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 是研究酒精暴露如何导致肝脏中脂肪积聚的动态变化 脂肪储存细胞器的性质，即脂滴(LD)。具体地说，我们将研究酒精如何 暴露通过干扰信号损害肝细胞中的脂解和脂噬机制 级联和膜转运效应器，最终导致脂肪肝。在酒精性肝病期间， 几乎所有大量饮酒的人都会患上脂肪肝，其特点是体内脂肪的异常和显著堆积 肝细胞内甘油三酯以LDS的形式存在。了解导致这种脂肪的细胞过程 积累将为防止伤害的进一步发展提供重要信息，因为众所周知 酒精性脂肪肝是肝损伤的初始但完全可逆的阶段。我们在之前的研究- Funded R01研究了几种GTP酶(Dynamins和 尤其是RABS)，可以作为分子开关来调节细胞膜的运输。我们证明了乙醇- 这些GTP酶的诱导破坏显著增加了LDS在肝细胞中的积累。初步 支持这一继续应用的数据显示，肝细胞的脂解机制是由 CAMP激酶激动剂(异丙肾上腺素、Forskolin)，但这种反应被乙醇暴露明显抑制。 此外，我们还表明，肝细胞利用顺序机制来分解导致脂肪分解的低密度脂蛋白。 紧随其后的是脂噬。此外，似乎驻留在细胞表面的非受体酪氨酸激酶的激活 LD-自噬小体(AP)表面具有驱动吞脂性的功能，而酒精损害了这一过程。最后，在 除了AP吞噬LDS外，从LDS上移走或“采样”脂质似乎是通过 对酒精暴露敏感的瞬时、可测量的相互作用。这些最新的新发现提供了一个 为这一提议奠定了良好的基础，并支持我们的核心假设，即接触乙醇会损害 肝细胞中的脂解和脂噬机制通过破坏信号级联和 膜转运事件，导致肝脏脂肪变性。指导这一项目的两名主要调查人员 优势互补：凯西博士是一名生物化学家，他的专长是酒精诱导的肝脏 损坏。麦克尼文博士是一位细胞生物学家，他的专长是膜-细胞骨架动力学。这个独一无二的 事实证明，合作努力对酒精性肝病领域非常有益，并将继续导致 否则，单靠个人努力是无法实现的结果。拟议中的调查将利用最先进的技术 膜运输和成像技术，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乙醇如何影响提供新的见解 肝细胞中LD的动态变化，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 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.