Altered Lipid Droplet Trafficking: Role in Alcoholic Fatty Liver Disease

描述（由申请人提供）：本提案是对NIAAA题为“酒精引起的代谢和肝损伤（AIMHI）”的申请请求（RFA）的回应。这个多pi应用的目的是研究乙醇暴露如何导致肝细胞膜运输受损，导致脂肪积聚增加，这是由于被称为脂滴（ld）的大脂肪储存细胞器的动力学改变。在酒精性脂肪性肝病（AFLD）期间，几乎所有重度饮酒者都会发展为脂肪肝，其特征是肝细胞内脂肪酸以ld的形式异常而显著地积累。导致这些细胞器数量和大小显著增加的细胞过程被认为是治疗干预的主要目标，以阻止进一步的进展，因为它是损伤的初始阶段，因此是可逆的。ld的周期性形成、积累和随后的代谢似乎依赖于肝细胞中复杂的运输过程，该过程与内吞和分泌运输途径有明显的相似之处。我们发现肝细胞LD动力学的核心是几种gtpase（特别是动力蛋白和rabs），它们可以作为调节膜交通的分子开关。在博士获得的初步数据。Casey和McNiven在最近资助的“挑战基金”中表明，（通过乙醇或实验操作）破坏这些gtpase可以显著增加肝细胞中ld的积累。这些发现支持了我们的中心假设，即乙醇暴露会导致肝细胞的膜运输机制受损，从而减弱LD的分解，从而导致肝脂肪变性。参与这个拟议项目的两位主要研究人员具有互补的优势；一位是酒精性肝损伤方面的专家（Casey），另一位是肝细胞膜-细胞骨架动力学方面的专家（McNiven）。我们将利用各种最先进的膜运输和成像技术，这些技术在我们的研究领域中是新颖的，我们将研究肝细胞中乙醇如何影响LD的形成和利用。本提案所追求的新颖和创新的生物学概念包括：一，ETOH破坏通常用于帮助脂肪分解的LD的囊泡；二，肝细胞内吞机制在LD囊泡过程中被利用，并被ETOH暴露损害；三，ETOH显著减弱LD蛋白的泛素化，并有助于LD靶向溶酶体进行随后的降解。这些研究的成功完成将为乙醇如何影响肝脏中的LD动力学提供新技术和见解，并提供可能导致旨在降低脂肪变性严重程度和阻止脂肪性肝炎、纤维化和肝硬化进一步发展的治疗策略的信息。

DESCRIPTION (provided by applicant): This proposal is in response to the Request for Applications (RFA) from NIAAA entitled "Alcohol-Induced Metabolic and Hepatic Injury (AIMHI)". The goal of this multiple-PI application is to examine how ethanol exposure can lead to impaired membrane trafficking events in the liver hepatocyte that results in increased fat accumulation due to altered dynamics of large fat storage organelles termed lipid droplets (LDs). During alcoholic fatty liver disease (AFLD), almost all heavy drinkers develop fatty liver, which is marked by the aberrant and significant accumulation of intrahepatocellular fatty acids in the form of LDs. The cellular processes contributing to this marked increase in the number and size of these organelles is considered a prime target for therapeutic intervention to block further progression as it is an initial stage of the injury, and thus reversible. It appears that the cyclical formation, accumulation and subsequent metabolism of LDs are dependent on an intricate trafficking process in the hepatocyte that share marked similarities with the endocytic and secretory trafficking pathways. We have found that central to LD dynamics in hepatocytes are several GTPases (dynamins and rabs in particular) that can act as molecular switches to regulate membrane traffic. In preliminary data obtained by Drs. Casey and McNiven in a recently funded "Challenge Grant" it was shown that disruption of these GTPases (by ethanol or by experimental manipulation) could dramatically increase accumulation of LDs in the liver cell. These findings support our central hypothesis that ethanol exposure leads to an impairment of the membrane trafficking machinery in the hepatocyte that attenuates LD disassembly resulting in hepatic steatosis. The two principal investigators involved in this proposed project have complementary strengths; one is an expert in alcoholic-induced liver damage (Casey), and the other in hepatocyte membrane-cytoskeleton dynamics (McNiven). We will utilize a variety of state-of-the art membrane trafficking and imaging technologies that are novel to this area of research in our investigations of how LD formation and utilization is affected by ethanol in hepatocytes. Novel and innovative biological concepts pursued in this proposal include: one, ETOH disrupts vesiculation of LDs normally used to aid in lipolysis, two, the hepatocyte endocytic machinery is utilized in this LD vesiculation process and compromised by ETOH exposure, three, ubiquitinylation of LD proteins is markedly attenuated by ETOH and aids in targeting of the LDs to the lysosome for subsequent degradation. Successful completion of these studies will provide new technologies and insights as to how ethanol affects LD dynamics in the liver, and provide information which could lead to therapeutic strategies aimed at reducing the severity of steatosis and blocking the further progression to steatohepatitis, fibrosis and cirrhosis. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to examine how alcohol exposure can lead to impaired membrane trafficking events in the liver which would eventually result in increased fat due to altered dynamics of the large fat storage organelles, lipid droplets (LDs). We hypothesize that ethanol exposure leads to hepatic steatosis as a result of attenuated LD vesiculation and subsequent trafficking to lysosomes. This leads to altered LD disassembly which then contributes to hepatic steatosis. Successful completion of these studies will provide novel insights as to how ethanol affects LD dynamics in the liver cells, and provide information which could lead to therapeutic strategies that might reduce the severity of steatosis and block the further progression to steatohepatitis, fibrosis and cirrhosis.