Obesity has reached epidemic proportions both in the U.S. and abroad, creating rapidly increasing healthcare costs. Metabolic diseases associated with obesity, including type 2 diabetes, non-alcoholic fatty liver disease (NAFLD) and cardiovascular disease, have also seen concomitant increases in population incidence rates. NAFLD, the hepatic manifestation of metabolic syndrome, is characterized by chronic ectopic lipid accumulation in the liver and is estimated to affect over one-third of the U.S. general population and up to 80% of obese and diabetic individuals. An estimated 10-25% of NAFLD patients progress to a more severe condition known as non-alcoholic steatohepatitis (NASH), characterized by chronic inflammation and hepatic cell death that can eventually lead to cirrhosis. Despite its high prevalence, the molecular mechanisms underlying the progression from NAFLD to NASH are not well understood and represent a critical area of investigation in order to develop more targeted and effective therapeutics for fatty liver disease.
In vitro experiments in a wide variety of cell types including Chinese hamster ovary (CHO) cells, pancreatic beta cells, breast cancer cells, HeLa cells and hepatic cells have consistently demonstrated the acute lipotoxic effects of saturated fatty acids, such as palmitate (PA), but not unsaturated fatty acids. Palmitate-mediated lipotoxicity is characterized by endoplasmic reticulum (ER) stress, ER calcium release, reactive oxygen species (ROS) accumulation, caspase activation and cell death. Cells treated with monunsaturated fatty acids (MUFAs), such as oleate (OA), exhibit none of these features but instead demonstrate significant increases in triglyceride (TG) synthesis and accumulation. Adding OA to PA-treated cells can fully reverse PA lipotoxicity though mechanisms that are not well defined but which have been previously attributed to increased TG accumulation and sequestration of PA into neutral lipid stores. However, this hypothesis has never been directly tested by experimentally modulating the relative partitioning of PA/OA between TGs and other lipid fates in hepatocytes. In this study, we test the alternative hypothesis that the rescue effect of PA/OA co-treatment (relative to treatment with PA alone) is due to its ability to normalize the composition of phospholipid membranes, rather than its ability to divert PA into neutral TG species. This hypothesis was suggested by our prior lipidomic profiling study that showed dramatic accumulation of saturated phospholipid species in response to PA treatment of liver hepatocytes, which was completely reversed by PA/OA co-treatment.
The goal of this study was to better understand the mechanism by which oleate rescues palmitate-induced lipotoxicity in hepatic cells and the role that triglyceride and phospholipid metabolism plays in this phenomenon. We show that addition of OA to PA-treated hepatocytes decreases overall phospholipid saturation while rescuing PA-induced apoptotic cell death. As shown previously, this rescue effect is accompanied by enhanced TG synthesis and, specifically, increased incorporation of PA into TG stores. However, simultaneous knockdown of both liver isoforms of diacylglycerol acyltransferase (DGAT), the rate-limiting step in TG synthesis, significantly reduced TG accumulation but without altering the ability of OA to prevent PA lipotoxicity. In both wild-type and DGAT knockdown cells, co-treatment with OA significantly reduced overall phospholipid saturation in comparison to cells treated with PA alone. These data indicate that OA’s protective effects are not due to its ability to divert PA into inert TG stores, but instead may be due to OA’s ability to normalize the composition of cellular phospholipids in the presence of a lipotoxic PA load.
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