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Journal of Lipid Research, Vol. 42, 1231-1238, August 2001
Copyright © 2001 by Lipid Research, Inc.

Original Article

CD36 does not play a direct role in HDL or LDL metabolism

Correspondence to: Willem J. S. de Villiers, To whom correspondence should be addressed., wdevil0{at}pop.uky.edu (E-mail)

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

CD36 and scavenger receptor class B, type I (SR-BI) are both class B scavenger receptors that recognize a broad variety of ligands, including oxidized low density lipoprotein (oxLDL), HDL, anionic phospholipids, and apoptotic cells. In this study we investigated the role of mouse CD36 (mCD36) as a physiological lipoprotein receptor. We compared the association of various lipoprotein particles with mCD36 and mSR-BI expressed in COS cells by adenovirus-mediated gene transfer. mCD36 bound human oxLDL and mouse HDL with high affinity. Human LDL bound poorly to mCD36, indicating that mCD36 is unlikely to play a significant role in LDL metabolism. The ability of mCD36 to mediate the selective uptake of cholesteryl esters (CE) from receptor-bound HDL was assessed. In comparison with mSR-BI, mCD36 inefficiently mediated the selective uptake of CE. Hepatic overexpression of mCD36 in C57BL/6 mice by adenovirus-mediated gene transfer did not result in significant alterations in plasma LDL and HDL levels.

We conclude that mCD36, while able to bind HDL with high affinity, does not contribute significantly to HDL or LDL metabolism. — de Villiers, W. J. S., L. Cai, N. R. Webb, M. C. de Beer, D. R. van der Westhuyzen, and F. C. de Beer. CD36 does not play a direct role in HDL or LDL metabolism. J. Lipid Res. 2001. 42: 1231;–1238.

Supplementary key words: scavenger receptor, oxidized LDL, SR-BI, adenovirus

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

CD36 is an 88-kDa member of a family of cell surface glycoproteins characterized as class B scavenger receptors, which also includes scavenger receptor class B, type I (SR-BI), an HDL receptor (1). Its expression by monocyte-macrophages (2), platelets (3), microvascular endothelial cells (4), retinal pigment epithelium (5), and adipose tissue (6) contrasts with the predominant hepatic and steroido-genic tissue expression of SR-BI (1). As a scavenger receptor CD36 recognizes a broad variety of ligands, including anionic phospholipids (7), apoptotic cells (8), thrombospondin (9), collagen (10), long-chain fatty acids (11), and Plasmodium falciparum-infected erythrocytes (12).

CD36 was originally identified as an oxidized lipoprotein receptor by an expression cloning strategy used to isolate murine macrophage receptors that recognize oxidized low density lipoprotein (oxLDL), but not acetylated low density lipoprotein (AcLDL) (13) (14). A genetic polymorphism in the CD36 gene has been identified in Japanese subjects (15) and shown to result in deficient expression of CD36 (NAK^a- phenotype). Monocyte-derived macrophages isolated from these patients bind 40% less oxLDL and accumulate 40% less cholesteryl ester (CE) from oxLDL than do cells derived from normal controls (16), further implicating CD36 as a physiological oxLDL receptor. In addition, peritoneal macrophages from mice rendered CD36 deficient by homologous recombination show an impaired ability to bind and internalize oxLDL (17).

Another member of the class B scavenger receptor family, SR-BI, also binds oxLDL, but it has been better characterized as a lipoprotein receptor for HDL and native LDL. SR-BI mediates the selective uptake of HDL CE (1) (18) by a process in which HDL delivers CE to the cell without the internalization and lysosomal degradation of the whole HDL particle (19). SR-BI (509 amino acids) and CD36 (472 amino acids) share significant sequence homology and have been proposed to have similar membrane topologies (4) (20). Both molecules possess a large extracellular loop that is anchored to the plasma membrane on each side by transmembrane domains adjacent to short cytoplasmic N- and C-terminal domains. The extracellular domain of SR-BI facilitates the selective uptake of HDL CE (21) (22).

