01

    the flux entering the TCA cycle was low and acetate and ethanol started to be excreted to the extracellular medium. During recovery of cell growth the flux entering the TCA cycle increased again, and at t = 30 this flux exceeded the corresponding steady-state flux. During the pulse an enhanced level of Gal...

  20. Objective assessment of dietary patterns by use of metabolic phenotyping

    DEFF Research Database (Denmark)

    Garcia-Perez, Isabel; Posma, Joram M; Gibson, Rachel

    2017-01-01

    BACKGROUND: Accurate monitoring of changes in dietary patterns in response to food policy implementation is challenging. Metabolic profiling allows simultaneous measurement of hundreds of metabolites in urine, the concentrations of which can be affected by food intake. We hypothesised...... that metabolic profiles of urine samples developed under controlled feeding conditions reflect dietary intake and can be used to model and classify dietary patterns of free-living populations. METHODS: In this randomised, controlled, crossover trial, we recruited healthy volunteers (aged 21-65 years, BMI 20...... controlled environment can classify groups of free-living people into consumers of diets associated with lower or higher non-communicable disease risk on the basis of multivariate metabolite patterns. This approach enables objective monitoring of dietary patterns in population settings and enhances...

  1. Changes in muscle cell metabolism and mechanotransduction are associated with myopathic phenotype in a mouse model of collagen VI deficiency.

    Directory of Open Access Journals (Sweden)

    Sara De Palma

    Full Text Available This study identifies metabolic and protein phenotypic alterations in gastrocnemius, tibialis anterior and diaphragm muscles of Col6a1(-/- mice, a model of human collagen VI myopathies. All three muscles of Col6a1(-/- mice show some common changes in proteins involved in metabolism, resulting in decreased glycolysis and in changes of the TCA cycle fluxes. These changes lead to a different fate of α-ketoglutarate, with production of anabolic substrates in gastrocnemius and tibialis anterior, and with lipotoxicity in diaphragm. The metabolic changes are associated with changes of proteins involved in mechanotransduction at the myotendineous junction/costameric/sarcomeric level (TN-C, FAK, ROCK1, troponin I fast and in energy metabolism (aldolase, enolase 3, triose phosphate isomerase, creatine kinase, adenylate kinase 1, parvalbumin, IDH1 and FASN. Together, these change may explain Ca(2+ deregulation, impaired force development, increased muscle-relaxation-time and fiber damage found in the mouse model as well as in patients. The severity of these changes differs in the three muscles (gastrocnemius

  2. Changes in Muscle Cell Metabolism and Mechanotransduction Are Associated with Myopathic Phenotype in a Mouse Model of Collagen VI Deficiency

    Science.gov (United States)

    De Palma, Sara; Leone, Roberta; Grumati, Paolo; Vasso, Michele; Polishchuk, Roman; Capitanio, Daniele; Braghetta, Paola; Bernardi, Paolo; Bonaldo, Paolo; Gelfi, Cecilia

    2013-01-01

    This study identifies metabolic and protein phenotypic alterations in gastrocnemius, tibialis anterior and diaphragm muscles of Col6a1−/− mice, a model of human collagen VI myopathies. All three muscles of Col6a1−/− mice show some common changes in proteins involved in metabolism, resulting in decreased glycolysis and in changes of the TCA cycle fluxes. These changes lead to a different fate of α-ketoglutarate, with production of anabolic substrates in gastrocnemius and tibialis anterior, and with lipotoxicity in diaphragm. The metabolic changes are associated with changes of proteins involved in mechanotransduction at the myotendineous junction/costameric/sarcomeric level (TN-C, FAK, ROCK1, troponin I fast) and in energy metabolism (aldolase, enolase 3, triose phosphate isomerase, creatine kinase, adenylate kinase 1, parvalbumin, IDH1 and FASN). Together, these change may explain Ca2+ deregulation, impaired force development, increased muscle-relaxation-time and fiber damage found in the mouse model as well as in patients. The severity of these changes differs in the three muscles (gastrocnemiusmass-to-tendon (myotendineous junction) ratio and to muscle morphology. PMID:23437220

  3. Integration of a constraint-based metabolic model of Brassica napus developing seeds with 13C-Metabolic Flux Analysis

    Directory of Open Access Journals (Sweden)

    Jordan eHay

    2014-12-01

    Full Text Available The use of large-scale or genome-scale metabolic reconstructions for modeling and simulation of plant metabolism and integration of those models with large-scale omics and experimental flux data is becoming increasingly important in plant metabolic research. Here we report an updated version of bna572, a bottom-up reconstruction of oilseed rape (Brassica napus L.; Brassicaceae developing seeds with emphasis on representation of biomass-component biosynthesis. New features include additional seed-relevant pathways for isoprenoid, sterol, phenylpropanoid, flavonoid, and choline biosynthesis. Being now based on standardized data formats and procedures for model reconstruction, bna572+ is available as a COBRA-compliant Systems Biology Markup Language (SBML model and conforms to the Minimum Information Requested in the Annotation of Biochemical Models (MIRIAM standards for annotation of external data resources. Bna572+ contains 966 genes, 671 reactions, and 666 metabolites distributed among 11 subcellular compartments. It is referenced to the Arabidopsis thaliana genome, with gene-protein-reaction associations resolving subcellular localization. Detailed mass and charge balancing and confidence scoring were applied to all reactions. Using Brassica napus seed specific transcriptome data, expression was verified for 78% of bna572+ genes and 97% of reactions. Alongside bna572+ we also present a revised carbon centric model for 13C-Metabolic Flux Analysis (13C-MFA with all its reactions being referenced to bna572+ based on linear projections. By integration of flux ratio constraints obtained from 13C-MFA and by elimination of infinite flux bounds around thermodynamically infeasible loops based on COBRA loopless methods, we demonstrate improvements in predictive power of Flux Variability Analysis (FVA. Using this combined approach we characterize the difference in metabolic flux of developing seeds of two Brassica napus genotypes contrasting in starch and

  4. Dynamic metabolic flux analysis using B-splines to study the effects of temperature shift on CHO cell metabolism

    Directory of Open Access Journals (Sweden)

    Verónica S. Martínez

    2015-12-01

    Full Text Available Metabolic flux analysis (MFA is widely used to estimate intracellular fluxes. Conventional MFA, however, is limited to continuous cultures and the mid-exponential growth phase of batch cultures. Dynamic MFA (DMFA has emerged to characterize time-resolved metabolic fluxes for the entire culture period. Here, the linear DMFA approach was extended using B-spline fitting (B-DMFA to estimate mass balanced fluxes. Smoother fits were achieved using reduced number of knots and parameters. Additionally, computation time was greatly reduced using a new heuristic algorithm for knot placement. B-DMFA revealed that Chinese hamster ovary cells shifted from 37 °C to 32 °C maintained a constant IgG volume-specific productivity, whereas the productivity for the controls peaked during mid-exponential growth phase and declined afterward. The observed 42% increase in product titer at 32 °C was explained by a prolonged cell growth with high cell viability, a larger cell volume and a more stable volume-specific productivity. Keywords: Dynamic, Metabolism, Flux analysis, CHO cells, Temperature shift, B-spline curve fitting

  5. Analysis of polyhydroxybutyrate flux limitations by systematic genetic and metabolic perturbations.

    Science.gov (United States)

    Tyo, Keith E J; Fischer, Curt R; Simeon, Fritz; Stephanopoulos, Gregory

    2010-05-01

    Poly-3-hydroxybutyrate (PHB) titers in Escherichia coli have benefited from 10+ years of metabolic engineering. In the majority of studies, PHB content, expressed as percent PHB (dry cell weight), is increased, although this increase can be explained by decreases in growth rate or increases in PHB flux. In this study, growth rate and PHB flux were quantified directly in response to systematic manipulation of (1) gene expression in the product-forming pathway and (2) growth rates in a nitrogen-limited chemostat. Gene expression manipulation revealed acetoacetyl-CoA reductase (phaB) limits flux to PHB, although overexpression of the entire pathway pushed the flux even higher. These increases in PHB flux are accompanied by decreases in growth rate, which can be explained by carbon diversion, rather than toxic effects of the PHB pathway. In chemostats, PHB flux was insensitive to growth rate. These results imply that PHB flux is primarily controlled by the expression levels of the product forming pathway and not by the availability of precursors. These results confirm prior in vitro measurements and metabolic models and show expression level is a major affecter of PHB flux. 2009 Elsevier Inc. All rights reserved.

  6. Bacterial adaptation through distributed sensing of metabolic fluxes

    NARCIS (Netherlands)

    Kotte, Oliver; Zaugg, Judith B.; Heinemann, Matthias

    The recognition of carbon sources and the regulatory adjustments to recognized changes are of particular importance for bacterial survival in fluctuating environments. Despite a thorough knowledge base of Escherichia coli’s central metabolism and its regulation, fundamental aspects of the employed

  7. Pool size measurements facilitate the determination of fluxes at branching points in nonstationary metabolic flux analysis: The case of Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Robert eHeise

    2015-06-01

    Full Text Available Pool size measurements are important for the estimation of absolute intracellular fluxes in particular scenarios based on data from heavy carbon isotope experiments. Recently, steady-state fluxes estimates were obtained for central carbon metabolism in an intact illuminated rosette of Arabidopsis thaliana grown photoautotrophically (Szecowka et al., 2013; Heise et al., 2014. Fluxes were estimated therein by integrating mass-spectrometric data of the dynamics of the unlabeled metabolic fraction, data on metabolic pool sizes, partitioning of metabolic pools between cellular compartments and estimates of photosynthetically inactive pools, with a simplified model of plant central carbon metabolism. However, the fluxes were determined by treating the pool sizes as fixed parameters. Here we investigated whether and, if so, to what extent the treatment of pool sizes as parameters to be optimized in three scenarios may affect the flux estimates. The results are discussed in terms of benchmark values for canonical pathways and reactions, including starch and sucrose synthesis as well as the ribulose-1,5-bisphosphate carboxylation and oxygenation reactions. In addition, we discuss pathways emerging from a divergent branch point for which pool sizes are required for flux estimation, irrespective of the computational approach used for the simulation of the observable labelling pattern. Therefore, our findings indicate the necessity for development of techniques for accurate pool size measurements to improve the quality of flux estimates from nonstationary flux estimates in intact plant cells in the absence of alternative flux measurements.

  8. Impact of tumor microenvironment and epithelial phenotypes on metabolism in breast cancer.

    Science.gov (United States)

    Brauer, Heather Ann; Makowski, Liza; Hoadley, Katherine A; Casbas-Hernandez, Patricia; Lang, Lindsay J; Romàn-Pèrez, Erick; D'Arcy, Monica; Freemerman, Alex J; Perou, Charles M; Troester, Melissa A

    2013-02-01

    Cancer cells have altered metabolism, with increased glucose uptake, glycolysis, and biomass production. This study conducted genomic and metabolomic analyses to elucidate how tumor and stromal genomic characteristics influence tumor metabolism. Thirty-three breast tumors and six normal breast tissues were analyzed by gene expression microarray and by mass spectrometry for metabolites. Gene expression data and clinical characteristics were evaluated in association with metabolic phenotype. To evaluate the role of stromal interactions in altered metabolism, cocultures were conducted using breast cancer cells and primary cancer-associated fibroblasts (CAF). Across all metabolites, unsupervised clustering resulted in two main sample clusters. Normal breast tissue and a subset of tumors with less aggressive clinical characteristics had lower levels of nucleic and amino acids and glycolysis byproducts, whereas more aggressive tumors had higher levels of these Warburg-associated metabolites. While tumor-intrinsic subtype did not predict metabolic phenotype, metabolic cluster was significantly associated with expression of a wound response signature. In cocultures, CAFs from basal-like breast cancers increased glucose uptake and basal-like epithelial cells increased glucose oxidation and glycogen synthesis, suggesting interplay of stromal and epithelial phenotypes on metabolism. Cytokine arrays identified hepatocyte growth factor (HGF) as a potential mediator of stromal-epithelial interaction and antibody neutralization of HGF resulted in reduced expression of glucose transporter 1 (GLUT1) and decreased glucose uptake by epithelium. Both tumor/epithelial and stromal characteristics play important roles in metabolism. Warburg-like metabolism is influenced by changes in stromal-epithelial interactions, including altered expression of HGF/Met pathway and GLUT1 expression.

  9. Metabolic network reconstruction, growth characterization and 13C-metabolic flux analysis of the extremophile Thermus thermophilus HB8.

    Science.gov (United States)

    Swarup, Aditi; Lu, Jing; DeWoody, Kathleen C; Antoniewicz, Maciek R

    2014-07-01

    Thermus thermophilus is an extremely thermophilic bacterium with significant biotechnological potential. In this work, we have characterized aerobic growth characteristics of T. thermophilus HB8 at temperatures between 50 and 85°C, constructed a metabolic network model of its central carbon metabolism and validated the model using (13)C-metabolic flux analysis ((13)C-MFA). First, cells were grown in batch cultures in custom constructed mini-bioreactors at different temperatures to determine optimal growth conditions. The optimal temperature for T. thermophilus grown on defined medium with glucose was 81°C. The maximum growth rate was 0.25h(-1). Between 50 and 81°C the growth rate increased by 7-fold and the temperature dependence was described well by an Arrhenius model with an activation energy of 47kJ/mol. Next, we performed a (13)C-labeling experiment with [1,2-(13)C] glucose as the tracer and calculated intracellular metabolic fluxes using (13)C-MFA. The results provided support for the constructed network model and highlighted several interesting characteristics of T. thermophilus metabolism. We found that T. thermophilus largely uses glycolysis and TCA cycle to produce biosynthetic precursors, ATP and reducing equivalents needed for cells growth. Consistent with its proposed metabolic network model, we did not detect any oxidative pentose phosphate pathway flux or Entner-Doudoroff pathway activity. The biomass precursors erythrose-4-phosphate and ribose-5-phosphate were produced via the non-oxidative pentose phosphate pathway, and largely via transketolase, with little contribution from transaldolase. The high biomass yield on glucose that was measured experimentally was also confirmed independently by (13)C-MFA. The results presented here provide a solid foundation for future studies of T. thermophilus and its metabolic engineering applications. Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  10. Strategies for individual phenotyping of linoleic and arachidonic Acid metabolism using an oral glucose tolerance test

    NARCIS (Netherlands)

    Saccenti, E.; Duynhoven, van J.P.M.; Jacobs, D.M.; Smilde, A.K.; Hoefsloot, H.C.

    2015-01-01

    The ability to restore homeostasis upon environmental challenges has been proposed as a measure for health. Metabolic profiling of plasma samples during the challenge response phase should offer a profound view on the flexibility of a phenotype to cope with daily stressors. Current data modeling

  11. Host Genotype and Gut Microbiome Modulate Insulin Secretion and Diet-Induced Metabolic Phenotypes

    Directory of Open Access Journals (Sweden)

    Julia H. Kreznar

    2017-02-01

    Full Text Available Genetic variation drives phenotypic diversity and influences the predisposition to metabolic disease. Here, we characterize the metabolic phenotypes of eight genetically distinct inbred mouse strains in response to a high-fat/high-sucrose diet. We found significant variation in diabetes-related phenotypes and gut microbiota composition among the different mouse strains in response to the dietary challenge and identified taxa associated with these traits. Follow-up microbiota transplant experiments showed that altering the composition of the gut microbiota modifies strain-specific susceptibility to diet-induced metabolic disease. Animals harboring microbial communities with enhanced capacity for processing dietary sugars and for generating hydrophobic bile acids showed increased susceptibility to metabolic disease. Notably, differences in glucose-stimulated insulin secretion between different mouse strains were partially recapitulated via gut microbiota transfer. Our results suggest that the gut microbiome contributes to the genetic and phenotypic diversity observed among mouse strains and provide a link between the gut microbiome and insulin secretion.

  12. Host Genotype and Gut Microbiome Modulate Insulin Secretion and Diet-Induced Metabolic Phenotypes.

    Science.gov (United States)

    Kreznar, Julia H; Keller, Mark P; Traeger, Lindsay L; Rabaglia, Mary E; Schueler, Kathryn L; Stapleton, Donald S; Zhao, Wen; Vivas, Eugenio I; Yandell, Brian S; Broman, Aimee Teo; Hagenbuch, Bruno; Attie, Alan D; Rey, Federico E

    2017-02-14

    Genetic variation drives phenotypic diversity and influences the predisposition to metabolic disease. Here, we characterize the metabolic phenotypes of eight genetically distinct inbred mouse strains in response to a high-fat/high-sucrose diet. We found significant variation in diabetes-related phenotypes and gut microbiota composition among the different mouse strains in response to the dietary challenge and identified taxa associated with these traits. Follow-up microbiota transplant experiments showed that altering the composition of the gut microbiota modifies strain-specific susceptibility to diet-induced metabolic disease. Animals harboring microbial communities with enhanced capacity for processing dietary sugars and for generating hydrophobic bile acids showed increased susceptibility to metabolic disease. Notably, differences in glucose-stimulated insulin secretion between different mouse strains were partially recapitulated via gut microbiota transfer. Our results suggest that the gut microbiome contributes to the genetic and phenotypic diversity observed among mouse strains and provide a link between the gut microbiome and insulin secretion. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  13. Assessment of metabolic phenotypic variability in children’s urine using 1H NMR spectroscopy

    Science.gov (United States)

    Maitre, Léa; Lau, Chung-Ho E.; Vizcaino, Esther; Robinson, Oliver; Casas, Maribel; Siskos, Alexandros P.; Want, Elizabeth J.; Athersuch, Toby; Slama, Remy; Vrijheid, Martine; Keun, Hector C.; Coen, Muireann

    2017-04-01

    The application of metabolic phenotyping in clinical and epidemiological studies is limited by a poor understanding of inter-individual, intra-individual and temporal variability in metabolic phenotypes. Using 1H NMR spectroscopy we characterised short-term variability in urinary metabolites measured from 20 children aged 8-9 years old. Daily spot morning, night-time and pooled (50:50 morning and night-time) urine samples across six days (18 samples per child) were analysed, and 44 metabolites quantified. Intraclass correlation coefficients (ICC) and mixed effect models were applied to assess the reproducibility and biological variance of metabolic phenotypes. Excellent analytical reproducibility and precision was demonstrated for the 1H NMR spectroscopic platform (median CV 7.2%). Pooled samples captured the best inter-individual variability with an ICC of 0.40 (median). Trimethylamine, N-acetyl neuraminic acid, 3-hydroxyisobutyrate, 3-hydroxybutyrate/3-aminoisobutyrate, tyrosine, valine and 3-hydroxyisovalerate exhibited the highest stability with over 50% of variance specific to the child. The pooled sample was shown to capture the most inter-individual variance in the metabolic phenotype, which is of importance for molecular epidemiology study design. A substantial proportion of the variation in the urinary metabolome of children is specific to the individual, underlining the potential of such data to inform clinical and exposome studies conducted early in life.

  14. Modelling metabolic evolution on phenotypic fitness landscapes: a case study on C4 photosynthesis.

    Science.gov (United States)

    Heckmann, David

    2015-12-01

    How did the complex metabolic systems we observe today evolve through adaptive evolution? The fitness landscape is the theoretical framework to answer this question. Since experimental data on natural fitness landscapes is scarce, computational models are a valuable tool to predict landscape topologies and evolutionary trajectories. Careful assumptions about the genetic and phenotypic features of the system under study can simplify the design of such models significantly. The analysis of C4 photosynthesis evolution provides an example for accurate predictions based on the phenotypic fitness landscape of a complex metabolic trait. The C4 pathway evolved multiple times from the ancestral C3 pathway and models predict a smooth 'Mount Fuji' landscape accordingly. The modelled phenotypic landscape implies evolutionary trajectories that agree with data on modern intermediate species, indicating that evolution can be predicted based on the phenotypic fitness landscape. Future directions will have to include structural changes of metabolic fitness landscape structure with changing environments. This will not only answer important evolutionary questions about reversibility of metabolic traits, but also suggest strategies to increase crop yields by engineering the C4 pathway into C3 plants. © 2015 Authors; published by Portland Press Limited.

  15. Multiobjective flux balancing using the NISE method for metabolic network analysis.

    Science.gov (United States)

    Oh, Young-Gyun; Lee, Dong-Yup; Lee, Sang Yup; Park, Sunwon

    2009-01-01

    Flux balance analysis (FBA) is well acknowledged as an analysis tool of metabolic networks in the framework of metabolic engineering. However, FBA has a limitation for solving a multiobjective optimization problem which considers multiple conflicting objectives. In this study, we propose a novel multiobjective flux balance analysis method, which adapts the noninferior set estimation (NISE) method (Solanki et al., 1993) for multiobjective linear programming (MOLP) problems. NISE method can generate an approximation of the Pareto curve for conflicting objectives without redundant iterations of single objective optimization. Furthermore, the flux distributions at each Pareto optimal solution can be obtained for understanding the internal flux changes in the metabolic network. The functionality of this approach is shown by applying it to a genome-scale in silico model of E. coli. Multiple objectives for the poly(3-hydroxybutyrate) [P(3HB)] production are considered simultaneously, and relationships among them are identified. The Pareto curve for maximizing succinic acid production vs. maximizing biomass production is used for the in silico analysis of various combinatorial knockout strains. This proposed method accelerates the strain improvement in the metabolic engineering by reducing computation time of obtaining the Pareto curve and analysis time of flux distribution at each Pareto optimal solution. (c) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009.

  16. Detection of Metabolic Fluxes of O and H Atoms into Intracellular Water in Mammalian Cells

    Science.gov (United States)

    Kreuzer, Helen W.; Quaroni, Luca; Podlesak, David W.; Zlateva, Theodora; Bollinger, Nikki; McAllister, Aaron; Lott, Michael J.; Hegg, Eric L.

    2012-01-01

    Metabolic processes result in the release and exchange of H and O atoms from organic material as well as some inorganic salts and gases. These fluxes of H and O atoms into intracellular water result in an isotopic gradient that can be measured experimentally. Using isotope ratio mass spectroscopy, we revealed that slightly over 50% of the H and O atoms in the intracellular water of exponentially-growing cultured Rat-1 fibroblasts were isotopically distinct from growth medium water. We then employed infrared spectromicroscopy to detect in real time the flux of H atoms in these same cells. Importantly, both of these techniques indicate that the H and O fluxes are dependent on metabolic processes; cells that are in lag phase or are quiescent exhibit a much smaller flux. In addition, water extracted from the muscle tissue of rats contained a population of H and O atoms that were isotopically distinct from body water, consistent with the results obtained using the cultured Rat-1 fibroblasts. Together these data demonstrate that metabolic processes produce fluxes of H and O atoms into intracellular water, and that these fluxes can be detected and measured in both cultured mammalian cells and in mammalian tissue. PMID:22848359

  17. Prediction of Metabolic Flux Distribution from Gene Expression Data Based on the Flux Minimization Principle

    Science.gov (United States)

    2014-11-14

    Saccharomyces cerevisiae , and wild-type and mutant Escherichia coli strains, our method achieved high prediction accuracy, as gauged by correlation...the aerobic growth of S. cerevisiae . We used experimental data collected by Lee et al., which included RNA-seq transcriptomic data and exchange flux...measurements of S. cerevisiae aerobically growing in chemostat cultures. This study provides data under two different growth conditions, i.e., glucose

  18. Comparative Analyses of QTLs Influencing Obesity and Metabolic Phenotypes in Pigs and Humans.

    Directory of Open Access Journals (Sweden)

    Sameer D Pant

    Full Text Available The pig is a well-known animal model used to investigate genetic and mechanistic aspects of human disease biology. They are particularly useful in the context of obesity and metabolic diseases because other widely used models (e.g. mice do not completely recapitulate key pathophysiological features associated with these diseases in humans. Therefore, we established a F2 pig resource population (n = 564 designed to elucidate the genetics underlying obesity and metabolic phenotypes. Segregation of obesity traits was ensured by using breeds highly divergent with respect to obesity traits in the parental generation. Several obesity and metabolic phenotypes were recorded (n = 35 from birth to slaughter (242 ± 48 days, including body composition determined at about two months of age (63 ± 10 days via dual-energy x-ray absorptiometry (DXA scanning. All pigs were genotyped using Illumina Porcine 60k SNP Beadchip and a combined linkage disequilibrium-linkage analysis was used to identify genome-wide significant associations for collected phenotypes. We identified 229 QTLs which associated with adiposity- and metabolic phenotypes at genome-wide significant levels. Subsequently comparative analyses were performed to identify the extent of overlap between previously identified QTLs in both humans and pigs. The combined analysis of a large number of obesity phenotypes has provided insight in the genetic architecture of the molecular mechanisms underlying these traits indicating that QTLs underlying similar phenotypes are clustered in the genome. Our analyses have further confirmed that genetic heterogeneity is an inherent characteristic of obesity traits most likely caused by segregation or fixation of different variants of the individual components belonging to cellular pathways in different populations. Several important genes previously associated to obesity in human studies, along with novel genes were identified. Altogether, this study provides novel

  19. Comparative analyses of QTLs influencing obesity and metabolic phenotypes in pigs and humans

    DEFF Research Database (Denmark)

    Pant, Sameer Dinkar; Karlskov-Mortensen, Peter; Jacobsen, Mette Juul

    2015-01-01

    The pig is a well-known animal model used to investigate genetic and mechanistic aspects of human disease biology. They are particularly useful in the context of obesity and metabolic diseases because other widely used models (e.g. mice) do not completely recapitulate key pathophysiological...... features associated with these diseases in humans. Therefore, we established a F2 pig resource population (n = 564) designed to elucidate the genetics underlying obesity and metabolic phenotypes. Segregation of obesity traits was ensured by using breeds highly divergent with respect to obesity traits...... in the parental generation. Several obesity and metabolic phenotypes were recorded (n = 35) from birth to slaughter (242 ± 48 days), including body composition determined at about two months of age (63 ± 10 days) via dual-energy x-ray absorptiometry (DXA) scanning. All pigs were genotyped using Illumina Porcine...

  20. 13C Metabolic Flux Analysis for systematic metabolic engineering of S. cerevisiae for overproduction of fatty acids.

    Directory of Open Access Journals (Sweden)

    Amit Ghosh

    2016-10-01

    Full Text Available Efficient redirection of microbial metabolism into the abundant production of desired bioproducts remains non-trivial. Here we used flux-based modeling approaches to improve yields of fatty acids in S. cerevisiae. We combined 13C labeling data with comprehensive genome-scale models to shed light onto microbial metabolism and improve metabolic engineering efforts. We concentrated on studying the balance of acetyl-CoA, a precursor metabolite for the biosynthesis of fatty acids. A genome-wide acetyl-CoA balance study showed ATP citrate lyase from Y. lipolytica as a robust source of cytoplasmic acetyl-CoA and malate synthase as a desirable target for down-regulation in terms of acetyl-CoA consumption. These genetic modifications were applied to S. cerevisiae WRY2, a strain that is capable of producing 460 mg L of free fatty acids. With the addition of ATP citrate lyase and down-regulation of malate synthase the engineered strain produced 26 per cent more free fatty acids. Further increases in free fatty acid production of 33 per cent were obtained by knocking out the cytoplasmic glycerol-3-phosphate dehydrogenase, which flux analysis had shown was competing for carbon flux upstream with the carbon flux through the acetyl-CoA production pathway in the cytoplasm. In total, the genetic interventions applied in this work increased fatty acid production by 70 per cent.

  1. Metabolic flux profiling of recombinant protein secreting Pichia pastoris growing on glucose:methanol mixtures

    Science.gov (United States)

    2012-01-01

    Background The methylotrophic yeast Pichia pastoris has emerged as one of the most promising yeast hosts for the production of heterologous proteins. Mixed feeds of methanol and a multicarbon source instead of methanol as sole carbon source have been shown to improve product productivities and alleviate metabolic burden derived from protein production. Nevertheless, systematic quantitative studies on the relationships between the central metabolism and recombinant protein production in P. pastoris are still rather limited, particularly when growing this yeast on mixed carbon sources, thus hampering future metabolic network engineering strategies for improved protein production. Results The metabolic flux distribution in the central metabolism of P. pastoris growing on a mixed feed of glucose and methanol was analyzed by Metabolic Flux Analysis (MFA) using 13C-NMR-derived constraints. For this purpose, we defined new flux ratios for methanol assimilation pathways in P. pastoris cells growing on glucose:methanol mixtures. By using this experimental approach, the metabolic burden caused by the overexpression and secretion of a Rhizopus oryzae lipase (Rol) in P. pastoris was further analyzed. This protein has been previously shown to trigger the unfolded protein response in P. pastoris. A series of 13C-tracer experiments were performed on aerobic chemostat cultivations with a control and two different Rol producing strains growing at a dilution rate of 0.09 h−1 using a glucose:methanol 80:20 (w/w) mix as carbon source. The MFA performed in this study reveals a significant redistristribution of carbon fluxes in the central carbon metabolism when comparing the two recombinant strains vs the control strain, reflected in increased glycolytic, TCA cycle and NADH regeneration fluxes, as well as higher methanol dissimilation rates. Conclusions Overall, a further 13C-based MFA development to characterise the central metabolism of methylotrophic yeasts when growing on mixed

  2. Metabolic flux profiling of recombinant protein secreting Pichia pastoris growing on glucose:methanol mixtures

    Directory of Open Access Journals (Sweden)

    Jordà Joel

    2012-05-01

    Full Text Available Abstract Background The methylotrophic yeast Pichia pastoris has emerged as one of the most promising yeast hosts for the production of heterologous proteins. Mixed feeds of methanol and a multicarbon source instead of methanol as sole carbon source have been shown to improve product productivities and alleviate metabolic burden derived from protein production. Nevertheless, systematic quantitative studies on the relationships between the central metabolism and recombinant protein production in P. pastoris are still rather limited, particularly when growing this yeast on mixed carbon sources, thus hampering future metabolic network engineering strategies for improved protein production. Results The metabolic flux distribution in the central metabolism of P. pastoris growing on a mixed feed of glucose and methanol was analyzed by Metabolic Flux Analysis (MFA using 13C-NMR-derived constraints. For this purpose, we defined new flux ratios for methanol assimilation pathways in P. pastoris cells growing on glucose:methanol mixtures. By using this experimental approach, the metabolic burden caused by the overexpression and secretion of a Rhizopus oryzae lipase (Rol in P. pastoris was further analyzed. This protein has been previously shown to trigger the unfolded protein response in P. pastoris. A series of 13C-tracer experiments were performed on aerobic chemostat cultivations with a control and two different Rol producing strains growing at a dilution rate of 0.09 h−1 using a glucose:methanol 80:20 (w/w mix as carbon source. The MFA performed in this study reveals a significant redistristribution of carbon fluxes in the central carbon metabolism when comparing the two recombinant strains vs the control strain, reflected in increased glycolytic, TCA cycle and NADH regeneration fluxes, as well as higher methanol dissimilation rates. Conclusions Overall, a further 13C-based MFA development to characterise the central metabolism of methylotrophic

  3. Characterizing the roles of changing population size and selection on the evolution of flux control in metabolic pathways.

    Science.gov (United States)

    Orlenko, Alena; Chi, Peter B; Liberles, David A

    2017-05-25

    Understanding the genotype-phenotype map is fundamental to our understanding of genomes. Genes do not function independently, but rather as part of networks or pathways. In the case of metabolic pathways, flux through the pathway is an important next layer of biological organization up from the individual gene or protein. Flux control in metabolic pathways, reflecting the importance of mutation to individual enzyme genes, may be evolutionarily variable due to the role of mutation-selection-drift balance. The evolutionary stability of rate limiting steps and the patterns of inter-molecular co-evolution were evaluated in a simulated pathway with a system out of equilibrium due to fluctuating selection, population size, or positive directional selection, to contrast with those under stabilizing selection. Depending upon the underlying population genetic regime, fluctuating population size was found to increase the evolutionary stability of rate limiting steps in some scenarios. This result was linked to patterns of local adaptation of the population. Further, during positive directional selection, as with more complex mutational scenarios, an increase in the observation of inter-molecular co-evolution was observed. Differences in patterns of evolution when systems are in and out of equilibrium, including during positive directional selection may lead to predictable differences in observed patterns for divergent evolutionary scenarios. In particular, this result might be harnessed to detect differences between compensatory processes and directional processes at the pathway level based upon evolutionary observations in individual proteins. Detecting functional shifts in pathways reflects an important milestone in predicting when changes in genotypes result in changes in phenotypes.

  4. Metabolic Phenotype Characterization of Botrytis cinerea, the Causal Agent of Gray Mold

    Directory of Open Access Journals (Sweden)

    Han-Cheng Wang

    2018-03-01

    Full Text Available Botrytis cinerea, which causes gray mold, is an important pathogen in four important economic crops, tomato, tobacco, cucumber and strawberry, in China and worldwide. Metabolic phenomics data on B. cinerea isolates from these four crops were characterized and compared for 950 phenotypes with a BIOLOG Phenotype MicroArray (PM. The results showed that the metabolic fingerprints of the four B. cinerea isolates were similar to each other with minimal differences. B. cinerea isolates all metabolized more than 17% of the tested carbon sources, 63% of the amino acid nitrogen substrates, 80% of the peptide nitrogen substrates, 93% of the phosphorus substrates, and 97% of the sulfur substrates. Carbon substrates of organic acids and carbohydrates, and nitrogen substrates of amino acids and peptides were the significant utilization patterns for B. cinerea. Each B. cinerea isolate contained 94 biosynthetic pathways. These isolates showed a large range of adaptabilities and were still able to metabolize substrates in the presence of the osmolytes, including up to 6% potassium chloride, 10% sodium chloride, 5% sodium sulfate, 6% sodium formate, 20% ethylene glycol, and 3% urea. These isolates all showed active metabolism in environments with pH values from 3.5 to 8.5 and exhibited decarboxylase activities. These characterizations provide a theoretical basis for the study of B. cinerea in biochemistry and metabolic phenomics and provide valuable clues to finding potential new ways to manage gray mold.

  5. Metabolic Phenotype Characterization of Botrytis cinerea, the Causal Agent of Gray Mold

    Science.gov (United States)

    Wang, Han-Cheng; Li, Li-Cui; Cai, Bin; Cai, Liu-Ti; Chen, Xing-Jiang; Yu, Zhi-He; Zhang, Chuan-Qing

    2018-01-01

    Botrytis cinerea, which causes gray mold, is an important pathogen in four important economic crops, tomato, tobacco, cucumber and strawberry, in China and worldwide. Metabolic phenomics data on B. cinerea isolates from these four crops were characterized and compared for 950 phenotypes with a BIOLOG Phenotype MicroArray (PM). The results showed that the metabolic fingerprints of the four B. cinerea isolates were similar to each other with minimal differences. B. cinerea isolates all metabolized more than 17% of the tested carbon sources, 63% of the amino acid nitrogen substrates, 80% of the peptide nitrogen substrates, 93% of the phosphorus substrates, and 97% of the sulfur substrates. Carbon substrates of organic acids and carbohydrates, and nitrogen substrates of amino acids and peptides were the significant utilization patterns for B. cinerea. Each B. cinerea isolate contained 94 biosynthetic pathways. These isolates showed a large range of adaptabilities and were still able to metabolize substrates in the presence of the osmolytes, including up to 6% potassium chloride, 10% sodium chloride, 5% sodium sulfate, 6% sodium formate, 20% ethylene glycol, and 3% urea. These isolates all showed active metabolism in environments with pH values from 3.5 to 8.5 and exhibited decarboxylase activities. These characterizations provide a theoretical basis for the study of B. cinerea in biochemistry and metabolic phenomics and provide valuable clues to finding potential new ways to manage gray mold. PMID:29593701

  6. Regulation of amino-acid metabolism controls flux to lipid accumulation in Yarrowia lipolytica

    DEFF Research Database (Denmark)

    Kerkhoven, Eduard J.; Pomraning, Kyle R.; Baker, Scott E.

    2016-01-01

    accumulation in Y. lipolytica does not involve transcriptional regulation of lipid metabolism but is associated with regulation of amino-acid biosynthesis, resulting in redirection of carbon flux during nitrogen limitation from amino acids to lipids. Lipid accumulation in Y. lipolytica at nitrogen limitation...

  7. Optimization of 13C isotopic tracers for metabolic flux analysis in mammalian cells.

    Science.gov (United States)

    Walther, Jason L; Metallo, Christian M; Zhang, Jie; Stephanopoulos, Gregory

    2012-03-01

    Mammalian cells consume and metabolize various substrates from their surroundings for energy generation and biomass synthesis. Glucose and glutamine, in particular, are the primary carbon sources for proliferating cancer cells. While this combination of substrates generates static labeling patterns for use in (13)C metabolic flux analysis (MFA), the inability of single tracers to effectively label all pathways poses an obstacle for comprehensive flux determination within a given experiment. To address this issue we applied a genetic algorithm to optimize mixtures of (13)C-labeled glucose and glutamine for use in MFA. We identified tracer combinations that minimized confidence intervals in an experimentally determined flux network describing central carbon metabolism in tumor cells. Additional simulations were used to determine the robustness of the [1,2-(13)C(2)]glucose/[U-(13)C(5)]glutamine tracer combination with respect to perturbations in the network. Finally, we experimentally validated the improved performance of this tracer set relative to glucose tracers alone in a cancer cell line. This versatile method allows researchers to determine the optimal tracer combination to use for a specific metabolic network, and our findings applied to cancer cells significantly enhance the ability of MFA experiments to precisely quantify fluxes in higher organisms. Copyright © 2011 Elsevier Inc. All rights reserved.

  8. Aerobic glucose metabolism of Saccharomyces kluyveri: Growth, metabolite production, and quantification of metabolic fluxes

    DEFF Research Database (Denmark)

    Møller, Kasper; Christensen, B.; Förster, Jochen

    2002-01-01

    growth in aerobic glucose-limited continuous cultivation. It was found that in S. kluyveri the flux into the pentose phosphate pathway was 18.8 mmole per 100 mmole glucose consumed during respiratory growth in aerobic glucose-limited continuous cultivation. Such a low flux into the pentose phosphate...

  9. The Protein Cost of Metabolic Fluxes: Prediction from Enzymatic Rate Laws and Cost Minimization.

    Directory of Open Access Journals (Sweden)

    Elad Noor

    2016-11-01

    Full Text Available Bacterial growth depends crucially on metabolic fluxes, which are limited by the cell's capacity to maintain metabolic enzymes. The necessary enzyme amount per unit flux is a major determinant of metabolic strategies both in evolution and bioengineering. It depends on enzyme parameters (such as kcat and KM constants, but also on metabolite concentrations. Moreover, similar amounts of different enzymes might incur different costs for the cell, depending on enzyme-specific properties such as protein size and half-life. Here, we developed enzyme cost minimization (ECM, a scalable method for computing enzyme amounts that support a given metabolic flux at a minimal protein cost. The complex interplay of enzyme and metabolite concentrations, e.g. through thermodynamic driving forces and enzyme saturation, would make it hard to solve this optimization problem directly. By treating enzyme cost as a function of metabolite levels, we formulated ECM as a numerically tractable, convex optimization problem. Its tiered approach allows for building models at different levels of detail, depending on the amount of available data. Validating our method with measured metabolite and protein levels in E. coli central metabolism, we found typical prediction fold errors of 4.1 and 2.6, respectively, for the two kinds of data. This result from the cost-optimized metabolic state is significantly better than randomly sampled metabolite profiles, supporting the hypothesis that enzyme cost is important for the fitness of E. coli. ECM can be used to predict enzyme levels and protein cost in natural and engineered pathways, and could be a valuable computational tool to assist metabolic engineering projects. Furthermore, it establishes a direct connection between protein cost and thermodynamics, and provides a physically plausible and computationally tractable way to include enzyme kinetics into constraint-based metabolic models, where kinetics have usually been ignored or

  10. Understanding the control of acyl flux through the lipid metabolic network of plant oil biosynthesis.

    Science.gov (United States)

    Bates, Philip D

    2016-09-01

    Plant oil biosynthesis involves a complex metabolic network with multiple subcellular compartments, parallel pathways, cycles, and pathways that have a dual function to produce essential membrane lipids and triacylglycerol. Modern molecular biology techniques provide tools to alter plant oil compositions through bioengineering, however with few exceptions the final composition of triacylglycerol cannot be predicted. One reason for limited success in oilseed bioengineering is the inadequate understanding of how to control the flux of fatty acids through various fatty acid modification, and triacylglycerol assembly pathways of the lipid metabolic network. This review focuses on the mechanisms of acyl flux through the lipid metabolic network, and highlights where uncertainty resides in our understanding of seed oil biosynthesis. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. The association between metabolic health, obesity phenotype and the risk of breast cancer.

    Science.gov (United States)

    Park, Yong-Moon Mark; White, Alexandra J; Nichols, Hazel B; O'Brien, Katie M; Weinberg, Clarice R; Sandler, Dale P

    2017-06-15

    Beyond the current emphasis on body mass index (BMI), it is unknown whether breast cancer risk differs between metabolically healthy and unhealthy normal weight or overweight/obese women. The Sister Study is a nationwide prospective cohort study. Data came from 50,884 cohort participants aged 35 to 74 years enrolled from 2003 through 2009. Cox proportional hazards models were used to estimate multivariable adjusted hazard ratios (HR) and 95% confidence intervals (CIs) for breast cancer risk. Metabolic abnormalities considered included: high waist circumference (≥88 cm); elevated blood pressure (≥130/85 mm Hg or antihypertensive medication); previously diagnosed diabetes or antidiabetic drug treatment; and cholesterol-lowering medication use. During follow-up (mean, 6.4 years), 1,388 invasive breast cancers were diagnosed at least 1 year after enrollment. Compared to women with BMI women with a BMI women with a BMI ≥25 kg/m 2 and no metabolic abnormalities (metabolically healthy overweight/obese phenotype) (HR = 1.24, 95% CI: 0.99-1.55). Furthermore, risk of postmenopausal breast cancer was consistently elevated in women with normal BMI and central obesity (normal weight central obesity phenotype) regardless of the criterion used to define central obesity, with HR for waist circumference ≥88 cm, waist circumference ≥80 cm, and waist-hip ratio ≥0.85 of 1.58, 95% CI: 1.02-2.46; 1.38, 95% CI: 1.09-1.75; and 1.38, 95% CI: 1.02-1.85, respectively. There was an inverse association between premenopausal breast cancer and metabolically healthy overweight/obese phenotype (HR = 0.71, 95% CI: 0.52-0.97). Our findings suggest that postmenopausal women who are metabolically unhealthy or have central adiposity may be at increased risk for breast cancer despite normal BMI. © 2017 UICC.

  12. Changes of in vivo fluxes through central metabolic pathways during the production of nystatin by Streptomyces noursei in batch culture

    DEFF Research Database (Denmark)

    Jonsbu, E.; Christensen, Bjarke; Nielsen, Jens

    2001-01-01

    of the amino acids and calculated fluxes of the central metabolism showed that changes in the primary and secondary metabolisms occurred simultaneously. Changes in the profiles for the integrated fluxes showed a decreased flux through the pentose phosphate pathway and an increased flux in the tricarboxylic...... acid cycle relative to the glucose uptake rate when the culture entered a phase with reduced specific growth rate and enhanced nystatin yield. The flux through the pentose phosphate pathway seemed to be adjusted according to the NADPH requirement during the different phases of the batch fermentation....

  13. Graded Maximal Exercise Testing to Assess Mouse Cardio-Metabolic Phenotypes.

    Directory of Open Access Journals (Sweden)

    Jennifer M Petrosino

    Full Text Available Functional assessments of cardiovascular fitness (CVF are needed to establish animal models of dysfunction, test the effects of novel therapeutics, and establish the cardio-metabolic phenotype of mice. In humans, the graded maximal exercise test (GXT is a standardized diagnostic for assessing CVF and mortality risk. These tests, which consist of concurrent staged increases in running speed and inclination, provide diagnostic cardio-metabolic parameters, such as, VO2max, anaerobic threshold, and metabolic crossover. Unlike the human-GXT, published mouse treadmill tests have set, not staged, increases in inclination as speed progress until exhaustion (PXT. Additionally, they often lack multiple cardio-metabolic parameters. Here, we developed a mouse-GXT with the intent of improving mouse-exercise testing sensitivity and developing translatable parameters to assess CVF in healthy and dysfunctional mice. The mouse-GXT, like the human-GXT, incorporated staged increases in inclination, speed, and intensity; and, was designed by considering imitations of the PXT and differences between human and mouse physiology. The mouse-GXT and PXTs were both tested in healthy mice (C57BL/6J, FVBN/J to determine their ability to identify cardio-metabolic parameters (anaerobic threshold, VO2max, metabolic crossover observed in human-GXTs. Next, theses assays were tested on established diet-induced (obese-C57BL/6J and genetic (cardiac isoform Casq2-/- models of cardiovascular dysfunction. Results showed that both tests reported VO2max and provided reproducible data about performance. Only the mouse-GXT reproducibly identified anaerobic threshold, metabolic crossover, and detected impaired CVF in dysfunctional models. Our findings demonstrated that the mouse-GXT is a sensitive, non-invasive, and cost-effective method for assessing CVF in mice. This new test can be used as a functional assessment to determine the cardio-metabolic phenotype of various animal models or

  14. Graded Maximal Exercise Testing to Assess Mouse Cardio-Metabolic Phenotypes.

    Science.gov (United States)

    Petrosino, Jennifer M; Heiss, Valerie J; Maurya, Santosh K; Kalyanasundaram, Anuradha; Periasamy, Muthu; LaFountain, Richard A; Wilson, Jacob M; Simonetti, Orlando P; Ziouzenkova, Ouliana

    2016-01-01

    Functional assessments of cardiovascular fitness (CVF) are needed to establish animal models of dysfunction, test the effects of novel therapeutics, and establish the cardio-metabolic phenotype of mice. In humans, the graded maximal exercise test (GXT) is a standardized diagnostic for assessing CVF and mortality risk. These tests, which consist of concurrent staged increases in running speed and inclination, provide diagnostic cardio-metabolic parameters, such as, VO2max, anaerobic threshold, and metabolic crossover. Unlike the human-GXT, published mouse treadmill tests have set, not staged, increases in inclination as speed progress until exhaustion (PXT). Additionally, they often lack multiple cardio-metabolic parameters. Here, we developed a mouse-GXT with the intent of improving mouse-exercise testing sensitivity and developing translatable parameters to assess CVF in healthy and dysfunctional mice. The mouse-GXT, like the human-GXT, incorporated staged increases in inclination, speed, and intensity; and, was designed by considering imitations of the PXT and differences between human and mouse physiology. The mouse-GXT and PXTs were both tested in healthy mice (C57BL/6J, FVBN/J) to determine their ability to identify cardio-metabolic parameters (anaerobic threshold, VO2max, metabolic crossover) observed in human-GXTs. Next, theses assays were tested on established diet-induced (obese-C57BL/6J) and genetic (cardiac isoform Casq2-/-) models of cardiovascular dysfunction. Results showed that both tests reported VO2max and provided reproducible data about performance. Only the mouse-GXT reproducibly identified anaerobic threshold, metabolic crossover, and detected impaired CVF in dysfunctional models. Our findings demonstrated that the mouse-GXT is a sensitive, non-invasive, and cost-effective method for assessing CVF in mice. This new test can be used as a functional assessment to determine the cardio-metabolic phenotype of various animal models or the effects of

  15. An integrative machine learning strategy for improved prediction of essential genes in Escherichia coli metabolism using flux-coupled features.

    Science.gov (United States)

    Nandi, Sutanu; Subramanian, Abhishek; Sarkar, Ram Rup

    2017-07-25

    Prediction of essential genes helps to identify a minimal set of genes that are absolutely required for the appropriate functioning and survival of a cell. The available machine learning techniques for essential gene prediction have inherent problems, like imbalanced provision of training datasets, biased choice of the best model for a given balanced dataset, choice of a complex machine learning algorithm, and data-based automated selection of biologically relevant features for classification. Here, we propose a simple support vector machine-based learning strategy for the prediction of essential genes in Escherichia coli K-12 MG1655 metabolism that integrates a non-conventional combination of an appropriate sample balanced training set, a unique organism-specific genotype, phenotype attributes that characterize essential genes, and optimal parameters of the learning algorithm to generate the best machine learning model (the model with the highest accuracy among all the models trained for different sample training sets). For the first time, we also introduce flux-coupled metabolic subnetwork-based features for enhancing the classification performance. Our strategy proves to be superior as compared to previous SVM-based strategies in obtaining a biologically relevant classification of genes with high sensitivity and specificity. This methodology was also trained with datasets of other recent supervised classification techniques for essential gene classification and tested using reported test datasets. The testing accuracy was always high as compared to the known techniques, proving that our method outperforms known methods. Observations from our study indicate that essential genes are conserved among homologous bacterial species, demonstrate high codon usage bias, GC content and gene expression, and predominantly possess a tendency to form physiological flux modules in metabolism.

  16. THE RELEVANCE OF METABOLIC PHENOTYPES OF OBESITY IN CHILDHOOD AND ADOLESCENCE

    Directory of Open Access Journals (Sweden)

    S. I. Malyavskaya

    2015-01-01

    Full Text Available Rationale: The study  on  specifics of metabolic phenotypes of obesity in children and adolescents seems be highly relevant for a comprehensive assessment  of causal and  pathophysiological  roles of obesity in the  atherogenesis. Aim: To identify particulars of metabolic  phenotypes of obesity in the  population of the  school children in the  city of Arkhangelsk. Materials and methods: We examined 102 patients aged from 10 to 15 years with obesity, abdominal type (boys, 44.6%, girls, 55.4%. According to the results of a comprehensive clinical and laboratory assessments, the patients  were divided  into  the  group  of metabolically  healthy obese   (children  and  adolescents  with  obesity, but without any metabolic abnormalities and the group of metabolically unhealthy obese (having at least 1 metabolic abnormality. The list of metabolic abnormalities  included  high triglyceride levels, low levels of high density lipoprotein  cholesterol (HDL-C, high blood pressure, impaired fasting glucose, increased  C-reactive protein  levels. Results: The  group  comparison   showed  that  the  mean levels  of  all studied   parameters  of  pro-atherogenic  metabolic  abnormalities  were significantly higher  in the  patients  with  metabolically  active obesity (the mean triglyceride levels in the groups of metabolically active and metabolically healthy obesity were 1.31 vs 0.74 mmol/L, glucose levels, 4.92  vs 4.54  mmol/L,  C-reactive protein,  3.15  vs 2.30 mg/mL, systolic and diastolic blood pressure, 118.97 vs 110.23 mmHg and 72.90 vs 68.58 mmHg, respectively; p < 0.001, with the  exclusion of the   mean level of anti-atherogenic HDL-C, which was lower (1.27 vs 1.49 mmol/L; p < 0.001. Also, in addition to abdominal obesity, 21.43% of school children with metabolically active obesity had ≥ 2 atherogenic factors, as well as some pro-inflammatory abnormalities (C-reactive protein levels were

  17. Metabolic flux of the oxidative pentose phosphate pathway under low light conditions in Synechocystis sp. PCC 6803.

    Science.gov (United States)

    Ueda, Kentaro; Nakajima, Tsubasa; Yoshikawa, Katsunori; Toya, Yoshihiro; Matsuda, Fumio; Shimizu, Hiroshi

    2018-02-27

    The role of the oxidative pentose phosphate pathway (oxPPP) in Synechocystis sp. PCC 6803 under mixotrophic conditions was investigated by 13 C metabolic flux analysis. Cells were cultured under low (10 μmol m -2  s -1 ) and high light intensities (100 μmol m -2  s -1 ) in the presence of glucose. The flux of CO 2 fixation by ribulose bisphosphate carboxylase/oxygenase under the high light condition was approximately 3-fold higher than that under the low light condition. Although no flux of the oxPPP was observed under the high light condition, flux of 0.08-0.19 mmol gDCW -1  h -1 in the oxPPP was observed under the low light condition. The balance between the consumption and production of NADPH suggested that approximately 10% of the total NADPH production was generated by the oxPPP under the low light condition. The growth phenotype of a mutant with deleted zwf, which encodes glucose-6-phosphate dehydrogenase in the oxPPP, was compared to that of the parental strain under low and high light conditions. Growth of the Δzwf mutant nearly stopped during the late growth phase under the low light condition, whereas the growth rates of the two strains were identical under the high light condition. These results indicate that NADPH production in the oxPPP is essential for anabolism under low light conditions. The oxPPP appears to play an important role in producing NADPH from glucose and ATP to compensate for NADPH shortage under low light conditions. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  18. Proteomic analysis uncovers a metabolic phenotype in C. elegans after nhr-40 reduction of function

    International Nuclear Information System (INIS)

    Pohludka, Michal; Simeckova, Katerina; Vohanka, Jaroslav; Yilma, Petr; Novak, Petr; Krause, Michael W.; Kostrouchova, Marta; Kostrouch, Zdenek

    2008-01-01

    Caenorhabditis elegans has an unexpectedly large number (284) of genes encoding nuclear hormone receptors, most of which are nematode-specific and are of unknown function. We have exploited comparative two-dimensional chromatography of synchronized cultures of wild type C. elegans larvae and a mutant in nhr-40 to determine if proteomic approaches will provide additional insight into gene function. Chromatofocusing, followed by reversed-phase chromatography and mass spectrometry, identified altered chromatographic patterns for a set of proteins, many of which function in muscle and metabolism. Prompted by the proteomic analysis, we find that the penetrance of the developmental phenotypes in the mutant is enhanced at low temperatures and by food restriction. The combination of our phenotypic and proteomic analysis strongly suggests that NHR-40 provides a link between metabolism and muscle development. Our results highlight the utility of comparative two-dimensional chromatography to provide a relatively rapid method to gain insight into gene function

  19. Metabolic profiles characterizing different phenotypes of polycystic ovary syndrome: plasma metabolomics analysis

    OpenAIRE

    Zhao, Yue; Fu, Li; Li, Rong; Wang, Li-Na; Yang, Yan; Liu, Na-Na; Zhang, Chun-Mei; Wang, Ying; Liu, Ping; Tu, Bin-Bin; Zhang, Xue; Qiao, Jie

    2012-01-01

    Background Polycystic ovary syndrome (PCOS) is a heterogeneous endocrine disorder accompanied with an increased risk of developing type 2 diabetes mellitus and cardiovascular disease; despite being a common condition, the pathogenesis of PCOS remains unclear. Our aim was to investigate the potential metabolic profiles for different phenotypes of PCOS, as well as for the early prognosis of complications. Methods A total of 217 women with PCOS and 48 healthy women as normal controls were studie...

  20. Metabolic phenotyping of various tea (Camellia sinensis L.) cultivars and understanding of their intrinsic metabolism.

    Science.gov (United States)

    Ji, Hyang-Gi; Lee, Yeong-Ran; Lee, Min-Seuk; Hwang, Kyeong Hwan; Kim, Eun-Hee; Park, Jun Seong; Hong, Young-Shick

    2017-10-15

    Recently, we selected three tea (Camellia sinensis) cultivars that are rich in taste, epigallocatechin-3-O-gallate (EGCG) and epigallocatechin-3-O-(3-O-methyl)-gallate (EGCG3″Me) and then cultivated them through asexual propagation by cutting in the same region. In the present study, proton nuclear magnetic resonance ( 1 H NMR)-based metabolomics was applied to characterize the metabotype and to understand the metabolic mechanism of these tea cultivars including wild type tea. Of the tea leaf metabolite variations, reverse associations of amino acid metabolism with catechin compound metabolism were found in the rich-taste, and EGCG- and EGCG3″Me-rich tea cultivars. Indeed, the metabolism of individual catechin compounds in the EGCG3″Me-rich cultivar differed from those of other tea cultivars. The current study highlights the distinct metabolism of various tea cultivars newly selected for cultivation and the important role of metabolomics in understanding the metabolic mechanism. Thus, comprehensive metabotyping is a useful method to assess and then develop a new plant cultivar. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Development of Computational Tools for Metabolic Model Curation, Flux Elucidation and Strain Design

    Energy Technology Data Exchange (ETDEWEB)

    Maranas, Costas D

    2012-05-21

    An overarching goal of the Department of Energy mission is the efficient deployment and engineering of microbial and plant systems to enable biomass conversion in pursuit of high energy density liquid biofuels. This has spurred the pace at which new organisms are sequenced and annotated. This torrent of genomic information has opened the door to understanding metabolism in not just skeletal pathways and a handful of microorganisms but for truly genome-scale reconstructions derived for hundreds of microbes and plants. Understanding and redirecting metabolism is crucial because metabolic fluxes are unique descriptors of cellular physiology that directly assess the current cellular state and quantify the effect of genetic engineering interventions. At the same time, however, trying to keep pace with the rate of genomic data generation has ushered in a number of modeling and computational challenges related to (i) the automated assembly, testing and correction of genome-scale metabolic models, (ii) metabolic flux elucidation using labeled isotopes, and (iii) comprehensive identification of engineering interventions leading to the desired metabolism redirection.

  2. To be certain about the uncertainty: Bayesian statistics for 13 C metabolic flux analysis.

    Science.gov (United States)

    Theorell, Axel; Leweke, Samuel; Wiechert, Wolfgang; Nöh, Katharina

    2017-11-01

    13 C Metabolic Fluxes Analysis ( 13 C MFA) remains to be the most powerful approach to determine intracellular metabolic reaction rates. Decisions on strain engineering and experimentation heavily rely upon the certainty with which these fluxes are estimated. For uncertainty quantification, the vast majority of 13 C MFA studies relies on confidence intervals from the paradigm of Frequentist statistics. However, it is well known that the confidence intervals for a given experimental outcome are not uniquely defined. As a result, confidence intervals produced by different methods can be different, but nevertheless equally valid. This is of high relevance to 13 C MFA, since practitioners regularly use three different approximate approaches for calculating confidence intervals. By means of a computational study with a realistic model of the central carbon metabolism of E. coli, we provide strong evidence that confidence intervals used in the field depend strongly on the technique with which they were calculated and, thus, their use leads to misinterpretation of the flux uncertainty. In order to provide a better alternative to confidence intervals in 13 C MFA, we demonstrate that credible intervals from the paradigm of Bayesian statistics give more reliable flux uncertainty quantifications which can be readily computed with high accuracy using Markov chain Monte Carlo. In addition, the widely applied chi-square test, as a means of testing whether the model reproduces the data, is examined closer. © 2017 Wiley Periodicals, Inc.

  3. Metabolic flux analysis of Cyanothece sp. ATCC 51142 under mixotrophic conditions.

    Science.gov (United States)

    Alagesan, Swathi; Gaudana, Sandeep B; Sinha, Avinash; Wangikar, Pramod P

    2013-11-01

    Cyanobacteria are a group of photosynthetic prokaryotes capable of utilizing solar energy to fix atmospheric carbon dioxide to biomass. Despite several "proof of principle" studies, low product yield is an impediment in commercialization of cyanobacteria-derived biofuels. Estimation of intracellular reaction rates by (13)C metabolic flux analysis ((13)C-MFA) would be a step toward enhancing biofuel yield via metabolic engineering. We report (13)C-MFA for Cyanothece sp. ATCC 51142, a unicellular nitrogen-fixing cyanobacterium, known for enhanced hydrogen yield under mixotrophic conditions. Rates of reactions in the central carbon metabolism under nitrogen-fixing and -non-fixing conditions were estimated by monitoring the competitive incorporation of (12)C and (13)C from unlabeled CO2 and uniformly labeled glycerol, respectively, into terminal metabolites such as amino acids. The observed labeling patterns suggest mixotrophic growth under both the conditions, with a larger fraction of unlabeled carbon in nitrate-sufficient cultures asserting a greater contribution of carbon fixation by photosynthesis and an anaplerotic pathway. Indeed, flux analysis complements the higher growth observed under nitrate-sufficient conditions. On the other hand, the flux through the oxidative pentose phosphate pathway and tricarboxylic acid cycle was greater in nitrate-deficient conditions, possibly to supply the precursors and reducing equivalents needed for nitrogen fixation. In addition, an enhanced flux through fructose-6-phosphate phosphoketolase possibly suggests the organism's preferred mode under nitrogen-fixing conditions. The (13)C-MFA results complement the reported predictions by flux balance analysis and provide quantitative insight into the organism's distinct metabolic features under nitrogen-fixing and -non-fixing conditions.

  4. Tissue-specific metabolic reprogramming drives nutrient flux in diabetic complications

    Science.gov (United States)

    Sas, Kelli M.; Kayampilly, Pradeep; Byun, Jaeman; Nair, Viji; Hinder, Lucy M.; Zhang, Hongyu; Lin, Chengmao; Qi, Nathan R.; Michailidis, George; Groop, Per-Henrik; Nelson, Robert G.; Darshi, Manjula; Sharma, Kumar; Schelling, Jeffrey R.; Sedor, John R.; Pop-Busui, Rodica; Weinberg, Joel M.; Soleimanpour, Scott A.; Abcouwer, Steven F.; Gardner, Thomas W.; Burant, Charles F.; Feldman, Eva L.; Kretzler, Matthias; Brosius, Frank C.

