Altered Lipid Droplet Trafficking: Role in Alcoholic Fatty Liver Disease

摘要 这项提案是通过NIAAA资助的多PI R01的竞争性更新。应用程序的目标是 是研究乙醇(Etoh)暴露如何由于动态变化而促进肝脏中的脂肪积累 脂滴(LD)是一种脂肪储存细胞器。几乎所有酗酒者都会患上脂肪肝，这就是 以储存在LDS内的肝细胞内甘油三酯的异常和显著积累为特征。 了解导致脂肪堆积的细胞过程将为 防止进一步损害进展，因为众所周知，酒精性脂肪肝是酒精性脂肪肝的初始但可逆阶段 肝脏损伤。在这里，我们建议提供对这些关键问题的分子见解，旨在 逆转或预防乙醇诱导的脂肪变性的潜在治疗方法。这项建议结合了 两位资深研究员因在乙醇诱导的细胞损伤研究中的贡献而受到表彰 和肝脏LD生物学。我们相信，这一合作努力对ALD领域是有益的，并将产生 仅靠个人努力是无法取得成果的。这项提议包括两个很好地整合的目标。目标 一：乙醇阻断内质网相关的吞脂作用。我们做了一个有趣的观察， 新生的大小保守的LDs(170 Nm)聚集在乙醇损伤的肝细胞内质网表面，并 溶酶体/自噬间隔随后分解这些内质网相关的LDs。值得注意的是，两者 慢性乙醇暴露和溶酶体功能的药物破坏可减弱这一过程，导致 肝细胞脂肪变性。目标二：Etoh对LD蛋白质组的破坏性改变。我们发现有很多 LD“表面蛋白质组”的组成部分在翻译后被泛素修饰，这是我们假设的一种途径 将选定的蛋白质直接移至蛋白酶体进行降解，或将整个LD定向至溶酶体进行降解 分解代谢。重要的是，我们发现慢性乙醇暴露显著增加了泛素化的 LD蛋白质组，从而扰乱正常的LD降解/分解代谢，导致脂肪变性。这些观察结果和 特定的目标将使我们能够追寻Etoh推动的这项研究的中心假说 通过LD蛋白质组抑制的改变积累成熟和新生的内质网相关的LDS 脂肪吞噬和脂肪分解代谢导致肝细胞脂肪变性。拟议的调查将利用 最先进的成像和蛋白质组学技术可以量化导致酒精的特定分子事件- 诱导性脂肪肝。这些研究的成功完成将为研究乙醇如何影响学习成绩提供新的见解 肝细胞的动态和重要的信息，旨在减少或消除的治疗策略 严重的脂肪变性并阻止其进一步发展为酒精性脂肪性肝炎、纤维化和肝硬变。

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.