Perilipin and cellular triacylglycerol metabolism

描述（由申请人提供）：脂肪细胞以三酰基甘油（TAGs）的形式储存身体的主要能量供应，这些标签被包装成脂肪周包被的脂滴。肥胖个体脂肪TAG代谢失调导致过量脂肪酸释放到循环中，从而导致外周胰岛素抵抗和肝脂肪变性等健康并发症的发生。脂肪TAG储存和水解的调控是复杂的；我们对这些过程的理解是基本的和不完整的。本研究旨在探讨perilipin控制和协调TAG代谢和脂滴动力学的分子机制。这些研究的首要假设是，脂周蛋白在脂肪细胞的脂滴表面形成支架，作为脂代谢酶和运输因子的组织中心。在基础条件下，当TAG储存高于低水平的基础脂肪分解时，perilipin支架结合CGI- 58，一种辅酶a依赖性溶血磷脂酸酰基转移酶和脂肪甘油三酯脂肪酶（ATGL）的共激活剂。当细胞表面肾上腺素能受体受到刺激时，细胞cAMP水平升高，蛋白激酶A （PKA）被激活。Perilipin可被PKA磷酸化多达6个丝氨酸残基；这些位点的磷酸化通过几种不同的机制促进脂肪分解。PKA位点1、2和3的氨基端磷酸化促进PKA磷酸化的激素敏感脂肪酶（HSL）通过蛋白-蛋白相互作用与脂滴对接，HSL获得TAG和二酰基甘油底物的通路。羧基末端PKA位点4、5和6的磷酸化促进了脂质分解，其机制尚不清楚，其中包括促进脂滴分裂成无数脂质微滴，增加脂酶（ATGL）结合的表面积。本研究的目标是：1)利用细胞和分子生物学技术研究丝氨酸517（位于PKA位点6）在脂滴与脂脂素的关联和脂解控制中的作用；2)研究PKA介导的丝氨酸492（位于PKA位点5）磷酸化在培养细胞模型中的脂滴重塑和细胞和小鼠中的脂解中的作用；3)研究gi -58协同激活ATGL的机制。而perilipin则是ATGL调控TAG水解的平台。研究人员将在培养细胞和一种新的转基因小鼠模型中研究突变的脂质蛋白和CGI-58的突变变体，该模型在脂质蛋白缺失的背景下对突变的脂质蛋白进行脂肪选择性表达。从这些研究中获得的信息将有助于确定脂肪细胞中控制TAG代谢的脂滴相关因子的长期目标。

DESCRIPTION (provided by applicant): Adipocytes store the body's major energy supply in the form of triacylglycerols (TAGs) packaged into perilipin- coated lipid droplets. Dysregulation of adipose TAG metabolism in obese individuals leads to release of excess fatty acids into circulation, which contributes to the development of health complications including peripheral insulin resistance and hepatic steatosis. The regulation of adipose TAG storage and hydrolysis is complex; our understanding of these processes is rudimentary and incomplete. The proposed studies investigate the molecular mechanisms by which perilipin controls and coordinates TAG metabolism and lipid droplet dynamics. The overarching hypothesis of these studies is that perilipin forms a scaffold at the surfaces of lipid droplets in adipocytes that serves as an organizing center for lipid metabolic enzymes and trafficking factors. Under basal conditions, when TAG storage predominates over a low level of basal lipolysis, the perilipin scaffold binds CGI- 58, a Coenzyme A-dependent lysophosphatidic acid acyltransferase and co-activator of adipose triglyceride lipase (ATGL). When cell surface ¿-adrenergic receptors are stimulated, cellular cAMP levels increase and protein kinase A (PKA) is activated. Perilipin is phosphorylated by PKA on as many as 6 serine residues; phosphorylation of these sites promotes lipolysis through several different mechanisms. Phosphorylation of PKA sites 1, 2, and 3 in the amino terminus of perilipin promotes the docking of PKA-phosphorylated hormone- sensitive lipase (HSL) on lipid droplets through a protein-protein interaction with perilipin, and HSL gains access to TAG and diacylglycerol substrates. Phosphorylation of carboxyl terminal PKA sites 4, 5, and 6 facilitates lipolysis by as yet poorly understood mechanisms, which include the promotion of lipid droplet fragmentation into myriad lipid micro-droplets with increased surface area for lipase (ATGL) binding. The goals of the proposed study are to 1) investigate the role of serine 517 (within PKA site 6) in lipid droplet association of perilipin and control of lipolysis using techniques of cellular and molecular biology, 2) investigate the role of PKA-mediated phosphorylation of serine 492 (within PKA site 5) in lipid droplet remodeling in a cultured cell model and lipolysis in both cells and mice, and 3) investigate the mechanisms by which CGI-58 co-activates ATGL, and perilipin serves as a platform for regulation of TAG hydrolysis by ATGL. Mutated variants of peri- lipin and CGI-58 will be studied in cultured cells and a novel transgenic mouse model of adipose-selective expression of mutated perilipin on a perilipin null background. The information gained from these studies will contribute to a long-term goal of defining lipid droplet-associated factors that control TAG metabolism in adipocytes.