    2016-01-01

    Diabetes is associated with altered cellular metabolism, but how altered metabolism contributes to the development of diabetic complications is unknown. We used the BKS db/db diabetic mouse model to investigate changes in carbohydrate and lipid metabolism in kidney cortex, peripheral nerve, and retina. A systems approach using transcriptomics, metabolomics, and metabolic flux analysis identified tissue-specific differences, with increased glucose and fatty acid metabolism in the kidney, a moderate increase in the retina, and a decrease in the nerve. In the kidney, increased metabolism was associated with enhanced protein acetylation and mitochondrial dysfunction. To confirm these findings in human disease, we analyzed diabetic kidney transcriptomic data and urinary metabolites from a cohort of Southwestern American Indians. The urinary findings were replicated in 2 independent patient cohorts, the Finnish Diabetic Nephropathy and the Family Investigation of Nephropathy and Diabetes studies. Increased concentrations of TCA cycle metabolites in urine, but not in plasma, predicted progression of diabetic kidney disease, and there was an enrichment of pathways involved in glycolysis and fatty acid and amino acid metabolism. Our findings highlight tissue-specific changes in metabolism in complication-prone tissues in diabetes and suggest that urinary TCA cycle intermediates are potential prognostic biomarkers of diabetic kidney disease progression. PMID:27699244

  5. Lansoprazole Is Associated with Worsening Asthma Control in Children with the CYP2C19 Poor Metabolizer Phenotype.

    Science.gov (United States)

    Lang, Jason E; Holbrook, Janet T; Mougey, Edward B; Wei, Christine Y; Wise, Robert A; Teague, W Gerald; Lima, John J

    2015-06-01

    Gastric acid blockade in children with asymptomatic acid reflux has not improved asthma control in published studies. There is substantial population variability regarding metabolism of and response to proton pump inhibitors based on metabolizer phenotype. How metabolizer phenotype affects asthma responses to acid blockage is not known. To determine how metabolizer phenotype based on genetic analysis of CYP2C19 affects asthma control among children treated with a proton pump inhibitor. Asthma control as measured by the Asthma Control Questionnaire (ACQ) and other questionnaires from a 6-month clinical trial of lansoprazole in children with asthma was analyzed for associations with surrogates of lansoprazole exposure (based on treatment assignment and metabolizer phenotype). Groups included placebo-treated children; lansoprazole-treated extensive metabolizers (EMs); and lansoprazole-treated poor metabolizers (PMs). Metabolizer phenotypes were based on CYP2C19 haplotypes. Carriers of the CYP2C19*2, *3, *8, *9, or *10 allele were PMs; carriers of two wild-type alleles were extensive metabolizers (EMs). Asthma control through most of the treatment period was unaffected by lansoprazole exposure or metabolizer phenotype. At 6 months, PMs displayed significantly worsened asthma control compared with EMs (+0.16 vs. -0.13; P = 0.02) and placebo-treated children (+0.16 vs. -0.23; P lansoprazole-treated PMs. Children with the PM phenotype developed worse asthma control after 6 months of lansoprazole treatment for poorly controlled asthma. Increased exposure to proton pump inhibitor may worsen asthma control by altering responses to respiratory infections. Clinical trial registered with www.clinicaltrials.gov (NCT00604851).

  6. Flux analysis of central metabolic pathways in Geobactermetallireducens during reduction of solubleFe(III)-NTA

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Yinjie J.; Chakraborty, Romy; Garcia-Martin, Hector; Chu,Jeannie; Hazen, Terry C.; Keasling, Jay D.

    2007-01-01

    We analyzed the carbon fluxes in the central metabolism ofGeobacter metallireducens strain GS-15 using 13C isotopomer modeling.Acetate labeled in the 1st or 2nd position was the sole carbon source,and Fe-NTA was the sole terminal electron acceptor. The measured labeledacetate uptake rate was 21 mmol/gdw/h in the exponential growth phase.The resulting isotope labeling pattern of amino acids allowed an accuratedetermination of the in vivo global metabolic reaction rates (fluxes)through the central metabolic pathways using a computational isotopomermodel. The tracer experiments showed that G. metallireducens containedcomplete biosynthesis pathways for essential metabolism, and this strainmight also have an unusual isoleucine biosynthesis route (usingacetyl-CoA and pyruvate as the precursors). The model indicated that over90 percent of the acetate was completely oxidized to CO2 via a completetricarboxylic acid (TCA) cycle while reducing iron. Pyruvate carboxylaseand phosphoenolpyruvate carboxykinase were present under theseconditions, but enzymes in the glyoxylate shunt and malic enzyme wereabsent. Gluconeogenesis and the pentose phosphate pathway were mainlyemployed for biosynthesis and accounted for less than 3 percent of totalcarbon consumption. The model also indicated surprisingly highreversibility in the reaction between oxoglutarate and succinate. Thisstep operates close to the thermodynamic equilibrium possibly becausesuccinate is synthesized via a transferase reaction, and the conversionof oxoglutarate to succinate is a rate limiting step for carbonmetabolism. These findings enable a better understanding of therelationship between genome annotation and extant metabolic pathways inG. metallireducens.

  7. Combining inferred regulatory and reconstructed metabolic networks enhances phenotype prediction in yeast.

    Science.gov (United States)

    Wang, Zhuo; Danziger, Samuel A; Heavner, Benjamin D; Ma, Shuyi; Smith, Jennifer J; Li, Song; Herricks, Thurston; Simeonidis, Evangelos; Baliga, Nitin S; Aitchison, John D; Price, Nathan D

    2017-05-01

    Gene regulatory and metabolic network models have been used successfully in many organisms, but inherent differences between them make networks difficult to integrate. Probabilistic Regulation Of Metabolism (PROM) provides a partial solution, but it does not incorporate network inference and underperforms in eukaryotes. We present an Integrated Deduced And Metabolism (IDREAM) method that combines statistically inferred Environment and Gene Regulatory Influence Network (EGRIN) models with the PROM framework to create enhanced metabolic-regulatory network models. We used IDREAM to predict phenotypes and genetic interactions between transcription factors and genes encoding metabolic activities in the eukaryote, Saccharomyces cerevisiae. IDREAM models contain many fewer interactions than PROM and yet produce significantly more accurate growth predictions. IDREAM consistently outperformed PROM using any of three popular yeast metabolic models and across three experimental growth conditions. Importantly, IDREAM's enhanced accuracy makes it possible to identify subtle synthetic growth defects. With experimental validation, these novel genetic interactions involving the pyruvate dehydrogenase complex suggested a new role for fatty acid-responsive factor Oaf1 in regulating acetyl-CoA production in glucose grown cells.

  8. 13CFLUX2--high-performance software suite for (13)C-metabolic flux analysis.

    Science.gov (United States)

    Weitzel, Michael; Nöh, Katharina; Dalman, Tolga; Niedenführ, Sebastian; Stute, Birgit; Wiechert, Wolfgang

    2013-01-01

    (13)C-based metabolic flux analysis ((13)C-MFA) is the state-of-the-art method to quantitatively determine in vivo metabolic reaction rates in microorganisms. 13CFLUX2 contains all tools for composing flexible computational (13)C-MFA workflows to design and evaluate carbon labeling experiments. A specially developed XML language, FluxML, highly efficient data structures and simulation algorithms achieve a maximum of performance and effectiveness. Support of multicore CPUs, as well as compute clusters, enables scalable investigations. 13CFLUX2 outperforms existing tools in terms of universality, flexibility and built-in features. Therewith, 13CFLUX2 paves the way for next-generation high-resolution (13)C-MFA applications on the large scale. 13CFLUX2 is implemented in C++ (ISO/IEC 14882 standard) with Java and Python add-ons to run under Linux/Unix. A demo version and binaries are available at www.13cflux.net.

  9. OptFlux: an open-source software platform for in silico metabolic engineering

    DEFF Research Database (Denmark)

    Rocha, I.; Maia, P.; Evangelista, P.

    2010-01-01

    software aimed at being the reference computational application in the field. It is the first tool to incorporate strain optimization tasks, i.e., the identification of Metabolic Engineering targets, using Evolutionary Algorithms/Simulated Annealing metaheuristics or the previously proposed Opt...... to address industrial goals. However, the use of these methods has been restricted to bioinformaticians or other expert researchers. The main aim of this work is, therefore, to provide a user-friendly computational tool for Metabolic Engineering applications. Results: OptFlux is an open-source and modular...... algorithms. The software supports importing/exporting to several flat file formats and it is compatible with the SBML standard. OptFlux has a visualization module that allows the analysis of the model structure that is compatible with the layout information of Cell Designer, allowing the superimposition...

  10. Magnetic Resonance Spectroscopic Imaging of Tumor Metabolic Markers for Cancer Diagnosis, Metabolic Phenotyping, and Characterization of Tumor Microenvironment

    Directory of Open Access Journals (Sweden)

    Qiuhong He

    2004-01-01

    Full Text Available Cancer cells display heterogeneous genetic characteristics, depending on the tumor dynamic microenvironment. Abnormal tumor vasculature and poor tissue oxygenation generate a fraction of hypoxic tumor cells that have selective advantages in metastasis and invasion and often resist chemo- and radiation therapies. The genetic alterations acquired by tumors modify their biochemical pathways, which results in abnormal tumor metabolism. An elevation in glycolysis known as the “Warburg effect” and changes in lipid synthesis and oxidation occur. Magnetic resonance spectroscopy (MRS has been used to study tumor metabolism in preclinical animal models and in clinical research on human breast, brain, and prostate cancers. This technique can identify specific genetic and metabolic changes that occur in malignant tumors. Therefore, the metabolic markers, detectable by MRS, not only provide information on biochemical changes but also define different metabolic tumor phenotypes. When combined with the contrast-enhanced Magnetic Resonance Imaging (MRI, which has a high sensitivity for cancer diagnosis, in vivo magnetic resonance spectroscopic imaging (MRSI improves the diagnostic specificity of malignant human cancers and is becoming an important clinical tool for cancer management and care. This article reviews the MRSI techniques as molecular imaging methods to detect and quantify metabolic changes in various tumor tissue types, especially in extracranial tumor tissues that contain high concentrations of fat. MRI/MRSI methods have been used to characterize tumor microenvironments in terms of blood volume and vessel permeability. Measurements of tissue oxygenation and glycolytic rates by MRS also are described to illustrate the capability of the MR technology in probing molecular information non-invasively in tumor tissues and its important potential for studying molecular mechanisms of human cancers in physiological conditions.

  11. Impaired TCA cycle flux in mitochondria in skeletal muscle from type 2 diabetic subjects: marker or maker of the diabetic phenotype?

    Science.gov (United States)

    Gaster, Michael; Nehlin, Jan O; Minet, Ariane D

    2012-07-01

    The diabetic phenotype is complex, requiring elucidation of key initiating defects. Recent research has shown that diabetic myotubes express a primary reduced tricarboxylic acid (TCA) cycle flux. A reduced TCA cycle flux has also been shown both in insulin resistant offspring of T2D patients and exercising T2D patients in vivo. This review will discuss the latest advances in the understanding of the molecular mechanisms regulating the TCA cycle with focus on possible underlying mechanism which could explain the impaired TCA flux in insulin resistant human skeletal muscle in type 2 diabetes. A reduced TCA is both a marker and a maker of the diabetic phenotype.

  12. Identifying model error in metabolic flux analysis - a generalized least squares approach.

    Science.gov (United States)

    Sokolenko, Stanislav; Quattrociocchi, Marco; Aucoin, Marc G

    2016-09-13

    The estimation of intracellular flux through traditional metabolic flux analysis (MFA) using an overdetermined system of equations is a well established practice in metabolic engineering. Despite the continued evolution of the methodology since its introduction, there has been little focus on validation and identification of poor model fit outside of identifying "gross measurement error". The growing complexity of metabolic models, which are increasingly generated from genome-level data, has necessitated robust validation that can directly assess model fit. In this work, MFA calculation is framed as a generalized least squares (GLS) problem, highlighting the applicability of the common t-test for model validation. To differentiate between measurement and model error, we simulate ideal flux profiles directly from the model, perturb them with estimated measurement error, and compare their validation to real data. Application of this strategy to an established Chinese Hamster Ovary (CHO) cell model shows how fluxes validated by traditional means may be largely non-significant due to a lack of model fit. With further simulation, we explore how t-test significance relates to calculation error and show that fluxes found to be non-significant have 2-4 fold larger error (if measurement uncertainty is in the 5-10 % range). The proposed validation method goes beyond traditional detection of "gross measurement error" to identify lack of fit between model and data. Although the focus of this work is on t-test validation and traditional MFA, the presented framework is readily applicable to other regression analysis methods and MFA formulations.

  13. Advantages of dynamic "closed loop" stable isotope flux phenotyping over static "open loop" clamps in detecting silent genetic and dietary phenotypes.

    Science.gov (United States)

    Vaitheesvaran, Bhavapriya; Chueh, Fu-Yu; Xu, Jun; Trujillo, Chuck; Saad, M F; Lee, W N P; McGuinness, Owen P; Kurland, Irwin J

    2010-06-01

    In vivo insulin sensitivity can be assessed using "open loop" clamp or "closed loop" methods. Open loop clamp methods are static, and fix plasma glucose independently from plasma insulin. Closed loop methods are dynamic, and assess glucose disposal in response to a stable isotope labeled glucose tolerance test. Using PPARalpha(-/-) mice, open and closed loop assessments of insulin sensitivity/glucose disposal were compared. Indirect calorimetry done for the assessment of diurnal substrate utilization/metabolic flexibility showed that chow fed PPARalpha(-/-) mice had increased glucose utilization during the light (starved) cycle. Euglycemic clamps showed no differences in insulin stimulated glucose disposal, whether for chow or high fat diets, but did show differences in basal glucose clearance for chow fed PPARalpha(-/-) versus SV129J-wt mice. In contrast, the dynamic stable isotope labeled glucose tolerance tests reveal enhanced glucose disposal for PPARalpha(-/-) versus SV129J-wt, for chow and high fat diets. Area under the curve for plasma labeled and unlabeled glucose for PPARalpha(-/-) was approximately 1.7-fold lower, P difference on a high fat diet (0.30 +/- 0.1 for SV129J-wt vs. 0.13 +/- 0.10 for PPARalpha(-/-), P = 0.28). This study demonstrates that dynamic stable isotope labeled glucose tolerance test can assess "silent" metabolic phenotypes, not detectable by the static, "open loop", euglycemic or hyperglycemic clamps. Both open loop and closed loop methods may describe different aspects of metabolic inflexibility and insulin sensitivity.

  14. Metabolic flux analysis during the exponential growth phase of Saccharomyces cerevisiae in wine fermentations.

    Science.gov (United States)

    Quirós, Manuel; Martínez-Moreno, Rubén; Albiol, Joan; Morales, Pilar; Vázquez-Lima, Felícitas; Barreiro-Vázquez, Antonio; Ferrer, Pau; Gonzalez, Ramon

    2013-01-01

    As a consequence of the increase in global average temperature, grapes with the adequate phenolic and aromatic maturity tend to be overripe by the time of harvest, resulting in increased sugar concentrations and imbalanced C/N ratios in fermenting musts. This fact sets obvious additional hurdles in the challenge of obtaining wines with reduced alcohols levels, a new trend in consumer demands. It would therefore be interesting to understand Saccharomyces cerevisiae physiology during the fermentation of must with these altered characteristics. The present study aims to determine the distribution of metabolic fluxes during the yeast exponential growth phase, when both carbon and nitrogen sources are in excess, using continuous cultures. Two different sugar concentrations were studied under two different winemaking temperature conditions. Although consumption and production rates for key metabolites were severely affected by the different experimental conditions studied, the general distribution of fluxes in central carbon metabolism was basically conserved in all cases. It was also observed that temperature and sugar concentration exerted a higher effect on the pentose phosphate pathway and glycerol formation than on glycolysis and ethanol production. Additionally, nitrogen uptake, both quantitatively and qualitatively, was strongly influenced by environmental conditions. This work provides the most complete stoichiometric model used for Metabolic Flux Analysis of S. cerevisiae in wine fermentations employed so far, including the synthesis and release of relevant aroma compounds and could be used in the design of optimal nitrogen supplementation of wine fermentations.

  15. Metabolic flux analysis during the exponential growth phase of Saccharomyces cerevisiae in wine fermentations.

    Directory of Open Access Journals (Sweden)

    Manuel Quirós

    Full Text Available As a consequence of the increase in global average temperature, grapes with the adequate phenolic and aromatic maturity tend to be overripe by the time of harvest, resulting in increased sugar concentrations and imbalanced C/N ratios in fermenting musts. This fact sets obvious additional hurdles in the challenge of obtaining wines with reduced alcohols levels, a new trend in consumer demands. It would therefore be interesting to understand Saccharomyces cerevisiae physiology during the fermentation of must with these altered characteristics. The present study aims to determine the distribution of metabolic fluxes during the yeast exponential growth phase, when both carbon and nitrogen sources are in excess, using continuous cultures. Two different sugar concentrations were studied under two different winemaking temperature conditions. Although consumption and production rates for key metabolites were severely affected by the different experimental conditions studied, the general distribution of fluxes in central carbon metabolism was basically conserved in all cases. It was also observed that temperature and sugar concentration exerted a higher effect on the pentose phosphate pathway and glycerol formation than on glycolysis and ethanol production. Additionally, nitrogen uptake, both quantitatively and qualitatively, was strongly influenced by environmental conditions. This work provides the most complete stoichiometric model used for Metabolic Flux Analysis of S. cerevisiae in wine fermentations employed so far, including the synthesis and release of relevant aroma compounds and could be used in the design of optimal nitrogen supplementation of wine fermentations.

  16. Hepatic Transporter Expression in Metabolic Syndrome: Phenotype, Serum Metabolic Hormones, and Transcription Factor Expression.

    Science.gov (United States)

    Donepudi, Ajay C; Cheng, Qiuqiong; Lu, Zhenqiang James; Cherrington, Nathan J; Slitt, Angela L

    2016-04-01

    Metabolic syndrome is a multifactorial disease associated with obesity, insulin resistance, diabetes, and the alteration of multiple metabolic hormones. Obesity rates have been rising worldwide, which increases our need to understand how this population will respond to drugs and exposure to other chemicals. The purpose of this study was to determine in lean and obese mice the ontogeny of clinical biomarkers such as serum hormone and blood glucose levels as well as the physiologic markers that correlate with nuclear receptor- and transporter-related pathways. Livers from male and female wild-type (WT) (C57BL/6) and ob/ob mice littermates were collected before, during, and after the onset of obesity. Serum hormone and mRNA levels were analyzed. Physiologic changes and gene expression during maturation and progression to obesity were performed and correlation analysis was performed using canonical correlations. Significant ontogenic changes in both WT and ob/ob mice were observed and these ontogenic changes differ in ob/ob mice with the development of obesity. In males and females, the ontogenic pattern of the expression of genes such as Abcc3, 4, Abcg2, Cyp2b10, and 4a14 started to differ from week 3, and became significant at weeks 4 and 8 in ob/ob mice compared with WT mice. In obese males, serum resistin, glucagon, and glucose levels correlated with the expression of most hepatic ATP-binding cassette (Abc) transporters, whereas in obese females, serum glucagon-like peptide 1 levels were correlated with most hepatic uptake transporters and P450 enzymes. Overall, the correlation between physiologic changes and gene expression indicate that metabolism-related hormones may play a role in regulating the genes involved in drug metabolism and transport. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  17. Investigation of useful carbon tracers for 13C-metabolic flux analysis of Escherichia coli by considering five experimentally determined flux distributions.

    Science.gov (United States)

    Maeda, Kousuke; Okahashi, Nobuyuki; Toya, Yoshihiro; Matsuda, Fumio; Shimizu, Hiroshi

    2016-12-01

    The 13 C-MFA experiments require an optimal design since the precision or confidence intervals of the estimated flux levels depends on factors such as the composition of 13 C-labeled carbon sources, as well as the metabolic flux distribution of interest. In this study, useful compositions of 13 C-labeled glucose for 13 C-metabolic flux analysis ( 13 C-MFA) of Escherichia coli are investigated using a computer simulation of the stable isotope labeling experiment. Following the generation of artificial mass spectra datasets of amino acid fragments using five literature-reported flux distributions of E. coli , the best fitted flux distribution and the 95% confidence interval were estimated by the 13 C-MFA procedure. A comparison of the precision scores showed that [1, 2- 13 C]glucose and a mixture of [1- 13 C] and [U- 13 C]glucose at 8:2 are one of the best carbon sources for a precise estimation of flux levels of the pentose phosphate pathway, glycolysis and the TCA cycle. Although the precision scores of the anaplerotic and glyoxylate pathway reactions were affected by both the carbon source and flux distribution, it was also shown that the mixture of non-labeled, [1- 13 C], and [U- 13 C]glucose at 4:1:5 was specifically effective for the flux estimation of the glyoxylate pathway reaction. These findings were confirmed by wet 13 C-MFA experiments.

  18. Strategies for individual phenotyping of linoleic and arachidonic acid metabolism using an oral glucose tolerance test.

    Directory of Open Access Journals (Sweden)

    Edoardo Saccenti

    Full Text Available The ability to restore homeostasis upon environmental challenges has been proposed as a measure for health. Metabolic profiling of plasma samples during the challenge response phase should offer a profound view on the flexibility of a phenotype to cope with daily stressors. Current data modeling approaches, however, struggle to extract biological descriptors from time-resolved metabolite profiles that are able to discriminate between different phenotypes. Thus, for the case of oxylipin responses in plasma upon an oral glucose tolerance test we developed a modeling approach that incorporates a priori biological pathway knowledge. The degradation pathways of arachidonic and linoleic acids were modeled using a regression model based on a pseudo-steady-state approximated model, resulting in a parameter A that summarizes the relative enzymatic activity in these pathways. Analysis of the phenotypic parameters As suggests that different phenotypes can be discriminated according to preferred relative activity of the arachidonic and linoleic pathway. Correlation analysis shows that there is little or no competition between the arachidonic and linoleic acid pathways, although they share the same enzymes.

  19. Modeling Rice Metabolism: From Elucidating Environmental Effects on Cellular Phenotype to Guiding Crop Improvement.

    Science.gov (United States)

    Lakshmanan, Meiyappan; Cheung, C Y Maurice; Mohanty, Bijayalaxmi; Lee, Dong-Yup

    2016-01-01

    Crop productivity is severely limited by various biotic and abiotic stresses. Thus, it is highly needed to understand the underlying mechanisms of environmental stress response and tolerance in plants, which could be addressed by systems biology approach. To this end, high-throughput omics profiling and in silico modeling can be considered to explore the environmental effects on phenotypic states and metabolic behaviors of rice crops at the systems level. Especially, the advent of constraint-based metabolic reconstruction and analysis paves a way to characterize the plant cellular physiology under various stresses by combining the mathematical network models with multi-omics data. Rice metabolic networks have been reconstructed since 2013 and currently six such networks are available, where five are at genome-scale. Since their publication, these models have been utilized to systematically elucidate the rice abiotic stress responses and identify agronomic traits for crop improvement. In this review, we summarize the current status of the existing rice metabolic networks and models with their applications. Furthermore, we also highlight future directions of rice modeling studies, particularly stressing how these models can be used to contextualize the affluent multi-omics data that are readily available in the public domain. Overall, we envisage a number of studies in the future, exploiting the available metabolic models to enhance the yield and quality of rice and other food crops.

  20. Amino Acid Flux from Metabolic Network Benefits Protein Translation: the Role of Resource Availability.

    Science.gov (United States)

    Hu, Xiao-Pan; Yang, Yi; Ma, Bin-Guang

    2015-06-09

    Protein translation is a central step in gene expression and affected by many factors such as codon usage bias, mRNA folding energy and tRNA abundance. Despite intensive previous studies, how metabolic amino acid supply correlates with protein translation efficiency remains unknown. In this work, we estimated the amino acid flux from metabolic network for each protein in Escherichia coli and Saccharomyces cerevisiae by using Flux Balance Analysis. Integrated with the mRNA expression level, protein abundance and ribosome profiling data, we provided a detailed description of the role of amino acid supply in protein translation. Our results showed that amino acid supply positively correlates with translation efficiency and ribosome density. Moreover, with the rank-based regression model, we found that metabolic amino acid supply facilitates ribosome utilization. Based on the fact that the ribosome density change of well-amino-acid-supplied genes is smaller than poorly-amino-acid-supply genes under amino acid starvation, we reached the conclusion that amino acid supply may buffer ribosome density change against amino acid starvation and benefit maintaining a relatively stable translation environment. Our work provided new insights into the connection between metabolic amino acid supply and protein translation process by revealing a new regulation strategy that is dependent on resource availability.

  1. Human breath analysis may support the existence of individual metabolic phenotypes.

    Directory of Open Access Journals (Sweden)

    Pablo Martinez-Lozano Sinues

    Full Text Available The metabolic phenotype varies widely due to external factors such as diet and gut microbiome composition, among others. Despite these temporal fluctuations, urine metabolite profiling studies have suggested that there are highly individual phenotypes that persist over extended periods of time. This hypothesis was tested by analyzing the exhaled breath of a group of subjects during nine days by mass spectrometry. Consistent with previous metabolomic studies based on urine, we conclude that individual signatures of breath composition exist. The confirmation of the existence of stable and specific breathprints may contribute to strengthen the inclusion of breath as a biofluid of choice in metabolomic studies. In addition, the fact that the method is rapid and totally non-invasive, yet individualized profiles can be tracked, makes it an appealing approach.

  2. Flux Balance Analysis of Cyanobacterial Metabolism.The Metabolic Network of Synechocystis sp. PCC 6803

    Czech Academy of Sciences Publication Activity Database

    Knoop, H.; Gründel, M.; Zilliges, Y.; Lehmann, R.; Hoffmann, S.; Lockau, W.; Steuer, Ralf

    2013-01-01

    Roč. 9, č. 6 (2013), e1003081-e1003081 ISSN 1553-7358 R&D Projects: GA MŠk(CZ) EE2.3.20.0256 Institutional support: RVO:67179843 Keywords : SP STRAIN PCC-6803 * SP ATCC 51142 * photoautotrophic metabolism * anacystis-nidulans * reconstructions * pathway * plants * models * growth Subject RIV: EI - Biotechnology ; Bionics Impact factor: 4.829, year: 2013

  3. The Prevalence of Metabolic Syndrome and Different Obesity Phenotype in Iranian Male Military Personnel.

    Science.gov (United States)

    Payab, Moloud; Hasani-Ranjbar, Shirin; Merati, Yaser; Esteghamati, Alireza; Qorbani, Mostafa; Hematabadi, Mahboobeh; Rashidian, Hoda; Shirzad, Nooshin

    2017-03-01

    Obesity, especially when concentrated in the abdominal area, is often associated with the presence of metabolic syndrome. Stress, particularly occupational stress, is one of the most important factors contributing to the increased prevalence of metabolic syndrome components among different populations. This study aimed to investigate the prevalence of overweight and obesity as well as the criteria for metabolic syndrome and its risk factors and different obesity phenotype in a population of military personnel aged 20 to 65 years. This study is a retrospective cross-sectional study in which data are extracted from the database of a military hospital (2,200 participants). The records of participants contained information such as age, marital status, educational level, weight, height, body mass index, blood pressure, waist circumference, history of drug use and smoking, as well as the results of tests including lipid profile and fasting blood glucose. The Adult Treatment Panel III criteria as well as two national criteria were used to identify metabolic syndrome among participants. Data analysis was p1erformed using SPSS version 16. The average age of participants was 33.37 (7.75) years. The prevalence of metabolic syndrome according to Iranian cutoff was 26.6% for the waist circumference >90 cm (585 persons) and 19.6% for the waist circumference >95 cm (432 persons). The rate of metabolic syndrome was identified as 11.1% (432 cases) according to Adult Treatment Panel III criteria. Results of the current study identified that the prevalence of metabolic syndrome among military individuals is less than other populations, but the prevalence of the syndrome is higher than other military personnel in other countries.

  4. A procedure for the estimation over time of metabolic fluxes in scenarios where measurements are uncertain and/or insufficient

    Directory of Open Access Journals (Sweden)

    Picó Jesús

    2007-10-01

    Full Text Available Abstract Background An indirect approach is usually used to estimate the metabolic fluxes of an organism: couple the available measurements with known biological constraints (e.g. stoichiometry. Typically this estimation is done under a static point of view. Therefore, the fluxes so obtained are only valid while the environmental conditions and the cell state remain stable. However, estimating the evolution over time of the metabolic fluxes is valuable to investigate the dynamic behaviour of an organism and also to monitor industrial processes. Although Metabolic Flux Analysis can be successively applied with this aim, this approach has two drawbacks: i sometimes it cannot be used because there is a lack of measurable fluxes, and ii the uncertainty of experimental measurements cannot be considered. The Flux Balance Analysis could be used instead, but the assumption of optimal behaviour of the organism brings other difficulties. Results We propose a procedure to estimate the evolution of the metabolic fluxes that is structured as follows: 1 measure the concentrations of extracellular species and biomass, 2 convert this data to measured fluxes and 3 estimate the non-measured fluxes using the Flux Spectrum Approach, a variant of Metabolic Flux Analysis that overcomes the difficulties mentioned above without assuming optimal behaviour. We apply the procedure to a real problem taken from the literature: estimate the metabolic fluxes during a cultivation of CHO cells in batch mode. We show that it provides a reliable and rich estimation of the non-measured fluxes, thanks to considering measurements uncertainty and reversibility constraints. We also demonstrate that this procedure can estimate the non-measured fluxes even when there is a lack of measurable species. In addition, it offers a new method to deal with inconsistency. Conclusion This work introduces a procedure to estimate time-varying metabolic fluxes that copes with the insufficiency of

  5. The nutritional status of Methanosarcina acetivorans regulates glycogen metabolism and gluconeogenesis and glycolysis fluxes.

    Science.gov (United States)

    Santiago-Martínez, Michel Geovanni; Encalada, Rusely; Lira-Silva, Elizabeth; Pineda, Erika; Gallardo-Pérez, Juan Carlos; Reyes-García, Marco Antonio; Saavedra, Emma; Moreno-Sánchez, Rafael; Marín-Hernández, Alvaro; Jasso-Chávez, Ricardo

    2016-05-01

    Gluconeogenesis is an essential pathway in methanogens because they are unable to use exogenous hexoses as carbon source for cell growth. With the aim of understanding the regulatory mechanisms of central carbon metabolism in Methanosarcina acetivorans, the present study investigated gene expression, the activities and metabolic regulation of key enzymes, metabolite contents and fluxes of gluconeogenesis, as well as glycolysis and glycogen synthesis/degradation pathways. Cells were grown with methanol as a carbon source. Key enzymes were kinetically characterized at physiological pH/temperature. Active consumption of methanol during exponential cell growth correlated with significant methanogenesis, gluconeogenic flux and steady glycogen synthesis. After methanol exhaustion, cells reached the stationary growth phase, which correlated with the rise in glycogen consumption and glycolytic flux, decreased methanogenesis, negligible acetate production and an absence of gluconeogenesis. Elevated activities of carbon monoxide dehydrogenase/acetyl-CoA synthetase complex and pyruvate: ferredoxin oxidoreductase suggested the generation of acetyl-CoA and pyruvate for glycogen synthesis. In the early stationary growth phase, the transcript contents and activities of pyruvate phosphate dikinase, fructose 1,6-bisphosphatase and glycogen synthase decreased, whereas those of glycogen phosphorylase, ADP-phosphofructokinase and pyruvate kinase increased. Therefore, glycogen and gluconeogenic metabolites were synthesized when an external carbon source was provided. Once such a carbon source became depleted, glycolysis and methanogenesis fed by glycogen degradation provided the ATP supply. Weak inhibition of key enzymes by metabolites suggested that the pathways evaluated were mainly transcriptionally regulated. Because glycogen metabolism and glycolysis/gluconeogenesis are not present in all methanogens, the overall data suggest that glycogen storage might represent an environmental

  6. Metabolic and functional phenotypic profiling of Drosophila melanogaster reveals reduced sex differentiation under stressful environmental conditions

    DEFF Research Database (Denmark)

    Ørsted, Michael; Malmendal, Anders; Muñoz, Joaquin

    2017-01-01

    Strong sexual dimorphism is commonly observed across species and e.g. trade-offs between reproduction and maintenance are thought to explain this dimorphism. Here we test how the metabolic and functional phenotypic responses to varying types of environmental stress differ in male and female...... rearing regimes were investigated using NMR metabolomics and assessed for body mass and viability. Our results showed that environmental stress leads to reduced sexual dimorphism in both metabolic composition and body mass compared to the level of dimorphism observed at benign conditions. This reduced...... Drosophila melanogaster (Diptera: Drosophilidae), and how this impacts the magnitude of sexual dimorphism. Experimental stressors that we exposed flies to during development were heat stress, poor nutrition, high acidity, high levels of ammonia and ethanol. Emerged male and female flies from the different...

  7. In vitro residual activity of phenylalanine hydroxylase variants and correlation with metabolic phenotypes in PKU.

    Science.gov (United States)

    Trunzo, Roberta; Santacroce, Rosa; Shen, Nan; Jung-Klawitter, Sabine; Leccese, Angelica; De Girolamo, Giuseppe; Margaglione, Maurizio; Blau, Nenad

    2016-12-05

    Hyperphenylalaninemias (HPAs) are genetic diseases predominantly caused by a wide range of variants in the phenylalanine hydroxylase (PAH) gene. In vitro expression analysis of PAH variants offers the opportunity to elucidate the molecular mechanisms involved in HPAs and to clarify whether a disease-associated variant is genuinely pathogenic, while investigating the severity of a metabolic phenotype, and determining how a variant exerts its deleterious effects on the PAH enzyme. To study the effects of gene variants on PAH activity, we investigated eight variants: c.611A>G (p.Y204C), c.635T>C (p.L212P), c.746T>C (p.L249P), c.745C>T (p.L249F), c.809G>A (p.R270K), c.782G>C (p.R261P), c.587C>A (p.S196Y) and c.1139C>T (p.T380M), associated with different phenotypic groups. Transient expression of mutant full-length cDNAs in COS-7 cells yielded PAH proteins with PAH activity levels between 7% and 51% compared to the wild-type enzyme. With one exception (p.Y204C, which had no significant impact on PAH function), lower PAH activity was associated with a more severe phenotype (e.g. p.L249P with 7% PAH activity, 100% of classic PKU and no BH 4 responsiveness), while higher activity correlated with milder phenotypes (e.g. p.T380M with 28% PAH activity, 97% of mild HPA and 83% of BH 4 responsiveness). The results of the in vitro residual PAH activity have major implications, both for our understanding of genotype-phenotype correlations, and thereby existing inconsistencies, but also for the elucidation of the molecular basis of tetrahydrobiopterin (BH 4 ) responsiveness. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Concept mapping One-Carbon Metabolism to model future ontologies for nutrient-gene-phenotype interactions.

    Science.gov (United States)

    Joslin, A C; Green, R; German, J B; Lange, M C

    2014-09-01

    Advances in the development of bioinformatic tools continue to improve investigators' ability to interrogate, organize, and derive knowledge from large amounts of heterogeneous information. These tools often require advanced technical skills not possessed by life scientists. User-friendly, low-barrier-to-entry methods of visualizing nutrigenomics information are yet to be developed. We utilized concept mapping software from the Institute for Human and Machine Cognition to create a conceptual model of diet and health-related data that provides a foundation for future nutrigenomics ontologies describing published nutrient-gene/polymorphism-phenotype data. In this model, maps containing phenotype, nutrient, gene product, and genetic polymorphism interactions are visualized as triples of two concepts linked together by a linking phrase. These triples, or "knowledge propositions," contextualize aggregated data and information into easy-to-read knowledge maps. Maps of these triples enable visualization of genes spanning the One-Carbon Metabolism (OCM) pathway, their sequence variants, and multiple literature-mined associations including concepts relevant to nutrition, phenotypes, and health. The concept map development process documents the incongruity of information derived from pathway databases versus literature resources. This conceptual model highlights the importance of incorporating information about genes in upstream pathways that provide substrates, as well as downstream pathways that utilize products of the pathway under investigation, in this case OCM. Other genes and their polymorphisms, such as TCN2 and FUT2, although not directly involved in OCM, potentially alter OCM pathway functionality. These upstream gene products regulate substrates such as B12. Constellations of polymorphisms affecting the functionality of genes along OCM, together with substrate and cofactor availability, may impact resultant phenotypes. These conceptual maps provide a foundational

  9. Hypoxia-inducible factors: coupling glucose metabolism and redox regulation with induction of the breast cancer stem cell phenotype.

    Science.gov (United States)

    Semenza, Gregg L

    2017-02-01

    Reduced oxygen availability (hypoxia) leads to increased production of reactive oxygen species (ROS) by the electron transport chain. Here, I review recent work delineating mechanisms by which hypoxia-inducible factor 1 (HIF-1) mediates adaptive metabolic responses to hypoxia, including increased flux through the glycolytic pathway and decreased flux through the tricarboxylic acid cycle, in order to decrease mitochondrial ROS production. HIF-1 also mediates increased flux through the serine synthesis pathway and mitochondrial one-carbon (folate cycle) metabolism to increase mitochondrial antioxidant production (NADPH and glutathione). Dynamic maintenance of ROS homeostasis is required for induction of the breast cancer stem cell phenotype in response to hypoxia or cytotoxic chemotherapy. Consistently, inhibition of phosphoglycerate dehydrogenase, the first enzyme of the serine synthesis pathway, in breast cancer cells impairs tumor initiation, metastasis, and response to cytotoxic chemotherapy. I discuss how these findings have important implications for understanding the logic of the tumor microenvironment and for improving therapeutic responses in women with breast cancer. © 2016 The Author.

  10. Metabolic phenotypes associated with high-temperature tolerance of Porphyra haitanensis strains.

    Science.gov (United States)

    Ye, Yangfang; Zhang, Limin; Yang, Rui; Luo, Qijun; Chen, Haimin; Yan, Xiaojun; Tang, Huiru

    2013-09-04

    Colored mutants of Porphyra haitanensis have superior production and quality characteristics, with two mutants, Shengfu 1 (SF-1) and Shengfu 2 (SF-2), having good high-temperature tolerances. To understand the molecular aspects of high-temperature tolerance, this study comprehensively investigated the metabolic differences between the high-temperature tolerant strains and wild type. Nuclear magnetic resonance (NMR) methods identified 35 algal metabolites, including sugars, amino acids, carboxylic acids, aldehydes, amines, and nucleotides. The results indicated that the high-temperature tolerant strains had significantly different metabolic phenotypes from the wild type. The high-temperature tolerant mutants had significantly higher levels in a set of osmolytes consisting of betaine, taurine, laminitol, and isofloridoside than the wild type, indicating the particular importance of efficient osmoregulation for high-temperature resistance. These findings provided essential metabolic information about high-temperature adaptation for P. haitanensis and demonstrated NMR-based metabolomics as a useful tool for understanding the metabolic features related to resistance to stressors.

  11. Rapamycin Suppresses Tumor Growth and Alters the Metabolic Phenotype in T-Cell Lymphoma.

    Science.gov (United States)

    Kittipongdaja, Wasakorn; Wu, Xuesong; Garner, Justine; Liu, Xiping; Komas, Steven M; Hwang, Sam T; Schieke, Stefan M

    2015-09-01

    The mTOR pathway is a master regulator of cellular growth and metabolism. The biosynthetic and energetic demand of rapidly proliferating cells such as cancer cells is met by metabolic adaptations such as an increased glycolytic rate known as the Warburg effect. Herein, we characterize the anti-tumor effect of rapamycin in a mouse model of T-cell lymphoma and examine the metabolic effects in vitro. The murine T-cell lymphoma line, MBL2, and human cutaneous T-cell lymphoma (CTCL) lines, HH and Hut78, were used in syngeneic or standard NSG mouse models to demonstrate a marked suppression of tumor growth by rapamycin accompanied by inhibition of mTORC1/2. Analysis of the metabolic phenotype showed a substantial reduction in the glycolytic rate and glucose utilization in rapamycin-treated lymphoma cells. This was associated with reduced expression of glucose transporters and glycolytic enzymes in cultured cells and xenograft tumors. As a result of the decrease in glycolytic state, rapamycin-treated cells displayed reduced sensitivity to low-glucose conditions but continued to rely on mitochondrial oxidative phosphorylation (OXPHOS) with sensitivity to inhibition of OXPHOS. Taken together, we demonstrate that rapamycin suppresses growth of T-cell lymphoma tumors and leads to a reduction in aerobic glycolysis counteracting the Warburg effect of cancer cells.

  12. Metabolic flux ratio analysis and multi-objective optimization revealed a globally conserved and coordinated metabolic response of E. coli to paraquat-induced oxidative stress.

    Science.gov (United States)

    Shen, Tie; Rui, Bin; Zhou, Hong; Zhang, Ximing; Yi, Yin; Wen, Han; Zheng, Haoran; Wu, Jihui; Shi, Yunyu

    2013-01-27

    The ability of a microorganism to adapt to changes in the environment, such as in nutrient or oxygen availability, is essential for its competitive fitness and survival. The cellular objective and the strategy of the metabolic response to an extreme environment are therefore of tremendous interest and, thus, have been increasingly explored. However, the cellular objective of the complex regulatory structure of the metabolic changes has not yet been fully elucidated and more details regarding the quantitative behaviour of the metabolic flux redistribution are required to understand the systems-wide biological significance of this response. In this study, the intracellular metabolic flux ratios involved in the central carbon metabolism were determined by fractional (13)C-labeling and metabolic flux ratio analysis (MetaFoR) of the wild-type E. coli strain JM101 at an oxidative environment in a chemostat. We observed a significant increase in the flux through phosphoenolpyruvate carboxykinase (PEPCK), phosphoenolpyruvate carboxylase (PEPC), malic enzyme (MEZ) and serine hydroxymethyltransferase (SHMT). We applied an ε-constraint based multi-objective optimization to investigate the trade-off relationships between the biomass yield and the generation of reductive power using the in silico iJR904 genome-scale model of E. coli K-12. The theoretical metabolic redistribution supports that the trans-hydrogenase pathway should not play a direct role in the defence mounted by E. coli against oxidative stress. The agreement between the measured ratio and the theoretical redistribution established the significance of NADPH synthesis as the goal of the metabolic reprogramming that occurs in response to oxidative stress. Our work presents a framework that combines metabolic flux ratio analysis and multi-objective optimization to investigate the metabolic trade-offs that occur under varied environmental conditions. Our results led to the proposal that the metabolic response of E

  13. A minimal growth medium for the basidiomycetePleurotus sapidusfor metabolic flux analysis.

    Science.gov (United States)

    Fraatz, Marco A; Naeve, Stefanie; Hausherr, Vanessa; Zorn, Holger; Blank, Lars M

    2014-01-01

    Pleurotus sapidus secretes a huge enzymatic repertoire including hydrolytic and oxidative enzymes and is an example for higher basidiomycetes being interesting for biotechnology. The complex growth media used for submerged cultivation limit basic physiological analyses of this group of organisms. Using undefined growth media, only little insights into the operation of central carbon metabolism and biomass formation, i.e. , the interplay of catabolic and anabolic pathways, can be gained. The development of a chemically defined growth medium allowed rapid growth of P. sapidus in submerged cultures. As P. sapidus grew extremely slow in salt medium, the co-utilization of amino acids using 13 C-labelled glucose was investigated by gas chromatography-mass spectrometry (GC-MS) analysis. While some amino acids were synthesized up to 90% in vivo from glucose ( e.g., alanine), asparagine and/or aspartate were predominantly taken up from the medium. With this information in hand, a defined yeast free salt medium containing aspartate and ammonium nitrate as a nitrogen source was developed. The observed growth rates of P. sapidus were well comparable with those previously published for complex media. Importantly, fast growth could be observed for 4 days at least, up to cell wet weights (CWW) of 400 g L -1 . The chemically defined medium was used to carry out a 13 C-based metabolic flux analysis, and the in vivo reactions rates in the central carbon metabolism of P. sapidus were investigated. The results revealed a highly respiratory metabolism with high fluxes through the pentose phosphate pathway and TCA cycle. The presented chemically defined growth medium enables researchers to study the metabolism of P. sapidus , significantly enlarging the analytical capabilities. Detailed studies on the production of extracellular enzymes and of secondary metabolites of P. sapidus may be designed based on the reported data.

  14. Physiology and genetics of metabolic flux control in Zymomonas mobilis. Progress report

    Energy Technology Data Exchange (ETDEWEB)

    Conway, T.

    1992-08-01

    This work seeks to understand the role of gene expression in regulating glycolytic enzyme synthesis in a balance that allows proper glycoltic flux control. The seven genes targeted for study in this laboratory have been cloned and sequenced, and molecular details of regulation have been investigated. Clear that glycolytic enzyme synthesis is coordinated to prevent the build up of toxic metabolic intermediates. The genetic mechanisms responsible for regulating balanced expression of the EntnerDoudoroff and glycolytic genes in Z. mobilis are beginning to be understood. Several layers of genetic control, perhaps in a hierarchal arrangement act in concert to determine the relative abundance of the glycolytic enzymes. These genetic controls involve differential translational efficiency, highly conserved promoter sequences, transcription factors, differential mRNA stabilities, and nucleolytic mRNA processing. The serendipitous cloning of the glucose facilitator, glf, as a result of linkage to several other genes of interest will have a significant impact on the study of Z. mobilis metabolism. The glucose facilitator is being characterized in a genetically reconstituted system in E. coli. Molecular genetic studies indicate that the ratio of glf expression to that of glk, zmf, and edd is carefully regulated, and suggests a critical role in metabolic control. Regulation of glycolytic gene expression is now sufficiently well understood to allow use of the glycolytic genes as tools to manipulate specified enzyme levels for the purpose of analyzing metabolic flux control. The critical genes have been subcloned for stable expression in Z. mobilis and placed under control of a regulated promoter system involving the tac promoter, the lacI repressor, and gene induction in by IPTG. HPLC methods have been developed that allow quantitation of virtually all of the metabolic intermediates in the cell pool.

  15. Aerobic glucose metabolism of Saccharomyces kluyveri: Growth, metabolite production, and quantification of metabolic fluxes

    DEFF Research Database (Denmark)

    Møller, Kasper; Christensen, B.; Förster, Jochen

    2002-01-01

    The growth and product formation of Saccharomyces kluyveri was characterized in aerobic batch cultivation on glucose. At these conditions it was found that ethyl acetate was a major overflow metabolite in S. kluyveri. During the exponential-growth phase on glucose ethyl acetate was produced...... at a constant specific rate of 0.12 g ethyl acetate per g dry weight per hour. The aerobic glucose metabolism in S. kluyveri was found to be less fermentative than in S. cerevisiae, as illustrated by the comparably low yield of ethanol on glucose (0.08 +/- 0.02 g/g), and high yield of biomass on glucose (0...

  16. (13)C-metabolic flux analysis of lipid accumulation in the oleaginous fungus Mucor circinelloides.

    Science.gov (United States)

    Zhao, Lina; Zhang, Huaiyuan; Wang, Liping; Chen, Haiqin; Chen, Yong Q; Chen, Wei; Song, Yuanda

    2015-12-01

    The oleaginous fungus Mucor circinelloides is of industrial interest because it can produce high levels of polyunsaturated fatty acid γ-linolenic acid. M. circinelloides CBS 277.49 is able to accumulate less than 15% of cell dry weight as lipids, while M. circinelloides WJ11 can accumulate lipid up to 36%. In order to better understand the mechanisms behind the differential lipid accumulation in these two strains, tracer experiments with (13)C-glucose were performed with the growth of M. circinelloides and subsequent gas chromatography-mass spectrometric detection of (13)C-patterns in proteinogenic amino acids was carried out to identify the metabolic network topology and estimate intracellular fluxes. Our results showed that the high oleaginous strain WJ11 had higher flux of pentose phosphate pathway and malic enzyme, lower flux in tricarboxylic acid cycle, higher flux in glyoxylate cycle and ATP: citrate lyase, together, it might provide more NADPH and substrate acetyl-CoA for fatty acid synthesis. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. iMS2Flux – a high–throughput processing tool for stable isotope labeled mass spectrometric data used for metabolic flux analysis

    Directory of Open Access Journals (Sweden)

    Poskar C Hart

    2012-11-01

    Full Text Available Abstract Background Metabolic flux analysis has become an established method in systems biology and functional genomics. The most common approach for determining intracellular metabolic fluxes is to utilize mass spectrometry in combination with stable isotope labeling experiments. However, before the mass spectrometric data can be used it has to be corrected for biases caused by naturally occurring stable isotopes, by the analytical technique(s employed, or by the biological sample itself. Finally the MS data and the labeling information it contains have to be assembled into a data format usable by flux analysis software (of which several dedicated packages exist. Currently the processing of mass spectrometric data is time-consuming and error-prone requiring peak by peak cut-and-paste analysis and manual curation. In order to facilitate high-throughput metabolic flux analysis, the automation of multiple steps in the analytical workflow is necessary. Results Here we describe iMS2Flux, software developed to automate, standardize and connect the data flow between mass spectrometric measurements and flux analysis programs. This tool streamlines the transfer of data from extraction via correction tools to 13C-Flux software by processing MS data from stable isotope labeling experiments. It allows the correction of large and heterogeneous MS datasets for the presence of naturally occurring stable isotopes, initial biomass and several mass spectrometry effects. Before and after data correction, several checks can be performed to ensure accurate data. The corrected data may be returned in a variety of formats including those used by metabolic flux analysis software such as 13CFLUX, OpenFLUX and 13CFLUX2. Conclusion iMS2Flux is a versatile, easy to use tool for the automated processing of mass spectrometric data containing isotope labeling information. It represents the core framework for a standardized workflow and data processing. Due to its flexibility

  18. The role of inflammatory pathway genetic variation on maternal metabolic phenotypes during pregnancy.

    Directory of Open Access Journals (Sweden)

    Margrit Urbanek

    Full Text Available Since mediators of inflammation are associated with insulin resistance, and the risk of developing diabetes mellitus and gestational diabetes, we hypothesized that genetic variation in members of the inflammatory gene pathway impact glucose levels and related phenotypes in pregnancy. We evaluated this hypothesis by testing for association between genetic variants in 31 inflammatory pathway genes in the Hyperglycemia and Adverse Pregnancy Outcome (HAPO cohort, a large multiethnic multicenter study designed to address the impact of glycemia less than overt diabetes on pregnancy outcome.Fasting, 1-hour, and 2-hour glucose, fasting and 1-hour C-peptide, and HbA1c levels were measured in blood samples obtained from HAPO participants during an oral glucose tolerance test at 24-32 weeks gestation. We tested for association between 458 SNPs mapping to 31 genes in the inflammatory pathway and metabolic phenotypes in 3836 European ancestry and 1713 Thai pregnant women. The strongest evidence for association was observed with TNF alpha and HbA1c (rs1052248; 0.04% increase per allele C; p-value = 4.4×10(-5, RETN and fasting plasma glucose (rs1423096; 0.7 mg/dl decrease per allele A; p-value = 1.1×10(-4, IL8 and 1 hr plasma glucose (rs2886920; 2.6 mg/dl decrease per allele T; p-value = 1.3×10(-4, ADIPOR2 and fasting C-peptide (rs2041139; 0.55 ug/L decrease per allele A; p-value = 1.4×10(-4, LEPR and 1-hour C-peptide (rs1171278; 0.62 ug/L decrease per allele T; p-value = 2.4×10(-4, and IL6 and 1-hour plasma glucose (rs6954897; -2.29 mg/dl decrease per allele G, p-value = 4.3×10(-4.Based on the genes surveyed in this study the inflammatory pathway is unlikely to have a strong impact on maternal metabolic phenotypes in pregnancy although variation in individual members of the pathway (e.g. RETN, IL8, ADIPOR2, LEPR, IL6, and TNF alpha, may contribute to metabolic phenotypes in pregnant women.

  19. Reconstruction and flux analysis of coupling between metabolic pathways of astrocytes and neurons: application to cerebral hypoxia

    Directory of Open Access Journals (Sweden)

    Akιn Ata

    2007-12-01

    Full Text Available Abstract Background It is a daunting task to identify all the metabolic pathways of brain energy metabolism and develop a dynamic simulation environment that will cover a time scale ranging from seconds to hours. To simplify this task and make it more practicable, we undertook stoichiometric modeling of brain energy metabolism with the major aim of including the main interacting pathways in and between astrocytes and neurons. Model The constructed model includes central metabolism (glycolysis, pentose phosphate pathway, TCA cycle, lipid metabolism, reactive oxygen species (ROS detoxification, amino acid metabolism (synthesis and catabolism, the well-known glutamate-glutamine cycle, other coupling reactions between astrocytes and neurons, and neurotransmitter metabolism. This is, to our knowledge, the most comprehensive attempt at stoichiometric modeling of brain metabolism to date in terms of its coverage of a wide range of metabolic pathways. We then attempted to model the basal physiological behaviour and hypoxic behaviour of the brain cells where astrocytes and neurons are tightly coupled. Results The reconstructed stoichiometric reaction model included 217 reactions (184 internal, 33 exchange and 216 metabolites (183 internal, 33 external distributed in and between astrocytes and neurons. Flux balance analysis (FBA techniques were applied to the reconstructed model to elucidate the underlying cellular principles of neuron-astrocyte coupling. Simulation of resting conditions under the constraints of maximization of glutamate/glutamine/GABA cycle fluxes between the two cell types with subsequent minimization of Euclidean norm of fluxes resulted in a flux distribution in accordance with literature-based findings. As a further validation of our model, the effect of oxygen deprivation (hypoxia on fluxes was simulated using an FBA-derivative approach, known as minimization of metabolic adjustment (MOMA. The results show the power of the

  20. Flux

    DEFF Research Database (Denmark)

    Ravn, Ib

    . FLUX betegner en flyden eller strømmen, dvs. dynamik. Forstår man livet som proces og udvikling i stedet for som ting og mekanik, får man et andet billede af det gode liv end det, som den velkendte vestlige mekanicisme lægger op til. Dynamisk forstået indebærer det gode liv den bedst mulige...... kanalisering af den flux eller energi, der strømmer igennem os og giver sig til kende i vore daglige aktiviteter. Skal vores tanker, handlinger, arbejde, samvær og politiske liv organiseres efter stramme og faste regelsæt, uden slinger i valsen? Eller skal de tværtimod forløbe ganske uhindret af regler og bånd...

  1. A robust and efficient method for estimating enzyme complex abundance and metabolic flux from expression data.

    Science.gov (United States)

    Barker, Brandon E; Sadagopan, Narayanan; Wang, Yiping; Smallbone, Kieran; Myers, Christopher R; Xi, Hongwei; Locasale, Jason W; Gu, Zhenglong

    2015-12-01

    A major theme in constraint-based modeling is unifying experimental data, such as biochemical information about the reactions that can occur in a system or the composition and localization of enzyme complexes, with high-throughput data including expression data, metabolomics, or DNA sequencing. The desired result is to increase predictive capability and improve our understanding of metabolism. The approach typically employed when only gene (or protein) intensities are available is the creation of tissue-specific models, which reduces the available reactions in an organism model, and does not provide an objective function for the estimation of fluxes. We develop a method, flux assignment with LAD (least absolute deviation) convex objectives and normalization (FALCON), that employs metabolic network reconstructions along with expression data to estimate fluxes. In order to use such a method, accurate measures of enzyme complex abundance are needed, so we first present an algorithm that addresses quantification of complex abundance. Our extensions to prior techniques include the capability to work with large models and significantly improved run-time performance even for smaller models, an improved analysis of enzyme complex formation, the ability to handle large enzyme complex rules that may incorporate multiple isoforms, and either maintained or significantly improved correlation with experimentally measured fluxes. FALCON has been implemented in MATLAB and ATS, and can be downloaded from: https://github.com/bbarker/FALCON. ATS is not required to compile the software, as intermediate C source code is available. FALCON requires use of the COBRA Toolbox, also implemented in MATLAB. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Dietary glycemic index is associated with less favorable anthropometric and metabolic profiles in polycystic ovary syndrome women with different phenotypes.

    Science.gov (United States)

    Graff, Scheila Karen; Mário, Fernanda Missio; Alves, Bruna Cherubini; Spritzer, Poli Mara

    2013-10-01

    To compare glycemic index (GI) in the usual diet of polycystic ovary syndrome (PCOS) and control women and to investigate whether dietary GI is associated with body composition and anthropometric and metabolic variables across PCOS phenotypes. Cross-sectional study. University hospital outpatient clinic. Sixty-one women with PCOS and 44 nonhirsute women with ovulatory cycles. Metabolic work-up, biochemical and hormonal assays, assessment of body composition and rest metabolic rate, physical activity (pedometer), and food consumption (food frequency questionnaire). GI, glycemic load, dietary intake, and hormone and metabolic profile in PCOS versus control and in PCOS women stratified by tertiles of GI and PCOS phenotype. Mean age was 23.7 ± 6.3 years. Participants with PCOS had higher body fat percentage, fasting insulin, insulin resistance, lipid accumulation product, and androgen levels compared with control women. PCOS and control women in the highest tertile of GI had higher body mass index and waist circumference than those in the lowest tertile. Dietary GI was higher in the classic PCOS group. Obesity and this more severe PCOS phenotype explained 28.3% of variance in dietary GI. Dietary GI is increased in the classic PCOS phenotype and associated with a less favorable anthropometric and metabolic profile. Obesity and classic PCOS phenotype are age-independent predictors of higher dietary GI. Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  3. Multi-objective experimental design for (13)C-based metabolic flux analysis.

    Science.gov (United States)

    Bouvin, Jeroen; Cajot, Simon; D'Huys, Pieter-Jan; Ampofo-Asiama, Jerry; Anné, Jozef; Van Impe, Jan; Geeraerd, Annemie; Bernaerts, Kristel

    2015-10-01

    (13)C-based metabolic flux analysis is an excellent technique to resolve fluxes in the central carbon metabolism but costs can be significant when using specialized tracers. This work presents a framework for cost-effective design of (13)C-tracer experiments, illustrated on two different networks. Linear and non-linear optimal input mixtures are computed for networks for Streptomyces lividans and a carcinoma cell line. If only glucose tracers are considered as labeled substrate for a carcinoma cell line or S. lividans, the best parameter estimation accuracy is obtained by mixtures containing high amounts of 1,2-(13)C2 glucose combined with uniformly labeled glucose. Experimental designs are evaluated based on a linear (D-criterion) and non-linear approach (S-criterion). Both approaches generate almost the same input mixture, however, the linear approach is favored due to its low computational effort. The high amount of 1,2-(13)C2 glucose in the optimal designs coincides with a high experimental cost, which is further enhanced when labeling is introduced in glutamine and aspartate tracers. Multi-objective optimization gives the possibility to assess experimental quality and cost at the same time and can reveal excellent compromise experiments. For example, the combination of 100% 1,2-(13)C2 glucose with 100% position one labeled glutamine and the combination of 100% 1,2-(13)C2 glucose with 100% uniformly labeled glutamine perform equally well for the carcinoma cell line, but the first mixture offers a decrease in cost of $ 120 per ml-scale cell culture experiment. We demonstrated the validity of a multi-objective linear approach to perform optimal experimental designs for the non-linear problem of (13)C-metabolic flux analysis. Tools and a workflow are provided to perform multi-objective design. The effortless calculation of the D-criterion can be exploited to perform high-throughput screening of possible (13)C-tracers, while the illustrated benefit of multi

  4. The Relation between Diverse Phenotypes of PCOS with Clinical Manifestations, Anthropometric Indices and Metabolic Characteristics.

    Science.gov (United States)

    Shahrami, Seyedeh Hajar; Abbasi Ranjbar, Zahra; Milani, Forozan; Kezem-Nejad, Ehsan; Hassanzadeh Rad, Afagh; Dalil Heirat, Seyedeh Fatemeh

    2016-02-01

    Critical issue regarding to variation of findings based on different phenotypes led investigators to define whether they are distinct features or overlapping ones. Therefore, we aimed to investigate the association between diverse phenotypes of PCOS (Poly Cystic Ovary Syndrome) with clinical manifestations, anthropometric indices, and metabolic characteristics. This was a descriptive cross-sectional study conducted in 15-39 years old women with PCOS referred to infertility clinics in the north part of Iran, Rasht during 2010-2011. Data were gathered through an interview by a form consisted of demographic characteristics, laboratory findings, ovarian volume and anthropometric indices. A total of 214 patients consisted of 161 PCOS (cases) and 53 normal women (controls) participated in this study. The most prevalent phenotype in PCOS population was IM/PCO/HA (54%), followed by IM/HA (28%) and IM/PCO (13%). PCO/HA was present only in 6 PCOS patients (5%). PCOS patients were significantly younger than controls (P=0.07). Results showed that increased ovarian volume were higher in PCOS group in comparison with controls and IM/PCO/HA, and IM/PCO had respectively the largest ovarian volumes. Also, a significant relation was observed based on Cholesterol, 17OHP, LH, TG, 2hpp, and LH/FSH between patients with PCOS and control groups. There were significant differences in demographic, anthropometric, hormonal and ultrasound findings between PCOS and controls. Therefore, it seems that classification of the characteristics of each phenotype could offer an appropriate guide for screening risks of PCOS and may facilitate performing most favorable treatment for these complications.

  5. Litter size variation in hypothalamic gene expression determines adult metabolic phenotype in Brandt's voles (Lasiopodomys brandtii.

    Directory of Open Access Journals (Sweden)

    Xue-Ying Zhang

    Full Text Available Early postnatal environments may have long-term and potentially irreversible consequences on hypothalamic neurons involved in energy homeostasis. Litter size is an important life history trait and negatively correlated with milk intake in small mammals, and thus has been regarded as a naturally varying feature of the early developmental environment. Here we investigated the long-term effects of litter size on metabolic phenotype and hypothalamic neuropeptide mRNA expression involved in the regulation of energy homeostasis, using the offspring reared from large (10-12 and small (3-4 litter sizes, of Brandt's voles (Lasiopodomys brandtii, a rodent species from Inner Mongolia grassland in China.Hypothalamic leptin signaling and neuropeptides were measured by Real-Time PCR. We showed that offspring reared from small litters were heavier at weaning and also in adulthood than offspring from large litters, accompanied by increased food intake during development. There were no significant differences in serum leptin levels or leptin receptor (OB-Rb mRNA in the hypothalamus at weaning or in adulthood, however, hypothalamic suppressor of cytokine signaling 3 (SOCS3 mRNA in adulthood increased in small litters compared to that in large litters. As a result, the agouti-related peptide (AgRP mRNA increased in the offspring from small litters.These findings support our hypothesis that natural litter size has a permanent effect on offspring metabolic phenotype and hypothalamic neuropeptide expression, and suggest central leptin resistance and the resultant increase in AgRP expression may be a fundamental mechanism underlying hyperphagia and the increased risk of overweight in pups of small litters. Thus, we conclude that litter size may be an important and central determinant of metabolic fitness in adulthood.

