Quantitative analysis of SR-BI-dependent HDL retroendocytosis in hepatocytes and fibroblasts.
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 (125)I-HDL(3) 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 approximately 10 min). In COS-7 cells, the calculated rate of HDL secretion ( approximately 0.5 ng HDL/mg/min) was >30-fold slower than the rate of SR-BI-dependent selective cholesteryl ester (CE) uptake ( approximately 17 ng HDL/mg/min), whereas the rate of release of HDL from the cell surface ( approximately 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.
Sun
Bing
B
Department of Internal Medicine, Graduate Center for Nutritional Sciences, University of Kentucky Medical Center, Lexington, 40536, USA.
Eckhardt
Erik R M
ER
Shetty
Shoba
S
van der Westhuyzen
Deneys R
DR
Webb
Nancy R
NR
D000818:Animals; D019921:Biotinylation; D019556:COS Cells; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D002788:Cholesterol Esters / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology*; D005347:Fibroblasts / Q000378:metabolism*; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D050356:Lipid Metabolism; D008075:Lipoproteins, HDL / Q000378:metabolism*; D051379:Mice; D018613:Microscopy, Confocal; D051122:Scavenger Receptors, Class B / Q000235:genetics / Q000378:metabolism* / Q000502:physiology
D002788:Cholesterol Esters; D008075:Lipoproteins, HDL; D051122:Scavenger Receptors, Class B