(760f) Calcium Stimulated Metabolism Promotes Oxidative Stress in Hepatic Lipotoxicity

The increasing prevalence of obesity in Western society has resulted in higher rates of associated diseases such as type-2 diabetes, atherosclerosis, and nonalcoholic fatty liver disease (NAFLD).  NAFLD is a chronic liver disease resulting from excess lipid accumulation (i.e., steatosis) and affects up to 30% of the US population.  Although simple steatosis does not always lead to complications, around 10% of NAFLD patients are at increased risk of developing more serious liver injuries such as nonalcoholic steatohepatitis  (NASH) and hepatocellular carcinoma.  Free fatty acid (FFA) levels are present in higher concentrations in the plasma of these individuals, suggesting that in vivo alterations in FFA metabolism are linked to corresponding changes in disease severity.  In vitro experiments have demonstrated that saturated fatty acid (SFA) overexposure stimulates acute apoptosis in a diverse range of cell types, although the mechanism of SFA-induced apoptosis may vary depending upon the tissue of origin.  In particular, SFA lipotoxicity in hepatic cells is associated with increased reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress but is independent of ceramide synthesis.  Furthermore, the response to SFA treatment is altogether different from that of monounsaturated fatty acid (MUFA) treatment, which induces steatotic triglyceride formation without initiating ROS accumulation or apoptosis.

To identify mechanisms of palmitate lipotoxicity in hepatic cells, H4IIEC3 cells were treated with free fatty acids in combination with interventions intended to block specific events leading to palmitate-induced metabolic dysfunction and apoptosis.  These experiments indicate that palmitate lipoapoptosis is marked by increased oxygen consumption and accumulation of reactive oxygen species (ROS).  Supplementing culture media with the antioxidant N-acetyl-cysteine (NAC) reduced caspase activation and partially restored cell viability, suggesting that ROS accumulation is a critical factor necessary for activating lipoapoptosis.  However, NAC did not normalize oxygen uptake rates of palmitate-treated cells, indicating that elevated ROS are not required for palmitate-induced alterations to oxidative metabolism.  On the other hand, addition of the mitochondrial antagonist phenformin to palmitate-treated cells eliminated abnormal ROS accumulation, prevented the appearance of apoptotic markers, and normalized oxygen consumption rate.  Taken together, these results indicate that palmitate-induced deregulation of mitochondrial metabolism is the primary cause of ROS accumulation and apoptosis in H4IIEC3 cells.  Furthermore, chelation of intracellular calcium using BAPTA-AM attenuated ROS accumulation, oxygen consumption, caspase activation, and cell death in palmitate-treated cells.  Based on these results, we propose a novel mechanism of palmitate lipotoxicity that is dependent on calcium-stimulated overactivation of oxidative mitochondrial metabolism, which promotes ROS accumulation and apoptosis. 

Our presentation will discuss our experiments using modulators of calcium trafficking (BAPTA) and mitochondrial function (Phenformin, Rotenone) which alter ROS production and demonstrate a clear role for calcium activated mitochondrial metabolism in the context of palmitate lipotoxicity.  Additionally, we will highlight how these alterations are promising targets in modulating NAFLD and lipoapoptosis.