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Estimation of flux ratios without uptake or release data: Application to serine and methionine metabolism.

ABSTRACT: Model-based metabolic flux analysis (MFA) using isotope-labeled substrates has provided great insight into intracellular metabolic activities across a host of organisms. One challenge with applying MFA in mammalian systems, however, is the need for absolute quantification of nutrient uptake, biomass composition, and byproduct release fluxes. Such measurements are often not feasible in complex culture systems or in vivo. One way to address this issue is to estimate flux ratios, the fractional contribution of a flux to a metabolite pool, which are independent of absolute measurements and yet informative for cellular metabolism. Prior work has focused on "local" estimation of a handful of flux ratios for specific metabolites and reactions. Here, we perform systematic, model-based estimation of all flux ratios in a metabolic network using isotope labeling data, in the absence of uptake/release data. In a series of examples, we investigate what flux ratios can be well estimated with reasonably tight confidence intervals, and contrast this with confidence intervals on normalized fluxes. We find that flux ratios can provide useful information on the metabolic state, and is complementary to normalized fluxes: for certain metabolic reactions, only flux ratios can be well estimated, while for others normalized fluxes can be obtained. Simulation studies of a large human metabolic network model suggest that estimation of flux ratios is technically feasible for complex networks, but additional studies on data from actual isotopomer labeling experiments are needed to validate these results. Finally, we experimentally study serine and methionine metabolism in cancer cells using flux ratios. We find that, in these cells, the methionine cycle is truncated with little remethylation from homocysteine, and polyamine synthesis in the absence of methionine salvage leads to loss of 5-methylthioadenosine, suggesting a new mode of overflow metabolism in cancer cells. This work highlights the potential for flux ratio analysis in the absence of absolute quantification, which we anticipate will be important for both in vitro and in vivo studies of cancer metabolism.

SUBMITTER: Nilsson R

PROVIDER: S-EPMC5563485 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Estimation of flux ratios without uptake or release data: Application to serine and methionine metabolism.

Nilsson Roland R Roci Irena I Watrous Jeramie J Jain Mohit M

Metabolic engineering 20170220 Pt B

Model-based metabolic flux analysis (MFA) using isotope-labeled substrates has provided great insight into intracellular metabolic activities across a host of organisms. One challenge with applying MFA in mammalian systems, however, is the need for absolute quantification of nutrient uptake, biomass composition, and byproduct release fluxes. Such measurements are often not feasible in complex culture systems or in vivo. One way to address this issue is to estimate flux ratios, the fractional con ...[more]

PMID: 28232235

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Project description:IntroductionHypertensive nephrosclerosis is among the leading causes of end-stage renal disease, but its pathophysiology is poorly understood. We wanted to explore early metabolic changes using gene expression and targeted metabolomics analysis.MethodsWe analyzed gene expression in kidneys biopsied from 20 patients with nephrosclerosis and 31 healthy controls with an Affymetrix array. Thirty-one amino acids were measured by liquid chromatography coupled with mass spectrometry (LC-MS) in urine samples from 62 patients with clinical hypertensive nephrosclerosis and 33 age- and sex-matched healthy controls, and major findings were confirmed in an independent cohort of 45 cases and 15 controls.ResultsAmino acid catabolism and synthesis were strongly underexpressed in hypertensive nephrosclerosis (13- and 7-fold, respectively), and these patients also showed gene expression patterns indicating decreased fatty acid oxidation (12-fold) and increased interferon gamma (10-fold) and cellular defense response (8-fold). Metabolomics analysis revealed significant distribution differences in 11 amino acids in hypertensive nephrosclerosis, among them tyrosine, phenylalanine, dopamine, homocysteine, and serine, with 30% to 70% lower urine excretion. These findings were replicated in the independent cohort. Integrated gene-metabolite pathway analysis showed perturbations of renal dopamine biosynthesis. There were also significant differences in homocysteine/methionine homeostasis and the serine pathway, which have strong influence on 1-carbon metabolism. Several of these disturbances could be interconnected through reduced regeneration of tetrahydrofolate and tetrahydrobiopterin.ConclusionEarly hypertensive nephrosclerosis showed perturbations of intrarenal biosynthesis of dopamine, which regulates natriuresis and blood pressure. There were also disturbances in serine/glycine and methionine/homocysteine metabolism, which may contribute to endothelial dysfunction, atherosclerosis, and renal fibrosis.

