A novel role of cholesterol and SR-BI in adipocyte biology

项目摘要(30行文本) 脂肪细胞在能量平衡中起关键作用，以甘油三酯(TG)的形式储存能量。除了……之外 中性脂，脂肪细胞含有大量来自循环高密度脂蛋白胆固醇的游离胆固醇。有一个 脂肪细胞胆固醇含量与甘油三酯含量呈显著正相关，提示其机制 脂肪细胞胆固醇和甘油三酯调节之间的联系。然而，脂肪细胞胆固醇是如何通过 高密度脂蛋白胆固醇，以及脂肪细胞中胆固醇稳态是否和如何影响甘油三酯积聚 未知。B型清道夫受体(SR-BI)是一种高亲和力的高密度脂蛋白受体，由SCARB1编码。 富含脂肪细胞。我们最近发现，在脂肪细胞中选择性缺乏SR-BI的小鼠(SR-BI)。 BI∆AD)被保护不受高脂饮食诱导的肥胖的影响，它们的脂肪细胞更小，而且它们已经减少 白色脂肪组织(WAT)高密度脂蛋白胆固醇摄取和细胞胆固醇含量。该计划的总体目标 该项目旨在阐明高密度脂蛋白胆固醇和SR-BI如何影响脂肪细胞的甘油三酯含量，并确定 脂肪细胞SR-BI的表达在人类中受到基因调控，潜在地影响肥胖的风险。三 目标将在新的小鼠模型和培养的人类脂肪细胞中进行。目标1将确定高密度脂蛋白 胆固醇和SR-BI通过转录调控相关基因促进脂肪细胞TG蓄积 控制血脂动态平衡。我们已经确定在SR-BI∆AD Wat中肝X受体β(LXRβ)的表达。 其靶基因表达下调，LXR配体氧合甾醇含量降低。目标 1将检验这一假设，即脂肪细胞SR-BI通过摄取高密度脂蛋白促进甘油三酯的积累 为Cyp27a1提供底物，将胆固醇转化为氧化甾醇，导致LxRβ上调 以及它的目标基因。目标2将确定高密度脂蛋白和SR-BI是否通过以下途径促进脂肪细胞TG的积累 支持脂肪细胞游离脂肪酸(FFA)的摄取，这种摄取发生在富含胆固醇的血浆中的小窝中 膜结构域。通过电子显微镜观察，我们发现小窝的含量明显减少 在SR-BIAD脂肪细胞中。目标2将验证SR-BI介导的高密度脂蛋白胆固醇摄取促进这一假说 通过支持小窝的形成和维持来摄取FFA和随后的TG积累。目标3将 确定脂肪细胞SR-BI在人类中的转录调控方式，以及是否存在基因 肥胖倾向与脂肪细胞SR-BI表达有关。使用一种新的深度学习算法，我们已经 在CHR 12上含有高度相关SNP的区域内发现了SCARB1的潜在远端增强子 与肥胖有关。在培养的人脂肪细胞中，CRISPR/Cas9候选区域的缺失会降低SR-BI 表情。利用进一步的突变和转录因子结合位点的询问，我们将测试 假设存在SCARB1的远程增强子，该增强子含有SNP，通过以下方式影响肥胖风险 它们对脂肪细胞SR-BI表达的影响。总的来说，拟议的研究将揭示基本过程 通过高密度脂蛋白胆固醇和SR-BI调节脂肪细胞的功能，从而影响肥胖的发展。

Project Summary (30 lines of text) Adipocytes play a key role in energy homeostasis, storing energy in the form of triglyceride (TG). In addition to neutral lipids, adipocytes contain abundant free cholesterol derived from circulating HDL cholesterol. There is a strong positive correlation between adipocyte cholesterol content and TG content, suggesting mechanistic linkage between adipocyte cholesterol and TG regulation. However, how adipocyte cholesterol is modulated by HDL cholesterol, and if and how cholesterol homeostasis in adipocytes influences TG accumulation are unknown. Scavenger receptor class B type I (SR-BI, encoded by SCARB1) is a high-affinity HDL receptor that is abundant in adipocytes. We recently discovered that mice selectively deficient in SR-BI in adipocytes (SR- BI∆AD) are protected from high fat diet-induced adiposity, their adipocytes are smaller, and they have decreased white adipose tissue (WAT) HDL cholesterol uptake and cellular cholesterol content. The overall goal of the project is to elucidate how HDL cholesterol and SR-BI influence adipocyte TG content, and to determine if adipocyte SR-BI expression is genetically regulated in humans to potentially impact the risk of obesity. Three Aims will be pursued in novel mouse models and cultured human adipocytes. Aim 1 will determine if HDL cholesterol and SR-BI promote adipocyte TG accumulation through transcriptional regulation of genes that control lipid homeostasis. We have determined that in SR-BI∆AD WAT the expression of liver X receptor β (LXRβ) and its target genes is downregulated and the content of oxysterols, which are LXR ligands, is decreased. Aim 1 will test the hypothesis that via HDL cholesterol uptake, adipocyte SR-BI promotes TG accumulation by providing the substrate for Cyp27a1 which converts cholesterol to oxysterols, leading to upregulation of LXRβ and its target genes. Aim 2 will determine if HDL cholesterol and SR-BI promote adipocyte TG accumulation by supporting adipocyte free fatty acid (FFA) uptake, which occurs in caveolae, which are cholesterol-rich plasma membrane domains. By electron microscopy we have discovered that caveolae content is markedly decreased in SR-BIAD adipocytes. Aim 2 will test the hypothesis that SR-BI-mediated HDL cholesterol uptake promotes FFA uptake and subsequent TG accumulation by supporting caveolae formation and maintenance. Aim 3 will determine how adipocyte SR-BI expression is transcriptionally regulated in humans, and if there is a genetic predisposition to obesity related to adipocyte SR-BI expression. Using a novel deep learning algorithm, we have identified a potential distal enhancer for SCARB1 within a region on Chr 12 harboring SNPs highly associated with obesity. In cultured human adipocytes, CRISPR/Cas9 deletion of the candidate region decreases SR-BI expression. Using further mutagenesis and interrogation of transcription factor binding sites, we will test the hypothesis that there is a remote enhancer of SCARB1 that harbors SNPs that influence the risk of obesity by their impact on adipocyte SR-BI expression. Collectively the proposed studies will reveal fundamental processes by which HDL cholesterol and SR-BI govern adipocyte function and thereby impact the development of obesity.