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The mechanisms of HDL-mediated cholesterol transport from peripheral tissues to the liver are incompletely defined. Here the function of scavenger receptor cluster of differentiation 36 (CD36) for HDL uptake by the liver was investigated. CD36 knockout (KO) mice, which were the model, have a 37% increase (P = 0.008) of plasma HDL cholesterol compared with wild-type (WT) littermates. To explore the mechanism of this increase, HDL metabolism was investigated with HDL radiolabeled in the apolipoprotein (125I) and cholesteryl ester (CE, [3H]) moiety. Liver uptake of [3H] and 125I from HDL decreased in CD36 KO mice and the difference, i. e. hepatic selective CE uptake ([3H]125I), declined (–33%, P = 0.0003) in CD36 KO compared with WT mice. Hepatic HDL holo-particle uptake (125I) decreased (–29%, P = 0.0038) in CD36 KO mice. In vitro, uptake of 125I-/[3H]HDL by primary liver cells from WT or CD36 KO mice revealed a diminished HDL uptake in CD36-deficient hepatocytes. Adenovirus-mediated expression of CD36 in cells induced an increase in selective CE uptake from HDL and a stimulation of holo-particle internalization. In conclusion, CD36 plays a role in HDL uptake in mice and by cultured cells. A physiologic function of CD36 in HDL metabolism in vivo is suggested.
High density lipoprotein plays a critical role in cholesterol homeostasis (1). This lipoprotein fraction presumably removes cholesterol from peripheral tissues, such as the vessel wall. After esterification in plasma, HDL-associated cholesteryl esters (CE) are delivered to other lipoprotein fractions or tissues (1, 2). One mechanism that mediates the direct delivery of HDL-associated CE to organs is the selective lipid uptake pathway (2). In this process, HDL CE is internalized by cells independently of the uptake of the HDL holo-particle. This selective lipid uptake appears to be important for the transport of cholesterol to steroidogenic tissues for hormone biosynthesis and to the liver. In the liver, HDL-derived cholesterol can be secreted into bile, used for bile acid synthesis, or packaged and secreted in newly synthesized lipoproteins.
Scavenger receptors mediate the cellular uptake of lipoproteins (3, 4). Scavenger receptor class B type I (SR-BI) and cluster of differentiation 36 (CD36) are members of class B of this family (3, 5). As plasma membrane-associated proteins, structurally both molecules comprise extracellular loops that are anchored in the membrane by transmembrane domains adjacent to short cytoplasmic tails. A high degree of sequence homology is detected throughout the extracellular loop domains between SR-BI and CD36; however, the cytoplasmic and the transmembrane domains have limited sequence similarity (4).
SR-BI is expressed abundantly in the liver and steroidogenic tissues (3). This receptor binds HDL with high affinity and mediates selective uptake of HDL CE in the liver and adrenals (6). In mice, hepatic SR-BI overexpression promotes selective HDL CE uptake by the liver (7). In contrast, mice with a targeted mutation in the SR-BI gene have a decrease in selective CE uptake (6, 8, 9). Thus SR-BI is a physiologically relevant HDL receptor with a function in cholesterol homeostasis in vivo.
The role of CD36 in lipoprotein metabolism is less well defined (4). This molecule is expressed in skeletal and heart muscle, liver, lungs, adipose tissue, spleen, small intestine, testis, capillary endothelium, microglia, and elicited peritoneal macrophages (10, 11). The ligands of CD36 include native and oxidized lipoproteins, anionic phospholipids, thrombospondin, collagen, amyloid β, and plasmodium falciparum-infected erythrocytes (4, 5, 11, 12). With respect to metabolism, CD36 facilitates the transfer of fatty acids into myocytes (10). Recently a role of CD36 for selective CE uptake from low density lipoprotein was proposed (11). On the basis of this broad ligand specificity and its widespread expression, diverse functions of CD36 have been suggested, including atherogenesis and lipoprotein metabolism (4).
In vitro, the function of scavenger receptor CD36 in HDL metabolism was compared with the respective role of SR-BI (12–14). CD36 and SR-BI bind HDL with high affinity, and both receptors mediate selective HDL CE uptake. However, the rate of selective uptake mediated by CD36 is lower compared with the uptake mediated by SR-BI. These studies were performed in cell lines overexpressing CD36, and therefore, the relevance of these in vitro experiments for the role of CD36 in HDL metabolism in vivo was not established.
