Influence of HDL-cholesterol-elevating drugs on the in vitro activity of the HDL receptor SR-BI

Treatment of atherosclerotic disease often focuses on reducing plasma LDL-cholesterol or increasing plasma HDL-cholesterol. We examined in vitro the effects on HDL receptor [scavenger receptor class B type I (SR-BI)] activity of three classes of clinical and experimental plasma HDL-cholesterol-elevating compounds: niacin, fibrates, and HDL376. Fenofibrate (FF) and HDL376 were potent (IC(50) approximately 1 microM), direct inhibitors of SR-BI-mediated lipid transport in cells and in liposomes reconstituted with purified SR-BI. FF, a prodrug, was a more potent inhibitor of SR-BI than an activator of peroxisome proliferator-activated receptor alpha, a target of its active fenofibric acid (FFA) derivative. Nevertheless, FFA, four other fibrates (clofibrate, gemfibrozil, ciprofibrate, and bezafibrate), and niacin had little, if any, effect on SR-BI, suggesting that they do not directly target SR-BI in vivo. However, similarities of HDL376 treatment and SR-BI gene knockout on HDL metabolism in vivo (increased HDL-cholesterol and HDL particle sizes) and structure-activity relationship analysis suggest that SR-BI may be a target of HDL376 in vivo. HDL376 and other inhibitors may help elucidate SR-BI function in diverse mammalian models and determine the therapeutic potential of SR-BI-directed pharmaceuticals.     

Negatively cooperative binding of high-density lipoprotein to the HDL receptor SR-BI

Scavenger receptor class B, type I (SR-BI), is a high-density lipoprotein (HDL) receptor, which also binds low-density lipoprotein (LDL), and mediates the cellular selective uptake of cholesteryl esters from lipoproteins. SR-BI also is a coreceptor for hepatitis C virus and a signaling receptor that regulates cell metabolism. Many investigators have reported that lipoproteins bind to SR-BI via a single class of independent (not interacting), high-affinity binding sites (one site model). We have reinvestigated the ligand concentration dependence of (125)I-HDL binding to SR-BI and SR-BI-mediated specific uptake of [(3)H]CE from [(3)H]CE-HDL using an expanded range of ligand concentrations (<1 μg of protein/mL, lower than previously reported). Scatchard and nonlinear least-squares model fitting analyses of the binding and uptake data were both inconsistent with a single class of independent binding sites binding univalent lipoprotein ligands. The data are best fit by models in which SR-BI has either two independent classes of binding sites or one class of sites exhibiting negative cooperativity due to either classic allostery or ensemble effects ("lattice model"). Similar results were observed for LDL. Application of the "infinite dilution" dissociation rate method established that the binding of (125)I-HDL to SR-BI at 4 °C exhibits negative cooperativity. The unexpected complexity of the interactions of lipoproteins with SR-BI should be taken into account when interpreting the results of experiments that explore the mechanism(s) by which SR-BI mediates ligand binding, lipid transport, and cell signaling.     

Identification of the molecular target of small molecule inhibitors of HDL receptor SR-BI activity

Scavenger receptor, class B, type I (SR-BI), controls high-density lipoprotein (HDL) metabolism by mediating cellular selective uptake of lipids from HDL without the concomitant degradation of the lipoprotein particle. We previously identified in a high-throughput chemical screen of intact cells five compounds (BLT-1-5) that inhibit SR-BI-dependent lipid transport from HDL, but do not block HDL binding to SR-BI on the cell surface. Although these BLTs are widely used to examine the diverse functions of SR-BI, their direct target(s), SR-BI itself or some other component of the SR-BI pathway, has not been identified. Here we show that SR-BI in the context of a membrane lipid environment is the target of BLT-1, -3, -4, and -5. The analysis using intact cells and an in vitro system of purified SR-BI reconstituted into liposomes was aided by information derived from structure-activity relationship (SAR) analysis of the most potent of these BLTs, the thiosemicarbazone BLT-1. We found that the sulfur atom of BLT-1 was crucially important for its inhibitory activity, because changing it to an oxygen atom resulted in the isostructural, but essentially inactive, semicarbazone derivative BLT-1sc. SAR analysis also established the importance of BLT-1's hydrophobic tail. BLTs and their corresponding inactive compounds can be used to explore the mechanism and function of SR-BI-mediated selective lipid uptake in diverse mammalian experimental models. Consequently, BLTs may help determine the therapeutic potential of SR-BI-targeted pharmaceutical drugs.     

