In vivo cholesteryl ester selective uptake of mildly and standardly oxidized LDL occurs by both parenchymal and nonparenchymal mouse hepatic cells but SR-BI is only responsible for standardly oxidized LDL selective uptake by nonparenchymal cells

In blood circulation, low density lipoproteins (LDL) can undergo modification, such as oxidation, and become key factors in the development of atherosclerosis. Although the liver is the major organ involved in the elimination of oxidized LDL (oxLDL), the identity of the receptor(s) involved remains to be defined. Our work aims to clarify the role of the scavenger receptor class B type I (SR-BI) in the hepatic metabolism of mildly and standardly oxLDL as well as the relative contribution of parenchymal (hepatocytes) and nonparenchymal liver cells with a special emphasis on CE-selective uptake. The association of native LDL and mildly or standardly oxLDL labeled either in proteins or in cholesteryl esters (CE) was measured on primary cultures of mouse hepatocytes from normal and SR-BI knock-out (KO) mice. These in vitro assays demonstrated that hepatocytes are able to mediate CE-selective uptake from both LDL and oxLDL and that SR-BI KO hepatocytes have a 60% reduced ability to selectively take CE from LDL but not towards mildly or standardly oxLDL. When lipoproteins were injected in the mouse inferior vena cava, parenchymal and nonparenchymal liver cells accumulated more CE than proteins from native, mildly and standardly oxLDL, indicating that selective uptake of CE from these lipoproteins occurs in vivo in these two cell types. The parenchymal cells contribute near 90% of the LDL-CE selective uptake and SR-BI for 60% of this pathway. Nonparenchymal cells capture mainly standardly oxLDL while parenchymal and nonparenchymal cells equally take up mildly oxLDL. An 82% reduction of standardly oxLDL-CE selective uptake by the nonparenchymal cells of SR-BI KO mice allowed emphasizing the contribution of SR-BI in hepatic metabolism of standardly oxLDL. However, SR-BI is not responsible for mildly oxLDL metabolism. Thus, SR-BI is involved in LDL- and standardly oxLDL-CE selective uptake in parenchymal and nonparenchymal cells, respectively.     

Apolipoproteins C-II and C-III inhibit selective uptake of low- and high-density lipoprotein cholesteryl esters in HepG2 cells

Plasma low- and high-density lipoproteins (LDL and HDL) are cleared from the circulation by specific receptors and are either totally degraded or their cholesteryl esters (CE) are selectively delivered to cells by receptors such as the scavenger receptor class B type I (SR-BI). The aim of the present study was to define the effect of apoC-II and apoC-III on the uptake of LDL and HDL by HepG2 cells. Stable transformants were obtained with sense or antisense strategies that secrete 47-294% the normal level of apoC-II or 60-200% that of apoC-III. Different levels of secreted apoC-II or apoC-III had little effect on LDL and HDL protein degradation by HepG2 cells. However, compared to controls, cells under-expressing apoC-II showed a 160% higher capacity to selectively take up HDL-CE, while cells under-expressing apoC-III demonstrated 70 and 160% higher capacity to take up CE from LDL and HDL, respectively. In experiments conducted with exogenously added apoC-II or apoC-III, no significant effect was observed on lipoprotein-protein association/degradation; however, LDL-CE and HDL-CE selective uptake was significantly reduced in a dose-dependent manner. These results indicate that apoC-II and apoC-III inhibit CE-selective uptake.     

Uptake and fate of class B scavenger receptor ligands in HepG2 cells.

