Cytoskeletal Regulation Of Vesicle Transport In Liver

本提案代表一项过渡性资金申请（R56），以便于我们收集初步数据，从而加强R 01申请在未来向NIDDK的应用。其长期目标一直集中在确定关键的细胞骨架成分，协调膜和受体运输途径的肝脏生理学，如脂质储存和利用的关键。非酒精性脂肪性肝病（NAFLD），与潜在的肝脏脂肪变性和炎症，已成为一个突出的健康问题。这些疾病的一个定义特征是称为脂滴（LD）的独特的富含磷脂酰胆碱的细胞器的积累。了解调节肝细胞储存，分解和LD的catelation的基本机制对于有效预防，减少和治疗NAFLD至关重要，也是本提案的重点。基于我们的工作和其他人的重要证据表明，在一个称为脂肪吞噬的过程中，自噬机制对肝脏LD的选择性靶向和分解。我们最近发现了一种新的自噬过程，称为“microlipophagy”（MiLi），溶酶体融合，吞噬，并直接降解LD。我们的证据表明MiLi是肝细胞分解代谢LD的主要机制。我们还发现，巨胞饮作用（MP;“大细胞饮水”）通过脂肪酸的直接内化显著促进肝细胞脂质储存。此外，MP似乎通过从质膜形成大的大胞饮体而在MiLi过程中发挥作用，所述大胞饮体进入细胞以与LD和溶酶体融合。我们和其他人已经证明，这些基本细胞过程的重要组成部分是大（发动蛋白）和小鸟苷三磷酸酶（Rab GTPases）。根据这些观察结果，该提议的中心假设预测MP和MiLi过程一起在肝细胞脂质catalysis中发挥核心作用，并且都受到特定Rab和发动蛋白GTP酶的协同作用的支持和调节，这些GTP酶被过量脂质和类固醇状态改变和破坏。该策略利用最先进的肝细胞成像方法，结合电子显微镜和膜生物化学，与从患者和新型敲除小鼠模型收集的数据相关。目的1将通过检验MP驱动脂肪酸摄取的假设来定义MP对肝细胞脂质储存和脂肪变性的生理贡献，从而通过Rab 5、Rab 10、大GT3 Dyn 2和新的内吞衔接子（SH 3D 19）导致从头LD生物合成和MiLi。目的2将定义溶酶体靶向和catalysts的新生LD通过测试的假设，溶酶体吞噬和分解代谢新生LD形成在ER的新机制，利用肌动蛋白细胞骨架，自噬受体，和Rab 10。这些研究的完成将为肝细胞脂质代谢、肝脂肪变性的基础以及NAFLD治疗干预的潜在新策略提供有价值的见解。

This proposal represents a bridge funding request (R56) to facilitate our collection of preliminary data that will strengthen a future application of an R01 submission to NIDDK. Its long-term objective has been centered on identifying key cytoskeletal components that orchestrate membrane and receptor trafficking pathways critical to liver physiology such as lipid storage and utilization. Non-alcoholic fatty liver disease (NAFLD), with underlying hepatic steatosis and inflammation, has become a prominent health issue. A defining feature of these diseases is the accumulation of unique triglyceride-rich organelles called lipid droplets (LDs). Understanding the fundamental mechanisms that regulate the hepatocellular storage, breakdown, and catabolism of LDs is essential to effectively prevent, reduce, and treat NAFLD and is the focus of this proposal. Significant evidence, based on our work and others, implicates the selective targeting and breakdown of hepatic LDs by the autophagic machinery during a process called lipophagy. We have recently identified a novel autophagic process termed “microlipophagy” (MiLi), by which lysosomes fuse, engulf, and degrade LDs directly. Our evidence indicates that MiLi is the predominant mechanism by which the hepatocyte catabolizes LDs. We also have found that macropinocytosis (MP; “large cellular drinking”) contributes significantly to hepatocellular lipid stores by a direct internalization of fatty acids. Further, MP appears to play a role in the MiLi process by forming large macropinosomes from the plasmalemma that traffic into the cell to fuse with LDs and the lysosome. We, and others, have demonstrated that important components of these essential cellular process are large (Dynamin) and small guanosine triphosphatases (Rab GTPases). From these observations, the central hypothesis of this proposal predicts that together the MP and MiLi processes play a central role in hepatocellular lipid catabolism and are both supported and regulated by the synergistic actions of specific Rab and Dynamin GTPases, which are altered and disrupted by excess lipids and the steototic condition. The strategy utilizes state of the art hepatocellular imaging approaches, coupled with electron microscopy, and membrane biochemistry, correlated with data gleaned from patients, and novel knock out mouse models. Aim 1 will define the physiological contributions of MP to hepatocellular lipid stores and steatosis by testing the hypothesis that MP drives the uptake of fatty acids, leading to de novo LD biogenesis and MiLi via Rab5, Rab10, the large GTPase Dyn2, and a new endocytic adapter (SH3D19). Aim 2 will define the lysosomal targeting and catabolism of nascent LDs by testing the hypothesis that lysosomes engulf and catabolize nascent LDs forming at the ER by novel mechanisms that utilize the actin cytoskeleton, autophagy receptors, and Rab10. Completion of these studies will provide valuable insights into hepatocellular lipid metabolism, the underlying basis for hepatic steatosis, and potential novel strategies for therapeutic intervention in NAFLD.