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

Department of Internal Medicine, University of Kentucky, Lexington, KY 40536

Corresponding Author: nrwebb1{at}uky.edu

Previous studies have suggested that HDL retroendocytosis may play a role in SR-BI-dependent selective lipid uptake in a cell-specific manner. To investigate this possibility, we developed methods to quantitatively measure HDL uptake and re-secretion 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 minutes 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 minutes (t1/2 ~10 minutes). In COS-7 cells, the calculated rate of HDL secretion (~0.5 ng HDL/mg/minute) was more than 30-fold slower than the rate of SR-BI-dependent selective CE uptake (~17 ng HDL/mg/minute), whereas the rate of release of HDL from the cell surface (~19 ng HDL/mg/minute) was similar to the rate of selective CE uptake. Notably, the rate of SR-BI-dependent HDL re-secretion in COS-7 and HepG2 cells was similar. BLT-1, 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 the vast majority of SR-BI-dependent selective uptake occurs at the cell surface in both cell types.

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