Class B scavenger receptors (SR-Bs) interact with native, acetylated and oxidized low-density lipoprotein (LDL, AcLDL and OxLDL), high-density lipoprotein (HDL3) and maleylated BSA (M-BSA). The aim of this study was to analyze the catabolism of CD36- and LIMPII-analogous-1 (CLA-1), the human orthologue for the scavenger receptor class B type I (SR-BI), and CD36 ligands in HepG2 (human hepatoma) cells. Saturation binding experiments revealed moderate-affinity binding sites for all the SR-B ligands tested with dissociation constants ranging from 20 to 30 microg.mL-1. Competition binding studies at 4 degrees C showed that HDL and modified and native LDL share common binding site(s), as OxLDL competed for the binding of 125I-LDL and 125I-HDL3 and vice versa, and that only M-BSA and LDL may have distinct binding sites. Degradation/association ratios for SR-B ligands show that LDL is very efficiently degraded, while M-BSA and HDL3 are poorly degraded. The modified LDL degradation/association ratio is equivalent to 60% of the LDL degradation ratio, but is three times higher than that of HDL3. All lipoproteins were good cholesteryl ester (CE) donors to HepG2 cells, as a 3.6-4.7-fold CE-selective uptake ([3H]CE association/125I-protein association) was measured. M-BSA efficiently competed for the CE-selective uptake of LDL-, OxLDL-, AcLDL- and HDL3-CE. All other lipoproteins tested were also good competitors with some minor variations. Hydrolysis of [3H]CE-lipoproteins in the presence of chloroquine demonstrated that modified and native LDL-CE were mainly hydrolyzed in lysosomes, whereas HDL3-CE was hydrolyzed in both lysosomal and extralysosomal compartments. Inhibition of the selective uptake of CE from HDL and native modified LDL by SR-B ligands clearly suggests that CLA-1 and/or CD36 are involved at least partially in this process in HepG2 cells.     

Overexpression of SR-BI by adenoviral vector promotes clearance of apoA-I,but not apoB,in human apoB transgenic mice

Scavenger receptor BI (SR-BI) is a multi-ligand lipoprotein receptor that mediates selective lipid uptake from HDL, and plays a central role in hepatic HDL metabolism. In this report, we investigated the extent to which SR-BI selective lipid uptake contributes to LDL metabolism. As has been reported for human LDL, mouse SR-BI expressed in transfected cells mediated selective lipid uptake from mouse LDL. However, LDL-cholesteryl oleoyl ester (CE) transfer relative to LDL-CE bound to the cell surface (fractional transfer) was approximately 18-fold lower compared with HDL-CE. Adenoviral vector-mediated SR-BI overexpression in livers of human apoB transgenic mice ( approximately 10-fold increased expression) reduced plasma HDL-cholesterol (HDL-C) and apolipoprotein (apo)A-I concentrations to nearly undetectable levels 3 days after adenovirus infusion. Increased hepatic SR-BI expression resulted in only a modest depletion in LDL-C that was restricted to large LDL particles, and no change in steady-state concentrations of human apoB. Kinetic studies showed a 19% increase in the clearance rate of LDL-CE in mice with increased SR-BI expression, but no change in LDL apolipoprotein clearance. Quantification of hepatic uptake of LDL-CE and LDL-apolipoprotein showed selective uptake of LDL-CE in livers of human apo B transgenic mice. However, such uptake was not significantly increased in mice over-expressing SR-BI. We conclude that SR-BI-mediated selective uptake from LDL plays a minor role in LDL metabolism in vivo.     

Specific targeting of high density lipoproteins to liver hepatocytes by incorporation of a tris-galactoside-terminated cholesterol derivative