In CD36-deficient mice, the function of CD36 for HDL metabolism was explored (15, 16). Mice with a targeted mutation in the CD36 gene have a significant increase in plasma HDL cholesterol compared with wildtype (WT) littermates. Therefore, a role of CD36 in HDL metabolism in vivo was suggested (15, 16). With respect to the mechanism of this increase in HDL, a recent study proposed that a deficiency of CD36 may promote HDL formation. A lack of CD36 may be associated with increased hepatic cholesterol and phospholipid efflux and a stimulated hepatic secretion of apoplipoproteins. These changes may represent a mechanism that mediates an increase in plasma HDL cholesterol. However, in these studies, the role of CD36 for HDL catabolism in mice was not exhaustively addressed (16).
In this study, the role of CD36 for HDL catabolism was investigated in mice. Metabolic experiments were performed using murine HDL which was radiolabeled in the apolipoprotein (125I) as well as in the lipid (CE, [3H]) moiety (2). In CD36-deficient mutant mice, experiments with 125I-/[3H]HDL showed diminished selective HDL CE uptake and reduced HDL holo-particle internalization by liver and adrenal glands compared with WT littermates. In cultured hepatocytes from CD36-deficient liver, selective CE uptake and HDL holo-particle internalization from 125I-/[3H]HDL declined. In contrast, adenovirus-mediated overexpression of CD36 in cultured cells stimulated cellular HDL uptake. In summary, evidence is presented that CD36 mediates HDL uptake by tissues and thus plays a physiologic role in the catabolism of this atheroprotective lipoprotein fraction in vivo.
Primers were purchased from Metabion. Taq-DNA polymerase, a fluorometric cholesterol assay, culture media, sera, and supplements for cell culture were supplied by Invitrogen. The 6-well tissue culture plates were obtained from Becton Dickinson. Collagenases were from Worthington (hepatocytes) or from Serva (non-parenchymal liver cells). 125Iodine, [3H]cholesteryl oleyl ether, [14C]oleate, and ECL reagent were purchased from GE Healthcare. Heparin, protease inhibitor cocktail “complete,” and enzymatic assays for cholesterol, HDL cholesterol, and triglyceride were supplied by Roche. Assays for phospholipid, unesterified cholesterol, and fatty acids were obtained from Wako. BSA, iodixanol gradient medium, and standard laboratory chemicals were purchased from Sigma Aldrich. Horseradish peroxidase-conjugated secondary antibodies were supplied by Dianova. Scintillation cocktail was from PerkinElmer. Nitrocellulose membrane was obtained from Schleicher and Schuell. Films were supplied by Kodak. Rodent chow was purchased from Ssniff.
Male mice with a targeted mutation in the CD36 gene [CD36−/−, knockout (KO), homozygous] and the respective male littermate controls (WT) were used in this study (17). All animals were on a C57BL/6J genetic background. The genotype of each mouse was analyzed by PCR from genomic DNA isolated from tail biopsies (17). Mice were maintained on a standard laboratory chow diet and had unlimited access to food and water. The age of the rodents used in this study was between 20 and 50 weeks. All animal protocols were approved by the Institutional Animal Care and Use Committee of the University Hospital Hamburg.
Mice were fasted for 4 h before blood harvest. HDL (d = 1.063-1.21 g/ml) was isolated from WT murine plasma by sequential ultracentrifugation (18).
Murine HDL was doubly labeled with 125I-tyramine cellobiose (125I-TC) in the apolipoprotein and with [3H]cholesteryl oleyl ether ([3H]CEt) in the CE moiety (9, 19). [3H]CEt was introduced in 125I-TC-HDL by exchange from donor liposomal particles, which contained [3H]CEt, using partially purified human plasma cholesteryl ester transfer protein (CETP) (20). The final 125I-TC-/[3H]CEt-HDL preparation was extensively dialyzed against PBS (pH 7.4) containing EDTA (1 mmol/l). Both HDL tracers were intracellularly trapped after uptake by tissues and cells and were not released thereafter (19).
For plasma decay analysis of 125I-TC-/[3H]CEt-HDL, the mice were fasted for 4 h before tracer injection (2). Then 125I-TC-/[3H]CEt-HDL (30 µg HDL protein per mouse) was injected in a tail vein. Thereafter, blood samples (30 µl per time point) were collected periodically (10 and 30 min; 2, 5, 9, 22, and 24 h) after injection. Animals were fasted throughout the 24 h study period but had unlimited access to water. Plasma aliquots were directly assayed for 125I radioactivity, and [3H] was analyzed after lipid extraction (21). Computer modeling was used to fit (by method of least squares) multiexponential curves arising from a common two-pool model simultaneously to both tracers’ plasma decay data and to calculate plasma fractional catabolic rates (plasma FCRs) for the two tracers (22). The modeling was done separately for the data from each mouse, so that individual plasma FCRs for both tracers were calculated for each animal.