Susceptibility to murine cholesterol gallstone formation is not affected by partial disruption of the HDL receptor SR-BI

High density lipoprotein (HDL) promotes reverse cholesterol transport from peripheral tissues to the liver where its cholesterol is secreted preferentially into bile. The scavenger receptor class B type I (SR-BI) is believed to play a pivotal role in unloading HDL cholesterol and its ester to hepatocytes. Here, using male SR-BI "att" mice with a dysfunctional mutation in the Sr-b1 promoter, we studied whether approximately 50% of normal SR-BI expression influences gallstone susceptibility in these mice fed a lithogenic diet containing 1% cholesterol, 0.5% cholic acid and 15% butterfat. Our results showed that the disruption of SR-BI expression reduced cholesterol secretion by 37% in the chow-fed state and 10% on the lithogenic diet, and while delaying incidence slightly, did not influence cumulative susceptibility to cholesterol gallstones. The lithogenic diet induced marked increases in biliary cholesterol and phospholipid secretion rates but not of bile salts. Basal expression of hepatic SR-BI protein was dissimilar in both wild-type and SR-BI mice, and remained unaltered in response to the lithogenic diet. By two independent dual isotope methods, intestinal cholesterol absorption was unimpaired by attenuation of the SR-BI which also displays low-density expression on small intestinal enterocytes. We conclude that although HDL cholesterol is a principal source of biliary cholesterol in the basal state, uptake of cholesterol from chylomicron remnants appears to be the major contributor to biliary cholesterol hypersecretion during diet-induced cholelithogenesis in the mouse.     

Identification of the N-linked glycosylation sites on the high density lipoprotein (HDL) receptor SR-BI and assessment of their effects on HDL binding and selective lipid uptake

The murine class B, type I scavenger receptor mSR-BI, a high density lipoprotein (HDL) receptor that mediates selective uptake of HDL lipids, contains 11 potential N-linked glycosylation sites and unknown numbers of both endoglycosidase H-sensitive and -resistant oligosaccharides. We have examined the consequences of mutating each of these sites (Asn --> Gln or Thr --> Ala) on post-translational processing of mSR-BI, cell surface expression, and HDL binding and lipid transport activities. All 11 sites were glycosylated; however, disruption of only two (Asn-108 and Asn-173) substantially altered expression and function. There was very little detectable post-translational processing of these two mutants to endoglycosidase H resistance and very low cell surface expression, suggesting that oligosaccharide modification at these sites apparently plays an important role in endoplasmic reticulum folding and/or intracellular transport. Strikingly, although the low levels of the 108 and 173 mutants that were expressed on the cell surface exhibited a marked reduction in their ability to transfer lipids from HDL to cells, they nevertheless bound nearly normal amounts of HDL. Indeed, the affinity of (125)I-HDL binding to the 173 mutant was similar to that of the wild-type receptor. Thus, N-linked glycosylation can influence both the intracellular transport and lipid-transporter activity of SR-BI. The ability to uncouple the HDL binding and lipid transport activities of mSR-BI by in vitro mutagenesis should provide a powerful tool for further analysis of the mechanism of SR-BI-mediated selective lipid uptake.     

Scavenger receptor BI (SR-BI) mediates free cholesterol flux independently of HDL tethering to the cell surface

In addition to its effect on high density lipoprotein (HDL) cholesteryl ester (CE) uptake, scavenger receptor BI (SR-BI) was recently reported to stimulate free cholesterol (FC) flux from Chinese hamster ovary (CHO) cells stably expressing mouse SR-BI, a novel function of SR-BI that may play a role in cholesterol removal from the vessel wall where the receptor can be found. It is possible that SR-BI stimulates flux simply by tethering acceptor HDL particles in close apposition to the cell surface thereby facilitating the movement of cholesterol between the plasma membrane and HDL. To test this, we used transiently transfected cells and compared the closely related class B scavenger receptors mouse SR-BI and rat CD36 for their ability to stimulate cholesterol efflux as both receptors bind HDL with high affinity. The results showed that, although acceptor binding to SR-BI may contribute to efflux to a modest extent, the major stimulation of FC efflux occurs independently of acceptor binding to cell surface receptors. Instead our data indicate that SR-BI mediates alterations to membrane FC domains which provoke enhanced bidirectional FC flux between cells and extracellular acceptors.     

