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

项目摘要（30行文本） 脂肪细胞在能量稳态中起关键作用，以甘油三酯（TG）的形式储存能量。除了 中性脂质，脂肪细胞含有丰富的游离胆固醇来源于循环HDL胆固醇。有一个 脂肪细胞胆固醇含量和TG含量之间存在强正相关，提示机制 脂肪细胞胆固醇和TG调节之间的联系。然而，脂肪细胞胆固醇是如何被 高密度脂蛋白胆固醇，以及脂肪细胞中胆固醇稳态是否以及如何影响甘油三酯的积累， 未知I型B类清道夫受体（SR-BI，由SCARB 1编码）是高亲和力HDL受体， 在脂肪细胞中含量丰富。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）受到高脂饮食诱导的肥胖症的保护，它们的脂肪细胞较小， 白色脂肪组织（WAT）HDL胆固醇摄取和细胞胆固醇含量。的总体目标 项目是阐明HDL胆固醇和SR-BI如何影响脂肪细胞TG含量，并确定是否 脂肪细胞SR-BI表达在人体中受到遗传调节，从而潜在地影响肥胖的风险。三 将在新型小鼠模型和培养的人脂肪细胞中追求目标。目标1将确定HDL是否 胆固醇和SR-BI通过基因的转录调节促进脂肪细胞TG积累， 控制脂质体内平衡。我们已经确定，在SR-BI大鼠WAT中，肝X受体β（LXRβ）的表达 并且其靶基因下调，并且作为LXR配体的氧固醇的含量降低。目的 1将检验通过HDL胆固醇摄取，脂肪细胞SR-BI通过以下方式促进TG积累的假设： 为Cyp 27 a1提供底物，Cyp 27 a1将胆固醇转化为氧化固醇，导致LXRβ上调 及其靶基因。目的2将确定HDL胆固醇和SR-BI是否通过以下方式促进脂肪细胞TG积累： 支持脂肪细胞游离脂肪酸（FFA）摄取，发生在小窝中，是富含胆固醇的血浆 膜结构域电镜观察发现，细胞膜小窝含量明显减少， 在SR-BI脂肪细胞中。目的2将检验SR-BI介导的HDL胆固醇摄取促进高脂血症的假设。 FFA摄取和随后的TG积累通过支持小窝的形成和维持。目标3将 确定脂肪细胞SR-BI表达在人类中是如何转录调节的，以及是否存在遗传学上的差异。 与脂肪细胞SR-BI表达相关的肥胖倾向。使用一种新的深度学习算法， 在Chr 12上的一个区域内鉴定了SCARB 1的一个潜在远端增强子，该区域具有高度相关的SNP， 肥胖症在培养的人脂肪细胞中，候选区域的CRISPR/Cas9缺失降低了SR-BI 表情通过进一步诱变和询问转录因子结合位点，我们将测试 假设存在SCARB 1的远程增强子，其含有通过以下方式影响肥胖风险的SNP： 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的过程 HDL胆固醇和SR-BI通过其控制脂肪细胞功能，从而影响肥胖症的发展。

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.