Mouse SR-BI and human and rat CD36 have been compared with regard to their respective abilities to bind human HDL and mediate cellular uptake of HDL lipids (21) (22). Although CD36 was shown to bind HDL with high affinity, it was less efficient than mSR-BI in mediating selective CE uptake from HDL. This raises a question concerning whether selective lipid uptake mediated by CD36 is of physiological relevance with respect to HDL metabolism. CD36-deficient mice have significantly increased HDL cholesterol levels, possibly because of decreased binding and clearance of HDL. Alternatively, changes in fatty acid metabolism leading to markedly increased VLDL-associated triacylglycerol could result in elevated HDL levels in this model (17).

The role of CD36 as an LDL receptor is contentious. It was suggested that human CD36 may, in addition to oxLDL and HDL, also be a receptor for LDL (23). Others have indicated otherwise (13) (14) (20). The aim of this study was to determine the significance of mCD36 in HDL and LDL metabolism and to compare mSR-BI and mCD36 directly with regard to their interaction with oxLDL, LDL, and HDL. We developed an adenoviral vector-mediated system of mCD36 overexpression and compared the relative efficiency of mCD36 and mSR-BI in mediating lipoprotein binding and selective lipid uptake. We further determined the physiological effect of hepatic mCD36 overexpression on plasma lipoprotein levels in mice.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Materials
FBS, BSA (essentially fatty acid free), penicillin, and streptomycin were purchased from Sigma (St. Louis, MO). Organic solvents were products of Fisher Scientific (Pittsburgh, PA). Tissue culture flasks and plates were obtained through Corning (Corning, NY) or Falcon (Lincoln, NJ). [1,2(n)-³H]cholesteryl oleyl ether and ¹²⁵I were purchased from Amersham (Piscataway, NJ). Tissue culture media were obtained from GIBCO (Grand Island, NY). Mouse SR-BI antibody was prepared as previously described (24).

Cell culture
COS-7 cells were routinely grown in DMEM supplemented with 10% FBS and 1% penicillin and streptomycin in 75-cm² flasks and subcultured once a week, using a 1:6 split ratio.

Preparation of adenoviral vectors
Sequence encoding mCD36 or mSR-BI was inserted into adenovirus expression vector pAdCMV.link (25) (26) containing the cytomegalovirus immediate-early enhancer promoter element, yielding pAdCD36 or pAdSR-BI. The expression plasmids were cotransfected into 293 cells with replication-defective adenoviral DNA as described previously (26) to generate recombinant adenoviruses expressing mCD36 or mSR-BI. Adnull (provided by D. J. Rader, University of Pennsylvania, Philadelphia, PA) is a recombinant virus with analogous adenoviral sequences but containing no transgene.

Antibody preparation
Spontaneously hypertensive rats, which lack CD36 (27), were injected intravenously with 2 x 10¹² recombinant adenoviral particles expressing mCD36. The rats were bled after 6 weeks and the sera were tested for anti-mCD36 reactivity. Nonimmune sera from spontaneously hypertensive rats served as negative controls. The polyclonal rat anti-mouse CD36 antiserum showed monospecificity against unfractionated extracts of murine macrophage-like cell lines and primary peritoneal macrophages (our unpublished observations). Furthermore, on tissues sections, there was a clear difference between immunostaining in the presence of the mCD36 antisera compared with nonimmune sera from spontaneously hypertensive rats.

Lipoprotein isolation
VLDL (d < 1.019), LDL (d 1.019 to 1.063 g/ml), and HDL (d 1.063 to 1.21 g/ml) fractions were isolated from fresh human or C57BL/6 mouse plasma by density gradient ultracentrifugation as previously described (28). Human HDL₃ (d 1.13 to 1.18) was obtained from total HDL by gradient fractionation. All isolated fractions were dialyzed against 150 mM NaCl-0.01% EDTA, sterile filtered, and stored under nitrogen at 4°C. Protein concentrations were determined by the method of Lowry et al. (29) and cholesterol concentrations were determined enzymatically (Wako Chemicals, Osaka, Japan).

Lipoprotein oxidation
Native LDL (2 mg/ml) was dialyzed against 150 mM NaCl to remove EDTA and oxidized overnight at 4°C, with 5 µM CuSO₄, and subsequently at room temperature for 6 h with minimal stirring. Oxidation was terminated with 10 mM EDTA and dialysis against 150 mM NaCl-0.01% EDTA to remove CuSO₄. Oxidation was assessed by electrophoretic mobility in agarose gels (30) and measurement of the amount of thiobarbituric acid-reactive substance (TBARS) generated with a colorimetric assay for malondialdehyde (MDA) (31). MDA was used as a standard, and the TBARS values were recorded as equivalents of MDA per milligram of LDL protein. The degree of oxidation of the various LDL preparations was as follows: native LDL and oxLDL, 1.7 ± 0.3 and 20 ± 5.2 MDA equivalents per milligram of LDL protein, respectively.