Tissue sites of uptake of HDL-associated tracers were determined 24 h after injection of 125I-TC-/[3H]CEt-HDL, when both tracers were predominantly cleared from plasma (2). Finally the animals were anesthetized, the abdomen and chest were opened, and a catheter was inserted into the heart. The inferior vena cava was cut and the mice were perfused extensively with saline (50 ml per animal). After perfusion, the liver, adrenals, kidneys, brain, heart, lungs, spleen, stomach, intestine, and carcass of each mouse were harvested and homogenized. Homogenates from each tissue and the carcass were directly assayed for 125I radioactivity, and aliquots were analyzed for [3H] after lipid extraction (21).
Total radioactivity recovered from all tissues and the carcass of each mouse was calculated (2). The fraction of total tracer uptake attributed to a specific organ was calculated as the radioactivity recovered in that organ divided by the total radioactivity recovered from all tissues and carcass. Thus the percentage of recovered extravascular radioactivity in tissues was determined 24 h after injection of labeled HDL.
To allow comparison of the specific activities of various tissues in HDL internalization and to directly compare the rates of uptake of the apolipoprotein component and the CE moiety of HDL, the data were expressed as organ fractional catabolic rates (organ FCR) (2). These rates were calculated as follows: (Organ FCR in Tissue X) = (Plasma FCR) × (Fraction [%] of Total Body Tracer Recovery in Tissue X). This organ FCR represents the fraction of the plasma pool of either HDL tracer cleared by an organ per h. 125I-TC represents the uptake of HDL holo-particles by tissues (19). Selective HDL CE uptake is calculated as the difference in organ FCR between [3H]CEt and 125I-TC.
Primary hepatocytes were isolated from murine liver by perfusion (37°C, 18 minutes) with Hanks balanced salt solution supplemented with collagenase (0.3 mg/ml, type I), Hepes (10 mMol/l), and protease inhibitor mixture “complete” (23). The cells then were cultured (37°C, 2.0 h) in DMEM containing FBS (5%, v/v), penicillin (100 µg/ml), and streptomycin (100 µg/ml). Finally the culture medium was aspirated, and the cells were washed three times in PBS. Hepatocytes were used for 125I-TC-/[3H]CEt-HDL uptake assays or for preparation of postnuclear supernatants (24, 25).
Non-parenchymal hepatic cells were prepared from murine liver as described (26). Briefly, the liver was perfused (37°C) through the portal vein with calcium-free phosphate buffer supplemented with collagenase (0.05%, w/v, type A). Then the organ was mechanically disrupted and incubated (37°C, 30 minutes) in Gray's balanced saline containing collagenase (0.05%, w/v, type IV) with constant rotation (240 rpm). The cell suspension was passed through a cell sieve to remove debris. Nonparenchymal liver cells were recovered by centrifugation using an iodixanol density gradient (30%). Finally, cells were cultured (37°C, 2.0 h) in DMEM containing FBS (5%, v/v), penicillin (100 µg/ml), and streptomycin (100 µg/ml). The culture medium was aspirated, and the cells were washed three times in PBS. These cells were used for 125I-TC-/[3H]CEt-HDL uptake assays (25). Nonparenchymal liver cells were predominantly composed of Kupffer cells (KC) and liver sinusoidal endothelial cells (LSEC). No hepatocytes were detected in this preparation (26).
The recombinant, replication-defective adenoviruses Ad-mCD36 and Ad-GFP were generated as outlined (27). Briefly, Ad-mCD36 contains an expression cassette encoding the murine CD36 cDNA; the sequence was inserted into vector AdEasy. Ad-GFP (control) contains no transgene expression cassette.
H1299 cells were incubated (37°C) in DMEM supplemented with FBS (10%, v/v), penicillin (100 µg/ml), and streptomycin (100 µg/ml) (28). At 24 h after plating H1299 cells in wells (35 mm), no virus (mock, DMEM), Ad-GFP, or Ad-mCD36 were added. H1299 cells were exposed for 72 h to a multiplicity of infection (MOI) of 200 virus particles per cell.
Hepatocytes, nonparenchymal liver cells, or H1299 cells were incubated (37°C, 2.0 or 4.0 h) in DMEM containing BSA (5 mg/ml), penicillin (100 µg/ml), streptomycin (100 µg/ml), and the respective 125I-TC-/[3H]CEt-HDL (25). Finally cells were harvested by trypsin/EDTA (1×, trypsin 0.05%, EDTA 0.53 mmol/l) treatment and cellular uptake of HDL tracers was measured. 125I was directly radioassayed and [3H] was analyzed after lipid extraction (21). Uptake of 125I-TC-/[3H]CEt-HDL by cells was shown in terms of apparent HDL particle uptake, expressed as HDL protein (2, 25), to compare the uptake of both tracers on a common basis. The uptake of HDL holo-particles is represented by 125I-TC, and the difference between [3H]CEt and 125I-TC yields apparent selective HDL CE uptake by cells (19).