A triantennary galactose-terminated cholesterol derivative, N-(tris(beta-D-galactopyranosyloxymethyl) methyl)-N alpha-(4(5-cholesten-3 beta-yloxy)succinyl)glycinamide (Tris-Gal-Chol), which dissolves easily in water, was added to human apolipoprotein E-free high density lipoproteins (HDL) in varying quantities. Incorporation of 5 or 13 micrograms of Tris-Gal-Chol into HDL (20 micrograms of protein) stimulates the liver association of the HDL apoprotein radioactivity 24- and 55-fold, respectively, at 10 min after intravenous injection into rats. The increased interaction of Tris-Gal-Chol HDL with the liver is blocked by preinjection of asialofetuin or N-acetylgalactosamine but not influenced by N-acetylglucosamine. The parenchymal liver cell uptake of HDL is stimulated 42- or 105-fold, respectively, by incorporation of 5 or 13 micrograms of Tris-Gal-Chol into HDL (20 micrograms of protein), while the association with nonparenchymal cells is stimulated only 1.7- or 5-fold. It can be calculated that 98.0% of the Tris-Gal-Chol HDL is associated with parenchymal cells. In contrast, incorporation of 13 micrograms of Tris-Gal-Chol into LDL (20 micrograms of protein) leads to a selective association of LDL with nonparenchymal cells (92.3% of the total liver uptake). It is concluded that Tris-Gal-Chol incorporation into HDL leads to a specific interaction of HDL with the asialoglycoprotein (galactose) receptor on parenchymal cells whereas Tris-Gal-Chol incorporation into LDL leads mainly to an interaction with a galactose receptor from Kupffer cells. Probably this highly selective cellular targeting of LDL and HDL by Tris-Gal-Chol is caused by the difference in size between these lipoproteins. The increased interaction of HDL with the parenchymal cells upon Tris-Gal-Chol incorporation is followed by degradation of the apolipoprotein in the lysosomes. It is concluded that Tris-Gal-Chol incorporation into LDL or HDL leads to a markedly increased catabolism of LDL by way of the Kupffer cells and HDL by parenchymal cells which might be used for lowering serum cholesterol levels. The use of Tris-Gal-Chol might also find application for targeting drugs or other compounds of interest to either Kupffer or parenchymal liver cells.     

Opposite effect of caveolin-1 in the metabolism of high-density and low-density lipoproteins

Receptors of the scavenger class B family were reported to be localized in caveolae, the cell surface microdomains rich in free cholesterol and glycosphyngolipids, which are characterized by the presence of caveolin-1. Parenchymal hepatic and hepatoma HepG2 cells express very low levels of caveolin-1. In the present study, stable transformants of HepG2 cells expressing caveolin-1 were generated to address the effect of caveolin-1 on receptor activity. Compared to normal cells, these cells show higher (125)I-bovine serum albumin (BSA) uptake and cholesterol efflux, two indicators of functional caveolae. By immunoprecipitation, cell fractionation and confocal analyses, we found that caveolin-1 is well colocalized with the cluster of differentiation-36 (CD36) and the low-density lipoprotein (LDL) receptor (LDLr) but to a lesser extent with the scavenger receptor class B type I (SR-BI) in HepG2 cells expressing caveolin-1. However, caveolin-1 expression favors the dimerization of SR-BI. Two clones of cells expressing caveolin-1 were investigated for their lipoprotein metabolism activity. Compared to normal cells, these cells show a 71-144% increase in (125)I-LDL degradation. The analysis of the cholesteryl esters (CE)-selective uptake (CE association minus protein association) revealed that the expression of caveolin-1 in HepG2 cells decreases by 59%-73% LDL-CE selective uptake and increases high-density lipoprotein (HDL)-CE selective uptake by 44%-66%. We conclude that the expression of caveolin-1 in HepG2 cells moves the balance of LDL degradation/CE selective uptake towards degradation and favors HDL-CE selective uptake. Thus, in the normal hepatic parenchymal situation where caveolin-1 is poorly expressed, LDL-CE selective uptake is the preferred pathway.     

Low and high density lipoprotein metabolism in primary cultures of hepatic cells from normal and apolipoprotein E knockout mice.

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 (Bmax) 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.     

