Perilipin and cellular triacylglycerol metabolism

描述（由申请人提供）：脂肪细胞以三酰甘油（TAG）的形式储存身体的主要能量供应，三酰甘油（TAG）包装在周脂蛋白包被的脂滴中。肥胖个体的脂肪 TAG 代谢失调会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初级的和不完整的。拟议的研究调查了 perilipin 控制和协调 TAG 代谢和脂滴动力学的分子机制。这些研究的总体假设是，周脂质在脂肪细胞的脂滴表面形成支架，作为脂质代谢酶和运输因子的组织中心。在基础条件下，当 TAG 储存超过低水平的基础脂肪分解时，周脂质支架会结合 CGI-58，CGI-58 是一种辅酶 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 β-肾上腺素能受体受到刺激时，细胞 cAMP 水平增加，蛋白激酶 A (PKA) 被激活。 Perilipin 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。 Perilipin 氨基末端 PKA 位点 1、2 和 3 的磷酸化可通过与 Perilipin 的蛋白质-蛋白质相互作用促进 PKA 磷酸化激素敏感脂肪酶 (HSL) 在脂滴上的对接，并且 HSL 可以获得 TAG 和二酰基甘油底物。羧基末端 PKA 位点 4、5 和 6 的磷酸化通过迄今知之甚少的机制促进脂肪分解，其中包括促进脂滴破碎成无数脂质微滴，并增加脂肪酶 (ATGL) 结合的表面积。拟议研究的目标是 1) 使用细胞和分子生物学技术研究丝氨酸 517（PKA 位点 6 内）在 perilipin 脂滴关联和脂肪分解控制中的作用，2）研究 PKA 介导的丝氨酸 492（PKA 位点 5 内）磷酸化在培养细胞模型中脂滴重塑以及细胞和小鼠脂肪分解中的作用，以及 3）研究CGI-58 共同激活 ATGL，而 perilipin 充当 ATGL 调节 TAG 水解的平台。将在培养细胞和新型转基因小鼠模型中研究 perilipin 和 CGI​​-58 的突变变体，该模型在 perilipin 无效背景下脂肪选择性表达突变的 perilipin。从这些研究中获得的信息将有助于确定控制脂肪细胞中 TAG 代谢的脂滴相关因子的长期目标。 公共健康相关性：脂肪组织中的脂肪细胞将身体的主要能量储备储存为脂滴结构中的三酰甘油。与瘦人相比，肥胖者脂肪细胞中的脂滴增大。脂滴上覆盖有周脂质，这是一种控制三酰甘油代谢的蛋白质，从而将脂肪酸释放到血液中，用作身体各组织的能量来源。肥胖的特点是脂肪酸不适当且过量地释放到循环中；这些脂肪酸被肌肉和肝脏吸收，导致胰岛素抵抗（发生在 II 型糖尿病中）和脂肪肝。我们对 perilipin 控制脂肪细胞中三酰甘油（脂肪）代谢的机制的理解并不完整；拟议的研究将增进对这些机制的理解，并确定潜在的新目标进行治疗干预，以控制脂肪组织中脂肪酸的释放。

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. PUBLIC HEALTH RELEVANCE: Adipocytes (fat cells) in adipose tissue store the major energy reserves of the body as triacylglycerols in struc- tures called lipid droplets. Obese individuals have enlarged lipid droplets in adipocytes when compared to lean individuals. Lipid droplets are covered with perilipin, a protein which controls the metabolism of triacylglycerols, and hence, the release of fatty acids into the blood for use as a source of energy by various tissues of the body. Obesity is characterized by inappropriate and excessive release of fatty acids into circulation; these fatty acids are taken up by muscle and liver and contribute to the development of insulin resistance (which occurs in type II diabetes) and fatty liver. Our understanding of the mechanisms by which perilipin controls triacylglycerol (fat) metabolism in adipocytes is incomplete; the proposed research will increase understanding of these mechanisms, and identify potential new targets for therapeutic intervention to control release of fatty acids from adipose tissue.