Lipoprotein radiolabeling
Both the protein and lipid moieties of lipoproteins were labeled. Lipoproteins were iodinated by the iodine monochloride method (32). HDL-associated CE was traced with nonhydrolyzable, intracellularly trapped [1, 2(n)-³H]cholesteryl oleyl ether prepared according to the method of Gwynne and Mahaffee (33), using partially purified CETP. Labeled lipoproteins were separated from CETP by ultracentrifugal flotation at a density of 1.21 g/ml. The integrity of the labeled lipoproteins was verified by SDS-PAGE and gradient gel electrophoresis and the lipid compositions were determined.

Western blot analysis
Cultured cells were lysed in 150 mM NaCl, 10 mM EDTA, 10 mM NaN₃, 10 mM Tris (pH 8.0), and 1% Nonidet P-40, containing 1 mM PMSF and 5 mM iodoacetamide. After incubation on ice for 30 min the cell lysates were spun at 13,000 g for 5 min and 10 µg aliquots of the supernatant were stored at -80°C. Membranes were prepared from mouse livers as described (34). Cell and tissue lysates were subjected to Western blot analyses using nonreducing 5;–20% SDS-PAGE. Samples were transferred onto nitrocellulose membrane and incubated with anti-mCD36 and anti-mSR-BI (34) antibodies, followed by enhanced chemiluminescence detection (ECL; Amersham Pharmacia Biotech) and autoradiography. Apolipoproteins were quantified by a combination of SDS-PAGE and immunoblotting according to modifications of procedures described previously (35) (36). Films were analyzed by densitometric scanning (Molecular Dynamics, Sunnyvale, CA). The resulting signals were shown to be in the semilinear portion of the response curve, using apolipoprotein standards run in parallel at various dilutions.

Ligand-binding and uptake assays
COS cells were seeded in 6-well plates 48 h before assays (2.5 x 10⁵ cells per well). Preliminary experiments with adenovirus expressing green fluorescent protein were performed to determine the viral dose required to achieve gene transfer in more than 95% of treated cells. This level of expression was confirmed for AdCD36 and AdSR-BI by indirect immunofluorescence. Adenoviral vector-mediated gene overexpression was performed by addition of Adnull, AdCD36, or AdSR-BI at a viral dose of 1,000 particles per cell 36 h before assay. Cell association assays, as previously described (34), were performed at 4 or 37°C in Ham's F-12 containing penicillin (100 units/ml), streptomycin (100 units/ml), 2 mM glutamine, 0.5% essentially fatty acid-free BSA, and radiolabeled lipoprotein. After incubating for the indicated times, unbound ligand were removed from cells by washing four times with 50 mM Tris-HCl-150 mM NaCl (pH 7.4) containing fatty acid-free BSA (2 mg/ml) followed by two washes in the same buffer without BSA. All washes were performed at 4°C with prechilled solutions. Cells were solubilized in 0.1 N NaOH for 60 min at room temperature before protein and radioactivity quantitation. Receptor-specific cell association values were calculated as the difference between the total for AdCD36- and AdSR-BI-expressing cells and corresponding values for Adnull control cells. K_d values were determined by nonlinear regression analysis of receptor-specific cell association values, using Prism^® software (GraphPad Software, San Diego, CA). For cell association of double-labeled HDL, the values of each tracer were shown in terms of apparent HDL particle association, expressed as lipoprotein protein (37). This was done to compare association of both tracers on the same basis. Therefore, ¹²⁵I represented apparent HDL association according to the protein tracer, ³H that due to the CE tracer, and ³H - ¹²⁵I showed the difference in lipoprotein particle association, that is, apparent selective CE uptake. The receptor-specific efficiency of HDL CE-selective uptake was determined by expressing the amount of selective uptake relative to the amount of cell-associated ¹²⁵I-labeled HDL (¹²⁵I-HDL).