Cholesteryl ester transfer protein expression partially attenuates the adverse effects of SR-BI receptor deficiency on cholesterol metabolism and atherosclerosis

Scavenger receptor SR-BI significantly contributes to HDL cholesterol metabolism and atherogenesis in mice. However, the role of SR-BI may not be as pronounced in humans due to cholesteryl ester transfer protein (CETP) activity. To address the impact of CETP expression on the adverse effects associated with SR-BI deficiency, we cross-bred our SR-BI conditional knock-out mouse model with CETP transgenic mice. CETP almost completely restored the abnormal HDL-C distribution in SR-BI-deficient mice. However, it did not normalize the elevated plasma free to total cholesterol ratio characteristic of hepatic SR-BI deficiency. Red blood cell and platelet count abnormalities observed in mice liver deficient for SR-BI were partially restored by CETP, but the elevated erythrocyte cholesterol to phospholipid ratio remained unchanged. Complete deletion of SR-BI was associated with diminished adrenal cholesterol stores, whereas hepatic SR-BI deficiency resulted in a significant increase in adrenal gland cholesterol content. In both mouse models, CETP had no impact on adrenal cholesterol metabolism. In diet-induced atherosclerosis studies, hepatic SR-BI deficiency accelerated aortic lipid lesion formation in both CETP-expressing (4-fold) and non-CETP-expressing (8-fold) mice when compared with controls. Impaired macrophage to feces reverse cholesterol transport in mice deficient for SR-BI in liver, which was not corrected by CETP, most likely contributed by such an increase in atherosclerosis susceptibility. Finally, comparison of the atherosclerosis burden in SR-BI liver-deficient and fully deficient mice demonstrated that SR-BI exerted an atheroprotective activity in extra-hepatic tissues whether CETP was present or not. These findings support the contention that the SR-BI pathway contributes in unique ways to cholesterol metabolism and atherosclerosis susceptibility even in the presence of CETP.     

Detergent-mediated phospholipidation of plasma lipoproteins increases HDL cholesterophilicity and cholesterol efflux via SR-BI

Cellular cholesterol efflux is an early, obligatory step in reverse cholesterol transport, the putative antiatherogenic mechanism by which human plasma high-density lipoproteins (HDL) transport cholesterol from peripheral tissue to the liver for recycling or disposal. HDL-phospholipid content is the essential cholesterol-binding component of lipoproteins and therefore a major determinant of cholesterol efflux. Thus, increased phospholipidation of lipoproteins, particularly HDL, is one strategy for increasing cholesterol efflux. This study validates a simple, new detergent perturbation method for the phospholipidation of plasma lipoproteins; we have quantified the cholesterophilicity of human plasma lipoproteins and the effects of lipoprotein phospholipidation on cholesterophilicity and cellular cholesterol efflux mediated by the class B type I scavenger receptor (SR-BI). We determined that low-density lipoproteins (LDL) are more cholesterophilic than HDL and that LDL has a higher affinity for phospholipids than HDL whereas HDL has a higher phospholipid capacity than LDL. Phospholipidation of total human plasma lipoproteins enhances cholesterol efflux, an effect that occurs largely through the preferential phospholipidation of HDL. We conclude that increasing HDL phospholipid increases its cholesterophilicity, thereby making it a better acceptor of cellular cholesterol efflux. Phospholipidation of lipoproteins by detergent perturbation is a simple way to increase HDL cholesterophilicity and cholesterol efflux in a way that may be clinically useful.     

