Cytoskeletal Regulation Of Vesicle Transport In Liver

肝脏囊泡运输的细胞骨架调节

• 批准号: 10664266
• 负责人: MARK A. MC NIVEN
• 金额: $ 39.75万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-02-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10664266
• 关键词: Actins; Affect; Albumins; Automobile Driving; Autophagocytosis; Biochemistry; Biogenesis; Catabolism; Cell membrane; Cell physiology; Cells; Cellular Stress; Collection; Complex; Consequentialism; Coupled; Critical Pathways; Cytoskeleton; Data; Diabetes Mellitus; Disease; Dynamin; Electron Microscopy; Electrons; Endoplasmic Reticulum; Failure; Fatty Acids; Fatty Liver; Fatty acid glycerol esters; Funding; Future; Generations; Glean; Goals; Guanosine; Guanosine Triphosphate Phosphohydrolases; Health; Hepatic; Hepatic Tissue; Hepatocyte; In Vitro; Incidence; Knockout Mice; Label; Lead; Light Cell; Lipids; Liver; Lysosomes; Membrane; Metabolic Diseases; Microscopic; Molecular; Mus; National Institute of Diabetes and Digestive and Kidney Diseases; Non-Insulin-Dependent Diabetes Mellitus; Organelles; Patients; Pharmacology; Phospholipids; Physiological; Physiology; Play; Population; Primary carcinoma of the liver cells; Process; Proteins; Regulation; Resolution; Role; Societies; Stress; Testing; Therapeutic Intervention; Triglycerides; Visualization; Work; base; drinking; evidence base; extracellular; imaging approach; in vivo; inhibitor; insight; light microscopy; lipid metabolism; liver inflammation; monolayer; mouse model; non-alcoholic fatty liver disease; novel; novel therapeutic intervention; prevent; protein complex; ras Proteins; receptor; response; trafficking; uptake; vesicle transport

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.

这项提案是一项过渡性资金申请(R56)，以促进我们收集初步数据，以加强未来向NIDDK提交R01的申请。它的长期目标一直集中在识别关键的细胞骨架成分，这些成分协调膜和受体运输途径，对肝脏生理至关重要，如脂类的储存和利用。非酒精性脂肪性肝病(NAFLD)以肝脏脂肪变性和炎症为基础，已成为一个突出的健康问题。这些疾病的一个明显特征是聚集了独特的富含甘油三酯的细胞器，称为脂滴(LDS)。了解LDS调节肝细胞储存、分解和分解代谢的基本机制对于有效预防、减少和治疗NAFLD是至关重要的，也是本提案的重点。基于我们的工作和其他工作的重要证据表明，在一个称为脂噬的过程中，自噬机制选择性地靶向和分解了肝脏LDS。我们最近发现了一种新的自噬过程，称为“微脂噬”(MILI)，通过它，溶酶体直接融合、吞噬和降解LDS。我们的证据表明，MILI是肝细胞分解LDS的主要机制。我们还发现，巨噬细胞增多(MP；“大细胞饮酒”)通过脂肪酸的直接内化显著促进了肝细胞的脂肪储存。此外，MP似乎在MILI过程中发挥作用，从质膜形成大的大管胞小体，进入细胞与LDS和溶酶体融合。我们和其他人已经证明了这些基本细胞过程的重要组成部分是大的(动力蛋白)和小的鸟苷三磷酸酶(RAB GTP酶)。从这些观察中，这一建议的中心假设预测，MP和MILI过程一起在肝细胞脂质分解代谢中发挥中心作用，并且都受到特定的Rab和Dynamin GTP酶的协同作用的支持和调节，这两种酶会被过量的脂质和硬脂酸条件改变和破坏。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，与从患者收集的数据和新的基因敲除小鼠模型相关联。目的1通过验证MP驱动脂肪酸摄取的假说，确定MP对肝细胞脂肪储存和脂肪变性的生理贡献，导致从头开始LD的生物发生和通过Rab5、Rab10、大GTP酶Dy2和一个新的内吞适配器(SH3D19)的MILI。目的2通过利用肌动蛋白细胞骨架、自噬受体和Rab10的新机制，验证溶酶体吞噬并分解内质网形成的新生LDS的假说，从而确定新生LDS的溶酶体靶向和分解代谢。这些研究的完成将对肝细胞脂代谢、肝脏脂肪变性的潜在基础以及治疗NAFLD的潜在新策略提供有价值的见解。