  6. Flux coupling and transcriptional regulation within the metabolic network of the photosynthetic bacterium Synechocystis sp. PCC6803

    DEFF Research Database (Denmark)

    Montagud, Arnau; Zelezniak, Aleksej; Navarro, Emilio

    2011-01-01

    Synechocystis sp. PCC6803 is a model cyanobacterium capable of producing biofuels with CO2 as carbon source and with its metabolism fueled by light, for which it stands as a potential production platform of socio-economic importance. Compilation and characterization of Synechocystis genome...... networks, surrounded by a stable core of pathways leading to biomass building blocks. This analysis identified potential bottlenecks for hydrogen and ethanol production. Integration of transcriptomic data with the Synechocystis flux coupling networks lead to identification of reporter flux coupling pairs...... and reporter flux coupling groups - regulatory hot spots during metabolic shifts triggered by the availability of light. Overall, flux coupling analysis provided insight into the structural organization of Synechocystis sp. PCC6803 metabolic network toward designing of a photosynthesis-based production...

  7. Computational analysis of storage synthesis in developing Brassica napus L. (oilseed rape) embryos: Flux variability analysis in relation to 13C-metabolic flux analysis

    Energy Technology Data Exchange (ETDEWEB)

    Hay, J.; Schwender, J.

    2011-08-01

    Plant oils are an important renewable resource, and seed oil content is a key agronomical trait that is in part controlled by the metabolic processes within developing seeds. A large-scale model of cellular metabolism in developing embryos of Brassica napus (bna572) was used to predict biomass formation and to analyze metabolic steady states by flux variability analysis under different physiological conditions. Predicted flux patterns are highly correlated with results from prior 13C metabolic flux analysis of B. napus developing embryos. Minor differences from the experimental results arose because bna572 always selected only one sugar and one nitrogen source from the available alternatives, and failed to predict the use of the oxidative pentose phosphate pathway. Flux variability, indicative of alternative optimal solutions, revealed alternative pathways that can provide pyruvate and NADPH to plastidic fatty acid synthesis. The nutritional values of different medium substrates were compared based on the overall carbon conversion efficiency (CCE) for the biosynthesis of biomass. Although bna572 has a functional nitrogen assimilation pathway via glutamate synthase, the simulations predict an unexpected role of glycine decarboxylase operating in the direction of NH4+ assimilation. Analysis of the light-dependent improvement of carbon economy predicted two metabolic phases. At very low light levels small reductions in CO2 efflux can be attributed to enzymes of the tricarboxylic acid cycle (oxoglutarate dehydrogenase, isocitrate dehydrogenase) and glycine decarboxylase. At higher light levels relevant to the 13C flux studies, ribulose-1,5-bisphosphate carboxylase activity is predicted to account fully for the light-dependent changes in carbon balance.

  8. Nutrient fluxes and net metabolism in a coastal lagoon SW peninsula of Baja California, Mexico

    Directory of Open Access Journals (Sweden)

    Cervantes Duarte, R.

    2016-09-01

    Full Text Available Fluxes of nutrients and net metabolism were estimated in coastal lagoon Magdalena Bay using LOICZ biogeochemical model. In situ data were obtained from 14 sites in the lagoon and also from a fixed site in the adjacent ocean area. Intense upwelling (February to July and faint upwelling (August to January were analyzed from monthly time series. The Temperature, nitrite + nitrate, ammonium and phosphate within the lagoon showed significant differences (p<0.05 between the two periods. Salinity (p=0.408 was more homogeneous (no significantly different due to mixing processes. During the intense upwelling period, nutrients increased in and out of the lagoon due to the influence of Transitional Water and Subartic Water transported by the California Current. However, during the faint upwelling, from August to January, the Transition Water and Subtropical Surface Water were predominant. Magdalena Bay showed denitrification processes of throughout the year as it occurred in other semi-arid coastal lagoons. It also showed a net autotrophic metabolism during intense upwelling and heterotrophic metabolism during faint upwelling. Understanding nutrient flows and net metabolism through simple biogeochemical models can provide tools for better management of the coastal zone.

  9. Random sampling of elementary flux modes in large-scale metabolic networks.

    Science.gov (United States)

    Machado, Daniel; Soons, Zita; Patil, Kiran Raosaheb; Ferreira, Eugénio C; Rocha, Isabel

    2012-09-15

    The description of a metabolic network in terms of elementary (flux) modes (EMs) provides an important framework for metabolic pathway analysis. However, their application to large networks has been hampered by the combinatorial explosion in the number of modes. In this work, we develop a method for generating random samples of EMs without computing the whole set. Our algorithm is an adaptation of the canonical basis approach, where we add an additional filtering step which, at each iteration, selects a random subset of the new combinations of modes. In order to obtain an unbiased sample, all candidates are assigned the same probability of getting selected. This approach avoids the exponential growth of the number of modes during computation, thus generating a random sample of the complete set of EMs within reasonable time. We generated samples of different sizes for a metabolic network of Escherichia coli, and observed that they preserve several properties of the full EM set. It is also shown that EM sampling can be used for rational strain design. A well distributed sample, that is representative of the complete set of EMs, should be suitable to most EM-based methods for analysis and optimization of metabolic networks. Source code for a cross-platform implementation in Python is freely available at http://code.google.com/p/emsampler. dmachado@deb.uminho.pt Supplementary data are available at Bioinformatics online.

  10. Metabolic network rewiring of propionate flux compensates vitamin B12 deficiency in C. elegans

    Science.gov (United States)

    Watson, Emma; Olin-Sandoval, Viridiana; Hoy, Michael J; Li, Chi-Hua; Louisse, Timo; Yao, Victoria; Mori, Akihiro; Holdorf, Amy D; Troyanskaya, Olga G; Ralser, Markus; Walhout, Albertha JM

    2016-01-01

    Metabolic network rewiring is the rerouting of metabolism through the use of alternate enzymes to adjust pathway flux and accomplish specific anabolic or catabolic objectives. Here, we report the first characterization of two parallel pathways for the breakdown of the short chain fatty acid propionate in Caenorhabditis elegans. Using genetic interaction mapping, gene co-expression analysis, pathway intermediate quantification and carbon tracing, we uncover a vitamin B12-independent propionate breakdown shunt that is transcriptionally activated on vitamin B12 deficient diets, or under genetic conditions mimicking the human diseases propionic- and methylmalonic acidemia, in which the canonical B12-dependent propionate breakdown pathway is blocked. Our study presents the first example of transcriptional vitamin-directed metabolic network rewiring to promote survival under vitamin deficiency. The ability to reroute propionate breakdown according to B12 availability may provide C. elegans with metabolic plasticity and thus a selective advantage on different diets in the wild. DOI: http://dx.doi.org/10.7554/eLife.17670.001 PMID:27383050

  11. Metabolic flux analysis of Saccharomyces cerevisiae in a sealed winemaking fermentation system.

    Science.gov (United States)

    Li, Hua; Su, Jing; Ma, Wen; Guo, Anque; Shan, Zuhua; Wang, Hua

    2015-03-01

    A sealed fermentation (SF) system and an anaerobic fermentation (AF) system (under normal atmospheric pressure conditions) were employed to study the influence of endogenous carbon dioxide (CO2) on the metabolism of Saccharomyces cerevisiae. The results showed that the fermentation stopped when 82.0 g L(-1) glucose was consumed and the endogenously produced CO2: pressure reached to 14.3 MPa in SF system, while the sugar was used up during AF. The total yeast viable count in the end of AF was higher than that of SF. It was also observed that the ethanol yield in AF and SF was similar, the glycerol yield in AF was 1.26 times higher than that in SF, while the succinic acid and acetic acid yields in SF were 24.7 and 26 times higher than that in AF, respectively. Additionally, this work provides a stoichiometric model used for metabolic flux analysis of S. cerevisiae to compare the flux distribution in SF and AF. The results showed that CO2 had an important effect on the pathways of oxaloacetic acid formation from pyruvic acid and ribose-5-phosphate formation from glucose-6-phosphate. However, the pathway of ethanol formation from pyruvic acid (decarboxylation reaction), catalyzed by pyruvate decarboxylase, was insensitive to CO2. © FEMS 2014. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.

  12. Looking at the metabolic consequences of the colchicine-based in vivo autophagic flux assay

    Science.gov (United States)

    Seiliez, Iban; Belghit, Ikram; Gao, Yujie; Skiba-Cassy, Sandrine; Dias, Karine; Cluzeaud, Marianne; Rémond, Didier; Hafnaoui, Nordine; Salin, Bénédicte; Camougrand, Nadine; Panserat, Stéphane

    2016-01-01

    ABSTRACT Monitoring autophagic flux in vivo or in organs remains limited and the ideal methods relative to the techniques possible with cell culture may not exist. Recently, a few papers have demonstrated the feasibility of measuring autophagic flux in vivo by intraperitoneal (IP) injection of pharmacological agents (chloroquine, leupeptin, vinblastine, and colchicine). However, the metabolic consequences of the administration of these drugs remain largely unknown. Here, we report that 0.8 mg/kg/day IP colchicine increased LC3-II protein levels in the liver of fasted trout, supporting the usefulness of this drug for studying autophagic flux in vivo in our model organism. This effect was accompanied by a decrease of plasma glucose concentration associated with a fall in the mRNA levels of gluconeogenesis-related genes. Concurrently, triglycerides and lipid droplets content in the liver increased. In contrast, transcript levels of β-oxidation-related gene Cpt1a dropped significantly. Together, these results match with the reported role of autophagy in the regulation of glucose homeostasis and intracellular lipid stores, and highlight the importance of considering these effects when using colchicine as an in vivo “autophagometer.” PMID:26902586

  13. Metabolic flux analysis of the hydrogen production potential in Synechocystis sp. PCC6803

    Energy Technology Data Exchange (ETDEWEB)

    Navarro, E. [Departamento de Lenguajes y Ciencias de la Computacion, Campus de Teatrinos, Universidad de Malaga, 29071 Malaga (Spain); Montagud, A.; Fernandez de Cordoba, P.; Urchueguia, J.F. [Instituto Universitario de Matematica Pura y Aplicada, Universidad Politecnica de Valencia, Camino de Vera 14, 46022 Valencia (Spain)

    2009-11-15

    Hydrogen is a promising energy vector; however, finding methods to produce it from renewable sources is essential to allow its wide-scale use. In that line, biological hydrogen production, although it is considered as a possible alternative, requires substantial improvements to overcome its present low yields. In that direction, genetic manipulation probably will play a central role and from that point of view metabolic flux analysis (MFA) constitutes an important tool to guide a priori most suitable genetic modifications oriented to a hydrogen yield increase. In this work MFA has been applied to analyze hydrogen photoproduction of Synechocystis sp. PCC6803. Flux analysis was carried out based on literature data and several basic fluxes were estimated in different growing conditions of the system. From this analysis, an upper limit for hydrogen photoproduction has been determined indicating a wide margin for improvement. MFA was also used to find a feasible operating space for hydrogen production, which avoids oxygen inhibition, one of the most important limitations to make hydrogen production cost effective. In addition, a set of biotechnological strategies are proposed that would be consistent with the performed mathematical analysis. (author)

  14. Differential phenotyping of Brucella species using a newly developed semi-automated metabolic system

    Directory of Open Access Journals (Sweden)

    Appel Bernd

    2010-10-01

    Full Text Available Abstract Background A commercial biotyping system (Taxa Profile™, Merlin Diagnostika testing the metabolization of various substrates by bacteria was used to determine if a set of phenotypic features will allow the identification of members of the genus Brucella and their differentiation into species and biovars. Results A total of 191 different amines, amides, amino acids, other organic acids and heterocyclic and aromatic substrates (Taxa Profile™ A, 191 different mono-, di-, tri- and polysaccharides and sugar derivates (Taxa Profile™ C and 95 amino peptidase- and protease-reactions, 76 glycosidase-, phosphatase- and other esterase-reactions, and 17 classic reactions (Taxa Profile™ E were tested with the 23 reference strains representing the currently known species and biovars of Brucella and a collection of 60 field isolates. Based on specific and stable reactions a 96-well "Brucella identification and typing" plate (Micronaut™ was designed and re-tested in 113 Brucella isolates and a couple of closely related bacteria. Brucella species and biovars revealed characteristic metabolic profiles and each strain showed an individual pattern. Due to their typical metabolic profiles a differentiation of Brucella isolates to the species level could be achieved. The separation of B. canis from B. suis bv 3, however, failed. At the biovar level, B. abortus bv 4, 5, 7 and B. suis bv 1-5 could be discriminated with a specificity of 100%. B. melitensis isolates clustered in a very homogenous group and could not be resolved according to their assigned biovars. Conclusions The comprehensive testing of metabolic activity allows cluster analysis within the genus Brucella. The biotyping system developed for the identification of Brucella and differentiation of its species and biovars may replace or at least complement time-consuming tube testing especially in case of atypical strains. An easy to handle identification software facilitates the

  15. Fat oxidation at rest predicts peak fat oxidation during exercise and metabolic phenotype in overweight men

    DEFF Research Database (Denmark)

    Rosenkilde, M; Nordby, P; Nielsen, L B

    2010-01-01

    OBJECTIVE: To elucidate if fat oxidation at rest predicts peak fat oxidation during exercise and/or metabolic phenotype in moderately overweight, sedentary men. DESIGN: Cross-sectional study.Subjects:We measured respiratory exchange ratio (RER) at rest in 44 moderately overweight, normotensive...... and normoglycemic men and selected 8 subjects with a low RER (L-RER, body mass index (BMI): 27.9+/-0.9 kg m(-2), RER: 0.76+/-0.02) and 8 with a high RER (H-RER; BMI 28.1+/-1.1 kg m(-2), RER: 0.89+/-0.02). After an overnight fast, a venous blood sample was obtained and a graded exercise test was performed. Fat...... oxidation during exercise was quantified using indirect calorimetry. RESULTS: Peak fat oxidation during exercise was higher in L-RER than in H-RER (0.333+/-0.096 vs 0.169+/-0.028 g min(-1); P

  16. Recovery of Phenotypes Obtained by Adaptive Evolution through Inverse Metabolic Engineering

    DEFF Research Database (Denmark)

    Hong, Kuk-Ki; Nielsen, Jens

    2012-01-01

    In a previous study, system level analysis of adaptively evolved yeast mutants showing improved galactose utilization revealed relevant mutations. The governing mutations were suggested to be in the Ras/PKA signaling pathway and ergosterol metabolism. Here, site-directed mutants having one...... of the mutations RAS2Lys77, RAS2Tyr112, and ERG5Pro370 were constructed and evaluated. The mutants were also combined with overexpression of PGM2, earlier proved as a beneficial target for galactose utilization. The constructed strains were analyzed for their gross phenotype, transcriptome and targeted metabolites......, and the results were compared to those obtained from reference strains and the evolved strains. The RAS2Lys77 mutation resulted in the highest specific galactose uptake rate among all of the strains with an increased maximum specific growth rate on galactose. The RAS2Tyr112 mutation also improved the specific...

  17. Optimal tracers for parallel labeling experiments and13C metabolic flux analysis: A new precision and synergy scoring system.

    Science.gov (United States)

    Crown, Scott B; Long, Christopher P; Antoniewicz, Maciek R

    2016-11-01

    13 C-Metabolic flux analysis ( 13 C-MFA) is a widely used approach in metabolic engineering for quantifying intracellular metabolic fluxes. The precision of fluxes determined by 13 C-MFA depends largely on the choice of isotopic tracers and the specific set of labeling measurements. A recent advance in the field is the use of parallel labeling experiments for improved flux precision and accuracy. However, as of today, no systemic methods exist for identifying optimal tracers for parallel labeling experiments. In this contribution, we have addressed this problem by introducing a new scoring system and evaluating thousands of different isotopic tracer schemes. Based on this extensive analysis we have identified optimal tracers for 13 C-MFA. The best single tracers were doubly 13 C-labeled glucose tracers, including [1,6- 13 C]glucose, [5,6- 13 C]glucose and [1,2- 13 C]glucose, which consistently produced the highest flux precision independent of the metabolic flux map (here, 100 random flux maps were evaluated). Moreover, we demonstrate that pure glucose tracers perform better overall than mixtures of glucose tracers. For parallel labeling experiments the optimal isotopic tracers were [1,6- 13 C]glucose and [1,2- 13 C]glucose. Combined analysis of [1,6- 13 C]glucose and [1,2- 13 C]glucose labeling data improved the flux precision score by nearly 20-fold compared to widely use tracer mixture 80% [1- 13 C]glucose +20% [U- 13 C]glucose. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  18. Metabolic flux ratio analysis and cell staining suggest the existence of C4 photosynthesis in Phaeodactylum tricornutum.

    Science.gov (United States)

    Huang, A; Liu, L; Zhao, P; Yang, C; Wang, G C

    2016-03-01

    Mechanisms for carbon fixation via photosynthesis in the diatom Phaeodactylum tricornutum Bohlin were studied recently but there remains a long-standing debate concerning the occurrence of C4 photosynthesis in this species. A thorough investigation of carbon metabolism and the evidence for C4 photosynthesis based on organelle partitioning was needed. In this study, we identified the flux ratios between C3 and C4 compounds in P. tricornutum using (13)C-labelling metabolic flux ratio analysis, and stained cells with various cell-permeant fluorescent probes to investigate the likely organelle partitioning required for single-cell C4 photosynthesis. Metabolic flux ratio analysis indicated the C3/C4 exchange ratios were high. Cell staining indicated organelle partitioning required for single-cell C4 photosynthesis might exist in P. tricornutum. The results of (13)C-labelling metabolic flux ratio analysis and cell staining suggest single-cell C4 photosynthesis exists in P. tricornutum. This study provides insights into photosynthesis patterns of P. tricornutum and the evidence for C4 photosynthesis based on (13)C-labelling metabolic flux ratio analysis and organelle partitioning. © 2015 The Society for Applied Microbiology.

  19. Metabolic mechanisms behind the type 2 diabetes susceptible phenotype in low birth weight individuals

    DEFF Research Database (Denmark)

    Ribel-Madsen, Amalie

    have 1) an increased, incomplete fatty acid beta-oxidation in mitochondria, 2) an altered amino acid metabolism to ensure an adequate supply of tricarboxylic acid (TCA) cycle intermediates and thereby enable an efficient acetyl-CoA oxidation, and 3) an increased fatty acid flux into lipogenesis......, including de novo ceramide synthesis, in non-adipose tissue. Methods: Fasting plasma levels of 45 acylcarnitines, 15 amino acids, and 27 ceramides were measured in the young, healthy, LBW (≤ 10th percentile) and NBW (50-90th percentile) men of the above mentioned study population after the isocaloric...... available for lipogenesis, including the synthesis of lipotoxic lipids such as ceramides and diacylglycerols that impair insulin signalling. In the second study, we demonstrated that LBW men had higher plasma alanine, proline, methionine, citrulline, and total amino acid levels after the HFHC diet compared...

  20. Modeling the Contribution of Allosteric Regulation for Flux Control in the Central Carbon Metabolism of E. coli

    DEFF Research Database (Denmark)

    Machado, Daniel; Herrgard, Markus; Rocha, Isabel

    2015-01-01

    contribution to the metabolic flux changes. Inspired by these results, we develop a constraint-based method (arFBA) for simulation of metabolic flux distributions that accounts for allosteric interactions. This method can be used for systematic prediction of potential allosteric regulation under the given...... the metabolic flux. Accounting for allosteric interactions in metabolic reconstructions reveals a hidden topology in metabolic networks, improving our understanding of cellular metabolism and fostering the development of novel simulation methods that account for this type of regulation....

  1. Rhabdomyosarcoma cells show an energy producing anabolic metabolic phenotype compared with primary myocytes

    Directory of Open Access Journals (Sweden)

    Higashi Richard M

    2008-10-01

    Full Text Available Abstract Background The functional status of a cell is expressed in its metabolic activity. We have applied stable isotope tracing methods to determine the differences in metabolic pathways in proliferating Rhabdomysarcoma cells (Rh30 and human primary myocytes in culture. Uniformly 13C-labeled glucose was used as a source molecule to follow the incorporation of 13C into more than 40 marker metabolites using NMR and GC-MS. These include metabolites that report on the activity of glycolysis, Krebs' cycle, pentose phosphate pathway and pyrimidine biosynthesis. Results The Rh30 cells proliferated faster than the myocytes. Major differences in flux through glycolysis were evident from incorporation of label into secreted lactate, which accounts for a substantial fraction of the glucose carbon utilized by the cells. Krebs' cycle activity as determined by 13C isotopomer distributions in glutamate, aspartate, malate and pyrimidine rings was considerably higher in the cancer cells than in the primary myocytes. Large differences were also evident in de novo biosynthesis of riboses in the free nucleotide pools, as well as entry of glucose carbon into the pyrimidine rings in the free nucleotide pool. Specific labeling patterns in these metabolites show the increased importance of anaplerotic reactions in the cancer cells to maintain the high demand for anabolic and energy metabolism compared with the slower growing primary myocytes. Serum-stimulated Rh30 cells showed higher degrees of labeling than serum starved cells, but they retained their characteristic anabolic metabolism profile. The myocytes showed evidence of de novo synthesis of glycogen, which was absent in the Rh30 cells. Conclusion The specific 13C isotopomer patterns showed that the major difference between the transformed and the primary cells is the shift from energy and maintenance metabolism in the myocytes toward increased energy and anabolic metabolism for proliferation in the Rh30 cells

  2. Comprehensive analysis of glucose and xylose metabolism in Escherichia coli under aerobic and anaerobic conditions by13C metabolic flux analysis.

    Science.gov (United States)

    Gonzalez, Jacqueline E; Long, Christopher P; Antoniewicz, Maciek R

    2017-01-01

    Glucose and xylose are the two most abundant sugars derived from the breakdown of lignocellulosic biomass. While aerobic glucose metabolism is relatively well understood in E. coli, until now there have been only a handful of studies focused on anaerobic glucose metabolism and no 13 C-flux studies on xylose metabolism. In the absence of experimentally validated flux maps, constraint-based approaches such as MOMA and RELATCH cannot be used to guide new metabolic engineering designs. In this work, we have addressed this critical gap in current understanding by performing comprehensive characterizations of glucose and xylose metabolism under aerobic and anaerobic conditions, using recent state-of-the-art techniques in 13 C metabolic flux analysis ( 13 C-MFA). Specifically, we quantified precise metabolic fluxes for each condition by performing parallel labeling experiments and analyzing the data through integrated 13 C-MFA using the optimal tracers [1,2- 13 C]glucose, [1,6- 13 C]glucose, [1,2- 13 C]xylose and [5- 13 C]xylose. We also quantified changes in biomass composition and confirmed turnover of macromolecules by applying [U- 13 C]glucose and [U- 13 C]xylose tracers. We demonstrated that under anaerobic growth conditions there is significant turnover of lipids and that a significant portion of CO 2 originates from biomass turnover. Using knockout strains, we also demonstrated that β-oxidation is critical for anaerobic growth on xylose. Quantitative analysis of co-factor balances (NADH/FADH 2 , NADPH, and ATP) for different growth conditions provided new insights regarding the interplay of energy and redox metabolism and the impact on E. coli cell physiology. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  3. Specific Metabolic Markers Are Associated with Future Waist-Gaining Phenotype in Women.

    Directory of Open Access Journals (Sweden)

    Benedikt Merz

    Full Text Available Our study aims to identify metabolic markers associated with either a gain in abdominal (measured by waist circumference or peripheral (measured by hip circumference body fat mass.Data of 4 126 weight-gaining adults (18-75 years from three population-based, prospective German cohort studies (EPIC, KORA, DEGS were analysed regarding a waist-gaining (WG or hip-gaining phenotype (HG. The phenotypes were obtained by calculating the differences of annual changes in waist minus hip circumference. The difference was displayed for all cohorts. The highest 10% of this difference were defined as WG whereas the lowest 10% were defined as HG. A total of 121 concordant metabolite measurements were conducted using Biocrates AbsoluteIDQ® kits in EPIC and KORA. Sex-specific associations with metabolite concentration as independent and phenotype as the dependent variable adjusted for confounders were calculated. The Benjamini-Hochberg method was used to correct for multiple testing.Across studies both sexes gained on average more waist than hip circumference. We could identify 12 metabolites as being associated with the WG (n = 8 or HG (n = 4 in men, but none were significant after correction for multiple testing; 45 metabolites were associated with the WG (n = 41 or HG (n = 4 in women. For WG, n = 21 metabolites remained significant after correction for multiple testing. Respective odds ratios (OR ranged from 0.66 to 0.73 for tryptophan, the diacyl-phosphatidylcholines (PC C32:3, C36:0, C38:0, C38:1, C42:2, C42:5, the acyl-alkyl-PCs C32:2, C34:0, C36:0, C36:1, C36:2, C38:0, C38:2, C40:1, C40:2, C40:5, C40:6, 42:2, C42:3 and lyso-PC C17:0.Both weight-gaining men and women showed a clear tendency to gain more abdominal than peripheral fat. Gain of abdominal fat seems to be related to an initial metabolic state reflected by low concentrations of specific metabolites, at least in women. Thus, higher levels of specific PCs may play a protective role in gaining

  4. Consequences of phosphoenolpyruvate:sugar phosphotranferase system and pyruvate kinase isozymes inactivation in central carbon metabolism flux distribution in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Meza Eugenio

    2012-09-01

    Full Text Available Abstract Background In Escherichia coli phosphoenolpyruvate (PEP is a key central metabolism intermediate that participates in glucose transport, as precursor in several biosynthetic pathways and it is involved in allosteric regulation of glycolytic enzymes. In this work we generated W3110 derivative strains that lack the main PEP consumers PEP:sugar phosphotransferase system (PTS- and pyruvate kinase isozymes PykA and PykF (PTS-pykA- and PTS-pykF-. To characterize the effects of these modifications on cell physiology, carbon flux distribution and aromatics production capacity were determined. Results When compared to reference strain W3110, strain VH33 (PTS- displayed lower specific rates for growth, glucose consumption and acetate production as well as a higher biomass yield from glucose. These phenotypic effects were even more pronounced by the additional inactivation of PykA or PykF. Carbon flux analysis revealed that PTS inactivation causes a redirection of metabolic flux towards biomass formation. A cycle involving PEP carboxylase (Ppc and PEP carboxykinase (Pck was detected in all strains. In strains W3110, VH33 (PTS- and VH35 (PTS-, pykF-, the net flux in this cycle was inversely correlated with the specific rate of glucose consumption and inactivation of Pck in these strains caused a reduction in growth rate. In the PTS- background, inactivation of PykA caused a reduction in Ppc and Pck cycling as well as a reduction in flux to TCA, whereas inactivation of PykF caused an increase in anaplerotic flux from PEP to OAA and an increased flux to TCA. The wild-type and mutant strains were modified to overproduce L-phenylalanine. In resting cells experiments, compared to reference strain, a 10, 4 and 7-fold higher aromatics yields from glucose were observed as consequence of PTS, PTS PykA and PTS PykF inactivation. Conclusions Metabolic flux analysis performed on strains lacking the main activities generating pyruvate from PEP revealed the high

  5. Genetic and phenotypic analysis of carbohydrate metabolism and transport in Lactobacillus reuteri.

    Science.gov (United States)

    Zhao, Xin; Gänzle, Michael G

    2018-05-02

    Lactobacilli derive metabolic energy mainly from carbohydrate fermentation. Homofermentative and heterofermentative lactobacilli exhibit characteristic differences in carbohydrate transport and regulation of metabolism, however, enzymes for carbohydrate transport in heterofermentative lactobacilli are poorly characterized. This study aimed to identify carbohydrate active enzymes in the L. reuteri strains LTH2584, LTH5448, TMW1.656, TMW1.112, 100-23, mlc3, and lpuph by phenotypic analysis and comparative genomics. Sourdough and intestinal isolates of L. reuteri displayed no difference in the number and type of carbohydrate-active enzymes encoded in the genome. Predicted sugar transporters encoded by genomes of L. reuteri strains were secondary carriers and most belong to the major facilitator superfamily. The quantification of gene expression during growth in sourdough and in chemically defined media corresponded to the predicted function of the transporters MalT, ScrT and LacS as carriers for maltose, sucrose, and lactose or raffinose, respectively. The genotype for sugar utilization matched the fermentation profile of 39 sugars for L. reuteri strains, and indicated preference for maltose, sucrose, raffinose and (iso)-malto-oligosaccharides, which are available in sourdough and in the upper intestine of rodents. Pentose utilization in L. reuteri species was strain-specific but independent of the origin or phylogenetic position of isolates. Two glycosyl hydrolases, licheninase (EC 3.2.1.73) and endo-1, 4-β-galactosidase (EC 3.2.1.89) were identified based on conserved domains. In conclusion, the study identified the lack of PTS systems, preference for secondary carriers for carbohydrate transport, and absence of carbon catabolite repression as characteristic features of the carbohydrate metabolism in the heterofermentative L. reuteri. Copyright © 2018 Elsevier B.V. All rights reserved.

  6. Anthropometric, clinical, and metabolic comparisons of the four Rotterdam PCOS phenotypes: A prospective study of PCOS women

    Directory of Open Access Journals (Sweden)

    Sujata Kar

    2013-01-01

    Full Text Available Aims: 1. To study the distribution of various Rotterdam classified phenotypes of polycystic ovarian syndrome (PCOS women, in our population. 2. To compare the four phenotypes with respect to anthropometric, clinical, and metabolic parameters. 3. To report the prevalence of insulin resistance (IR and metabolic syndrome in these women. Settings and Design: Private practice, Prospective cross-sectional comparative study. Materials and Methods: Women attending gynecology outpatient with the primary complains of irregular menses and/or infertility were evaluated. Each of them underwent detailed clinical examination, transvaginal sonography, and biochemical and hormonal assays. Four hundred and ten women with a clinical diagnosis of PCOS based on Rotterdam criteria were included in the study. The four phenotypes were 1 PCO complete, that is oligo/anovulation (O + polycystic ovaries (P + hyperandrogenism (H 2 P + O, 3 P + H, and 4 O + H. All women were also evaluated for metabolic syndrome (American Heart Association/National Heart, Lung, and Blood Institute (AHA/NHLBI, modified Adult Treatment Panel (ATP III 2005 guidelines and IR (homeostatic model assessment-IR (HOMA-IR. Statistical Analysis: Statistical Package for Social Sciences (SPSS version 18. Results: Largest group was PCOS complete (65.6% followed by P + O (22.2%; H + O (11.2%; and P + H (0.9%. Overall prevalence of metabolic syndrome was 35.07%. Hyperandrogenic phenotyptes; H + O (50% and P + H + O (37.04%, had significantly higher prevalence of metabolic syndrome than normoandrogenic P + O phenotype (10% (P ≤ 0.001. Body mass index (BMI ≥ 25 (P = 0.0004; odds ratio (OR = 3.07 (1.6574-5.7108, 95% CI, waist circumference (WC ≥ 80 cm (P = 0.001; OR = 3.68 (1.6807-8.0737, 95% CI and family history of diabetes (P = 0.019; OR 1.82 (1.1008-3.0194, 95% CI, were strongly associated with the presence of metabolic syndrome. The overall prevalence of IR in PCOS women was 30.44% (HOMA-IR cutoff

  7. A holistic view of dietary carbohydrate utilization in lobster: digestion, postprandial nutrient flux, and metabolism.

    Directory of Open Access Journals (Sweden)

    Leandro Rodríguez-Viera

    Full Text Available Crustaceans exhibit a remarkable variation in their feeding habits and food type, but most knowledge on carbohydrate digestion and utilization in this group has come from research on few species. The aim of this study was to make an integrative analysis of dietary carbohydrate utilization in the spiny lobster Panulirus argus. We used complementary methodologies such as different assessments of digestibility, activity measurements of digestive and metabolic enzymes, and post-feeding flux of nutrients and metabolites. Several carbohydrates were well digested by the lobster, but maize starch was less digestible than all other starches studied, and its inclusion in diet affected protein digestibility. Most intense hydrolysis of carbohydrates in the gastric chamber of lobster occurred between 2-6 h after ingestion and afterwards free glucose increased in hemolymph. The inclusion of wheat in diet produced a slow clearance of glucose from the gastric fluid and a gradual increase in hemolymph glucose. More intense hydrolysis of protein in the gastric chamber occurred 6-12 h after ingestion and then amino acids tended to increase in hemolymph. Triglyceride concentration in hemolymph rose earlier in wheat-fed lobsters than in lobsters fed other carbohydrates, but it decreased the most 24 h later. Analyses of metabolite levels and activities of different metabolic enzymes revealed that intermolt lobsters had a low capacity to store and use glycogen, although it was slightly higher in wheat-fed lobsters. Lobsters fed maize and rice diets increased amino acid catabolism, while wheat-fed lobsters exhibited higher utilization of fatty acids. Multivariate analysis confirmed that the type of carbohydrate ingested had a profound effect on overall metabolism. Although we found no evidence of a protein-sparing effect of dietary carbohydrate, differences in the kinetics of their digestion and absorption impacted lobster metabolism determining the fate of other

  8. Metabolic and transcriptional responses of gilthead sea bream (Sparus aurata L.) to environmental stress: New insights in fish mitochondrial phenotyping

    NARCIS (Netherlands)

    Bermejo-Nogales, A.; Nederlof, M.A.J.; Benedito-Palos, L.; Ballester-Lozano, G.F.; Folkedal, O.; Olsen, R.E.; Sitjà-Bobadilla, A.; Pérez-Sánchez, J.

    2014-01-01

    The aim of the current study was to phenotype fish metabolism and the transcriptionally-mediated response of hepatic mitochondria of gilthead sea bream to intermittent and repetitive environmental stressors: (i) changes in water temperature (T-ST), (ii) changes in water level and chasing (C-ST) and

  9. Resuscitation of ischemic donor livers with normothermic machine perfusion: a metabolic flux analysis of treatment in rats.

    Directory of Open Access Journals (Sweden)

    Maria-Louisa Izamis

    Full Text Available Normothermic machine perfusion has previously been demonstrated to restore damaged warm ischemic livers to transplantable condition in animal models. However, the mechanisms of recovery are unclear, preventing rational optimization of perfusion systems and slowing clinical translation of machine perfusion. In this study, organ recovery time and major perfusate shortcomings were evaluated using a comprehensive metabolic analysis of organ function in perfusion prior to successful transplantation. Two groups, Fresh livers and livers subjected to 1 hr of warm ischemia (WI received perfusion for a total preservation time of 6 hrs, followed by successful transplantation. 24 metabolic fluxes were directly measured and 38 stoichiometrically-related fluxes were estimated via a mass balance model of the major pathways of energy metabolism. This analysis revealed stable metabolism in Fresh livers throughout perfusion while identifying two distinct metabolic states in WI livers, separated at t = 2 hrs, coinciding with recovery of oxygen uptake rates to Fresh liver values. This finding strongly suggests successful organ resuscitation within 2 hrs of perfusion. Overall perfused livers regulated metabolism of perfusate substrates according to their metabolic needs, despite supraphysiological levels of some metabolites. This study establishes the first integrative metabolic basis for the dynamics of recovery during perfusion treatment of marginal livers. Our initial findings support enhanced oxygen delivery for both timely recovery and long-term sustenance. These results are expected to lead the optimization of the treatment protocols and perfusion media from a metabolic perspective, facilitating translation to clinical use.

  10. [Metabolic mechanisms underlying reparative action of metal-dependent spectral light flux from a hollow cathode lamp (experimental study)].

    Science.gov (United States)

    Pusyreva, G A; Frolkov, V K; Bobrovnitskiĭ, I P

    2010-01-01

    The objective of the present study was to evaluate the influence of serial irradiation of experimental animals with a visible light flow showing spectral lines of copper and manganese on the rate of reparative processes and the associated metabolic events. The cathode of the lamp that contained both microelements generated the light flux possessed of significant biological activity. Specifically, it accelerated reduction of the wound surface area and optimized regulation of carbohydrate metabolism in the lipid peroxidation system by insulin and cotisol. The light flux emitted by the cathode containing only one of the two elements (either copper or manganese) produced a similar but less pronounced effect.

  11. Impact of CYP2C19 phenotypes on escitalopram metabolism and an evaluation of pupillometry as a serotonergic biomarker

    DEFF Research Database (Denmark)

    Noehr-Jensen, L; Zwisler, S; Larsen, F

    2009-01-01

    PURPOSE: To investigate the impact of cytochrome P450 2C19 (CYP2C19) phenotypes on escitalopram metabolism and to evaluate pupillometry as a serotonergic biomarker. METHODS: This was a double-blind, crossover design study with single and multiple doses of 10 mg escitalopram and placebo in panels...... of CYP2C19 extensive (EM) and poor metabolisers (PM). Pupillometry was measured by a NeurOptics Pupillometer-PLR. RESULTS: Five PM and eight EM completed the study. The CYP2C19 phenotype significantly affected the metabolism of escitalopram. The area under the time-plasma concentration curve (AUC(0......-24)) was 1.8-fold higher in PM than in EM after both single and multiple doses. Escitalopram treatment did not affect the maximum pupil size, but it did statistically significantly decrease the relative amplitude of the pupil light reflex compared to the placebo; this effect was equal in both phenotype...

  12. Producing Acetic Acid of Acetobacter pasteurianus by Fermentation Characteristics and Metabolic Flux Analysis.

    Science.gov (United States)

    Wu, Xuefeng; Yao, Hongli; Liu, Qing; Zheng, Zhi; Cao, Lili; Mu, Dongdong; Wang, Hualin; Jiang, Shaotong; Li, Xingjiang

    2018-03-19

    The acetic acid bacterium Acetobacter pasteurianus plays an important role in acetic acid fermentation, which involves oxidation of ethanol to acetic acid through the ethanol respiratory chain under specific conditions. In order to obtain more suitable bacteria for the acetic acid industry, A. pasteurianus JST-S screened in this laboratory was compared with A. pasteurianus CICC 20001, a current industrial strain in China, to determine optimal fermentation parameters under different environmental stresses. The maximum total acid content of A. pasteurianus JST-S was 57.14 ± 1.09 g/L, whereas that of A. pasteurianus CICC 20001 reached 48.24 ± 1.15 g/L in a 15-L stir stank. Metabolic flux analysis was also performed to compare the reaction byproducts. Our findings revealed the potential value of the strain in improvement of industrial vinegar fermentation.

  13. Integrating flux balance analysis into kinetic models to decipher the dynamic metabolism of Shewanella oneidensis MR-1.

    Directory of Open Access Journals (Sweden)

    Xueyang Feng

    2012-02-01

    Full Text Available Shewanella oneidensis MR-1 sequentially utilizes lactate and its waste products (pyruvate and acetate during batch culture. To decipher MR-1 metabolism, we integrated genome-scale flux balance analysis (FBA into a multiple-substrate Monod model to perform the dynamic flux balance analysis (dFBA. The dFBA employed a static optimization approach (SOA by dividing the batch time into small intervals (i.e., ∼400 mini-FBAs, then the Monod model provided time-dependent inflow/outflow fluxes to constrain the mini-FBAs to profile the pseudo-steady-state fluxes in each time interval. The mini-FBAs used a dual-objective function (a weighted combination of "maximizing growth rate" and "minimizing overall flux" to capture trade-offs between optimal growth and minimal enzyme usage. By fitting the experimental data, a bi-level optimization of dFBA revealed that the optimal weight in the dual-objective function was time-dependent: the objective function was constant in the early growth stage, while the functional weight of minimal enzyme usage increased significantly when lactate became scarce. The dFBA profiled biologically meaningful dynamic MR-1 metabolisms: 1. the oxidative TCA cycle fluxes increased initially and then decreased in the late growth stage; 2. fluxes in the pentose phosphate pathway and gluconeogenesis were stable in the exponential growth period; and 3. the glyoxylate shunt was up-regulated when acetate became the main carbon source for MR-1 growth.

  14. Pyruvate modifies metabolic flux and nutrient sensing during extracorporeal membrane oxygenation in an immature swine model

    Energy Technology Data Exchange (ETDEWEB)

    Ledee, Dolena R.; Kajimoto, Masaki; O' Kelly-Priddy, Colleen M.; Olson, Aaron; Isern, Nancy G.; Robillard Frayne, Isabelle; Des Rosiers, Christine; Portman, Michael A.

    2015-07-01

    Extracorporeal membrane oxygenation (ECMO) provides mechanical circulatory support for infants and children with postoperative cardiopulmonary failure. Nutritional support is mandatory during ECMO, although specific actions for substrates on the heart have not been delineated. Prior work shows that enhancing pyruvate oxidation promotes successful weaning from ECMO. Accordingly, we closely examined the role of prolonged systemic pyruvate supplementation in modifying metabolic parameters during the unique conditions of ventricular unloading provided by ECMO. Twelve male mixed breed Yorkshire piglets (age 30-49 days) received systemic infusion of either normal saline (Group C) or pyruvate (Group P) during ECMO for 8 hours. Over the final hour piglets received [2-13C] pyruvate, and [13C6]-L-leucine, as an indicator for oxidation and protein synthesis. A significant increase in lactate and pyruvate concentrations occurred, along with an increase in the absolute concentration of all measured CAC intermediates. Group P showed greater anaplerotic flux through pyruvate carboxylation although pyruvate oxidation relative to citrate synthase flux was similar to Group C. The groups demonstrated similar leucine fractional contributions to acetyl-CoA and fractional protein synthesis rates. Pyruvate also promoted an increase in the phosphorylation state of several nutrient sensitive enzymes, such as AMPK and ACC, and promoted O-GlcNAcylation through the hexosamine biosynthetic pathway (HBP). In conclusion, prolonged pyruvate supplementation during ECMO modified anaplerotic pyruvate flux and elicited changes in important nutrient and energy sensitive pathways, while preserving protein synthesis. Therefore, the observed results support the further study of nutritional supplementation and its downstream effects on cardiac adaptation during ventricular unloading.

  15. Differential association between sarcopenia and metabolic phenotype in Korean young and older adults with and without obesity.

    Science.gov (United States)

    Hwang, You-Cheol; Cho, In-Jin; Jeong, In-Kyung; Ahn, Kyu Jeung; Chung, Ho Yeon

    2017-01-01

    To determine whether sarcopenia was associated with metabolic phenotype in subjects with and without obesity. A total of 6,021 participants (2,592 men, 3,429 women) aged 30 to 93 years were assessed using data from the 2009 Korea National Health and Nutrition Examination Survey. Sarcopenia was defined as appendicular skeletal muscle mass divided by weight (%) that is young adults. Metabolically unhealthy was defined as ≥2 components of metabolic syndrome or the presence of hypertension, diabetes, or cardiovascular disease. Obesity was defined as body mass index ≥25.0 kg/m 2 . Sarcopenia was associated with a metabolically unhealthy phenotype in nonobese men independent of age, smoking, regular physical activity, daily energy intake, total body fat, fasting insulin, non-HDL cholesterol, white blood cell count, ferritin level, and 25(OH) vitamin D level (OR per 1 SD increment (95% CI) 1.88 (1.28-2.75), P obesity. Sarcopenia was independently associated with a metabolically unhealthy phenotype in nonobese men, but this association was not evident in nonobese women or subjects with obesity. © 2016 The Obesity Society.

  16. Rerouting of carbon flux in a glycogen mutant of cyanobacteria assessed via isotopically non-stationary13C metabolic flux analysis.

    Science.gov (United States)

    Hendry, John I; Prasannan, Charulata; Ma, Fangfang; Möllers, K Benedikt; Jaiswal, Damini; Digmurti, Madhuri; Allen, Doug K; Frigaard, Niels-Ulrik; Dasgupta, Santanu; Wangikar, Pramod P

    2017-10-01

    Cyanobacteria, which constitute a quantitatively dominant phylum, have attracted attention in biofuel applications due to favorable physiological characteristics, high photosynthetic efficiency and amenability to genetic manipulations. However, quantitative aspects of cyanobacterial metabolism have received limited attention. In the present study, we have performed isotopically non-stationary 13 C metabolic flux analysis (INST- 13 C-MFA) to analyze rerouting of carbon in a glycogen synthase deficient mutant strain (glgA-I glgA-II) of the model cyanobacterium Synechococcus sp. PCC 7002. During balanced photoautotrophic growth, 10-20% of the fixed carbon is stored in the form of glycogen via a pathway that is conserved across the cyanobacterial phylum. Our results show that deletion of glycogen synthase gene orchestrates cascading effects on carbon distribution in various parts of the metabolic network. Carbon that was originally destined to be incorporated into glycogen gets partially diverted toward alternate storage molecules such as glucosylglycerol and sucrose. The rest is partitioned within the metabolic network, primarily via glycolysis and tricarboxylic acid cycle. A lowered flux toward carbohydrate synthesis and an altered distribution at the glucose-1-phosphate node indicate flexibility in the network. Further, reversibility of glycogen biosynthesis reactions points toward the presence of futile cycles. Similar redistribution of carbon was also predicted by Flux Balance Analysis. The results are significant to metabolic engineering efforts with cyanobacteria where fixed carbon needs to be re-routed to products of interest. Biotechnol. Bioeng. 2017;114: 2298-2308. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  17. Sleep duration independently influences metabolic body size phenotype in children and adolescents: a population-based study.

    Science.gov (United States)

    Lim, Han Hyuk

    2018-02-01

    Epidemiologic studies have found an association between sleep duration and obesity. However, the relationship between sleep duration and metabolic body size phenotype in children and adolescents remains unknown. In this cross-sectional study, 3650 participants (1946 boys and 1704 girls) from the Korean National Health and Nutrition Examination Survey 2007-2012 were classified into four phenotypes according to body mass index and metabolic heath status based on the definition of metabolic syndrome by NCEP-ATP III or the International Diabetes Federation. The four phenotypes were: metabolically healthy normal weight (MHNW), metabolically unhealthy normal weight (MUNW), metabolically healthy overweight (MHO), and metabolically unhealthy overweight (MUO). The associations between the four metabolic body size phenotypes and sleep duration, categorized as very short (≤5 h), short (6-7 h), normal (8-10 h), or long (≥11 h), were evaluated. Sleep duration was shorter in the MUO group (7.0 ± 1.5 h) than in the MHNW (7.3 ± 1.4 h) or MUNW (7.8 ± 1.6 h) groups. After adjusting for age, sex, household income, and physical activity, compared with a normal sleep duration, very short sleep duration (≤5 h) was associated with a higher prevalence of being overweight/obese (26.4% vs 17.4%, p = 0.001), lower risk of being MHNW (0.711 (0.538-0.940), p = 0.017) or MUNW (0.478 (0.237-0.962), p = 0.039), and higher risk of being MHO (1.702 (1.193-2.428), p = 0.003). By contrast, long sleep duration (≥11 h) was associated with a higher risk of being MUNW (2.581 (1.124-5.928), p = 0.025). Sleep duration may be independently associated with metabolic body size phenotype in children and adolescents. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Effects of drugs in subtoxic concentrations on the metabolic fluxes in human hepatoma cell line Hep G2

    International Nuclear Information System (INIS)

    Niklas, Jens; Noor, Fozia; Heinzle, Elmar

    2009-01-01

    Commonly used cytotoxicity assays assess the toxicity of a compound by measuring certain parameters which directly or indirectly correlate to the viability of the cells. However, the effects of a given compound at concentrations considerably below EC 50 values are usually not evaluated. These subtoxic effects are difficult to identify but may eventually cause severe and costly long term problems such as idiosyncratic hepatotoxicity. We determined the toxicity of three hepatotoxic compounds, namely amiodarone, diclofenac and tacrine on the human hepatoma cell line Hep G2 using an online kinetic respiration assay and analysed the effects of subtoxic concentrations of these drugs on the cellular metabolism by using metabolic flux analysis. Several changes in the metabolism could be detected upon exposure to subtoxic concentrations of the test compounds. Upon exposure to diclofenac and tacrine an increase in the TCA-cycle activity was observed which could be a signature of an uncoupling of the oxidative phosphorylation. The results indicate that metabolic flux analysis could serve as an invaluable novel tool for the investigation of the effects of drugs. The described methodology enables tracking the toxicity of compounds dynamically using the respiration assay in a range of concentrations and the metabolic flux analysis permits interesting insights into the changes in the central metabolism of the cell upon exposure to drugs.

  19. Elucidation of the Metabolic Network of Helicobacter pylori J99 and Malaysian Clinical Strains by Phenotype Microarray.

    Science.gov (United States)

    Lee, Woon Ching; Goh, Khean Lee; Loke, Mun Fai; Vadivelu, Jamuna

    2017-02-01

    Helicobacter pylori colonizes almost half of the human population worldwide. H. pylori strains are genetically diverse, and the specific genotypes are associated with various clinical manifestations including gastric adenocarcinoma, peptic ulcer disease (PUD), and nonulcer dyspepsia (NUD). However, our current knowledge of the H. pylori metabolism is limited. To understand the metabolic differences among H. pylori strains, we investigated four Malaysian H. pylori clinical strains, which had been previously sequenced, and a standard strain, H. pylori J99, at the phenotypic level. The phenotypes of the H. pylori strains were profiled using the Biolog Phenotype Microarray system to corroborate genomic data. We initiated the analyses by predicting carbon and nitrogen metabolic pathways from the H. pylori genomic data from the KEGG database. Biolog PM aided the validation of the prediction and provided a more intensive analysis of the H. pylori phenomes. We have identified a core set of metabolic nutrient sources that was utilized by all strains tested and another set that was differentially utilized by only the local strains. Pentose sugars are the preferred carbon nutrients utilized by H. pylori. The amino acids l-aspartic acid, d-alanine, and l-asparagine serve as both carbon and nitrogen sources in the metabolism of the bacterium. The phenotypic profile based on this study provides a better understanding on the survival of H. pylori in its natural host. Our data serve as a foundation for future challenges in correlating interstrain metabolic differences in H. pylori. © 2016 The Authors. Helicobacter Published by John Wiley & Sons Ltd.

  20. Metabolic engineering of Synechocystis sp. PCC 6803 for enhanced ethanol production based on flux balance analysis.

    Science.gov (United States)

    Yoshikawa, Katsunori; Toya, Yoshihiro; Shimizu, Hiroshi

    2017-05-01

    Synechocystis sp. PCC 6803 is an attractive host for bio-ethanol production due to its ability to directly convert atmospheric carbon dioxide into ethanol using photosystems. To enhance ethanol production in Synechocystis sp. PCC 6803, metabolic engineering was performed based on in silico simulations, using the genome-scale metabolic model. Comprehensive reaction knockout simulations by flux balance analysis predicted that the knockout of NAD(P)H dehydrogenase enhanced ethanol production under photoautotrophic conditions, where ammonium is the nitrogen source. This deletion inhibits the re-oxidation of NAD(P)H, which is generated by ferredoxin-NADP + reductase and imposes re-oxidation in the ethanol synthesis pathway. The effect of deleting the ndhF1 gene, which encodes NADH dehydrogenase subunit 5, on ethanol production was experimentally evaluated using ethanol-producing strains of Synechocystis sp. PCC 6803. The ethanol titer of the ethanol-producing ∆ndhF1 strain increased by 145%, compared with that of the control strain.

  1. Direct calculation of elementary flux modes satisfying several biological constraints in genome-scale metabolic networks.

    Science.gov (United States)

    Pey, Jon; Planes, Francisco J

    2014-08-01

    The concept of Elementary Flux Mode (EFM) has been widely used for the past 20 years. However, its application to genome-scale metabolic networks (GSMNs) is still under development because of methodological limitations. Therefore, novel approaches are demanded to extend the application of EFMs. A novel family of methods based on optimization is emerging that provides us with a subset of EFMs. Because the calculation of the whole set of EFMs goes beyond our capacity, performing a selective search is a proper strategy. Here, we present a novel mathematical approach calculating EFMs fulfilling additional linear constraints. We validated our approach based on two metabolic networks in which all the EFMs can be obtained. Finally, we analyzed the performance of our methodology in the GSMN of the yeast Saccharomyces cerevisiae by calculating EFMs producing ethanol with a given minimum carbon yield. Overall, this new approach opens new avenues for the calculation of EFMs in GSMNs. Matlab code is provided in the supplementary online materials fplanes@ceit.es. Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  2. Histone Deacetylase AtSRT1 Links Metabolic Flux and Stress Response in Arabidopsis.

    Science.gov (United States)

    Liu, Xiaoyun; Wei, Wei; Zhu, Wenjun; Su, Lufang; Xiong, Zeyang; Zhou, Man; Zheng, Yu; Zhou, Dao-Xiu

    2017-12-04

    How plant metabolic flux alters gene expression to optimize plant growth and response to stress remains largely unclear. Here, we report that Arabidopsis thaliana NAD + -dependent histone deacetylase AtSRT1 negatively regulates plant tolerance to stress and glycolysis but stimulates mitochondrial respiration. We found that AtSRT1 interacts with Arabidopsis cMyc-Binding Protein 1 (AtMBP-1), a transcriptional repressor produced by alternative translation of the cytosolic glycolytic enolase gene LOS2/ENO2. We demonstrated that AtSRT1 could associate with the chromatin of AtMBP-1 targets LOS2/ENO2 and STZ/ZAT10, both of which encode key stress regulators, and reduce the H3K9ac levels at these genes to repress their transcription. Overexpression of both AtSRT1 and AtMBP-1 had synergistic effects on the expression of glycolytic genes, glycolytic enzymatic activities, and mitochondrial respiration. Furthermore, we found that AtMBP-1 is lysine-acetylated and vulnerable to proteasomal protein degradation, while AtSRT1 could remove its lysine acetylation and significantly enhance its stability in vivo. Taken together, these results indicate that AtSRT1 regulates primary metabolism and stress response by both epigenetic regulation and modulation of AtMBP-1 transcriptional activity in Arabidopsis. Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.

  3. 13C Metabolic Flux Analysis for Systematic Metabolic Engineering of S. cerevisiae for Overproduction of Fatty Acids

    DEFF Research Database (Denmark)

    Ghosh, Amit; Ando, David; Gin, Jennifer

    2016-01-01

    Efficient redirection of microbial metabolism into the abundant production of desired bioproducts remains non-trivial. Here, we used flux-based modeling approaches to improve yields of fatty acids in Saccharomyces cerevisiae. We combined 13C labeling data with comprehensive genome-scale models to...

  4. Quantitative Metabolomics and Instationary 13C-Metabolic Flux Analysis Reveals Impact of Recombinant Protein Production on Trehalose and Energy Metabolism in Pichia pastoris

    NARCIS (Netherlands)

    Jorda, J.; Cueto Rojas, H.F.; Carnicer, M.; Wahl, S.A.; Ferrer, P.; Albiol, J.

    2014-01-01

    Pichia pastoris has been recognized as an effective host for recombinant protein production. In this work, we combine metabolomics and instationary 13C metabolic flux analysis (INST 13C-MFA) using GC-MS and LC-MS/MS to evaluate the potential impact of the production of a Rhizopus oryzae lipase (Rol)

  5. Simultaneous characterization of metabolic, cardiac, vascular and renal phenotypes of lean and obese SHHF rats.

    Directory of Open Access Journals (Sweden)

    Gina Youcef

    Full Text Available Individuals with metabolic syndrome (MetS are prone to develop heart failure (HF. However, the deleterious effects of MetS on the continuum of events leading to cardiac remodeling and subsequently to HF are not fully understood. This study characterized simultaneously MetS and cardiac, vascular and renal phenotypes in aging Spontaneously Hypertensive Heart Failure lean (SHHF(+/? regrouping (+/+ and (+/cp rats and obese (SHHF(cp/cp, "cp" defective mutant allele of the leptin receptor gene rats. We aimed to refine the milestones and their onset during the progression from MetS to HF in this experimental model. We found that SHHF(cp/cp but not SHHF(+/? rats developed dyslipidemia, as early as 1.5 months of age. This early alteration in the lipidic profile was detectable concomitantly to impaired renal function (polyuria, proteinuria but no glycosuria and reduced carotid distensibility as compared to SHHF(+/? rats. By 3 months of age SHHFcp/cp animals developed severe obesity associated with dislipidemia and hypertension defining the onset of MetS. From 6 months of age, SHHF(+/? rats developed concentric left ventricular hypertrophy (LVH while SHHF(cp/cp rats developed eccentric LVH apparent from progressive dilation of the LV dimensions. By 14 months of age only SHHF(cp/cp rats showed significantly higher central systolic blood pressure and a reduced ejection fraction resulting in systolic dysfunction as compared to SHHF(+/?. In summary, the metabolic and hemodynamic mechanisms participating in the faster decline of cardiac functions in SHHF(cp/cp rats are established long before their physiological consequences are detectable. Our results suggest that the molecular mechanisms triggered within the first three months after birth of SHHF(cp/cp rats should be targeted preferentially by therapeutic interventions in order to mitigate the later HF development.

  6. Metabolic profiles characterizing different phenotypes of polycystic ovary syndrome: plasma metabolomics analysis

    Science.gov (United States)

    2012-01-01

    Background Polycystic ovary syndrome (PCOS) is a heterogeneous endocrine disorder accompanied with an increased risk of developing type 2 diabetes mellitus and cardiovascular disease; despite being a common condition, the pathogenesis of PCOS remains unclear. Our aim was to investigate the potential metabolic profiles for different phenotypes of PCOS, as well as for the early prognosis of complications. Methods A total of 217 women with PCOS and 48 healthy women as normal controls were studied. Plasma samples of subjects were tested using two different analytical platforms of metabolomics: 1H nuclear magnetic resonance (NMR) and gas chromatography/time-of-flight mass spectrometry (GC/TOF-MS). Results Our results showed that carbohydrate, lipid and amino acid metabolisms were influenced in PCOS. The levels of lactate, long-chain fatty acids, triglyceride and very low-density lipoprotein were elevated, while glucose, phosphatidylcholine and high-density lipoprotein (HDL) concentrations were reduced in PCOS patients as compared with controls. Additionally, the levels of alanine, valine, serine, threonine, ornithine, phenylalanine, tyrosine and tryptophan were generally increased, whereas the levels of glycine and proline were significantly reduced in PCOS samples compared to controls. Furthermore, the ratio of branched-chain amino acid to aromatic amino acid concentrations (BCAA/AAA) in PCOS plasma was significantly reduced in PCOS patients and was insusceptible to obesity and insulin sensitivity. Conclusions Our results suggested that the enhanced glycolysis and inhibited tricarboxylic acid cycle (TAC) in women with PCOS. Decrease of BCAA/AAA ratio was directly correlated with the development of PCOS. Ovulatory dysfunction of PCOS patients was associated with raised production of serine, threonine, phenylalanine, tyrosine and ornithine. Elevated levels of valine and leucine, and decreased concentrations of glycine in PCOS plasma could contribute to insulin

  7. Beaver-mediated lateral hydrologic connectivity, fluvial carbon and nutrient flux, and aquatic ecosystem metabolism

    Science.gov (United States)

    Wegener, Pam; Covino, Tim; Wohl, Ellen

    2017-06-01

    River networks that drain mountain landscapes alternate between narrow and wide valley segments. Within the wide segments, beaver activity can facilitate the development and maintenance of complex, multithread planform. Because the narrow segments have limited ability to retain water, carbon, and nutrients, the wide, multithread segments are likely important locations of retention. We evaluated hydrologic dynamics, nutrient flux, and aquatic ecosystem metabolism along two adjacent segments of a river network in the Rocky Mountains, Colorado: (1) a wide, multithread segment with beaver activity; and, (2) an adjacent (directly upstream) narrow, single-thread segment without beaver activity. We used a mass balance approach to determine the water, carbon, and nutrient source-sink behavior of each river segment across a range of flows. While the single-thread segment was consistently a source of water, carbon, and nitrogen, the beaver impacted multithread segment exhibited variable source-sink dynamics as a function of flow. Specifically, the multithread segment was a sink for water, carbon, and nutrients during high flows, and subsequently became a source as flows decreased. Shifts in river-floodplain hydrologic connectivity across flows related to higher and more variable aquatic ecosystem metabolism rates along the multithread relative to the single-thread segment. Our data suggest that beaver activity in wide valleys can create a physically complex hydrologic environment that can enhance hydrologic and biogeochemical buffering, and promote high rates of aquatic ecosystem metabolism. Given the widespread removal of beaver, determining the cumulative effects of these changes is a critical next step in restoring function in altered river networks.

  8. Metabolic flux rearrangement in the amino acid metabolism reduces ammonia stress in the α1-antitrypsin producing human AGE1.HN cell line.

    Science.gov (United States)

    Priesnitz, Christian; Niklas, Jens; Rose, Thomas; Sandig, Volker; Heinzle, Elmar

    2012-03-01

    This study focused on metabolic changes in the neuronal human cell line AGE1.HN upon increased ammonia stress. Batch cultivations of α(1)-antitrypsin (A1AT) producing AGE1.HN cells were carried out in media with initial ammonia concentrations ranging from 0mM to 5mM. Growth, A1AT production, metabolite dynamics and finally metabolic fluxes calculated by metabolite balancing were compared. Growth and A1AT production decreased with increasing ammonia concentration. The maximum A1AT concentration decreased from 0.63g/l to 0.51g/l. Central energy metabolism remained relatively unaffected exhibiting only slightly increased glycolytic flux at high initial ammonia concentration in the medium. However, the amino acid metabolism was significantly changed. Fluxes through transaminases involved in amino acid degradation were reduced concurrently with a reduced uptake of amino acids. On the other hand fluxes through transaminases working in the direction of amino acid synthesis, i.e., alanine and phosphoserine, were increased leading to increased storage of excess nitrogen in extracellular alanine and serine. Glutamate dehydrogenase flux was reversed increasingly fixing free ammonia with increasing ammonia concentration. Urea production additionally observed was associated with arginine uptake by the cells and did not increase at high ammonia stress. It was therefore not used as nitrogen sink to remove excess ammonia. The results indicate that the AGE1.HN cell line can adapt to ammonia concentrations usually present during the cultivation process to a large extent by changing metabolism but with slightly reduced A1AT production and growth. Copyright © 2012 Elsevier Inc. All rights reserved.