Physiological importance of SR-BI in the in vivo metabolism of human HDL and LDL in male and female mice

The physiological role of murine scavenger receptor class B type I (SR-BI) was evaluated by in vivo clearances of human HDL3 and LDL in normal and SR-BI knockout (KO) mice. In normal mice, cholesteryl esters (CEs) were removed faster than proteins, indicating a selective uptake process from both HDL3 and LDL. SR-BI KO mice showed 80% losses of HDL-CE selective uptake and the complete loss of LDL-CE selective uptake in the first phase of clearance. However, the second phase was characterized by an acceleration of CE disappearance in SR-BI KO mice. Thus, SR-BI is the only murine receptor mediating HDL-CE selective uptake, whereas a SR-BI-independent pathway specific to LDL can rescue SR-BI deficiency. The analysis of LDL recovered 3 h after injection in mice from different genotypes revealed that LDLs are significantly depleted in CE (reduction from 19% to 50% of the CE/protein ratios). A smaller LDL size in comparison with that of noninjected LDL was also detectable but was more evident for LDL recovered from normal mice. All LDL preparations migrate faster than noninjected LDL on agarose-barbital gels. Thus, both SR-BI-dependent and -independent pathways lead to substantial changes in LDL.     

Selective uptake of cholesteryl esters from various classes of lipoproteins by HepG2 cells.

Selective uptake of cholesteryl esters (CE) from lipoproteins by cells has been extensively studied with high density lipoproteins (HDL). It is only recently that such a mechanism has been attributed to intermediate and low density lipoproteins (IDL and LDL). Here, we compare the association of proteins and CE from very low density lipoproteins (VLDL), IDL, LDL and HDL3 to HepG2 cells. These lipoproteins were either labelled in proteins with 125I or in CE with 3H-cholesteryl oleate. We show that, at any lipoprotein concentration, protein association to the cells is significantly smaller for IDL, LDL, and HDL3 than CE association, but not for VLDL. At a concentration of 20 microg lipoprotein/mL, these associations reveal CE-selective uptake in the order of 2-, 4-, and 11-fold for IDL, LDL, and HDL3, respectively. These studies reveal that LDL and HDL3 are good selective donors of CE to HepG2 cells, while IDL is a poor donor and VLDL is not a donor. A significant inverse correlation (r2 = 0.973) was found between the total lipid/protein ratios of the four classes of lipoproteins and the extent of CE-selective uptake by HepG2 cells. The fate of 3H-CE of the two best CE donors (LDL and HDL3) was followed in HepG2 cells after 3 h of incubation. Cells were shown to hydrolyze approximately 25% of the 3H-CE of both lipoproteins. However, when the cells were treated with 100 microM of chloroquine, a lysosomotropic agent, 85 and 40% of 3H-CE hydrolysis was lost for LDL and HDL3, respectively. The fate of LDL and HDL3-CE in HepG2 cells deficient in LDL-receptor was found to be the same, indicating that the portion of CE hydrolysis sensitive to chloroquine is not significantly linked to LDL-receptor activity. Thus, in HepG2 cells, the magnitude of CE-selective uptake is inversely correlated with the total lipid/protein ratios of the lipoproteins and CE-selective uptake from the two best CE donors (LDL and HDL3) appears to follow different pathways.     

Scavenger receptor class B, type I, mediates selective uptake of low density lipoprotein cholesteryl ester.

Scavenger receptor, class B, type I (SR-BI) is a cell-surface glycoprotein that mediates selective uptake of high density lipoprotein cholesteryl ester (CE) without the concomitant uptake and degradation of the particle. We have investigated the endocytic and selective uptake of low density lipoprotein (LDL)-CE by SR-BI using COS-7 cells transiently transfected with mouse SR-BI. Analysis of lipoprotein uptake data showed a concentration-dependent LDL-CE-selective uptake when doubly labeled LDL particles were incubated with SR-BI-expressing COS-7 cells. In contrast to vector-transfected cells, SR-BI-expressing COS-7 cells showed marked increases in LDL cell association and CE uptake by the selective uptake pathway, but only a modest increase in CE uptake by the endocytic pathway. SR-BI-mediated LDL-CE-selective uptake exceeded LDL endocytic uptake by 50-100-fold. SR-BI-mediated LDL-CE-selective uptake was not inhibited by the proteoglycan synthesis inhibitor, p-nitrophenyl-beta-D-xylopyranoside or by the sulfation inhibitor sodium chlorate, indicating that SR-BI-mediated LDL-CE uptake occurs independently of LDL interaction with cell-surface proteoglycan. Analyses with subclones of Y1 adrenocortical cells showed that LDL-CE-selective uptake was proportional to the level of SR-BI expression. Furthermore, antibody directed to the extracellular domain of SR-BI blocked LDL-CE-selective uptake in adrenocortical cells. Thus, in cells that normally express SR-BI and in transfected COS-7 cells SR-BI mediates the efficient uptake of LDL-CE via the selective uptake mechanism. These results suggest that SR-BI may influence the metabolism of apoB-containing lipoproteins in vivo by mediating LDL-CE uptake into SR-BI-expressing cells.     

