Progression from steatosis to steatohepatitis in alcoholic liver disease is enhanced by the risk allele of PNPLA3 via remodeling of lipid droplets and disruption of bioactive lipid composition

项目摘要/摘要： 酒精性肝病(ALD)是世界范围内慢性肝病的主要原因之一，占全世界 在美国，与终末期肝病相关的死亡比例为48%1.与其他类型的肝脏相比 目前，ALD的药理学治疗进展甚微。导致以下问题的因素 缺乏具体的治疗目标，包括目前不太理想的疾病模型和我们对 慢性酒精暴露的易感性和耐受性机制。我们建议通过使用以下方式弥合这一差距 人类干细胞和基因编辑技术研究涉及的最重要的遗传易感因素 在ALD的进展中，PNPLA32-4基因的风险变异多态性。PNPLA3(148异亮氨酸)变异等位基因 对蛋氨酸蛋白变异；I148M)诱导异常脂解并导致肝脏脂肪变性加重 由于长期饮酒以及更具侵袭性的炎症和纤维化进展 这是ALD2，4高级阶段的特征。我们已经产生了人类诱导的 多能干细胞系将被用于这一提议，我们已经优化了一种方案来分化 这些干细胞转化为肝细胞。我们已经完成了PNPLA3变异肝细胞的基本特征和 证实它们会积聚富含甘油三酯的脂滴，并表达炎性细胞因子 被认为与从脂肪变性到肝炎的进展有关。为了了解 变异的PNPLA3增加了对酒精相关性肝损伤的易感性，我们将进行详细的分析 来自人类干细胞系的肝细胞脂滴的表达 PNPLA3.我们将测定脂质成分、蛋白质成分和特异性炎症的特征。 决定ALD患者从脂肪变性到炎症进展的介质。我们将使用乙醇和 棕榈酸可以模拟酒精消费和脂肪毒性引起的环境应激。我们的主要目标是 就是直接在活体人肝细胞中确定可用于治疗的新的治疗靶点 对ALD的影响。

Project Summary/Abstract: Alcoholic liver disease (ALD) is one of the main causes of chronic liver disease worldwide and accounts for up to 48% of deaths associated with end stage liver disease in the United States 1. Compared to other types of liver disease, there has been little progress in the pharmacological management of ALD. Factors contributing to a lack of specific therapeutic targets include current suboptimal disease models and the gap in our knowledge of mechanisms of susceptibility and tolerance to chronic alcohol exposure. We propose to bridge this gap by using human stem cell and gene editing technology to study the most important genetic susceptibility factor involved in ALD progression; the risk variant polymorphism of PNPLA3 2-4. The variant allele of PNPLA3 (148 isoleucine to methionine protein variant; I148M) induces abnormal lipolysis and contributes to enhanced hepatic steatosis as a result of chronic alcohol consumption, as well as more aggressive progression to inflammation and fibrosis that are characteristic of the advanced stages of ALD2,4. We have already generated the human induced pluripotent stem cell lines that will be used for this proposal, and we have optimized a protocol to differentiate these stem cells to hepatocytes. We have performed basic characterization of PNPLA3 variant hepatocytes and confirmed that they accumulate triglyceride-rich lipid droplets and express inflammatory cytokines that are thought to be involved in progression from steatosis to hepatitis. To understand the mechanism by which the variant PNPLA3 confers increased susceptibility to alcohol-related liver injury, we will perform detailed profiling of lipid droplets of hepatocytes derived from human stem cell lines engineered to express the risk variant of PNPLA3. We will determine the lipid composition, protein composition and profile of specific inflammatory mediators that determine progression from steatosis to inflammation in ALD patients. We will use ethanol and palmitic acid to mimic the environmental stress induced by alcohol consumption and lipotoxicity. Our main goal is to identify novel therapeutic targets directly in live human hepatocytes that can be exploited for the treatment of ALD.