  9. High-fat diets exaggerate endocrine and metabolic phenotypes in a rat model of DHEA-induced PCOS.

    Science.gov (United States)

    Zhang, Haolin; Yi, Ming; Zhang, Yan; Jin, Hongyan; Zhang, Wenxin; Yang, Jingjing; Yan, Liying; Li, Rong; Zhao, Yue; Qiao, Jie

    2016-04-01

    Polycystic ovary syndrome (PCOS) is a complex endocrine and metabolic disorder with unclear etiology and unsatisfactory management. Effects of diets on the phenotype of PCOS were not fully understood. In the present study, we applied 45 and 60% high-fat diets (HFDs) on a rat model of PCOS induced by postnatal DHEA injection. We found that both DHEA and DHEA+HFDs rats exhibited reproductive abnormalities, including hyperandrogenism, irregular cycles and polycystic ovaries. The addition of HFDs, especially 60% HFDs, exaggerated morphological changes of ovaries and a number of metabolic changes, including increased body weight and body fat content, impaired glucose tolerance and increased serum insulin levels. Results from qPCR showed that DHEA-induced increased expression of hypothalamic androgen receptor and LH receptor were reversed by the addition of 60% HFDs. In contrast, the ovarian expression of LH receptor and insulin receptor mRNA was upregulated only with the addition of 60% HFDs. These findings indicated that DHEA and DHEA+HFDs might influence PCOS phenotypes through distinct mechanisms: DHEA affects the normal function of hypothalamus-pituitary-ovarian axis through LH, whereas the addition of HFDs exaggerated endocrine and metabolic dysfunction through ovarian responses to insulin-related mechanisms. We concluded that the addition of HFDs yielded distinct phenotypes of DHEA-induced PCOS and could be used for studies on both reproductive and metabolic features of the syndrome. © 2016 Society for Reproduction and Fertility.

  10. 5α-Reductase Type 2 Regulates Glucocorticoid Action and Metabolic Phenotype in Human Hepatocytes.

    Science.gov (United States)

    Nasiri, Maryam; Nikolaou, Nikolaos; Parajes, Silvia; Krone, Nils P; Valsamakis, George; Mastorakos, George; Hughes, Beverly; Taylor, Angela; Bujalska, Iwona J; Gathercole, Laura L; Tomlinson, Jeremy W

    2015-08-01

    Glucocorticoids and androgens have both been implicated in the pathogenesis of nonalcoholic fatty liver disease (NAFLD); androgen deficiency in males, androgen excess in females, and glucocorticoid excess in both sexes are associated with NAFLD. Glucocorticoid and androgen action are regulated at a prereceptor level by the enzyme 5α-reductase type 2 (SRD5A2), which inactivates glucocorticoids to their dihydrometabolites and converts T to DHT. We have therefore explored the role of androgens and glucocorticoids and their metabolism by SRD5A2 upon lipid homeostasis in human hepatocytes. In both primary human hepatocytes and human hepatoma cell lines, glucocorticoids decreased de novo lipogenesis in a dose-dependent manner. Whereas androgen treatment (T and DHT) increased lipogenesis in cell lines and in primary cultures of human hepatocytes from female donors, it was without effect in primary hepatocyte cultures from men. SRD5A2 overexpression reduced the effects of cortisol to suppress lipogenesis and this effect was lost following transfection with an inactive mutant construct. Conversely, pharmacological inhibition using the 5α-reductase inhibitors finasteride and dutasteride augmented cortisol action. We have demonstrated that manipulation of SRD5A2 activity can regulate lipogenesis in human hepatocytes in vitro. This may have significant clinical implications for those patients prescribed 5α-reductase inhibitors, in particular augmenting the actions of glucocorticoids to modulate hepatic lipid flux.

  11. Effects of temperature and UVR on organic matter fluxes and the metabolic activity of Acropora muricata

    Directory of Open Access Journals (Sweden)

    Lucile Courtial

    2017-08-01

    Full Text Available Coral bleaching events are predicted to occur more frequently in the coming decades with global warming. The susceptibility of corals to bleaching during thermal stress episodes depends on many factors, including the magnitude of thermal stress and irradiance. The interactions among these two factors, and in particular with ultra-violet radiation (UVR, the most harmful component of light, are more complex than assumed, and are not yet well understood. This paper explores the individual and combined effects of temperature and UVR on the metabolism of Acropora muricata, one of the most abundant coral species worldwide. Particulate and dissolved organic matter (POM/DOM fluxes and organic matter (OM degradation by the mucus-associated bacteria were also monitored in all conditions. The results show that UVR exposure exacerbated the temperature-induced bleaching, but did not affect OM fluxes, which were only altered by seawater warming. Temperature increase induced a shift from POM release and DOM uptake in healthy corals to POM uptake and DOM release in stressed ones. POM uptake was linked to a significant grazing of pico- and nanoplankton particles during the incubation, to fulfil the energetic requirements of A. muricata in the absence of autotrophy. Finally, OM degradation by mucus-associated bacterial activity was unaffected by UVR exposure, but significantly increased under high temperature. Altogether, our results demonstrate that seawater warming and UVR not only affect coral physiology, but also the way corals interact with the surrounding seawater, with potential consequences for coral reef biogeochemical cycles and food webs.

  12. Quantitative Metabolomics and Instationary 13C-Metabolic Flux Analysis Reveals Impact of Recombinant Protein Production on Trehalose and Energy Metabolism in Pichia pastoris

    Directory of Open Access Journals (Sweden)

    Joel Jordà

    2014-05-01

    Full Text Available Pichia pastoris has been recognized as an effective host for recombinant protein production. In this work, we combine metabolomics and instationary 13C metabolic flux analysis (INST 13C-MFA using GC-MS and LC-MS/MS to evaluate the potential impact of the production of a Rhizopus oryzae lipase (Rol on P. pastoris central carbon metabolism. Higher oxygen uptake and CO2 production rates and slightly reduced biomass yield suggest an increased energy demand for the producing strain. This observation is further confirmed by 13C-based metabolic flux analysis. In particular, the flux through the methanol oxidation pathway and the TCA cycle was increased in the Rol-producing strain compared to the reference strain. Next to changes in the flux distribution, significant variations in intracellular metabolite concentrations were observed. Most notably, the pools of trehalose, which is related to cellular stress response, and xylose, which is linked to methanol assimilation, were significantly increased in the recombinant strain.

  13. Metabolic engineering of Synechococcus elongatus PCC 7942 for improvement of 1,3-propanediol and glycerol production based on in silico simulation of metabolic flux distribution.

    Science.gov (United States)

    Hirokawa, Yasutaka; Matsuo, Shingo; Hamada, Hiroyuki; Matsuda, Fumio; Hanai, Taizo

    2017-11-25

    Production directly from carbon dioxide by engineered cyanobacteria is one of the promising technologies for sustainable future. Previously, we have successfully achieved 1,3-propanediol (1,3-PDO) production using Synechococcus elongatus PCC 7942 with a synthetic metabolic pathway. The strain into which the synthetic metabolic pathway was introduced produced 3.48 mM (0.265 g/L) 1,3-PDO and 14.3 mM (1.32 g/L) glycerol during 20 days of incubation. In this study, the productivities of 1,3-PDO were improved by gene disruption selected by screening with in silico simulation. First, a stoichiometric metabolic model was applied to prediction of cellular metabolic flux distribution in a 1,3-PDO-producing strain of S. elongatus PCC 7942. A genome-scale model of S. elongatus PCC 7942 constructed by Knoop was modified by the addition of a synthetic metabolic pathway for 1,3-PDO production. Next, the metabolic flux distribution predicted by metabolic flux balance analysis (FBA) was used for in silico simulation of gene disruption. As a result of gene disruption simulation, NADPH dehydrogenase 1 (NDH-1) complexes were found by screening to be the most promising candidates for disruption to improve 1,3-PDO production. The effect of disruption of the gene encoding a subunit of the NDH-1 complex was evaluated in the 1,3-PDO-producing strain. During 20 days of incubation, the ndhF1-null 1,3-PDO-producing strain showed the highest titers: 4.44 mM (0.338 g/L) 1,3-PDO and 30.3 mM (2.79 g/L) glycerol. In this study, we successfully improved 1,3-PDO productivity on the basis of in silico simulation of gene disruption.

  14. Responses to high-fat challenges varying in fat type in subjects with different metabolic risk phenotypes: a randomized trial.

    Directory of Open Access Journals (Sweden)

    Susan J van Dijk

    Full Text Available The ability of subjects to respond to nutritional challenges can reflect the flexibility of their biological system. Nutritional challenge tests could be used as an indicator of health status but more knowledge on metabolic and immune responses of different subjects to nutritional challenges is needed. The aim of this study was to compare the responses to high-fat challenges varying in fat type in subjects with different metabolic risk phenotypes.In a cross-over design 42 men (age 50-70 y consumed three high-fat shakes containing saturated fat (SFA, monounsaturated fat (MUFA or n-3 polyunsaturated (PUFA. Men were selected on BMI and health status (lean, obese or obese diabetic and phenotyped with MRI for adipose tissue distribution. Before and 2 and 4 h after shake consumption blood was drawn for measurement of expression of metabolic and inflammation-related genes in peripheral blood mononuclear cells (PBMCs, plasma triglycerides (TAG, glucose, insulin, cytokines and ex vivo PBMC immune response capacity. The MUFA and n-3 PUFA challenge, compared to the SFA challenge, induced higher changes in expression of inflammation genes MCP1 and IL1β in PBMCs. Obese and obese diabetic subjects had different PBMC gene expression and metabolic responses to high-fat challenges compared to lean subjects. The MUFA challenge induced the most pronounced TAG response, mainly in obese and obese diabetic subjects.The PBMC gene expression response and metabolic response to high-fat challenges were affected by fat type and metabolic risk phenotype. Based on our results we suggest using a MUFA challenge to reveal differences in response capacity of subjects.ClinicalTrials.gov NCT00977262.

  15. Semi-automated curation of metabolic models via flux balance analysis: a case study with Mycoplasma gallisepticum.

    Directory of Open Access Journals (Sweden)

    Eddy J Bautista

    Full Text Available Primarily used for metabolic engineering and synthetic biology, genome-scale metabolic modeling shows tremendous potential as a tool for fundamental research and curation of metabolism. Through a novel integration of flux balance analysis and genetic algorithms, a strategy to curate metabolic networks and facilitate identification of metabolic pathways that may not be directly inferable solely from genome annotation was developed. Specifically, metabolites involved in unknown reactions can be determined, and potentially erroneous pathways can be identified. The procedure developed allows for new fundamental insight into metabolism, as well as acting as a semi-automated curation methodology for genome-scale metabolic modeling. To validate the methodology, a genome-scale metabolic model for the bacterium Mycoplasma gallisepticum was created. Several reactions not predicted by the genome annotation were postulated and validated via the literature. The model predicted an average growth rate of 0.358±0.12[Formula: see text], closely matching the experimentally determined growth rate of M. gallisepticum of 0.244±0.03[Formula: see text]. This work presents a powerful algorithm for facilitating the identification and curation of previously known and new metabolic pathways, as well as presenting the first genome-scale reconstruction of M. gallisepticum.

  16. Interaction of storage carbohydrates and other cyclic fluxes with central metabolism: A quantitative approach by non-stationary 13C metabolic flux analysis

    Directory of Open Access Journals (Sweden)

    C.A. Suarez-Mendez

    2016-12-01

    Full Text Available 13C labeling experiments in aerobic glucose limited cultures of Saccharomyces cerevisiae at four different growth rates (0.054; 0.101, 0.207, 0.307 h−1 are used for calculating fluxes that include intracellular cycles (e.g., storage carbohydrate cycles, exchange fluxes with amino acids, which are rearranged depending on the growth rate. At low growth rates the impact of the storage carbohydrate recycle is relatively more significant than at high growth rates due to a higher concentration of these materials in the cell (up to 560-fold and higher fluxes relative to the glucose uptake rate (up to 16%. Experimental observations suggest that glucose can be exported to the extracellular space, and that its source is related to storage carbohydrates, most likely via the export and subsequent extracellular breakdown of trehalose. This hypothesis is strongly supported by 13C-labeling experimental data, measured extracellular trehalose, and the corresponding flux estimations. Keywords: Non-stationary 13C labeling, Flux estimation, Trehalose, Glycogen, Amino acids

  17. Genealogy profiling through strain improvement by using metabolic network analysis: metabolic flux genealogy of several generations of lysine-producing corynebacteria.

    Science.gov (United States)

    Wittmann, Christoph; Heinzle, Elmar

    2002-12-01

    A comprehensive approach of metabolite balancing, (13)C tracer studies, gas chromatography-mass spectrometry, matrix-assisted laser desorption ionization-time of flight mass spectrometry, and isotopomer modeling was applied for comparative metabolic network analysis of a genealogy of five successive generations of lysine-producing Corynebacterium glutamicum. The five strains examined (C. glutamicum ATCC 13032, 13287, 21253, 21526, and 21543) were previously obtained by random mutagenesis and selection. Throughout the genealogy, the lysine yield in batch cultures increased markedly from 1.2 to 24.9% relative to the glucose uptake flux. Strain optimization was accompanied by significant changes in intracellular flux distributions. The relative pentose phosphate pathway (PPP) flux successively increased, clearly corresponding to the product yield. Moreover, the anaplerotic net flux increased almost twofold as a consequence of concerted regulation of C(3) carboxylation and C(4) decarboxylation fluxes to cover the increased demand for lysine formation; thus, the overall increase was a consequence of concerted regulation of C(3) carboxylation and C(4) decarboxylation fluxes. The relative flux through isocitrate dehydrogenase dropped from 82.7% in the wild type to 59.9% in the lysine-producing mutants. In contrast to the NADPH demand, which increased from 109 to 172% due to the increasing lysine yield, the overall NADPH supply remained constant between 185 and 196%, resulting in a decrease in the apparent NADPH excess through strain optimization. Extrapolated to industrial lysine producers, the NADPH supply might become a limiting factor. The relative contributions of PPP and the tricarboxylic acid cycle to NADPH generation changed markedly, indicating that C. glutamicum is able to maintain a constant supply of NADPH under completely different flux conditions. Statistical analysis by a Monte Carlo approach revealed high precision for the estimated fluxes, underlining the

  18. Dynamic regulation of metabolic flux in engineered bacteria using a pathway-independent quorum-sensing circuit.

    Science.gov (United States)

    Gupta, Apoorv; Reizman, Irene M Brockman; Reisch, Christopher R; Prather, Kristala L J

    2017-03-01

    Metabolic engineering of microorganisms to produce desirable products on an industrial scale can result in unbalanced cellular metabolic networks that reduce productivity and yield. Metabolic fluxes can be rebalanced using dynamic pathway regulation, but few broadly applicable tools are available to achieve this. We present a pathway-independent genetic control module that can be used to dynamically regulate the expression of target genes. We apply our module to identify the optimal point to redirect glycolytic flux into heterologous engineered pathways in Escherichia coli, resulting in titers of myo-inositol increased 5.5-fold and titers of glucaric acid increased from unmeasurable to >0.8 g/L, compared to the parent strains lacking dynamic flux control. Scaled-up production of these strains in benchtop bioreactors resulted in almost ten- and fivefold increases in specific titers of myo-inositol and glucaric acid, respectively. We also used our module to control flux into aromatic amino acid biosynthesis to increase titers of shikimate in E. coli from unmeasurable to >100 mg/L.

  19. Lipid mediator metabolic profiling demonstrates differences in eicosanoid patterns in two phenotypically distinct mast cell populations[S

    Science.gov (United States)

    Lundström, Susanna L.; Saluja, Rohit; Adner, Mikael; Haeggström, Jesper Z.; Nilsson, Gunnar; Wheelock, Craig E.

    2013-01-01

    Mast cells are inflammatory cells that play key roles in health and disease. They are distributed in all tissues and appear in two main phenotypes, connective tissue and mucosal mast cells, with differing capacities to release inflammatory mediators. A metabolic profiling approach was used to obtain a more comprehensive understanding of the ability of mast cell phenotypes to produce eicosanoids and other lipid mediators. A total of 90 lipid mediators (oxylipins) were characterized using liquid chromatography-tandem mass spectrometry (LC-MS/MS), representing the cyclooxygenase (COX), lipoxygenase (LO), and cytochrome P450 (CYP) metabolic pathways. In vitro-derived murine mucosal-like mast cells (MLMC) and connective tissue-like mast cells (CTLMC) exhibited distinct mRNA expression patterns of enzymes involved in oxylipin biosynthesis. Oxylipins produced by 5-LO and COX pathways were the predominant species in both phenotypes, with 5-LO products constituting 90 ± 2% of the CTLMCs compared with 58 ± 8% in the MLMCs. Multivariate analyses demonstrated that CTLMCs and MLMCs secrete differing oxylipin profiles at baseline and following calcium ionophore stimulation, evidencing specificity in both a time- and biosynthetic pathway-dependent manner. In addition to the COX-regulated prostaglandin PGD2 and 5-LO-regulated cysteinyl-leukotrienes (e.g., LTC4), several other mediators evidenced phenotype-specificity, which may have biological implications in mast cell-mediated regulation of inflammatory responses. PMID:23034214

  20. Identifying postmenopausal women at risk for cognitive decline within a healthy cohort using a panel of clinical metabolic indicators: Potential for detecting an at-Alzheimer?s risk metabolic phenotype

    OpenAIRE

    Rettberg, Jamaica R.; Dang, Ha; Hodis, Howard N.; Henderson, Victor W.; St. John, Jan A.; Mack, Wendy J.; Brinton, Roberta Diaz

    2016-01-01

    Detecting at-risk individuals within a healthy population is critical for preventing or delaying Alzheimer?s disease. The systems biology integration of brain and body metabolism enables peripheral metabolic biomarkers to serve as reporters of brain bioenergetic status. Using clinical metabolic data derived from healthy postmenopausal women in the ELITE trial, we conducted principal components and k-means clustering analyses of nine biomarkers to define metabolic phenotypes. Metabolic cluster...

  1. Influence of the late winter bloom on migrant zooplankton metabolism and its implications on export fluxes

    Science.gov (United States)

    Putzeys, S.; Yebra, L.; Almeida, C.; Bécognée, P.; Hernández-León, S.

    2011-12-01

    Studies on carbon active fluxes due to diel migrants are scarce and critical for carbon flux models and biogeochemical estimates. We studied the temporal variability and vertical distribution of biomass, indices of feeding and respiration of the zooplanktonic community north off the Canary Islands during the end of the late winter bloom, in order to assess vertical carbon fluxes in this area. Biomass distribution during the day presented two dense layers of organisms at 0-200 m and around 500 m, whereas at night, most of the biomass concentrated in the epipelagic layer. The gut pigment flux (0.05-0.18 mgC·m - 2 ·d - 1 ) represented 0.22% of the estimated passive export flux (POC flux) while potential ingestion represented 3.91% of the POC (1.24-3.40 mgC·m - 2 ·d - 1 ). The active respiratory flux (0.50-1.36 mgC·m - 2 ·d - 1 ) was only 1.57% of the POC flux. The total carbon flux mediated by diel migrants (respiration plus potential ingestion) ranged between 3.37 and 9.22% of the POC flux; which is three-fold higher than calculating ingestion fluxes from gut pigments. Our results suggest that the fluxes by diel migrants play a small role in the downward flux of carbon in the open ocean during the post-bloom period.

  2. Profiling metabolic networks to study cancer metabolism.

    Science.gov (United States)

    Hiller, Karsten; Metallo, Christian M

    2013-02-01

    Cancer is a disease of unregulated cell growth and survival, and tumors reprogram biochemical pathways to aid these processes. New capabilities in the computational and bioanalytical characterization of metabolism have now emerged, facilitating the identification of unique metabolic dependencies that arise in specific cancers. By understanding the metabolic phenotype of cancers as a function of their oncogenic profiles, metabolic engineering may be applied to design synthetically lethal therapies for some tumors. This process begins with accurate measurement of metabolic fluxes. Here we review advanced methods of quantifying pathway activity and highlight specific examples where these approaches have uncovered potential opportunities for therapeutic intervention. Copyright © 2012 Elsevier Ltd. All rights reserved.

  3. Integrated analysis of gene expression and metabolic fluxes in PHA-producing Pseudomonas putida grown on glycerol.

    Science.gov (United States)

    Beckers, Veronique; Poblete-Castro, Ignacio; Tomasch, Jürgen; Wittmann, Christoph

    2016-05-03

    Given its high surplus and low cost, glycerol has emerged as interesting carbon substrate for the synthesis of value-added chemicals. The soil bacterium Pseudomonas putida KT2440 can use glycerol to synthesize medium-chain-length poly(3-hydroxyalkanoates) (mcl-PHA), a class of biopolymers of industrial interest. Here, glycerol metabolism in P. putida KT2440 was studied on the level of gene expression (transcriptome) and metabolic fluxes (fluxome), using precisely adjusted chemostat cultures, growth kinetics and stoichiometry, to gain a systematic understanding of the underlying metabolic and regulatory network. Glycerol-grown P. putida KT2440 has a maintenance energy requirement [0.039 (mmolglycerol (gCDW h)(-1))] that is about sixteen times lower than that of other bacteria, such as Escherichia coli, which provides a great advantage to use this substrate commercially. The shift from carbon (glycerol) to nitrogen (ammonium) limitation drives the modulation of specific genes involved in glycerol metabolism, transport electron chain, sensors to assess the energy level of the cell, and PHA synthesis, as well as changes in flux distribution to increase the precursor availability for PHA synthesis (Entner-Doudoroff pathway and pyruvate metabolism) and to reduce respiration (glyoxylate shunt). Under PHA-producing conditions (N-limitation), a higher PHA yield was achieved at low dilution rate (29.7 wt% of CDW) as compared to a high rate (12.8 wt% of CDW). By-product formation (succinate, malate) was specifically modulated under these regimes. On top of experimental data, elementary flux mode analysis revealed the metabolic potential of P. putida KT2440 to synthesize PHA and identified metabolic engineering targets towards improved production performance on glycerol. This study revealed the complex interplay of gene expression levels and metabolic fluxes under PHA- and non-PHA producing conditions using the attractive raw material glycerol as carbon substrate. This

  4. Metabolic differences underlying two distinct rat urinary phenotypes, a suggested role for gut microbial metabolism of phenylalanine and a possible connection to autism.

    Science.gov (United States)

    Clayton, T Andrew

    2012-04-05

    A novel explanation is proposed for the metabolic differences underlying two distinct rat urinary compositional phenotypes i.e. that these may arise from differences in the gut microbially-mediated metabolism of phenylalanine. As part of this hypothesis, it is further suggested that elements of the mammalian gut microbiota may convert phenylalanine to cinnamic acid, either by means of an ammonia lyase-type reaction or by means of a three step route via phenylpyruvate and phenyllactate. The wider significance of such conversions is discussed with similar metabolism of tryptophan and subsequent glycine conjugation potentially explaining the origin of trans-indolylacryloylglycine, a postulated marker for autism. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  5. Integrating Kinetic Model of E. coli with Genome Scale Metabolic Fluxes Overcomes Its Open System Problem and Reveals Bistability in Central Metabolism.

    Directory of Open Access Journals (Sweden)

    Ahmad A Mannan

    Full Text Available An understanding of the dynamics of the metabolic profile of a bacterial cell is sought from a dynamical systems analysis of kinetic models. This modelling formalism relies on a deterministic mathematical description of enzyme kinetics and their metabolite regulation. However, it is severely impeded by the lack of available kinetic information, limiting the size of the system that can be modelled. Furthermore, the subsystem of the metabolic network whose dynamics can be modelled is faced with three problems: how to parameterize the model with mostly incomplete steady state data, how to close what is now an inherently open system, and how to account for the impact on growth. In this study we address these challenges of kinetic modelling by capitalizing on multi-'omics' steady state data and a genome-scale metabolic network model. We use these to generate parameters that integrate knowledge embedded in the genome-scale metabolic network model, into the most comprehensive kinetic model of the central carbon metabolism of E. coli realized to date. As an application, we performed a dynamical systems analysis of the resulting enriched model. This revealed bistability of the central carbon metabolism and thus its potential to express two distinct metabolic states. Furthermore, since our model-informing technique ensures both stable states are constrained by the same thermodynamically feasible steady state growth rate, the ensuing bistability represents a temporal coexistence of the two states, and by extension, reveals the emergence of a phenotypically heterogeneous population.

  6. Integrating Kinetic Model of E. coli with Genome Scale Metabolic Fluxes Overcomes Its Open System Problem and Reveals Bistability in Central Metabolism

    Science.gov (United States)

    Mannan, Ahmad A.; Toya, Yoshihiro; Shimizu, Kazuyuki; McFadden, Johnjoe; Kierzek, Andrzej M.; Rocco, Andrea

    2015-01-01

    An understanding of the dynamics of the metabolic profile of a bacterial cell is sought from a dynamical systems analysis of kinetic models. This modelling formalism relies on a deterministic mathematical description of enzyme kinetics and their metabolite regulation. However, it is severely impeded by the lack of available kinetic information, limiting the size of the system that can be modelled. Furthermore, the subsystem of the metabolic network whose dynamics can be modelled is faced with three problems: how to parameterize the model with mostly incomplete steady state data, how to close what is now an inherently open system, and how to account for the impact on growth. In this study we address these challenges of kinetic modelling by capitalizing on multi-‘omics’ steady state data and a genome-scale metabolic network model. We use these to generate parameters that integrate knowledge embedded in the genome-scale metabolic network model, into the most comprehensive kinetic model of the central carbon metabolism of E. coli realized to date. As an application, we performed a dynamical systems analysis of the resulting enriched model. This revealed bistability of the central carbon metabolism and thus its potential to express two distinct metabolic states. Furthermore, since our model-informing technique ensures both stable states are constrained by the same thermodynamically feasible steady state growth rate, the ensuing bistability represents a temporal coexistence of the two states, and by extension, reveals the emergence of a phenotypically heterogeneous population. PMID:26469081

  7. A metabolic and body-size scaling framework for parasite within-host abundance, biomass, and energy flux.

    Science.gov (United States)

    Hechinger, Ryan F

    2013-08-01

    Energetics may provide a useful currency for studying the ecology of parasite assemblages within individual hosts. Parasite assemblages may also provide powerful models to study general principles of ecological energetics. Yet there has been little ecological research on parasite-host energetics, probably due to methodological difficulties. However, the scaling relationships of individual metabolic rate with body or cell size and temperature may permit us to tackle the energetics of parasite assemblages in hosts. This article offers the foundations and initial testing of a metabolic theory of ecology (MTE) framework for parasites in hosts. I first provide equations to estimate energetic flux through observed parasite assemblages. I then develop metabolic scaling theory for parasite abundance, energetics, and biomass in individual hosts. In contrast to previous efforts, the theory factors in both host and parasite metabolic scaling, how parasites use host space, and whether energy or space dictates carrying capacity. Empirical tests indicate that host energetic flux can set parasite carrying capacity, which decreases as predicted considering the scaling of host and parasite metabolic rates. The theory and results also highlight that the phenomenon of "energetic equivalence" is not an assumption of MTE but a possible outcome contingent on how species partition resources. Hence, applying MTE to parasites can lend mechanistic, quantitative, predictive insight into the nature of parasitism and can inform general ecological theory.

  8. A Novel Letrozole Model Recapitulates Both the Reproductive and Metabolic Phenotypes of Polycystic Ovary Syndrome in Female Mice1

    Science.gov (United States)

    Kauffman, Alexander S.; Thackray, Varykina G.; Ryan, Genevieve E.; Tolson, Kristen P.; Glidewell-Kenney, Christine A.; Semaan, Sheila J.; Poling, Matthew C.; Iwata, Nahoko; Breen, Kellie M.; Duleba, Antoni J.; Stener-Victorin, Elisabet; Shimasaki, Shunichi; Webster, Nicholas J.; Mellon, Pamela L.

    2015-01-01

    Polycystic ovary syndrome (PCOS) pathophysiology is poorly understood, due partly to lack of PCOS animal models fully recapitulating this complex disorder. Recently, a PCOS rat model using letrozole (LET), a nonsteroidal aromatase inhibitor, mimicked multiple PCOS phenotypes, including metabolic features absent in other models. Given the advantages of using genetic and transgenic mouse models, we investigated whether LET produces a similar PCOS phenotype in mice. Pubertal female C57BL/6N mice were treated for 5 wk with LET, which resulted in increased serum testosterone and normal diestrus levels of estradiol, similar to the hyperandrogenemia and follicular phase estrogen levels of PCOS women. As in PCOS, ovaries from LET mice were larger, polycystic, and lacked corpora lutea versus controls. Most LET females were acyclic, and all were infertile. LET females displayed elevated serum LH levels and higher Lhb mRNA in the pituitary. In contrast, serum FSH and Fshb were significantly reduced in LET females, demonstrating differential effects on gonadotropins, as in PCOS. Within the ovary, LET females had higher Cyp17, Cyp19, and Fsh receptor mRNA expression. In the hypothalamus, LET females had higher kisspeptin receptor mRNA expression but lower progesterone receptor mRNA levels. LET females also gained more weight than controls, had increased abdominal adiposity and adipocyte size, elevated adipose inflammatory mRNA levels, and impaired glucose tolerance, mirroring the metabolic phenotype in PCOS women. This is the first report of a LET paradigm in mice that recapitulates both reproductive and metabolic PCOS phenotypes and will be useful to genetically probe the PCOS condition. PMID:26203175

  9. Optimized Phenotypic Biomarker Discovery and Confounder Elimination via Covariate-Adjusted Projection to Latent Structures from Metabolic Spectroscopy Data.

    Science.gov (United States)

    Posma, Joram M; Garcia-Perez, Isabel; Ebbels, Timothy M D; Lindon, John C; Stamler, Jeremiah; Elliott, Paul; Holmes, Elaine; Nicholson, Jeremy K

    2018-02-27

    Metabolism is altered by genetics, diet, disease status, environment, and many other factors. Modeling either one of these is often done without considering the effects of the other covariates. Attributing differences in metabolic profile to one of these factors needs to be done while controlling for the metabolic influence of the rest. We describe here a data analysis framework and novel confounder-adjustment algorithm for multivariate analysis of metabolic profiling data. Using simulated data, we show that similar numbers of true associations and significantly less false positives are found compared to other commonly used methods. Covariate-adjusted projections to latent structures (CA-PLS) are exemplified here using a large-scale metabolic phenotyping study of two Chinese populations at different risks for cardiovascular disease. Using CA-PLS, we find that some previously reported differences are actually associated with external factors and discover a number of previously unreported biomarkers linked to different metabolic pathways. CA-PLS can be applied to any multivariate data where confounding may be an issue and the confounder-adjustment procedure is translatable to other multivariate regression techniques.

  10. Analysis of Metabolic Pathways and Fluxes in a Newly Discovered Thermophilic and Ethanol-Tolerant Geobacillus Strain

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Yinjie J.; Sapra, Rajat; Joyner, Dominique; Hazen, Terry C.; Myers, Samuel; Reichmuth, David; Blanch, Harvey; Keasling, Jay D.

    2009-01-20

    A recently discovered thermophilic bacterium, Geobacillus thermoglucosidasius M10EXG, ferments a range of C5 (e.g., xylose) and C6 sugars (e.g., glucose) and istolerant to high ethanol concentrations (10percent, v/v). We have investigated the central metabolism of this bacterium using both in vitro enzyme assays and 13C-based flux analysis to provide insights into the physiological properties of this extremophile and explore its metabolism for bio-ethanol or other bioprocess applications. Our findings show that glucose metabolism in G. thermoglucosidasius M10EXG proceeds via glycolysis, the pentose phosphate pathway, and the TCA cycle; the Entner?Doudoroff pathway and transhydrogenase activity were not detected. Anaplerotic reactions (including the glyoxylate shunt, pyruvate carboxylase, and phosphoenolpyruvate carboxykinase) were active, but fluxes through those pathways could not be accuratelydetermined using amino acid labeling. When growth conditions were switched from aerobic to micro-aerobic conditions, fluxes (based on a normalized glucose uptake rate of 100 units (g DCW)-1 h-1) through the TCA cycle and oxidative pentose phosphate pathway were reduced from 64+-3 to 25+-2 and from 30+-2 to 19+-2, respectively. The carbon flux under micro-aerobic growth was directed formate. Under fully anerobic conditions, G. thermoglucosidasius M10EXG used a mixed acid fermentation process and exhibited a maximum ethanol yield of 0.38+-0.07 mol mol-1 glucose. In silico flux balance modeling demonstrates that lactate and acetate production from G. thermoglucosidasius M10EXG reduces the maximum ethanol yieldby approximately threefold, thus indicating that both pathways should be modified to maximize ethanol production.

  11. A kinetic model describes metabolic response to perturbations and distribution of flux control in the benzenoid network of Petunia hybrida.

    Science.gov (United States)

    Colón, Amy Marshall; Sengupta, Neelanjan; Rhodes, David; Dudareva, Natalia; Morgan, John

    2010-04-01

    In recent years there has been much interest in the genetic enhancement of plant metabolism; however, attempts at genetic modification are often unsuccessful due to an incomplete understanding of network dynamics and their regulatory properties. Kinetic modeling of plant metabolic networks can provide predictive information on network control and response to genetic perturbations, which allow estimation of flux at any concentration of intermediate or enzyme in the system. In this research, a kinetic model of the benzenoid network was developed to simulate whole network responses to different concentrations of supplied phenylalanine (Phe) in petunia flowers and capture flux redistributions caused by genetic manipulations. Kinetic parameters were obtained by network decomposition and non-linear least squares optimization of data from petunia flowers supplied with either 75 or 150 mm(2)H(5)-Phe. A single set of kinetic parameters simultaneously accommodated labeling and pool size data obtained for all endogenous and emitted volatiles at the two concentrations of supplied (2)H(5)-Phe. The generated kinetic model was validated using flowers from transgenic petunia plants in which benzyl CoA:benzyl alcohol/phenylethanol benzoyltransferase (BPBT) was down-regulated via RNAi. The determined in vivo kinetic parameters were used for metabolic control analysis, in which flux control coefficients were calculated for fluxes around the key branch point at Phe and revealed that phenylacetaldehyde synthase activity is the primary controlling factor for the phenylacetaldehyde branch of the benzenoid network. In contrast, control of flux through the beta-oxidative and non-beta-oxidative pathways is highly distributed.

  12. Development of metabolic and inflammatory mediator biomarker phenotyping for early diagnosis and triage of pediatric sepsis.

    Science.gov (United States)

    Mickiewicz, Beata; Thompson, Graham C; Blackwood, Jaime; Jenne, Craig N; Winston, Brent W; Vogel, Hans J; Joffe, Ari R

    2015-09-09

    The first steps in goal-directed therapy for sepsis are early diagnosis followed by appropriate triage. These steps are usually left to the physician's judgment, as there is no accepted biomarker available. We aimed to determine biomarker phenotypes that differentiate children with sepsis who require intensive care from those who do not. We conducted a prospective, observational nested cohort study at two pediatric intensive care units (PICUs) and one pediatric emergency department (ED). Children ages 2-17 years presenting to the PICU or ED with sepsis or presenting for procedural sedation to the ED were enrolled. We used the judgment of regional pediatric ED and PICU attending physicians as the standard to determine triage location (PICU or ED). We performed metabolic and inflammatory protein mediator profiling with serum and plasma samples, respectively, collected upon presentation, followed by multivariate statistical analysis. Ninety-four PICU sepsis, 81 ED sepsis, and 63 ED control patients were included. Metabolomic profiling revealed clear separation of groups, differentiating PICU sepsis from ED sepsis with accuracy of 0.89, area under the receiver operating characteristic curve (AUROC) of 0.96 (standard deviation [SD] 0.01), and predictive ability (Q(2)) of 0.60. Protein mediator profiling also showed clear separation of the groups, differentiating PICU sepsis from ED sepsis with accuracy of 0.78 and AUROC of 0.88 (SD 0.03). Combining metabolomic and protein mediator profiling improved the model (Q(2) =0.62), differentiating PICU sepsis from ED sepsis with accuracy of 0.87 and AUROC of 0.95 (SD 0.01). Separation of PICU sepsis or ED sepsis from ED controls was even more accurate. Prespecified age subgroups (2-5 years old and 6-17 years old) improved model accuracy minimally. Seventeen metabolites or protein mediators accounted for separation of PICU sepsis and ED sepsis with 95% confidence. In children ages 2-17 years, combining metabolomic and

  13. Irisin and the Metabolic Phenotype of Adults with Prader-Willi Syndrome.

    Directory of Open Access Journals (Sweden)

    Harry J Hirsch

    Full Text Available Hyperphagia, low resting energy expenditure, and abnormal body composition contribute to severe obesity in Prader Willi syndrome (PWS. Irisin, a circulating myokine, stimulates "browning" of white adipose tissue resulting in increased energy expenditure and improved insulin sensitivity. Irisin has not been previously studied in PWS.Compare plasma and salivary irisin in PWS adults and normal controls. Examine the relationship of irisin to insulin sensitivity and plasma lipids.A fasting blood sample for glucose, lipids, insulin, leptin, adinopectin, and irisin was obtained from 22 PWS adults and 54 healthy BMI-matched volunteers. Saliva was collected for irisin assay in PWS and controls.Fasting glucose (77 ± 9 vs 83 ± 7 mg/dl, p = 0.004, insulin (4.1 ± 2.0 vs 7.9 ± 4.7 μU/ml, p<0.001, and triglycerides (74 ± 34 vs 109 ± 71 mg/dl, p = 0.007 were lower in PWS than in controls. Insulin resistance (HOMA-IR was lower (0.79 ± 0.041 vs 1.63 ± 1.02, p<0.001 and insulin sensitivity (QUICKI was higher (0.41 ± 0.04 vs 0.36 ± 0.03, p<0.001 in PWS. Plasma irisin was similar in both groups, but salivary irisin (64.5 ± 52.0 vs 33.0 ± 12.1ng/ml, plasma leptin (33.5 ± 24.2 vs 19.7 ± 19.3 ng/ml and plasma adinopectin (13.0 ± 10.8 vs 7.6 ± 4.5μg/ml were significantly greater in PWS (p<0.001. In PWS, plasma irisin showed positive Pearson correlations with total cholesterol (r = 0.58, p = 0.005, LDL-cholesterol (r = 0.59, p = 0.004, and leptin (r = 0.43, p = 0.045. Salivary irisin correlated negatively with HDL-cholesterol (r = -0.50, p = 0.043 and positively with LDL-cholesterol (r = 0.51, p = 0.037 and triglycerides (r = 0.50, p = 0.041.Salivary irisin was markedly elevated in PWS although plasma irisin was similar to levels in controls. Significant associations with plasma lipids suggest that irisin may contribute to the metabolic phenotype of PWS.

  14. In Vivo and Ex Vivo Inflammatory Markers of Common Metabolic Phenotypes in Humans.

    Science.gov (United States)

    Mærkedahl, Rasmus Baadsgaard; Frøkiær, Hanne; Stenbæk, Marie Grøntved; Nielsen, Camilla Betak; Lind, Mads V; Lundtoft, Christian; Bohr, Marietta Boje; Ibrügger, Sabine; Metzdorff, Stine Broeng; Vestergaard, Henrik; Pedersen, Oluf; Lauritzen, Lotte

    2018-02-01

    Low-grade systemic inflammation (LGSI) is often characterized by elevated levels of interleukin (IL)6, tumor necrosis factor (TNF)α, and C-reactive protein (CRP). Other serum proteins, ex vivo-stimulated cytokine production, and leukocyte count have, however, also been suggested LGSI-markers, but their associations with the metabolic syndrome (MS) are less clear. We aimed to evaluate mutual relationships between in vivo and ex vivo inflammatory markers and their association with MS and its subcomponents. A cross-sectional study of 118 overweight adults with one or several features of MS. Inflammatory markers included fasting serum levels of IL6, TNFα, CRP, and pentraxin-3 (PTX3), IL1-receptors, leukocytes, and whole-blood ex vivo-produced IL1β, IL6, TNFα, and IL8 after lipopolysaccharide stimulation. All classical serum LGSI-markers correlated with each other, and IL6 and CRP were also correlated with leukocyte count. Ex vivo-produced cytokines were intercorrelated and correlated with leukocyte count, but did not correlate with the serum immune markers. MS score, body mass index, and glycated hemoglobin (HbA1c) were associated with 8%-16% higher inflammatory score per standard deviation increment (P = 0.030, 0.001, and 0.034, respectively), primarily driven by higher serum IL6. Serum PTX3 was only significantly associated with high-density lipoprotein cholesterol (1.19[1.04; 1.37], P = 0.013). HbA1c was inversely associated with surface expression of IL1R1 on monocytes and IL1R2 on granulocytes (P vivo production of cytokines when adjusting for leukocyte count, as were plasma triacylglycerol (9%-10% lower IL1β and IL6). Leukocyte count was most consistently associated with MS and its subcomponents, although not with HbA1. The classical fasting serum markers of LGSI and leukocyte counts associated best with measures of MS-associated LGSI, whereas ex vivo cytokine production was only associated with prevailing glycemia and dyslipidemia. Taken together

  15. Ethanol-metabolizing pathways in deermice. Estimation of flux calculated from isotope effects

    International Nuclear Information System (INIS)

    Alderman, J.; Takagi, T.; Lieber, C.S.

    1987-01-01

    The apparent deuterium isotope effects on Vmax/Km (D(V/K] of ethanol oxidation in two deermouse strains (one having and one lacking hepatic alcohol dehydrogenase (ADH] were used to calculate flux through the ADH, microsomal ethanol-oxidizing system (MEOS), and catalase pathways. In vitro, D(V/K) values were 3.22 for ADH, 1.13 for MEOS, and 1.83 for catalase under physiological conditions of pH, temperature, and ionic strength. In vivo, in deermice lacking ADH (ADH-), D(V/K) was 1.20 +/- 0.09 (mean +/- S.E.) at 7.0 +/- 0.5 mM blood ethanol and 1.08 +/- 0.10 at 57.8 +/- 10.2 mM blood ethanol, consistent with ethanol oxidation principally by MEOS. Pretreatment of ADH- animals with the catalase inhibitor 3-amino-1,2,4-triazole did not significantly change D(V/K). ADH+ deermice exhibited D(V/K) values of 1.87 +/- 0.06 (untreated), 1.71 +/- 0.13 (pretreated with 3-amino-1,2,4-triazole), and 1.24 +/- 0.13 (after the ADH inhibitor, 4-methylpyrazole) at 5-7 mM blood ethanol levels. At elevated blood ethanol concentrations (58.1 +/- 2.4 mM), a D(V/K) of 1.37 +/- 0.21 was measured in the ADH+ strain. For measured D(V/K) values to accurately reflect pathway contributions, initial reaction conditions are essential. These were shown to exist by the following criteria: negligible fractional conversion of substrate to product and no measurable back reaction in deermice having a reversible enzyme (ADH). Thus, calculations from D(V/K) indicate that, even when ADH is present, non-ADH pathways (mostly MEOS) participate significantly in ethanol metabolism at all concentrations tested and play a major role at high levels

  16. Metabolic flux analysis of a phenol producing mutant of Pseudomonas putida S12: verification and complementation of hypotheses derived from transcriptomics.

    Science.gov (United States)

    Wierckx, Nick; Ruijssenaars, Harald J; de Winde, Johannes H; Schmid, Andreas; Blank, Lars M

    2009-08-20

    The physiological effects of genetic and transcriptional changes observed in a phenol producing mutant of the solvent-tolerant Pseudomonas putida S12 were assessed with metabolic flux analysis. The upregulation of a malate/lactate dehydrogenase encoding gene could be connected to a flux increase from malate to oxaloacetate. A mutation in the pykA gene decreased in vitro pyruvate kinase activity, which is consistent with a lower flux from phosphoenolpyruvate to pyruvate. Changes in the oprB-1, gntP and gnuK genes, encoding a glucose-selective porin, gluconokinase and a gluconate transporter respectively, altered the substrate uptake profile. Metabolic flux analysis furthermore revealed cellular events not predicted by the transcriptome analysis. Gluconeogenic formation of glucose-6-phosphate from triose-3-phosphate was abolished, in favour of increased phosphoenolpyruvate production. An increased pentose phosphate pathway flux resulted in higher erythrose-4-phosphate production. Thus, the availability of these two central phenol precursors was improved. Furthermore, metabolic fluxes were redistributed such that the overall TCA cycle flux was unaffected and energy production increased. Engineering P. putida S12 for phenol production has yielded a strain that channels carbon fluxes to previously unfavourable routes to reconcile the drain on metabolites required for phenol production, while maintaining basal flux levels through central carbon metabolism.

  17. Characterization of the metabolic shift between oxidative and fermentative growth in Saccharomyces cerevisiae by comparative 13C flux analysis

    Directory of Open Access Journals (Sweden)

    Wittmann Christoph

    2005-11-01

    Full Text Available Abstract Background One of the most fascinating properties of the biotechnologically important organism Saccharomyces cerevisiae is its ability to perform simultaneous respiration and fermentation at high growth rate even under fully aerobic conditions. In the present work, this Crabtree effect called phenomenon was investigated in detail by comparative 13C metabolic flux analysis of S. cerevisiae growing under purely oxidative, respiro-fermentative and predominantly fermentative conditions. Results The metabolic shift from oxidative to fermentative growth was accompanied by complex changes of carbon flux throughout the whole central metabolism. This involved a flux redirection from the pentose phosphate pathway (PPP towards glycolysis, an increased flux through pyruvate carboxylase, the fermentative pathways and malic enzyme, a flux decrease through the TCA cycle, and a partial relocation of alanine biosynthesis from the mitochondrion to the cytosol. S. cerevisiae exhibited a by-pass of pyruvate dehydrogenase in all physiological regimes. During oxidative growth this by-pass was mainly provided via pyruvate decarboxylase, acetaldehyde dehydrogenase, acetyl-CoA synthase and transport of acetyl-CoA into the mitochondrion. During fermentative growth this route, however, was saturated due to limited enzyme capacity. Under these conditions the cells exhibited high carbon flux through a chain of reactions involving pyruvate carboxylase, the oxaloacetate transporter and malic enzyme. During purely oxidative growth the PPP alone was sufficient to completely supply NADPH for anabolism. During fermentation, it provided only 60 % of the required NADPH. Conclusion We conclude that, in order to overcome the limited capacity of pyruvate dehydrogenase, S. cerevisiae possesses different metabolic by-passes to channel carbon into the mitochondrion. This involves the conversion of cytosolic pyruvate either into acetyl CoA or oxaloacetate followed by

  18. Hyperandrogenemia in Polycystic Ovary Syndrome: Exploration of the Role of Free Testosterone and Androstenedione in Metabolic Phenotype

    Science.gov (United States)

    Lerchbaum, Elisabeth; Schwetz, Verena; Rabe, Thomas; Giuliani, Albrecht; Obermayer-Pietsch, Barbara

    2014-01-01

    Objective To evaluate the association between androstenedione, testosterone, and free testosterone and metabolic disturbances in polycystic ovary syndrome. Methods We analyzed the association between androstenedione, testosterone, and free testosterone and metabolic parameters in a cross-sectional study including 706 polycystic ovary syndrome and 140 BMI-matched healthy women. Polycystic ovary syndrome women were categorized into 4 groups: normal androstenedione and normal free testosterone (NA/NFT), elevated androstenedione and normal free testosterone (HA/NFT), normal androstenedione and elevated free testosterone (NA/HFT), elevated androstenedione and free testosterone (HA/HFT). Results Polycystic ovary syndrome women with elevated free testosterone levels (HA/HFT and NA/HFT) have an adverse metabolic profile including 2 h glucose, HbA1c, fasting and 2 h insulin, area under the insulin response curve, insulin resistance, insulin sensitivity index (Matsuda), triglycerides, total and high density lipoprotein cholesterol levels compared to NA/NFT (pmetabolic disorders in polycystic ovary syndrome women with HA/NFT. In multiple linear regression analyses (age- and BMI-adjusted), we found a significant negative association between androstenedione/free testosterone-ratio and area under the insulin response curve, insulin resistance, and total cholesterol/high density lipoprotein cholesterol-ratio and a positive association with Matsuda-index, and high density lipoprotein cholesterol (ptestosterone levels but not with isolated androstenedione elevation have an adverse metabolic phenotype. Further, a higher androstenedione/free testosterone-ratio was independently associated with a beneficial metabolic profile. PMID:25310562

  19. Shigella reroutes host cell central metabolism to obtain high-flux nutrient supply for vigorous intracellular growth

    Science.gov (United States)

    Kentner, David; Martano, Giuseppe; Callon, Morgane; Chiquet, Petra; Brodmann, Maj; Burton, Olga; Wahlander, Asa; Nanni, Paolo; Delmotte, Nathanaël; Grossmann, Jonas; Limenitakis, Julien; Schlapbach, Ralph; Kiefer, Patrick; Vorholt, Julia A.; Hiller, Sebastian; Bumann, Dirk

    2014-01-01

    Shigella flexneri proliferate in infected human epithelial cells at exceptionally high rates. This vigorous growth has important consequences for rapid progression to life-threatening bloody diarrhea, but the underlying metabolic mechanisms remain poorly understood. Here, we used metabolomics, proteomics, and genetic experiments to determine host and Shigella metabolism during infection in a cell culture model. The data suggest that infected host cells maintain largely normal fluxes through glycolytic pathways, but the entire output of these pathways is captured by Shigella, most likely in the form of pyruvate. This striking strategy provides Shigella with an abundant favorable energy source, while preserving host cell ATP generation, energy charge maintenance, and survival, despite ongoing vigorous exploitation. Shigella uses a simple three-step pathway to metabolize pyruvate at high rates with acetate as an excreted waste product. The crucial role of this pathway for Shigella intracellular growth suggests targets for antimicrobial chemotherapy of this devastating disease. PMID:24958876

  20. Shigella reroutes host cell central metabolism to obtain high-flux nutrient supply for vigorous intracellular growth.

    Science.gov (United States)

    Kentner, David; Martano, Giuseppe; Callon, Morgane; Chiquet, Petra; Brodmann, Maj; Burton, Olga; Wahlander, Asa; Nanni, Paolo; Delmotte, Nathanaël; Grossmann, Jonas; Limenitakis, Julien; Schlapbach, Ralph; Kiefer, Patrick; Vorholt, Julia A; Hiller, Sebastian; Bumann, Dirk

    2014-07-08

    Shigella flexneri proliferate in infected human epithelial cells at exceptionally high rates. This vigorous growth has important consequences for rapid progression to life-threatening bloody diarrhea, but the underlying metabolic mechanisms remain poorly understood. Here, we used metabolomics, proteomics, and genetic experiments to determine host and Shigella metabolism during infection in a cell culture model. The data suggest that infected host cells maintain largely normal fluxes through glycolytic pathways, but the entire output of these pathways is captured by Shigella, most likely in the form of pyruvate. This striking strategy provides Shigella with an abundant favorable energy source, while preserving host cell ATP generation, energy charge maintenance, and survival, despite ongoing vigorous exploitation. Shigella uses a simple three-step pathway to metabolize pyruvate at high rates with acetate as an excreted waste product. The crucial role of this pathway for Shigella intracellular growth suggests targets for antimicrobial chemotherapy of this devastating disease.

  1. Bacterial persistence is an active σS stress response to metabolic flux limitation

    NARCIS (Netherlands)

    Radzikowski, Jakub Leszek; Vedelaar, Silke; Siegel, David; Ortega, Álvaro Dario; Schmidt, Alexander; Heinemann, Matthias

    While persisters are a health threat due to their transient antibiotic tolerance, little is known about their phenotype and what actually causes persistence. Using a new method for persister generation and high-throughput methods, we comprehensively mapped the molecular phenotype of Escherichia coli

  2. Identifying postmenopausal women at risk for cognitive decline within a healthy cohort using a panel of clinical metabolic indicators: potential for detecting an at-Alzheimer's risk metabolic phenotype.

    Science.gov (United States)

    Rettberg, Jamaica R; Dang, Ha; Hodis, Howard N; Henderson, Victor W; St John, Jan A; Mack, Wendy J; Brinton, Roberta Diaz

    2016-04-01

    Detecting at-risk individuals within a healthy population is critical for preventing or delaying Alzheimer's disease. Systems biology integration of brain and body metabolism enables peripheral metabolic biomarkers to serve as reporters of brain bioenergetic status. Using clinical metabolic data derived from healthy postmenopausal women in the Early versus Late Intervention Trial with Estradiol (ELITE), we conducted principal components and k-means clustering analyses of 9 biomarkers to define metabolic phenotypes. Metabolic clusters were correlated with cognitive performance and analyzed for change over 5 years. Metabolic biomarkers at baseline generated 3 clusters, representing women with healthy, high blood pressure, and poor metabolic phenotypes. Compared with healthy women, poor metabolic women had significantly lower executive, global and memory cognitive performance. Hormone therapy provided metabolic benefit to women in high blood pressure and poor metabolic phenotypes. This panel of well-established clinical peripheral biomarkers represents an initial step toward developing an affordable, rapidly deployable, and clinically relevant strategy to detect an at-risk phenotype of late-onset Alzheimer's disease. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  3. Revealing differences in metabolic flux distributions between a mutant strain and its parent strain Gluconacetobacter xylinus CGMCC 2955.

    Directory of Open Access Journals (Sweden)

    Cheng Zhong

    Full Text Available A better understanding of metabolic fluxes is important for manipulating microbial metabolism toward desired end products, or away from undesirable by-products. A mutant strain, Gluconacetobacter xylinus AX2-16, was obtained by combined chemical mutation of the parent strain (G. xylinus CGMCC 2955 using DEC (diethyl sulfate and LiCl. The highest bacterial cellulose production for this mutant was obtained at about 11.75 g/L, which was an increase of 62% compared with that by the parent strain. In contrast, gluconic acid (the main byproduct concentration was only 5.71 g/L for mutant strain, which was 55.7% lower than that of parent strain. Metabolic flux analysis indicated that 40.1% of the carbon source was transformed to bacterial cellulose in mutant strain, compared with 24.2% for parent strain. Only 32.7% and 4.0% of the carbon source were converted into gluconic acid and acetic acid in mutant strain, compared with 58.5% and 9.5% of that in parent strain. In addition, a higher flux of tricarboxylic acid (TCA cycle was obtained in mutant strain (57.0% compared with parent strain (17.0%. It was also indicated from the flux analysis that more ATP was produced in mutant strain from pentose phosphate pathway (PPP and TCA cycle. The enzymatic activity of succinate dehydrogenase (SDH, which is one of the key enzymes in TCA cycle, was 1.65-fold higher in mutant strain than that in parent strain at the end of culture. It was further validated by the measurement of ATPase that 3.53-6.41 fold higher enzymatic activity was obtained from mutant strain compared with parent strain.

  4. Annual benthic metabolism and organic carbon fluxes in a semi-enclosed Mediterranean bay dominated by the macroalgae Caulerpa prolifera.

    Directory of Open Access Journals (Sweden)

    Sergio eRuiz-Halpern

    2014-12-01

    Full Text Available Coastal areas play an important role on carbon cycling. Elucidating the dynamics on the production, transport and fate of organic carbon is relevant to gain a better understanding of the role coastal areas play in the global carbon budget. Here, we assess the metabolic status and associated organic carbon fluxes of a semi-enclosed Mediterranean bay supporting a meadow of Caulerpa prolifera. We test whether the EDOC pool is a significant component of the organic carbon pool and associated fluxes in this ecosystem. The Bay of Portocolom was in net metabolic balance on a yearly basis, but heterotrophic during the summer months. Community respiration (CR was positively correlated to C. prolifera biomass, while net community production (NCP had a negative correlation. The benthic compartment represented, on average, 72.6 ± 5.2 % of CR and 86.8 ± 4.5 % of gross primary production (GPP. Dissolved organic carbon (DOC production peaked in summer and was always positive, with the incubations performed in the dark almost doubling the flux of those performed in the light. Exchangeable dissolved organic carbon (EDOC, however, oscillated between production and uptake, being completely recycled within the system and representing around 14% of the DOC flux. The pools of bottom and surface DOC were high for an oligotrophic environment, and were positively correlated to the pool of EDOC. Thus, despite being in metabolic balance, this ecosystem acted as a conduit for organic carbon (OC, as it is able to export OC to adjacent areas derived from allochtonous inputs during heterotrophic conditions. These inputs likely come from groundwater discharge, human activity in the watershed, delivered to the sediments through the high capacity of C. prolifera to remove particles from the water column, and from the air-water exchange of EDOC, demonstrating that these communities are a major contributor to the cycling of OC in coastal embayments.

  5. C-13 Tracer experiments and metabolite balancing for metabolic flux analysis

    DEFF Research Database (Denmark)

    Schmidt, Karsten; Marx, A.; de Graaf, A. A.

    1998-01-01

    through the pentose phosphate pathway. Hence, wrong assumptions on the presence or activity of transhydrogenation reactions will result in wrong estimations of the intracellular flux distribution. Using C-13 tracer experiments and NMR analysis, flux analysis can be performed on the basis of only well...

  6. Phenotypic flexibility of traits related to energy acquisition in mice divergently selected for basal metabolic rate (BMR).

    Science.gov (United States)

    Ksiazek, Aneta; Czerniecki, Jan; Konarzewski, Marek

    2009-03-01

    Theoretical considerations suggest that one of the main factors determining phenotypic flexibility of the digestive system is the size (mass) of internal organs. To test this, we used mice from two lines selected for high and low levels of basal metabolic rate (BMR). Mice with higher BMRs also have larger internal organs and higher daily food consumption (C) under non-stressful conditions. We exposed animals from both lines to a sudden cold exposure by transferring them (without prior acclimation) from an ambient temperature of 23 degrees C to 5 degrees C. Cold exposure elicited a twofold increase in C and a 25% reduction of apparent digestive efficiency. For the same body mass-corrected C, small intestine, kidneys, heart and liver of cold-exposed low-BMR mice were smaller than those of the high-BMR line. Therefore, the internal organs of low-BMR animals were burdened with substantially higher metabolic loads (defined as C or digestible food intake per total mass of a particular organ). The mass-specific activity of citrate synthase (CS) in the liver and kidneys (but not heart) was also lower in the low-BMR mice. The magnitude of phenotypic flexibility of internal organ size and CS activity was strictly proportional to the organ mass (in the case of kidneys and liver, also mass-specific CS activity) prior to an increased energy demand. Thus, phenotypic flexibility had additive rather than multiplicative dynamics. Our results also suggest that variation in BMR positively correlates with the magnitude of an immediate spare capacity that fuels the initial response of internal organs to a sudden metabolic stress.

  7. Physical Fitness can Partly Explain the Metabolically Healthy Obese Phenotype in Women

    NARCIS (Netherlands)

    Poelkens, F.; Eijsvogels, T.M.H.; Brussee, P.; Verheggen, R.J.H.M.; Tack, C.J.J.; Hopman, M.T.E.

    2014-01-01

    To investigate whether physical fitness and/or fat distribution and inflammation profile may explain why approximately 30% of the women with obesity are protected against obesity-related disorders.10 metabolically healthy obese women and 10 age- and weight-matched women with the metabolic syndrome

  8. Genome-Wide Meta-Analysis Unravels Interactions between Magnesium Homeostasis and Metabolic Phenotypes.

    NARCIS (Netherlands)

    Corre, T.; Arjona, F.J.; Hayward, C.; Youhanna, S.; Baaij, J.H.F. de; Belge, H.; Nagele, N.; Debaix, H.; Blanchard, M.G.; Traglia, M.; Harris, S.E.; Ulivi, S.; Rueedi, R.; Lamparter, D.; Mace, A.; Sala, C.; Lenarduzzi, S.; Ponte, B.; Pruijm, M.; Ackermann, D.; Ehret, G.; Baptista, D.; Polasek, O.; Rudan, I.; Hurd, T.W.; Hastie, N.D.; Vitart, V.; Waeber, G.; Kutalik, Z.; Bergmann, S.; Vargas-Poussou, R.; Konrad, M.; Gasparini, P.; Deary, I.J.; Starr, J.M.; Toniolo, D.; Vollenweider, P.; Hoenderop, J.G.J.; Bindels, R.J.M.; Bochud, M.; Devuyst, O.

    2018-01-01

    Magnesium (Mg(2+)) homeostasis is critical for metabolism. However, the genetic determinants of the renal handling of Mg(2+), which is crucial for Mg(2+) homeostasis, and the potential influence on metabolic traits in the general population are unknown. We obtained plasma and urine parameters from

  9. Environmental spread of microbes impacts the development of metabolic phenotypes in mice transplanted with microbial communities from humans

    DEFF Research Database (Denmark)

    Zhang, Li; Bahl, Martin Iain; Roager, Henrik Munch

    2017-01-01

    -derived microbiotas in mice, factors affecting this process and resulting impact on metabolic health. We thus transplanted faecal microbiotas from humans (16 obese and 16 controls) separately into 64 germ-free Swiss Webster mice caged in pairs within four isolators, with two isolators assigned to each phenotype......Microbiota transplantation to germ-free animals is a powerful method to study involvement of gut microbes in the aetiology of metabolic syndrome. Owing to large interpersonal variability in gut microbiota, studies with broad coverage of donors are needed to elucidate the establishment of human......, thereby allowing us to explore the extent of microbial spread between cages in a well-controlled environment. Despite high group-wise similarity between obese and control human microbiotas, transplanted mice in the four isolators developed distinct gut bacterial composition and activity, body mass gain...

  10. Symptom patterns and phenotypic subgrouping of women with polycystic ovary syndrome: association between endocrine characteristics and metabolic aberrations.