Inherited disorders of HDL metabolism and atherosclerosis

PURPOSE OF REVIEW: Genetic disorders of HDL metabolism are rare and, as a result, the assessment of atherosclerosis risk in individuals suffering from these disorders has been difficult. Ultrasound imaging of carotid arteries has provided a tool to assess the risk in hereditary hypo and hyperalphalipoproteinemia. This review gives a comprehensive summary. RECENT FINDINGS: Epidemiological studies have unequivocally shown that HDL cholesterol levels are inversely related to coronary artery disease risk, but the literature concerning genetic disorders of HDL metabolism provides less convincing information. Fortuitously, we were able to directly compare carotid intima media thickness data of substantial numbers of individuals with mutations in either apolipoprotein A-I (apoA-I), ATP binding cassette AI (ABCA1), lecithin: cholesterol acyltransferase (LCAT) or cholesteryl ester transfer protein. These data show that carriers of an apoA-I mutation exhibit the most pronounced accelerated atherosclerosis compared with those carrying mutations in ABCA1 and LCAT. Heterozygosity for a non-sense mutation in cholesteryl ester transfer protein did, by contrast, not distinguish carriers from controls in terms of intima media thickness progression. We will discuss these results in the context of the current literature. SUMMARY: Intima media thickness studies have provided evidence that hypoalphalipoproteinemia due to mutations in apoA-I, ABCA1, and LCAT is associated with increased progression of atherosclerosis. In contrast, hyperalphalipoproteinemia as a result of loss of cholesteryl ester transfer protein function is associated with unaltered atherosclerosis progression compared with family controls. This insight is of interest, since it can assist in the prioritizing of antiatherogenic therapy by increasing HDL cholesterol levels.     

ApoA-II modulates the association of HDL with class B scavenger receptors SR-BI and CD36

The class B scavenger receptors SR-BI and CD36 exhibit a broad ligand binding specificity. SR-BI is well characterized as a HDL receptor that mediates selective cholesteryl ester uptake from HDL. CD36, a receptor for oxidized LDL, also binds HDL and mediates selective cholesteryl ester uptake, although much less efficiently than SR-BI. Apolipoprotein A-II (apoA-II), the second most abundant HDL protein, is considered to be proatherogenic, but the underlying mechanisms are unclear. We previously showed that apoA-II modulates SR-BI-dependent binding and selective uptake of cholesteryl ester from reconstituted HDL. To investigate the effect of apoA-II in naturally occurring HDL on these processes, we compared HDL without apoA-II (from apoA-II null mice) with HDLs containing differing amounts of apoA-II (from C57BL/6 mice and transgenic mice expressing a mouse apoA-II transgene). The level of apoA-II in HDL was inversely correlated with HDL binding and selective cholesteryl ester uptake by both scavenger receptors, particularly CD36. Interestingly, for HDL lacking apoA-II, the efficiency with which CD36 mediated selective uptake reached a level similar to that of SR-BI. These results demonstrate that apoA-II exerts a marked effect on HDL binding and selective lipid uptake by the class B scavenger receptors and establishes a potentially important relationship between apoA-II and CD36.     

Polyunsaturated fatty acids up-regulate hepatic scavenger receptor B1 (SR-BI) expression and HDL cholesteryl ester uptake in the hamster.

Diets rich in polyunsaturated fatty acids lower plasma HDL cholesterol concentrations when compared to diets rich in saturated fatty acids. We investigated the mechanistic basis for this effect in the hamster and sought to determine whether reduced plasma HDL cholesterol concentrations resulting from a high polyunsaturated fat diet are associated with a decrease in reverse cholesterol transport. Animals were fed semisynthetic diets enriched with polyunsaturated or saturated fatty acids for 6 weeks. We then determined the effect of these diets on the following parameters: 1) hepatic scavenger receptor B1 (SR-BI) mRNA and protein levels, 2) the rate of hepatic HDL cholesteryl ester uptake, and 3) the rate of cholesterol acquisition by the extrahepatic tissues (from de novo synthesis, LDL and HDL) as a measure of the rate of reverse cholesterol transport. Compared to saturated fatty acids, dietary polyunsaturated fatty acids up-regulated hepatic SR-BI expression by approximately 50% and increased HDL cholesteryl ester transport to the liver; as a consequence, plasma HDL cholesteryl ester concentrations were reduced. Although dietary polyunsaturated fatty acids increased hepatic HDL cholesteryl ester uptake and lowered plasma HDL cholesterol concentrations, there was no change in the cholesterol content or in the rate of cholesterol acquisition (via de novo synthesis and lipoprotein uptake) by the extrahepatic tissues.These studies indicate that substitution of polyunsaturated for saturated fatty acids in the diet increases SR-BI expression and lowers plasma HDL cholesteryl ester concentrations but does not affect reverse cholesterol transport.     

