Genetic and Metabolic Basis of Fatty Liver Disease

这个项目的总体目标是确定脂肪性肝病（FLD）的代谢和分子基础，这是一个新兴的健康问题，几乎没有治疗选择。2004年，我们的研究小组在达拉斯心脏研究（DHS）中对多种族人群的肝脏甘油三酯（TG）含量进行了首次调查。该调查显示，相对于欧裔美国人（EA），肝脂肪变性在西班牙裔美国人（AA）中更为常见，而在非洲裔美国人（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方法确定了与FLD可重复相关的第一个遗传变异（PNPLA3-148M）。在这3个族群中，HTGC的大部分遗传相关差异都是由这种变异造成的。该变异不仅与脂肪变性有关，还与脂肪性肝炎、肝硬化和肝细胞癌有关。酒精性肝病进展的风险相当。随后，我们在TM6SF2中发现了一个变体，尽管通过完全不同的机制引起脂肪变性，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。在这个应用中，我们建立在这些发现的基础上，阐明这些变异的致病机制，并为治疗干预提供原理证明实验。在目标1中，我们将确定PNPLA3-148M是如何导致肝脂肪变性的，并制定逆转这一过程的策略。先前我们发现PNPLA3-148M在脂滴（LD）上积累。在本研究中，我们将使用148M敲入小鼠来模拟与该变体相关的人类病理生理，以确定PNPLA3在LD上的积累如何促进肝脂肪变性。腺相关病毒（AAV）传递的短发夹（sh） rna和sirna将用于确定敲除PNPLA3-148M是否能逆转脂肪变性。我们还将针对肝脏中控制PNPLA3表达和TG合成的调节机制。在目的2中，我们将确定TM6SF2 167K相关肝脂肪变性的分子基础。先前，我们发现TM6SF2是一种内质网和高尔基蛋白，是VLDL大量脂化所必需的，并且TM6SF2 -/-小鼠复制了人类表型。我们将使用TM6SF2敲除肝细胞系来检测TM6SF2在VLDL组装、运输和分泌中的作用。我们将利用基于赖氨酸的蛋白c端靶向序列来确定TM6SF2促进新生VLDL颗粒脂化的亚细胞位点。我们将验证TM6SF2作为一个支架，在分泌途径中协调中性脂质的添加到VLDL的假设。在Aim 3中，我们将重点关注HSD17B13的一种变体，这种变体在AA中很常见，并在不改变HTGC的情况下赋予FLD进展抗性。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.