    Science.gov (United States)

    Huang, Chu-Chun; Tien, Yin-Jing; Chen, Mei-Jou; Chen, Chun-Houh; Ho, Hong-Nerng; Yang, Yu-Shih

    2015-04-01

    What are the potential endocrine characteristics related to risk and severity of metabolic disturbances in women with polycystic ovary syndrome (PCOS)? Women with PCOS could be subtyped into four subgroups according to heterogeneous endocrine characteristics and the major predictive endocrine factors for metabolic aberrations among different subgroups were free androgen index (FAI) and luteinizing hormone (LH) levels. Women diagnosed with PCOS present with highly heterogeneous phenotypes, including endocrine and metabolic aberrations. Different strategies have been proposed to predict the metabolic outcomes but whether the endocrine factors can solely predict the metabolic aberrations is still inconclusive. A cross-sectional study including 460 patients recruited from a reproductive endocrinology outpatient clinic of a tertiary medical center. Patients with PCOS diagnosed according to the 2003 Rotterdam criteria were studied. Clinical history recorded by questionnaires, anthropometric measurements, biochemistry tests after an overnight fast, and pelvic ultrasonography were collected from all patients. Applying a matrix visualization and clustering approach (generalized association plots), the patients were divided into four distinct clusters according to the correlation with four endocrine parameters. Each cluster exhibited specific endocrine characteristics and the prevalence of metabolic syndrome (MS) was significantly different among the clusters (P characteristic of these two metabolically unhealthy clusters was relatively lower LH level. Contrarily, higher LH level (≧15 mIU/ml) during early follicular phase was found to be the best indicator of the metabolically healthy cluster (cluster 1). While high FAI level did correlate with more severe metabolic aberrations, high LH level showed better predictive value than low FAI level to become a metabolically healthy cluster. The results should be applied to other populations with caution due to racial or

  11. Metabolic flux analysis of a phenol producing mutant of Pseudomonas putida S12: Verification and complementation of hypotheses derived from transcriptomics

    NARCIS (Netherlands)

    Wierckx, N.; Ruijssenaars, H.J.; Winde, J.H.de; Schmid, A.; Blank, L.M.

    2009-01-01

    The physiological effects of genetic and transcriptional changes observed in a phenol producing mutant of the solvent-tolerant Pseudomonas putida S12 were assessed with metabolic flux analysis. The upregulation of a malate/lactate dehydrogenase encoding gene could be connected to a flux increase

  12. Carbon-flux distribution within Streptomyces coelicolor metabolism: a comparison between the actinorhodin-producing strain M145 and its non-producing derivative M1146.

    Directory of Open Access Journals (Sweden)

    Fabien Coze

    Full Text Available Metabolic Flux Analysis is now viewed as essential to elucidate the metabolic pattern of cells and to design appropriate genetic engineering strategies to improve strain performance and production processes. Here, we investigated carbon flux distribution in two Streptomyces coelicolor A3 (2 strains: the wild type M145 and its derivative mutant M1146, in which gene clusters encoding the four main antibiotic biosynthetic pathways were deleted. Metabolic Flux Analysis and (13C-labeling allowed us to reconstruct a flux map under steady-state conditions for both strains. The mutant strain M1146 showed a higher growth rate, a higher flux through the pentose phosphate pathway and a higher flux through the anaplerotic phosphoenolpyruvate carboxylase. In that strain, glucose uptake and the flux through the Krebs cycle were lower than in M145. The enhanced flux through the pentose phosphate pathway in M1146 is thought to generate NADPH enough to face higher needs for biomass biosynthesis and other processes. In both strains, the production of NADPH was higher than NADPH needs, suggesting a key role for nicotinamide nucleotide transhydrogenase for redox homeostasis. ATP production is also likely to exceed metabolic ATP needs, indicating that ATP consumption for maintenance is substantial.Our results further suggest a possible competition between actinorhodin and triacylglycerol biosynthetic pathways for their common precursor, acetyl-CoA. These findings may be instrumental in developing new strategies exploiting S. coelicolor as a platform for the production of bio-based products of industrial interest.

  13. Metabolic and transcriptional responses of gilthead sea bream (Sparus aurata L.) to environmental stress: new insights in fish mitochondrial phenotyping.

    Science.gov (United States)

    Bermejo-Nogales, Azucena; Nederlof, Marit; Benedito-Palos, Laura; Ballester-Lozano, Gabriel F; Folkedal, Ole; Olsen, Rolf Eric; Sitjà-Bobadilla, Ariadna; Pérez-Sánchez, Jaume

    2014-09-01

    The aim of the current study was to phenotype fish metabolism and the transcriptionally-mediated response of hepatic mitochondria of gilthead sea bream to intermittent and repetitive environmental stressors: (i) changes in water temperature (T-ST), (ii) changes in water level and chasing (C-ST) and (iii) multiple sensory perception stressors (M-ST). Gene expression profiling was done using a quantitative PCR array of 60 mitochondria-related genes, selected as markers of transcriptional regulation, oxidative metabolism, respiration uncoupling, antioxidant defense, protein import/folding/assembly, and mitochondrial dynamics and apoptosis. The mitochondrial phenotype mirrored changes in fish performance, haematology and lactate production. T-ST especially up-regulated transcriptional factors (PGC1α, NRF1, NRF2), rate limiting enzymes of fatty acid β-oxidation (CPT1A) and tricarboxylic acid cycle (CS), membrane translocases (Tim/TOM complex) and molecular chaperones (mtHsp10, mtHsp60, mtHsp70) to improve the oxidative capacity in a milieu of a reduced feed intake and impaired haematology. The lack of mitochondrial response, increased production of lactate and negligible effects on growth performance in C-ST fish were mostly considered as a switch from aerobic to anaerobic metabolism. A strong down-regulation of PGC1α, NRF1, NRF2, CPT1A, CS and markers of mitochondrial dynamics and apoptosis (BAX, BCLX, MFN2, MIRO2) occurred in M-ST fish in association with the greatest circulating cortisol concentration and a reduced lactate production and feed efficiency, which represents a metabolic condition with the highest allostatic load score. These findings evidence a high mitochondrial plasticity against stress stimuli, providing new insights to define the threshold level of stress condition in fish. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. In vivo metabolic flux profiling with stable isotopes discriminates sites and quantifies effects of mitochondrial dysfunction in C. elegans

    Science.gov (United States)

    Ostrovsky, Julian; Clarke, Colleen; Preston, Judith; Bennett, Michael J.; Yudkoff, Marc; Xiao, Rui; Falk, Marni J.

    2014-01-01

    Mitochondrial respiratory chain (RC) disease diagnosis is complicated both by an absence of biomarkers that sufficiently divulge all cases and limited capacity to quantify adverse effects across intermediary metabolism. We applied high performance liquid chromatography (HPLC) and mass spectrometry (MS) studies of stable-isotope based precursor–product relationships in the nematode, C. elegans, to interrogate in vivo differences in metabolic flux among distinct genetic models of primary RC defects and closely related metabolic disorders. Methods C. elegans strains studied harbor single nuclear gene defects in complex I, II, or III RC subunits (gas-1, mev-1, isp-1); enzymes involved in coenzyme Q biosynthesis (clk-1), the tricarboxylic acid cycle (TCA, idh-1), or pyruvate metabolism (pdha-1); and central nodes of the nutrient-sensing signaling network that involve insulin response (daf-2) or the sirtuin homologue (sir-2.1). Synchronous populations of 2000 early larval stage worms were fed standard Escherichia coli on nematode growth media plates containing 1,6-13C2-glucose throughout their developmental period, with samples extracted on the first day of adult life in 4% perchloric acid with an internal standard. Quantitation of whole animal free amino acid concentrations and isotopic incorporation into amino and organic acids throughout development was performed in all strains by HPLC and isotope ratio MS, respectively. GC/MS analysis was also performed to quantify absolute isotopic incorporation in all molecular species of key TCA cycle intermediates in gas-1 and N2 adult worms. Results Genetic mutations within different metabolic pathways displayed distinct metabolic profiles. RC complex I (gas-1) and III (isp-1) subunit mutants, together with the coenzyme Q biosynthetic mutant (clk-1), shared a similar amino acid profile of elevated alanine and decreased glutamate. The metabolic signature of the complex II mutant (mev-1) was distinct from that of the other RC

  15. Magnetic Resonance Spectroscopic Imaging of Tumor Metabolic Markers for Cancer Diagnosis, Metabolic Phenotyping, and Characterization of Tumor Microenvironment

    OpenAIRE

    He, Qiuhong; Xu, Ray Z.; Shkarin, Pavel; Pizzorno, Giuseppe; Lee-French, Carol H.; Rothman, Douglas L.; Shungu, Dikoma C.; Shim, Hyunsuk

    2004-01-01

    Cancer cells display heterogeneous genetic characteristics, depending on the tumor dynamic microenvironment. Abnormal tumor vasculature and poor tissue oxygenation generate a fraction of hypoxic tumor cells that have selective advantages in metastasis and invasion and often resist chemo- and radiation therapies. The genetic alterations acquired by tumors modify their biochemical pathways, which results in abnormal tumor metabolism. An elevation in glycolysis known as the “Warburg effect” and ...

  16. Flux Balance Analysis Inspired Bioprocess Upgrading for Lycopene Production by a Metabolically Engineered Strain of Yarrowia lipolytica

    Directory of Open Access Journals (Sweden)

    Komi Nambou

    2015-12-01

    Full Text Available Genome-scale metabolic models embody a significant advantage of systems biology since their applications as metabolic flux simulation models enable predictions for the production of industrially-interesting metabolites. The biotechnological production of lycopene from Yarrowia lipolytica is an emerging scope that has not been fully scrutinized, especially for what concerns cultivation conditions of newly generated engineered strains. In this study, by combining flux balance analysis (FBA and Plackett-Burman design, we screened chemicals for lycopene production from a metabolically engineered strain of Y. lipolytica. Lycopene concentrations of 126 and 242 mg/L were achieved correspondingly from the FBA-independent and the FBA-assisted designed media in fed-batch cultivation mode. Transcriptional studies revealed upregulations of heterologous genes in media designed according to FBA, thus implying the efficiency of model predictions. Our study will potentially support upgraded lycopene and other terpenoids production from existing or prospect bioengineered strains of Y. lipolytica and/or closely related yeast species.

  17. Flux Balance Analysis Inspired Bioprocess Upgrading for Lycopene Production by a Metabolically Engineered Strain of Yarrowia lipolytica

    Science.gov (United States)

    Nambou, Komi; Jian, Xingxing; Zhang, Xinkai; Wei, Liujing; Lou, Jiajia; Madzak, Catherine; Hua, Qiang

    2015-01-01

    Genome-scale metabolic models embody a significant advantage of systems biology since their applications as metabolic flux simulation models enable predictions for the production of industrially-interesting metabolites. The biotechnological production of lycopene from Yarrowia lipolytica is an emerging scope that has not been fully scrutinized, especially for what concerns cultivation conditions of newly generated engineered strains. In this study, by combining flux balance analysis (FBA) and Plackett-Burman design, we screened chemicals for lycopene production from a metabolically engineered strain of Y. lipolytica. Lycopene concentrations of 126 and 242 mg/L were achieved correspondingly from the FBA-independent and the FBA-assisted designed media in fed-batch cultivation mode. Transcriptional studies revealed upregulations of heterologous genes in media designed according to FBA, thus implying the efficiency of model predictions. Our study will potentially support upgraded lycopene and other terpenoids production from existing or prospect bioengineered strains of Y. lipolytica and/or closely related yeast species. PMID:26703753

  18. Flux analysis of central metabolic pathways in the Fe(III)-reducing organism Geobacter metallireducens via 13C isotopiclabeling

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Yinjie J.; Chakraborty, Romy; Martin, Hector Garcia; Chu,Jeannie; Hazen, Terry C.; Keasling, Jay D.

    2007-08-13

    We analyzed the carbon fluxes in the central metabolism ofGeobacter metallireducens strain GS-15 using 13C isotopomer modeling.Acetate labeled in the 1st or 2nd position was the sole carbon source,and Fe-NTA was the sole terminal electron acceptor. The measured labeledacetate uptake rate was 21 mmol/gdw/h in the exponential growth phase.The resulting isotope labeling pattern of amino acids allowed an accuratedetermination of the in vivo global metabolic reaction rates (fluxes)through the central metabolic pathways using a computational isotopomermodel. The model indicated that over 90 percent of the acetate wascompletely oxidized to CO2 via a complete tricarboxylic acid (TCA) cyclewhile reducing iron. Pyruvate carboxylase and phosphoenolpyruvatecarboxykinase were present under these conditions, but enzymes in theglyoxylate shunt and malic enzyme were absent. Gluconeogenesis and thepentose phosphate pathway were mainly employed for biosynthesis andaccounted for less than 3 percent of total carbon consumption. The modelalso indicated surprisingly high reversibility in the reaction betweenoxoglutarate and succinate. This step operates close to the thermodynamicequilibrium possibly because succinate is synthesized via a transferasereaction, and its product, acetyl-CoA, inhibits the conversion ofoxoglutarate to succinate. These findings enable a better understandingof the relationship between genome annotation and extant metabolicpathways in G. metallireducens.

  19. Control of fluxes towards antibiotics and the role of primary metabolism in production of antibiotics

    DEFF Research Database (Denmark)

    Gunnarsson, Nina; Eliasson Lantz, Anna; Nielsen, Jacob

    2004-01-01

    Yield improvements in antibiotic-producing strains have classically been obtained through random mutagenesis and screening. An attractive alternative to this strategy is the rational design of producer strains via metabolic engineering, an approach that offers the possibility to increase yields...... in the metabolic network. Here we describe and discuss available methods for identification of these steps, both in antibiotic biosynthesis pathways and in the primary metabolism, which serves as the supplier of precursors and cofactors for the secondary metabolism. Finally, the importance of precursor...... and cofactor supply from primary metabolism in the biosynthesis of different types of antibiotics is discussed and recent developments in metabolic engineering towards increased product yields in antibiotic producing strains are reviewed....

  20. Control of fluxes towards antibiotics and the role of primary metabolism in production of antibiotics

    DEFF Research Database (Denmark)

    Gunnarsson, Nina; Eliasson Lantz, Anna; Nielsen, Jacob

    2004-01-01

    in the metabolic network. Here we describe and discuss available methods for identification of these steps, both in antibiotic biosynthesis pathways and in the primary metabolism, which serves as the supplier of precursors and cofactors for the secondary metabolism. Finally, the importance of precursor...... and cofactor supply from primary metabolism in the biosynthesis of different types of antibiotics is discussed and recent developments in metabolic engineering towards increased product yields in antibiotic producing strains are reviewed.......Yield improvements in antibiotic-producing strains have classically been obtained through random mutagenesis and screening. An attractive alternative to this strategy is the rational design of producer strains via metabolic engineering, an approach that offers the possibility to increase yields...

  1. Annual cycles of metabolic rate are genetically determined but can be shifted by phenotypic flexibility

    NARCIS (Netherlands)

    Versteegh, M. A.; Helm, B.; Gwinner, E.; Tieleman, B. I.

    2012-01-01

    Birds have adjusted their life history and physiological traits to the characteristics of the seasonally changing environments they inhabit. Annual cycles in physiology can result from phenotypic flexibility or from variation in its genetic basis. A key physiological trait that shows seasonal

  2. Phenotypic analysis of mutant and overexpressing strains of lipid metabolism genes in Saccharomyces cerevisiae: implication in growth at low temperatures.

    Science.gov (United States)

    López-Malo, María; Chiva, Rosana; Rozes, Nicolas; Guillamon, José Manuel

    2013-03-01

    The growing demand for wines with a more pronounced aromatic profile calls for low temperature alcoholic fermentations (10-15°C). However, there are certain drawbacks to low temperature fermentations such as reduced growth rate, long lag phase and sluggish or stuck fermentations. The lipid metabolism of Saccharomyces cerevisiae plays a central role in low temperature adaptation. The aim of this study was to detect lipid metabolism genes involved in cold adaptation. To do so, we analyzed the growth of knockouts in phospholipids, sterols and sphingolipids, from the EUROSCARF collection S. cerevisiae BY4742 strain at low and optimal temperatures. Growth rate of these knockouts, compared with the control, enabled us to identify the genes involved, which were also deleted or overexpressed in a derivative haploid of a commercial wine strain. We identified genes involved in the phospholipid (PSD1 and OPI3), sterol (ERG3 and IDI1) and sphingolipid (LCB3) pathways, whose deletion strongly impaired growth at low temperature and whose overexpression reduced generation or division time by almost half. Our study also reveals many phenotypic differences between the laboratory strain and the commercial wine yeast strain, showing the importance of constructing mutant and overexpressing strains in both genetic backgrounds. The phenotypic differences in the mutant and overexpressing strains were correlated with changes in their lipid composition. Copyright © 2013. Published by Elsevier B.V.

  3. Metabolic phenotyping for discovery of urinary biomarkers of diet, xenobiotics and blood pressure in the INTERMAP Study: an overview.

    Science.gov (United States)

    Chan, Queenie; Loo, Ruey Leng; Ebbels, Timothy M D; Van Horn, Linda; Daviglus, Martha L; Stamler, Jeremiah; Nicholson, Jeremy K; Holmes, Elaine; Elliott, Paul

    2017-04-01

    The etiopathogenesis of cardiovascular diseases (CVDs) is multifactorial. Adverse blood pressure (BP) is a major independent risk factor for epidemic CVD affecting ~40% of the adult population worldwide and resulting in significant morbidity and mortality. Metabolic phenotyping of biological fluids has proven its application in characterizing low-molecular-weight metabolites providing novel insights into gene-environmental-gut microbiome interaction in relation to a disease state. In this review, we synthesize key results from the INTERnational study of MAcro/micronutrients and blood Pressure (INTERMAP) Study, a cross-sectional epidemiologic study of 4680 men and women aged 40-59 years from Japan, the People's Republic of China, the United Kingdom and the United States. We describe the advancements we have made regarding the following: (1) analytical techniques for high-throughput metabolic phenotyping; (2) statistical analyses for biomarker identification; (3) discovery of unique food-specific biomarkers; and (4) application of metabolome-wide association studies to gain a better understanding into the molecular mechanisms of cross-cultural and regional BP differences.

  4. Interaction of ibuprofen and probenecid with drug metabolizing enzyme phenotyping procedures using caffeine as the probe drug

    Science.gov (United States)

    Vrtic, Fatima; Haefeli, Walter E; Drewe, Jürgen; Krähenbühl, Stephan; Wenk, Markus

    2003-01-01

    Aim To examine the suspected inhibitory potential of the over-the-counter (OTC) drug ibuprofen on N-acetyltransferase 2 (NAT2) in vitro and in vivo and the possible implications for phenotyping procedures using caffeine as probe drug. Methods We first studied the inhibitory effect of ibuprofen on NAT2 in vitro, using human liver cytosol and sulfamethazine as substrate. In vivo 15 fast and 15 slow acetylating healthy volunteers were treated with a single dose of ibuprofen (800 mg) orally and phenotyped for NAT2, CYP1A2, and xanthine oxidase (XO) with caffeine as probe drug before and during drug treatment. Because of unexpected in vivo results with ibuprofen this study was repeated in 20 healthy volunteers with probenecid, a model substrate of renal organic anion transport (OAT). For phenotyping tests a urine sample was collected 6 h after caffeine (200 mg) intake. The caffeine metabolites acetyl-6-formylamino-3-methyluracil (AFMU), 1-methylxanthine (1MX), 1-methyluric acid (1MU), and 1,7-dimethyluric acid (17MU) were quantified by HPLC, and the corresponding metabolic ratios for CYP1A2, NAT2, and XO were then calculated. Genotyping for NAT2 was performed with standard PCR-RFLP methods. Results In vitro, with human liver cytosol an inhibition by ibuprofen of the acetylation of sulfamethazine with Ki values between 2.2 and 3.1 mm was observed. Surprisingly, in vivo a significant (P probenecid, a model substrate of the renal OAT system. Again, a prominent elevation of the AFMU/1MX ratio from 0.97 ± 0.21 to 1.53 ± 0.35 was found (P < 0.002; 95% CI on the difference 0.237, 0.876), but also the XO ratio 1MU/1MX was significantly (P < 0.0001) increased from 1.34 ± 0.09 to 2.24 ± 0.14 (95% CI on difference 0.735, 1.059) due to a reduction of 1MX excretion. Conclusions Substrates of OAT interact with renal excretion of caffeine metabolites and may falsify NAT2 and XO phenotyping results. Other phenotyping procedures, which are based on urinary metabolic ratios, should

  5. Metabolic and Behavioural Phenotypes in Nestin-Cre Mice Are Caused by Hypothalamic Expression of Human Growth Hormone.

    Directory of Open Access Journals (Sweden)

    Jeroen Declercq

    Full Text Available The Nestin-Cre driver mouse line has mild hypopituitarism, reduced body weight, a metabolic phenotype and reduced anxiety. Although several causes have been suggested, a comprehensive explanation is still lacking. In this study we examined the molecular mechanisms leading to this compound phenotype. Upon generation of the Nestin-Cre mice, the human growth hormone (hGH minigene was inserted downstream of the Cre recombinase to ensure efficient transgene expression. As a result, hGH is expressed in the hypothalamus. This results in the auto/paracrine activation of the GH receptor as demonstrated by the increased phosphorylation of signal transducer and activator of transcription 5 (STAT5 and reduced expression of growth hormone releasing hormone (Ghrh. Low Ghrh levels cause hypopituitarism consistent with the observed mouse growth hormone (mGH deficiency. mGH deficiency caused reduced activation of the GH receptor and hence reduced phosphorylation of STAT5 in the liver. This led to decreased levels of hepatic Igf-1 mRNA and consequently postnatal growth retardation. Furthermore, genes involved in lipid uptake and synthesis, such as CD36 and very low-density lipoprotein receptor were upregulated, resulting in liver steatosis. In conclusion, this study demonstrates the unexpected expression of hGH in the hypothalamus of Nestin-Cre mice which is able to activate both the GH receptor and the prolactin receptor. Increased hypothalamic GH receptor signaling explains the observed hypopituitarism, reduced growth and metabolic phenotype of Nestin-Cre mice. Activation of either receptor is consistent with reduced anxiety.

  6. Flux balance analysis of genome-scale metabolic model of rice ...

    Indian Academy of Sciences (India)

    2015-09-28

    Sep 28, 2015 ... producing vitamin A–enriched golden rice (Beyer et al. 2002). On the other hand, metabolic engineering has been proven as a powerful technique for over or less production of specific metabolites in microbes (Peralta-Yahya et al. 2012). Modelling and analysis of the cellular metabolism is a key step in the ...

  7. Transorgan fluxes in a porcine model reveal a central role for liver in acylcarnitine metabolism

    NARCIS (Netherlands)

    Schooneman, Marieke G.; ten Have, Gabriella A. M.; van Vlies, Naomi; Houten, Sander M.; Deutz, Nicolaas E. P.; Soeters, Maarten R.

    2015-01-01

    Acylcarnitines are derived from mitochondrial acyl-CoA metabolism and have been associated with diet-induced insulin resistance. However, plasma acylcarnitine profiles have been shown to poorly reflect whole body acylcarnitine metabolism. We aimed to clarify the individual role of different organ

  8. Role of androgen ratios in the prediction of the metabolic phenotype in polycystic ovary syndrome.

    Science.gov (United States)

    Minooee, Sonia; Ramezani Tehrani, Fahimeh; Tohidi, Maryam; Azizi, Fereidoun

    2017-05-01

    To identify the androgen ratio that best predicts insulin resistance and metabolic syndrome among women with polycystic ovary syndrome (PCOS). Data for 180 women with PCOS and 180 healthy controls were extracted from two previous studies in Iran (conducted during 2008-2010 and 2011-2013). The diagnosis of PCOS was based on the Rotterdam criteria. The serum concentration of different androgens was measured. Receiver operating characteristic curve analysis was used to assess the ability of various androgen ratios to predict insulin resistance and metabolic syndrome. Among women with PCOS, the testosterone-to-androstenedione ratio was the best predictor of insulin resistance (sensitivity 0.83, specificity 0.42) and metabolic syndrome (sensitivity 0.85, specificity 0.70). Among healthy controls, the ratio of free androgen index to testosterone was the best predictor of insulin resistance (sensitivity 0.84, specificity 0.33) and metabolic syndrome (sensitivity 0.91, specificity 0.17). The prediction of insulin resistance and metabolic syndrome among women with PCOS was best accomplished with the testosterone-to-androstenedione ratio. © 2017 International Federation of Gynecology and Obstetrics.

  9. Radiopharmaceuticals for Assessment of Altered Metabolism and Biometal Fluxes in Brain Aging and Alzheimer’s Disease with Positron Emission Tomography

    OpenAIRE

    Xie, Fang; Peng, Fangyu

    2017-01-01

    Aging is a risk factor for Alzheimer’s disease (AD). There are changes of brain metabolism and biometal fluxes due to brain aging, which may play a role in pathogenesis of AD. Positron emission tomography (PET) is a versatile tool for tracking alteration of metabolism and biometal fluxes due to brain aging and AD. Age-dependent changes in cerebral glucose metabolism can be tracked with PET using 2-deoxy-2-[18F]-fluoro-D-glucose (18F-FDG), a radiolabeled glucose analogue, as a radiotracer. Bas...

  10. Metabolic flux pattern of glucose utilization by Xanthomonas campestris pv. campestris: prevalent role of the Entner-Doudoroff pathway and minor fluxes through the pentose phosphate pathway and glycolysis.

    Science.gov (United States)

    Schatschneider, Sarah; Huber, Claudia; Neuweger, Heiko; Watt, Tony Francis; Pühler, Alfred; Eisenreich, Wolfgang; Wittmann, Christoph; Niehaus, Karsten; Vorhölter, Frank-Jörg

    2014-10-01

    The well-studied plant pathogenic bacterium Xanthomonas campestris pv. campestris (Xcc) synthesizes the biotechnologically important polysaccharide xanthan gum, which is also regarded as a virulence factor in plant interactions. In Xcc, sugars like glucose are utilized as a source to generate energy and biomass for growth and pathogenicity. In this study, we used [1-(13)C]glucose as a tracer to analyze the fluxes in the central metabolism of the bacterium growing in a minimal medium. (13)C-Metabolic flux analysis based on gas chromatography-mass spectrometry (GC-MS) confirmed the prevalent catabolic role of the Entner-Doudoroff pathway. Comparative nuclear magnetic resonance (NMR)-based isotopologue profiling of a mutant deficient in glycolysis gave evidence for a moderate flux via glycolysis in the wild-type. In addition to reconfirming the Entner-Doudoroff pathway as a catabolic main route, this approach affirmed a numerically minor but important flux via the pentose phosphate pathway.

  11. Integrative field scale phenotyping for investigating metabolic components of water stress within a vineyard

    Directory of Open Access Journals (Sweden)

    Jorge Gago

    2017-10-01

    Full Text Available Abstract Background There is currently a high requirement for field phenotyping methodologies/technologies to determine quantitative traits related to crop yield and plant stress responses under field conditions. Methods We employed an unmanned aerial vehicle equipped with a thermal camera as a high-throughput phenotyping platform to obtain canopy level data of the vines under three irrigation treatments. High-resolution imagery (< 2.5 cm/pixel was employed to estimate the canopy conductance (g c via the leaf energy balance model. In parallel, physiological stress measurements at leaf and stem level as well as leaf sampling for primary and secondary metabolome analysis were performed. Results Aerial g c correlated significantly with leaf stomatal conductance (g s and stem sap flow, benchmarking the quality of our remote sensing technique. Metabolome profiles were subsequently linked with g c and g s via partial least square modelling. By this approach malate and flavonols, which have previously been implicated to play a role in stomatal function under controlled greenhouse conditions within model species, were demonstrated to also be relevant in field conditions. Conclusions We propose an integrative methodology combining metabolomics, organ-level physiology and UAV-based remote sensing of the whole canopy responses to water stress within a vineyard. Finally, we discuss the general utility of this integrative methodology for broad field phenotyping.

  12. Hydrogen Peroxide-Induced Root Ca2+ and K+ Fluxes Correlate with Salt Tolerance in Cereals: Towards the Cell-Based Phenotyping

    Directory of Open Access Journals (Sweden)

    Haiyang Wang

    2018-03-01

    Full Text Available Salinity stress-induced production of reactive oxygen species (ROS and associated oxidative damage is one of the major factors limiting crop production in saline soils. However, the causal link between ROS production and stress tolerance is not as straightforward as one may expect, as ROS may also play an important signaling role in plant adaptive responses. In this study, the causal relationship between salinity and oxidative stress tolerance in two cereal crops—barley (Hordeum vulgare and wheat (Triticum aestivum—was investigated by measuring the magnitude of ROS-induced net K+ and Ca2+ fluxes from various root tissues and correlating them with overall whole-plant responses to salinity. We have found that the association between flux responses to oxidative stress and salinity stress tolerance was highly tissue specific, and was also dependent on the type of ROS applied. No correlation was found between root responses to hydroxyl radicals and the salinity tolerance. However, when oxidative stress was administered via H2O2 treatment, a significant positive correlation was found for the magnitude of ROS-induced K+ efflux and Ca2+ uptake in barley and the overall salinity stress tolerance, but only for mature zone and not the root apex. The same trends were found for wheat. These results indicate high tissue specificity of root ion fluxes response to ROS and suggest that measuring the magnitude of H2O2-induced net K+ and Ca2+ fluxes from mature root zone may be used as a tool for cell-based phenotyping in breeding programs aimed to improve salinity stress tolerance in cereals.

  13. Hydrogen Peroxide-Induced Root Ca2+and K⁺ Fluxes Correlate with Salt Tolerance in Cereals: Towards the Cell-Based Phenotyping.

    Science.gov (United States)

    Wang, Haiyang; Shabala, Lana; Zhou, Meixue; Shabala, Sergey

    2018-03-01

    Salinity stress-induced production of reactive oxygen species (ROS) and associated oxidative damage is one of the major factors limiting crop production in saline soils. However, the causal link between ROS production and stress tolerance is not as straightforward as one may expect, as ROS may also play an important signaling role in plant adaptive responses. In this study, the causal relationship between salinity and oxidative stress tolerance in two cereal crops-barley ( Hordeum vulgare ) and wheat ( Triticum aestivum )-was investigated by measuring the magnitude of ROS-induced net K⁺ and Ca 2+ fluxes from various root tissues and correlating them with overall whole-plant responses to salinity. We have found that the association between flux responses to oxidative stress and salinity stress tolerance was highly tissue specific, and was also dependent on the type of ROS applied. No correlation was found between root responses to hydroxyl radicals and the salinity tolerance. However, when oxidative stress was administered via H₂O₂ treatment, a significant positive correlation was found for the magnitude of ROS-induced K⁺ efflux and Ca 2+ uptake in barley and the overall salinity stress tolerance, but only for mature zone and not the root apex. The same trends were found for wheat. These results indicate high tissue specificity of root ion fluxes response to ROS and suggest that measuring the magnitude of H₂O₂-induced net K⁺ and Ca 2+ fluxes from mature root zone may be used as a tool for cell-based phenotyping in breeding programs aimed to improve salinity stress tolerance in cereals.

  14. Insulin sensitivity and metabolic flexibility following exercise training among different obese insulin resistant phenotypes

    DEFF Research Database (Denmark)

    Malin, Steven K; Haus, Jacob M; Solomon, Thomas

    2013-01-01

    ) and were instructed to maintain a eucaloric diet. A euglycemic-hyperinsulinemic clamp (40 mU/m(2)/min) with [6,6-(2)H]-glucose was used to determine peripheral and hepatic insulin sensitivity. Non-oxidative glucose disposal and metabolic flexibility (insulin-stimulated respiratory quotient [RQ] minus...

  15. The prevalence of the metabolically healthy obese phenotype in an aging population and its association with subclinical cardiovascular disease: The Brazilian study on healthy aging.

    Science.gov (United States)

    Roberson, Lara; Shaharyar, Sameer; Aneni, Ehimen; Freitas, Wladimir; Blaha, Michael; Agatston, Arthur; Blumenthal, Roger; Santos, Raul D; Feiz, Hamid; Nasir, Khurram; Sposito, Andrei

    2014-01-01

    Current literature has elucidated a new phenotype, metabolically healthy obese (MHO), with risks of cardiovascular disease similar to that of normal weight individuals. Few studies have examined the MHO phenotype in an aging population, especially in association with subclinical CVD. This cross sectional study population consisted of 208 octogenarians and older. Anthropometrics, biochemical, and radiological parameters were measured to assess obesity, metabolic health (assessed by the National Cholesterol Education Program -Adult Treatment Panel (NCEP-ATP III) criteria), and subclinical measures of CVD. The prevalence of MHO was 13.5% (N = 28). No significant association with MHO was noted for age, coronary artery calcium score, cIMT, or hs-CRP > 3 mg/dl (p = NS). Our results suggest that the MHO phenotype exists in the elderly; however, subclinical CVD measures were not different in sub-group analysis suggesting traditional metabolic risk factor algorithms may not be accurate in the very elderly.

  16. Cross-sectional surveillance study to phenotype lorry drivers' sedentary behaviours, physical activity and cardio-metabolic health.

    Science.gov (United States)

    Varela-Mato, Veronica; O'Shea, Orlagh; King, James A; Yates, Thomas; Stensel, David J; Biddle, Stuart Jh; Nimmo, Myra A; Clemes, Stacy A

    2017-06-21

    Elevated risk factors for a number of chronic diseases have been identified in lorry drivers. Unhealthy lifestyle behaviours such as a lack of physical activity (PA) and high levels of sedentary behaviour (sitting) likely contribute to this elevated risk. This study behaviourally phenotyped UK lorry drivers' sedentary and non-sedentary behaviours during workdays and non-workdays and examined markers of drivers cardio-metabolic health. A transport company from the East Midlands, UK. A sample of 159 male heavy goods vehicle drivers (91% white European; (median (range)) age: 50 (24, 67) years) completed the health assessments. 87 (age: 50.0 (25.0, 65.0); body mass index (BMI): 27.7 (19.6, 43.4) kg/m 2 ) provided objective information on sedentary and non-sedentary time. Participants self-reported their sociodemographic information. Primary outcomes: sedentary behaviour and PA, assessed over 7 days using an activPAL3 inclinometer. Cardio-metabolic markers included: blood pressure (BP), heart rate, waist circumference (WC), hip circumference, body composition and fasted capillary blood glucose, triglycerides, high-density lipopreotein cholesterol, low-density lipoprotein cholesterol (LDL-C) and total cholesterol (TC) levels. These cardio-metabolic markers were treated as secondary outcomes. Lorry drivers presented an unhealthy cardio-metabolic health profile (median (IQR) systolic BP: 129 (108.5, 164) mm Hg; diastolic BP: 81 (63, 104) mm Hg; BMI: 29 (20, 47) kg/m 2 ; WC: 102 (77.5, 146.5) cm; LDL-C: 3 (1, 6) mmol/L; TC: 4.9 (3, 7.5) mmol/L). 84% were overweight or obese, 43% had type 2 diabetes or prediabetes and 34% had the metabolic syndrome. The subsample of lorry drivers with objective postural data (n=87) accumulated 13 hours/day and 8 hours/day of sedentary behaviour on workdays and non-workdays (pcardio-metabolic profile and are highly sedentary and physically inactive. Interventions to reduce sitting and increase MVPA during breaks and leisure time to

  17. Cofactor engineering through heterologous expression of an NADH oxidase and its impact on metabolic flux redistribution in Klebsiella pneumoniae

    Directory of Open Access Journals (Sweden)

    Ji Xiao-Jun

    2013-01-01

    Full Text Available Abstract Background Acetoin is an important bio-based platform chemical. However, it is usually existed as a minor byproduct of 2,3-butanediol fermentation in bacteria. Results The present study reports introducing an exogenous NAD+ regeneration sysytem into a 2,3-butanediol producing strain Klebsiella pneumoniae to increse the accumulation of acetoin. Batch fermentation suggested that heterologous expression of the NADH oxidase in K. pneumoniae resulted in large decreases in the intracellular NADH concentration (1.4 fold and NADH/NAD+ ratio (2.0 fold. Metabolic flux analysis revealed that fluxes to acetoin and acetic acid were enhanced, whereas, production of lactic acid and ethanol were decreased, with the accumualation of 2,3-butanediol nearly unaltered. By fed-batch culture of the recombinant, the highest reported acetoin production level (25.9 g/L by Klebsiella species was obtained. Conclusions The present study indicates that microbial production of acetoin could be improved by decreasing the intracellular NADH/NAD+ ratio in K. pneumoniae. It demonstrated that the cofactor engineering method, which is by manipulating the level of intracellular cofactors to redirect cellular metabolism, could be employed to achieve a high efficiency of producing the NAD+-dependent microbial metabolite.

  18. A Nested Case-Control Study of Metabolically Defined Body Size Phenotypes and Risk of Colorectal Cancer in the European Prospective Investigation into Cancer and Nutrition (EPIC.

    Directory of Open Access Journals (Sweden)

    Neil Murphy

    2016-04-01

    Full Text Available Obesity is positively associated with colorectal cancer. Recently, body size subtypes categorised by the prevalence of hyperinsulinaemia have been defined, and metabolically healthy overweight/obese individuals (without hyperinsulinaemia have been suggested to be at lower risk of cardiovascular disease than their metabolically unhealthy (hyperinsulinaemic overweight/obese counterparts. Whether similarly variable relationships exist for metabolically defined body size phenotypes and colorectal cancer risk is unknown.The association of metabolically defined body size phenotypes with colorectal cancer was investigated in a case-control study nested within the European Prospective Investigation into Cancer and Nutrition (EPIC study. Metabolic health/body size phenotypes were defined according to hyperinsulinaemia status using serum concentrations of C-peptide, a marker of insulin secretion. A total of 737 incident colorectal cancer cases and 737 matched controls were divided into tertiles based on the distribution of C-peptide concentration amongst the control population, and participants were classified as metabolically healthy if below the first tertile of C-peptide and metabolically unhealthy if above the first tertile. These metabolic health definitions were then combined with body mass index (BMI measurements to create four metabolic health/body size phenotype categories: (1 metabolically healthy/normal weight (BMI < 25 kg/m2, (2 metabolically healthy/overweight (BMI ≥ 25 kg/m2, (3 metabolically unhealthy/normal weight (BMI < 25 kg/m2, and (4 metabolically unhealthy/overweight (BMI ≥ 25 kg/m2. Additionally, in separate models, waist circumference measurements (using the International Diabetes Federation cut-points [≥80 cm for women and ≥94 cm for men] were used (instead of BMI to create the four metabolic health/body size phenotype categories. Statistical tests used in the analysis were all two-sided, and a p-value of <0.05 was

  19. Metabolomic and 13C-Metabolic Flux Analysis of a Xylose-Consuming Saccharomyces cerevisiae Strain Expressing Xylose Isomerase

    Science.gov (United States)

    Wasylenko, Thomas M.; Stephanopoulos, Gregory

    2016-01-01

    Over the past two decades significant progress has been made in the engineering of xylose-consuming Saccharomyces cerevisiae strains for production of lignocellulosic biofuels. However, the ethanol productivities achieved on xylose are still significantly lower than those observed on glucose for reasons that are not well understood. We have undertaken an analysis of central carbon metabolite pool sizes and metabolic fluxes on glucose and on xylose under aerobic and anaerobic conditions in a strain capable of rapid xylose assimilation via xylose isomerase in order to investigate factors that may limit the rate of xylose fermentation. We find that during xylose utilization the flux through the non-oxidative PPP is high but the flux through the oxidative PPP is low, highlighting an advantage of the strain employed in this study. Furthermore, xylose fails to elicit the full carbon catabolite repression response that is characteristic of glucose fermentation in S. cerevisiae. We present indirect evidence that the incomplete activation of the fermentation program on xylose results in a bottleneck in lower glycolysis, leading to inefficient re-oxidation of NADH produced in glycolysis. PMID:25311863

  20. Metabolomic and (13)C-metabolic flux analysis of a xylose-consuming Saccharomyces cerevisiae strain expressing xylose isomerase.

    Science.gov (United States)

    Wasylenko, Thomas M; Stephanopoulos, Gregory

    2015-03-01

    Over the past two decades, significant progress has been made in the engineering of xylose-consuming Saccharomyces cerevisiae strains for production of lignocellulosic biofuels. However, the ethanol productivities achieved on xylose are still significantly lower than those observed on glucose for reasons that are not well understood. We have undertaken an analysis of central carbon metabolite pool sizes and metabolic fluxes on glucose and on xylose under aerobic and anaerobic conditions in a strain capable of rapid xylose assimilation via xylose isomerase in order to investigate factors that may limit the rate of xylose fermentation. We find that during xylose utilization the flux through the non-oxidative Pentose Phosphate Pathway (PPP) is high but the flux through the oxidative PPP is low, highlighting an advantage of the strain employed in this study. Furthermore, xylose fails to elicit the full carbon catabolite repression response that is characteristic of glucose fermentation in S. cerevisiae. We present indirect evidence that the incomplete activation of the fermentation program on xylose results in a bottleneck in lower glycolysis, leading to inefficient re-oxidation of NADH produced in glycolysis. © 2014 Wiley Periodicals, Inc.

  1. SU-G-TeP3-10: Radiation Induces Prompt Live-Cell Metabolic Fluxes

    Energy Technology Data Exchange (ETDEWEB)

    Campos, D [University of Wisconsin Madison, Madison, WI (United States); Peeters, W; Bussink, J [Radboud University Medical Center, Nijmegen, GA (United States); Nickel, K [University of Wisconsin - Madison, Madison, Wisconsin (United States); Burkel, B; Kimple, R; Kogel, A van der; Eliceiri, K [University of Wisconsin - Madison, Madison, WI (United States); Kissick, M [University of Wisconsin, Madison, WI (United States)

    2016-06-15

    Purpose: To compare metabolic dynamics and HIF-1α expression following radiation between a cancerous cell line (UM-SCC-22B) and a normal, immortalized cell line, NOK (Normal Oral Keratinocyte). HIF-1 is a key factor in metabolism and radiosensitivity. A better understanding of how radiation affects the interplay of metabolism and HIF-1 might give a better understanding of the mechanisms responsible for radiosensitivity. Methods: Changes in cellular metabolism in response to radiation are tracked by fluorescence lifetime of NADH. Expression of HIF-1α was measured by immunofluorescence for both cell lines with and without irradiation. Radiation response is also monitored with additional treatment of a HIF-1α inhibitor (chrysin) as well as a radical scavenger (glutathione). Changes in oxygen consumption and respiratory capacity are also monitored using the Seahorse XF analyzer. Results: An increase in HIF-1α was found to be in response to radiation for the cancer cell line, but not the normal cell line. Radiation was found to shift metabolism toward glycolytic pathways in cancer cells as measured by oxygen consumption and respiratory capacity. Radiation response was found to be muted by addition of glutathione to cell media. HIF-1α inhibition similarly muted radiation response in cancer. Conclusion: The HIF-1 protein complex is a key regulator cellular metabolism through the regulation of glycolysis and glucose transport enzymes. Moreover, HIF-1 has shown radio-protective effects in tumor vascular endothelia, and has been implicated in metastatic aggression. Monitoring interplay between metabolism and the HIF-1 protein complex can give a more fundamental understanding of radiotherapy response.

  2. Diacylglycerol lipase a knockout mice demonstrate metabolic and behavioral phenotypes similar to those of cannabinoid receptor 1 knockout mice

    Directory of Open Access Journals (Sweden)

    David R Powell

    2015-06-01

    Full Text Available After creating >4650 knockouts (KOs of independent mouse genes, we screened them by high-throughput phenotyping and found that cannabinoid receptor 1 (Cnr1 KO mice had the same lean phenotype published by others. We asked if our KOs of DAG lipase a or b (Dagla or Daglb, which catalyze biosynthesis of the endocannabinoid (EC 2-Arachidonoylglycerol (2-AG, or Napepld, which catalyzes biosynthesis of the EC anandamide, shared the lean phenotype of Cnr1 KO mice. We found that Dagla KO mice, but not Daglb or Napepld KO mice, were among the leanest of 3651 chow-fed KO lines screened. In confirmatory studies, chow- or high fat diet-fed Dagla and Cnr1 KO mice were leaner than wild type (WT littermates; when data from multiple cohorts of adult mice were combined, body fat was 47% and 45% lower in Dagla and Cnr1 KO mice, respectively, relative to WT values. In contrast, neither Daglb nor Napepld KO mice were lean. Weanling Dagla KO mice ate less than WT mice and had body weight similar to pair-fed WT mice, and adult Dagla KO mice had normal activity and VO2 levels, similar to Cnr1 KO mice. Our Dagla and Cnr1 KO mice also had low fasting insulin, triglyceride and total cholesterol levels, and after a glucose challenge had normal glucose but very low insulin levels. Dagla and Cnr1 KO mice also showed similar responses to a battery of behavioral tests. These data suggest: 1 the lean phenotype of young Dagla and Cnr1 KO mice is mainly due to hypophagia; 2 in pathways where ECs signal through Cnr1 to regulate food intake and other metabolic and behavioral phenotypes observed in Cnr1 KO mice, Dagla alone provides the 2-AG that serves as the EC signal; and 3 small molecule Dagla inhibitors with a pharmacokinetic profile similar to that of Cnr1 inverse agonists are likely to mirror the ability of these Cnr1 inverse agonists to lower body weight and improve glycemic control in obese patients with type 2 diabetes, but may also induce undesirable neuropsychiatric

  3. Early Life Exposure to Fructose and Offspring Phenotype: Implications for Long Term Metabolic Homeostasis

    Directory of Open Access Journals (Sweden)

    Deborah M. Sloboda

    2014-01-01

    Full Text Available The consumption of artificially sweetened processed foods, particularly high in fructose or high fructose corn syrup, has increased significantly in the past few decades. As such, interest into the long term outcomes of consuming high levels of fructose has increased significantly, particularly when the exposure is early in life. Epidemiological and experimental evidence has linked fructose consumption to the metabolic syndrome and associated comorbidities—implicating fructose as a potential factor in the obesity epidemic. Yet, despite the widespread consumption of fructose-containing foods and beverages and the rising incidence of maternal obesity, little attention has been paid to the possible adverse effects of maternal fructose consumption on the developing fetus and long term effects on offspring. In this paper we review studies investigating the effects of fructose intake on metabolic outcomes in both mother and offspring using human and experimental studies.

  4. CeCaFDB: a curated database for the documentation, visualization and comparative analysis of central carbon metabolic flux distributions explored by 13C-fluxomics.

    Science.gov (United States)

    Zhang, Zhengdong; Shen, Tie; Rui, Bin; Zhou, Wenwei; Zhou, Xiangfei; Shang, Chuanyu; Xin, Chenwei; Liu, Xiaoguang; Li, Gang; Jiang, Jiansi; Li, Chao; Li, Ruiyuan; Han, Mengshu; You, Shanping; Yu, Guojun; Yi, Yin; Wen, Han; Liu, Zhijie; Xie, Xiaoyao

    2015-01-01

    The Central Carbon Metabolic Flux Database (CeCaFDB, available at http://www.cecafdb.org) is a manually curated, multipurpose and open-access database for the documentation, visualization and comparative analysis of the quantitative flux results of central carbon metabolism among microbes and animal cells. It encompasses records for more than 500 flux distributions among 36 organisms and includes information regarding the genotype, culture medium, growth conditions and other specific information gathered from hundreds of journal articles. In addition to its comprehensive literature-derived data, the CeCaFDB supports a common text search function among the data and interactive visualization of the curated flux distributions with compartmentation information based on the Cytoscape Web API, which facilitates data interpretation. The CeCaFDB offers four modules to calculate a similarity score or to perform an alignment between the flux distributions. One of the modules was built using an inter programming algorithm for flux distribution alignment that was specifically designed for this study. Based on these modules, the CeCaFDB also supports an extensive flux distribution comparison function among the curated data. The CeCaFDB is strenuously designed to address the broad demands of biochemists, metabolic engineers, systems biologists and members of the -omics community. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  5. Interactions between genotype and environment drive the metabolic phenotype within Escherichia coli isolates.

    Science.gov (United States)

    Sabarly, Victor; Aubron, Cécile; Glodt, Jérémy; Balliau, Thierry; Langella, Olivier; Chevret, Didier; Rigal, Odile; Bourgais, Aurélie; Picard, Bertrand; de Vienne, Dominique; Denamur, Erick; Bouvet, Odile; Dillmann, Christine

    2016-01-01

    To gain insights into the adaptation of the Escherichia coli species to different environments, we monitored protein abundances using quantitative proteomics and measurements of enzymatic activities of central metabolism in a set of five representative strains grown in four contrasted culture media including human urine. Two hundred and thirty seven proteins representative of the genome-scale metabolic network were identified and classified into pathway categories. We found that nutrient resources shape the general orientation of metabolism through coordinated changes in the average abundances of proteins and in enzymatic activities that all belong to the same pathway category. For example, each culture medium induces a specific oxidative response whatever the strain. On the contrary, differences between strains concern isolated proteins and enzymes within pathway categories in single environments. Our study confirms the predominance of genotype by environment interactions at the proteomic and enzyme activity levels. The buffering of genetic variation when considering life-history traits suggests a multiplicity of evolutionary strategies. For instance, the uropathogenic isolate CFT073 shows a deregulation of iron demand and increased oxidative stress response. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

  6. Consequences of gas flux model choice on the interpretation of metabolic balance across 15 lakes

    Science.gov (United States)

    Dugan, Hilary; Woolway, R. Iestyn; Santoso, Arianto; Corman, Jessica; Jaimes, Aline; Nodine, Emily; Patil, Vijay; Zwart, Jacob A.; Brentrup, Jennifer A.; Hetherington, Amy; Oliver, Samantha K.; Read, Jordan S.; Winters, Kirsten; Hanson, Paul; Read, Emily; Winslow, Luke; Weathers, Kathleen

    2016-01-01

    Ecosystem metabolism and the contribution of carbon dioxide from lakes to the atmosphere can be estimated from free-water gas measurements through the use of mass balance models, which rely on a gas transfer coefficient (k) to model gas exchange with the atmosphere. Theoretical and empirically based models of krange in complexity from wind-driven power functions to complex surface renewal models; however, model choice is rarely considered in most studies of lake metabolism. This study used high-frequency data from 15 lakes provided by the Global Lake Ecological Observatory Network (GLEON) to study how model choice of kinfluenced estimates of lake metabolism and gas exchange with the atmosphere. We tested 6 models of k on lakes chosen to span broad gradients in surface area and trophic states; a metabolism model was then fit to all 6 outputs of k data. We found that hourly values for k were substantially different between models and, at an annual scale, resulted in significantly different estimates of lake metabolism and gas exchange with the atmosphere.

  7. Radiopharmaceuticals for Assessment of Altered Metabolism and Biometal Fluxes in Brain Aging and Alzheimer's Disease with Positron Emission Tomography.

    Science.gov (United States)

    Xie, Fang; Peng, Fangyu

    2017-01-01

    Aging is a risk factor for Alzheimer's disease (AD). There are changes of brain metabolism and biometal fluxes due to brain aging, which may play a role in pathogenesis of AD. Positron emission tomography (PET) is a versatile tool for tracking alteration of metabolism and biometal fluxes due to brain aging and AD. Age-dependent changes in cerebral glucose metabolism can be tracked with PET using 2-deoxy-2-[18F]-fluoro-D-glucose (18F-FDG), a radiolabeled glucose analogue, as a radiotracer. Based on different patterns of altered cerebral glucose metabolism, 18F-FDG PET was clinically used for differential diagnosis of AD and Frontotemporal dementia (FTD). There are continued efforts to develop additional radiopharmaceuticals or radiotracers for assessment of age-dependent changes of various metabolic pathways and biometal fluxes due to brain aging and AD with PET. Elucidation of age-dependent changes of brain metabolism and altered biometal fluxes is not only significant for a better mechanistic understanding of brain aging and the pathophysiology of AD, but also significant for identification of new targets for the prevention, early diagnosis, and treatment of AD.

  8. Exhaustive Analysis of a Genotype Space Comprising 10(15 Central Carbon Metabolisms Reveals an Organization Conducive to Metabolic Innovation.

    Directory of Open Access Journals (Sweden)

    Sayed-Rzgar Hosseini

    2015-08-01

    Full Text Available All biological evolution takes place in a space of possible genotypes and their phenotypes. The structure of this space defines the evolutionary potential and limitations of an evolving system. Metabolism is one of the most ancient and fundamental evolving systems, sustaining life by extracting energy from extracellular nutrients. Here we study metabolism's potential for innovation by analyzing an exhaustive genotype-phenotype map for a space of 10(15 metabolisms that encodes all possible subsets of 51 reactions in central carbon metabolism. Using flux balance analysis, we predict the viability of these metabolisms on 10 different carbon sources which give rise to 1024 potential metabolic phenotypes. Although viable metabolisms with any one phenotype comprise a tiny fraction of genotype space, their absolute numbers exceed 10(9 for some phenotypes. Metabolisms with any one phenotype typically form a single network of genotypes that extends far or all the way through metabolic genotype space, where any two genotypes can be reached from each other through a series of single reaction changes. The minimal distance of genotype networks associated with different phenotypes is small, such that one can reach metabolisms with novel phenotypes--viable on new carbon sources--through one or few genotypic changes. Exceptions to these principles exist for those metabolisms whose complexity (number of reactions is close to the minimum needed for viability. Increasing metabolic complexity enhances the potential for both evolutionary conservation and evolutionary innovation.

  9. The key to acetate: metabolic fluxes of acetic acid bacteria under cocoa pulp fermentation-simulating conditions.

    Science.gov (United States)

    Adler, Philipp; Frey, Lasse Jannis; Berger, Antje; Bolten, Christoph Josef; Hansen, Carl Erik; Wittmann, Christoph

    2014-08-01

    Acetic acid bacteria (AAB) play an important role during cocoa fermentation, as their main product, acetate, is a major driver for the development of the desired cocoa flavors. Here, we investigated the specialized metabolism of these bacteria under cocoa pulp fermentation-simulating conditions. A carefully designed combination of parallel 13C isotope labeling experiments allowed the elucidation of intracellular fluxes in the complex environment of cocoa pulp, when lactate and ethanol were included as primary substrates among undefined ingredients. We demonstrate that AAB exhibit a functionally separated metabolism during coconsumption of two-carbon and three-carbon substrates. Acetate is almost exclusively derived from ethanol, while lactate serves for the formation of acetoin and biomass building blocks. Although this is suboptimal for cellular energetics, this allows maximized growth and conversion rates. The functional separation results from a lack of phosphoenolpyruvate carboxykinase and malic enzymes, typically present in bacteria to interconnect metabolism. In fact, gluconeogenesis is driven by pyruvate phosphate dikinase. Consequently, a balanced ratio of lactate and ethanol is important for the optimum performance of AAB. As lactate and ethanol are individually supplied by lactic acid bacteria and yeasts during the initial phase of cocoa fermentation, respectively, this underlines the importance of a well-balanced microbial consortium for a successful fermentation process. Indeed, AAB performed the best and produced the largest amounts of acetate in mixed culture experiments when lactic acid bacteria and yeasts were both present.

  10. Human taurine metabolism: fluxes and fractional extraction rates of the gut, liver, and kidneys

    NARCIS (Netherlands)

    van Stijn, Mireille F. M.; Vermeulen, Mechteld A. R.; Siroen, Michiel P. C.; Wong, Leanne N.; van den Tol, M. Petrousjka; Ligthart-Melis, Gerdien C.; Houdijk, Alexander P. J.; van Leeuwen, Paul A. M.

    2012-01-01

    Taurine is involved in numerous biological processes. However, taurine plasma level decreases in response to pathological conditions, suggesting an increased need. Knowledge on human taurine metabolism is scarce and only described by arterial-venous differences across a single organ. Here we present

  11. Enzyme allocation problems in kinetic metabolic networks: Optimal solutions are elementary flux modes

    Czech Academy of Sciences Publication Activity Database

    Müller, Stefan; Regensburger, G.; Steuer, Ralf

    2014-01-01

    Roč. 347, APR 2014 (2014), s. 182-190 ISSN 0022-5193 R&D Projects: GA MŠk(CZ) EE2.3.20.0256 Institutional support: RVO:67179843 Keywords : metabolic optimization * enzyme kinetics * oriented matroid * elementary vector * conformal sum Subject RIV: EI - Biotechnology ; Bionics Impact factor: 2.116, year: 2014

  12. Meta-analysis of melanin-concentrating hormone signaling-deficient mice on behavioral and metabolic phenotypes.

    Directory of Open Access Journals (Sweden)

    Kenkichi Takase

    Full Text Available The demand for meta-analyses in basic biomedical research has been increasing because the phenotyping of genetically modified mice does not always produce consistent results. Melanin-concentrating hormone (MCH has been reported to be involved in a variety of behaviors that include feeding, body-weight regulation, anxiety, sleep, and reward behavior. However, the reported behavioral and metabolic characteristics of MCH signaling-deficient mice, such as MCH-deficient mice and MCH receptor 1 (MCHR1-deficient mice, are not consistent with each other. In the present study, we performed a meta-analysis of the published data related to MCH-deficient and MCHR1-deficient mice to obtain robust conclusions about the role of MCH signaling. Overall, the meta-analysis revealed that the deletion of MCH signaling enhanced wakefulness, locomotor activity, aggression, and male sexual behavior and that MCH signaling deficiency suppressed non-REM sleep, anxiety, responses to novelty, startle responses, and conditioned place preferences. In contrast to the acute orexigenic effect of MCH, MCH signaling deficiency significantly increased food intake. Overall, the meta-analysis also revealed that the deletion of MCH signaling suppressed the body weight, fat mass, and plasma leptin, while MCH signaling deficiency increased the body temperature, oxygen consumption, heart rate, and mean arterial pressure. The lean phenotype of the MCH signaling-deficient mice was also confirmed in separate meta-analyses that were specific to sex and background strain (i.e., C57BL/6 and 129Sv. MCH signaling deficiency caused a weak anxiolytic effect as assessed with the elevated plus maze and the open field test but also caused a weak anxiogenic effect as assessed with the emergence test. MCH signaling-deficient mice also exhibited increased plasma corticosterone under non-stressed conditions, which suggests enhanced activity of the hypothalamic-pituitary-adrenal axis. To the best of our

  13. {sup 13}C dynamic nuclear polarization for measuring metabolic flux in endothelial progenitor cells

    Energy Technology Data Exchange (ETDEWEB)

    Nielsen, Nathalie; Laustsen, Christoffer; Bertelsen, Lotte Bonde, E-mail: Lotte@clin.au.dk

    2016-11-15

    Endothelial progenitor cells (EPCs) represent a heterogeneous cell population that is believed to be involved in vasculogenesis. With the purpose of enhancing endothelial repair, EPCs could have a potential for future cell therapies. Due to the low amount of EPCs in the peripheral circulating blood, in vitro expansion is needed before administration to recipients and the effects of in vitro culturing is still an under-evaluated field with little knowledge of how the cells change over time in culture. The aim of this study was to use hyperpolarised carbon-13 magnetic resonance spectroscopy to profile important metabolic pathways in a population of progenitor cells and to show that cell culturing in 3D scaffolds seem to block the metabolic processes that leads to cell senescence. The metabolic breakdown of hyperpolarized [1-{sup 13}C]pyruvate was followed after injection of the substrate to a bioreactor system with EPCs either adhered to 3D printed scaffolds or kept in cell suspension. The pyruvate-to-lactate conversion was elevated in suspension of EPCs compared to the EPCs adhered to scaffolds. Furthermore in the setup with EPCs in suspension, an increase in lactate production was seen over time indicating that the older the cultures of EPCs was before using the cells for cell suspension experiments, the more lactate they produce, compared to a constant lactate level in the cells adhered to scaffolds. It could therefore be stated that cells grown first in 2D culture and subsequent prepared for cell suspension show a metabolism with higher lactate production consistent with cells senescence processes compared to cells grown first at 2D culture and subsequent in the 3D printed scaffolds, where metabolism shows no sign of metabolic shifting during the monitored period. - Highlights: • Hyperpolarized 13C MRS detects EPCs metabolic changes associated with ageing and cultivating conditions. • Increased lactate production in EPC’s correlates positively with aging.

  14. Energy expenditure in the etiology of human obesity: spendthrift and thrifty metabolic phenotypes and energy-sensing mechanisms.

    Science.gov (United States)

    Piaggi, P; Vinales, K L; Basolo, A; Santini, F; Krakoff, J

    2018-01-01

    The pathogenesis of human obesity is the result of dysregulation of the reciprocal relationship between food intake and energy expenditure (EE), which influences daily energy balance and ultimately leads to weight gain. According to principles of energy homeostasis, a relatively lower EE in a setting of energy balance may lead to weight gain; however, results from different study groups are contradictory and indicate a complex interaction between EE and food intake which may differentially influence weight change in humans. Recently, studies evaluating the adaptive response of one component to perturbations of the other component of energy balance have revealed both the existence of differing metabolic phenotypes ("spendthrift" and "thrifty") resulting from overeating or underfeeding, as well as energy-sensing mechanisms linking EE to food intake, which might explain the propensity of an individual to weight gain. The purpose of this review is to debate the role that human EE plays on body weight regulation and to discuss the physiologic mechanisms linking EE and food intake. An increased understanding of the complex interplay between human metabolism and food consumption may provide insight into pathophysiologic mechanisms underlying weight gain, which may eventually lead to prevention and better treatment of human obesity.

  15. MicrO: an ontology of phenotypic and metabolic characters, assays, and culture media found in prokaryotic taxonomic descriptions.

