(283e) Metabolic Flux Determinants of Hepatic Lipotoxicity

Nonalcoholic fatty liver disease (NAFLD) is present in up to 30% of the American population, and is commonly associated with obesity and insulin resistance. A subset of these individuals (about 5%) develop hepatic inflammation, a condition referred to as nonalcoholic steatohepatitis (NASH), which can further progress to cirrhosis, chronic liver disease and hepatocellular carcinoma. Hepatic apoptosis is a prominent feature of NASH and correlates with disease severity. Prior in vitro studies have demonstrated that saturated fatty acids (SFAs) induce apoptosis, whereas monounsaturated fatty acids (MUFAs) predominantly induce steatosis 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 and to prevent its progression toward NASH. It also obscures our view of how NAFLD/NASH as well as other forms of lipotoxicity fit into the broader picture of obesity, insulin resistance, and metabolic syndrome.

To identify metabolic pathways causing hepatic lipoapoptosis, 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 or both palmitate and oleate in combination. Our results indicate that exposure to elevated SFA (400 μM palmitate) 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 oxidation of glutamine as an energy substrate (see Figure). 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 MUFA (50 μM 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 hypothesize that the observed metabolic flux alterations are responsible for triggering mitochondrial ROS generation and apoptotic cell death in palmitate-treated cells.