(683b) Phosphoplipid Alterations and ER Calcium Efflux Promote Metabolic Dysfunction in the Context of Hepatic Lipotoxicity

Lipotoxicity from free fatty acid overload has been implicated in a variety of disease states including type II diabetes and non-alcoholic fatty liver disease (NAFLD).  Incubation of hepatic cells with saturated fatty acids (SFAs) induces apoptosis characterized by the accumulation of reactive oxygen species (ROS), metabolic dysfunction, and apoptosis.  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.  Our central hypothesis is that SFA overexposure leads to increased saturation of ER membrane phospholipids, which alters membrane stability, induces endoplasmic reticulum (ER) calcium efflux leading to metabolic dysfunction, and ultimately causes lipoapoptosis.

Our objective was to first determine how perturbations of lipid metabolic pathways influence SFA-induced lipotoxicity.  We measured tritiated palmitate incorporation into total cellular phospholipids in H4IIEC3 rat hepatoma cells.  We found increased saturation of phospholipids which correlated to decreased ER calcium stores.  Interestingly, co-treatment with the mono-unsaturated fatty acid oleate prevented phospholipid oversaturation by redirecting palmitate carbon to triglyceride stores, which blocked apoptosis.  We hypothesized that the observed increases in ER saturation could disrupt membrane homeostasis and enhance calcium efflux.  In fact, after six hours of palmitate treatment we observed decreases in ER calcium indicating a net efflux of calcium from the ER.

To determine if intracellular calcium flux affects ROS accumulation and apoptosis, we co-treated  H4IIEC3 cells with both palmitate and the intracellular calcium chelator BAPTA.  BAPTA co-treatment suppressed ROS accumulation and caspase activation, indicating that these events are calcium dependent.  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 cells treated with either palmitate alone or both palmitate and BAPTA in combination.  Our results indicate that palmitate treatment leads to increased channeling of pyruvate into TCA cycle and  increased mitochondrial oxidation of glutamine as an energy substrate. We hypothesizd that these metabolic alterations are the primary cause of toxic ROS accumulation.  Co-feeding BAPTA restored most mitochondrial fluxes to their control levels while suppressing ROS and apoptosis.  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.  Additionally, our results indicate that the observed metabolic uncoupling is a primary effect of palmitate overexposure due to lipid-induced ER calcium efflux.  These findings shed light on the mechanisms of hepatic lipotoxicity and may suggest novel strategies for the treatment and prevention of NAFLD.