Analysis of the Spatiotemporal Regulation of Lipolysis

脂肪分解的时空调控分析

• 批准号: 8231445
• 负责人: Lisa M. DiPilato
• 金额: $ 5.39万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-01 至 2013-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8231445
• 关键词: A kinase anchoring protein; Address; Adipocytes; Adipose tissue; Binding; Biochemical; Biological Assay; Biosensor; Blood; Cardiovascular Diseases; Catecholamines; Caveolins; Cells; Clinical; Co-Immunoprecipitations; Complex; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cytosol; Defect; Density Gradient Centrifugation; Development; Diabetes Mellitus; Disease Progression; Dominant-Negative Mutation; Dyslipidemias; Ensure; Fasting; Fatty Acids; Fatty acid glycerol esters; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; Functional disorder; Genetic; Glucose; Glycerol; Goals; Heart; Homeostasis; Hydrolysis; Hypertension; Immunofluorescence Immunologic; Impairment; Individual; Insulin; Insulin Resistance; Lead; Life; Lipids; Lipolysis; Liver; Measures; Mediating; Methods; Molecular; Monitor; Muscle; Myocardium; Neurons; Non-Insulin-Dependent Diabetes Mellitus; Nonesterified Fatty Acids; Nutrient; Obesity; Pathway interactions; Phosphorylation; Process; Protein Isoforms; Proteins; Public Health; RNA Interference; Regulation; Research; Resistance; Resolution; Risk; Role; Scaffolding Protein; Signal Pathway; Signal Transduction; Signaling Protein; Site; Specificity; Staining method; Stains; Stimulus; Stress; Structure of beta Cell of islet; Time; Tissues; Triglycerides; base; caveolin 1; cell growth regulation; design; diabetic; energy balance; fluorescence imaging; glucose uptake; insight; insulin signaling; novel therapeutic intervention; novel therapeutics; perilipin; phosphoric diester hydrolase; research study; spatiotemporal; sterol esterase; time use

DESCRIPTION (provided by applicant): Obesity and diabetes are related public health issues that have become increasingly prominent in the past few decades. Obese individuals have an increased risk for developing Type II diabetes suggesting shared pathophysiology. In fact, obese individuals develop diabetes when they develop insulin resistance, in which tissues no longer respond appropriately to insulin. In particular, adipose tissue is an important site of insulin action, thus resistance in this tissue has major repercussions for whole body energy homeostasis. During fasting, adipocytes, cells of adipose tissue, break down their triglyceride stores (lipolysis) to provide energy for other tissues. Conversely, glucose uptake and suppression of lipolysis is mediated by insulin signaling. However, the mechanism by which insulin opposes lipolysis is still not fully understood. In preliminary studies, we have shown this regulation to be highly localized to lipid droplets, the site of triglyceride storage in fat cells. In this proposal, I set out to elucidate signaling complexes formed by key scaffolding proteins, caveolins and A kinase anchoring proteins (AKAPs) at lipid droplets that organize the spatiotemporal regulation of lipolysis upon stimulation with catecholamine (lipolytic) and insulin (anti- lipolytic). Specifically, I will isolate lipid droplets from 3T3L1 adipocytes via density gradient centrifugation method and probe the fractions for specific isoforms of caveolins and AKAPs upon lipolytic and anti-lipolytic stimuli in order to characterize the conditions under which these proteins dynamically associate with lipid droplets. Furthermore, I will identify the proteins that associate with these scaffolders via co-immunoprecipitation experiments and immunofluorescence staining. Finally, I set out to determine the functional role of these complexes at lipid droplets in the context of lipolysis by measuring glycerol release and PKA activity upon genetic knockdown of caveolin and AKAPs using RNAi approach, as well as upon inhibition and displacement of the components in the complexes. For example, I will generate a dominant negative form of PDE3B that will compete with the endogenous PDE3B for binding to caveolin-1, thus dissociating the protein from signaling complexes at the lipid droplet without effecting the overall PDE activity within the cell. While important information can be collected from these proposed experiments, they only provide a snapshot of what is going on in the cell. To achieve a higher level of spatial and temporal resolution, I will use FRET-based biosensors of cAMP and PKA activity targeted to lipid droplets to monitor the effect of such perturbations on cAMP/PKA signaling in real time using fluorescence microscopy. Ultimately, I aim to address existing gaps in our understanding of the molecular mechanisms that are responsible for the dynamic regulation of lipolysis in hopes to provide key insights into the defects in insulin resistant tissue that contribute to disease progression. The information generated by this study will aid in the development of new therapeutics as well as our understanding of the mechanisms behind the spatiotemporal regulation of cellular signaling.

描述（由申请人提供）：肥胖和糖尿病是相关的公共卫生问题，在过去几十年中变得越来越突出。肥胖个体患 II 型糖尿病的风险增加，这表明有共同的病理生理学。事实上，肥胖者在出现胰岛素抵抗时就会患上糖尿病，此时组织不再对胰岛素做出适当的反应。特别是，脂肪组织是胰岛素作用的重要部位，因此该组织的抵抗力对全身能量稳态具有重大影响。禁食期间，脂肪细胞（脂肪组织细胞）会分解其甘油三酯储存（脂肪分解），为其他组织提供能量。相反，葡萄糖的摄取和脂肪分解的抑制是由胰岛素信号介导的。然而，胰岛素对抗脂肪分解的机制仍不完全清楚。在初步研究中，我们已经证明这种调节高度集中于脂滴，即脂肪细胞中甘油三酯的储存部位。在本提案中，我着手阐明由关键支架蛋白、小窝蛋白和 A 激酶锚定蛋白 (AKAP) 在脂滴上形成的信号复合物，这些复合物在儿茶酚胺（解脂）和胰岛素（抗脂解）刺激下组织脂肪分解的时空调节。 。具体来说，我将通过密度梯度离心法从 3T3L1 脂肪细胞中分离脂滴，并在脂解和抗脂解刺激下探测小窝蛋白和 AKAP 特定亚型的分数，以表征这些蛋白质与脂滴动态结合的条件。此外，我将通过免疫共沉淀实验和免疫荧光染色来鉴定与这些支架相关的蛋白质。最后，我着手确定这些复合物在脂解背景下对脂滴的功能作用，通过使用 RNAi 方法测量小窝蛋白和 AKAP 基因敲除后的甘油释放和 PKA 活性，以及​​抑制和置换中的成分复合体。例如，我将生成 PDE3B 的显性失活形式，它将与内源性 PDE3B 竞争与 Caveolin-1 的结合，从而使该蛋白质与脂滴处的信号复合物解离，而不影响细胞内的整体 PDE 活性。虽然可以从这些拟议的实验中收集重要信息，但它们仅提供细胞中正在发生的情况的快照。为了实现更高水平的空间和时间分辨率，我将使用基于 FRET 的 cAMP 和 PKA 活性生物传感器，针对脂滴，使用荧光显微镜实时监测此类扰动对 cAMP/PKA 信号传导的影响。最终，我的目标是解决我们对负责脂肪分解动态调节的分子机制的理解中存在的差距，希望为有助于疾病进展的胰岛素抵抗组织缺陷提供关键见解。这项研究产生的信息将有助于新疗法的开发以及我们对细胞信号传导时空调节背后机制的理解。