Hepatic lipase promotes the selective uptake of high density lipoprotein-cholesteryl esters via the scavenger receptor B1.

Hepatic lipase (HL) plays a major role in high-density lipoprotein (HDL) metabolism both as a lipolytic enzyme and as a ligand. To investigate whether HL enhances the uptake of HDL-cholesteryl ester (CE) via the newly described scavenger receptor BI (SR-BI), we measured the effects of expressing HL and SR-BI on HDL-cell association as well as uptake of 125I-labeled apoA-I and [3H]CE-HDL, by embryonal kidney 293 cells. As expected, HDL cell association and CE selective uptake were increased in SR-BI transfected cells by 2- and 4-fold, respectively, compared to controls (P < 0.001). Cells transfected with HL alone or in combination with SR-BI expressed similar amounts of HL, 20% of which was bound to cell surface proteoglycans. HL alone increased HDL cell association by 2-fold but had no effect on HDL-CE uptake in 293 cells. However, in cells expressing SR-BI, HL further enhanced the selective uptake of CE from HDL by 3-fold (P < 0.001). To determine whether the lipolytic and/or ligand function of HL are required in this process, we generated a catalytically inactive form of HL (HL-145G). Cells co-transfected with HL-145G and SR-BI increased their HDL cell association and HDL-CE selective uptake by 1.4-fold compared to cells expressing SR-BI only (P < 0.03). Heparin abolished the effect of HL-145G on SR-BI-mediated HDL-CE selective uptake.Thus, the enhanced uptake of HDL-CE by HL is mediated by both its ligand role, which requires interaction with proteoglycans, and by lipolysis with subsequent HDL particle remodeling. These results establish HL as a major modulator of SR-BI mediated selective uptake of HDL-CE.     

SR-BI-directed HDL-cholesteryl ester hydrolysis

We have examined the metabolic fate of HDL cholesteryl ester (CE) delivered to cells expressing scavenger receptor class B type I (SR-BI). Comparison of SR-BI with a related class B scavenger receptor, CD36, showed a greater uptake and a more rapid and extensive hydrolysis of HDL-CE when delivered by SR-BI. In addition, hydrolysis of HDL-CE delivered by both receptors was via a neutral CE hydrolase. These data indicate that SR-BI, but not CD36, can efficiently direct HDL-CE to a neutral CE hydrolytic pathway. In contrast, LDL-CE was delivered and hydrolyzed equally well by SR-BI and CD36. Hydrolysis of LDL-CE delivered by SR-BI was via a neutral CE hydrolase but that delivered by CD36 occurred via an acidic CE hydrolase, indicating that SR-BI and CD36 deliver LDL-CE to different metabolic pathways. Comparison of inhibitor sensitivities in Y1-BS1 adrenal, Fu5AH hepatoma, and transfected cells suggests that hydrolysis of HDL-CE delivered by SR-BI occurs via cell type-specific neutral CE hydrolases. Furthermore, HDL-CE hydrolytic activity was recovered in a membrane fraction of Y1-BS1 cells. These findings suggest that SR-BI efficiently delivers HDL-CE to a metabolically active membrane compartment where CE is hydrolyzed by a neutral CE hydrolase.     