SR-BI inhibits ABCG1-stimulated net cholesterol efflux from cells to plasma HDL

This study compares the roles of ABCG1 and scavenger receptor class B type I (SR-BI) singly or together in promoting net cellular cholesterol efflux to plasma HDL containing active LCAT. In transfected cells, SR-BI promoted free cholesterol efflux to HDL, but this was offset by an increased uptake of HDL cholesteryl ester (CE) into cells, resulting in no net efflux. Coexpression of SR-BI with ABCG1 inhibited the ABCG1-mediated net cholesterol efflux to HDL, apparently by promoting the reuptake of CE from medium. However, ABCG1-mediated cholesterol efflux was not altered in cholesterol-loaded, SR-BI-deficient (SR-BI(-/-)) macrophages. Briefly cultured macrophages collected from SR-BI(-/-) mice loaded with acetylated LDL in the peritoneal cavity did exhibit reduced efflux to HDL. However, this was attributable to reduced expression of ABCG1 and ABCA1, likely reflecting increased macrophage cholesterol efflux to apolipoprotein E-enriched HDL during loading in SR-BI(-/-) mice. In conclusion, cellular SR-BI does not promote net cholesterol efflux from cells to plasma HDL containing active LCAT as a result of the reuptake of HDL-CE into cells. Previous findings of increased atherosclerosis in mice transplanted with SR-BI(-/-) bone marrow probably cannot be explained by a defect in macrophage cholesterol efflux.     

C323 of SR-BI is required for SR-BI-mediated HDL binding and cholesteryl ester uptake

Scavenger receptor BI (SR-BI) is an HDL receptor. It binds HDL and mediates the uptake of cholesteryl ester from HDL. Early studies have pointed out that the extracellular domain of SR-BI is critical for SR-BI-mediated cholesteryl ester uptake. However, the extracellular loop of SR-BI is large: it contains 403 amino acids. The HDL binding site and the modulation of SR-BI-mediated cholesteryl ester uptake remain to be identified. In this study, using C323G mutant SR-BI, we showed that C323G mutant SR-BI lost its HDL binding and cholesteryl ester uptake activity, indicating that the highly conserved C323 is required for SR-BI-mediated HDL binding and cholesteryl ester uptake. Using a blocking antibody against C323 region, we demonstrated that C323 is directly involved in HDL binding and likely an HDL binding site. Using C323G mutant transgenic mouse model, we further demonstrated that C323 of SR-BI is required for regulating plasma cholesterol levels in vivo. Using redox reagents, we showed that physiological relevant levels of H(2)O(2) upregulated the SR-BI-mediated cholesteryl ester uptake activity by 65%, whereas GSH or DTT significantly downregulated SR-BI-mediated cholesteryl ester uptake activity by 45%. C323 of SR-BI is critical for SR-BI-mediated HDL binding and cholesteryl ester uptake, and changes in redox status may be a regulatory factor modulating SR-BI-mediated cholesterol transport.     

Selective uptake of HDL cholesteryl esters and cholesterol efflux from mouse peritoneal macrophages independent of SR-BI

Scavenger receptor class B type I (SR-BI) mediates the selective uptake of HDL cholesteryl esters (CEs) and facilitates the efflux of unesterified cholesterol. SR-BI expression in macrophages presumably plays a role in atherosclerosis. The role of SR-BI for selective CE uptake and cholesterol efflux in macrophages was explored. Macrophages and HDL originated from wild-type (WT) or SR-BI knockout (KO; homozygous) mice. For uptake, macrophages were incubated in medium containing 125I-/3H-labeled HDL. For lipid removal, [3H]cholesterol efflux was analyzed using HDL as acceptor. Selective uptake of HDL CE ([3H]cholesteryl oleyl ether - 125I-tyramine cellobiose) was similar in WT and SR-BI KO macrophages. Radiolabeled SR-BI KO-HDL yielded a lower rate of selective uptake compared with WT-HDL in WT and SR-BI KO macrophages. Cholesterol efflux was similar in WT and SR-BI KO cells using HDL as acceptor. SR-BI KO-HDL more efficiently promoted cholesterol removal compared with WT-HDL from both types of macrophages. Macrophages selectively take up HDL CE independently of SR-BI. Additionally, in macrophages, there is substantial cholesterol efflux that is not mediated by SR-BI. Therefore, SR-BI-independent mechanisms mediate selective CE uptake and cholesterol removal. SR-BI KO-HDL is an inferior donor for selective CE uptake compared with WT-HDL, whereas SR-BI KO-HDL more efficiently promotes cholesterol efflux.     