    Science.gov (United States)

    Blank, Carrine E; Cui, Hong; Moore, Lisa R; Walls, Ramona L

    2016-01-01

    MicrO is an ontology of microbiological terms, including prokaryotic qualities and processes, material entities (such as cell components), chemical entities (such as microbiological culture media and medium ingredients), and assays. The ontology was built to support the ongoing development of a natural language processing algorithm, MicroPIE (or, Microbial Phenomics Information Extractor). During the MicroPIE design process, we realized there was a need for a prokaryotic ontology which would capture the evolutionary diversity of phenotypes and metabolic processes across the tree of life, capture the diversity of synonyms and information contained in the taxonomic literature, and relate microbiological entities and processes to terms in a large number of other ontologies, most particularly the Gene Ontology (GO), the Phenotypic Quality Ontology (PATO), and the Chemical Entities of Biological Interest (ChEBI). We thus constructed MicrO to be rich in logical axioms and synonyms gathered from the taxonomic literature. MicrO currently has ~14550 classes (~2550 of which are new, the remainder being microbiologically-relevant classes imported from other ontologies), connected by ~24,130 logical axioms (5,446 of which are new), and is available at (http://purl.obolibrary.org/obo/MicrO.owl) and on the project website at https://github.com/carrineblank/MicrO. MicrO has been integrated into the OBO Foundry Library (http://www.obofoundry.org/ontology/micro.html), so that other ontologies can borrow and re-use classes. Term requests and user feedback can be made using MicrO's Issue Tracker in GitHub. We designed MicrO such that it can support the ongoing and future development of algorithms that can leverage the controlled vocabulary and logical inference power provided by the ontology. By connecting microbial classes with large numbers of chemical entities, material entities, biological processes, molecular functions, and qualities using a dense array of logical axioms, we

  16. Prenatal Metformin Exposure in Mice Programs the Metabolic Phenotype of the Offspring during a High Fat Diet at Adulthood

    Science.gov (United States)

    Salomäki, Henriikka; Vähätalo, Laura H.; Laurila, Kirsti; Jäppinen, Norma T.; Penttinen, Anna-Maija; Ailanen, Liisa; Ilyasizadeh, Juan; Pesonen, Ullamari; Koulu, Markku

    2013-01-01

    Aims The antidiabetic drug metformin is currently used prior and during pregnancy for polycystic ovary syndrome, as well as during gestational diabetes mellitus. We investigated the effects of prenatal metformin exposure on the metabolic phenotype of the offspring during adulthood in mice. Methods Metformin (300 mg/kg) or vehicle was administered orally to dams on regular diet from the embryonic day E0.5 to E17.5. Gene expression profiles in liver and brain were analysed from 4-day old offspring by microarray. Body weight development and several metabolic parameters of offspring were monitored both during regular diet (RD-phase) and high fat diet (HFD-phase). At the end of the study, two doses of metformin or vehicle were given acutely to mice at the age of 20 weeks, and Insig-1 and GLUT4 mRNA expressions in liver and fat tissue were analysed using qRT-PCR. Results Metformin exposed fetuses were lighter at E18.5. There was no effect of metformin on the maternal body weight development or food intake. Metformin exposed offspring gained more body weight and mesenteric fat during the HFD-phase. The male offspring also had impaired glucose tolerance and elevated fasting glucose during the HFD-phase. Moreover, the expression of GLUT4 mRNA was down-regulated in epididymal fat in male offspring prenatally exposed to metformin. Based on the microarray and subsequent qRT-PCR analyses, the expression of Insig-1 was changed in the liver of neonatal mice exposed to metformin prenatally. Furthermore, metformin up-regulated the expression of Insig-1 later in development. Gene set enrichment analysis based on preliminary microarray data identified several differentially enriched pathways both in control and metformin exposed mice. Conclusions The present study shows that prenatal metformin exposure causes long-term programming effects on the metabolic phenotype during high fat diet in mice. This should be taken into consideration when using metformin as a therapeutic agent during

  17. Prenatal metformin exposure in mice programs the metabolic phenotype of the offspring during a high fat diet at adulthood.

    Directory of Open Access Journals (Sweden)

    Henriikka Salomäki

    Full Text Available AIMS: The antidiabetic drug metformin is currently used prior and during pregnancy for polycystic ovary syndrome, as well as during gestational diabetes mellitus. We investigated the effects of prenatal metformin exposure on the metabolic phenotype of the offspring during adulthood in mice. METHODS: Metformin (300 mg/kg or vehicle was administered orally to dams on regular diet from the embryonic day E0.5 to E17.5. Gene expression profiles in liver and brain were analysed from 4-day old offspring by microarray. Body weight development and several metabolic parameters of offspring were monitored both during regular diet (RD-phase and high fat diet (HFD-phase. At the end of the study, two doses of metformin or vehicle were given acutely to mice at the age of 20 weeks, and Insig-1 and GLUT4 mRNA expressions in liver and fat tissue were analysed using qRT-PCR. RESULTS: Metformin exposed fetuses were lighter at E18.5. There was no effect of metformin on the maternal body weight development or food intake. Metformin exposed offspring gained more body weight and mesenteric fat during the HFD-phase. The male offspring also had impaired glucose tolerance and elevated fasting glucose during the HFD-phase. Moreover, the expression of GLUT4 mRNA was down-regulated in epididymal fat in male offspring prenatally exposed to metformin. Based on the microarray and subsequent qRT-PCR analyses, the expression of Insig-1 was changed in the liver of neonatal mice exposed to metformin prenatally. Furthermore, metformin up-regulated the expression of Insig-1 later in development. Gene set enrichment analysis based on preliminary microarray data identified several differentially enriched pathways both in control and metformin exposed mice. CONCLUSIONS: The present study shows that prenatal metformin exposure causes long-term programming effects on the metabolic phenotype during high fat diet in mice. This should be taken into consideration when using metformin as a

  18. Assessment of energetic costs of AhR activation by β-naphthoflavone in rainbow trout (Oncorhynchus mykiss) hepatocytes using metabolic flux analysis

    Energy Technology Data Exchange (ETDEWEB)

    Nault, Rance, E-mail: naultran@msu.edu [Ottawa-Carleton Institute of Biology, Department of Biology and Centre for Advanced Research in Environmental Genomics, University of Ottawa, Ottawa, Ontario, K1N 6N5 (Canada); Abdul-Fattah, Hiba [Ottawa-Carleton Institute of Biology, Department of Biology and Centre for Advanced Research in Environmental Genomics, University of Ottawa, Ottawa, Ontario, K1N 6N5 (Canada); Mironov, Gleb G.; Berezovski, Maxim V. [Ottawa-Carleton Institute of Biology, Department of Biology and Centre for Advanced Research in Environmental Genomics, University of Ottawa, Ottawa, Ontario, K1N 6N5 (Canada); Department of Chemistry, University of Ottawa, Ottawa, Ontario, K1N 6N5 (Canada); Moon, Thomas W. [Ottawa-Carleton Institute of Biology, Department of Biology and Centre for Advanced Research in Environmental Genomics, University of Ottawa, Ottawa, Ontario, K1N 6N5 (Canada)

    2013-08-15

    Exposure to environmental contaminants such as activators of the aryl hydrocarbon receptor (AhR) leads to the induction of defense and detoxification mechanisms. While these mechanisms allow organisms to metabolize and excrete at least some of these environmental contaminants, it has been proposed that these mechanisms lead to significant energetic challenges. This study tests the hypothesis that activation of the AhR by the model agonist β-naphthoflavone (βNF) results in increased energetic costs in rainbow trout (Oncorhynchus mykiss) hepatocytes. To address this hypothesis, we employed traditional biochemical approaches to examine energy allocation and metabolism including the adenylate energy charge (AEC), protein synthesis rates, Na{sup +}/K{sup +}-ATPase activity, and enzyme activities. Moreover, we have used for the first time in a fish cell preparation, metabolic flux analysis (MFA) an in silico approach for the estimation of intracellular metabolic fluxes. Exposure of trout hepatocytes to 1 μM βNF for 48 h did not alter hepatocyte AEC, protein synthesis, or Na{sup +}/K{sup +}-ATPase activity but did lead to sparing of glycogen reserves and changes in activities of alanine aminotransferase and citrate synthase suggesting altered metabolism. Conversely, MFA did not identify altered metabolic fluxes, although we do show that the dynamic metabolism of isolated trout hepatocytes poses a significant challenge for this type of approach which should be considered in future studies. - Highlights: • Energetic costs of AhR activation by βNF was examined in rainbow trout hepatocytes. • Metabolic flux analysis was performed on a fish cell preparation for the first time. • Exposure to βNF led to sparing of glycogen reserves and altered enzyme activities. • Adenylate energy charge was maintained despite temporal changes in metabolism.

  19. Assessment of energetic costs of AhR activation by β-naphthoflavone in rainbow trout (Oncorhynchus mykiss) hepatocytes using metabolic flux analysis

    International Nuclear Information System (INIS)

    Nault, Rance; Abdul-Fattah, Hiba; Mironov, Gleb G.; Berezovski, Maxim V.; Moon, Thomas W.

    2013-01-01

    Exposure to environmental contaminants such as activators of the aryl hydrocarbon receptor (AhR) leads to the induction of defense and detoxification mechanisms. While these mechanisms allow organisms to metabolize and excrete at least some of these environmental contaminants, it has been proposed that these mechanisms lead to significant energetic challenges. This study tests the hypothesis that activation of the AhR by the model agonist β-naphthoflavone (βNF) results in increased energetic costs in rainbow trout (Oncorhynchus mykiss) hepatocytes. To address this hypothesis, we employed traditional biochemical approaches to examine energy allocation and metabolism including the adenylate energy charge (AEC), protein synthesis rates, Na + /K + -ATPase activity, and enzyme activities. Moreover, we have used for the first time in a fish cell preparation, metabolic flux analysis (MFA) an in silico approach for the estimation of intracellular metabolic fluxes. Exposure of trout hepatocytes to 1 μM βNF for 48 h did not alter hepatocyte AEC, protein synthesis, or Na + /K + -ATPase activity but did lead to sparing of glycogen reserves and changes in activities of alanine aminotransferase and citrate synthase suggesting altered metabolism. Conversely, MFA did not identify altered metabolic fluxes, although we do show that the dynamic metabolism of isolated trout hepatocytes poses a significant challenge for this type of approach which should be considered in future studies. - Highlights: • Energetic costs of AhR activation by βNF was examined in rainbow trout hepatocytes. • Metabolic flux analysis was performed on a fish cell preparation for the first time. • Exposure to βNF led to sparing of glycogen reserves and altered enzyme activities. • Adenylate energy charge was maintained despite temporal changes in metabolism

  20. Applications of computational modeling in metabolic engineering of yeast

    DEFF Research Database (Denmark)

    Kerkhoven, Eduard J.; Lahtvee, Petri-Jaan; Nielsen, Jens

    2015-01-01

    Generally, a microorganism's phenotype can be described by its pattern of metabolic fluxes. Although fluxes cannot be measured directly, inference of fluxes is well established. In biotechnology the aim is often to increase the capacity of specific fluxes. For this, metabolic engineering methods...... a preferred flux distribution. These methods point to strategies for altering gene expression; however, fluxes are often controlled by post-transcriptional events. Moreover, GEMs are usually not taking into account metabolic regulation, thermodynamics and enzyme kinetics. To facilitate metabolic engineering......, tools from synthetic biology have emerged, enabling integration and assembly of naturally nonexistent, but well-characterized components into a living organism. To describe these systems kinetic models are often used and to integrate these systems with the standard metabolic engineering approach...

  1. A nuclear-directed human pancreatic ribonuclease (PE5) targets the metabolic phenotype of cancer cells.

    Science.gov (United States)

    Vert, Anna; Castro, Jessica; Ribó, Marc; Benito, Antoni; Vilanova, Maria

    2016-04-05

    Ribonucleases represent a new class of antitumor RNA-damaging drugs. However, many wild-type members of the vertebrate secreted ribonuclease family are not cytotoxic because they are not able to evade the cytosolic ribonuclease inhibitor. We previously engineered the human pancreatic ribonuclease to direct it to the cell nucleus where the inhibitor is not present. The best characterized variant is PE5 that kills cancer cells through apoptosis mediated by the p21(WAF1/CIP1) induction and the inactivation of JNK. Here, we have used microarray-derived transcriptional profiling to identify PE5 regulated genes on the NCI/ADR-RES ovarian cancer cell line. RT-qPCR analyses have confirmed the expression microarray findings. The results show that PE5 cause pleiotropic effects. Among them, it is remarkable the down-regulation of multiple genes that code for enzymes involved in deregulated metabolic pathways in cancer cells.

  2. d3-Growth hormone receptor polymorphism in acromegaly: effects on metabolic phenotype.

    Science.gov (United States)

    Montefusco, Laura; Filopanti, Marcello; Ronchi, Cristina L; Olgiati, Luca; La-Porta, Carmen; Losa, Marco; Epaminonda, Paolo; Coletti, Francesca; Beck-Peccoz, Paolo; Spada, Anna; Lania, Andrea G; Arosio, Maura

    2010-05-01

    A common polymorphic variant of the growth hormone receptor (GHR) is because of genomic deletion of exon 3 and has been linked with increased responsiveness to exogenous GH. The impact of this polymorphism in acromegaly, a disease characterized by endogenous excess of GH and partial loss of IGF-I feedback on tumoural GH secretion, is not clear. The aim of this study was to investigate possible influences of d3GHR on the GH/IGF-I relationship and metabolic parameters in acromegaly. Retrospective study on 76 acromegalic patients. Genotype analysis was carried out on leucocyte DNA by multiplex PCR assay. Clinical, hormonal and biochemical parameters at diagnosis were collected from patients' medical records. Forty-two patients (55.3%) were homozygotes for the allele encoding the full-length GHR (fl/flGHR), 27 patients were heterozygotes (fl/d3) and seven homozygotes (d3/d3) for the genomic deletion of exon 3. Heterozygotes and homozygotes for the d3 allele were considered together (d3GHR) and compared with fl/flGHR patients. d3GHR and fl/flGHR patients showed no difference in GH and IGF-I levels or in the relationship between these two parameters. Patients bearing d3GHR had a lower body mass index (BMI) than patients bearing fl/flGHR (25.8 +/- 2.1 vs. 28.1 +/- 4.8 kg/m(2), P d3GHR patients had a normal glucose tolerance (66.7%vs. 56.3%, P d3GHR allele, and not BMI or age, was a significant negative predictor of insulin levels 120 min after oral glucose load (beta = -80.8, P d3GHR is functionally different from the fl/fl variant mostly for the effects on body weight regulation and on glucose metabolism.

  3. Simple and robust determination of the activity signature of key carbohydrate metabolism enzymes for physiological phenotyping in model and crop plants

    Czech Academy of Sciences Publication Activity Database

    Jammer, A.; Gapserl, A.; Luschin-Ebengreuth, N.; Heyneke, E.; Chu, H.; Cantero-Navarro, E.; Grosskinsky, D. K.; Albacete, A.; Stabentheiner, E.; Franzaring, J.; Fangmeier, A.; van der Graaff, E.; Roitsch, Thomas

    2015-01-01

    Roč. 66, č. 18 (2015), s. 5531-5542 ISSN 0022-0957 Institutional support: RVO:67179843 Keywords : Carbohydrate metabolism * dialysis * enzyme activities * kinetic assay * physiological phenotyping * physiological state * protein extraction * signatures Subject RIV: EH - Ecology, Behaviour Impact factor: 5.677, year: 2015

  4. Metabolic Flux Distribution during Defatting of Steatotic Human Hepatoma (HepG2 Cells

    Directory of Open Access Journals (Sweden)

    Gabriel Yarmush

    2016-01-01

    Full Text Available Methods that rapidly decrease fat in steatotic hepatocytes may be helpful to recover severely fatty livers for transplantation. Defatting kinetics are highly dependent upon the extracellular medium composition; however, the pathways involved are poorly understood. Steatosis was induced in human hepatoma cells (HepG2 by exposure to high levels of free fatty acids, followed by defatting using plain medium containing no fatty acids, or medium supplemented with a cocktail of defatting agents previously described before. We measured the levels of 28 extracellular metabolites and intracellular triglyceride, and fed the data into a steady-state mass balance model to estimate strictly intracellular fluxes. We found that during defatting, triglyceride content decreased, while beta-oxidation, the tricarboxylic acid cycle, and the urea cycle increased. These fluxes were augmented by defatting agents, and even more so by hyperoxic conditions. In all defatting conditions, the rate of extracellular glucose uptake/release was very small compared to the internal supply from glycogenolysis, and glycolysis remained highly active. Thus, in steatotic HepG2 cells, glycolysis and fatty acid oxidation may co-exist. Together, these pathways generate reducing equivalents that are supplied to mitochondrial oxidative phosphorylation.

  5. The link between phenotype and fatty acid metabolism in advanced chronic kidney disease.

    Science.gov (United States)

    Chen, Dan-Qian; Chen, Hua; Chen, Lin; Vaziri, Nosratola D; Wang, Ming; Li, Xiang-Ri; Zhao, Ying-Yong

    2017-07-01

    The kidney plays a central role in elimination of metabolic waste products and regulation of low-molecular weight metabolites via glomerular filtration, tubular secretion and reabsorption. Disruption of these processes results in profound changes in the biochemical milieu of the body fluids, which contribute to complications of chronic kidney disease (CKD) by inducing cytotoxicity and inflammation. Insight into the changes of the composition of metabolites and dysregulation of target genes and proteins enhances the understanding of the pathophysiology of CKD and its complications, and the development of novel therapeutic strategies. Chronic interstitial nephropathy is a common cause of CKD. The present study was designed to determine the effect of chronic interstitial nephropathy on the composition of serum metabolites and regulation of oxidative, inflammatory, fibrotic and cytoprotective pathways. Male Sprague-Dawley rats were randomized to the CKD and control groups ( n  = 8/group). CKD was induced by administration of adenine (200 mg/kg body weight/day) by oral gavage for 3 weeks. The control group was treated with the vehicle alone. The animals were then observed for an additional 3 weeks, at which point they were sacrificed and kidney and serum samples were collected. Serum metabolomic and lipidomic analyses were performed using ultra-performance liquid chromatography-quadrupole time-of-flight high-definition mass spectrometry. Kidney tissues were processed for histological and molecular biochemical analyses. CKD rats exhibited increased plasma urea and creatinine concentrations, renal interstitial fibrosis, tubular damage and up-regulation of pro-inflammatory, pro-oxidant and pro-fibrotic pathways. Comparison of serum from CKD and control rats revealed significant differences in concentrations of amino acids and lipids including 33 metabolites and 35 lipid species. This was associated with marked abnormalities of fatty acid oxidation, and

  6. Fermentation Process and Metabolic Flux of Ethanol Production from the Detoxified Hydrolyzate of Cassava Residue

    Directory of Open Access Journals (Sweden)

    Xingjiang Li

    2017-08-01

    Full Text Available With the growth of the world population, energy problems are becoming increasingly severe; therefore, sustainable energy sources have gained enormous importance. With respect to ethanol fuel production, biomass is gradually replacing grain as the main raw material. In this study, we explored the fermentation of five- and six-carbon sugars, the main biomass degradation products, into alcohol. We conducted mutagenic screening specifically for Candida tropicalis CICC1779 to obtain a strain that effectively used xylose (Candida tropicalis CICC1779-Dyd. By subsequently studying fermentation conditions under different initial liquid volume oxygen transfer coefficients (kLα, and coupling control of the aeration rate and agitation speed under optimal conditions, the optimal dissolved oxygen change curve was obtained. In addition, we constructed metabolic flow charts and equations to obtain a better understanding of the fermentation mechanism and to improve the ethanol yield. In our experiment, the ethanol production of the wild type stain was 17.58 g·L−1 at a kLα of 120. The highest ethanol yield of the mutagenic strains was 24.85 g·L−1. The ethanol yield increased to 26.56 g·L−1 when the dissolved oxygen content was optimized, and the conversion of sugar into alcohol reached 0.447 g·g−1 glucose (the theoretical titer of yeast-metabolized xylose was 0.46 g ethanol/g xylose and the glucose ethanol fermentation titer was 0.51 g ethanol/g glucose. Finally, the detected activity of xylose reductase and xylose dehydrogenase was higher in the mutant strain than in the original, which indicated that the mutant strain (CICC1779-Dyd could effectively utilize xylose for metabolism.

  7. Metabolic profiling uncovers a phenotypic signature of small for gestational age in early pregnancy.

    LENUS (Irish Health Repository)

    Horgan, Richard P

    2012-01-31

    Being born small for gestational age (SGA) confers increased risks of perinatal morbidity and mortality and increases the risk of cardiovascular complications and diabetes in later life. Accumulating evidence suggests that the etiology of SGA is usually associated with poor placental vascular development in early pregnancy. We examined metabolomic profiles using ultra performance liquid chromatography-mass spectrometry (UPLC-MS) in three independent studies: (a) venous cord plasma from normal and SGA babies, (b) plasma from a rat model of placental insufficiency and controls, and (c) early pregnancy peripheral plasma samples from women who subsequently delivered a SGA baby and controls. Multivariate analysis by cross-validated Partial Least Squares Discriminant Analysis (PLS-DA) of all 3 studies showed a comprehensive and similar disruption of plasma metabolism. A multivariate predictive model combining 19 metabolites produced by a Genetic Algorithm-based search program gave an Odds Ratio for developing SGA of 44, with an area under the Receiver Operator Characteristic curve of 0.9. Sphingolipids, phospholipids, carnitines, and fatty acids were among this panel of metabolites. The finding of a consistent discriminatory metabolite signature in early pregnancy plasma preceding the onset of SGA offers insight into disease pathogenesis and offers the promise of a robust presymptomatic screening test.

  8. Ruxolitinib combined with vorinostat suppresses tumor growth and alters metabolic phenotype in hematological diseases.

    Science.gov (United States)

    Civallero, Monica; Cosenza, Maria; Pozzi, Samantha; Sacchi, Stefano

    2017-11-28

    JAK-2 dysregulation plays an important role as an oncogenic driver, and is thus a promising therapeutic target in hematological malignancies. Ruxolitinib is a pyrrolo[2.3-d]pyrimidine derivative with inhibitory activity against JAK1 and JAK2, moderate activity against TYK2, and minor activity against JAK3. Vorinostat is an HDAC inhibitor that reduces JAK-2 expression, thus affecting JAK-2 mRNA expression and increasing JAK-2 proteasomal deterioration. Here we hypothesized that the combination of ruxolitinib and vorinostat could have synergistic effects against hematological disease. We tested combinations of low doses of ruxolitinib and vorinostat in 12 cell lines, and observed highly synergistic cytotoxic action in six cell lines, which was maintained for up to 120 h in the presence of stromal cells. The sensitivity of the six cell lines may be explained by the broad effects of the drug combination, which can affect various targets. Treatment with the combination of ruxolitinib and vorinostat appeared to induce a possible reversal of the Warburg effect, with associated ROS production, apoptotic events, and growth inhibition. Decreased glucose metabolism may have markedly sensitized the six more susceptible cell lines to combined treatment. Therapeutic inhibition of the JAK/STAT pathway seems to offer substantial anti-tumor benefit, and combined therapy with ruxolitinib and vorinostat may represent a promising novel therapeutic modality for hematological neoplasms.

  9. Development of a screening approach for exploring cell factory potential through metabolic flux analysis and physiology

    DEFF Research Database (Denmark)

    Knudsen, Peter Boldsen; Nielsen, Kristian Fog; Thykær, Jette

    2012-01-01

    of the recombinant strains in order to evaluate their potential as fungal cell factories and for guiding further metabolic engineering strategies. To meet the demand for a fast and reliable method for physiological characterisation of fungal strains, a screening approach using a micro titer format was developed...... of the validation, already well described fungal strains were selected and tested using the described method and the developed method was subsequently used to test recombinant fungal strains producing the model polyketide 6-methylsalicylic acid. Diligent application of this strategy significantly reduces the cost...

  10. [Anthropometric indices to identify metabolic syndrome and hypertriglyceridemic waist phenotype: a comparison between the three stages of adolescence].

    Science.gov (United States)

    Pereira, Patrícia Feliciano; Faria, Franciane Rocha de; Faria, Eliane Rodrigues de; Hermsdorff, Helen Hermana Miranda; Peluzio, Maria do Carmo Gouveia; Franceschini, Sylvia do Carmo Castro; Priore, Silvia Eloiza

    2015-01-01

    To determine the prevalence of metabolic syndrome (MS) and the hypertriglyceridemic waist phenotype (HW) in a representative adolescent sample; as well as to establish which anthropometric indicator better identifies MS and HW, according to gender and adolescent age. This cross sectional study had the participation of 800 adolescents (414 girls) from 10-19 years old. Anthropometric indicators (body mass index, waist perimeter, waist/stature ratio, waist/hip ratio, and central/peripheral skinfolds) were determined by standard protocols. For diagnosis of MS, the criteria proposed by de Ferranti et al. (2004) were used. HW was defined by the simultaneous presence of increased waist perimeter (>75th percentile for age and sex) and high triglycerides (>100mg/dL). The ability of anthropometric indicators was evaluated by Receiver Operating Characteristic curve. The prevalence of MS was identical to HW (6.4%), without differences between genders and the adolescence phases. The waist perimeter showed higher area under the curve for the diagnosis of MS, except for boys with 17-19 years old, for whom the waist/stature ratio exhibited better performance. For diagnosing HW, waist perimeter also showed higher area under the curve, except for boys in initial and final phases, in which the waist/stature ratio obtained larger area under the curve. The central/peripheral skinfolds had the lowest area under the curve for the presence of both MS and HW phenotype. The waist perimeter and the waist/stature showed a better performance to identify MS and HW in both genders and in all three phases of adolescence. Copyright © 2015 Associação de Pediatria de São Paulo. Publicado por Elsevier Editora Ltda. All rights reserved.

  11. Prevalence of Desloratadine Slow-metabolizer Phenotype and Food-dependent Pharmacokinetics of Desloratadine in Healthy Chinese Volunteers.

    Science.gov (United States)

    Wang, Ting; Zhang, Kun; Li, Tingting; He, Lin; Xie, Huiru; Jiang, Xuehua; Wang, Ling

    2015-12-01

    Desloratadine, the major active metabolite of loratadine, is a non-sedating long-acting antihistamine that is widely used in the treatment of allergic rhinitis and chronic idiopathic urticaria. This study aimed to investigate the prevalence of desloratadine slow-metabolizer (DSM) phenotype and the effects of food on the pharmacokinetics of desloratadine and its active metabolite 3-OH-desloratadine in healthy Chinese volunteers. A total of 46 healthy Chinese male volunteers were included in this investigation. All subjects received a single dose of a 5-mg desloratadine tablet under fasting or fed conditions and the plasma concentrations of desloratadine and 3-OH-desloratadine were measured by liquid chromatography-tandem mass spectrometry. The pharmacokinetic profiles were analyzed using a non-compartmental method in the Phoenix WinNonlin program. The individuals with a 3-OH-desloratadine-to-desloratadine exposure ratio lower than 10 % or a desloratadine half-life (t 1/2) of ≥50 h were supposed to be DSM. There was only one DSM among the 46 volunteers, with a prevalence of 2.2 %. Moreover, administration in a fed state resulted in 34.07 and 32.06 % decreases in maximum plasma concentration and area under the concentration-time curve from time zero to infinity for desloratadine and 47.26 and 48.46 % for 3-OH-desloratadine compared with those values under fasting conditions. Taken together, these results indicated that the incidence of the DSM phenotype in the Chinese population was low and that food intake could significantly decrease the absorption rate and extent of desloratadine.

  12. Oxygen-enriched fermentation of sodium gluconate by Aspergillus niger and its impact on intracellular metabolic flux distributions.

    Science.gov (United States)

    Shen, Yuting; Tian, Xiwei; Zhao, Wei; Hang, Haifeng; Chu, Ju

    2018-01-01

    Different concentrations of oxygen-enriched air were utilized for sodium gluconate (SG) fermentation by Aspergillus niger. The fermentation time shortened from 20 to 15.5 h due to the increase of volumetric oxygen transfer coefficient (K L a) and the formation of more dispersed mycelia when inlet oxygen concentration ascended from 21 to 32%. According to metabolic flux analysis, during the growth phase, extracellular glucose for SG synthesis accounted for 79.0 and 85.3% with air and oxygen-enriched air (25%), respectively, whereas the proportions were 89.4 and 93.0% in the stationary phase. Intracellular glucose consumption decreased in oxygen-enriched fermentation, as cell respiration was more high-efficiently performed. Metabolic profiling indicated that most intermediates in TCA cycle and EMP pathway had smaller pool sizes in oxygen-enriched fermentations. Moreover, the main by-product of citric acid dramatically decreased from 1.36 to 0.34 g L -1 in oxygen-enriched fermentation. And the sodium gluconate yield increased from 0.856 to 0.903 mol mol -1 .

  13. Ketosis-prone atypical diabetes in Cameroonian people with hyperglycaemic crisis: frequency, clinical and metabolic phenotypes.

    Science.gov (United States)

    Lontchi-Yimagou, E; Nguewa, J L; Assah, F; Noubiap, J J; Boudou, P; Djahmeni, E; Balti, E V; Atogho-Tiedeu, B; Gautier, J F; Mbanya, J C; Sobngwi, E

    2017-03-01

    It is unclear whether ketosis-prone diabetes is a specific type or a subtype of Type 2 diabetes. We aimed to describe the clinical and metabolic features of ketosis-prone diabetes in a sub-Saharan population. We consecutively enrolled and characterized 173 people with non-autoimmune diabetes admitted for hyperglycaemic crisis at the Yaoundé Central Hospital, Cameroon. Blood samples were collected for fasting glucose, HbA 1c , lipid profile and C-peptide assays with insulin resistance and secretion estimation by homeostasis model assessment. People were classified as having Type 2 diabetes (n = 124) or ketosis-prone diabetes (n = 49). Ketosis-prone diabetes was sub-classified as new-onset ketotic phase (n = 34) or non-ketotic phase (n = 15). Ketosis-prone diabetes was found in 28.3% of the hyperglycaemic crises. Age at diabetes diagnosis was comparable in Type 2 and ketosis-prone diabetes [48 ± 14 vs 47 ± 11 years; P = 0.13] with a similar sex distribution. Overall BMI was 27.7 ± 13.4 kg/m 2 and was ≥ 25 kg/m 2 in 55.8% of those taking part, however, 73.5% of those with ketosis-prone diabetes reported weight loss of > 5% at diagnosis. Blood pressure and lipid profile were comparable in both types. Ketosis-prone diabetes in the ketotic phase was characterized by lower insulin secretion and higher serum triglycerides compared with non-ketotic ketosis prone and Type 2 diabetes. Type 2 and ketosis prone diabetes in the non-ketotic phase were comparable in terms of lipid profile, blood pressure, waist-to-hip ratio, BMI and fat mass, insulin secretion and insulin resistance indices. Ketosis-prone diabetes is likely to be a subtype of Type 2 diabetes with the potential to develop acute insulinopenic episodes. © 2016 Diabetes UK.

  14. Studies of metabolic phenotypic correlates of 15 obesity associated gene variants.

    Directory of Open Access Journals (Sweden)

    Camilla Helene Sandholt

    Full Text Available Genome-wide association studies have identified novel BMI/obesity associated susceptibility loci. The purpose of this study is to determine associations with overweight, obesity, morbid obesity and/or general adiposity in a Danish population. Moreover, we want to investigate if these loci associate with type 2 diabetes and to elucidate potential underlying metabolic mechanisms.15 gene variants in 14 loci including TMEM18 (rs7561317, SH2B1 (rs7498665, KCTD15 (rs29941, NEGR1 (rs2568958, ETV5 (rs7647305, BDNF (rs4923461, rs925946, SEC16B (rs10913469, FAIM2 (rs7138803, GNPDA2 (rs10938397, MTCH2 (rs10838738, BAT2 (rs2260000, NPC1 (rs1805081, MAF (rs1424233, and PTER (rs10508503 were genotyped in 18,014 middle-aged Danes.Five of the 15 gene variants associated with overweight, obesity and/or morbid obesity. Per allele ORs ranged from 1.15-1.20 for overweight, 1.10-1.25 for obesity, and 1.41-1.46 for morbid obesity. Five of the 15 variants moreover associated with increased measures of adiposity. BDNF rs4923461 displayed a borderline BMI-dependent protective effect on type 2 diabetes (0.87 (0.78-0.96, p = 0.008, whereas SH2B1 rs7498665 associated with nominally BMI-independent increased risk of type 2 diabetes (1.16 (1.07-1.27, p = 7.8×10(-4.Associations with overweight and/or obesity and measures of obesity were confirmed for seven out of the 15 gene variants. The obesity risk allele of BDNF rs4923461 protected against type 2 diabetes, which could suggest neuronal and peripheral distinctive ways of actions for the protein. SH2B1 rs7498665 associated with type 2 diabetes independently of BMI.

  15. Biochemical phenotyping unravels novel metabolic abnormalities and potential biomarkers associated with treatment of GLUT1 deficiency with ketogenic diet.

    Directory of Open Access Journals (Sweden)

    Gerarda Cappuccio

    Full Text Available Global metabolomic profiling offers novel opportunities for the discovery of biomarkers and for the elucidation of pathogenic mechanisms that might lead to the development of novel therapies. GLUT1 deficiency syndrome (GLUT1-DS is an inborn error of metabolism due to reduced function of glucose transporter type 1. Clinical presentation of GLUT1-DS is heterogeneous and the disorder mirrors patients with epilepsy, movement disorders, or any paroxysmal events or unexplained neurological manifestation triggered by exercise or fasting. The diagnostic biochemical hallmark of the disease is a reduced cerebrospinal fluid (CSF/blood glucose ratio and the only available treatment is ketogenic diet. This study aimed at advancing our understanding of the biochemical perturbations in GLUT1-DS pathogenesis through biochemical phenotyping and the treatment of GLUT1-DS with a ketogenic diet. Metabolomic analysis of three CSF samples from GLUT1-DS patients not on ketogenic diet was feasible inasmuch as CSF sampling was used for diagnosis before to start with ketogenic diet. The analysis of plasma and urine samples obtained from GLUT1-DS patients treated with a ketogenic diet showed alterations in lipid and amino acid profiles. While subtle, these were consistent findings across the patients with GLUT1-DS on ketogenic diet, suggesting impacts on mitochondrial physiology. Moreover, low levels of free carnitine were present suggesting its consumption in GLUT1-DS on ketogenic diet. 3-hydroxybutyrate, 3-hydroxybutyrylcarnitine, 3-methyladipate, and N-acetylglycine were identified as potential biomarkers of GLUT1-DS on ketogenic diet. This is the first study to identify CSF, plasma, and urine metabolites associated with GLUT1-DS, as well as biochemical changes impacted by a ketogenic diet. Potential biomarkers and metabolic insights deserve further investigation.

  16. A Maternal Low-Fiber Diet Predisposes Offspring to Improved Metabolic Phenotypes in Adulthood in an Herbivorous Rodent.

    Science.gov (United States)

    Zhang, Xue-Ying; Lou, Mei-Fang; Shen, Wei; Fu, Rong-Shu; Wang, De-Hua

    The maternal or paternal dietary composition can have important effects on various aspects of their offspring's physiology. Studies from animal models and humans showed that a maternal high-fiber diet protected offspring against fat accumulation. However, little is known about how a maternal low-fiber diet modifies the metabolism of offspring in herbivorous rodents. We hypothesized that a maternal low-fiber diet would confer long-lasting beneficial effects on offspring metabolic phenotypes in herbivorous Brandt's vole (Lasiopodomys brandtii). Female voles were fed either a control (12.4% fiber) or a low-fiber (3.5% fiber) diet throughout pregnancy and lactation, and all offspring were fed the control diet after weaning till 14 wk old. Offspring were sampled from each litter at 18 d and 14 wk of age. Another subset of adult offspring at 15 wk of age was fed a high-fat diet for 8 wk. We found that there was no difference in litter size, litter mass, or pup mass before weaning between the two maternal diet groups. Offspring from the maternal low-fiber diet increased energy intake, body mass, and lean mass; suppressed fat accumulation; and improved glucose tolerance compared with those from the control diet. Moreover, the maternal low-fiber diet alleviated high-fat diet-induced obesity in the adult offspring. Serum leptin concentration and uncoupling protein 1 content in brown adipose tissue of offspring were not affected by a maternal low-fiber diet. We demonstrate that herbivorous females fed a low-fiber diet during pregnancy and lactation may predispose their offspring to accelerated growth of lean tissue, which may increase the opportunity for survival and reproduction in offspring.

  17. Biochemical phenotyping unravels novel metabolic abnormalities and potential biomarkers associated with treatment of GLUT1 deficiency with ketogenic diet.

    Science.gov (United States)

    Cappuccio, Gerarda; Pinelli, Michele; Alagia, Marianna; Donti, Taraka; Day-Salvatore, Debra-Lynn; Veggiotti, Pierangelo; De Giorgis, Valentina; Lunghi, Simona; Vari, Maria Stella; Striano, Pasquale; Brunetti-Pierri, Nicola; Kennedy, Adam D; Elsea, Sarah H

    2017-01-01

    Global metabolomic profiling offers novel opportunities for the discovery of biomarkers and for the elucidation of pathogenic mechanisms that might lead to the development of novel therapies. GLUT1 deficiency syndrome (GLUT1-DS) is an inborn error of metabolism due to reduced function of glucose transporter type 1. Clinical presentation of GLUT1-DS is heterogeneous and the disorder mirrors patients with epilepsy, movement disorders, or any paroxysmal events or unexplained neurological manifestation triggered by exercise or fasting. The diagnostic biochemical hallmark of the disease is a reduced cerebrospinal fluid (CSF)/blood glucose ratio and the only available treatment is ketogenic diet. This study aimed at advancing our understanding of the biochemical perturbations in GLUT1-DS pathogenesis through biochemical phenotyping and the treatment of GLUT1-DS with a ketogenic diet. Metabolomic analysis of three CSF samples from GLUT1-DS patients not on ketogenic diet was feasible inasmuch as CSF sampling was used for diagnosis before to start with ketogenic diet. The analysis of plasma and urine samples obtained from GLUT1-DS patients treated with a ketogenic diet showed alterations in lipid and amino acid profiles. While subtle, these were consistent findings across the patients with GLUT1-DS on ketogenic diet, suggesting impacts on mitochondrial physiology. Moreover, low levels of free carnitine were present suggesting its consumption in GLUT1-DS on ketogenic diet. 3-hydroxybutyrate, 3-hydroxybutyrylcarnitine, 3-methyladipate, and N-acetylglycine were identified as potential biomarkers of GLUT1-DS on ketogenic diet. This is the first study to identify CSF, plasma, and urine metabolites associated with GLUT1-DS, as well as biochemical changes impacted by a ketogenic diet. Potential biomarkers and metabolic insights deserve further investigation.

  18. Metabolic flux distributions in Corynebacterium glutamicum during growth and lysine overproduction. Reprinted from Biotechnology and Bioengineering, Vol. 41, Pp 633-646 (1993).

    Science.gov (United States)

    Vallino, J J; Stephanopoulos, G

    2000-03-20

    The two main contributions of this article are the solidification of Corynebacterium glutamicum biochemistry guided by bioreaction network analysis, and the determination of basal metabolic flux distributions during growth and lysine synthesis. Employed methodology makes use of stoichiometrically based mass balances to determine flux distributions in the C. glutamicum metabolic network. Presented are a brief description of the methodology, a thorough literature review of glutamic acid bacteria biochemistry, and specific results obtained through a combination of fermentation studies and analysis-directed intracellular assays. The latter include the findings of the lack of activity of glyoxylate shunt, and that phosphoenolpyruvate carboxylase (PPC) is the only anaplerotic reaction expressed in C. glutamicum cultivated on glucose minimal media. Network simplifications afforded by the above findings facilitated the determination of metabolic flux distributions under a variety of culture conditions and led to the following conclusions. Both the pentose phosphate pathway and PPC support significant fluxes during growth and lysine overproduction, and that flux partitioning at the glucosa-6-phosphate branch point does not appear to limit lysine synthesis. Copyright 1993 John Wiley & Sons, Inc.

  19. Simultaneous investigation of cardiac pyruvate dehydrogenase flux, Krebs cycle metabolism and pH, using hyperpolarized [1,2-(13)C2]pyruvate in vivo.

    Science.gov (United States)

    Chen, Albert P; Hurd, Ralph E; Schroeder, Marie A; Lau, Angus Z; Gu, Yi-ping; Lam, Wilfred W; Barry, Jennifer; Tropp, James; Cunningham, Charles H

    2012-02-01

    (13)C MR spectroscopy studies performed on hearts ex vivo and in vivo following perfusion of prepolarized [1-(13)C]pyruvate have shown that changes in pyruvate dehydrogenase (PDH) flux may be monitored non-invasively. However, to allow investigation of Krebs cycle metabolism, the (13)C label must be placed on the C2 position of pyruvate. Thus, the utilization of either C1 or C2 labeled prepolarized pyruvate as a tracer can only afford a partial view of cardiac pyruvate metabolism in health and disease. If the prepolarized pyruvate molecules were labeled at both C1 and C2 positions, then it would be possible to observe the downstream metabolites that were the results of both PDH flux ((13)CO(2) and H(13)CO(3)(-)) and Krebs cycle flux ([5-(13)C]glutamate) with a single dose of the agent. Cardiac pH could also be monitored in the same experiment, but adequate SNR of the (13)CO(2) resonance may be difficult to obtain in vivo. Using an interleaved selective RF pulse acquisition scheme to improve (13)CO(2) detection, the feasibility of using dual-labeled hyperpolarized [1,2-(13)C(2)]pyruvate as a substrate for dynamic cardiac metabolic MRS studies to allow simultaneous investigation of PDH flux, Krebs cycle flux and pH, was demonstrated in vivo. Copyright © 2011 John Wiley & Sons, Ltd.

  20. Metabolic Flux Analysis of Lipid Biosynthesis in the Yeast Yarrowia lipolytica Using 13C-Labled Glucose and Gas Chromatography-Mass Spectrometry.

    Science.gov (United States)

    Zhang, Huaiyuan; Wu, Chao; Wu, Qingyu; Dai, Junbiao; Song, Yuanda

    2016-01-01

    The oleaginous yeast Yarrowia lipolytica has considerable potential for producing single cell oil, which can be converted to biodiesel, a sustainable alternative to fossil fuels. However, extensive fundamental and engineering efforts must be carried out before commercialized production become cost-effective. Therefore, in this study, metabolic flux analysis of Y. lipolytica was performed using 13C-labeled glucose as a sole carbon source in nitrogen sufficient and insufficient media. The nitrogen limited medium inhibited cell growth while promoting lipid accumulation (from 8.7% of their biomass to 14.3%). Metabolic flux analysis showed that flux through the pentose phosphate pathway was not significantly regulated by nitrogen concentration, suggesting that NADPH generation is not the limiting factor for lipid accumulation in Y. lipolytica. Furthermore, metabolic flux through malic enzyme was undetectable, confirming its non-regulatory role in lipid accumulation in this yeast. Nitrogen limitation significantly increased flux through ATP:citrate lyase (ACL), implying that ACL plays a key role in providing acetyl-CoA for lipid accumulation in Y. lipolytica.

  1. Metabolic Flux Analysis of Lipid Biosynthesis in the Yeast Yarrowia lipolytica Using 13C-Labled Glucose and Gas Chromatography-Mass Spectrometry.

    Directory of Open Access Journals (Sweden)

    Huaiyuan Zhang

    Full Text Available The oleaginous yeast Yarrowia lipolytica has considerable potential for producing single cell oil, which can be converted to biodiesel, a sustainable alternative to fossil fuels. However, extensive fundamental and engineering efforts must be carried out before commercialized production become cost-effective. Therefore, in this study, metabolic flux analysis of Y. lipolytica was performed using 13C-labeled glucose as a sole carbon source in nitrogen sufficient and insufficient media. The nitrogen limited medium inhibited cell growth while promoting lipid accumulation (from 8.7% of their biomass to 14.3%. Metabolic flux analysis showed that flux through the pentose phosphate pathway was not significantly regulated by nitrogen concentration, suggesting that NADPH generation is not the limiting factor for lipid accumulation in Y. lipolytica. Furthermore, metabolic flux through malic enzyme was undetectable, confirming its non-regulatory role in lipid accumulation in this yeast. Nitrogen limitation significantly increased flux through ATP:citrate lyase (ACL, implying that ACL plays a key role in providing acetyl-CoA for lipid accumulation in Y. lipolytica.

  2. Novel use of Tat components to increase metabolic flux in light-driven biosynthesis

    DEFF Research Database (Denmark)

    Henriques de Jesus, Maria Perestrello Ramos

    Oxygenic photosynthesis provides plants, algae and cyanobacteria the ability to convert solar energy and CO2 into complex organic molecules. Thus tapping into photosynthesis for synthetic biology efforts has a huge potential for the industrial production of fuels and high value bioactive compounds...... relocating a biosynthetic pathway into the chloroplast, as well as possible solutions. Paper I compares the potential of chloroplasts and cyanobacteria as sustainable biosynthetic compartments and hosts for metabolic engineering. An estimation of the percentage of photosynthetic electrons that can...... be redirected into the biosynthesis of products is given. The availability of photosynthetic reducing power appears to be non-limiting whereas the redirection of the fixed carbon seems to be main challenge for achieving higher product yields. On paper II a novel strategy where the membrane anchors...

  3. Rational Design of a Corynebacterium glutamicum Pantothenate Production Strain and Ins Characterization by Metabolic Flux Analysis and Genome-Wide Transcriptional Profiling

    Czech Academy of Sciences Publication Activity Database

    Hüser, A.T.; Chassagnole, Ch.; Lindley, N.D.; Merkamm, M.; Guyonvarch, A.; Elišáková, Veronika; Pátek, Miroslav; Kalinowski, J.; Brune, I.; Pühler, A.; Tauch, A.

    2005-01-01

    Roč. 71, č. 6 (2005), s. 3255-3268 ISSN 0099-2240 Institutional research plan: CEZ:AV0Z50200510 Keywords : corynebacterium glutamicum * metabolic flux Subject RIV: EE - Microbiology, Virology Impact factor: 3.818, year: 2005

  4. Co-factor engineering in lactobacilli: Effects of uncoupled ATPase activity on metabolic fluxes in Lactobacillus (L.) plantarum and L. sakei

    DEFF Research Database (Denmark)

    Rud, Ida; Solem, Christian; Jensen, Peter Ruhdal

    2008-01-01

    The hydrolytic F-1-part of the F1F0-ATPase was over-expressed in Lactobacillus (L.) plantarum NC8 and L. sakei Lb790x during fermentation of glucose or ribose, in order to study how changes in the intracellular levels of ATP and ADP affect the metabolic fluxes. The uncoupled ATPase activity resul...

  5. Dissolved organic matter and lake metabolism: Biogeochemistry and controls of nutrient flux dynamics to fresh waters. Technical progress report, January 1, 1990--December 31, 1991

    Energy Technology Data Exchange (ETDEWEB)

    Wetzel, R.G.

    1992-12-31

    The land-water interface region consists of two major components: the wetland, and the down-gradient adjacent littoral floating-leaved and submersed, macrophyte communities. Because of the importance of very high production and nutrient turnover of attached microbiota, a major emphasis of this investigation was placed upon these biota and their metabolic capacities for assimilation and release of organic compounds and nutrient retention and cycling. Examination of the capacities of wetland littoral communities to regulate fluxes of nutrients and organic compounds often has been limited to input-output analyses. These input-output data are an integral part of these investigations, but most of the research effort concentrated on the biotic and metabolic mechanisms that control fluxes and retention capacities and their effects upon biota in the down-gradient waters. The important regulatory capacities of dissolved organic compounds on enzyme reactivity was examined experimentally and coupled to the wetland-littoral organic carbon flux budgets.

  6. Is it the resistance training itself or the combined associated weight loss that improves the metabolic syndrome-related phenotypes in postmenopausal women?

    Directory of Open Access Journals (Sweden)

    Soyluk O

    2015-10-01

    Full Text Available Ozlem Soyluk,1 Gulistan Bahat21Division of Endocrinology and Metabolism, Department of Internal Medicine, Istanbul Medical School, Istanbul University, Capa, Istanbul, Turkey; 2Division of Geriatrics, Department of Internal Medicine, Istanbul Medical School, Istanbul University, Capa, Istanbul, TurkeyWe read the article entitled “Resistance training improves isokinetic strength and metabolic syndrome-related phenotypes in postmenopausal women” by Oliveira et al1 with great interest. In the study, the authors examined the effects of 12 weeks of resistance training (RT on metabolic syndrome-related phenotypes in postmenopausal women. They reported that total cholesterol, low-density lipoprotein cholesterol levels, total cholesterol/high-density lipoprotein cholesterol ratio, blood glucose, basal insulin, and homeostatic model assessment of insulin resistance were all significantly reduced with RT (P<0.01. Accordingly, they concluded that a 12-week progressive RT program induces beneficial alterations on metabolic syndrome-related phenotypes in postmenopausal women. View original paper by Oliveira and colleagues.

  7. Continuous and batch cultures of Escherichia coli KJ134 for succinic acid fermentation: metabolic flux distributions and production characteristics.

    Science.gov (United States)

    van Heerden, Carel D; Nicol, Willie

    2013-09-17

    Succinic acid (SA) has become a prominent biobased platform chemical with global production quantities increasing annually. Numerous genetically modified E. coli strains have been developed with the main aim of increasing the SA yield of the organic carbon source. In this study, a promising SA-producing strain, E. coli KJ134 [Biotechnol. Bioeng. 101:881-893, 2008], from the Department of Microbiology and Cell Science of the University of Florida was evaluated under continuous and batch conditions using D-glucose and CO2 in a mineral salt medium. Production characteristics entailing growth and maintenance rates, growth termination points and metabolic flux distributions under growth and non-growth conditions were determined. The culture remained stable for weeks under continuous conditions. Under growth conditions the redox requirements of the reductive tricarboxylic acid (TCA) cycle was solely balanced by acetic acid (AcA) production via the pyruvate dehydrogenase route resulting in a molar ratio of SA:AcA of two. A maximum growth rate of 0.22 h(-1) was obtained, while complete growth inhibition occurred at a SA concentration of 18 g L(-1). Batch culture revealed that high-yield succinate production (via oxidative TCA or glyoxylate redox balancing) occurred under non-growth conditions where a SA:AcA molar ratio of up to five was attained, with a final SA yield of 0.94 g g(-1). Growth termination of the batch culture was in agreement with that of the continuous culture. The maximum maintenance production rate of SA under batch conditions was found to be 0.6 g g(-1) h(-1). This is twice the maintenance rate observed in the continuous runs. The study revealed that the metabolic flux of E. coli KJ134 differs significantly for growth and non-growth conditions, with non-growth conditions resulting in higher SA:AcA ratios and SA yields. Bioreaction characteristics entailing growth and maintenance rates, as well as growth termination markers will guide future fermentor

  8. Measuring and modeling C flux rates through the central metabolic pathways in microbial communities using position-specific 13C-labeled tracers

    Science.gov (United States)

    Dijkstra, P.; van Groenigen, K.; Hagerty, S.; Salpas, E.; Fairbanks, D. E.; Hungate, B. A.; KOCH, G. W.; Schwartz, E.

    2012-12-01

    The production of energy and metabolic precursors occurs in well-known processes such as glycolysis and Krebs cycle. We use position-specific 13C-labeled metabolic tracers, combined with models of microbial metabolic organization, to analyze the response of microbial community energy production, biosynthesis, and C use efficiency (CUE) in soils, decomposing litter, and aquatic communities. The method consists of adding position-specific 13C -labeled metabolic tracers to parallel soil incubations, in this case 1-13C and 2,3-13C pyruvate and 1-13C and U-13C glucose. The measurement of CO2 released from the labeled tracers is used to calculate the C flux rates through the various metabolic pathways. A simplified metabolic model consisting of 23 reactions is solved using results of the metabolic tracer experiments and assumptions of microbial precursor demand. This new method enables direct estimation of fundamental aspects of microbial energy production, CUE, and soil organic matter formation in relatively undisturbed microbial communities. We will present results showing the range of metabolic patterns observed in these communities and discuss results from testing metabolic models.

  9. Moderate DNA damage promotes metabolic flux into PPP via PKM2 Y-105 phosphorylation: a feature that favours cancer cells.

    Science.gov (United States)

    Kumar, Bhupender; Bamezai, Rameshwar N K

    2015-08-01

    Pyruvate kinase M2, an important metabolic enzyme, promotes aerobic glycolysis (Warburg effect) to facilitate cancer cell proliferation. Unravelling the status of this important glycolytic pathway enzyme under sub-lethal doses of etoposide, a commonly used anti-proliferative genotoxic drug to induce mild/moderate DNA damage in HeLa cells as a model system and discern its effect on: PKM2 expression, phosphorylation, dimer: tetramer ratio, activity and associated effects, was pertinent. Protein expression and phosphorylation of PKM2 from HeLa cells was estimated using Western blotting. Same protein lysate was also used to estimate total pyruvate kinase activity and the total dimer: tetramer content evaluated using glycerol gradient ultra-centrifugation. Intracellular PEP was estimated manually using standard curve; while NADPH was assessed by NADPH estimation kit. Unpaired t test and two-way-ANOVA was used for statistical analysis. A relative decrease in PKM2 expression and a subsequent dose and time dependent increase in Y105-phosphorylation were observed. A concomitant increase in PKM2 dimer content and Y105-phosphorylation responsible for reduced PKM2 activity promoted PEP accumulation and NADPH production, representing increased metabolic flux into PPP, a feature that favours cancer cells. It was apparent that the sub-lethal doses of etoposide induced inadequate damage to DNA in cancer cells in culture promoted pro-survival conditions due to Y105-phosphorylation of PKM2, its stable dimerization and inactivation, a unique association not known earlier, indicating what might happen in tumour revivals or recurrences.

  10. Metabolic-flux analysis of continuously cultured hybridoma cells using 13CO2 mass spectrometry in combination with 13C-lactate nuclear magnetic resonance spectroscopy and metabolic balancing

    NARCIS (Netherlands)

    Bonarius, H.P.J.; Ozemre, A.; Timmerarends, B.; Skrabal, P.; Tramper, J.; Schmid, G.; Heinzle, E.

    2001-01-01

    Protein production of mammalian-cell culture is limited due to accumulation of waste products such as lactate, CO2, and ammonia. In this study, the intracellular fluxes of hybridoma cells are measured to determine the amount by which various metabolic pathways contribute to the secretion of waste

  11. Determination of metabolic fluxes during glucose catabolism in Propionibacterium freudenreichii subsp. shermanii.

    Science.gov (United States)

    Meurice, G; Bondon, A; Deborde, C; Boyaval, P

    2001-01-01

    In vivo 13C Nuclear Magnetic Resonance (NMR) spectroscopy was used to investigate the pathways of glucose metabolism, non-invasively, in living cell suspensions of Propionibacterium freudenreichii subsp. shermanii. This species is the main ripening flora of the Swiss-type cheeses and is widely used as propionic acid and vitamin B12 industrial producer. The flow of labelled [1-13C]glucose was monitored in living cell suspensions and enrichment was detected in main products like [1-13C]glycogen, [6-13C]lycogen, [1-13C]trehalose, [6-13C]trehalose, [1-13C]propionate, [2-13C]propionate, [3-13C]propionate, [1-13C]acetate, [2-13C]acetate, [1-13C]succinate, [2-13C]succinate and [1-13C]CO2. alpha and beta glucose consumption could be examined separately and were catabolized at the same rate. Three intermediates were also found out, namely [1-13C]glucose-6-phosphate, [6-13C]glucose-6-phosphate and [1-13C]glucose-1-phosphate. From the formation of intermediates such as [6-3C]glucose-6-phosphate and products like [6-13C]glycogen from [1-13C]glucose we concluded the bidirectionality of reactions in the first part of glycolysis and the isomerization at the triose-phosphate level. Comparison of spectra obtained after addition of [1-13C]glucose or [U-12C]glucose revealed production of [1-13C]CO2 which means that pentose phosphates pathway is active under our experimental conditions. From the isotopic pattern of trehalose, it could be postulated that trehalose biosynthesis occurred either by direct condensation of two glucose molecules or by gluconeogenesis. A chemically defined medium was elaborated for the study and trehalose was the main osmolyte found in the intracellular fraction of P. shermanii grown in this medium.

  12. Validation of the doubly-labeled water (H3H18O) method for measuring water flux and energy metabolism in tenebrionid beetles

    International Nuclear Information System (INIS)

    Cooper, P.D.

    1981-01-01

    Doubly-labeled water (H 3 H 18 O) has been used to determine water flux and energy metabolism in a variety of vertebrates. This study examines the applicability of this technique to arthropods. The theory of the technique depends upon the assumption that doubly-labeled water introduced into the animal's body water equilibrates with water and carbon dioxide by the action of carbonic anhydrase. Tritium ( 3 H) is lost from the animal only with water while oxygen-18 is lost with both water and carbon dioxide. The difference bwtween the rates of loss of the two isotopes is proportional to CO 2 loss rate. Validation of the use of tritiated water for measuring water flux was accomplished by comparing gravimetric measurements of water gain with flux rates determined by loss of tritiated water. At room humidity, an overestimate for influx calculated from labeled water calculations was found, averaging 12 mg H 2 O (g.d) -1 . Comparison of CO 2 loss rate determined isotopically with rates of CO 2 loss determined by standard metabolic rates also yielded overestimates for the isotopic technique, overestimates ranging between 20 and 30%. The relevance of this for studies using labeled water for studying water fluxes and free metabolism of free-ranging arthropods is discussed

  13. Gill lipid metabolism and unidirectional Na+ flux in the European eel (Anguilla anguilla) after transfer to dilute media

    DEFF Research Database (Denmark)

    Hansen, H.J.M.; Grosell, M.; Rosenkilde, P.

    1999-01-01

    positive linear correlation of percentage (14C) wax alcohols with log [22Na efflux]. Based on the observed parallel between Naf flux and gill lipid metabolism, it is suggested that the eel reacts at first to a loss of Na+ by synthesizing wax alcohols that can tighten the gill membrane. (C) 1999 Elsevier......+ uptake rate was 12 mu mol kg(-1) h(-1), i.e., the general picture in DW was a net Na+ loss. In another similar experiment, groups of three FW-adapted eels were incubated in vivo for up to 24 h with (C-14) acetate added as lipid precursor to the ambient water. Incubation in FW showed about 20......% of the total C-14-activity incorporated into gill lipids as (C-14) wax alcohols (WA; octadecanol and eicosanol). This percentage went up to 50% shortly after transfer to DW and came down again to about 20% after 2 weeks in DW. Single eels labelled with Na-22 in the plasma showed a statistically significant...

  14. Genetic and phenotypic variation in UGT2B17, a testosterone-metabolizing enzyme, is associated with BMI in males.

    Science.gov (United States)

    Zhu, Andy Z X; Cox, Lisa S; Ahluwalia, Jasjit S; Renner, Caroline C; Hatsukami, Dorothy K; Benowitz, Neal L; Tyndale, Rachel F

    2015-05-01

    A number of candidate gene and genome-wide association studies have identified significant associations between single nucleotide polymorphisms, particularly in FTO and MC4R, and body weight. However, the association between copy number variation and body weight is less understood. Anabolic androgenic steroids, such as testosterone, can regulate body weight. In humans, UDP-glucuronosyltransferase 2B17 (UGT2B17) metabolizes testosterone to a metabolite, which is readily excreted in urine. We investigate the association between genetic and phenotypic variation in UGT2B17 and body weight. UGT2B17 deletion was genotyped and in-vivo UGT2B17 enzymatic activity (as measured by the 3-hydroxycotinine glucuronide to free 3-hydroxycotinine ratio) was measured in 400 Alaska Native individuals and 540 African Americans. In Alaska Native people, UGT2B17 deletion was strongly associated with lower BMI in male individuals (P<0.001), but not in female individuals, consistent with testosterone being a male dominant steroid. The sex-specific association between UGT2B17 deletion and lower BMI was also observed in African Americans (P=0.01 in male individuals). In both populations, UGT2B17 deletion was significantly associated with lower measured in-vivo UGT2B17 activity. In male individuals, lower in-vivo UGT2B17 activity was associated with lower BMI, as observed in the sex-specific genotypic association. These data suggest that UGT2B17 deletion leads to reduced UGT2B17 activity, and lower BMI in male individuals. This is consistent with the hypothesis that reduced UGT2B17-mediated testosterone excretion results in higher testosterone levels. Future studies could confirm this hypothesis by directly measuring serum testosterone levels.

  15. Responses of nitrogen metabolism and seed nutrition to drought stress in soybean genotypes differing in slow-wilting phenotype

    Directory of Open Access Journals (Sweden)

    Nacer eBellaloui

    2013-12-01

    Full Text Available Recent advances in soybean breeding have resulted in genotypes that express the slow-wilting phenotype (trait under drought stress conditions. The physiological mechanisms of this trait remain unknown due to the complexity of trait × environment interactions. The objective of this research was to investigate nitrogen metabolism and leaf and seed nutrients composition of the slow-wilting soybean genotypes under drought stress conditions. A repeated greenhouse experiment was conducted using check genotypes: NC-Roy (fast wilting, Boggs (intermediate in wilting; and NTCPR94-5157 and N04-9646 (slow-wilting, SLW genotypes. Plants were either well-watered or drought stressed. Results showed that under well-watered conditions, nitrogen fixation (NF, nitrogen assimilation (NA, and leaf and seed composition differed between genotypes. Under drought stress, NF and NA were higher in NTCPR94-5157 and N04-9646 than in NC-Roy and Boggs. Under severe water stress, however, NA was low in all genotypes. Leaf water potential was significantly lower in checks (-2.00 MPa than in the SLW genotypes (-1.68 MPa. Leaf and seed concentrations of K, P, Ca, Cu, Na, B were higher in SLW genotypes than in the checks under drought stress conditions. Seed protein, oleic acid, and sugars were higher in SLW genotypes, and oil, linoleic and linolenic acids were lower in SLW genotypes. This research demonstrated that K, P, Ca, Cu, Na, and B may be involved in SLW trait by maintaining homeostasis and osmotic regulation. Maintaining higher leaf water potential in NTCPR94-5157 and N04-9646 under drought stress could be a possible water conservation mechanism to maintain leaf turgor pressure. The increase in osmoregulators such as minerals, raffinose and stachyose, and oleic acid could be beneficial for soybean breeders in selecting for drought stress tolerance.

  16. Assessment of metabolic flux distribution in the thermophilic hydrogen producer Caloramator celer as affected by external pH and hydrogen partial pressure.

