Investigating folliculin as a regulator of VLDL-TG secretion in non-alcoholic fatty liver disease and steatohepatitis

摘要 非酒精性脂肪性肝病(NAFLD)是一种迅速出现的公共健康风险。约35%的 美国人患有NAFLD，如果不治疗，NAFLD可能会发展为非酒精性脂肪性肝炎(NASH)， 肝硬变和肝细胞癌。尽管肥胖和II型糖尿病已经成为危险因素， 该病的发病机制尚不清楚。因此，没有FDA批准的药物疗法 目前适用于NAFLD/NASH患者。理想的治疗方法是针对多发性肝脂 动态平衡过程，包括从头脂肪生成(DNL)、脂肪酸氧化(FAO)和极低密度 脂蛋白(VLDL)-甘油三酯(TG)分泌，因为调节单一脂质平衡过程的尝试已经 往往导致来自剩余路径的不受欢迎的补偿。我们的实验室已经鉴定出毛囊蛋白 (FLCN)作为潜在的治疗靶点。肝脏中Flcn基因缺失的小鼠受到强有力的保护 脂肪变性时，NAFLD诱导的饮食通过FLCN-mTORC1-TFE3信号轴进行攻击。TFE3 抑制DNL，诱导FAO相关基因降低肝脏中的甘油三酯含量。vt.给出 极低密度脂蛋白-甘油三酯的分泌在很大程度上受到甘油三酯可用性的调节，我们预计 Flcn基因敲除小鼠的VLDL-TG分泌率。然而，引人注目的是，我们看到极低密度脂蛋白-甘油三酯显著上调。 Flcn缺陷小鼠的分泌物，这使得Flcn作为治疗靶点更具吸引力。我假设 这种上调的极低密度脂蛋白-甘油三酯分泌对于肝脏缺失所提供的强大的预防脂肪变性至关重要。 激活TFE3是VLDL-TG分泌的必要条件和充分条件。为了测试这一点，我将生成 Flcn基因敲除(KO)、TFE3KO和Flcn/TFE3双基因敲除(DKO)小鼠 费率。我将在野生型小鼠中过表达具有结构性活性的TFE3，以测试TFE3是否足以产生VLDL-TG 分泌物。我的初步数据进一步让我假设，在FLCN中上调VLDL-TG分泌- 依赖TFE3的CTP：磷酸胆碱胞苷转移酶转录激活介导的零肝脏 阿尔法(CCTA)。CCTA是磷脂酰胆碱合成过程中的限速酶，是磷脂酰胆碱合成的关键 分泌极低密度脂蛋白-甘油三酯的成分。我的初步数据显示，FLCN中CCTA的mRNA和蛋白水平增加 基因敲除老鼠。我将产生FLCN KO、CCTA KO、FLCN/CCTA DKO小鼠。我会评估他们对 NAFLD/NASH，并进行VLDL-TG分泌分析。这项工作的完成将提供对 NAFLD/NASH影响的肝脏脂质平衡过程的调节，以及Flcn是 一个很有希望的治疗靶点。

Abstract Non-alcoholic fatty liver disease (NAFLD) is a rapidly emerging public health risk. Approximately 35% of Americans have NAFLD, and if left untreated, NAFLD can progress to non-alcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma. Although obesity and Type II diabetes have emerged as risk factors, disease pathogenesis is poorly understood. Accordingly, there are no FDA-approved pharmacotherapies currently available for patients with NAFLD/NASH. An ideal therapeutic would target multiple hepatic lipid homeostatic processes, including de novo lipogenesis (DNL), fatty acid oxidation (FAO), and very-low-density lipoprotein (VLDL)-triglyceride (TG) secretion, as attempts to modulate a single lipid homeostatic process have often led to undesirable compensation from the remaining pathways. Our lab has identified the protein folliculin (FLCN) as a potential therapeutic target. Mice with hepatic deletion of FLCN are robustly protected against steatosis when challenged with NAFLD-inducing diets via an FLCN-mTORC1-TFE3 signaling axis. TFE3 suppresses DNL and induces genes involved in FAO to decrease hepatic TG content in FLCN-null livers. Given that VLDL-TG secretion is heavily regulated by TG availability, we expected to see a compensatory decrease in VLDL-TG secretion rate in FLCN-null mice. Strikingly, however, we saw marked upregulation of VLDL-TG secretion in FLCN deficient mice, which makes FLCN even more appealing as a therapeutic target. I hypothesize that upregulated VLDL-TG secretion is critical to the robust protection against steatosis afforded by liver deletion of FLCN, and that activated TFE3 is necessary and sufficient for VLDL-TG secretion. To test this, I will generate FLCN knockout (KO), TFE3 KO, and FLCN/TFE3 double knockout (DKO) mice and assess VLDL-TG secretion rates. I will overexpress constitutively active TFE3 in wild-type mice to test if TFE3 is sufficient for VLDL-TG secretion. My preliminary data further lead me to hypothesize that the upregulated VLDL-TG secretion in FLCN- null livers is mediated by TFE3-dependent transcriptional activation of CTP:phosphocholine cytidylyltransferase alpha (CCTa). CCTa is the rate-limiting enzyme in the synthesis of phosphatidylcholine, a critical necessary component for VLDL-TG secretion. My preliminary data show increased CCTa mRNA and protein levels in FLCN knockout mice. I will generate FLCN KO, CCTa KO, FLCN/CCTa DKO mice. I will assess their susceptibility to NAFLD/NASH and perform VLDL-TG secretion assays. Completion of this work would provide insight into the regulation of hepatic lipid homeostatic processes affected in NAFLD/NASH, as well as demonstrate FLCN to be a promising therapeutic target for treatment.