Adenoviral vector treatments and analysis of plasma lipids
Male C57BL/6 mice (Jackson Laboratories, Bar harbor, ME) weighing at least 25 g were injected in the tail vein with 1 x 10¹¹ particles of recombinant adenovirus in 100 µl of PBS. Plasma samples were collected from mice fasted for 10 h before collection. Aliquots (100 µl) were spun through a 0.22-µm pore size filter before fast protein liquid chromatography (FPLC) on a Superose 6 column (Amersham Pharmacia Biotech). The column was eluted at a flow rate of 0.5 ml/min in buffer containing 150 mM NaCl, 10 mM Tris-HCl (pH 7.4), and 0.01% sodium azide. The cholesterol content of individual FPLC fractions (0.5 ml) was determined enzymatically (Wako Chemicals). Plasma HDL cholesterol was measured enzymatically after precipitation of LDL and VLDL by heparin and manganese.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Adenovirus-mediated mCD36 overexpression in COS cells
An adenovirus expressing mCD36 (AdCD36) was produced with a second-generation adenoviral system (26) as described in Materials and Methods. This recombinant adenovirus was used to generate a rat anti-mouse CD36 antibody (36S1) in CD36-deficient spontaneously hypertensive rats (27) and to express mCD36 in cultured cells. As shown in Fig 1, reproducible high level CD36 protein expression was achieved in COS cells by adenoviral vector-mediated gene transfer. Suitable titration of the viral dose allowed for expression in greater than 95% of cells as determined by indirect immunofluorescence microscopy using a secondary rhodamine-labeled anti-rat IgG antibody (data not shown). In contrast, no expression of mCD36 was observed in COS cells transfected with Adnull control virus, which produces no protein product.

View larger version (31K): [in this window] [in a new window]   Figure 1. Adenovirus-mediated overexpression of mCD36 in COS cells. Immunoblot analysis of mCD36 expression in COS cells treated with AdCD36 or Adnull control virus. Cells were exposed to a multiplicity of infection of 1,000 virus particles per cell and analyzed 24 h after treatment. Cell lysates were obtained from triplicate wells treated similarly, indicating comparable adenovirus-mediated protein expression. Mouse adipose tissue served as a positive control. Cell and tissue lysate protein loads were 5 µg per lane.

mCD36 binds human oxLDL and HDL, but not LDL
The ability of mCD36 to bind various human lipoprotein ligands was examined. Concentration-dependent ligand binding at 4°C of ¹²⁵I-labeled oxLDL, HDL, and LDL to COS cells expressing mCD36 is shown in Fig 2. Cell-bound radioactive ligand was measured as previously described (34) and CD36-specific values were calculated as the difference between binding to AdCD36- and Adnull-treated cells. Specific binding of ¹²⁵I-labeled oxLDL, HDL, and LDL to AdCD36 COS cells increased in a dose-dependent and saturable manner. The dissociation constant (K_d) calculated for the binding of oxLDL at 4°C to AdCD36 COS cells (6.9 ± 3.1 µg/ml) agrees with other reports for high-affinity oxLDL binding to CD36 expressed in mammalian cells (13) and indicates that mCD36 expressed in COS cells is functional. Human HDL bound AdCD36 COS cells with a lower affinity than oxLDL. Owing to the lower binding affinity of HDL, an accurate K_d could not be determined from these data, but the estimated K_d value (K_d, 40 µg/ml) is in agreement with other reports (1) (34). In contrast to oxLDL, LDL showed an extremely low binding affinity. An accurate K_d value could not be determined, but was estimated to be greater than 300 µg/ml. These results confirmed the ability of mCD36 to act as a receptor for human oxLDL and HDL (13) (14) and also indicated that human LDL is a poor ligand for mCD36.

View larger version (19K): [in this window] [in a new window]   Figure 2. Dose-dependent binding of human 125I-labeled oxLDL, HDL, and LDL to mCD36. COS cells overexpressing mCD36 by adenovirus-mediated gene transfer were incubated with increasing concentrations of 125I-labeled human oxLDL, HDL, or LDL at 4°C for 2 h. CD36-specific values are given and were calculated as the difference between the values in mCD36-expressing cells and control cells treated with Adnull. The data are representative of three separate experiments performed in triplicate using two different batches of ligand; mean values ± SD of a representative experiment are given.