Nascent HDL formation in hepatocytes and role of ABCA1, ABCG1, and SR-BI

To study the mechanisms of hepatic HDL formation, we investigated the roles of ABCA1, ABCG1, and SR-BI in nascent HDL formation in primary hepatocytes isolated from mice deficient in ABCA1, ABCG1, or SR-BI and from wild-type (WT) mice. Under basal conditions, in WT hepatocytes, cholesterol efflux to exogenous apoA-I was accompanied by conversion of apoA-I to HDL-sized particles. LXR activation by T0901317 markedly enhanced the formation of larger HDL-sized particles as well as cellular cholesterol efflux to apoA-I. Glyburide treatment completely abolished the formation of 7.4 nm diameter and greater particles but led to the formation of novel 7.2 nm-sized particles. However, cells lacking ABCA1 failed to form such particles. ABCG1-deficient cells showed similar capacity to efflux cholesterol to apoA-I and to form nascent HDL particles compared with WT cells. Cholesterol efflux to apoA-I and nascent HDL formation were slightly but significantly enhanced in SR-BI-deficient cells compared with WT cells under basal but not LXR activated conditions. As in WT but not in ABCA1-deficient hepatocytes, 7.2 nm-sized particles generated by glyburide treatment were also detected in ABCG1-deficient and SR-BI-deficient hepatocytes. Our data indicate that hepatic nascent HDL formation is highly dependent on ABCA1 but not on ABCG1 or SR-BI.     

The role of the high-density lipoprotein receptor SR-BI in cholesterol metabolism

The HDL receptor scavenger receptor class B type I (SR-BI), which mediates selective HDL cholesterol uptake, plays a role in murine HDL metabolism, reverse cholesterol transport and whole-body cholesterol homeostasis. SR-BI is found in the liver, where its expression is regulated by estrogen, dietary cholesterol and fat, and controls murine plasma HDL cholesterol levels and bile cholesterol secretion. SR-BI is also highly expressed in rodent steroidogenic cells, where it facilitates cholesterol uptake for storage or steroid hormone synthesis and where its expression is regulated by trophic hormones. The detailed mechanism(s) underlying SR-BI-mediated selective cholesterol uptake have not yet been elucidated. Further analysis of the molecular and cellular bases of SR-BI regulation and function should provide new insights into the physiology and pathophysiology of cholesterol metabolism.     

Binding of high density lipoprotein (HDL) and discoidal reconstituted HDL to the HDL receptor scavenger receptor class B type I. Effect of lipid association and APOA-I mutations on receptor binding

The binding of apoA-I-containing ligands to the HDL receptor scavenger receptor class B type I (SR-BI) was characterized using two different assays. The first employed conventional binding or competition assays with (125)I-labeled ligands. The second is a new nonradioactive ligand binding assay, in which the receptor-associated ligand is detected by quantitative immunoblotting ("immunoreceptor assay"). Using both methods, we observed that the K(d) value for spherical HDL (density = 1.1-1.13 g/ml) was approximately 16 microgram of protein/ml, while the values for discoidal reconstituted HDL (rHDL) containing proapoA-I or plasma apoA-I were substantially lower (approximately 0.4-5 microgram of protein/ml). We also observed reduced affinity and/or competition for spherical (125)I-HDL cell association by higher relative to lower density HDL and very poor competition by lipid-free apoA-I and pre-beta-1 HDL. Deletion of either 58 carboxyl-terminal or 59 amino-terminal residues from apoA-I, relative to full-length control apoA-I, resulted in little or no change in the affinity of corresponding rHDL particles. However, rHDL particles containing a double mutant lacking both terminal domains competed poorly with spherical (125)I-HDL for binding to SR-BI. These findings suggest an important role for apoA-I and its conformation/organization within particles in mediating HDL binding to SR-BI and indicate that the NH(2) and COOH termini of apoA-I directly or indirectly contribute independently to binding to SR-BI.     

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.