Genetic and Metabolic Basis of Fatty Liver Disease

该项目的总体目标是确定脂肪肝（FLD）的代谢和分子基础，这是一种新兴的健康问题，治疗选择很少。在2004年，我们的研究小组进行了第一次调查肝脏甘油三酯（TG）含量在多种族人口为基础的样本，达拉斯心脏研究（DHS）。这项调查显示，肝脂肪变性在西班牙裔美国人中更为常见，而在非洲裔美国人（AA）中相对于欧洲裔美国人（EA）较不常见。为了深入了解这种种族差异的分子基础，我们使用人类遗传学来鉴定与FLD可重复相关的第一个遗传变异（PNPLA 3 - 148 M）。该变异占这3个种族群体中HTGC的大部分祖先相关差异。该变异不仅与脂肪变性有关，而且与脂肪性肝炎、肝硬化和肝细胞癌有关。它赋予酒精性肝病进展的同等风险。随后，我们确定了TM 6SF 2的一个变体，该变体也与全谱FLD相关，尽管其通过完全不同的机制引起脂肪变性。最近，我们发现了第一个防止FLD进展的变异。在本申请中，我们基于这些发现来阐明这些变体的致病机制，并为治疗干预提供原理验证实验。在目标1中，我们将确定PNPLA 3 - 148 M如何引起肝脂肪变性，并制定逆转这一过程的策略。先前我们表明PNPLA 3 - 148 M在脂滴（LD）上积累。在这个目标中，我们将使用148 M“敲入”小鼠来确定PNPLA 3在LD上的积累如何促进肝脂肪变性，以模拟与该变体相关的人类病理生理学。通过腺相关病毒（AAV）递送的短发夹（sh）RNA和siRNA将用于确定敲低PNPLA 3 - 148 M是否逆转脂肪变性。我们还将靶向控制肝脏中PNPLA 3表达和TG合成的调节机制。在目标2中，我们将确定TM 6SF 2 167 K相关的肝脂肪变性的分子基础。以前，我们表明，TM 6SF 2是一个ER和高尔基体蛋白，是所需的大量脂化的VLDL和Tm 6Sf 2-/-小鼠复制人类表型。我们将使用TM 6SF 2基因敲除的肝细胞系来研究TM 6SF 2在VLDL组装、运输和分泌中的作用。我们将利用蛋白质C末端的赖氨酸靶向序列来确定TM 6SF 2促进新生VLDL颗粒脂化的亚细胞位点。我们将测试的假设，TM 6SF 2作为一个支架，以协调添加中性脂质VLDL的分泌途径。在目标3中，我们将重点关注在AA中常见的HSD 17 B13的变体，并且赋予对FLD进展的抗性而不改变HTGC。这3个目标的成功完成将为肝脏TG稳态提供新的见解，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.