Mechanistic Investigations on the Implications of mTORC1 and ER Stress Crosstalk in Hepatic Lipotoxicity

Non-alcoholic fatty liver disease (NAFLD) is a type of chronic liver disease intimately related to obesity and considered the hepatic manifestation of metabolic syndrome. A central mechanism underlying NAFLD pathogenesis is hepatic lipotoxicity, defined as the dysfunction and cell death caused by the accumulation of toxic lipids in hepatocytes. Despite intense investigations over last several decades, the exact mechanisms underscoring lipotoxicity remain elusive. Palmitate, a 16-C saturated fatty acid (SFA), is the most abundant SFA in human circulation and is widely used to induce lipotoxicity in cell culture systems. Using exogenous palmitate exposure of hepatocytes (AML-12 and primary hepatocytes) as our primary in vitro hepatolipotoxicity model, three Research Projects were conducted in this thesis to investigate the cellular and molecular mechanisms accounting for palmitate-induced cell death, with a focus on how endoplasmic reticulum (ER) stress and mTORC1 crosstalk to contribute to palmitate-induced lipotoxicity. We demonstrated that exogenous palmitate exposure induced ER stress and activated mTORC1 signaling pathway in hepatocytes, contributing to palmitate-elicited hepatocyte cell death. Further mechanistic investigations revealed that the cross-talk between these two pathways exists. We identified that mTORC1 activation was required for palmitate-elicited activation of IRE1alpha and ATF4, two of the three canonical signaling pathways activated by ER stress. Inhibition of either kinase activity or endoribonuclease activity of IRE1alpha ameliorated palmitate lipotoxicity, suggesting that the mTORC1-IRE1alpha pathway activation contributes to palmitate-induced lipotoxicity in hepatocytes (Project 1). We were the first to identify ATF4 as a transcription factor for hepatic nicotinamide N-methyltransferase (NNMT) expression. Here, we further demonstrated that NNMT upregulation derived from the mTORC1-ATF4 pathway activation contributes to palmitate-induced hepatocyte cell death via preserving PKA activity (Project 2). Finally, we showed that palmitate upregulated CD36 expression in a PPARγ-independent fashion in hepatocytes. Further mechanistic investigation unraveled that ATF4 activation plays a critical role in mediating palmitate-elicited hepatic CD36. Importantly, genetic knockdown of CD36 confers protection against palmitate-induced cell death, suggesting that the ATF4-CD36 pathway activation contributes to palmitate-induced hepatic lipotoxicity (Project 3). Collectively, studies in this thesis provide evidence that mTORC1 activation and ER stress induction coordinately implicate palmitate-induced hepatolipotoxicity.