    Science.gov (United States)

    Ciranna, Alessandro; Pawar, Sudhanshu S; Santala, Ville; Karp, Matti; van Niel, Ed W J

    2014-03-28

    Caloramator celer is a strict anaerobic, alkalitolerant, thermophilic bacterium capable of converting glucose to hydrogen (H2), carbon dioxide, acetate, ethanol and formate by a mixed acid fermentation. Depending on the growth conditions C. celer can produce H2 at high yields. For a biotechnological exploitation of this bacterium for H2 production it is crucial to understand the factors that regulate carbon and electron fluxes and therefore the final distribution of metabolites to channel the metabolic flux towards the desired product. Combining experimental results from batch fermentations with genome analysis, reconstruction of central carbon metabolism and metabolic flux analysis (MFA), this study shed light on glucose catabolism of the thermophilic alkalitolerant bacterium C. celer. Two innate factors pertaining to culture conditions have been identified to significantly affect the metabolic flux distribution: culture pH and partial pressures of H2 (PH2). Overall, at alkaline to neutral pH the rate of biomass synthesis was maximized, whereas at acidic pH the lower growth rate and the less efficient biomass formation are accompanied with more efficient energy recovery from the substrate indicating high cell maintenance possibly to sustain intracellular pH homeostasis. Higher H2 yields were associated with fermentation at acidic pH as a consequence of the lower synthesis of other reduced by-products such as formate and ethanol. In contrast, PH2 did not affect the growth of C. celer on glucose. At high PH2 the cellular redox state was balanced by rerouting the flow of carbon and electrons to ethanol and formate production allowing unaltered glycolytic flux and growth rate, but resulting in a decreased H2 synthesis. C. celer possesses a flexible fermentative metabolism that allows redistribution of fluxes at key metabolic nodes to simultaneously control redox state and efficiently harvest energy from substrate even under unfavorable conditions (i.e. low pH and high

  17. Metabolic profiling and flux analysis of MEL-2 human embryonic stem cells during exponential growth at physiological and atmospheric oxygen concentrations.

    Directory of Open Access Journals (Sweden)

    Jennifer Turner

    Full Text Available As human embryonic stem cells (hESCs steadily progress towards regenerative medicine applications there is an increasing emphasis on the development of bioreactor platforms that enable expansion of these cells to clinically relevant numbers. Surprisingly little is known about the metabolic requirements of hESCs, precluding the rational design and optimisation of such platforms. In this study, we undertook an in-depth characterisation of MEL-2 hESC metabolic behaviour during the exponential growth phase, combining metabolic profiling and flux analysis tools at physiological (hypoxic and atmospheric (normoxic oxygen concentrations. To overcome variability in growth profiles and the problem of closing mass balances in a complex environment, we developed protocols to accurately measure uptake and production rates of metabolites, cell density, growth rate and biomass composition, and designed a metabolic flux analysis model for estimating internal rates. hESCs are commonly considered to be highly glycolytic with inactive or immature mitochondria, however, whilst the results of this study confirmed that glycolysis is indeed highly active, we show that at least in MEL-2 hESC, it is supported by the use of oxidative phosphorylation within the mitochondria utilising carbon sources, such as glutamine to maximise ATP production. Under both conditions, glycolysis was disconnected from the mitochondria with all of the glucose being converted to lactate. No difference in the growth rates of cells cultured under physiological or atmospheric oxygen concentrations was observed nor did this cause differences in fluxes through the majority of the internal metabolic pathways associated with biogenesis. These results suggest that hESCs display the conventional Warburg effect, with high aerobic activity despite high lactate production, challenging the idea of an anaerobic metabolism with low mitochondrial activity. The results of this study provide new insight that

  18. Pharmacokinetics of diclofenac in healthy controls with wild-type phenotype for CYP2C9 shows metabolism variability

    Directory of Open Access Journals (Sweden)

    M. Martín-De Saro

    2017-04-01

    Conclusions: This data indicate that CYP2C9 is not the only enzyme responsible of the metabolism of diclofenac, so it is important to analyze other cytochromes and their variants potentially involved in the metabolism of this drug.

  19. Quantifying phenotypic flexibility as the response to a high-fat challenge test in different states of metabolic health

    NARCIS (Netherlands)

    Kardinaal, A.F.M.; Erk, M.J. van; Dutman, A.E.; Stroeve, J.H.M.; Steeg, E. van de; Bijlsma, S.; Kooistra, T.; Ommen, B. van; Wopereis, S.

    2015-01-01

    Metabolism maintains homeostasis at chronic hypercaloric conditions, activating postprandial response mechanisms, which come at the cost of adaptation processes such as energy storage, eventually with negative health consequences. This study quantified the metabolic adaptation capacity by studying

  20. Effects of experimental nitrogen fertilization on planktonic metabolism and CO2 flux in a hypereutrophic hardwater lake.

    Directory of Open Access Journals (Sweden)

    Matthew J Bogard

    Full Text Available Hardwater lakes are common in human-dominated regions of the world and often experience pollution due to agricultural and urban effluent inputs of inorganic and organic nitrogen (N. Although these lakes are landscape hotspots for CO2 exchange and food web carbon (C cycling, the effect of N enrichment on hardwater lake food web functioning and C cycling patterns remains unclear. Specifically, it is unknown if different eutrophication scenarios (e.g., modest non point vs. extreme point sources yield consistent effects on auto- and heterotrophic C cycling, or how biotic responses interact with the inorganic C system to shape responses of air-water CO2 exchange. To address this uncertainty, we induced large metabolic gradients in the plankton community of a hypereutrophic hardwater Canadian prairie lake by adding N as urea (the most widely applied agricultural fertilizer at loading rates of 0, 1, 3, 8 or 18 mg N L-1 week-1 to 3240-L, in-situ mesocosms. Over three separate 21-day experiments, all treatments of N dramatically increased phytoplankton biomass and gross primary production (GPP two- to six-fold, but the effects of N on autotrophs plateaued at ~3 mg N L-1. Conversely, heterotrophic metabolism increased linearly with N fertilization over the full treatment range. In nearly all cases, N enhanced net planktonic uptake of dissolved inorganic carbon (DIC, and increased the rate of CO2 influx, while planktonic heterotrophy and CO2 production only occurred in the highest N treatments late in each experiment, and even in these cases, enclosures continued to in-gas CO2. Chemical effects on CO2 through calcite precipitation were also observed, but similarly did not change the direction of net CO2 flux. Taken together, these results demonstrate that atmospheric exchange of CO2 in eutrophic hardwater lakes remains sensitive to increasing N loading and eutrophication, and that even modest levels of N pollution are capable of enhancing autotrophy and CO

  1. ¹³C metabolic flux analysis identifies an unusual route for pyruvate dissimilation in mycobacteria which requires isocitrate lyase and carbon dioxide fixation.

    Directory of Open Access Journals (Sweden)

    Dany J V Beste

    2011-07-01

    Full Text Available Mycobacterium tuberculosis requires the enzyme isocitrate lyase (ICL for growth and virulence in vivo. The demonstration that M. tuberculosis also requires ICL for survival during nutrient starvation and has a role during steady state growth in a glycerol limited chemostat indicates a function for this enzyme which extends beyond fat metabolism. As isocitrate lyase is a potential drug target elucidating the role of this enzyme is of importance; however, the role of isocitrate lyase has never been investigated at the level of in vivo fluxes. Here we show that deletion of one of the two icl genes impairs the replication of Mycobacterium bovis BCG at slow growth rate in a carbon limited chemostat. In order to further understand the role of isocitrate lyase in the central metabolism of mycobacteria the effect of growth rate on the in vivo fluxes was studied for the first time using ¹³C-metabolic flux analysis (MFA. Tracer experiments were performed with steady state chemostat cultures of BCG or M. tuberculosis supplied with ¹³C labeled glycerol or sodium bicarbonate. Through measurements of the ¹³C isotopomer labeling patterns in protein-derived amino acids and enzymatic activity assays we have identified the activity of a novel pathway for pyruvate dissimilation. We named this the GAS pathway because it utilizes the Glyoxylate shunt and Anapleurotic reactions for oxidation of pyruvate, and Succinyl CoA synthetase for the generation of succinyl CoA combined with a very low flux through the succinate--oxaloacetate segment of the tricarboxylic acid cycle. We confirm that M. tuberculosis can fix carbon from CO₂ into biomass. As the human host is abundant in CO₂ this finding requires further investigation in vivo as CO₂ fixation may provide a point of vulnerability that could be targeted with novel drugs. This study also provides a platform for further studies into the metabolism of M. tuberculosis using ¹³C-MFA.

  2. Flux control exerted by mitochondrial outer membrane carnitine palmitoyltransferase over beta-oxidation, ketogenesis and tricarboxylic acid cycle activity in hepatocytes isolated from rats in different metabolic states.

    OpenAIRE

    Drynan, L; Quant, P A; Zammit, V A

    1996-01-01

    The Flux Control Coefficients of mitochondrial outer membrane carnitine palmitoyltransferase (CPT I) with respect to the overall rates of beta-oxidation, ketogenesis and tricarboxylic acid cycle activity were measured in hepatocytes isolated from rats in different metabolic states (fed, 24 h-starved, starved-refed and starved/insulin-treated). These conditions were chosen because there is controversy as to whether, when significant control ceases to be exerted by CPT I over the rate of fatty ...

  3. Effects of ocean acidification and hydrodynamic conditions on carbon metabolism and dissolved organic carbon (DOC) fluxes in seagrass populations.

    Science.gov (United States)

    Egea, Luis G; Jiménez-Ramos, Rocío; Hernández, Ignacio; Bouma, Tjeerd J; Brun, Fernando G

    2018-01-01

    Global change has been acknowledged as one of the main threats to the biosphere and its provision of ecosystem services, especially in marine ecosystems. Seagrasses play a critical ecological role in coastal ecosystems, but their responses to ocean acidification (OA) and climate change are not well understood. There have been previous studies focused on the effects of OA, but the outcome of interactions with co-factors predicted to alter during climate change still needs to be addressed. For example, the impact of higher CO2 and different hydrodynamic regimes on seagrass performance remains unknown. We studied the effects of OA under different current velocities on productivity of the seagrass Zostera noltei, using changes in dissolved oxygen as a proxy for the seagrass carbon metabolism, and release of dissolved organic carbon (DOC) in a four-week experiment using an open-water outdoor mesocosm. Under current pH conditions, increasing current velocity had a positive effect on productivity, but this depended on shoot density. However, this positive effect of current velocity disappeared under OA conditions. OA conditions led to a significant increase in gross production rate and respiration, suggesting that Z. noltei is carbon-limited under the current inorganic carbon concentration of seawater. In addition, an increase in non-structural carbohydrates was found, which may lead to better growing conditions and higher resilience in seagrasses subjected to environmental stress. Regarding DOC flux, a direct and positive relationship was found between current velocity and DOC release, both under current pH and OA conditions. We conclude that OA and high current velocity may lead to favourable growth scenarios for Z. noltei populations, increasing their productivity, non-structural carbohydrate concentrations and DOC release. Our results add new dimensions to predictions on how seagrass ecosystems will respond to climate change, with important implications for the

  4. Effects of ocean acidification and hydrodynamic conditions on carbon metabolism and dissolved organic carbon (DOC fluxes in seagrass populations.

    Directory of Open Access Journals (Sweden)

    Luis G Egea

    Full Text Available Global change has been acknowledged as one of the main threats to the biosphere and its provision of ecosystem services, especially in marine ecosystems. Seagrasses play a critical ecological role in coastal ecosystems, but their responses to ocean acidification (OA and climate change are not well understood. There have been previous studies focused on the effects of OA, but the outcome of interactions with co-factors predicted to alter during climate change still needs to be addressed. For example, the impact of higher CO2 and different hydrodynamic regimes on seagrass performance remains unknown. We studied the effects of OA under different current velocities on productivity of the seagrass Zostera noltei, using changes in dissolved oxygen as a proxy for the seagrass carbon metabolism, and release of dissolved organic carbon (DOC in a four-week experiment using an open-water outdoor mesocosm. Under current pH conditions, increasing current velocity had a positive effect on productivity, but this depended on shoot density. However, this positive effect of current velocity disappeared under OA conditions. OA conditions led to a significant increase in gross production rate and respiration, suggesting that Z. noltei is carbon-limited under the current inorganic carbon concentration of seawater. In addition, an increase in non-structural carbohydrates was found, which may lead to better growing conditions and higher resilience in seagrasses subjected to environmental stress. Regarding DOC flux, a direct and positive relationship was found between current velocity and DOC release, both under current pH and OA conditions. We conclude that OA and high current velocity may lead to favourable growth scenarios for Z. noltei populations, increasing their productivity, non-structural carbohydrate concentrations and DOC release. Our results add new dimensions to predictions on how seagrass ecosystems will respond to climate change, with important

  5. A dynamic metabolic flux analysis of ABE (acetone-butanol-ethanol) fermentation by Clostridium acetobutylicum ATCC 824, with riboflavin as a by-product.

    Science.gov (United States)

    Zhao, Xinhe; Kasbi, Mayssa; Chen, Jingkui; Peres, Sabine; Jolicoeur, Mario

    2017-12-01

    The present study reveals that supplementing sodium acetate (NaAc) strongly stimulates riboflavin production in acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum ATCC 824 with xylose as carbon source. Riboflavin production increased from undetectable concentrations to ∼0.2 g L -1 (0.53 mM) when supplementing 60 mM NaAc. Of interest, solvents production and biomass yield were also promoted with fivefold acetone, 2.6-fold butanol, and 2.4-fold biomass adding NaAc. A kinetic metabolic model, developed to simulate ABE biosystem, with riboflavin production, revealed from a dynamic metabolic flux analysis (dMFA) simultaneous increase of riboflavin (ribA) and GTP (precursor of riboflavin) (PurM) synthesis flux rates under NaAc supplementation. The model includes 23 fluxes, 24 metabolites, and 72 kinetic parameters. It also suggested that NaAc condition has first stimulated the accumulation of intracellular metabolite intermediates during the acidogenic phase, which have then fed the solventogenic phase leading to increased ABE production. In addition, NaAc resulted in higher intracellular levels of NADH during the whole culture. Moreover, lower GTP-to-adenosine phosphates (ATP, ADP, AMP) ratio under NaAc supplemented condition suggests that GTP may have a minor role in the cell energetic metabolism compared to its contribution to riboflavin synthesis. © 2017 Wiley Periodicals, Inc.

  6. Genome and Phenotype Microarray Analyses of Rhodococcus sp. BCP1 and Rhodococcus opacus R7: Genetic Determinants and Metabolic Abilities with Environmental Relevance.

    Science.gov (United States)

    Orro, Alessandro; Cappelletti, Martina; D'Ursi, Pasqualina; Milanesi, Luciano; Di Canito, Alessandra; Zampolli, Jessica; Collina, Elena; Decorosi, Francesca; Viti, Carlo; Fedi, Stefano; Presentato, Alessandro; Zannoni, Davide; Di Gennaro, Patrizia

    2015-01-01

    In this paper comparative genome and phenotype microarray analyses of Rhodococcus sp. BCP1 and Rhodococcus opacus R7 were performed. Rhodococcus sp. BCP1 was selected for its ability to grow on short-chain n-alkanes and R. opacus R7 was isolated for its ability to grow on naphthalene and on o-xylene. Results of genome comparison, including BCP1, R7, along with other Rhodococcus reference strains, showed that at least 30% of the genome of each strain presented unique sequences and only 50% of the predicted proteome was shared. To associate genomic features with metabolic capabilities of BCP1 and R7 strains, hundreds of different growth conditions were tested through Phenotype Microarray, by using Biolog plates and plates manually prepared with additional xenobiotic compounds. Around one-third of the surveyed carbon sources was utilized by both strains although R7 generally showed higher metabolic activity values compared to BCP1. Moreover, R7 showed broader range of nitrogen and sulphur sources. Phenotype Microarray data were combined with genomic analysis to genetically support the metabolic features of the two strains. The genome analysis allowed to identify some gene clusters involved in the metabolism of the main tested xenobiotic compounds. Results show that R7 contains multiple genes for the degradation of a large set of aromatic and PAHs compounds, while a lower variability in terms of genes predicted to be involved in aromatic degradation was found in BCP1. This genetic feature can be related to the strong genetic pressure exerted by the two different environment from which the two strains were isolated. According to this, in the BCP1 genome the smo gene cluster involved in the short-chain n-alkanes degradation, is included in one of the unique regions and it is not conserved in the Rhodococcus strains compared in this work. Data obtained underline the great potential of these two Rhodococcus spp. strains for biodegradation and environmental decontamination

  7. Co-utilization of glucose and xylose by evolved Thermus thermophilus LC113 strain elucidated by 13C metabolic flux analysis and whole genome sequencing

    Science.gov (United States)

    Cordova, Lauren T.; Lu, Jing; Cipolla, Robert M.; Sandoval, Nicholas R.; Long, Christopher P.; Antoniewicz, Maciek R.

    2018-01-01

    We evolved Thermus thermophilus to efficiently co-utilize glucose and xylose, the two most abundant sugars in lignocellulosic biomass, at high temperatures without carbon catabolite repression. To generate the strain, T. thermophilus HB8 was first evolved on glucose to improve its growth characteristics, followed by evolution on xylose. The resulting strain, T. thermophilus LC113, was characterized in growth studies, by whole genome sequencing, and 13C-metabolic flux analysis (13C-MFA) with [1,6-13C]glucose, [5-13C]xylose, and [1,6-13C]glucose + [5-13C]xylose as isotopic tracers. Compared to the starting strain, the evolved strain had an increased growth rate (~ 2-fold), increased biomass yield, increased tolerance to high temperatures up to 90 °C, and gained the ability to grow on xylose in minimal medium. At the optimal growth temperature of 81 °C, the maximum growth rate on glucose and xylose was 0.44 and 0.46 h−1, respectively. In medium containing glucose and xylose the strain efficiently co-utilized the two sugars. 13C-MFA results provided insights into the metabolism of T. thermophilus LC113 that allows efficient co-utilization of glucose and xylose. Specifically, 13C-MFA revealed that metabolic fluxes in the upper part of metabolism adjust flexibly to sugar availability, while fluxes in the lower part of metabolism remain relatively constant. Whole genome sequence analysis revealed two large structural changes that can help explain the physiology of the evolved strain: a duplication of a chromosome region that contains many sugar transporters, and a 5× multiplication of a region on the pVV8 plasmid that contains xylose isomerase and xylulokinase genes, the first two enzymes of xylose catabolism. Taken together, 13C-MFA and genome sequence analysis provided complementary insights into the physiology of the evolved strain. PMID:27164561

  8. A simplified method for power-law modelling of metabolic pathways from time-course data and steady-state flux profiles

    Directory of Open Access Journals (Sweden)

    Sugimoto Masahiro

    2006-07-01

    Full Text Available Abstract Background In order to improve understanding of metabolic systems there have been attempts to construct S-system models from time courses. Conventionally, non-linear curve-fitting algorithms have been used for modelling, because of the non-linear properties of parameter estimation from time series. However, the huge iterative calculations required have hindered the development of large-scale metabolic pathway models. To solve this problem we propose a novel method involving power-law modelling of metabolic pathways from the Jacobian of the targeted system and the steady-state flux profiles by linearization of S-systems. Results The results of two case studies modelling a straight and a branched pathway, respectively, showed that our method reduced the number of unknown parameters needing to be estimated. The time-courses simulated by conventional kinetic models and those described by our method behaved similarly under a wide range of perturbations of metabolite concentrations. Conclusion The proposed method reduces calculation complexity and facilitates the construction of large-scale S-system models of metabolic pathways, realizing a practical application of reverse engineering of dynamic simulation models from the Jacobian of the targeted system and steady-state flux profiles.

  9. Metabolic parameters linked by Phenotype MicroArray to acid resistance profiles of poultry-associated Salmonella enterica.

    Science.gov (United States)

    Phenotype microarrays were analyzed for 51 datasets derived from Salmonella enterica. The top 4 serovars associated with poultry products and one associated with turkey, respectively Typhimurium, Enteritidis, Heidelberg, Infantis and Senftenberg, were represented. Datasets were clustered into two ...

  10. Expression of mutant huntingtin in leptin receptor-expressing neurons does not control the metabolic and psychiatric phenotype of the BACHD mouse.

    Directory of Open Access Journals (Sweden)

    Sofia Hult Lundh

    Full Text Available Metabolic and psychiatric disturbances occur early on in the clinical manifestation of Huntington's disease (HD, a neurodegenerative disorder caused by an expanded CAG repeat in the huntingtin (HTT gene. Hypothalamus has emerged as an important site of pathology and alterations in this area and its neuroendocrine circuits may play a role in causing early non-motor symptoms and signs in HD. Leptin is a hormone that controls energy homeostasis by signaling through leptin receptors in the hypothalamus. Disturbed leptin action is implicated in both obesity and depression and altered circulating levels of leptin have been reported in both clinical HD and rodent models of the disease. Pathological leptin signaling may therefore be involved in causing the metabolic and psychiatric disturbances of HD. Here we tested the hypothesis that expression of mutant HTT in leptin receptor carrying neurons plays a role in the development of the non-motor phenotype in the BACHD mouse model. Our results show that inactivation of mutant HTT in leptin receptor-expressing neurons in the BACHD mouse using cross-breeding based on a cre-loxP system did not have an effect on the metabolic phenotype or anxiety-like behavior. The data suggest that mutant HTT disrupts critical hypothalamic pathways by other mechanisms than interfering with intracellular leptin signaling.

  11. Metabolic flux analysis of Escherichia coli in glucose-limited continuous culture. I. Growth-rate-dependent metabolic efficiency at steady state.

    Science.gov (United States)

    Kayser, Anke; Weber, Jan; Hecht, Volker; Rinas, Ursula

    2005-03-01

    The Escherichia coli K-12 strain TG1 was grown at 28 degrees C in aerobic glucose-limited continuous cultures at dilution rates ranging from 0.044 to 0.415 h(-1). The rates of biomass formation, the specific rates of glucose, ammonium and oxygen uptake and the specific carbon dioxide evolution rate increased linearly with the dilution rate up to 0.3 h(-1). At dilution rates between 0.3 h(-1) and 0.4 h(-1), a strong deviation from the linear increase to lower specific oxygen uptake and carbon dioxide evolution rates occurred. The biomass formation rate and the specific glucose and ammonium uptake rates did not deviate that strongly from the linear increase up to dilution rates of 0.4 h(-1). An increasing percentage of glucose carbon flow towards biomass determined by a reactor mass balance and a decreasing specific ATP production rate concomitant with a decreasing adenylate energy charge indicated higher energetic efficiency of carbon substrate utilization at higher dilution rates. Estimation of metabolic fluxes by a stoichiometric model revealed an increasing activity of the pentose phosphate pathway and a decreasing tricarboxylic acid cycle activity with increasing dilution rates, indicative of the increased NADPH and precursor demand for anabolic purposes at the expense of ATP formation through catabolic activities. Thus, increasing growth rates first result in a more energy-efficient use of the carbon substrate for biomass production, i.e. a lower portion of the carbon substrate is channelled into the respiratory, energy-generating pathway. At dilution rates above 0.4 h(-1), close to the wash-out point, respiration rates dropped sharply and accumulation of glucose and acetic acid was observed. Energy generation through acetate formation yields less ATP compared with complete oxidation of the sugar carbon substrate, but is the result of maximized energy generation under conditions of restrictions in the tricarboxylic acid cycle or in respiratory NADH turnover

  12. ToMI-FBA: A genome-scale metabolic flux based algorithm to select optimum hosts and media formulations for expressing pathways of interest

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    Hadi Nazem-Bokaee

    2015-09-01

    Full Text Available The Total Membrane Influx constrained Flux Balance Analysis (ToMI-FBA algorithm was developed in this research as a new tool to help researchers decide which microbial host and medium formulation are optimal for expressing a new metabolic pathway. ToMI-FBA relies on genome-scale metabolic flux modeling and a novel in silico cell membrane influx constraint that specifies the flux of atoms (not molecules into the cell through all possible membrane transporters. The ToMI constraint is constructed through the addition of an extra row and column to the stoichiometric matrix of a genome-scale metabolic flux model. In this research, the mathematical formulation of the ToMI constraint is given along with four case studies that demonstrate its usefulness. In Case Study 1, ToMI-FBA returned an optimal culture medium formulation for the production of isobutanol from Bacillus subtilis. Significant levels of L-valine were recommended to optimize production, and this result has been observed experimentally. In Case Study 2, it is demonstrated how the carbon to nitrogen uptake ratio can be specified as an additional ToMI-FBA constraint. This was investigated for maximizing medium chain length polyhydroxyalkanoates (mcl-PHA production from Pseudomonas putida KT2440. In Case Study 3, ToMI-FBA revealed a strategy of adding cellobiose as a means to increase ethanol selectivity during the stationary growth phase of Clostridium acetobutylicum ATCC 824. This strategy was also validated experimentally. Finally, in Case Study 4, B. subtilis was identified as a superior host to Escherichia coli, Saccharomyces cerevisiae, and Synechocystis PCC6803 for the production of artemisinate.

  13. Urinary Metabolic Phenotyping Reveals Differences in the Metabolic Status of Healthy and Inflammatory Bowel Disease (IBD Children in Relation to Growth and Disease Activity

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    Francois-Pierre Martin

    2016-08-01

    Full Text Available Background: Growth failure and delayed puberty are well known features of children and adolescents with inflammatory bowel disease (IBD, in addition to the chronic course of the disease. Urinary metabonomics was applied in order to better understand metabolic changes between healthy and IBD children. Methods: 21 Pediatric patients with IBD (mean age 14.8 years, 8 males were enrolled from the Pediatric Gastroenterology Outpatient Clinic over two years. Clinical and biological data were collected at baseline, 6, and 12 months. 27 healthy children (mean age 12.9 years, 16 males were assessed at baseline. Urine samples were collected at each visit and subjected to 1H Nuclear Magnetic Resonance (NMR spectroscopy. Results: Using 1H NMR metabonomics, we determined that urine metabolic profiles of IBD children differ significantly from healthy controls. Metabolic differences include central energy metabolism, amino acid, and gut microbial metabolic pathways. The analysis described that combined urinary urea and phenylacetylglutamine—two readouts of nitrogen metabolism—may be relevant to monitor metabolic status in the course of disease. Conclusion: Non-invasive sampling of urine followed by metabonomic profiling can elucidate and monitor the metabolic status of children in relation to disease status. Further developments of omic-approaches in pediatric research might deliver novel nutritional and metabolic hypotheses.

  14. Individual Differences in the Phenotypic Flexibility of Basal Metabolic Rate in Siberian Hamsters Are Consistent on Short- and Long-Term Timescales.

    Science.gov (United States)

    Boratyński, Jan S; Jefimow, Małgorzata; Wojciechowski, Michał S

    Basal metabolic rate (BMR) correlates with the cost of life in endothermic animals. It usually differs consistently among individuals in a population, but it may be adjusted in response to predictable or unpredictable changes in the environment. The phenotypic flexibility of BMR is considered an adaptation to living in a stochastic environment; however, whether it is also repeatable it is still unexplored. Assuming that variations in phenotypic flexibility are evolutionarily important, we hypothesized that they are consistently different among individuals. We predicted that not only BMR but also its flexibility in response to changes in ambient temperature (T a ) are repeatable on short- and long-term timescales. To examine this, we acclimated Siberian hamsters (Phodopus sungorus) for 100 d to winterlike and then to summerlike conditions, and after each acclimation we exposed them interchangeably to 10° and 28°C for 14 d. The difference in BMR measured after each exposure defined an individual's phenotypic flexibility (ΔBMR). BMR was repeatable within and among seasons. It was also flexible in both seasons, but in winter this flexibility was lower in individuals responding to seasonal changes than in nonresponding ones. When we accounted for individual responsiveness, the repeatability of ΔBMR was significant in winter (τ = 0.48, P = 0.01) and in summer (τ = 0.55, P = 0.005). Finally, the flexibility of BMR in response to changes in T a was also repeatable on a long-term timescale, that is, among seasons (τ = 0.31, P = 0.008). Our results indicate the evolutionary importance of the phenotypic flexibility of energy metabolism and suggest that it may be subject to selection.

  15. CYP2C9 genotype vs. metabolic phenotype for individual drug dosing--a correlation analysis using flurbiprofen as probe drug.

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    Silvia Vogl

    Full Text Available Currently, genotyping of patients for polymorphic enzymes responsible for metabolic elimination is considered a possibility to adjust drug dose levels. For a patient to profit from this procedure, the interindividual differences in drug metabolism within one genotype should be smaller than those between different genotypes. We studied a large cohort of healthy young adults (283 subjects, correlating their CYP2C9 genotype to a simple phenotyping metric, using flurbiprofen as probe drug. Genotyping was conducted for CYP2C9*1, *2, *3. The urinary metabolic ratio MR (concentration of CYP2C9-dependent metabolite divided by concentration of flurbiprofen determined two hours after flurbiprofen (8.75 mg administration served as phenotyping metric. Linear statistical models correlating genotype and phenotype provided highly significant allele-specific MR estimates of 0.596 for the wild type allele CYP2C9*1, 0.405 for CYP2C9*2 (68 % of wild type, and 0.113 for CYP2C9*3 (19 % of wild type. If these estimates were used for flurbiprofen dose adjustment, taking 100 % for genotype *1/*1, an average reduction to 84 %, 60 %, 68 %, 43 %, and 19 % would result for genotype *1/*2, *1/*3, *2/*2, *2/*3, and *3/*3, respectively. Due to the large individual variation within genotypes with coefficients of variation ≥ 20 % and supposing the normal distribution, one in three individuals would be out of the average optimum dose by more than 20 %, one in 20 would be 40 % off. Whether this problem also applies to other CYPs and other drugs has to be investigated case by case. Our data for the given example, however, puts the benefit of individual drug dosing to question, if it is exclusively based on genotype.

  16. A Diel Flux Balance Model Captures Interactions between Light and Dark Metabolism during Day-Night Cycles in C3 and Crassulacean Acid Metabolism Leaves1[C][W][OPEN

    Science.gov (United States)

    Cheung, C.Y. Maurice; Poolman, Mark G.; Fell, David. A.; Ratcliffe, R. George; Sweetlove, Lee J.

    2014-01-01

    Although leaves have to accommodate markedly different metabolic flux patterns in the light and the dark, models of leaf metabolism based on flux-balance analysis (FBA) have so far been confined to consideration of the network under continuous light. An FBA framework is presented that solves the two phases of the diel cycle as a single optimization problem and, thus, provides a more representative model of leaf metabolism. The requirement to support continued export of sugar and amino acids from the leaf during the night and to meet overnight cellular maintenance costs forces the model to set aside stores of both carbon and nitrogen during the day. With only minimal constraints, the model successfully captures many of the known features of C3 leaf metabolism, including the recently discovered role of citrate synthesis and accumulation in the night as a precursor for the provision of carbon skeletons for amino acid synthesis during the day. The diel FBA model can be applied to other temporal separations, such as that which occurs in Crassulacean acid metabolism (CAM) photosynthesis, allowing a system-level analysis of the energetics of CAM. The diel model predicts that there is no overall energetic advantage to CAM, despite the potential for suppression of photorespiration through CO2 concentration. Moreover, any savings in enzyme machinery costs through suppression of photorespiration are likely to be offset by the higher flux demand of the CAM cycle. It is concluded that energetic or nitrogen use considerations are unlikely to be evolutionary drivers for CAM photosynthesis. PMID:24596328

  17. Knockout of arsenic (+3 oxidation state) methyltransferase is associated with adverse metabolic phenotype in mice: the role of sex and arsenic exposure.

    Science.gov (United States)

    Douillet, Christelle; Huang, Madelyn C; Saunders, R Jesse; Dover, Ellen N; Zhang, Chongben; Stýblo, Miroslav

    2017-07-01

    Susceptibility to toxic effects of inorganic arsenic (iAs) depends, in part, on efficiency of iAs methylation by arsenic (+3 oxidation state) methyltransferase (AS3MT). As3mt-knockout (KO) mice that cannot efficiently methylate iAs represent an ideal model to study the association between iAs metabolism and adverse effects of iAs exposure, including effects on metabolic phenotype. The present study compared measures of glucose metabolism, insulin resistance and obesity in male and female wild-type (WT) and As3mt-KO mice during a 24-week exposure to iAs in drinking water (0.1 or 1 mg As/L) and in control WT and As3mt-KO mice drinking deionized water. Results show that effects of iAs exposure on fasting blood glucose (FBG) and glucose tolerance in either WT or KO mice were relatively minor and varied during the exposure. The major effects were associated with As3mt KO. Both male and female control KO mice had higher body mass with higher percentage of fat than their respective WT controls. However, only male KO mice were insulin resistant as indicated by high FBG, and high plasma insulin at fasting state and 15 min after glucose challenge. Exposure to iAs increased fat mass and insulin resistance in both male and female KO mice, but had no significant effects on body composition or insulin resistance in WT mice. These data suggest that As3mt KO is associated with an adverse metabolic phenotype that is characterized by obesity and insulin resistance, and that the extent of the impairment depends on sex and exposure to iAs, including exposure to iAs from mouse diet.

  18. Characterization of the metabolic phenotype of rapamycin-treated CD8+ T cells with augmented ability to generate long-lasting memory cells.

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    Shan He

    Full Text Available BACKGROUND: Cellular metabolism plays a critical role in regulating T cell responses and the development of memory T cells with long-term protections. However, the metabolic phenotype of antigen-activated T cells that are responsible for the generation of long-lived memory cells has not been characterized. DESIGN AND METHODS: Using lymphocytic choriomeningitis virus (LCMV peptide gp33-specific CD8(+ T cells derived from T cell receptor transgenic mice, we characterized the metabolic phenotype of proliferating T cells that were activated and expanded in vitro in the presence or absence of rapamycin, and determined the capability of these rapamycin-treated T cells to generate long-lived memory cells in vivo. RESULTS: Antigen-activated CD8(+ T cells treated with rapamycin gave rise to 5-fold more long-lived memory T cells in vivo than untreated control T cells. In contrast to that control T cells only increased glycolysis, rapamycin-treated T cells upregulated both glycolysis and oxidative phosphorylation (OXPHOS. These rapamycin-treated T cells had greater ability than control T cells to survive withdrawal of either glucose or growth factors. Inhibition of OXPHOS by oligomycin significantly reduced the ability of rapamycin-treated T cells to survive growth factor withdrawal. This effect of OXPHOS inhibition was accompanied with mitochondrial hyperpolarization and elevation of reactive oxygen species that are known to be toxic to cells. CONCLUSIONS: Our findings indicate that these rapamycin-treated T cells may represent a unique cell model for identifying nutrients and signals critical to regulating metabolism in both effector and memory T cells, and for the development of new methods to improve the efficacy of adoptive T cell cancer therapy.

  19. Metabolic flexibility of d-ribose producer strain of Bacillus pumilus under environmental perturbations

    DEFF Research Database (Denmark)

    Srivastava, Rajesh K.; Maiti, Soumen K.; Das, Debasish

    2012-01-01

    The metabolic reaction rate vector is a bridge that links gene and protein expression alterations to the phenotypic endpoint. We present a simple approach for the estimation of flux distribution at key branch points in the metabolic network by using substrate uptake, metabolite secretion rate, an...

  20. Computational Flux Balance Analysis Predicts that Stimulation of Energy Metabolism in Astrocytes and their Metabolic Interactions with Neurons Depend on Uptake of K(+) Rather than Glutamate

    DEFF Research Database (Denmark)

    DiNuzzo, Mauro; Giove, Federico; Maraviglia, Bruno

    2017-01-01

    Brain activity involves essential functional and metabolic interactions between neurons and astrocytes. The importance of astrocytic functions to neuronal signaling is supported by many experiments reporting high rates of energy consumption and oxidative metabolism in these glial cells...... utilization. In order to examine the participation of astrocytic energy metabolism in brain ion homeostasis, here we attempted to devise a simple stoichiometric relation linking glutamatergic neurotransmission to Na(+) and K(+) ionic currents. To this end, we took into account ion pumps and voltage....../ligand-gated channels using the stoichiometry derived from available energy budget for neocortical signaling and incorporated this stoichiometric relation into a computational metabolic model of neuron-astrocyte interactions. We aimed at reproducing the experimental observations about rates of metabolic pathways...

  1. Co-utilization of glucose and xylose by evolved Thermus thermophilus LC113 strain elucidated by (13)C metabolic flux analysis and whole genome sequencing.

    Science.gov (United States)

    Cordova, Lauren T; Lu, Jing; Cipolla, Robert M; Sandoval, Nicholas R; Long, Christopher P; Antoniewicz, Maciek R

    2016-09-01

    We evolved Thermus thermophilus to efficiently co-utilize glucose and xylose, the two most abundant sugars in lignocellulosic biomass, at high temperatures without carbon catabolite repression. To generate the strain, T. thermophilus HB8 was first evolved on glucose to improve its growth characteristics, followed by evolution on xylose. The resulting strain, T. thermophilus LC113, was characterized in growth studies, by whole genome sequencing, and (13)C-metabolic flux analysis ((13)C-MFA) with [1,6-(13)C]glucose, [5-(13)C]xylose, and [1,6-(13)C]glucose+[5-(13)C]xylose as isotopic tracers. Compared to the starting strain, the evolved strain had an increased growth rate (~2-fold), increased biomass yield, increased tolerance to high temperatures up to 90°C, and gained the ability to grow on xylose in minimal medium. At the optimal growth temperature of 81°C, the maximum growth rate on glucose and xylose was 0.44 and 0.46h(-1), respectively. In medium containing glucose and xylose the strain efficiently co-utilized the two sugars. (13)C-MFA results provided insights into the metabolism of T. thermophilus LC113 that allows efficient co-utilization of glucose and xylose. Specifically, (13)C-MFA revealed that metabolic fluxes in the upper part of metabolism adjust flexibly to sugar availability, while fluxes in the lower part of metabolism remain relatively constant. Whole genome sequence analysis revealed two large structural changes that can help explain the physiology of the evolved strain: a duplication of a chromosome region that contains many sugar transporters, and a 5x multiplication of a region on the pVV8 plasmid that contains xylose isomerase and xylulokinase genes, the first two enzymes of xylose catabolism. Taken together, (13)C-MFA and genome sequence analysis provided complementary insights into the physiology of the evolved strain. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  2. 13C based proteinogenic amino acid (PAA) and metabolic flux ratio analysis of Lactococcus lactis reveals changes in pentose phosphate (PP) pathway in response to agitation and temperature related stresses

    Science.gov (United States)

    2017-01-01

    Lactococcus lactis subsp. cremoris MG1363 is an important starter culture for dairy fermentation. During industrial fermentations, L. lactis is constantly exposed to stresses that affect the growth and performance of the bacterium. Although the response of L. lactis to several stresses has been described, the adaptation mechanisms at the level of in vivo fluxes have seldom been described. To gain insights into cellular metabolism, 13C metabolic flux analysis and gas chromatography mass spectrometry (GC-MS) were used to measure the flux ratios of active pathways in the central metabolism of L. lactis when subjected to three conditions varying in temperature (30°C, 37°C) and agitation (with and without agitation at 150 rpm). Collectively, the concentrations of proteinogenic amino acids (PAAs) and free fatty acids (FAAs) were compared, and Pearson correlation analysis (r) was calculated to measure the pairwise relationship between PAAs. Branched chain and aromatic amino acids, threonine, serine, lysine and histidine were correlated strongly, suggesting changes in flux regulation in glycolysis, the pentose phosphate (PP) pathway, malic enzyme and anaplerotic reaction catalysed by pyruvate carboxylase (pycA). Flux ratio analysis revealed that glucose was mainly converted by glycolysis, highlighting the stability of L. lactis’ central carbon metabolism despite different conditions. Higher flux ratios through oxaloacetate (OAA) from pyruvate (PYR) reaction in all conditions suggested the activation of pyruvate carboxylate (pycA) in L. lactis, in response to acid stress during exponential phase. Subsequently, more significant flux ratio differences were seen through the oxidative and non-oxidative pentose phosphate (PP) pathways, malic enzyme, and serine and C1 metabolism, suggesting NADPH requirements in response to environmental stimuli. These reactions could play an important role in optimization strategies for metabolic engineering in L. lactis. Overall, the

  3. 13C based proteinogenic amino acid (PAA and metabolic flux ratio analysis of Lactococcus lactis reveals changes in pentose phosphate (PP pathway in response to agitation and temperature related stresses

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    Kamalrul Azlan Azizan

    2017-07-01

    Full Text Available Lactococcus lactis subsp. cremoris MG1363 is an important starter culture for dairy fermentation. During industrial fermentations, L. lactis is constantly exposed to stresses that affect the growth and performance of the bacterium. Although the response of L. lactis to several stresses has been described, the adaptation mechanisms at the level of in vivo fluxes have seldom been described. To gain insights into cellular metabolism, 13C metabolic flux analysis and gas chromatography mass spectrometry (GC-MS were used to measure the flux ratios of active pathways in the central metabolism of L. lactis when subjected to three conditions varying in temperature (30°C, 37°C and agitation (with and without agitation at 150 rpm. Collectively, the concentrations of proteinogenic amino acids (PAAs and free fatty acids (FAAs were compared, and Pearson correlation analysis (r was calculated to measure the pairwise relationship between PAAs. Branched chain and aromatic amino acids, threonine, serine, lysine and histidine were correlated strongly, suggesting changes in flux regulation in glycolysis, the pentose phosphate (PP pathway, malic enzyme and anaplerotic reaction catalysed by pyruvate carboxylase (pycA. Flux ratio analysis revealed that glucose was mainly converted by glycolysis, highlighting the stability of L. lactis’ central carbon metabolism despite different conditions. Higher flux ratios through oxaloacetate (OAA from pyruvate (PYR reaction in all conditions suggested the activation of pyruvate carboxylate (pycA in L. lactis, in response to acid stress during exponential phase. Subsequently, more significant flux ratio differences were seen through the oxidative and non-oxidative pentose phosphate (PP pathways, malic enzyme, and serine and C1 metabolism, suggesting NADPH requirements in response to environmental stimuli. These reactions could play an important role in optimization strategies for metabolic engineering in L. lactis. Overall

  4. Human peripheral late/exhausted memory B cells express a senescent-associated secretory phenotype and preferentially utilize metabolic signaling pathways.

    Science.gov (United States)

    Frasca, Daniela; Diaz, Alain; Romero, Maria; Blomberg, Bonnie B

    2017-01-01

    The percentage of late/exhausted memory (LM) B cells increases with age and we show here that this is associated with a lower influenza vaccine response. To identify novel contributors to the phenotypic and functional changes observed in aged B cells, we sorted the major peripheral B cell subsets [naïve, IgM memory, switched memory (swIg) and late/exhausted memory (LM)] and determined their percentages in the peripheral blood as well as their level of immune activation by measuring basal levels of expression of multiple senescence-associated secretory phenotype (SASP) markers, such as pro-inflammatory cytokines (TNF-α/IL-6/IL-8), inflammatory micro-RNAs (miRs, miR-155/16/93), cell cycle regulators (p16 INK4 ). We found that only memory B cells express SASP markers, and especially the LM B cell subset, which is also showing spontaneous activation of AMP-activated protein kinase (AMPK), the energy sensing enzyme which is ubiquitously expressed in mammalian cells. LM B cells, but not IgM memory B cells, activate a p38MAPK signaling pathway, downstream of AMPK, leading to the expression of SASP mediators, while class switch recombination is downregulated. These data show that some B cell subsets are more inflammatory than others, that they are pre-activated and that this signaling through metabolic pathways is associated with a senescence phenotype, demonstrating for the first time in human B lymphocytes the link between aging, cellular senescence, SASP and metabolism. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Obesity-induced discrepancy between contractile and metabolic phenotypes in slow- and fast-twitch skeletal muscles of female obese Zucker rats.

    Science.gov (United States)

    Acevedo, Luz M; Raya, Ana I; Ríos, Rafael; Aguilera-Tejero, Escolástico; Rivero, José-Luis L

    2017-07-01

    A clear picture of skeletal muscle adaptations to obesity and related comorbidities remains elusive. This study describes fiber-type characteristics (size, proportions, and oxidative enzyme activity) in two typical hindlimb muscles with opposite structure and function in an animal model of genetic obesity. Lesser fiber diameter, fiber-type composition, and histochemical succinic dehydrogenase activity (an oxidative marker) of muscle fiber types were assessed in slow (soleus)- and fast (tibialis cranialis)-twitch muscles of obese Zucker rats and compared with age (16 wk)- and sex (females)-matched lean Zucker rats ( n = 16/group). Muscle mass and lesser fiber diameter were lower in both muscle types of obese compared with lean animals even though body weights were increased in the obese cohort. A faster fiber-type phenotype also occurred in slow- and fast-twitch muscles of obese rats compared with lean rats. These adaptations were accompanied by a significant increment in histochemical succinic dehydrogenase activity of slow-twitch fibers in the soleus muscle and fast-twitch fiber types in the tibialis cranialis muscle. Obesity significantly increased plasma levels of proinflammatory cytokines but did not significantly affect protein levels of peroxisome proliferator-activated receptors PPARγ or PGC1α in either muscle. These data demonstrate that, in female Zucker rats, obesity induces a reduction of muscle mass in which skeletal muscles show a diminished fiber size and a faster and more oxidative phenotype. It was noteworthy that this discrepancy in muscle's contractile and metabolic features was of comparable nature and extent in muscles with different fiber-type composition and antagonist functions. NEW & NOTEWORTHY This study demonstrates a discrepancy between morphological (reduced muscle mass), contractile (shift toward a faster phenotype), and metabolic (increased mitochondrial oxidative enzyme activity) characteristics in skeletal muscles of female Zucker

  6. Genome and Phenotype Microarray Analyses of Rhodococcus sp. BCP1 and Rhodococcus opacus R7: Genetic Determinants and Metabolic Abilities with Environmental Relevance.

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    Alessandro Orro

    Full Text Available In this paper comparative genome and phenotype microarray analyses of Rhodococcus sp. BCP1 and Rhodococcus opacus R7 were performed. Rhodococcus sp. BCP1 was selected for its ability to grow on short-chain n-alkanes and R. opacus R7 was isolated for its ability to grow on naphthalene and on o-xylene. Results of genome comparison, including BCP1, R7, along with other Rhodococcus reference strains, showed that at least 30% of the genome of each strain presented unique sequences and only 50% of the predicted proteome was shared. To associate genomic features with metabolic capabilities of BCP1 and R7 strains, hundreds of different growth conditions were tested through Phenotype Microarray, by using Biolog plates and plates manually prepared with additional xenobiotic compounds. Around one-third of the surveyed carbon sources was utilized by both strains although R7 generally showed higher metabolic activity values compared to BCP1. Moreover, R7 showed broader range of nitrogen and sulphur sources. Phenotype Microarray data were combined with genomic analysis to genetically support the metabolic features of the two strains. The genome analysis allowed to identify some gene clusters involved in the metabolism of the main tested xenobiotic compounds. Results show that R7 contains multiple genes for the degradation of a large set of aromatic and PAHs compounds, while a lower variability in terms of genes predicted to be involved in aromatic degradation was found in BCP1. This genetic feature can be related to the strong genetic pressure exerted by the two different environment from which the two strains were isolated. According to this, in the BCP1 genome the smo gene cluster involved in the short-chain n-alkanes degradation, is included in one of the unique regions and it is not conserved in the Rhodococcus strains compared in this work. Data obtained underline the great potential of these two Rhodococcus spp. strains for biodegradation and

  7. Effects of high-intensity interval versus mild-intensity endurance training on metabolic phenotype and corticosterone response in rats fed a high-fat or control diet.

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    Youqing Shen

    Full Text Available The aim of the present study was to compare the effects of high-intensity interval training (HI to mild-intensity endurance training (ME, combined with a high-fat diet (HFD or control diet (CD on metabolic phenotype and corticosterone levels in rats. Fifty-three rats were randomized to 6 groups according to diet and training regimen as follows: CD and sedentary (CS, n = 11, CD and ME (CME, n = 8, CD and HI (CHI, n = 8, HFD and sedentary (HS, n = 10, HFD and ME (HME, n = 8, and HFD and HI (HHI, n = 8. All exercise groups were trained for 10 weeks and had matched running distances. Dietary intake, body composition, blood metabolites, and corticosterone levels were measured. Histological lipid droplets were observed in the livers. The HFD led to hyperglycemia, hyperlipidemia and higher body fat (all, P 0.06, as well as higher corticosterone levels (P < 0.01, η2 = 0.09 compared with the CD groups. Exercise training improved fat weight, glucose, and lipid profiles, and reduced corticosterone levels (P < 0.01, η2 = 0.123. Furthermore, body and fat weight, serum glucose and triglycerides, lipid content in the liver, and corticosterone levels (P < 0.05 were lower with HI training compared to ME training. Reductions in HFD-induced body weight gain, blood glucose and lipid profiles, and corticosterone levels, as well as improvements in QUICKI were better with HHI compared to HME. Correlation analyses revealed that corticosterone levels were significantly associated with phenotype variables (P < 0.01. Corticosterone level was inversely correlated with QUICKI (r = -0.38, P < 0.01. Altogether, these results indicate that HFD may elicit an exacerbated basal serum corticosterone level and thus producing a metabolic imbalance. Compared with ME training, HI training contributes to greater improvements in metabolic and corticosterone responses, leading to a greater reduction in susceptibility to HFD-induced disorders.

  8. Enzyme oscillation can enhance the thermodynamic efficiency of cellular metabolism: consequence of anti-phase coupling between reaction flux and affinity

    Science.gov (United States)

    Himeoka, Yusuke; Kaneko, Kunihiko

    2016-04-01

    Cells generally convert nutrient resources to products via energy transduction. Accordingly, the thermodynamic efficiency of this conversion process is one of the most essential characteristics of living organisms. However, although these processes occur under conditions of dynamic metabolism, most studies of cellular thermodynamic efficiency have been restricted to examining steady states; thus, the relevance of dynamics to this efficiency has not yet been elucidated. Here, we develop a simple model of metabolic reactions with anabolism-catabolism coupling catalyzed by enzymes. Through application of external oscillation in the enzyme abundances, the thermodynamic efficiency of metabolism was found to be improved. This result is in strong contrast with that observed in the oscillatory input, in which the efficiency always decreased with oscillation. This improvement was effectively achieved by separating the anabolic and catabolic reactions, which tend to disequilibrate each other, and taking advantage of the temporal oscillations so that each of the antagonistic reactions could progress near equilibrium. In this case, anti-phase oscillation between the reaction flux and chemical affinity through oscillation of enzyme abundances is essential. This improvement was also confirmed in a model capable of generating autonomous oscillations in enzyme abundances. Finally, the possible relevance of the improvement in thermodynamic efficiency is discussed with respect to the potential for manipulation of metabolic oscillations in microorganisms.

  9. Urinary phenotyping indicates weight loss-independent metabolic effects of Roux-en-Y gastric bypass in mice.

    Science.gov (United States)

    Seyfried, Florian; Li, Jia V; Miras, Alexander D; Cluny, Nina L; Lannoo, Matthias; Fenske, Wiebke K; Sharkey, Keith A; Nicholson, Jeremy K; le Roux, Carel W; Holmes, Elaine

    2013-03-01

    Patients with a body mass index (BMI) above 35 kg/m(2) with metabolic diseases benefit from Roux-en-Y gastric bypass (RYGB) independently of their final BMI and the amount of body weight lost. However, the weight loss independent metabolic effects induced by RYGB remain less well understood. To elucidate metabolic changes after RYGB, (1)H NMR spectroscopy-based urine metabolic profiles from RYGB (n = 7), ad libitum-fed sham (AL, n = 5), and body-weight-matched sham (BWM, n = 5) operated mice were obtained. Gut morphometry and fecal energy content were analyzed. Food intake and body weight of RYGB mice were significantly reduced (p = 0.001) compared to sham-AL. There was a strong tendency that BWM-shams required less food to maintain the same body weight as RYGB mice (p = 0.05). No differences were found in fecal energy content between the groups, excluding malabsorption in RYGB animals. Unlike RYGB-operated rats, gut hypertrophy was not observed in RYGB-operated mice. Urinary tricarboxylic acid cycle intermediates were higher in the sham groups, suggesting altered mitochondrial metabolism after RYGB surgery. Higher urinary levels of trimethylamine, hippurate and trigonelline in RYGB mice indicate that the RYGB operation caused microbial disturbance. Taken together, we demonstrate for the first time that there are RYGB specific metabolic effects, which are independent of food intake and body weight loss. Increased utilization of TCA cycle intermediates and altered gut microbial-host co-metabolites might indicate increased energy expenditure and microbial changes in the gut, respectively.

  10. Impact of genetic polymorphisms of SLC2A2, SLC2A5, and KHK on metabolic phenotypes in hypertensive individuals.

    Directory of Open Access Journals (Sweden)

    MyPhuong T Le

    Full Text Available In the past few decades, consumption of added sugars has increased dramatically. Studies have linked high sugar intake with increased risk for a number of diseases. Importantly, fructose, a component of sugar, has been linked with the development of features of metabolic syndrome. This study determined if single nucleotide polymorphisms in genes involved in fructose transport (solute carrier family 2 facilitated glucose transporter, member 2 (SLC2A2 and solute carrier family 2 facilitated glucose/fructose transporter, member 5 (SLC2A5 and metabolism (ketohexokinase (KHK affect inter-individual variability in metabolic phenotypes, such as increased serum uric acid levels.The influence of SLC2A2, SLC2A5, and KHK SNPs on metabolic phenotypes was tested in 237 European Americans and 167 African Americans from the Pharmacogenomic Evaluation and Antihypertensive Responses (PEAR study. Using baseline untreated fasting data, associations were considered significant if p≤0.005. These SNPs were then evaluated for potential replication (p≤0.05 using data from the Genetic Epidemiology of Responses to Antihypertensives (GERA studies.SLC2A5 rs5438 was associated with an increase in serum uric acid in European American males. However, we were unable to replicate the association in GERA. The minor allele of SLC2A2 rs8192675 showed an association with lower high-density lipoproteins in European Americans (A/A: 51.0 mg/dL, A/G: 47.0 mg/dL, G/G: 41.5 mg/dL, p = 0.0034 in PEAR. The association between rs8192675 and lower high-density lipoproteins was replicated in the combined European American GERA study samples (A/A: 47.6 mg/dL, A/G: 48.6 mg/dL, G/G: 41.9 mg/dL, p = 0.0315.The association between SLC2A2 rs8192675 and high-density lipoproteins suggests the polymorphism may play a role in influencing high-density lipoproteins and thus metabolic risk of cardiovascular disease.

  11. Coexpression network analysis in abdominal and gluteal adipose tissue reveals regulatory genetic loci for metabolic syndrome and related phenotypes

    DEFF Research Database (Denmark)

    Min, Josine L; Nicholson, George; Halgrimsdottir, Ingileif

    2012-01-01

    Metabolic Syndrome (MetS) is highly prevalent and has considerable public health impact, but its underlying genetic factors remain elusive. To identify gene networks involved in MetS, we conducted whole-genome expression and genotype profiling on abdominal (ABD) and gluteal (GLU) adipose tissue, ...

  12. Evaluating the role of a galanin enhancer genotype on a range of metabolic, depressive and addictive phenotypes

    NARCIS (Netherlands)

    Richardson, T.G.; Minica, C.C.; Heron, J.; Tavare, J.; MacKenzie, A.; Day, I.; Lewis, G.; Hickman, M.; Vink, J.M.; Gelernter, J.; Kranzler, H.R.; Farrer, L.A.; Munafò, M.; Wynick, D.

    2014-01-01

    There is a large body of pre-clinical and some clinical data to link the neuropeptide galanin to a range of physiological and pathological functions that include metabolism, depression, and addiction. An enhancer region upstream of the human GAL transcriptional start site has previously been

  13. Evaluating the role of a Galanin Enhancer Genotype on a range of metabolic, depressive and addictive phenotypes

    NARCIS (Netherlands)

    Richardson, T.G.; Minica, C.C.; Heron, J.; Tavare, J.; MacKenzie, A.; Day, I.; Lewis, G.; Hickman, M.; Vink, J.M.; Gelernter, J.; Kranzler, H.R.; Farrer, L.A.; Munafò, M.R.; Wynick, D.

    2014-01-01

    There is a large body of pre-clinical and some clinical data to link the neuropeptide galanin to a range of physiological and pathological functions that include metabolism, depression, and addiction. An enhancer region upstream of the human GAL transcriptional start site has previously been

  14. Studies of the common DIO2 Thr92Ala polymorphism and metabolic phenotypes in 7342 Danish white subjects

    DEFF Research Database (Denmark)

    Grarup, Niels; Andersen, Mette K; Andreasen, Camilla H

    2007-01-01

    The type 2 iodothyronine deiodinase (D2) catalyzes the conversion of T(4) to the active form of thyroid hormone, which is a critical regulator of thermogenesis and glucose metabolism. A Thr92Ala polymorphism in the gene encoding D2 (DIO2) has been reported to associate with insulin resistance....

  15. A comparative study on genetic and environmental influences on metabolic phenotypes in Eastern (Chinese) and Western (Danish) populations

    DEFF Research Database (Denmark)

    Li, Shuxia

    2015-01-01

    During the last decades metabolic disorders such as high blood pressure, impaired blood glycose, atherosclerotic lipid abnormalities, overweight/obesity and elevated blood pressure are among the leading risks for mortality and morbidity worldwide. Those risks are mostly responsible for raising...

  16. Squeezing Flux Out of Fat

    DEFF Research Database (Denmark)

    Gonzalez-Franquesa, Alba; Patti, Mary-Elizabeth

    2018-01-01

    Merging transcriptomics or metabolomics data remains insufficient for metabolic flux estimation. Ramirez et al. integrate a genome-scale metabolic model with extracellular flux data to predict and validate metabolic differences between white and brown adipose tissue. This method allows both metab...

  17. Pathway confirmation and flux analysis of central metabolic pathways in Desulfovibrio vulgaris Hildenborough using Gas Chromatography-Mass Spectrometry and Fourier Transform-Ion Cyclotron Resonance Mass Spectrometry

    International Nuclear Information System (INIS)

    Tang, Yinjie; Pingitore, Francesco; Mukhopadhyay, Aindrila; Phan, Richard; Hazen, Terry C.; Keasling, Jay D.

    2007-01-01

    Flux distribution in central metabolic pathways of Desulfovibrio vulgaris Hildenborough was examined using 13C tracer experiments. Consistent with the current genome annotation and independent evidence from enzyme activity assays, the isotopomer results from both GC-MS and Fourier Transform-Ion Cyclotron Resonance mass spectrometry (FT-ICR MS) indicate the lack of oxidatively functional TCA cycle and an incomplete pentose phosphate pathway. Results from this study suggest that fluxes through both pathways are limited to biosynthesis. The data also indicate that >80 percent of the lactate was converted to acetate and the reactions involved are the primary route of energy production (NAD(P)H and ATP production). Independent of the TCA cycle, direct cleavage of acetyl-CoA to CO and 5,10-methyl-THF also leads to production of NADH and ATP. Although the genome annotation implicates a ferredoxin-dependent oxoglutarate synthase, isotopic evidence does not support flux through this reaction in either the oxidative or reductive mode; therefore, the TCA cycle is incomplete. FT-ICR MS was used to locate the labeled carbon distribution in aspartate and glutamate and confirmed the presence of an atypical enzyme for citrate formation suggested in previous reports (the citrate synthesized by this enzyme is the isotopic antipode of the citrate synthesized by the (S)-citrate synthase). These findings enable a better understanding of the relation between genome annotation and actual metabolic pathways in D. vulgaris, and also demonstrate FT-ICR MS as a powerful tool for isotopomer analysis, overcoming problems in both GC-MS and NMR spectroscopy

  18. Integrated stress response activation by sleep fragmentation during late gestation in mice leads to emergence of adverse metabolic phenotype in offspring.

    Science.gov (United States)

    Trzepizur, Wojciech; Khalyfa, Abdelnaby; Qiao, Zhuanhong; Popko, Brian; Gozal, David

    2017-04-01

    Late gestational sleep fragmentation (SF) is highly prevalent particularly in obese women, and induces metabolic dysfunction in adult offspring mice. SF induces activation of the integrated stress response (ISR), which might be involved in metabolic disorders. We hypothesized that adult offspring of double mutant mice (DM) involving the critical ISR genes CHOP and GADD34 would be protected from developing obesity and insulin resistance following SF. Time-pregnant CHOP/GADD34 DM and wild type (WT) mice were randomly assigned to sleep control (SC) or SF conditions during the last 5days of gestation. At 24-weeks of age, body weight, fat mass, and HOMA-IR were assessed in the offspring. Tregs lymphocytes, Lyc6c high , M1 and M2 macrophages were examined in visceral white adipose tissues (vWAT) using flow cytometry. The effects of plasma exosomes on adipocyte cell line proliferation, differentiation and insulin sensitivity were also evaluated. SF-WT male showed significant increases in body weight, vWAT mass and HOMA-IR compared to SC-WT mice, while SF had no effect in SF-DM mice. Inflammatory macrophages (Ly-6c high ) and the ratio of M1/M2 macrophages were increased while FoxP3+ Tregs counts were decreased in SF-WT but not in SF-DM mice. Exosomes from SF-WT, but not from the SF-DM offspring increased pre-adipocyte proliferation and differentiation, and decreased in vitro adipocyte insulin sensitivity. Activation of the ISR during late gestation, as induced by late gestational SF, appears to underlie some of the transgenerational modifications in metabolic genes ultimately contributing to a metabolic syndrome phenotype in adult offspring. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. The new total Western diet for rodents does not induce an overweight phenotype or alter parameters of metabolic syndrome in mice.