Comparative binding of human oxLDL to mCD36 and mSR-BI
Adenovirus-mediated gene transfer was also used to overexpress the other class B scavenger receptor, SR-BI, in COS cells, which allowed for a functional comparison of mCD36 and mSR-BI. As shown in Fig 3A and Fig B, both SR-BI- and CD36-expressing cells bound human ¹²⁵I-oxLDL with high affinity in a concentration-dependent saturable manner. The similar maximal binding values at 4°C for mCD36 (895 ng/mg cell protein) and mSR-BI (863 ng/mg cell protein) are a reflection of the efficiency of gene transfer and indicate that approximately equal numbers of functional ligand-binding sites were expressed on the cell surface. In this setting the calculated dissociation constants (K_d) for the binding of oxLDL at 4°C to AdCD36 (3.1 µg/ml) and AdSR-BI (7.1 µg/ml) showed comparable affinity of these receptors for oxLDL. The apparent K_d values for oxLDL cell association at 37°C to AdCD36 (10 µg/ml) and AdSR-BI (20 µg/ml) demonstrated a similar trend. In the association studies carried out at 37°C, little degradation of oxLDL occurred with either of the receptors, as evidenced by the relatively small amount of degradation products detected in the medium (degradation products 6% of the cell-associated ligand). This, coupled with the fact that cell association of oxLDL to cells approached a maximum by 1 h of incubation, provides evidence that cell-associated ¹²⁵I-oxLDL values predominantly reflect cell surface binding, although internalization of ligand cannot be excluded.

View larger version (22K): [in this window] [in a new window]   Figure 3. Comparison of human oxLDL cell association mediated by mCD36 and mSR-BI. COS cells expressing mCD36 and mSR-BI by adenovirus-mediated gene transfer were treated with increasing concentrations of 125I-labeled human oxLDL for 2 h at (A) 4°C and (B) 37°C. CD36- and SR-BI-specific values are shown. The data are representative of two separate experiments performed in triplicate using two different batches of ligand; mean values ± SD of a representative experiment are given.

Comparative binding of mouse HDL to mCD36 and mSR-BI
To pursue the role of mCD36 in lipoprotein metabolism further in a physiologically relevant autologous setting, we examined its ability to bind mouse lipoprotein particles. We therefore investigated the cell association and selective lipid uptake of mouse HDL particles by cells expressing mCD36 and compared this with mSR-BI. Cells were incubated at 37°C with increasing concentrations of HDL double labeled with ¹²⁵I- and [³H]cholesteryl oleyl ether ( Fig 4). High-affinity, receptor-specific cell association of HDL was observed both in the case of CD36 and SR-BI (Fig 4A). Given the low level of ligand degradation observed (9% of cell-associated ligand) and the fact that ¹²⁵I-HDL association reached a maximum within 1 h for both receptors, cell association values are taken to predominantly reflect cell surface association. Apparent dissociation constants (K_d) for AdCD36 and AdSR-BI were 9.1 and 11.5 µg/ml, respectively. In Fig 4A, the B_max value for ¹²⁵I-HDL binding to AdCD36 (232 ng/mg cell protein) was significantly lower compared with AdSR-BI (706 ng/mg cell protein), which most likely reflects a difference in transfection efficiency. In summary, both mCD36 and mSR-BI bind HDL with high affinity.

View larger version (18K): [in this window] [in a new window]   Figure 4. Cell association and selective uptake of mouse HDL by mCD36 and mSR-BI. mCD36 and SR-BI were expressed in COS cells as described in text and incubated with increasing concentrations of HDL double labeled with 125I- and [3H]CE at 37°C for 2 h. CD36- and SR-BI-specific values are given and were calculated as the difference between the values in mCD36- and SR-BI-expressing cells and control cells treated with Adnull. Selective lipid uptake (3H - 125I) represents the difference between cell-associated [3H]CE (3H) and 125I-apolipoprotein (125I), all expressed as HDL protein. A: Cell-associated 125I-HDL. B: Selective uptake of HDL CE. C: Selective uptake efficiency. The efficiency of HDL CE-selective uptake was determined by normalizing the amount of HDL CE-selective uptake to the amount of cell-associated 125I-HDL. Values represent the means of duplicate determinations and are representative of three independent experiments.

