Asymmetric Distribution of Cholesterol in Membranes

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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.