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 是研究乙醇暴露如何导致肝脏中的脂肪积累， 脂肪储存细胞器，脂滴（LD）的性质。具体来说，我们将研究酒精如何 暴露通过干扰信号传导损害肝细胞中的脂解和脂肪吞噬机制 级联和膜运输效应，最终导致脂肪肝。在酒精性肝病期间， 几乎所有的重度饮酒者都会发生脂肪肝，其特征是异常和显著的 肝细胞内甘油三酯以LD形式存在。了解导致这种脂肪的细胞过程 积累将为防止损伤的进一步发展提供重要的信息，因为众所周知， 酒精性脂肪肝是肝损伤的初始阶段，但完全可逆。我们在以前的研究中- 资助的R 01研究了肝细胞中LD动力学如何受几种GTP酶（动力蛋白和 特别是RABS），可以作为分子开关来调节膜交通。我们发现乙醇- 这些GTP酶的诱导破坏显著增加了LD在肝细胞中的积累。初步 支持该继续申请的数据表明，肝细胞的脂解机制被以下物质激活： cAMP激酶激动剂（异丙肾上腺素、毛喉素），但乙醇暴露可显著抑制该反应。 此外，我们发现，肝细胞利用顺序机制分解代谢LD，需要脂解 接着是脂肪吞噬。此外，似乎存在于细胞膜上的非受体酪氨酸激酶的激活， LD-自噬体（AP）表面的功能，以驱动脂肪吞噬，酒精损害这一进程。最后在 除了AP对LD的吞噬外，从LD中去除或“取样”脂质似乎是通过 短暂的，可测量的相互作用，对乙醇暴露敏感。这些新的发现提供了一个 这一建议的良好基础，并支持我们的核心假设，即乙醇暴露损害 通过破坏信号级联，破坏肝细胞中的脂肪分解和脂肪吞噬机制， 膜运输事件，导致肝脂肪变性。指导这个项目的两位主要研究员 优势互补：凯西博士是一位生化学家，其专长是酒精诱导的肝脏 损害McNiven博士是一位细胞生物学家，其专长是膜细胞骨架动力学。这种独特 合作努力已被证明对酒精性肝病领域非常有益，并将继续导致 这些结果是个人努力无法达到的。拟议的调查将利用最先进的 膜运输和成像技术，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乙醇如何影响 肝细胞中的LD动力学，以及旨在减少或消除LD的治疗策略的重要信息 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 exposure contributes to fat accumulation in the liver due to altered dynamic properties of the fat storage organelle, the lipid droplet (LD). Specifically we will examine how alcohol exposure compromises both the lipolytic and lipophagic machineries in hepatocytes via disrupting signaling cascades and membrane trafficking effectors, ultimately leading to a fatty liver. During alcoholic liver disease, almost all heavy drinkers develop fatty liver, which is marked by the aberrant and significant accumulation of intrahepatocellular triglycerides in the form of LDs. Understanding the cellular processes contributing to this fat accumulation will provide important information for preventing further progression of injury, as it is known that alcoholic fatty liver is the initial, but entirely reversible stage of liver injury. Our research in the previously- funded R01 examined how LD dynamics in hepatocytes are regulated by several GTPases (dynamins and Rabs in particular) that can act as molecular switches to regulate membrane traffic. We showed that ethanol- induced disruption of these GTPases dramatically increases accumulation of LDs in the liver cell. Preliminary data in support of this continuation application show that the lipolytic machinery of hepatocytes, is activated by agonists of cAMP kinase (isoproterenol, forskolin) but this response is markedly inhibited by ethanol exposure. Additionally, we show that the hepatocyte utilizes sequential mechanisms to catabolize LDs that entail lipolysis followed by lipophagy. Further, it appears that activation of non-receptor tyrosine kinases that reside on the LD-autophagosome (AP) surface function to drive lipophagy, and that alcohol impairs this process. Finally, in addition to engulfment of LDs by APs, removal or “sampling” of lipids away from LDs seems to occur by a transient, measurable interaction that is sensitive to ethanol exposure. These recent, novel findings provide an excellent foundation for this proposal, and support our central hypothesis that ethanol exposure compromises both the lipolytic and lipophagic machineries in the hepatocyte by disrupting signaling cascades and membrane trafficking events, leading to hepatic steatosis. The two principal investigators directing this project have complementary strengths: Dr. Casey is a biochemist whose expertise is in alcoholic-induced liver damage. Dr. McNiven is a cell biologist whose specialty is membrane-cytoskeleton dynamics. This unique collaborative effort has proven very beneficial to the field of alcoholic liver disease and will continue to result in outcomes otherwise unattainable by individual efforts. The proposed investigation will utilize state-of-the-art membrane trafficking and imaging 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 ethanol 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.