Regulation of cholesterol transport in adipocytes by SR-BI and PCPE2

摘要 美国成年人和儿童的肥胖率每年都在继续上升。 肥胖是多种慢性病的危险因素，包括2型糖尿病和心血管疾病。 (CVD)，以及其他。脂肪细胞是脂肪积累的主要细胞，它含有在体内膨胀的脂滴 摄取过量循环甘油三酯(TG)作为器官保护机制的反应 脂肪毒性。脂肪细胞含有大约四分之一的体内游离胆固醇，但减少了 自身合成胆固醇的能力。因此，脂肪细胞在很大程度上依赖循环脂蛋白来获得 肥大扩张所需的胆固醇含量。这也意味着胆固醇的运输 脂肪细胞功能的重要决定因素。然而，关于以下方面的知识差距仍然很大 脂肪细胞胆固醇转运的机制和调控。在本应用程序中，我们建立在激动人心的基础上 初步数据表明，胆固醇双向转运功能的高密度脂蛋白受体，清道夫 B型受体I型(SR-BI)在缺乏前胶原肽酶内肽酶的情况下受损 增强子2(PCPE2)是一种细胞外基质蛋白，在小鼠脂肪组织和分化为 小鼠胚胎成纤维细胞。基于这些发现，这份奖学金提案中描述的实验 将检验PCPE2促进SR-BI的胆固醇运输功能以调节脂肪细胞的新假设 胆固醇动态平衡。AIM 1旨在确定PCPE2可能促进的机制 脂肪细胞中的SR-BI寡聚，这是胆固醇运输所需的过程。我们将使用全氟辛酸 酸性聚丙烯酰胺凝胶电泳法检测有无SR-BI齐聚反应 PCPE2。此外，利用尖端的质谱学技术，我们将验证SR-BI/PCPE2 在脂肪细胞中的相互作用，并确定该复合体是否包含其他蛋白质伙伴。AIM 2的设计目的是 研究PCPE2可能影响SR-BI介导的信号转导和膜定位的机制。 首先，我们将测量已知的触发高密度脂蛋白胆固醇酯摄取的信号网络的激活(例如，MAPK 路径)。接下来，我们将进行不连续的蔗糖梯度分离方法来分离脂肪细胞的脂肪 RAFT微域以可视化SR-BI在没有和存在PCPE2的情况下的膜定位。一起， 这些研究将确定(1)SR-BI和PCPE2物理相互作用的机制 双向胆固醇流动，以及(2)PCPE2如何影响SR-BI在双向调节中的作用 脂肪细胞中的胆固醇流动。我们预计，这些研究的结果将有助于识别小说 帮助我们更好地了解导致肥胖、心血管疾病和其他疾病的机制 由这些情况引起的并发症。

ABSTRACT The prevalence of obesity in the United States continues to rise in both adults and children on an annual basis. Obesity is a risk factor for a multitude of chronic diseases, including type 2 diabetes and cardiovascular disease (CVD), among others. Adipocytes, the primary cells for fat accumulation, contain lipid droplets that expand in response to taking up excess circulating triglycerides (TG) as a protective mechanism to prevent organ lipotoxicity. Adipocytes contain approximately a quarter of the body’s pool of free cholesterol, but have a reduced ability to synthesize its own cholesterol. Therefore, adipocytes rely heavily on circulating lipoproteins to acquire the necessary amount of cholesterol for hypertrophic expansion. This also means that cholesterol transport is an essential determinant for adipocyte function. However, there remains a significant knowledge gap regarding the mechanism and regulation of cholesterol transport in adipocytes. In this application, we build on exciting preliminary data that shows that the bi-directional cholesterol transport functions of the HDL receptor, scavenger receptor class B type I (SR-BI), are compromised in the absence of procollagen peptidase endopeptidase enhancer 2 (PCPE2), an extracellular matrix protein, in mouse adipose tissue and adipocytes differentiated from mouse embryonic fibroblasts (MEFs). Based on these findings, experiments described in this fellowship proposal will test the novel hypothesis that PCPE2 facilitates SR-BI’s cholesterol transport functions to regulate adipocyte cholesterol homeostasis. Aim 1 is designed to determine the mechanism by which PCPE2 may be facilitating SR-BI oligomerization in adipocytes, a process required for cholesterol transport. We will use perfluorooctanoic acid polyacrylamide gel electrophoresis [PFO-PAGE] to test SR-BI oligomerization in the presence or absence of PCPE2. Further, using cutting-edge mass spectrometry technology, we will validate the SR-BI/PCPE2 interaction in adipocytes and determine if this complex contains other protein partners. Aim 2 is designed to investigate the mechanisms by which PCPE2 may impact SR-BI-mediated signaling and membrane localization. First, we will measure activation of signaling networks known to trigger HDL-cholesterol ester uptake (e.g. MAPK pathway). Next, we will perform discontinuous sucrose gradient fractionation methods to isolate adipocyte lipid raft microdomains to visualize SR-BI’s membrane localization in the absence and presence of PCPE2. Together, these studies will identify (1) the mechanism by which SR-BI and PCPE2 may physically interact to facilitate bidirectional cholesterol flux, and (2) how PCPE2 may be impacting SR-BI’s role in mediating bidirectional cholesterol flux in adipocytes. We anticipate that the findings from these studies will help identify novel information that will help us better understand the mechanisms driving obesity, cardiovascular disease, and other complications that arise from these conditions.