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

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.