Analysis of low-density lipoprotein catabolism by primary cultures of hepatic cells from normal and low-density lipoprotein receptor knockout mice

Low-density lipoproteins (LDL) are taken up by LDL receptor (LDLr)-dependent and -independent pathways; the role and importance of the latest being less well defined. We analyzed the importance of these pathways in the mouse by comparing LDL binding to primary cultures of hepatocytes from LDLr knockout (LDLr KO) and normal C57BL/6J mice. Saturation curve analysis shows that (125)I-LDL bind specifically to normal and LDLr KO mouse hepatocytes with similar dissociation constants (K(d)) (31.2 and 22.9 microg LDL-protein/ml, respectively). The maximal binding capacity (B(max)) is, however, reduced by 48% in LDLr KO mouse hepatocytes in comparison to normal hepatocytes. Conducting the assay in the presence of a 200-fold excess of high-density lipoprotein-3 (HDL3) reduced by 39% the binding of (125)I-LDL to normal hepatocytes and abolished the binding to the LDLr KO mouse hepatocytes. These data indicate that in normal mouse hepatocytes, the LDLr is responsible for approximately half of the LDL binding while a lipoprotein binding site (LBS), interacting with both LDL and HDL3, is responsible for the other half. It can also be deduced that both receptors/sites have a similar affinity for LDL. The metabolism of LDL-protein and cholesteryl esters (CE) was analyzed in both types of cells. (125)I-LDL-protein degradation was reduced by 95% in LDLr KO hepatocytes compared to normal hepatocytes. Comparing the association of (125)I-LDL and (3)H-CE-LDL revealed a CE-selective uptake of 35.6- and 22-fold for normal and LDLr KO mouse hepatocytes, respectively. Adding a 200-fold excess of HDL3 in the assay reduced by 71% the CE-selective uptake in LDLr KO hepatocytes and by 96% in normal hepatocytes. This indicates that mouse hepatocytes are able to selectively take up CE from LDL by the LBS. The comparison of LDL-CE association also showed that the LBS pathway provides 5-fold more LDL-CE to the cell than the LDLr. Overall, our results indicate that in mouse hepatocytes, LDLr is almost completely responsible for LDL-protein degradation while the LBS is responsible for the major part of LDL-CE entry by a CE-selective uptake pathway.     

The role of human and mouse hepatic scavenger receptor class B type I (SR-BI) in the selective uptake of low-density lipoprotein-cholesteryl esters

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.     

Streptococcal serum opacity factor increases the rate of hepatocyte uptake of human plasma high-density lipoprotein cholesterol

Serum opacity factor (SOF), a virulence determinant of Streptococcus pyogenes, converts plasma high-density lipoproteins (HDL) to three distinct species: lipid-free apolipoprotein (apo) A-I, neo HDL, a small discoidal HDL-like particle, and a large cholesteryl ester-rich microemulsion (CERM) that contains the cholesterol esters (CE) of up to ～400000 HDL particles and apo E as its major protein. Similar SOF reaction products are obtained with HDL, total plasma lipoproteins, and whole plasma. We hypothesized that hepatic uptake of CERM-CE via multiple apo E-dependent receptors would be faster than that of HDL-CE. We tested our hypothesis using human hepatoma cells and lipoprotein receptor-specific Chinese hamster ovary (CHO) cells. The uptake of [(3)H]CE by HepG2 and Huh7 cells from HDL after SOF treatment, which transfers >90% of HDL-CE to CERM, was 2.4 and 4.5 times faster, respectively, than from control HDL. CERM-[(3)H]CE uptake was inhibited by LDL and HDL, suggestive of uptake by both the LDL receptor (LDL-R) and scavenger receptor class B type I (SR-BI). Studies in CHO cells specifically expressing LDL-R and SR-BI confirmed CERM-[(3)H]CE uptake by both receptors. RAP and heparin inhibit CERM-[(3)H]CE but not HDL-[(3)H]CE uptake, thereby implicating LRP-1 and cell surface proteoglycans in this process. These data demonstrate that SOF treatment of HDL increases the rate of CE uptake via multiple hepatic apo E receptors. In so doing, SOF might increase the level of hepatic disposal of plasma cholesterol in a way that is therapeutically useful.     

