Analysis of the Spatiotemporal Regulation of Lipolysis

描述（由申请人提供）：肥胖和糖尿病是近几十年来日益突出的公共卫生问题。肥胖个体患II型糖尿病的风险增加，这表明两者有共同的病理生理机制。事实上，肥胖个体在产生胰岛素抵抗时就会患上糖尿病，即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，因此该组织的抵抗对全身能量稳态有重大影响。在禁食期间，脂肪细胞，脂肪组织的细胞，分解它们储存的甘油三酯（脂肪分解），为其他组织提供能量。相反，葡萄糖摄取和脂肪分解的抑制是由胰岛素信号介导的。然而，胰岛素抵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局限于脂滴，即脂肪细胞中甘油三酯储存的部位。在本提案中，我着手阐明由关键支架蛋白、小洞蛋白和A激酶锚定蛋白（AKAPs）在脂滴上形成的信号复合物，它们在儿茶酚胺（脂溶）和胰岛素（抗脂溶）刺激下组织脂溶的时空调节。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，并探测在脂溶和抗脂溶刺激下的小泡蛋白和akap的特定同工型，以表征这些蛋白质与脂滴动态关联的条件。此外，我将通过共免疫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，我开始确定这些复合物在脂溶过程中在脂滴处的功能作用，通过使用RNAi方法测量甘油释放和基因敲低小窝蛋白和akap时的PKA活性，以及复合物中成分的抑制和位移。例如，我将生成PDE3B的显性阴性形式，它将与内源性PDE3B竞争与小窝蛋白-1的结合，从而将蛋白质与脂滴的信号复合物分离，而不会影响细胞内的整体PDE活性。虽然从这些拟议的实验中可以收集到重要的信息，但它们只提供了细胞中发生的事情的快照。为了获得更高的空间和时间分辨率，我将使用基于fret的靶向脂滴的cAMP和PKA活性生物传感器，利用荧光显微镜实时监测这种扰动对cAMP/PKA信号的影响。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的现有空白，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节背后的机制的理解。

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.