Regulation of Hepatic Phosphatidylcholine Synthesis by mTORC1

项目总结 非酒精性脂肪性肝病(NAFLD)是一种脂代谢改变的疾病，其特征是 脂肪在肝脏中堆积。NAFLD的患病率与肥胖、胰岛素抵抗、 和2型糖尿病。NALF可发展为一种更严重的肝病，称为非酒精性肝病 脂肪性肝炎，以肝脏炎症和纤维化为特征。尽管这些疾病很严重，但有 目前还没有FDA批准的治疗NAFLD和NASH的方法，这突显了澄清 这些疾病的潜在机制，以开发新的治疗方法。在肝脏中，合成代谢激素胰岛素 通过促进三酰甘油(TAG)合成，抑制脂肪酸，调节肝脏脂质稳态 通过极低密度脂蛋白(VLDL)促进标签出口。极低密度脂蛋白标签的分泌 肝脏由磷脂酰胆碱(PC)的生物合成控制，PC是包被脂蛋白的主要磷脂。 人类和啮齿动物模型中PC合成的缺陷与VLDL-Tag分泌减少有关，最终， 全美橄榄球联盟。我们的实验室最近在小鼠体内证明了胰岛素信号的下游，其机制靶点是 雷帕霉素复合体1通过调节Cctα调控极低密度脂蛋白的分泌 PC合成中的酶。因此，本研究的目的是(1)阐明mTORC_1与mTORC_1之间的关系 和Cctα蛋白，以及(2)评价它们对肝脏磷脂合成和脂质的调节作用 体内动态平衡。根据已发表的磷酸蛋白质组学研究，我假设mTORC1直接 在丝氨酸-315处磷酸化Cctα，这种磷酸化阻止泛素化和随后的 Cctα蛋白的降解。在我的初步数据中，我确定了cctα的mTORC1依赖的磷酸化 At丝氨酸-315是控制cctα活性的关键步骤，这种磷酸化调控cctα总蛋白 内容。因此，我将测试cctα是否被mTORc1直接磷酸化，并建立其机制 MTORc1通过其调节cctα的稳定性和活性。此外，我还将测试CCTα的效果 体内丝氨酸-315位的磷酸化，通过使用腺相关病毒载体携带Cctα磷酸化 变种人。根据我的初步数据显示Cctα在Ser315(S315D)模拟磷化突变体已经 Cctα活性增加，我假设Cctα模拟磷酸化突变体S315D增强PC合成和 TAG在小鼠体内的分泌。最后，我将测试CctαS315D突变体是否足以预防NASH 小鼠喂食高脂肪、低蛋氨酸、胆碱缺乏的饮食，这是一种常用的NASH诱导饮食。总而言之， 这项研究的发现将确定mTORC1在调节肝脏PC合成和TAG中的一个新角色 分泌，这可能有利于NAFLD和NASH新疗法的开发。

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.