Altered Lipid Droplet Trafficking: Role in Alcoholic Fatty Liver Disease

摘要 该提案是通过NIAAA资助的多PI R 01的竞争性更新。应用程序的目标 是研究乙醇（EtOH）暴露如何导致肝脏中的脂肪积累， 脂滴（LD）是一种脂肪储存细胞器。几乎所有的酗酒者都会患上脂肪肝， 以储存在LD内的肝细胞内甘油三酯的异常和显著蓄积为标志。 了解这种脂肪积累的细胞过程将为以下方面提供重要信息： 防止进一步的损伤进展，因为众所周知，酒精性脂肪肝是肝硬化的初始但可逆的阶段。 肝损伤在这里，我们建议为这些关键问题提供分子见解，旨在 逆转或预防EtOH诱导的脂肪变性的潜在治疗。该提案结合了 两名高级研究人员因其对EtOH诱导细胞损伤研究的贡献而受到表彰 和肝LD生物学。我们相信这种合作努力对ALD领域是有益的， 无法通过个人努力实现的结果。这项建议包括两个完整的目标。目的 一：EtOH阻断ER相关的脂肪吞噬作用。我们发现了一个有趣的现象， 保守大小（170 nm）的新生LD在EtOH损伤的肝细胞的ER表面积聚， 溶酶体/自噬区室随后分解代谢这些ER相关LD。值得注意的是， 慢性EtOH暴露和溶酶体功能的药理学破坏减弱了这一过程，导致 肝细胞脂肪变性目的二：乙醇对LD蛋白质组的破坏性改变。我们发现很多 LD“表面蛋白质组”的组成部分被泛素后修饰，这是我们发现的一种途径。 将选择的蛋白质引导至蛋白酶体进行降解，或将整个LD引导至溶酶体进行降解。 猫重要的是，我们已经发现慢性乙醇暴露显著增加了细胞的泛素化。 LD蛋白质组，从而破坏正常的LD降解/降解，导致脂肪变性。这些意见和 具体的目标将使我们能够追求EtOH促进的这项研究的中心假设 通过LD蛋白质组的改变抑制成熟和新生ER相关LD的积累， 导致肝细胞脂肪变性的脂肪吞噬和脂质代谢。拟议的调查将利用国家- 最先进的成像和蛋白质组学技术，以量化特定的分子事件，有助于酒精- 诱发脂肪肝这些研究的成功完成将为EtOH如何影响LD提供新的见解 肝细胞中的动力学和重要的信息，为治疗策略，旨在减少或消除 脂肪变性的严重程度，并阻止其进一步发展为酒精性脂肪性肝炎、纤维化和肝硬化。

ABSTRACT This proposal is a competitive renewal of a multiple-PI R01 funded through NIAAA. The goal of the application is to examine how ethanol (EtOH) exposure contributes to fat accumulation in the liver due to altered dynamic properties of the lipid droplet (LD), a fat storage organelle. Almost all heavy drinkers develop fatty liver, which is marked by the aberrant and significant accumulation of intrahepatocellular triglycerides stored within LDs. Understanding the cellular processes contributing to this fat accumulation will provide essential information for preventing further injury progression, as it is known that alcoholic fatty liver is the initial but reversible stage of liver injury. Here, we propose to provide molecular insights into these critical questions, aimed at potential treatments that reverse or prevent EtOH-induced steatosis. This proposal combines the expertise of two senior investigators recognized for their contributions to the study of EtOH-induced cell injury and hepatic LD biology. We believe this collaborative effort has been beneficial to the field of ALD and will result in outcomes not attainable by individual efforts alone. This proposal comprises two well-integrated aims. Aim One: Blockage of ER-Associated Lipophagy by EtOH. We have made the interesting observation that nascent LDs of a conserved size (170nm) accumulate at the ER surface of EtOH-damaged hepatocytes and that lysosomal/autophagic compartments subsequently catabolize these ER-associated LDs. Notably, both chronic EtOH exposure and pharmacologic disruption of lysosome function attenuate this process leading to hepatocyte steatosis. Aim Two: Disruptive Alterations of the LD Proteome by EtOH. We found that many components of the LD “surface proteome” are post-translationally modified by ubiquitin, a pathway we posit directs removal of select proteins to the proteasome for degradation or directs the entire LD to the lysosome for catabolism. Importantly, we have found that chronic EtOH exposure markedly increases the ubiquitination of the LD proteome and thus disrupts normal LD degradation/catabolism leading to steatosis. These observations and specific aims will allow us to pursue the CENTRAL HYPOTHESIS of this study that EtOH promotes accumulation of both mature and nascent, ER-associated, LDs through alterations of the LD proteome inhibiting lipophagy and lipid catabolism leading to hepatocyte steatosis. The proposed investigation will utilize state-of- the-art imaging and proteomic technologies to quantify specific molecular events that contribute to alcohol- induced fatty liver. Successful completion of these studies will provide novel insights as to how EtOH affects LD dynamics in liver cells and important information for therapeutic strategies aimed at reducing or eliminating the severity of steatosis and blocking its further progression to alcoholic steatohepatitis, fibrosis, and cirrhosis.