Selective uptake of cholesteryl ester from low density lipoprotein is involved in HepG2 cell cholesterol homeostasis.

Low density lipoprotein (LDL) can follow either a holoparticle uptake pathway, initiated by the LDL receptor (LDLr), and be completely degraded, or it can deliver its cholesteryl esters (CE) selectively to HepG2 cells. Although high density lipoprotein-CE selective uptake has been shown to be linked to cell cholesterol homeostasis in nonhepatic cells, there is no available information on the effect of LDL-CE selective uptake on hepatic cell cholesterol homeostasis. In order to define the role of the LDL-CE selective uptake pathway in hepatic cell cholesterol homeostasis, we used a cellular model that expresses constitutively a LDLr antisense mRNA and that shows LDLr activity at 31% the normal level (HepG2-all cells). The addition of a specific antibody anti-LDLr (IgG-C7) reduces LDL protein degradation (LDLr activity) to 7%. This cellular model therefore reflects, above all, LDL-CE selective uptake activity when incubated with LDL. The inactivation of LDLr reduces LDL-protein association by 78% and LDL-CE association by only 43%. The LDL-CE selective uptake was not reduced by the inactivation of LDLr. The activities of the various enzymes involved in cell cholesterol homeostasis were measured in normal and LDLr-deficient cells during incubation in the absence or presence of LDL as a cholesterol source. Essentially, 3-hydroxy-3-methylglutaryl coenzyme A reductase and acyl coenzyme A:cholesterol acyltransferase (ACAT) activities responded to LDL in LDLr-deficient cells as well as in normal HepG2 cells. Inhibition of lysosomal hydrolysis with chloroquine abolished the effect measured on ACAT activity in the presence of LDL, suggesting that CE of LDL, but not free cholesterol, maintains cell cholesterol homeostasis. Thus, in HepG2 cells, when LDLr function is virtually abolished, LDL-CE selective uptake is coupled to cell cholesterol homeostasis.     

In vivo uptake of human and rat low density and high density lipoprotein by parenchymal and nonparenchymal cells from rat liver.

The relative contribution of the parenchymal and nonparenchymal rat liver cells to the hepatic uptake of human and rat high density lipoprotein (HDL) and low density lipoprotein (LDL) was determined in vivo. Nonparenchymal cells, isolated 6 h after intravenous injection of iodinated human HDL and LDL, contained respectively 4.2 and 6.3 times the amount of trichloroacetic acid-precipitable radioactivity per mg cell protein as compared to parenchymal cells. For rat iodinated HDL and LDL these factors were 3.4 and 4.1, respectively. These results indicate that nonparenchymal liver cells play a substantial role in the hepatic uptake of human and rat HDL and LDL in vivo.     

Scavenger receptor class B type I is solely responsible for the selective uptake of cholesteryl esters from HDL by the liver and the adrenals in mice

Scavenger receptor class B type I (SR-BI) has been identified as a functional HDL binding protein that can mediate the selective uptake of cholesteryl ester (CE) from HDL. To quantify the in vivo role of SR-BI in the process of selective uptake, HDL was labeled with cholesteryl ether ([(3)H] CEt-HDL) and (125)I-tyramine cellobiose ([(125)I]TC-HDL) and injected into SR-BI knockout (KO) and wild-type (WT) mice. In SR-BI KO mice, the clearance of HDL-CE from the blood circulation was greatly diminished (0.043 +/- 0.004 pools/h for SR-BI KO mice vs. 0.106 +/- 0.004 pools/h for WT mice), while liver and adrenal uptake were greatly reduced. Utilization of double-labeled HDL ([(3)H]CEt and [(125)I]TC) indicated the total absence in vivo of the selective decay and liver uptake of CE from HDL in SR-BI KO mice. Parenchymal cells isolated from SR-BI KO mice showed similar association values for [(3)H]CEt and [(125)I]TC in contrast to WT cells, indicating that in parenchymal liver cells SR-BI is the only molecule exerting selective CE uptake from HDL. Thus, in vivo and in vitro, SR-BI is the sole molecule mediating the selective uptake of CE from HDL by the liver and the adrenals, making it the unique target to modulate reverse cholesterol transport.     

