(593e) Mitochondrial Dysfunction In Hepatic Lipotoxicity

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 vitro studies have demonstrated that saturated fatty acids (SFAs) are potent inducers of apoptosis, whereas monounsaturated fatty acids (MUFAs) predominantly induce triglyceride accumulation without triggering apoptosis.  SFAs can induce both endoplasmic reticulum (ER) stress and oxidative stress, both of which appear to play a causal role in triggering apoptosis. We have previously shown that SFAs alter central carbon metabolism by reducing glucose uptake and lactate production while increasing glutamine oxidation, and we hypothesize that this uncoupling of glycolytic and mitochondrial metabolism is linked to increased accumulation of reactive oxygen species (ROS) in SFA-treated cells.

Recently, we have investigated a possible mechanism in which calcium translocation from ER to mitochondria leads to overstimulation of oxidative metabolism, resulting in ROS accumulation and lipoapoptotic cell death.  Our approach to study SFA toxicity uses metabolic inhibitors, calcium chelators, antioxidants, and a [U-¹³C₅]glutamine isotopic tracer to quantify phenotypic changes in H4IIEC3 rat hepatoma cells treated with 200-400 μM palmitate.  First, to confirm that ROS is critical in promoting lipoapoptosis, the antioxidant n-acetyl cysteine (NAC) was added to cells treated with palmitate, which resulted in an approximately 50% increase in 24-hour viability.  Therefore, the overproduction of ROS is a critical step in initiating palmitate lipoapoptosis in hepatic cells and is a potential therapeutic target.  Next, we hypothesized that reducing mitochondrial metabolism would result in reduced ROS production and increased viability.  Through co-treatment with the mitochondrial antagonist phenformin, a complete reversal of palmitate-induced ROS was achieved.  Since phenformin is known to stimulate glucose uptake, we used insulin combined with palmitate to examine the effects of increased glucose uptake.  Insulin co-treatment enhanced glucose metabolism, but unlike phenformin resulted in enhanced oxidative stress and higher levels of cell death in SFA-treated cells.  Therefore, increased mitochondrial metabolism and not decreased glucose uptake is responsible for increased oxidative stress in hepatic lipoapoptosis. 

To explore how lipotoxic SFA treatments increase mitochondrial metabolism, we hypothesized a mechanism in which ER stress-induced calcium release overstimulates mitochondrial function.  To limit intracellular calcium trafficking, the membrane permeable calcium-specific chelator BAPTA was added to palmitate-treated cells.  Addition of BAPTA normalized SFA-induced oxidative stress and increased cell viability.  This supports our hypothesis that calcium has a role in promoting mitochondrial metabolism, ROS accumulation, and apoptosis resulting from SFA overexposure. 

In summary, our recent experiments using intracellular calcium chelators (BAPTA) and mitochondrial antagonists (Phenformin) to rescue viability while reducing ROS implicate a critical role for ER calcium release and mitochondrial overactivation in promoting oxidative stress and lipoapoptosis in SFA-treated hepatic cells.  Our presentation will highlight these results and discuss how mitochondrial metabolism and oxidative stress represent promising targets for treating NASH and lipotoxicity.