Asymmetric Distribution of Cholesterol in Membranes

虽然胆汁排泄HDL胆固醇是净胆固醇从体内清除的主要途径，但肝细胞如何如此迅速地摄取和转移HDL衍生的胆固醇用于胆汁流出尚不清楚。了解这一过程可能为降低血浆胆固醇以改善不良脂质代谢提供新的治疗靶点。HDL将胆固醇从组织转运到肝脏，与SRB 1结合以将胆固醇递送到肝细胞基底外侧膜。由于胆固醇自发解吸进入胞质溶胶非常缓慢，因此HDL-胆固醇的快速摄取、解吸和大量跨肝细胞转运必然存在未知机制。基因靶向研究表明，SCP-2和L-FABP增强胆汁胆固醇流出，而不上调ORP，START或NPC家族胆固醇运输蛋白。作为候选蛋白，SCP-2和L-FABP被假设为介导这些快速步骤：结合基底外侧SRB 1，以增强胆固醇摄取/解吸，并增加细胞溶质浓度或胆固醇转运到胆汁中。这项为期2年的ARRA R 01资助将测试这一假设的最重要方面，减少到两个目标，重点是SCP-2和L-FABP功能获得和功能丧失基因工程小鼠肝细胞的结构和功能分析： 1）。胆固醇摄取：SCP-2或L-FABP是否通过与基底外侧膜中的SRB 1结合来影响HDL胆固醇摄取？初步数据显示，SCP-2在体外与SRB 1结合，SCP-2过表达增加，SCP-2/SCP-x消融降低了肝细胞中SR-B1的水平。HDL增加SRB 1/SCP-2在基底外侧膜的共定位。 2）。胞质胆固醇转运：SCP-2或L-FABP能否通过增加HDL衍生胆固醇的胞浆浓度或转移速率促进跨肝细胞运动？SCP-2和L-FABP增加水溶性，增强胞质转运/扩散，并在肝细胞中共转运另一种配体（脂肪酸）。 这些实验将解决一个关键的悬而未决的问题，在肝胆固醇排泄HDL-胆固醇是如何如此迅速地采取了在基底外侧膜，并通过细胞质转移到肝细胞小管膜胆汁流出？通过检验SCP-2和L-FABP介导快速摄取和转移的假设，将获得关于介导胆汁胆固醇流出这些快速步骤的机制的基本新见解，胆汁胆固醇流出是从体内去除过量胆固醇的主要途径。如果理解了这一过程，就有可能开发出增加胆固醇流出以降低血浆胆固醇的新的治疗靶点。

Although biliary excretion of HDL cholesterol is the major route for net cholesterol removal from the body, how hepatocytes so rapidly take up and transfer HDL-derived cholesterol for biliary efflux is unknown. Understanding this process may provide new therapeutic targets for lowering plasma cholesterol to improve adverse lipid metabolism. HDL transports cholesterol from tissues to liver, binding to SRB1 for cholesterol delivery to the hepatocyte basolateral membrane. Since spontaneous cholesterol desorption into cytosol is very slow, an unknown mechanism must exist for rapid uptake, desorption and massive transhepatocyte transport of HDL-cholesterol. Gene targeting studies show that SCP-2 and L-FABP enhance biliary cholesterol efflux without upregulating ORP, START, or NPC family cholesterol-trafficking proteins. As candidate proteins, SCP-2 and L-FABP are hypothesized to mediate these rapid steps by: binding basolateral SRB1 to enhance cholesterol uptake/desorption and increase cytosolic concentration or transport of cholesterol for canalicular efflux into bile. This 2 year ARRA R01 grant will test the most important aspects of this hypothesis, reduced to two aims, focusing on structural and functional analyses of hepatocytes from SCP-2 and L-FABP gain-of function and loss-of-function genetically-engineered mice: 1). Cholesterol uptake: Does SCP-2 or L-FABP impact HDL cholesterol uptake by binding with SRB1 in the basolateral membrane? Preliminary data show that SCP-2 binds SRB1 in vitro, SCP-2 overexpression increases, and SCP-2/SCP-x ablation decreases SR-B1 levels in hepatocytes. HDL increases SRB1/SCP-2 colocalization at the basolateral membrane. 2). Cytosolic cholesterol transport: Can SCP-2 or L-FABP facilitate transhepatocyte movement by increasing cytoplasmic concentration or rate of transfer of HDL-derived cholesterol? SCP-2 and L-FABP increase aqueous solubility, enhance cytosolic transport/diffusion, and cotransport another ligand (fatty acid) in hepatocytes. These experiments will address a key unanswered question in hepatic cholesterol excretion—how is HDL-cholesterol so rapidly taken up at the basolateral membrane and transferred through the cytoplasm to the hepatocyte canalicular membrane for biliary efflux? By testing the hypothesis that SCP-2 and L-FABP mediate rapid uptake and transfer, fundamental new insights will be gained regarding mechanisms mediating these rapid steps of biliary cholesterol efflux—the primary route for excess cholesterol removal from the body. If this process were understood, new therapeutic targets for increasing cholesterol efflux for lowering plasma cholesterol could potentially be developed.