Analysis of the Spatiotemporal Regulation of Lipolysis

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。肥胖个体发生II型糖尿病的风险增加，提示有共同的病理生理学。事实上，当肥胖个体出现胰岛素抵抗时，他们就会患上糖尿病，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，因此该组织中的抵抗对全身能量稳态具有重大影响。在禁食期间，脂肪细胞，脂肪组织的细胞，分解它们的甘油三酯储存（脂解），为其他组织提供能量。相反，葡萄糖摄取和脂解抑制由胰岛素信号传导介导。然而，胰岛素对抗脂肪分解的机制仍然没有完全了解。 在初步研究中，我们已经表明这种调节高度定位于脂滴，脂肪细胞中甘油三酯储存的部位。在该提案中，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）在脂滴处形成的信号传导复合物，其在用儿茶酚胺（脂解）和胰岛素（抗脂解）刺激后组织脂解的时空调节。具体而言，我将通过密度梯度离心法从3 T3 L1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的特定亚型的组分，以表征这些蛋白质动态与脂滴结合的条件。此外，我将通过免疫共沉淀实验和免疫荧光染色鉴定与这些支架相关的蛋白质。最后，我开始确定这些复合物在脂滴中的功能作用，在脂解的背景下，通过测量甘油释放和PKA活性后，基因敲低小窝蛋白和AKAP使用RNAi方法，以及抑制和置换的复合物中的组件。例如，I将产生显性负性形式的PDE 3B，其将与内源性PDE 3B竞争结合小窝蛋白-1，从而使蛋白质从脂滴处的信号传导复合物解离，而不影响细胞内的总体PDE活性。虽然可以从这些拟议的实验中收集到重要的信息，但它们只能提供细胞中发生的事情的快照。为了实现更高水平的空间和时间分辨率，我将使用FRET为基础的cAMP和PKA活性的生物传感器，以脂滴监测cAMP/PKA信号转导的影响，在真实的时间使用荧光显微镜。 最终，我的目标是解决我们对负责脂解动态调节的分子机制的理解中存在的差距，希望为胰岛素抵抗组织中导致疾病进展的缺陷提供关键见解。这项研究产生的信息将有助于开发新的治疗方法，以及我们对细胞信号时空调控机制的理解。 公共卫生相关性：为了确保体内正常的能量平衡，相反的信号通路调节脂肪组织中甘油三酯的分解：儿茶酚胺刺激和胰岛素抑制这一过程。然而，在糖尿病和许多肥胖个体中，脂肪和其他组织变得对胰岛素无反应，导致血液中葡萄糖和脂肪酸的高水平。因此，了解胰岛素如何调节正常功能脂肪组织中脂肪的分解将为抵抗组织的缺陷提供重要的见解，并导致新疗法的开发。

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. PUBLIC HEALTH RELEVANCE: To ensure normal energy balance in the body, opposing signaling pathways regulate the breakdown of triglycerides in adipose tissue: catecholamines stimulate and insulin inhibits this process. However in diabetic and many obese individuals, fat and other tissues become unresponsive to insulin leading to high levels of glucose and fatty acids in the blood. Thus, understanding how insulin regulates the breakdown of fat in normal functioning adipose tissue will provide important insights into the defects of resistant tissue and lead to the development of new therapeutics.