Cellular and molecular mechanisms of fatty liver disease

项目摘要 富含甘油三酯（TG）的脂滴（LDs）在细胞内的积聚是非酒精性脂肪肝的标志 非酒精性脂肪肝（NAFLD）。许多LD相关蛋白参与了人类和啮齿类动物的发病机制 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（增厚的干燥鳞状皮肤）和大多数细胞类型中富含TG的LD积累。患者显示NAFLD 从单纯性脂肪变性到非酒精性脂肪性肝炎（NASH）、纤维化和肝硬化。CGI-58是 显示通过激活脂肪甘油三酯脂肪酶（ATGL）促进TG体外水解，但ATGL突变 不会引起鱼鳞病虽然肝脏特异性CGI-58基因敲除（LivKO）小鼠发生严重的肝损伤， 脂肪变性、NASH和纤维化，即使在常规食物饮食中，肝脏特异性ATGL KO小鼠也仅显示轻度的 无NASH和纤维化的肝脂肪变性。ATGL和CGI-58之间的这些表型差异 突变为我们提供了一个独特的机会，利用NAFLD进展的分子机制。两大 途径与细胞内LD分解有关：1）主要由ATGL介导的细胞溶质/中性脂解， 和2）由脂质特异性大自噬（脂肪吞噬）介导的溶酶体/酸性脂解。脂肪吞噬 胞质LDs的溶酶体降解的酸性脂肪酶，但其分子的细节是未知的。我们 初步数据表明，肝脏自噬相关蛋白减少，主要的上游自噬 抑制性信号传导途径（mTORC 1）被激活，并且LD和 CGI-58缺陷的肝脏和/或肝细胞中的酸性细胞器。最近的研究表明，围脂蛋白2，一种主要的LD 已知与CGI-58相互作用的外壳蛋白也结合70 kDa的热休克同源蛋白（hsc70）， 通过分子伴侣介导的自噬（CMA）降解，CMA的抑制降低了中性和酸性 脂肪分解，导致严重的肝脂肪变性和肝损伤。根据这些观察和我们的 根据初步数据，我们假设CGI-58可能与hsc70协调，通过以下方式刺激LD脱膜： 通过CMA促进周脂蛋白2降解，从而激活ATGL脂解和脂肪吞噬。食脂性 失败又可通过改变细胞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来检验这一中心假设： 比较肝特异性CGI-58和ATGL KO小鼠以确定肝CGI-58和ATGL是否差异， 调节脂肪吞噬和自噬。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脱包（周脂蛋白2的降解）。 最后，我们将确定mTORC 1抑制和自噬诱导是否可以防止NAFLD进展。 CGI-58 LivKO小鼠。鉴于CGI-58位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.