Membrane fractions (29) from murine liver or adrenals and postnuclear supernatants (24) from cultured primary hepatocytes and H1299 cells were prepared. Protease inhibitor cocktail “complete” was present during these preparations.
Membrane fractions or postnuclear supernatants were boiled (93°C, 10 minutes) and separated by SDS polyacrylamide gel (10%) electrophoresis under reducing conditions (mercaptoethanol) and then transferred to a nitrocellulose membrane (9). The blots were probed with anti-CD36 (rabbit polyclonal antiserum against murine CD36) (30), anti-SR-BI (rabbit polyclonal antibody to murine SR-BI, Novus Biologicals), anti-LRP1 (sheep anti-LRP1 antibody, raised against a synthetic peptide from human LRP1) (31), or anti-β-Actin (monoclonal anti-β-Actin, mouse, Sigma Aldrich) as primary antibodies. β-Actin was used as loading control. Finally the blots were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies and developed with ECL. Blots were exposed to Bio Max MR films and quantified using Image Quant software, version 5.2 (GE Healthcare).
Lipase activity was determined in pre- and postheparin plasma (32). After fasting for 4 h, blood was harvested from mice. Then heparin (100 units/kg body weight) was injected via a tail vein. After 5 min, blood was obtained by retro-orbital bleeding, and plasma was frozen at −80°C. Pre- and postheparin plasma lipase activity were assayed using an artificial glycerol tri-[1-14C]oleate-containing emulsion (33).
Mice were euthanized, and adrenal glands were harvested and immediately frozen (24). The glands were subsequently homogenized. Protein in the homogenates was measured using the method described by Lowry et al. (34). Cholesterol was analyzed with a commercial fluorometric assay. Cholesterol content is expressed as µg cholesterol/mg protein.
Routinely, all mice were fasted for 4 h before blood was harvested for analytical or preparative purposes. Total cholesterol, unesterified cholesterol, HDL cholesterol, triglycerides, and phospholipids from plasma and lipoproteins were measured using commercial enzymatic assays. Plasma lipoproteins were fractionated by fast protein liquid chromatography (FPLC) (35). Protein was analyzed as outlined (34).
For determination of free fatty acids, tetrahydrolipstatin (THL, 25 µg/ml) was added to murine blood (36). THL prevents in vitro lipolysis of triglycerides, which could lead to an overestimation of fatty acids. Plasma free fatty acids were determined using a commercial kit, which was adapted to microtiter plates.
Values are means ± SEM. All statistical analyses were performed using Student's t-test. Probability values less than 0.05 were considered statistically significant.
A targeted mutation in the gene encoding CD36 in mice induced an increase in plasma cholesterol (Table 1) (15, 16). Compared with WT mice, cholesterol increased by 33% in CD36 KO animals. To determine the lipoprotein fraction which was modified by the CD36 deficiency, FPLC was used (Fig. 1). Compared with WT mice, the increase in plasma cholesterol in CD36 KO littermates was predominantly due to a rise in the HDL fraction. Plasma HDL analysis was also performed by precipitation of apolipoprotein B-containing lipoproteins and subsequent cholesterol measurement in the supernatant (Table 1). Compared with WT mice, HDL cholesterol of CD36 KO animals increased by 37%.
CD36 can facilitate the cellular uptake of fatty acids (10), and CD36 KO mice may show an increase in plasma triacylglycerol and fatty acids (15, 37). Therefore triglycerides and free fatty acids were measured in WT and in CD36 KO mice (Table 1). No significant differences in plasma triglycerides and fasting fatty acids were detected either in the absence or presence of CD36.
Following preparation from plasma, the chemical composition of WT-HDL and of CD36 KO-HDL was investigated (Table 2). This analysis revealed no significant differences between WT-HDL and CD36 KO-HDL.
To explore a possible mechanism of the CD36 deficiency-induced increase in HDL, we investigated whether this scavenger receptor has a role in HDL internalization and catabolism. Metabolic studies were performed using murine HDL, which was radiolabeled in the protein (125I-TC) as well as in the CE ([3H]CEt) moiety (2, 9). HDL apolipoprotein-associated 125I-TC represents the metabolism of HDL holo-particles, and the difference between [3H]CEt and 125I-TC yields selective CE uptake from HDL (38).