    Science.gov (United States)

    Monsanto, Stephany P; Hintze, Korry J; Ward, Robert E; Larson, Deanna P; Lefevre, Michael; Benninghoff, Abby D

    2016-09-01

    In this study, we determined the impact of the total Western diet (TWD) for rodents and its macro- and micronutrient components on weight gain and biomarkers of metabolic function in mice compared to a 45% fat diet-induced obesity (DIO) diet and the standard AIN93G diet. We hypothesized that mice fed the TWD would have increased body fat with indicators of metabolic syndrome similar to mice consuming the DIO diet. As expected, DIO-fed mice acquired a metabolic syndrome phenotype typified by increased energy intake, increased body weight gain, increased fat mass, higher fasting glucose, impaired glucose tolerance, and higher plasma leptin relative to the AIN93G diet. Mice fed a macronutrient-modified (MM) diet (with standard vitamin and mineral composition) had a similar response, albeit to a lesser degree than mice fed the DIO diet. Mice fed a vitamin- and mineral-modified diet (with standard macronutrient composition) were not different from mice fed the AIN93G diet. Surprisingly, the TWD (with modified macronutrients, vitamins and minerals) did not significantly affect any of these parameters, despite the fact that the TWD macronutrient profile was identical to the MM diet. These data suggest that, in the context of the TWD, vitamin and mineral intakes in mice that reflect a Western dietary pattern inhibit the hyperphagia and resulting increased weight gain associated with the higher fat content of the TWD. In conclusion, these observations underscore the need to consider the influence of micronutrient intakes in pre-clinical models of obesity and metabolic syndrome. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Sublethal Concentrations Of Antibiotics Cause Shift To Anaerobic Metabolism In Listeria Monocytogenes And Induce Phenotypes Linked To Antibiotic Tolerance

    DEFF Research Database (Denmark)

    Knudsen, Gitte Maegaard; Ng, Yin; Gram, Lone

    2015-01-01

    to the coexistence with antibiotic-producing organisms during its saprophytic lifestyle. To determine if tolerance could be induced or potentially alter virulence, we investigated the transcriptome after exposure to sublethal antibiotic concentrations. Results: Four antibiotics caused induction of the alcohol...... dehydrogenase gene lmo1634 and repression of alsA and lmo1992, which are involved in acetoin production leading to more ethanol and less acetoin production. This shift in central metabolism indicates a shift from aerobic to anaerobic metabolism, that could reduce oxidative stress and be a survival strategy...... in response to antibiotics. We investigated the antibiotic tolerance of a Δlmo1634 mutant, however; it was comparable with the wild-type in a killing assay. L. monocytogenes encodes a second alcohol dehydrogenase lmo1179, which potentially could cause a redundant pathway and this is under further...

  1. Relationship between murine Ah phenotype and the hepatic metabolism of 2,3,7,8-tetrachlorodibenzo-P-dioxin (TCDD)

    International Nuclear Information System (INIS)

    Shen, E.S.; Olson, J.R.

    1986-01-01

    The Ah receptor has been correlated with the toxic effects of TCDD in C57BL/6J (B6) and DBA/2J (D2) mice. The B6 strain, which has a high affinity cytosolic Ah receptor, is more sensitive to TCDD than the D2 strain, which lacks this receptor. The metabolism of TCDD was studied by incubating 14 C-TCDD (2.2 μM) with hepatocytes from control and TCDD-pretreated B6 and D2 mice. Mice were pretreated with TCDD at doses that maximally induce ethoxyresorufin-O-deethylase (EROD) activity, a measure of Ah locus responsiveness to TCDD (B6, 3μg/kg, ip;D2, 30μg/kg, ip). Similar cytochrome P-450 content was detected in control B6 and D2 hepatocytes, however, TCDD pretreatment increased P-450 content 400% in B6 and 300% in D2 mice. No difference in hepatic EROD activity was found between control B6 and D2 mice (81.7 and 101.7 pmol/min/nmol P-450, respectively), but EROD activity was increased 17-fold in B6 and 10-fold in D2 mice after TCDD administration. The average rate of hepatic TCDD metabolism over two hours was similar in control B6 and D2 mice (1.103 and 0.945 pmol/hr/mg cell protein, respectively), although some qualitative differences in the metabolites were detected by HPLC. TCDD pretreatment produced no quantitative or qualitative changes in TCDD metabolism. These results suggest that the rate of hepatic TCDD metabolism does not correlate with genetic differences at the Ah locus

  2. Leaf Rolling and Stem Fasciation in Grass Pea (Lathyrus sativus L. Mutant Are Mediated through Glutathione-Dependent Cellular and Metabolic Changes and Associated with a Metabolic Diversion through Cysteine during Phenotypic Reversal

    Directory of Open Access Journals (Sweden)

    Dibyendu Talukdar

    2014-01-01

    Full Text Available A Lathyrus sativus L. mutant isolated in ethylmethane sulfonate-treated M2 progeny of mother variety BioL-212 and designated as rlfL-1 was characterized by inwardly rolled-leaf and stem and bud fasciations. The mutant exhibited karyomorphological peculiarities in both mitosis and meiosis with origin of aneuploidy. The mitosis was vigorous with high frequency of divisional cells and their quick turnover presumably steered cell proliferations. Significant transcriptional upregulations of cysteine and glutathione synthesis and concomitant stimulations of glutathione-mediated antioxidant defense helped rlfL-1 mutant to maintain balanced reactive oxygen species (ROS metabolisms, as deduced by ROS-imaging study. Glutathione synthesis was shut down in buthionine sulfoximine- (BSO- treated mother plant and mutant, and leaf-rolling and stems/buds fasciations in the mutant were reversed, accompanied by normalization of mitotic cell division process. Antioxidant defense was downregulated under low glutathione-redox but cysteine-desulfurations and photorespiratory glycolate oxidase transcripts were markedly overexpressed, preventing cysteine overaccumulation but resulted in excess H2O2 in BSO-treated mutant. This led to oxidative damage in proliferating cells, manifested by severe necrosis in rolled-leaf and fasciated stems. Results indicated vital role of glutathione in maintaining abnormal proliferations in plant organs, and its deficiency triggered phenotypic reversal through metabolic diversions of cysteine and concomitant cellular and metabolic modulations.

  3. Deleted in breast cancer 1 limits adipose tissue fat accumulation and plays a key role in the development of metabolic syndrome phenotype.

    Science.gov (United States)

    Escande, Carlos; Nin, Veronica; Pirtskhalava, Tamar; Chini, Claudia C S; Tchkonia, Tamar; Kirkland, James L; Chini, Eduardo N

    2015-01-01

    Obesity is often regarded as the primary cause of metabolic syndrome. However, many lines of evidence suggest that obesity may develop as a protective mechanism against tissue damage during caloric surplus and that it is only when the maximum fat accumulation capacity is reached and fatty acid spillover occurs into to peripheral tissues that metabolic diseases develop. In this regard, identifying the molecular mechanisms that modulate adipocyte fat accumulation and fatty acid spillover is imperative. Here we identify the deleted in breast cancer 1 (DBC1) protein as a key regulator of fat storage capacity of adipocytes. We found that knockout (KO) of DBC1 facilitated fat cell differentiation and lipid accumulation and increased fat storage capacity of adipocytes in vitro and in vivo. This effect resulted in a "healthy obesity" phenotype. DBC1 KO mice fed a high-fat diet, although obese, remained insulin sensitive, had lower free fatty acid in plasma, were protected against atherosclerosis and liver steatosis, and lived longer. We propose that DBC1 is part of the molecular machinery that regulates fat storage capacity in adipocytes and participates in the "turn-off" switch that limits adipocyte fat accumulation and leads to fat spillover into peripheral tissues, leading to the deleterious effects of caloric surplus. © 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

  4. Divergent Urinary Metabolic Phenotypes between Major Depressive Disorder and Bipolar Disorder Identified by a Combined GC-MS and NMR Spectroscopic Metabonomic Approach.

    Science.gov (United States)

    Chen, Jian-Jun; Zhou, Chan-Juan; Liu, Zhao; Fu, Yu-Ying; Zheng, Peng; Yang, De-Yu; Li, Qi; Mu, Jun; Wei, You-Dong; Zhou, Jing-Jing; Huang, Hua; Xie, Peng

    2015-08-07

    Bipolar disorder (BD) is a complex debilitating mental disorder that is often misdiagnosed as major depressive disorder (MDD). Therefore, a large percentage of BD subjects are incorrectly treated with antidepressants in clinical practice. To address this challenge, objective laboratory-based tests are needed to discriminate BD from MDD patients. Here, a combined gas chromatography-mass spectrometry (GC-MS)-based and nuclear magnetic resonance (NMR) spectroscopic-based metabonomic approach was performed to profile urine samples from 76 MDD and 43 BD subjects (training set) to identify the differential metabolites. Samples from 126 healthy controls were included as metabolic controls. A candidate biomarker panel was identified by further analyzing these differential metabolites. A testing set of, 50 MDD and 28 BD subjects was then used to independently validate the diagnostic efficacy of the identified panel using an area under the receiver operating characteristic curve (AUC). A total of 20 differential metabolites responsible for the discrimination between MDD and BD subjects were identified. A panel consisting of six candidate urinary metabolite biomarkers (propionate, formate, (R*,S*)2,3-dihydroxybutanoic acid, 2,4-dihydroxypyrimidine, phenylalanine, and β-alanine) was identified. This panel could distinguish BD from MDD subjects with an AUC of 0.913 and 0.896 in the training and testing sets, respectively. These results reveal divergent urinary metabolic phenotypes between MDD and BD. The identified urinary biomarkers can aid in the future development of an objective laboratory-based diagnostic test for distinguishing BD from MDD patients.

  5. Association of Markers of Proinflammatory Phenotype and Beige Adipogenesis with Metabolic Syndrome in Chinese Centrally Obese Adults

    Directory of Open Access Journals (Sweden)

    Wilson K. C. Leung

    2018-01-01

    Full Text Available Background. Visceral adiposity is associated with higher productions of C-reactive protein (CRP and interleukin-6 (IL-6. Inflammation of obese adipose tissues could contribute to systemic metabolic dysregulation, especially thermogenic activity of white adipose tissues, namely, beige adipogenesis, characterized by altered irisin expression. Thus, we investigated the roles of inflammation and adipocyte beiging in Chinese centrally obese (CO adults with metabolic syndrome (MetS. Methods. This cross-sectional study was conducted on 54 CO and 58 non-CO subjects drawn from 1492 Chinese people with age and sex matched during November 2010 and August 2013. Twenty (37.0% of the CO subjects fulfilled the IDF worldwide definition of MetS. Serum CRP, IL-6, and irisin levels were examined. Results. Higher CRP and IL-6, but lower irisin, levels were manifested in MetS versus non-MetS subjects with or without CO. Multiple linear regression identified high-density lipoprotein cholesterol level as the only independent risk factor for irisin level. Categorized by median of CRP and IL-6 levels, a lower irisin level was only observed in high CRP group. Conclusion. Under the condition of central obesity, chronic inflammation and impaired beige adipogenesis are associated with MetS in Chinese adults.

  6. Eplerenone ameliorates the phenotypes of metabolic syndrome with NASH in liver-specific SREBP-1c Tg mice fed high-fat and high-fructose diet.

    Science.gov (United States)

    Wada, Tsutomu; Miyashita, Yusuke; Sasaki, Motohiro; Aruga, Yusuke; Nakamura, Yuto; Ishii, Yoko; Sasahara, Masakiyo; Kanasaki, Keizo; Kitada, Munehiro; Koya, Daisuke; Shimano, Hitoshi; Tsuneki, Hiroshi; Sasaoka, Toshiyasu

    2013-12-01

    Because the renin-angiotensin-aldosterone system has been implicated in the development of insulin resistance and promotion of fibrosis in some tissues, such as the vasculature, we examined the effect of eplerenone, a selective mineralocorticoid receptor (MR) antagonist, on nonalcoholic steatohepatitis (NASH) and metabolic phenotypes in a mouse model reflecting metabolic syndrome in humans. We adopted liver-specific transgenic (Tg) mice overexpressing the active form of sterol response element binding protein-1c (SREBP-1c) fed a high-fat and fructose diet (HFFD) as the animal model in the present study. When wild-type (WT) C57BL/6 and liver-specific SREBP-1c Tg mice grew while being fed HFFD for 12 wk, body weight and epididymal fat weight increased in both groups with an elevation in blood pressure and dyslipidemia. Glucose intolerance and insulin resistance were also observed. Adipose tissue hypertrophy and macrophage infiltration with crown-like structure formation were also noted in mice fed HFFD. Interestingly, the changes noted in both genotypes fed HFFD were significantly ameliorated with eplerenone. HFFD-fed Tg mice exhibited the histological features of NASH in the liver, including macrovesicular steatosis and fibrosis, whereas HFFD-fed WT mice had hepatic steatosis without apparent fibrotic changes. Eplerenone effectively ameliorated these histological abnormalities. Moreover, the direct suppressive effects of eplerenone on lipopolysaccharide-induced TNFα production in the presence and absence of aldosterone were observed in primary-cultured Kupffer cells and bone marrow-derived macrophages. These results indicated that eplerenone prevented the development of NASH and metabolic abnormalities in mice by inhibiting inflammatory responses in both Kupffer cells and macrophages.

  7. Metabolism

    Science.gov (United States)

    ... functions: Anabolism (uh-NAB-uh-liz-um), or constructive metabolism, is all about building and storing. It ... in infants and young children. Hypothyroidism slows body processes and causes fatigue (tiredness), slow heart rate, excessive ...

  8. Metabolism

    Science.gov (United States)

    ... a particular food provides to the body. A chocolate bar has more calories than an apple, so ... acid phenylalanine, needed for normal growth and protein production). Inborn errors of metabolism can sometimes lead to ...

  9. Effects of Citrus aurantium (bitter orange) fruit extracts and p-synephrine on metabolic fluxes in the rat liver.

    Science.gov (United States)

    Peixoto, Jéssica Sereno; Comar, Jurandir Fernando; Moreira, Caroline Tessaro; Soares, Andréia Assunção; de Oliveira, Andrea Luiza; Bracht, Adelar; Peralta, Rosane Marina

    2012-05-16

    The fruit extracts of Citrus aurantium (bitter orange) are traditionally used as weight-loss products and as appetite supressants. An important fruit component is p-synephrine, which is structurally similar to the adrenergic agents. Weight-loss and adrenergic actions are always related to metabolic changes and this work was designed to investigate a possible action of the C. aurantium extract on liver metabolism. The isolated perfused rat liver was used to measure catabolic and anabolic pathways, including oxygen uptake and perfusion pressure. The C. aurantium extract and p-synephrine increased glycogenolysis, glycolysis, oxygen uptake and perfusion pressure. These changes were partly sensitive to α- and β-adrenergic antagonists. p-Synephrine (200 μM) produced an increase in glucose output that was only 15% smaller than the increment caused by the extract containing 196 μM p-synephrine. At low concentrations the C. aurantium extract tended to increase gluconeogenesis, but at high concentrations it was inhibitory, opposite to what happened with p-synephrine. The action of the C. aurantium extract on liver metabolism is similar to the well known actions of adrenergic agents and can be partly attributed to its content in p-synephrine. Many of these actions are catabolic and compatible with the weight-loss effects usually attributed to C. aurantium.

  10. Studies of association of AGPAT6 variants with type 2 diabetes and related metabolic phenotypes in 12,068 Danes

    DEFF Research Database (Denmark)

    Snogdal, Lena Sønder; Grarup, Niels; Banasik, Karina

    2013-01-01

    Type 2 diabetes, obesity and insulin resistance are characterized by hypertriglyceridemia and ectopic accumulation of lipids in liver and skeletal muscle. AGPAT6 encodes a novel glycerol-3 phosphate acyltransferase, GPAT4, which catalyzes the first step in the de novo triglyceride synthesis. AGPA......-deficient mice show lower weight and resistance to diet- and genetically induced obesity. Here, we examined whether common or low-frequency variants in AGPAT6 associate with type 2 diabetes or related metabolic traits in a Danish population.......Type 2 diabetes, obesity and insulin resistance are characterized by hypertriglyceridemia and ectopic accumulation of lipids in liver and skeletal muscle. AGPAT6 encodes a novel glycerol-3 phosphate acyltransferase, GPAT4, which catalyzes the first step in the de novo triglyceride synthesis. AGPAT6...

  11. Sublethal Concentrations of Antibiotics Cause Shift to Anaerobic Metabolism in Listeria monocytogenes and Induce Phenotypes Linked to Antibiotic Tolerance

    DEFF Research Database (Denmark)

    Knudsen, Gitte Maegaard; Fromberg, Arvid; Ng, Yin

    2016-01-01

    The human pathogenic bacterium Listeria monocytogenes is exposed to antibiotics both during clinical treatment and in its saprophytic lifestyle. As one of the keys to successful treatment is continued antibiotic sensitivity, the purpose of this study was to determine if exposure to sublethal...... antibiotic concentrations would affect the bacterial physiology and induce antibiotic tolerance. Transcriptomic analyses demonstrated that each of the four antibiotics tested caused an antibiotic-specific gene expression pattern related to mode-of-action of the particular antibiotic. All four antibiotics...... in Imo1179 (eutE) encoding an aldehyde oxidoreductase where rerouting caused increased ethanol production was tolerant to three of four antibiotics tested. This shift in metabolism could be a survival strategy in response to antibiotics to avoid generation of ROS production from respiration by oxidation...

  12. Lipid metabolic perturbation is an early-onset phenotype in adult spinster mutants: a Drosophila model for lysosomal storage disorders.

    Science.gov (United States)

    Hebbar, Sarita; Khandelwal, Avinash; Jayashree, R; Hindle, Samantha J; Chiang, Yin Ning; Yew, Joanne Y; Sweeney, Sean T; Schwudke, Dominik

    2017-12-15

    Intracellular accumulation of lipids and swollen dysfunctional lysosomes are linked to several neurodegenerative diseases, including lysosomal storage disorders (LSD). Detailed characterization of lipid metabolic changes in relation to the onset and progression of neurodegeneration is currently missing. We systematically analyzed lipid perturbations in spinster (spin) mutants, a Drosophila model of LSD-like neurodegeneration. Our results highlight an imbalance in brain ceramide and sphingosine in the early stages of neurodegeneration, preceding the accumulation of endomembranous structures, manifestation of altered behavior, and buildup of lipofuscin. Manipulating levels of ceramidase and altering these lipids in spin mutants allowed us to conclude that ceramide homeostasis is the driving force in disease progression and is integral to spin function in the adult nervous system. We identified 29 novel physical interaction partners of Spin and focused on the lipid carrier protein, Lipophorin (Lpp). A subset of Lpp and Spin colocalize in the brain and within organs specialized for lipid metabolism (fat bodies and oenocytes). Reduced Lpp protein was observed in spin mutant tissues. Finally, increased levels of lipid metabolites produced by oenocytes in spin mutants allude to a functional interaction between Spin and Lpp, underscoring the systemic nature of lipid perturbation in LSD. © 2017 Hebbar et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  13. Adaptive Evolution of Phosphorus Metabolism in Prochlorococcus

    DEFF Research Database (Denmark)

    Casey, John R; Mardinoglu, Adil; Nielsen, Jens

    2016-01-01

    Inorganic phosphorus is scarce in the eastern Mediterranean Sea, where the high-light-adapted ecotype HLI of the marine picocyanobacterium Prochlorococcus marinus thrives. Physiological and regulatory control of phosphorus acquisition and partitioning has been observed in HLI both in culture...... and in the field; however, the optimization of phosphorus metabolism and associated gains for its phosphorus-limited-growth (PLG) phenotype have not been studied. Here, we reconstructed a genome-scale metabolic network of the HLI axenic strain MED4 (iJC568), consisting of 568 metabolic genes in relation to 794...... reactions involving 680 metabolites distributed in 6 subcellular locations. iJC568 was used to quantify metabolic fluxes under PLG conditions, and we observed a close correspondence between experimental and computed fluxes. We found that MED4 has minimized its dependence on intracellular phosphate, not only...

  14. Metabolic flux analysis of the phenylpropanoid pathway in wound-healing potato tuber tissue using stable isotope-labeled tracer and LC-MS spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Matsuda, Fumio; Morino, Keiko; Miyashita, Masahiro; Miyagawa, Hisashi [Kyoto Univ. (Japan). Department of Agriculture

    2003-05-01

    The metabolic flux of two phenylpropanoid metabolites, N-p-coumaroyloctopamine (p-CO) and chlorogenic acid (CGA), in the wound-healing potato tuber tissue was quantitatively analyzed by a newly developed method based upon the tracer experiment using stable isotope-labeled compounds and LC-MS. Tuber disks were treated with aqueous solution of L-phenyl-d{sub 5}-alanine, and the change in the ratio of stable isotope-labeled compound to non-labeled (isotope abundance) was monitored for p-CO and CGA in the tissue extract by LC-MS. The time-dependent change in the isotope abundance of each metabolite was fitted to an equation that was derived from the formation and conversion kinetics of each compound. Good correlations were obtained between the observed and calculated isotope abundances for both p-CO and CGA. The rates of p-CO formation and conversion (i.e. fluxes) were 1.15 and 0.96 nmol (g FW){sup -1}h{sup -1}, respectively, and for CGA, the rates 4.63 and 0.42 nmol (g FW){sup -1}h{sup -1}, respectively. This analysis enabled a direct comparison of the biosynthetic activity between these two compounds. (author)

  15. Metabolic control analysis of the penicillin biosynthetic pathway: the influence of the LLD-ACV:bisACV ratio on the flux control.

    Science.gov (United States)

    Theilgaard, H A; Nielsen, J

    1999-01-01

    An extended kinetic model for the first two steps of the penicillin biosynthetic pathway in Penicillium chrysogenum is set up. It includes the formation and reduction of the dimer bis-delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (bisACV) from the first pathway intermediate LLD-ACV and their parallel inhibition of the enzyme ACV synthetase (ACVS). The kinetic model is based on Michaelis-Menten type kinetics, with non-competitive inhibition of the ACVS by both LLD-ACV and bisACV, and competitive inhibition of the isopenicillin N synthetase (IPNS) by glutathione. The inhibition constant of LLD-ACV, KACV is determined to be 0.54 mm. With the kinetic model metabolic control analysis is performed to identify the distribution of rate-control in the pathway at all ratios of LLD-ACV:bisACV. It is concluded that the flux control totally resides at the IPNS. This is a result of the regulation of the ACVS by both the LLD-ACV and bisACV demanding a higher flux through the IPNS enzyme to alleviate their inhibition. The measurement of an intracellular ratio of LLD-ACV:bisACV to be in the range of 1-2 moles per moles emphasises the importance of a fast conversion of LLD-ACV to IPN, and accumulation of LLD-ACV above the K(m)-value of the IPNS should therefore be avoided.

  16. Flight muscle enzymes and metabolic flux rates during hovering flight of the nectar bat, Glossophaga soricina: further evidence of convergence with hummingbirds.

    Science.gov (United States)

    Suarez, R K; Welch, K C; Hanna, S K; Herrera M, L G

    2009-06-01

    Given their high metabolic rates, nectarivorous diet, and ability to directly fuel their energetically-expensive flight using recently-ingested sugar, we tested the hypothesis that Pallas long tongued nectar bats (Glossophaga soricina) possess flight muscles similar to those of hummingbirds with respect to enzymatic flux capacities in bioenergetic pathways. In addition, we compared these biochemical capacities with flux rates achieved in vivo during hovering flight. Rates of oxygen consumption (V(O(2))) were measured during hover-feeding and used to estimate rates of ATP turnover, glucose and long-chain fatty acid oxidation per unit mass of flight muscle. Enzyme V(max) values at key steps in glucose and fatty acid oxidation obtained in vitro from pectoralis muscle samples exceed those found in the locomotory muscles of other species of small mammals and resemble data obtained from hummingbird flight muscles. The ability of nectar bats and hummingbirds to hover in fed and fasted states, fueled almost exclusively by carbohydrate or fat, respectively, allowed the estimation of fractional velocities (v/V(max)) at both the hexokinase and carnitine palmitoyltransferase-2 steps in glucose and fatty acid oxidation, respectively. The results further support the hypothesis of convergent evolution in biochemical and physiological traits in nectar bats and hummingbirds.

  17. Transcriptional-metabolic networks in beta-carotene-enriched potato tubers: the long and winding road to the Golden phenotype.

    Science.gov (United States)

    Diretto, Gianfranco; Al-Babili, Salim; Tavazza, Raffaela; Scossa, Federico; Papacchioli, Velia; Migliore, Melania; Beyer, Peter; Giuliano, Giovanni

    2010-10-01

    Vitamin A deficiency is a public health problem in a large number of countries. Biofortification of major staple crops (wheat [Triticum aestivum], rice [Oryza sativa], maize [Zea mays], and potato [Solanum tuberosum]) with β-carotene has the potential to alleviate this nutritional problem. Previously, we engineered transgenic "Golden" potato tubers overexpressing three bacterial genes for β-carotene synthesis (CrtB, CrtI, and CrtY, encoding phytoene synthase, phytoene desaturase, and lycopene β-cyclase, respectively) and accumulating the highest amount of β-carotene in the four aforementioned crops. Here, we report the systematic quantitation of carotenoid metabolites and transcripts in 24 lines carrying six different transgene combinations under the control of the 35S and Patatin (Pat) promoters. Low levels of B-I expression are sufficient for interfering with leaf carotenogenesis, but not for β-carotene accumulation in tubers and calli, which requires high expression levels of all three genes under the control of the Pat promoter. Tubers expressing the B-I transgenes show large perturbations in the transcription of endogenous carotenoid genes, with only minor changes in carotenoid content, while the opposite phenotype (low levels of transcriptional perturbation and high carotenoid levels) is observed in Golden (Y-B-I) tubers. We used hierarchical clustering and pairwise correlation analysis, together with a new method for network correlation analysis, developed for this purpose, to assess the perturbations in transcript and metabolite levels in transgenic leaves and tubers. Through a "guilt-by-profiling" approach, we identified several endogenous genes for carotenoid biosynthesis likely to play a key regulatory role in Golden tubers, which are candidates for manipulations aimed at the further optimization of tuber carotenoid content.

  18. Whole-system metabolism and CO2 fluxes in a Mediterranean Bay dominated by seagrass beds (Palma Bay, NW Mediterranean

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    F. Gazeau

    2005-01-01

    Full Text Available Planktonic and benthic incubations (bare and Posidonia oceanica vegetated sediments were performed at monthly intervals from March 2001 to October 2002 in a seagrass vegetated area of the Bay of Palma (Mallorca, Spain. Results showed a contrast between the planktonic compartment, which was on average near metabolic balance (−4.6±5.9 mmol O2 m-2 d-1 and the benthic compartment, which was autotrophic (17.6±8.5 mmol O2 m-2 d-1. During two cruises in March and June 2002, planktonic and benthic incubations were performed at several stations in the bay to estimate the whole-system metabolism and to examine its relationship with partial pressure of CO2 (pCO2 and apparent oxygen utilisation (AOU spatial patterns. Moreover, during the second cruise, when the residence time of water was long enough, net ecosystem production (NEP estimates based on incubations were compared, over the Posidonia oceanica meadow, to rates derived from dissolved inorganic carbon (DIC and oxygen (O2 mass balance budgets. These budgets provided NEP estimates in fair agreement with those derived from direct metabolic estimates based on incubated samples over the Posidonia oceanica meadow. Whereas the seagrass community was autotrophic, the excess organic carbon production therein could only balance the planktonic heterotrophy in shallow waters relative to the maximum depth of the bay (55 m. This generated a horizontal gradient from autotrophic or balanced communities in the shallow seagrass-covered areas, to strongly heterotrophic communities in deeper areas of the bay. It seems therefore that, on an annual scale in the whole bay, the organic matter production by the Posidonia oceanica may not be sufficient to fully compensate the heterotrophy of the planktonic compartment, which may require external organic carbon inputs, most likely from land.

  19. Effect of amino acid supplementation on titer and glycosylation distribution in hybridoma cell cultures-Systems biology-based interpretation using genome-scale metabolic flux balance model and multivariate data analysis.

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    Reimonn, Thomas M; Park, Seo-Young; Agarabi, Cyrus D; Brorson, Kurt A; Yoon, Seongkyu

    2016-09-01

    Genome-scale flux balance analysis (FBA) is a powerful systems biology tool to characterize intracellular reaction fluxes during cell cultures. FBA estimates intracellular reaction rates by optimizing an objective function, subject to the constraints of a metabolic model and media uptake/excretion rates. A dynamic extension to FBA, dynamic flux balance analysis (DFBA), can calculate intracellular reaction fluxes as they change during cell cultures. In a previous study by Read et al. (2013), a series of informed amino acid supplementation experiments were performed on twelve parallel murine hybridoma cell cultures, and this data was leveraged for further analysis (Read et al., Biotechnol Prog. 2013;29:745-753). In order to understand the effects of media changes on the model murine hybridoma cell line, a systems biology approach is applied in the current study. Dynamic flux balance analysis was performed using a genome-scale mouse metabolic model, and multivariate data analysis was used for interpretation. The calculated reaction fluxes were examined using partial least squares and partial least squares discriminant analysis. The results indicate media supplementation increases product yield because it raises nutrient levels extending the growth phase, and the increased cell density allows for greater culture performance. At the same time, the directed supplementation does not change the overall metabolism of the cells. This supports the conclusion that product quality, as measured by glycoform assays, remains unchanged because the metabolism remains in a similar state. Additionally, the DFBA shows that metabolic state varies more at the beginning of the culture but less by the middle of the growth phase, possibly due to stress on the cells during inoculation. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1163-1173, 2016. © 2016 American Institute of Chemical Engineers.

  20. Genome-wide association study identifies chromosome 10q24.32 variants associated with arsenic metabolism and toxicity phenotypes in Bangladesh.

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    Brandon L Pierce

    Full Text Available Arsenic contamination of drinking water is a major public health issue in many countries, increasing risk for a wide array of diseases, including cancer. There is inter-individual variation in arsenic metabolism efficiency and susceptibility to arsenic toxicity; however, the basis of this variation is not well understood. Here, we have performed the first genome-wide association study (GWAS of arsenic-related metabolism and toxicity phenotypes to improve our understanding of the mechanisms by which arsenic affects health. Using data on urinary arsenic metabolite concentrations and approximately 300,000 genome-wide single nucleotide polymorphisms (SNPs for 1,313 arsenic-exposed Bangladeshi individuals, we identified genome-wide significant association signals (P<5×10(-8 for percentages of both monomethylarsonic acid (MMA and dimethylarsinic acid (DMA near the AS3MT gene (arsenite methyltransferase; 10q24.32, with five genetic variants showing independent associations. In a follow-up analysis of 1,085 individuals with arsenic-induced premalignant skin lesions (the classical sign of arsenic toxicity and 1,794 controls, we show that one of these five variants (rs9527 is also associated with skin lesion risk (P = 0.0005. Using a subset of individuals with prospectively measured arsenic (n = 769, we show that rs9527 interacts with arsenic to influence incident skin lesion risk (P = 0.01. Expression quantitative trait locus (eQTL analyses of genome-wide expression data from 950 individual's lymphocyte RNA suggest that several of our lead SNPs represent cis-eQTLs for AS3MT (P = 10(-12 and neighboring gene C10orf32 (P = 10(-44, which are involved in C10orf32-AS3MT read-through transcription. This is the largest and most comprehensive genomic investigation of arsenic metabolism and toxicity to date, the only GWAS of any arsenic-related trait, and the first study to implicate 10q24.32 variants in both arsenic metabolism and arsenical

  1. Distribution of CYP2D6 and CYP2C19 polymorphisms associated with poor metabolizer phenotype in five Amerindian groups and western Mestizos from Mexico.

    Science.gov (United States)

    Salazar-Flores, Joel; Torres-Reyes, Luis A; Martínez-Cortés, Gabriela; Rubi-Castellanos, Rodrigo; Sosa-Macías, Martha; Muñoz-Valle, José F; González-González, César; Ramírez, Angélica; Román, Raquel; Méndez, José L; Barrera, Andrés; Torres, Alfredo; Medina, Rafael; Rangel-Villalobos, Héctor

    2012-09-01

    The distribution of polymorphisms in the CYP2D6 and CYP2C19 genes allows inferring the potential risk for specific adverse drug reactions and lack of therapeutic effects in humans. This variability shows differences among human populations. The aim of this study was to analyze single-nucleotide polymorphisms related to a poor metabolizer (PM) phenotype in nonpreviously studied Amerindian groups and Mestizos (general admixed population) from Mexico. We detected by SNaPshot(®) different polymorphisms located in CYP2D6 (*3, *4, *6, *7, and *8) and CYP2C19 (*2, *3, *4 and *5) in western Mestizos (n=145) and five Amerindian groups from Mexico: Tarahumaras from the North (n=88); Purépechas from the Center (n=101); and Tojolabales (n=68), Tzotziles (n=88), and Tzeltales (n=20) from the Southeast. Genotypes were observed by capillary electrophoresis. The genetic relationships among these populations were estimated based on these genes. The wild-type allele (*1) of both genes was predominant in the Mexican populations studied. The most widely observed alleles were CYP2C19*2 (range, 0%-31%) and CYP2D6*4 (range, 1.2%-7.3%), whereas CYP2D6*3 was exclusively detected in Mestizos. Conversely, CYP2C19*4 and *5, as well as CYP2D6*3, *6, *7, and *8, were not observed in the majority of the Mexican populations. The Tarahumaras presented a high frequency of the allele CYP2C19*2 (31%) and of homozygotes *2/*2 (10.7%), which represent a high frequency of potentially PM phenotypes in this Amerindian group. The genetic distances showed high differentiation of Tarahumaras (principally for CYP2C19 gene). In general, a relative proximity was observed between most of the Amerindian, Mexican-Mestizo, and Latin-American populations. In general, the wild-type allele (*1) predominates in Mexican populations, outlining a relatively homogeneous distribution for CYP2C19 and CYP2D6. The exception is the Tarahumara group that displays a potentially increased risk for adverse reactions to CYP2C19

  2. Coexpression network analysis in abdominal and gluteal adipose tissue reveals regulatory genetic loci for metabolic syndrome and related phenotypes.

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    Josine L Min

    Full Text Available Metabolic Syndrome (MetS is highly prevalent and has considerable public health impact, but its underlying genetic factors remain elusive. To identify gene networks involved in MetS, we conducted whole-genome expression and genotype profiling on abdominal (ABD and gluteal (GLU adipose tissue, and whole blood (WB, from 29 MetS cases and 44 controls. Co-expression network analysis for each tissue independently identified nine, six, and zero MetS-associated modules of coexpressed genes in ABD, GLU, and WB, respectively. Of 8,992 probesets expressed in ABD or GLU, 685 (7.6% were expressed in ABD and 51 (0.6% in GLU only. Differential eigengene network analysis of 8,256 shared probesets detected 22 shared modules with high preservation across adipose depots (D(ABD-GLU = 0.89, seven of which were associated with MetS (FDR P100,000 individuals; rs10282458, affecting expression of RARRES2 (encoding chemerin, was associated with body mass index (BMI (P = 6.0×10(-4; and rs2395185, affecting inter-depot differences of HLA-DRB1 expression, was associated with high-density lipoprotein (P = 8.7×10(-4 and BMI-adjusted waist-to-hip ratio (P = 2.4×10(-4. Since many genes and their interactions influence complex traits such as MetS, integrated analysis of genotypes and coexpression networks across multiple tissues relevant to clinical traits is an efficient strategy to identify novel associations.

  3. Reduction of DILP2 in Drosophila triages a metabolic phenotype from lifespan revealing redundancy and compensation among DILPs.

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    Susan Broughton

    Full Text Available The insulin/IGF-like signalling (IIS pathway has diverse functions in all multicellular organisms, including determination of lifespan. The seven insulin-like peptides (DILPs in Drosophila are expressed in a stage- and tissue-specific manner. Partial ablation of the median neurosecretory cells (mNSCs in the brain, which produce three DILPs, extends lifespan, reduces fecundity, alters lipid and carbohydrate metabolism and increases oxidative stress resistance. To determine if reduced expression of DILPs is causal in these effects, and to investigate possible functional diversification and redundancy between DILPs, we used RNA interference to lower specifically the transcript and protein levels of dilp2, the most highly expressed of the mNSC-derived DILPs. We found that DILP2 was limiting only for the increased whole-body trehalose content associated with mNSC-ablation. We observed a compensatory increase in dilp3 and 5 mRNA upon dilp2 knock down. By manipulation of dfoxo and dInR, we showed that the increase in dilp3 is regulated via autocrine insulin signaling in the mNSCs. Our study demonstrates that, despite the correlation between reduced dilp2 mRNA levels and lifespan-extension often observed, DILP2 reduction is not sufficient to extend lifespan. Nor is the increased trehalose storage associated with reduced IIS sufficient to extend lifespan. To understand the normal regulation of expression of the dilps and any functional diversification between them will require independent control of the expression of different dilps.

  4. Stage-Specific Fatty Acid Fluxes Play a Regulatory Role in Glycerolipid Metabolism during Seed Development in Jatropha curcas L.

    Science.gov (United States)

    Chaitanya, Bharatula Sri Krishna; Kumar, Sumit; Kaki, Shiva Shanker; Balakrishna, Marrapu; Karuna, Mallampalli Sri Lakshmi; Prasad, Rachapudi Badari Narayana; Sastry, Pidaparty Seshadri; Reddy, Attipalli Ramachandra

    2015-12-23

    The present study describes the changes in lipid profile as well as fatty acid fluxes during seed development in Jatropha curcas L. Endosperm from 34, 37, and 40 days after anthesis (DAA), incubated with [(14)C]acetate, showed significant synthesis of phosphatidylcholine (PC) at seed maturation. The fatty acid methyl ester profile showed PC from 34 DAA was rich in palmitic acid (16:0), whereas PC from 37 and 40 DAA was rich in oleic acid (18:1n-9). Molecular species analysis of diacylglycerol (DAG) indicated DAG (16:0/18:2n-6) was in abundance at 34 DAA, whereas DAG (18:1n-9/18:2n-6) was significantly high at 40 DAA. Triacylglycerol (TAG) analysis revealed TAG (16:0/18:2n-6/16:0) was abundant at 34 DAA, whereas TAG (18:1n-9/18:2n-6/18:1n-9) formed the majority at 40 DAA. Expression of two types of diacylglycerol acyltransferases varied with seed maturation. These data demonstrate stage-specific distinct pools of PC and DAG synthesis during storage TAG accumulation in Jatropha seed.

  5. Metabolic requirements for cancer cell proliferation.

    Science.gov (United States)

    Keibler, Mark A; Wasylenko, Thomas M; Kelleher, Joanne K; Iliopoulos, Othon; Vander Heiden, Matthew G; Stephanopoulos, Gregory

    2016-01-01

    The study of cancer metabolism has been largely dedicated to exploring the hypothesis that oncogenic transformation rewires cellular metabolism to sustain elevated rates of growth and division. Intense examination of tumors and cancer cell lines has confirmed that many cancer-associated metabolic phenotypes allow robust growth and survival; however, little attention has been given to explicitly identifying the biochemical requirements for cell proliferation in a rigorous manner in the context of cancer metabolism. Using a well-studied hybridoma line as a model, we comprehensively and quantitatively enumerate the metabolic requirements for generating new biomass in mammalian cells; this indicated a large biosynthetic requirement for ATP, NADPH, NAD(+), acetyl-CoA, and amino acids. Extension of this approach to serine/glycine and glutamine metabolic pathways suggested lower limits on serine and glycine catabolism to supply one-carbon unit synthesis and significant availability of glutamine-derived carbon for biosynthesis resulting from nitrogen demands alone, respectively. We integrated our biomass composition results into a flux balance analysis model, placing upper bounds on mitochondrial NADH oxidation to simulate metformin treatment; these simulations reproduced several empirically observed metabolic phenotypes, including increased reductive isocitrate dehydrogenase flux. Our analysis clarifies the differential needs for central carbon metabolism precursors, glutamine-derived nitrogen, and cofactors such as ATP, NADPH, and NAD(+), while also providing justification for various extracellular nutrient uptake behaviors observed in tumors. Collectively, these results demonstrate how stoichiometric considerations alone can successfully predict empirically observed phenotypes and provide insight into biochemical dynamics that underlie responses to metabolic perturbations.

  6. Pathway analysis of Pichia pastoris to elucidate methanol metabolism and its regulation for production of recombinant proteins.

    Science.gov (United States)

    Unrean, Pornkamol

    2014-01-01

    This research rationally analyzes metabolic pathways of Pichia pastoris to study the metabolic flux responses of this yeast under methanol metabolism. A metabolic model of P. pastoris was constructed and analyzed by elementary mode analysis (EMA). EMA was used to comprehensively identify the cell's metabolic flux profiles and its underlying regulation mechanisms for the production of recombinant proteins from methanol. Change in phenotypes and flux profiles during methanol adaptation with varying feed mixture of glycerol and methanol was examined. EMA identified increasing and decreasing fluxes during the glycerol-methanol metabolic shift, which well agreed with experimental observations supporting the validity of the metabolic network model. Analysis of all the identified pathways also led to the determination of the metabolic capacities as well as the optimum metabolic pathways for recombinant protein synthesis during methanol induction. The network sensitivity analysis revealed that the production of proteins can be improved by manipulating the flux ratios at the pyruvate branch point. In addition, EMA suggested that protein synthesis is optimum under hypoxic culture conditions. The metabolic modeling and analysis presented in this study could potentially form a valuable knowledge base for future research on rational design and optimization of P. pastoris by determining target genes, pathways, and culture conditions for enhanced recombinant protein synthesis. The metabolic pathway analysis is also of considerable value for production of therapeutic proteins by P. pastoris in biopharmaceutical applications. © 2013 American Institute of Chemical Engineers.

  7. Transcriptomics, Targeted Metabolomics and Gene Expression of Blackberry Leaves and Fruits Indicate Flavonoid Metabolic Flux from Leaf to Red Fruit.

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    Gutierrez, Enrique; García-Villaraco, Ana; Lucas, José A; Gradillas, Ana; Gutierrez-Mañero, F Javier; Ramos-Solano, Beatriz

    2017-01-01

    Blackberries ( Rubus spp.) are among the high added value food products relevant for human health due to the increasing evidence of the beneficial effects of polyphenols, which are very abundant in these fruits. Interestingly, these compounds also play a role on plant physiology, being especially relevant their role in plant defense against biotic and abiotic stress. Hence, we hypothesize that since blackberry fruits have high amounts of flavonols and anthocyanins, leaves would also have high amounts of these compounds, and can be studied as a source of active molecules; furthermore, leaf synthesis would support their high contents in fruits. To explore this hypothesis, the present study reports a de novo transcriptome analysis on field grown blackberry leaves and fruits at the same time point, to establish the metabolic relationship of these compounds in both organs. Transcripts were aligned against Fragaria vesca genome, and genes were identified and annotated in different databases; tissue expression pattern showed 20,463 genes common to leaves and fruits, while 6,604 genes were significantly overexpressed only in fruits, while another 6,599 genes were significantly overexpressed in leaves, among which flavonol-anthocyanin transporter genes were present. Bioactives characterization indicated that total phenolics in leaves were three-fold, and flavonols were six-fold than in fruits, while concentration of anthocyanins was higher in fruits; HPLC-MS analysis indicated different composition in leaves and fruits, with cyanidin-3-glucoside as the only common compound identified. Next, RT-qPCR of the core genes in the flavonol anthocyanin pathway and regulatory MYB genes were carried out. Interestingly, genes in the flavonol-anthocyanin pathway and flavonol-transport families were overexpressed in leaves, consistent with the higher bioactive levels. On the other hand, transcription factors were overexpressed in fruits anticipating an active anthocyanin biosynthesis

  8. GEN GEN: the genomic genetic analysis of androgen-metabolic genes and prostate cancer as a paradigm for the dissection of complex phenotypes.

    Science.gov (United States)

    Reichardt, J K

    1999-07-15

    Prostate cancer will be diagnosed in about 179,300 men in the US in 1999 alone. Some 37,000 individuals die of this disease annually. Prostate cancer is characterized by a substantial racial/ethnic variation in risk: highest in African-American men, lowest in Asian men and intermediate in Caucasian and Latino men. We set out to investigate as our central hypothesis that genetic variants of genes involved in androgen metabolism by themselves and in combination significantly contribute to prostate cancer progression and its racial/ethnic variation. Specifically, we examined the hypothesis that DNA sequence (allelic) variations in the type II (or prostatic) steroid 5alpha-reductase (SRD5A2) gene contribute substantially to the risk and progression of prostate cancer particularly across racial/ethnic lines. The "candidate gene", SRD5A2, was chosen because the reaction product [i.e. dihydrotestosterone (DHT)] of the enzyme encoded by this gene modulates directly cell division in the prostate. DHT binds to the androgen receptor (AR) and the DHT-AR complex leads to the transactivation of a variety of genes which ultimately modulates cell division in the prostate. Epidemiologic evidence suggests that variation in DHT levels play an important role in risk of prostate cancer. Thus, steroid 5alpha-reductase activity encoded by SRD5A2 variant alleles may be important in regulating intraprostatic DHT steady state levels by controlling its biosynthesis. A second candidate gene, the type II 3beta-hydroxysteroid dehydrogenase (HSD3B2) gene, encodes the enzyme that initiates the metabolic inactivation of testosterone (T) to DHT. We have identified allelic variants in this gene as well. Here I review our strategy for identifying candidate genes for prostate cancer, a multifactorial disease. I summarize the significant findings, particularly of allelic variants in the HSD3B2 and SRD5A2 genes and discuss how they by themselves, in combination and through interactions with the

  9. Generation of a multi-locus chicken introgression line to study the effects of genetic interactions on metabolic phenotypes in chickens

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    Weronica eEk

    2012-03-01

    Full Text Available Most biological traits are regulated by a complex interplay between genetic and environmental factors. By intercrossing divergent lines, it is possible to identify individual and interacting QTL involved in the genetic architecture of these traits. When the loci have been mapped, alternative strategies are needed for fine-mapping and studying the individual and interactive effects of the QTL in detail. We have previously identified, replicated and fine-mapped a four-locus QTL network that determines nearly half of the eight-fold difference in body-weight at 56 days of age between two divergently selected chicken lines. Here, we describe, to our knowledge, the first generation of a three-locus QTL introgression line in chickens to further study the effect of three of the interacting loci in this network on metabolic phenotypes. Recurrent marker assisted backcrossing was used to simultaneously transfer QTL alleles from the low-weight selected line into the high-weight selected line. Three generations of backcrossing and one generation of intercrossing resulted in an introgression line where all three introgressed QTL and several unlinked and linked control-loci were segregating at nearly expected allele frequencies. We show that marker-based sexing is an efficient method for sexing breeding populations and how intensive selection can be applied using artificial insemination to generate large half-sib families. Based on our empirical observations, we provide recommendations for future introgression-line breeding experiments. In the future, use of this confirmed introgression line will facilitate detailed studies of the effects of genetic interactions on complex traits.

  10. Sex-Specific Life Course Changes in the Neuro-Metabolic Phenotype of Glut3 Null Heterozygous Mice: Ketogenic Diet Ameliorates Electroencephalographic Seizures and Improves Sociability.

    Science.gov (United States)

    Dai, Yun; Zhao, Yuanzi; Tomi, Masatoshi; Shin, Bo-Chul; Thamotharan, Shanthie; Mazarati, Andrey; Sankar, Raman; Wang, Elizabeth A; Cepeda, Carlos; Levine, Michael S; Zhang, Jingjing; Frew, Andrew; Alger, Jeffry R; Clark, Peter M; Sondhi, Monica; Kositamongkol, Sudatip; Leibovitch, Leah; Devaskar, Sherin U

    2017-04-01

    We tested the hypothesis that exposure of glut3+/- mice to a ketogenic diet ameliorates autism-like features, which include aberrant behavior and electrographic seizures. We first investigated the life course sex-specific changes in basal plasma-cerebrospinal fluid (CSF)-brain metabolic profile, brain glucose transport/uptake, glucose and monocarboxylate transporter proteins, and adenosine triphosphate (ATP) in the presence or absence of systemic insulin administration. Glut3+/- male but not female mice (5 months of age) displayed reduced CSF glucose/lactate concentrations with no change in brain Glut1, Mct2, glucose uptake or ATP. Exogenous insulin-induced hypoglycemia increased brain glucose uptake in glut3+/- males alone. Higher plasma-CSF ketones (β-hydroxybutyrate) and lower brain Glut3 in females vs males proved protective in the former while enhancing vulnerability in the latter. As a consequence, increased synaptic proteins (neuroligin4 and SAPAP1) with spontaneous excitatory postsynaptic activity subsequently reduced hippocampal glucose content and increased brain amyloid β1-40 deposition in an age-dependent manner in glut3+/- males but not females (4 to 24 months of age). We then explored the protective effect of a ketogenic diet on ultrasonic vocalization, sociability, spatial learning and memory, and electroencephalogram seizures in male mice (7 days to 6 to 8 months of age) alone. A ketogenic diet partially restored sociability without affecting perturbed vocalization, spatial learning and memory, and reduced seizure events. We conclude that (1) sex-specific and age-dependent perturbations underlie the phenotype of glut3+/- mice, and (2) a ketogenic diet ameliorates seizures caused by increased cortical excitation and improves sociability, but fails to rescue vocalization and cognitive deficits in glut3+/- male mice. Copyright © 2017 Endocrine Society.

  11. A novel metabolism-based phenotypic drug discovery platform in zebrafish uncovers HDACs 1 and 3 as a potential combined anti-seizure drug target.

    Science.gov (United States)

    Ibhazehiebo, Kingsley; Gavrilovici, Cezar; de la Hoz, Cristiane L; Ma, Shun-Chieh; Rehak, Renata; Kaushik, Gaurav; Meza Santoscoy, Paola L; Scott, Lucas; Nath, Nandan; Kim, Do-Young; Rho, Jong M; Kurrasch, Deborah M

    2018-01-24

    Despite the development of newer anti-seizure medications over the past 50 years, 30-40% of patients with epilepsy remain refractory to treatment. One explanation for this lack of progress is that the current screening process is largely biased towards transmembrane channels and receptors, and ignores intracellular proteins and enzymes that might serve as efficacious molecular targets. Here, we report the development of a novel drug screening platform that harnesses the power of zebrafish genetics and combines it with in vivo bioenergetics screening assays to uncover therapeutic agents that improve mitochondrial health in diseased animals. By screening commercially available chemical libraries of approved drugs, for which the molecular targets and pathways are well characterized, we were able to reverse-identify the proteins targeted by efficacious compounds and confirm the physiological roles that they play by utilizing other pharmacological ligands. Indeed, using an 870-compound screen in kcna1-morpholino epileptic zebrafish larvae, we uncovered vorinostat (Zolinza™; suberanilohydroxamic acid, SAHA) as a potent anti-seizure agent. We further demonstrated that vorinostat decreased average daily seizures by ∼60% in epileptic Kcna1-null mice using video-EEG recordings. Given that vorinostat is a broad histone deacetylase (HDAC) inhibitor, we then delineated a specific subset of HDACs, namely HDACs 1 and 3, as potential drug targets for future screening. In summary, we have developed a novel phenotypic, metabolism-based experimental therapeutics platform that can be used to identify new molecular targets for future drug discovery in epilepsy. © The Author(s) (2018). Published by Oxford University Press on behalf of the Guarantors of Brain.

  12. Metabolic phenotype and adipose and liver features in a high-fat Western diet-induced mouse model of obesity-linked NAFLD.

    Science.gov (United States)

    Luo, Yuwen; Burrington, Christine M; Graff, Emily C; Zhang, Jian; Judd, Robert L; Suksaranjit, Promporn; Kaewpoowat, Quanhathai; Davenport, Samantha K; O'Neill, Ann Marie; Greene, Michael W

    2016-03-15

    nonalcoholic fatty liver disease (NAFLD), an obesity and insulin resistance associated clinical condition - ranges from simple steatosis to nonalcoholic steatohepatitis. To model the human condition, a high-fat Western diet that includes liquid sugar consumption has been used in mice. Even though liver pathophysiology has been well characterized in the model, little is known about the metabolic phenotype (e.g., energy expenditure, activity, or food intake). Furthermore, whether the consumption of liquid sugar exacerbates the development of glucose intolerance, insulin resistance, and adipose tissue dysfunction in the model is currently in question. In our study, a high-fat Western diet (HFWD) with liquid sugar [fructose and sucrose (F/S)] induced acute hyperphagia above that observed in HFWD-fed mice, yet without changes in energy expenditure. Liquid sugar (F/S) exacerbated HFWD-induced glucose intolerance and insulin resistance and impaired the storage capacity of epididymal white adipose tissue (eWAT). Hepatic TG, plasma alanine aminotransferase, and normalized liver weight were significantly increased only in HFWD+F/S-fed mice. HFWD+F/S also resulted in increased hepatic fibrosis and elevated collagen 1a2, collagen 3a1, and TGFβ gene expression. Furthermore, HWFD+F/S-fed mice developed more profound eWAT inflammation characterized by adipocyte hypertrophy, macrophage infiltration, a dramatic increase in crown-like structures, and upregulated proinflammatory gene expression. An early hypoxia response in the eWAT led to reduced vascularization and increased fibrosis gene expression in the HFWD+F/S-fed mice. Our results demonstrate that sugary water consumption induces acute hyperphagia, limits adipose tissue expansion, and exacerbates glucose intolerance and insulin resistance, which are associated with NAFLD progression. Copyright © 2016 the American Physiological Society.

  13. Controlling the transcription levels of argGH redistributed L-arginine metabolic flux in N-acetylglutamate kinase and ArgR-deregulated Corynebacterium crenatum.

    Science.gov (United States)

    Zhao, Qinqin; Luo, Yuchang; Dou, Wenfang; Zhang, Xian; Zhang, Xiaomei; Zhang, Weiwei; Xu, Meijuan; Geng, Yan; Rao, Zhiming; Xu, Zhenghong

    2016-01-01

    Corynebacterium crenatum SYPA5-5, an L-arginine high-producer obtained through multiple mutation-screening steps, had been deregulated by the repression of ArgR that inhibits L-arginine biosynthesis at genetic level. Further study indicated that feedback inhibition of SYPA5-5 N-acetylglutamate kinase (CcNAGK) by L-arginine, as another rate-limiting step, could be deregulated by introducing point mutations. Here, we introduced two of the positive mutations (H268N or R209A) of CcNAGK into the chromosome of SYPA5-5, however, resulting in accumulation of large amounts of the intermediates (L-citrulline and L-ornithine) and decreased production of L-arginine. Genetic and enzymatic levels analysis involved in L-arginine biosynthetic pathway of recombinants SYPA5-5-NAGKH268N (H-7) and SYPA5-5-NAGKR209A (R-8) showed that the transcription levels of argGH decreased accompanied with the reduction of argininosuccinate synthase and argininosuccinase activities, respectively, which led to the metabolic obstacle from L-citrulline to L-arginine. Co-expression of argGH with exogenous plasmid in H-7 and R-8 removed this bottleneck and increased L-arginine productivity remarkably. Compared with SYPA5-5, fermentation period of H-7/pDXW-10-argGH (H-7-GH) reduced to 16 h; meanwhile, the L-arginine productivity improved about 63.6%. Fed-batch fermentation of H-7-GH in 10 L bioreactor produced 389.9 mM L-arginine with the productivity of 5.42 mM h(-1). These results indicated that controlling the transcription of argGH was a key factor for regulating the metabolic flux toward L-arginine biosynthesis after deregulating the repression of ArgR and feedback inhibition of CcNAGK, and therefore functioned as another regulatory mode for L-arginine production. Thus, deregulating all these three regulatory modes was a powerful strategy to construct L-arginine high-producing C. crenatum.

  14. Enhanced H2 Production and Redirected Metabolic Flux via Overexpression of fhlA and pncB in Klebsiella HQ-3 Strain.

    Science.gov (United States)

    Jawed, Muhammad; Pi, Jian; Xu, Li; Zhang, Houjin; Hakeem, Abdul; Yan, Yunjun

    2016-03-01

    Genetic modifications are considered as one of the most important technologies for improving fermentative hydrogen yield. Herein, we overexpress fhlA and pncB genes from Klebsiella HQ-3 independently to enhance hydrogen molar yield. HQ-3-fhlA/pncB strain is developed by manipulation of pET28-Pkan/fhlA Kan(r) and pBBR1-MCS5/pncB Gm(r) as expression vectors to examine the synchronous effects of fhlA and pncB. Optimization of anaerobic batch fermentations is achieved and the maximum yield of biohydrogen (1.42 mol H2/mol of glucose) is produced in the range of pH 6.5-7.0 at 33-37 °C. Whole cell H2 yield is increased up to 40 % from HQ-3-fhlA/pncB, as compared with HQ-3-fhlA 20 % and HQ-3-pncB 12 % keeping HQ-3-C as a control. Mechanism of improved H2 yield is studied in combination with metabolic flux analysis by measuring glucose consumption and other metabolites including formate, succinate, 2,3 butanediol, lactate, acetate, ethanol, and hydrogen. The results suggest that under transient conditions, the increase in the total level of NAD by NAPRTase can enhance the rate of NADH-dependent pathways, and therefore, final distribution of metabolites is changed. Combined overexpression of fhlA and pncB eventually modifies the energy and carbon balance leading to enhanced H2 production from FHL as well as by NADH pathway.

  15. Microalgal Metabolic Network Model Refinement through High-Throughput Functional Metabolic Profiling

    International Nuclear Information System (INIS)

    Chaiboonchoe, Amphun; Dohai, Bushra Saeed; Cai, Hong; Nelson, David R.; Jijakli, Kenan; Salehi-Ashtiani, Kourosh

    2014-01-01

    Metabolic modeling provides the means to define metabolic processes at a systems level; however, genome-scale metabolic models often remain incomplete in their description of metabolic networks and may include reactions that are experimentally unverified. This shortcoming is exacerbated in reconstructed models of newly isolated algal species, as there may be little to no biochemical evidence available for the metabolism of such isolates. The phenotype microarray (PM) technology (Biolog, Hayward, CA, USA) provides an efficient, high-throughput method to functionally define cellular metabolic activities in response to a large array of entry metabolites. The platform can experimentally verify many of the unverified reactions in a network model as well as identify missing or new reactions in the reconstructed metabolic model. The PM technology has been used for metabolic phenotyping of non-photosynthetic bacteria and fungi, but it has not been reported for the phenotyping of microalgae. Here, we introduce the use of PM assays in a systematic way to the study of microalgae, applying it specifically to the green microalgal model species Chlamydomonas reinhardtii. The results obtained in this study validate a number of existing annotated metabolic reactions and identify a number of novel and unexpected metabolites. The obtained information was used to expand and refine the existing COBRA-based C. reinhardtii metabolic network model iRC1080. Over 254 reactions were added to the network, and the effects of these additions on flux distribution within the network are described. The novel reactions include the support of metabolism by a number of d-amino acids, l-dipeptides, and l-tripeptides as nitrogen sources, as well as support of cellular respiration by cysteamine-S-phosphate as a phosphorus source. The protocol developed here can be used as a foundation to functionally profile other microalgae such as known microalgae mutants and novel isolates.

  16. Microalgal Metabolic Network Model Refinement through High Throughput Functional Metabolic Profiling

    Directory of Open Access Journals (Sweden)

    Amphun eChaiboonchoe

    2014-12-01

    Full Text Available Metabolic modeling provides the means to define metabolic processes at a systems level; however, genome-scale metabolic models often remain incomplete in their description of metabolic networks and may include reactions that are experimentally unverified. This shortcoming is exacerbated in reconstructed models of newly isolated algal species, as there may be little to no biochemical evidence available for the metabolism of such isolates. The Phenotype Microarray (PM technology (Biolog, Hayward, CA, USA provides an efficient, high throughput method to functionally define cellular metabolic activities in response to a large array of entry metabolites. The platform can experimentally verify many of the unverified reactions in a network model as well as identify missing or new reactions in the reconstructed metabolic model. The PM technology has been used for metabolic phenotyping of non-photosynthetic bacteria and fungi but it has not been reported for the phenotyping of microalgae. Here we introduce the use of PM assays in a systematic way to the study of microalgae, applying it specifically to the green microalgal model species Chlamydomonas reinhardtii. The results obtained in this study validate a number of existing annotated metabolic reactions and identify a number of novel and unexpected metabolites. The obtained information was used to expand and refine the existing COBRA-based C. reinhardtii metabolic network model iRC1080. Over 254 reactions were added to the network, and the effects of these additions on flux distribution within the network are described. The novel reactions include the support of metabolism by a number of D-amino acids, L-dipeptides, and L-tripeptides as nitrogen sources, as well as support of cellular respiration by cysteamine-S-phosphate as a phosphorus source. The protocol developed here can be used as a foundation to functionally profile other microalgae such as known microalgae mutants and novel isolates.

  17. Microalgal Metabolic Network Model Refinement through High-Throughput Functional Metabolic Profiling.

    Science.gov (United States)

    Chaiboonchoe, Amphun; Dohai, Bushra Saeed; Cai, Hong; Nelson, David R; Jijakli, Kenan; Salehi-Ashtiani, Kourosh

    2014-01-01

    Metabolic modeling provides the means to define metabolic processes at a systems level; however, genome-scale metabolic models often remain incomplete in their description of metabolic networks and may include reactions that are experimentally unverified. This shortcoming is exacerbated in reconstructed models of newly isolated algal species, as there may be little to no biochemical evidence available for the metabolism of such isolates. The phenotype microarray (PM) technology (Biolog, Hayward, CA, USA) provides an efficient, high-throughput method to functionally define cellular metabolic activities in response to a large array of entry metabolites. The platform can experimentally verify many of the unverified reactions in a network model as well as identify missing or new reactions in the reconstructed metabolic model. The PM technology has been used for metabolic phenotyping of non-photosynthetic bacteria and fungi, but it has not been reported for the phenotyping of microalgae. Here, we introduce the use of PM assays in a systematic way to the study of microalgae, applying it specifically to the green microalgal model species Chlamydomonas reinhardtii. The results obtained in this study validate a number of existing annotated metabolic reactions and identify a number of novel and unexpected metabolites. The obtained information was used to expand and refine the existing COBRA-based C. reinhardtii metabolic network model iRC1080. Over 254 reactions were added to the network, and the effects of these additions on flux distribution within the network are described. The novel reactions include the support of metabolism by a number of d-amino acids, l-dipeptides, and l-tripeptides as nitrogen sources, as well as support of cellular respiration by cysteamine-S-phosphate as a phosphorus source. The protocol developed here can be used as a foundation to functionally profile other microalgae such as known microalgae mutants and novel isolates.

  18. Phenotypic characterization of glucose repression mutants of Saccharomyce cerevisiae usinge experimen