(536b) Uncoupling of Central Metabolism Is Responsible for Palmitate-Induced ROS Accumulation and Hepatic Apoptosis

Excess fat accumulation in the liver results in nonalcoholic fatty liver disease (NAFLD), which is closely associated with obesity and insulin resistance. NAFLD is present in up to 30% of the American population, and can progress to a more severe form of liver dysfunction known as nonalcoholic steatohepatitis (NASH). Hepatocyte apoptosis is a prominent feature of NASH and correlates with disease severity. Prior in vitrostudies have demonstrated that saturated fatty acids (SFAs) are potent inducers of apoptosis, whereas monounsaturated fatty acids (MUFAs) predominantly induce triglyceride accumulation without triggering apoptosis. Several factors including ceramide accumulation, reactive oxygen species (ROS) formation and endoplasmic reticulum (ER) stress have been proposed to explain the unique ability of SFAs to activate apoptotic signaling. However, a consensus mechanism linking SFA-induced metabolic alterations to apoptosis has been difficult to establish. Lack of such knowledge represents an important problem, because it limits the ability of researchers to develop novel nutritional and/or pharmacologic interventions to combat the effects of NAFLD.

To identify metabolic pathways causing hepatic lipotoxicity, we applied metabolic flux analysis (MFA) using [U-¹³C₅]glutamine as an isotopic tracer to quantify phenotypic changes in H4IIEC3 rat hepatoma cells treated with either palmitate alone (SFA) or both palmitate and oleate in combination (SFA + MUFA). Our results indicate that exposure to elevated SFA leads to (1) inhibition of glycolysis, (2) drastically reduced lactate formation, (3) altered cytosolic redox, (4) increased channeling of pyruvate into TCA cycle and (5) increased mitochondrial oxidation of glutamine as an energy substrate. All of these metabolic changes occur over the course of 6-9 hours following palmitate exposure and precede the onset of various apoptotic markers (increased ROS, caspase activation, DNA laddering, cell death) that occur 12-24 hours post-exposure. Co-feeding oleate restored most fluxes to their control levels, resulting in pronounced lipid accumulation while suppressing ROS and apoptosis. In addition, while palmitate strongly increased the cytosolic NAD+/NADH ratio, oleate co-treatment had the opposite effect on cytosolic redox state. Based on the timing of these events, we hypothesized that the observed uncoupling between glycolytic and TCA cycle fluxes is responsible for triggering mitochondrial ROS generation and apoptotic cell death in palmitate-treated cells.

Our hypothesis is supported by recent evidence derived from co-treating H4IIEC3 cells with palmitate and the anti-oxidant N-acetyl cysteine (NAC). Although metabolic uncoupling was still observed in the presence of NAC, the cells did not accumulate ROS or undergo apoptosis. This indicates that the observed metabolic uncoupling is not a secondary effect of ROS or apoptosis, but is a primary effect of palmitate overexposure. We will outline these results and describe their importance for the treatment and prevention of NAFLD and NASH.