Perilipin and cellular triacylglycerol metabolism

Perilipin 和细胞三酰甘油代谢

• 批准号: 8470621
• 负责人: DAWN L BRASAEMLE
• 金额: $ 30.55万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-15 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8470621
• 关键词: 1,2-diacylglycerol; Abbreviations; Address; Adipocytes; Adipose tissue; Adrenergic Receptor; Affect; Alanine; Amino Acids; Anisotropy; Area; Binding; Blood; Blood Circulation; Bovine Serum Albumin; CAR receptor; Cell Culture Techniques; Cell model; Cell surface; Cells; Coenzyme A; Complex; Cultured Cells; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cytoplasm; DEXA; Development; Diglycerides; Docking; Drops; Dual-Energy X-Ray Absorptiometry; Embryo; Energy Supply; Energy Transfer; Energy-Generating Resources; Enzyme Tests; Enzymes; Exercise; Fasting; Fatty Acids; Fatty Liver; Fibroblasts; Fluorescence Recovery After Photobleaching; Forms Controls; Glycerides; Goals; Health; Hydrolase; Hydrolysis; Individual; Insulin Resistance; Knockout Mice; Lipase; Lipids; Lipolysis; Liver; Mediating; Metabolic; Metabolic Control; Metabolism; Modeling; Molecular; Molecular and Cellular Biology; Mus; Muscle; Mutate; Mutation; NIH 3T3 Cells; Non-Insulin-Dependent Diabetes Mellitus; Obesity; Peptides; Peripheral; Phospholipid Metabolism; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Polyacrylamide Gel Electrophoresis; Positioning Attribute; Process; Protein Kinase C; Proteins; Recombinants; Recruitment Activity; Regulation; Research; Reverse Transcriptase Polymerase Chain Reaction; Role; Serine; Site; Sodium Dodecyl Sulfate-PAGE; Structure; Surface; Syndrome; Techniques; Testing; Therapeutic Intervention; Tissues; Transgenic Mice; Triglycerides; United States National Institutes of Health; Variant; Work; controlled release; enzyme activity; feeding; in vivo; insulin sensitivity; lipid metabolism; lipine; lysophosphatidic acid acyltransferase; methylxanthine; mouse model; novel; perilipin; perilipin A; protein protein interaction; public health relevance; research study; scaffold; selective expression; sterol esterase; trafficking

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.

描述(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，这些三酰甘油(TAG)被包装成Perilipin包被的脂滴。肥胖者脂肪标签代谢的失调会导致过量的脂肪酸进入循环，从而导致外周胰岛素抵抗和肝脏脂肪变性等健康并发症的发生。脂肪标签的储存和水解的调节是复杂的；我们对这些过程的理解是初步的和不完整的。拟议的研究探讨了Perilipin控制和协调Tag代谢和脂滴动力学的分子机制。这些研究的主要假设是，Perilipin在脂肪细胞中的脂滴表面形成一个支架，作为脂代谢酶和运输因子的组织中心。在基础条件下，当标签储存超过低水平的基础脂解时，Perilipin支架与CGI-58结合，CGI-58是一种依赖于辅酶A的溶血磷脂酸酰基转移酶，也是脂肪甘油三酯脂肪酶(ATGL)的共激活因子。当细胞表面肾上腺素能受体被刺激时，细胞内cAMP水平升高，蛋白激酶A(PKA)被激活。Perilipin被PKA在多达6个丝氨酸残基上磷酸化；这些位点的磷酸化通过几种不同的机制促进脂解。Perilipin氨基末端的PKA位点1、2和3的磷酸化通过与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，HSL获得TAG和二酰甘油底物。羧基末端PKA的4、5和6位的磷酸化促进了脂解，其机制尚不清楚，其中包括促进脂滴碎裂成无数的脂微滴，增加了脂肪酶(ATGL)的结合表面积。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内)在Perilipin的脂滴结合和控制脂解反应中的作用；2)研究PKA介导的丝氨酸492(位于PKA位点5内)的磷酸化在培养细胞模型的脂滴重塑以及细胞和小鼠的脂解中的作用；以及3)研究CGI-58共同激活ATGL的机制，Perilipin可作为调节ATGL TAG水解的平台。Peri-Lipin和CGI-58的突变变体将在培养细胞中进行研究，并建立一种新的转基因小鼠模型，在Perilipin零背景上选择性表达突变的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。