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Journal of Lipid Research, Vol. 47, 1700-1713, August 2006
Copyright © 2006 by American Society for Biochemistry and Molecular Biology

Quantitative analysis of SR-BI-dependent HDL retroendocytosis in hepatocytes and fibroblasts

Bing Sun^*, Erik R. M. Eckhardt^*, Shoba Shetty^*, Deneys R. van der Westhuyzen^*, and Nancy R. Webb^1,*

^* Graduate Center for Nutritional Sciences and Department of Internal Medicine, University of Kentucky Medical Center, Lexington, KY 40536
Department of Molecular and Cellular Biochemistry, University of Kentucky Medical Center, Lexington, KY 40536

Published, JLR Papers in Press, May 16, 2006.

¹ To whom correspondence should be addressed. e-mail: nrwebb1{at}uky.edu

Previous studies have suggested that HDL retroendocytosis may play a role in scavenger receptor class B type I (SR-BI)-dependent selective lipid uptake in a cell-specific manner. To investigate this possibility, we developed methods to quantitatively measure HDL uptake and resecretion in fibroblast (COS-7) and hepatocyte (HepG2) cells expressing exogenous SR-BI. Approximately 17% and 24% of HDL associated in an SR-BI-dependent manner with COS-7 and HepG2 cells, respectively, accumulates intracellularly after a 10 min incubation. To determine whether this intracellular HDL undergoes retroendocytosis, we developed a pulse-chase assay whereby internalized biotinylated ¹²⁵I-HDL₃ secreted from cells is quantitatively precipitated from cell supernatants using immobilized streptavidin. Our results show a rapid secretion of a portion of intracellular HDL from both cell types (representing 4–7% of the total cell-associated HDL) that is almost complete within 30 min (half-life 10 min). In COS-7 cells, the calculated rate of HDL secretion (0.5 ng HDL/mg/min) was >30-fold slower than the rate of SR-BI-dependent selective cholesteryl ester (CE) uptake (17 ng HDL/mg/min), whereas the rate of release of HDL from the cell surface (19 ng HDL/mg/min) was similar to the rate of selective CE uptake. Notably, the rate of SR-BI-dependent HDL resecretion in COS-7 and HepG2 cells was similar. BLT1, a compound that inhibits selective CE uptake, does not alter the amount of SR-BI-mediated HDL retroendocytosis in COS-7 cells. From these data, we conclude that HDL retroendocytosis in COS-7 and HepG2 cells is similar and that the vast majority of SR-BI-dependent selective uptake occurs at the cell surface in both cell types.

Supplementary key words selective cholesteryl ester uptake • biotinylation • scavenger receptor class B type I

Abbreviations: CE, cholesteryl ester; MFI, mean fluorescence intensity; MOI, multiplicity of infection; SR-BI, scavenger receptor class B type I

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