Fellowship/Shetty: PI3-Kinase (P13K) Regulation of SR-BI Distribution and Selective Lipid Uptakes in Hepatocytes

9 Project title (limit to 120 characters or less): PI3-Kinase(PI3K) regulation of SR-BI distribution and selective lipid uptake in hepatocytes. 10.Project summary (must be completed on this page): HDL is considered atheroprotective largely due to its ability to promote reverse cholesterol transport (RCT), a pathway by which HDL transports excess cholesterol from the peripheral tissues to the liver for excretion. SRBI, the HDL receptor, plays a major role in the terminal step of the RCT pathway, by selectively transporting cholesterol esters from HDL into hepatocytes. Our unpublished studies showed that PI3K determines cell surface localization of SR-BI in HepG2 cells. PI3K inhibition markedly decreased (>70%) cell surface expression of SR-BI whereas activation increased it PI3K also correspondingly affected SR-BI dependent selective lipid uptake from HDL. Thus, our data suggeststhat SR-Bllocalization to the plasma membrane,and consequently selective lipid uptake, is dependent on the PI3K activity in the liver and that SR-BI function may be significantly compromised in insulin resistance, when PI3K is less active. The central hypothesis of this proposal is that PI3K regulates SR-BI function, by regulating the cellular localization of SR-B!. Our finding may explain the defective processing of HDL observed in the severely insulin resistant ob/ob (Ieptin deficient) mice. These mice have increased HDL cholesterol levels and large lipid-rich HDL particles due to less efficient hepatic selective lipid uptake despite unchanged total SR-Bllevels. This phenotype was reversed by administration of leptin, which is known to activate PI3K. These findings and the fact that total SR-Bllevels were apparently unchanged suggest a possible regulation of SR-BI by PI3K in the ob/ob mice. This proposal will investigate PI3K regulation of SR-BI in primary hepatocytes and the possible dysregulation of SR-BI due to less active PI3K in the insulin resistant ob/ob mice. The PI3K responsiveness of SR-BI may be due to a putative pleckstrin homology (PH) domain in SR-BI identified by our group (unpublished data). This domain falls in a region (amino acids 360-440), which has previously been shown to affect HDL binding and selective uptake from HDL. In this proposal we will also determine the importance of this domain in the PI3K dependent cell-surface targeting of SR-BI. Specific Aim 1: To test the hypothesis that PI3K regulates cell surface localization of SR-BI in primary hepatocytes of C57BU6 and ob/ob mice. Specific Aim 2: To test the hypothesis that PI3K responsiveness of SR-BI is dependent on the putative PH domain.