Streptococcal serum opacity factor promotes cholesterol ester metabolism and bile acid secretion in vitro and in vivo

Plasma high density lipoprotein-cholesterol (HDL-C) concentrations negatively correlate with atherosclerotic cardiovascular disease. HDL is thought to have several atheroprotective functions, which are likely distinct from the epidemiological inverse relationship between HDL-C levels and risk. Specifically, strategies that reduce HDL-C while promoting reverse cholesterol transport (RCT) may have therapeutic value. The major product of the serum opacity factor (SOF) reaction versus HDL is a cholesteryl ester (CE)-rich microemulsion (CERM), which contains apo E and the CE of ~ 400,000 HDL particles. Huh7 hepatocytes take up CE faster when delivered as CERM than as HDL, in part via the LDL-receptor (LDLR). Here we compared the final RCT step, hepatic uptake and subsequent intracellular processing to cholesterol and bile salts for radiolabeled HDL-, CERM- and LDL-CE by Huh7 cells and in vivo in C57BL/6J mice. In Huh7 cells, uptake from LDL was greater than from CERM (2–4X) and HDL (5–10X). Halftimes for [¹⁴C]CE hydrolysis were 3.0 ± 0.2, 4.4 ± 0.6 and 5.4 ± 0.7 h respectively for HDL, CERM and LDL-CE. The fraction of sterols secreted as bile acids was ~ 50% by 8 h for all three particles. HDL, CERM and LDL-CE metabolism in mice showed efficient plasma clearance of CERM-CE, liver uptake and metabolism, and secretion as bile acids into the gall bladder. This work supports the therapeutic potential of the SOF reaction, which diverts HDL-CE to the LDLR, thereby increasing hepatic CE uptake, and sterol disposal as bile acids.     

Mechanism of the cholesteryl ester transfer protein-mediated uptake of high density lipoprotein cholesteryl esters by Hep G2 cells

Plasma cholesteryl ester transfer protein (CETP) mediates the transfer of cholesteryl esters (CE) between lipoproteins and was reported to also directly mediate the uptake of high density lipoprotein (HDL) CE by human Hep G2 cells and fibroblasts. The present study investigates that uptake and its relationship to a pathway for "selective uptake" of HDL CE that does not require CETP. HDL3 labeled in both the CE and apoprotein moieties was incubated with Hep G2 cells. During 4-h incubations, CE tracer was selectively taken up from doubly labeled HDL3 in excess of apoA-I tracer, and added CETP did not modify that uptake. However, during 18-20-h incubations, CETP stimulated the uptake of CE tracer more than 4-fold without modifying the uptake of apoA-I tracer. This suggested that secreted products, perhaps lipoproteins, might be required for the CETP effect. Four inhibitors of lipoprotein uptake via low density lipoprotein (LDL) receptors (heparin, monensin, an antibody against the LDL receptor, and antibodies against the receptor binding domains of apoB and apoE) effectively blocked the CETP stimulation of CE tracer uptake. Heparin caused an increase in CE tracer in a d less than 1.063 g/ml fraction of the medium that more than accounted for the heparin blockade of CETP-stimulated CE uptake. CETP did not affect the uptake of doubly labeled HDL3 by human fibroblasts, even at twice plasma levels of activity, and heparin did not modify uptake of HDL3 tracers. Thus the CETP effect on Hep G2 cells can be accounted for by transfer of HDL CE to secreted lipoproteins which are then retaken up, and there is no evidence for a direct effect of CETP on cellular uptake of HDL CE.