Project description:A study was conducted to investigate effects of different methionine (Met) to cysteine (Cys) supplementation ratios (MCR) on growth performance, oxidative status, intestinal health, immune responses, and methionine metabolism in broilers under Eimeria challenge. A total of 720 male Cobb500 broilers (14-day-old) were allocated in a 2 × 5 factorial arrangement (5 diets, with or without challenge) with 6 replicates per treatment. The total sulfur amino acid concentrations were consistent across treatments meeting the breeder's recommendation, only MCR varied. The diets were labeled as MET100; MET75; MET50; MET25; and MET0, representing MCR of 100:0; 75:25; 50:50; 25:75; and 0:100, respectively. Data were analyzed by 2-way ANOVA and orthogonal polynomial contrast. Growth performance declined linearly or quadratically as MCR decreased (P < 0.01). On 6-day postinoculation (DPI), interaction effects (P < 0.01) were found; BW and body weight gain were lower in MET0 compared to the other treatments in the nonchallenged groups, whereas not in the challenged groups. On 6 and 9 DPI, serum total antioxidant capacity linearly decreased as MCR decreased (P < 0.05). Hepatic activities of glutathione peroxidase on 6 DPI and superoxide dismutase on 9 DPI changed quadratically as MCR decreased (P < 0.05). The digestibility of Met linearly decreased whereas the digestibility of Cys linearly increased as MCR decreased. The ileal crypt depth linearly decreased as MCR decreased (P < 0.01) on 6 DPI. The expression of transforming growth factor beta on 6 and 9 DPI, tumor necrotic factor alpha and interleukin 10 on 9 DPI changed quadratically as MCR decreased (P < 0.05). Eimeria challenge increased expression of Met adenosyltransferase and cystathionine gamma-lyase, whereas decreasing the expression of other Met metabolism genes (P < 0.01) on 6 DPI. Expression of Met metabolism genes linearly increased as MCR decreased (P < 0.05). In conclusion, different Met to Cys supplementation ratios exerted linearly or quadratically effects on the growth performance, oxidative status, intestinal health, and metabolism of Met in broiler chickens under Eimeria infection.

Project description:Induction of cell death represents a primary goal of most anticancer treatments. Despite the efficacy of such approaches, a small population of "persisters" develop evasion strategies to therapy-induced cell death. While previous studies have identified mechanisms of resistance to apoptosis, the mechanisms by which persisters dampen other forms of cell death, such as pyroptosis, remain to be elucidated. Pyroptosis is a form of inflammatory cell death that involves formation of membrane pores, ion gradient imbalance, water inflow, and membrane rupture. Herein, we investigate mechanisms by which cancer persisters resist pyroptosis, survive, then proliferate in the presence of tyrosine kinase inhibitors (TKI). Lung, prostate, and esophageal cancer persister cells remaining after treatments exhibited several hallmarks indicative of pyroptosis resistance. The inflammatory attributes of persisters included chronic activation of inflammasome, STING, and type I interferons. Comprehensive metabolomic characterization uncovered that TKI-induced pyroptotic persisters display high methionine consumption and excessive taurine production. Elevated methionine flux or exogenous taurine preserved plasma membrane integrity via osmolyte-mediated effects. Increased dependency on methionine flux decreased the level of one carbon metabolism intermediate S-(5'-adenosyl)-L-homocysteine, a determinant of cell methylation capacity. The consequent increase in methylation potential induced DNA hypermethylation of genes regulating metal ion balance and intrinsic immune response. This enabled thwarting TKI resistance by using the hypomethylating agent decitabine. In summary, the evolution of resistance to pyroptosis can occur via a stepwise process of physical acclimation and epigenetic changes without existing or recurrent mutations.SignificanceMethionine enables cancer cells to persist by evading pyroptotic osmotic lysis, which leads to genome-wide hypermethylation that allows persisters to gain proliferative advantages.

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Estimation of flux ratios without uptake or release data: Application to serine and methionine metabolism.

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Estimation of flux ratios without uptake or release data: Application to serine and methionine metabolism.

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