Cellular and molecular mechanisms of fatty liver disease

项目摘要 富含甘油三酯的脂滴(LDS)胞浆积聚是非酒精性脂肪肝的标志 疾病(NAFLD)。许多LD相关蛋白与人类和啮齿动物的发病有关 NAFLD，包括CGI-58(比较基因鉴定-58)。人类CGI-58基因突变导致 鱼鳞病(变厚的干燥鳞状皮肤)和富含甘油三酯的LD在大多数细胞类型中聚集。患者表现为非酒精性脂肪肝 从单纯性脂肪变性到非酒精性脂肪性肝炎(NASH)、纤维化和肝硬变。CGI-58是 在体外通过激活脂肪甘油三酯脂肪酶(ATGL)促进甘油三酯的水解，但ATGL突变 在人类身上不会引起鱼鳞病。而肝脏特异性CGI-58基因敲除(LivKO)小鼠则发展为严重的肝脏 脂肪变性、NASH和纤维化即使在常规饮食中，肝脏特异的ATGL KO小鼠也只表现出轻微的 肝脏脂肪变性，无NASH和纤维化。ATGL和CGI-58的这些表型差异 突变为我们提供了一个独特的机会来探索NAFLD进展的分子机制。两大 参与细胞内LD降解的途径有：1)主要由ATGL介导的胞浆/中性脂解作用； 2)脂类特异性大自噬(脂噬)介导的溶酶体/酸性脂解作用。吞脂术带来 胞质低密度脂蛋白被酸性脂肪酶降解为溶酶体，但其分子细节尚不清楚。我们的 初步数据显示，肝脏自噬相关蛋白减少，这是主要的上游自噬 抑制信号通路(MTORC1)被激活，LDS和LDS之间存在关联缺陷 CGI-58缺乏的肝脏和/或肝细胞中的酸性细胞器。最近有研究表明，Perilipin 2，一种主要的LD 已知与CGI-58相互作用的外壳蛋白也与70 kDa的热休克同源蛋白(Hsc70)结合 通过伴侣介导的自噬(CMA)降解和抑制CMA可减少中性和酸性 脂肪分解，导致严重的肝脏脂肪变性和肝脏损伤。基于这些观察和我们的 初步数据，我们假设CGI-58可能与hsc70配位，通过以下方式刺激LD脱涂层 通过CMA促进Perilipin 2的降解，从而激活ATGL脂解和脂噬。吞脂症 失败可能反过来通过改变细胞的能量平衡来抑制自噬。这一假设可以解释为什么 ATGL和CGI-58突变导致重叠但不同的表型。我们将通过以下方式验证这一中心假设 比较肝脏特异性CGI-58和ATGL KO小鼠，以确定肝脏CGI-58和ATGL是否存在差异， 调节脂噬和自噬。然后，我们将进行详细的蛋白质-蛋白质相互作用研究 检查CGI-58和hsc70是否协调CMA介导的LD去涂层(Perilipin 2的降解)。 最后，我们将确定mTORC1抑制和自噬诱导是否对NAFLD的进展具有保护作用 CGI-58LivKO小鼠。鉴于CGI-58处于细胞脂肪分解的关键十字路口，该项目 有望揭示NAFLD进展的一般机制，NAFLD是一个主要的公共卫生问题。

Project Summary Cytosolic accumulation of triglyceride (TG)-rich lipid droplets (LDs) is a hallmark of nonalcoholic fatty liver disease (NAFLD). Many LD-associated proteins are implicated in the pathogenesis of human and rodent NAFLD, including CGI-58 (Comparative Gene Identification-58). Mutations in human CGI-58 gene cause ichthyosis (thickened dry scaly skin) and TG-rich LD accumulation in most cell types. Patients display NAFLD ranging from simple steatosis to nonalcoholic steatohepatitis (NASH), fibrosis and cirrhosis. CGI-58 was shown to promote TG hydrolysis in vitro by activating Adipose Triglyceride Lipase (ATGL), but ATGL mutations in humans cause no ichthyosis. While liver-specific CGI-58 knockout (LivKO) mice develop severe hepatic steatosis, NASH and fibrosis even on a regular chow diet, liver-specific ATGL KO mice display only mild hepatic steatosis without NASH and fibrosis. These phenotypic differences between ATGL and CGI-58 mutations provide us a unique opportunity to exploit molecular mechanisms of NAFLD progression. Two major pathways are implicated in intracellular LD breakdown: 1) Cytosolic/Neutral Lipolysis mainly mediated by ATGL, and 2) Lysosomal/Acidic Lipolysis mediated by a lipid-specific macroautophagy (lipophagy). Lipophagy brings cytosolic LDs to lysosomes for degradation by acidic lipases, but its molecular details are unknown. Our preliminary data suggest that hepatic autophagy-related proteins are reduced, the major upstream autophagy inhibitory signaling pathway (mTORC1) is activated, and there is a defect in association between LDs and acidic organelles in CGI-58-deficient liver and/or hepatocytes. It was recently shown that perilipin 2, a major LD coat protein known to interact with CGI-58, also binds the heat shock cognate protein of 70 kDa (hsc70) for degradation via chaperone-mediated autophagy (CMA), and inhibition of CMA reduces both Neutral and Acidic Lipolysis, leading to severe hepatic steatosis and liver damage. Based on these observations and our preliminary data, we hypothesize that CGI-58 may coordinate with hsc70 to stimulate LD uncoating by promoting perilipin 2 degradation via CMA, thereby activating both ATGL lipolysis and lipophagy. Lipophagy failure may in turn inhibit autophagy by altering cellular energy balance. This hypothesis may explain why ATGL and CGI-58 mutations cause overlapping yet distinct phenotypes. We will test this central hypothesis by comparing liver-specific CGI-58 and ATGL KO mice to determine whether liver CGI-58 and ATGL differentially, regulates lipophagy and autophagy. We will then perform detailed protein-protein interaction studies to examine whether CGI-58 and hsc70 coordinate CMA-mediated LD uncoating (degradation of perilipin 2). Finally we will determine if mTORC1 inhibition and autophagy induction protect against NAFLD progression in CGI-58 LivKO mice. Given that CGI-58 resides at the critical crossroad of cellular fat breakdown, this project holds promise of revealing general mechanisms for progression of NAFLD, a major public health problem.