The ability of CD36 and SR-BI to mediate selective CE uptake was also measured (Fig 4B). As expected, SR-BI mediates significant selective uptake as is evident from the relative amounts of receptor-specific cell association of the protein and cholesterol components of double-labeled HDL after 2 h. Although mCD36 also exhibited selective uptake of the lipid label, this occurred to a much lesser extent than for mSR-BI. The receptor-specific efficiency of HDL CE-selective uptake was determined by expressing the amount of selective uptake relative to the amount of cell-associated ¹²⁵I-HDL (Fig 4C). This analysis showed that at different concentrations of the double-labeled mouse HDL, mSR-BI mediated HDL CE-selective uptake with a significantly greater efficiency (approximately 10-fold) than did mCD36. These results showed that CD36 is less efficient than SR-BI on a per-HDL particle bound basis in mediating selective CE uptake.

Hepatic mCD36 overexpression does not change lipoprotein profiles
To examine and compare the in vivo effects of hepatic mCD36 and mSR-BI overexpression on plasma lipoprotein profiles, C57BL/6 mice were administered 1 x 10¹¹ particles of AdCD36, AdSR-BI, or Adnull. Immunoblot analyses of tissue lysates confirmed that the expression of either mCD36 or mSR-BI was increased more than 20-fold over endogenous levels 3 days after adenoviral vector treatments ( Fig 5B). Plasma was collected and subsequently fractionated on a Superose 6 column. The cholesterol content of individual 0.5-ml fractions was determined. As depicted in Fig 5A, AdSR-BI produced a drastic reduction in HDL cholesterol to undetectable levels, consistent with previous reports (34) (38). In contrast, hepatic overexpression of CD36, as verified by tissue immunoblot analysis in the same experiment, did not alter lipoprotein profiles. ApoA-I (data not shown) and HDL cholesterol levels were not altered by AdCD36 treatment. This is consistent with the low efficiency of mCD36 selective lipid uptake in cultured cells and represents strong evidence that this receptor does not play a significant role in hepatic HDL metabolism in vivo.

View larger version (19K): [in this window] [in a new window]   Figure 5. Effect of hepatic mCD36 overexpression on plasma lipoprotein profiles in mice. C57BL/6 mice were administered 1 x 1011 particles of a replication-deficient adenovirus expressing either mSR-BI (AdSR-BI) or CD36 (AdCD36). Control mice were injected with Adnull, which contains no transgene. A: FPLC lipoprotein profiles. Plasma was collected and subsequently separated on a Superose 6 column. The cholesterol content of individual 0.5-ml fractions was determined. Profile of a single animal in each group is shown. No significant differences were observed in the profiles between the four animals in each adenoviral vector treatment group. B: Hepatic expression. Immunoblot analysis of mCD36 and SR-BI expression in liver lysates from mice treated with AdCD36, AdSR-BI, or Adnull control. Representative data are shown from two animals in each treatment group.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In this study the two class B scavenger receptors, mCD36 and mSR-BI, were compared directly for their ability to serve as receptors for the lipoprotein ligands oxLDL, LDL, and HDL using the same adenoviral vector expression system. The results clearly show marked differences in the activity of these two receptors toward different lipoprotein ligands. The data indicate the following: i) LDL is a poor ligand for mCD36. The data from 4°C binding studies with human LDL imply that CD36 cannot be considered an important LDL receptor; ii) mCD36 and SR-BI bind human oxLDL with comparable high affinity; iii) mCD36 is much less efficient than mSR-BI in mediating selective uptake of CE from HDL; and iv) hepatic overexpression of mCD36 indicates a lack of a significant role for CD36 in HDL metabolism in vivo.

Characterization of the role of CD36 in mouse lipid metabolism has been hampered by a lack of specific anti-mCD36 antibodies. NIH-derived strains of spontaneously hypertensive rats, a model for human insulin resistance, were shown to lack CD36 (27) (39). We therefore adopted an approach of immunizing spontaneously hypertensive rats with adenovirus expressing mCD36 and obtained high titers of specific rat anti-mCD36 polyclonal sera. This reagent proved useful in immunoblot analysis of cells and tissues and acted as a neutralizing antibody to the binding of oxLDL by CD36 overexpressed in COS cells (data not shown).

