Regulation of Hepatic Phosphatidylcholine Synthesis by mTORC1

项目摘要 非酒精性脂肪性肝病（NAFLD）是一种脂质代谢改变的疾病，其特征在于： 脂肪在肝脏中的积累。NAFLD的患病率与肥胖、胰岛素抵抗、 和2型糖尿病。NALF可以进展为更严重的肝病形式，称为非酒精性肝病。 脂肪性肝炎，以肝脏的炎症和纤维化为特征。尽管这些疾病的严重性， 目前还没有FDA批准的NAFLD和NASH治疗方法，这突出了阐明NAFLD和NASH的关键需求。 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 通过促进甘油三酯（TAG）合成，抑制脂肪酸合成， 分解，并通过极低密度脂蛋白（VLDL）促进TAG输出。VLDL-TAG分泌 肝脏受磷脂酰胆碱（PC）的生物合成控制，PC是主要的磷脂包被脂蛋白。 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， NAFL。我们的实验室最近证明，小鼠胰岛素信号的下游， 雷帕霉素复合物1（mTORC 1）通过调节CCTα控制VLDL-TAG分泌，CCT α是VLDL-TAG分泌的限速因子。 PC合成中的酶因此，本研究的目的是：（1）阐明mTORC 1与 和CCTα蛋白，以及（2）评估其随后对肝磷脂合成和脂质的控制 体内稳态。基于已发表的磷酸化蛋白质组学研究，我假设mTORC 1直接 使CCTα在丝氨酸-315处磷酸化，这种磷酸化阻止了泛素化和随后的 CCTα蛋白降解。在我的初步数据中，我确定了CCTα的mTORC 1依赖性磷酸化， 在丝氨酸-315作为控制CCTα活性的关键步骤，这种磷酸化调节总CCTα蛋白 内容因此，我将测试CCTα是否被mTORC 1直接磷酸化，并建立其机制。 mTORC 1通过其调节CCTα的稳定性和活性。此外，我将测试CCTα的效果， 利用携带CCTα磷酸化的腺相关病毒构建体， 变种人根据我的初步数据，CCTα磷酸模拟突变体S315 D具有 CCTα活性增加，我推测CCTα磷酸化模拟突变体S315 D增强PC合成， 小鼠体内TAG分泌。最后，我将测试CCTα S315 D突变体是否足以预防NASH。 给小鼠喂食高脂肪、低甲硫氨酸、缺乏胆碱的饮食，这是一种常用的NASH诱导饮食。总之， 这项研究的结果将确定mTORC 1在调节肝脏PC合成和TAG中的新作用。 分泌，这可能有利于开发NAFLD和NASH的新疗法。

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.