Cell dysfunction and death induced by elevated serum free fatty acid (FFA) concentrations is a key contributor to the pathogenesis of obesity and type 2 diabetes. In particular, fat accumulation in the liver can lead to a condition known as non-alcoholic steatohepatitis (NASH), which involves tissue inflammation and apoptotic cell death. To identify metabolic signatures associated with hepatic lipoapoptosis, we applied metabolic flux analysis (MFA) using [U-¹³C₅]glutamine as an isotopic tracer to quantify system-wide phenotypic changes in H4IIEC3 hepatoma cells treated with either palmitate alone or both palmitate and oleate in combination. Our results indicate that palmitate inhibited glycolysis and lactate dehydrogenase fluxes while activating TCA cycle flux. Surprisingly, the increased TCA cycle flux was driven by increased glutamine oxidation rather than increased fatty acid catabolism. Oleate co-treatment restored most fluxes to their control levels, resulting in pronounced lipid accumulation while preventing apoptosis. In addition, palmitate strongly increased the cytosolic NAD⁺/NADH ratio, while oleate co-treatment had the opposite effect on cellular redox. Our results therefore indicate that FFA-induced apoptosis is preceded by the decoupling of glycolysis and TCA cycle fluxes leading to abnormal cytosolic redox states. Future studies to uncover the molecular basis of these systemic effects may lead to improved strategies to prevent NASH in obese and diabetic patients.

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