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

项目概要/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， 在美国，48%的死亡与终末期肝病相关1。与其他类型的肝脏相比 尽管如此，在ALD的药理学管理方面几乎没有进展。导致a的因素 缺乏特异性治疗靶点包括目前的次优疾病模型和我们对以下疾病的认识存在差距： 慢性酒精暴露的易感性和耐受性机制。我们建议通过以下方式来弥补这一差距： 人类干细胞和基因编辑技术，以研究最重要的遗传易感因素参与 在ALD进展中; PNPLA 3 2-4的风险变体多态性。PNPLA 3的变异等位基因（148异亮氨酸 甲硫氨酸蛋白变体; I148 M）诱导异常脂解，并有助于增强肝脂肪变性 由于长期饮酒，以及更积极的炎症和纤维化进展， 这是晚期ALD2.4的特征。我们已经产生了人类诱导的 多能干细胞系，将用于这一建议，我们已经优化了一个协议，以分化 这些干细胞转化为肝细胞。我们已经进行了PNPLA 3变体肝细胞的基本表征， 证实他们积累了富含磷脂酰肌醇的脂滴，并表达炎性细胞因子， 被认为参与了从脂肪变性到肝炎的进展。为了理解 PNPLA 3变异增加了酒精相关肝损伤的易感性，我们将进行详细的分析， 来自人类干细胞系的肝细胞的脂滴，这些干细胞系被工程化以表达 PNPLA3.我们将确定脂质组成，蛋白质组成和特定炎症的概况， 在ALD患者中决定脂肪变性进展为炎症的介质。我们将使用乙醇， 棕榈酸来模拟由酒精消耗和脂毒性诱导的环境应激。我们的主要目标 是直接在人肝细胞中鉴定新的治疗靶点， 的ALD。

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.