CD36 was found to bind LDL poorly, suggesting that this receptor should not be considered a physiological receptor for LDL. Our findings were based on 4°C binding assays using freshly prepared human LDL stored under nitrogen gas in the presence of EDTA. These data are in general agreement with published studies. Endemann and colleagues (13) reported that LDL did not compete for oxLDL binding to mCD36 transiently transfected into human embryonic kidney 293 cells. A minimum 4-h period of oxidation was required to transform native LDL to an effective ligand. Two studies using human CD36 transfected into COS (20) and mouse fibroblast NIH 3T3 (14) cell lines also did not show evidence of high affinity LDL binding. In contrast, Calvo and colleagues (23) found human CD36 expressed in COS mammalian and Sf9 insect cells to be a high-affinity receptor for LDL. The differences in the various observations may be explained by differences in the procedures used for lipoprotein isolation and labeling that could lead to minor oxidative modifications in normal LDL. In contrast to unmodified LDL, oxLDL was bound by mCD36 with high affinity, supporting evidence that mCD36 is a physiological receptor for oxLDL (17).

Our studies further show that mCD36 binds HDL with an affinity comparable to that of mSR-BI but does not mediate efficient selective lipid uptake. Studies using CD36-overexpressing transfected cell lines demonstrated that CD36 can bind HDL (21) (22) (23). However, in one case the human CD36 protein that was expressed was of a different size than endogenous CD36 (23). Gu and colleagues (21) showed that although both mSR-BI and hCD36 bound HDL, only mSR-BI efficiently mediated the transfer of CE intracellularly. Connelly and colleagues (22) found that rat CD36 mediated CE selective uptake from human HDL but with 7-fold less efficiency than mSR-BI. In an autologous mouse system we show that CD36 binds HDL with high affinity but mediates selective CE uptake with much less (10-fold) efficiency than SR-BI.

A key question concerns whether the binding and selective CE uptake from HDL by CD36 is of physiological relevance. CD36-deficient mice have increased HDL cholesterol levels, which could possibly result from decreased receptor-mediated binding and uptake (17). The animals, however, also have abnormal fatty acid metabolism with significantly increased nonesterified free fatty acids and triacylglycerol levels and triacylglycerol-enriched VLDL fractions. It is feasible that inefficient fatty acid utilization in peripheral tissues in CD36-deficient animals increases hepatic fatty acid loading and triacylglycerol-enriched VLDL. HDL cholesterol may thus be increased as a result of increased levels or catabolism of VLDL. To assess the potential role of CD36 in lipoprotein metabolism we compared the effects of adenovirus-mediated hepatic overexpression of mCD36 and mSR-BI on HDL cholesterol levels in vivo. As previously reported (34) (38) hepatic overexpression of mSR-BI decreased HDL cholesterol levels dramatically, supporting its role as a physiological HDL receptor. In contrast, hepatic overexpression of mCD36 had no effect on the murine lipoprotein profile and specifically HDL cholesterol levels, despite greatly induced (more than 20-fold) CD36 expression. There is little evidence, therefore, to support a significant role as a HDL receptor in vivo.

The presence of multiple functional domains in the CD36 extracellular region could explain different abilities for binding, internalization, or selective uptake of different lipoprotein ligands such as oxLDL and HDL. Future studies identifying the structural motifs in the extracellular domains of CD36 and SR-BI required for selective lipid uptake will be crucial to determine the underlying molecular mechanisms involved in this process. In summary, our data obtained from in vitro and in vivo experiments using adenovirus-mediated overexpression of mCD36 indicate that this class B scavenger receptor does not play a significant role in either HDL or LDL metabolism.

	FOOTNOTES

Abbreviations: CE, cholesteryl esters; oxLDL, oxidized low density lipoprotein; SR-BI, scavenger receptor class B, type I.

	ACKNOWLEDGMENTS

This work was supported in part by National Institutes of Health grants AA00292 (W.J.S.d.V.), AG14668 (F.C.d.B.), and HL59376 (D.R.v.d.W.) and by an Atorvastatin Research Award (W.J.S.d.V.).

Manuscript received November 28, 2000; and in revised form February 20, 2001; and in revised form March 22, 2001

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