Low-density lipoprotein (LDL)-cholesteryl ester (CE) selective uptake has been demonstrated in nonhepatic cells overexpressing the scavenger receptor class B type I (SR-BI). The role of hepatic SR-BI toward LDL, the main carrier of plasma CE in humans, remains unclear. The aim of this study was to determine if SR-BI, expressed at its normal level, is implicated in LDL-CE selective uptake in human HepG2 hepatoma cells and mouse hepatic cells, to quantify its contribution and to determine if LDL-CE selective uptake is likely to occur in the presence of human HDL. First, antibody blocking experiments were conducted on normal HepG2 cells. SR-BI/BII antiserum inhibited (125)I-LDL and (125)I-HDL(3) binding (10 microg of protein/mL) by 45% (p < 0.05) and CE selective uptake by more than 85% (p < 0.01) for both ligands. Second, HepG2 cells were stably transfected with a eukaryotic vector expressing a 400-bp human SR-BI antisense cDNA fragment. Clone 17 (C17) has a 70% (p < 0.01) reduction in SR-BI expression. In this clone, (3)H-CE-LDL and (3)H-CE-HDL(3) association (10 microg of protein/mL) was 54 +/- 6% and 45 +/- 7% of control values, respectively, while (125)I-LDL and (125)I-HDL(3) protein association was 71 +/- 3% and 58 +/- 5% of controls, resulting in 46% and 55% (p < 0.01) decreases in LDL- and HDL(3)-CE selective uptake. Normalizing CE selective uptake for SR-BI expression reveals that SR-BI is responsible for 68% and 74% of LDL- and HDL(3)-CE selective uptake, respectively. Thus, both approaches show that, in HepG2 cells, SR-BI is responsible for 68-85% of CE selective uptake. Other pathways for selective uptake in HepG2 cells do not require CD36, as shown by anti-CD36 antibody blocking experiments, or class A scavenger receptors, as shown by the lack of competition by poly(inosinic acid). However, CD36 is a functional oxidized LDL receptor on HepG2 cells, as shown by antibody blocking experiments. Similar results for CE selective uptake were obtained with primary cultures of hepatic cells from normal (+/+), heterozygous (-/+), and homozygous (-/-) SR-BI knockout mice. Flow cytometry experiments show that SR-BI accounts for 75% of DiI-LDL uptake, the LDL receptor for 14%, and other pathways for 11%. CE selective uptake from LDL and HDL(3) is likely to occur in the liver, since unlabeled HDL (total and apoE-free HDL(3)) and LDL, when added in physiological proportions, only partially competed for LDL- and HDL(3)-CE selective uptake. In this setting, human hepatic SR-BI may be a crucial molecule in the turnover of both LDL- and HDL(3)-cholesterol.

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The aim of this study was to quantify the abilities of mouse liver parenchymal and nonparenchymal cells with respect to (i) cholesteryl ester (CE) selective uptake from low-density lipoproteins (LDL), oxidized LDL (OxLDL), and high-density lipoprotein (HDL); and (ii) their free cholesterol efflux to HDL. The preparations of cells were incubated with lipoproteins labelled either in protein with iodine-125 or in CE with 3H-cholesterol oleate, and lipoprotein-protein and lipoprotein-CE associations were measured. The associations of LDL-protein and LDL-CE with nonparenchymal cells were 5- and 2-fold greater, respectively, than with parenchymal cells. However, in terms of CE-selective uptake (CE association minus protein association) both types of cell were equivalent. Similar results were obtained with OxLDL, but both types of cell showed higher abilities in OxLDL-CE than in LDL-CE selective uptake (on average by 3.4-fold). The association of HDL-protein with nonparenchymal cells was 3x that with parenchymal cells; however, nonparenchymal cells associated 45% less HDL-CE. Contrary to parenchymal cells, nonparenchymal cells did not show HDL-CE selective uptake activity. Thus parenchymal cells selectively take CE from the 3 types of lipoproteins, whereas nonparenchymal cells exert this function only on LDL and OxLDL. Efflux was 3.5-fold more important in nonparenchymal than in parenchymal cells.

Apolipoprotein E (apoE) plays a major role in lipoprotein metabolism by mediating the binding of apoE-containing lipoproteins to receptors. The role of hepatic apoE in the catabolism of apoE-free lipoproteins such as low density lipoprotein (LDL) and high density lipoprotein-3 (HDL 3 ) is however, unclear. We analyzed the importance of hepatic apoE by comparing human LDL and HDL 3 metabolism in primary cultures of hepatic cells from control C57BL/6J and apoE knockout (KO) mice. Binding analysis showed that the maximal binding capacity (B max ) of LDL, but not of HDL 3 , is increased by twofold in the absence of apoE synthesis/secretion. Compared to control hepatic cells, LDL and HDL 3 holoparticle uptake by apoE KO hepatic cells, as monitored by protein degradation, is reduced by 54 and 77%, respectively. Cleavage of heparan sulfate proteoglycans (HSPG) by treatment with heparinase I reduces LDL association by 21% in control hepatic cells. Thus, HSPG alone or a hepatic apoE-HSPG complex is partially involved in LDL association with mouse hepatic cells. In apoE KO, but not in normal hepatic cells, the same treatment increases LDL uptake/degradation by 2.4-fold suggesting that in normal hepatic cells, hepatic apoE increases LDL degradation by masking apoB-100 binding sites on proteoglycans. Cholesteryl ester (CE) association and CE selective uptake (CE/protein association ratio) from LDL and HDL 3 by mouse hepatic cells were not affected by the absence of apoE expression. We also show that 69 and 72% of LDL-CE hydrolysis in control and apoE KO hepatic cells, respectively, is sensitive to chloroquine revealing the importance of a pathway linked to lysosomes. In contrast, HDL 3 -CE hydrolysis is only mediated by a nonlysosomal pathway in both control and apoE KO hepatic cells. Overall, our results indicate that hepatic apoE increases the holoparticle uptake pathway of LDL and HDL 3 by mouse hepatic cells, that HSPG devoid of apoE favors LDL binding/association but impairs LDL uptake/degradation and that apoE plays no significant role in CE selective uptake from either human LDL or HDL 3 lipoproteins. [7]. The LDLr and the LRP are two receptors that mediate the endocytosis and degradation of lipoproteins particles (holoparticle uptake pathway), while the LBS mediates cholesteryl ester (CE) selective uptake [8]. CE selective uptake refers to a transfer of CE into the cells without the complete internalization of the lipoprotein. The LBS shares many characteristics with the scavenger receptor class B type I (SR-BI) identified in rodents [9] and in human as CD36-and LIMPII-analogous-1 (CLA-1) [10]. Localized at the cell-surface of hepatocytes, both receptors can bind various types of apolipoproteins and can mediate CE selective uptake from HDL [11,12]