Genetic and Metabolic Basis of Fatty Liver Disease

这个项目的总体目标是确定脂肪肝(FLD)的代谢和分子基础，这是一种几乎没有治疗选择的新兴健康问题。2004年，我们小组在达拉斯心脏研究(DHS)的多种族人群样本中进行了第一次肝脏甘油三酯(TG)含量的调查。这项调查显示，与欧洲裔美国人(EA)相比，肝脏脂肪变性在西班牙裔美国人中更常见，在非裔美国人(AA)中较少见。为了深入了解这种种族差异的分子基础，我们利用人类遗传学鉴定了与FLD相关的第一个基因变异(PNPLA3-148M)。该变异是这3个民族之间HTGC的祖先相关差异的主要原因。该变异不仅与脂肪变性有关，还与脂肪性肝炎、肝硬变和肝细胞癌有关。它对酒精性肝病的进展具有同等的风险。随后，我们在TM6SF2中发现了一个变异，尽管通过完全不同的机制导致脂肪变性，但它也与FLD的全谱相关。最近，我们发现了第一个防止FLD进展的变异。在这项应用中，我们以这些发现为基础来阐明这些变异的致病机制，并为治疗干预提供原则性实验。在目标1中，我们将确定PNPLA3-148m如何导致肝脏脂肪变性，并制定逆转这一过程的策略。此前，我们证明了PNPLA3-148M积聚在脂滴(LD)上。在这个目标中，我们将使用一只148m的“敲击”小鼠来模拟与这种变异相关的人类病理生理学，来确定PNPLA3在LD上的积累如何促进肝脏脂肪变性。用腺相关病毒(AAV)传递的短发夹状(Sh)RNA和siRNAs来确定敲除PNPLA3-148m是否能逆转脂肪变性。我们还将针对控制肝脏中PNPLA3表达和TG合成的调控机制。在目标2中，我们将确定TM6SF2 167K相关的肝脏脂肪变性的分子基础。此前，我们证明了TM6SF2是一种ER和高尔基蛋白，是VLDL大量脂化所必需的，并且TM6SF2-/-小鼠复制了人类的表型。我们将使用TM6SF2基因敲除的肝细胞株来研究TM6SF2在极低密度脂蛋白的组装、运输和分泌中的作用。我们将利用蛋白质C-末端基于赖氨酸的靶向序列来确定TM6SF2促进新生极低密度脂蛋白颗粒脂化的亚细胞位置(S)。我们将检验这一假设，即TM6SF2作为支架，协调中性脂类在分泌途径中向VLDL添加。在目标3中，我们将重点研究HSD17B13的一个变异，该变异在再生障碍性贫血中很常见，可以在不改变HTGC的情况下对FLD进展产生抵抗。这3个目标的成功完成将为肝脏TG稳态的研究提供新的见解，并为慢性肝病的治疗提供新的策略和靶点。

The overall goal of this project is to define the metabolic and molecular basis of fatty liver disease (FLD), a burgeoning health problem with few therapeutic options. In 2004, our group performed the first survey of hepatic triglyceride (TG) content in a multiethnic population-based sample, the Dallas Heart Study (DHS). This survey showed that hepatic steatosis is much more common in Hispanics and less common in African- Americans (AA) relative to European-Americans (EA). To glean insights into the molecular underpinnings of this ethnic disparity we used human genetics to identify the first genetic variation (PNPLA3-148M) associated reproducibly with FLD. The variant accounts for a majority of the ancestry-related differences in HTGC among these 3 ethnic groups. The variant is not only associated with steatosis, but also steatohepatitis, cirrhosis and hepatocellular carcinoma. It confers equivalent risk for progression of alcoholic liver disease. Subsequently we identified a variant in TM6SF2 that also is associated with the full spectrum of FLD despite causing steatosis by a completely different mechanism. More recently we discovered the first variation that protects against progression of FLD. In this application we build on these discoveries to elucidate the pathogenic mechanisms of these variants and provide proof-of-principle experiments for therapeutic intervention. In Aim 1 we will determine how PNPLA3-148M causes hepatic steatosis and develop strategies to reverse this process. Previously we showed that PNPLA3-148M accumulates on lipid droplets (LD). In this Aim we will determine how accumulation of PNPLA3 on LD promotes hepatic steatosis using a 148M “knockin” mouse to model the human pathophysiology associated with this variant. Short hairpin(sh) RNAs delivered by adeno-associated virus (AAV), and siRNAs will be used to determine if knocking down PNPLA3-148M reverses steatosis. We will also target the regulatory machinery that controls both PNPLA3 expression and TG synthesis in the liver. In Aim 2, we will determine the molecular basis of TM6SF2 167K- associated hepatic steatosis. Previously, we showed that TM6SF2 is an ER and Golgi protein that is required for bulk lipidation of VLDL and that Tm6Sf2-/- mice replicate the human phenotype. We will use a TM6SF2 knockout liver cell line to examine the role of TM6SF2 in VLDL assembly, trafficking, and secretion. We will take advantage of lysine-based targeting sequences at the C-terminal end of the protein to define the subcellular site(s) at which TM6SF2 promotes lipidation of nascent VLDL particles. We will test the hypothesis that TM6SF2 serves as a scaffold to coordinate addition of neutral lipids to VLDL in the secretory pathway. In Aim 3 we will focus on a variant in HSD17B13 that is common in AA and confers resistance to FLD progression without altering HTGC. Successful completion of these 3 aims will provide new insights into hepatic TG homeostasis and new strategies and targets for the treatment of chronic liver disease.