Regulation of Hepatic Phosphatidylcholine Synthesis by mTORC1

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PROJECT SUMMARY Non-alcoholic fatty liver disease (NAFLD) is a disease of altered lipid metabolism characterized by the accumulation of fat in the liver. The prevalence of NAFLD is closely correlated with obesity, insulin resistance, and type 2 diabetes mellitus. NALF can progress to a more severe form of liver disease, known as non-alcoholic steatohepatitis, hallmarked by inflammation and fibrosis of the liver. Despite the severity of these diseases, there are currently no FDA approved treatments for NAFLD and NASH, highlighting the critical need to elucidate the mechanism underlying these diseases to develop new therapies. In the liver, the anabolic hormone insulin regulates hepatic lipid homeostasis by promoting triacylglyceride (TAG) synthesis, suppressing fatty acid breakdown, and promoting TAG export via very low-density lipoproteins (VLDL). VLDL-TAG secretion from the liver is controlled by the biosynthesis of phosphatidylcholine (PC), the main phospholipid coating lipoproteins. Defects in PC synthesis in human and rodent models are linked to decreased VLDL-TAG secretion and ultimately, NAFL. Our lab recently demonstrated that downstream of insulin signaling in mice, the mechanistic target of rapamycin complex 1 (mTORC1) controls VLDL-TAG secretion through regulation of CCTα, the rate-limiting enzyme in PC synthesis. Therefore, the goals of this study are (1) to elucidate the relationship between mTORC1 and CCTα protein, and (2) to evaluate their subsequent control of hepatic phospholipid synthesis and lipid homeostasis in vivo. Based on published phosphoproteomic studies, I hypothesize that mTORC1 directly phosphorylates CCTα at Serine-315, and this phosphorylation prevents ubiquitination and subsequent degradation of CCTα protein. In my preliminary data, I identify a mTORC1-dependent phosphorylation of CCTα at Serine-315 as a critical step in controlling CCTα activity and this phosphorylation regulates total CCTα protein content. Thus, I will test whether CCTα is directly phosphorylated by mTORC1 and establish the mechanism through which mTORC1 regulates CCTα stability and activity. Furthermore, I will test the effects of CCTα phosphorylation at Serine-315 in vivo, by utilizing adeno-associated virus constructs carry CCTα phosphorylation mutants. On the basis of my preliminary data revealing CCTα phosphomimetic mutant at Ser315 (S315D) has increased CCTα activity, I hypothesize that CCTα phosphomimetic mutant, S315D, enhances PC synthesis and TAG secretion in vivo in mice. Lastly, I will test whether CCTα S315D mutant is sufficient to prevent NASH in mice fed a high fat, low methionine, choline deficient diet, a commonly used NASH-inducing diet. Altogether, findings from this study will identify a novel role of mTORC1 in regulating hepatic PC synthesis and TAG secretion, which may be beneficial for the development of new therapies for NAFLD and NASH.