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Application of metabolic flux analysis to identify the mechanisms of free fatty acid toxicity to human hepatoma cell line.

ABSTRACT: Chronic exposure to elevated levels of free fatty acids (FFAs) has been shown to cause cell death (lipotoxicity), but the underlying mechanisms of lipotoxicity in hepatocytes remain unclear. We have previously shown that the saturated FFAs cause much greater toxicity to human hepatoma cells (HepG2) than the unsaturated ones (Srivastava and Chan, 2007). In this study, metabolic flux analysis (MFA) was applied to identify the metabolic changes associated with the cytotoxicity of saturated FFA. Measurements of the fluxes revealed that the saturated FFA, palmitate, was oxidized to a greater extent than the non-toxic oleate and had comparatively less triglyceride synthesis and reduced cystine uptake. Although fatty acid oxidation had a high positive correlation to the cytotoxicity, inhibitor experiments indicated that the cytotoxicity was not due to the higher fatty acid oxidation. Application of MFA revealed that cells exposed to palmitate also had a consistently reduced flux of glutathione (GSH) synthesis but greater de novo ceramide synthesis. These predictions were experimentally confirmed. In silico sensitivity analyses identified that the GSH synthesis was limited by the uptake of cysteine. Western blot analyses revealed that the levels of the cystine transporter xCT, but not that of the GSH-synthesis enzyme glutamyl-cysteine synthase (GCS), were reduced in the palmitate cultures, suggesting the limitation of cysteine import as the cause of the reduced GSH synthesis. Finally, supplementing with N-acetyl L-cysteine (NAC), a cysteine-provider whose uptake does not depend on xCT levels, reduced the FFA-toxicity significantly. Thus, the metabolic alterations that contributed to the toxicity and suggested treatments to reduce the toxicity were identified, which were experimentally validated.

SUBMITTER: Srivastava S

PROVIDER: S-EPMC4059351 | biostudies-literature | 2008 Feb

REPOSITORIES: biostudies-literature

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Application of metabolic flux analysis to identify the mechanisms of free fatty acid toxicity to human hepatoma cell line.

Srivastava Shireesh S Chan Christina C

Biotechnology and bioengineering 20080201 2

Chronic exposure to elevated levels of free fatty acids (FFAs) has been shown to cause cell death (lipotoxicity), but the underlying mechanisms of lipotoxicity in hepatocytes remain unclear. We have previously shown that the saturated FFAs cause much greater toxicity to human hepatoma cells (HepG2) than the unsaturated ones (Srivastava and Chan, 2007). In this study, metabolic flux analysis (MFA) was applied to identify the metabolic changes associated with the cytotoxicity of saturated FFA. Mea ...[more]

PMID: 17615559

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Project description:IntroductionFlux phenotypes from different organisms and growth conditions allow better understanding of differential metabolic networks functions. Fluxes of metabolic reactions represent the integrated outcome of transcription, translation, and post-translational modifications, and directly affect growth and fitness. However, fluxes of intracellular metabolic reactions cannot be directly measured, but are estimated via metabolic flux analysis (MFA) that integrates data on isotope labeling patterns of metabolites with metabolic models. While the application of metabolomics technologies in photosynthetic organisms have resulted in unprecedented data from 13CO2-labeling experiments, the bottleneck in flux estimation remains the application of isotopically nonstationary MFA (INST-MFA). INST-MFA entails fitting a (large) system of coupled ordinary differential equations, with metabolite pools and reaction fluxes as parameters. Here, we focus on the Calvin-Benson cycle (CBC) as a key pathway for carbon fixation in photosynthesizing organisms and ask if approaches other than classical INST-MFA can provide reliable estimation of fluxes for reactions comprising this pathway.MethodsFirst, we show that flux estimation with the labeling patterns of all CBC intermediates can be formulated as a single constrained regression problem, avoiding the need for repeated simulation of time-resolved labeling patterns.ResultsWe then compare the flux estimates of the simulation-free constrained regression approach with those obtained from the classical INST-MFA based on labeling patterns of metabolites from the microalgae Chlamydomonas reinhardtii, Chlorella sorokiniana and Chlorella ohadii under different growth conditions.DiscussionOur findings indicate that, in data-rich scenarios, simulation-free regression-based approaches provide a suitable alternative for flux estimation from classical INST-MFA since we observe a high qualitative agreement (rs=0.89) to predictions obtained from INCA, a state-of-the-art tool for INST-MFA.

Dataset Information

Application of metabolic flux analysis to identify the mechanisms of free fatty acid toxicity to human hepatoma cell line.

Publications

Application of metabolic flux analysis to identify the mechanisms of free fatty acid toxicity to human hepatoma cell line.

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