125I-TC-/[3H]CEt-HDL was injected in WT and CD36 KO mice (Fig. 2A). Over a 24 h, periodic blood samples were collected for analysis of 125I-TC and [3H]CEt. In WT animals, the plasma decay of HDL-associated [3H]CEt was faster than that of 125I-TC. The difference between [3H]CEt and 125I-TC yields selective CE clearance from HDL in controls (2). In CD36 KO mice, the decay of 125I-TC was decreased compared with WT, suggesting a reduced rate of HDL holo-particle catabolism. In CD36 KO mice, selective CE removal ([3H]CEt − 125I-TC) from plasma HDL was diminished compared with WT rodents.
Plasma FCRs were calculated (2, 22) from HDL decay curves (Fig. 2A). In WT mice, the plasma FCR for HDL-associated [3H]CEt was higher than that for 125I-TC (Fig. 2B). The difference in plasma FCRs ([3H]CEt − 125I-TC) yields selective CE clearance from plasma HDL by tissues, and the respective rate was substantial in WT mice (9). In CD36 KO animals, a decrease of 28% in plasma FCR for 125I-TC was observed, which suggests a reduction in HDL holo-particle catabolism compared with WT (corresponding to 100%). Selective CE removal from circulating HDL ([3H]CEt − 125I-TC) was significantly diminished by 29% in CD36 KO animals compared with WT (corresponding to 100%).
To investigate the function of CD36 in tissue HDL metabolism, mice were euthanized 24 h after 125I-TC-/[3H]CEt-HDL injection, and tissues were harvested for tracer content analysis (2). Based on plasma FCRs and fractional tracer recovery of individual tissues, the organ FCRs for each HDL tracer were calculated. These organ FCRs represent the fraction of the plasma pool of the traced HDL component cleared by a tissue per h.
For the liver of WT mice, the organ FCR for HDL-associated [3H]CEt was higher than the respective rate for 125I-TC (Fig. 3A). Selective HDL CE uptake ([3H]CEt − 125I-TC) by the liver was substantial compared with hepatic HDL holo-particle internalization (125I-TC) in WT animals (9). In contrast, in CD36 KO mice, the hepatic organ FCR for HDL-associated 125I-TC decreased by 29% compared with WT, which suggests a decline in HDL holo-particle uptake by the mutant liver (Fig. 3A). In the CD36-deficient liver, the organ FCR for [3H]CEt was reduced by 32% and selective CE uptake from HDL ([3H]CEt − 125I-TC) was diminished by 33% (WT corresponds to 100%).
125I-TC-/[3H]CEt-HDL uptake by adrenals in WT mice revealed a higher organ FCR for [3H]CEt compared with 125I-TC, and selective CE uptake ([3H]CEt − 125I-TC) was substantial in normal glands compared with HDL holo-particle (125I-TC) internalization (Fig. 3B) (9). In adrenals from CD36 KO mice, the organ FCRs for 125I-TC and [3H]CEt decreased by 37% and 40%, respectively. This decline indicated a decrease of 37% of HDL holo-particle uptake and yielded a significant reduction of 41% of selective CE uptake ([3H]CEt − 125I-TC) by adrenal glands from CD36-deficient mice compared with WT (corresponding to 100%). In summary, these experiments provide evidence for a role of CD36 in hepatic and adrenal HDL metabolism.
125I-TC-/[3H]CEt-HDL uptake by nonhepatic and nonsteroidogenic tissues was explored. In kidneys of WT mice, the organ FCR for HDL-associated 125I-TC was higher than the rate for [3H]CEt; this result is consistent with a role of these organs in HDL apolipoprotein catabolism (Fig. 3C) (39). In kidneys of CD36 KO animal, the organ FCR for HDL-associated 125I-TC decreased by 35%; this observation is line with a role of CD36 in renal HDL apolipoprotein catabolism. Kidney organ FCRs for HDL-associated [3H]CEt were similar in WT and CD36 KO mice.
Uptake of 125I-TC-/[3H]CEt-HDL by spleen, stomach, intestine, brain, heart, lungs, and carcass was investigated (supplementary Table I). Compared with the liver, the organ FCRs for all tissues except carcass were quantitatively small. This result reinforces a dominant role of the liver in HDL catabolism in mice (2, 9). In WT rodents, some selective CE uptake ([3H]CEt − 125I-TC) from HDL was detected in spleen, stomach, brain, heart, lungs, and carcass. In the intestine of WT mice, no selective CE uptake from HDL was measured. In CD36 KO animals, selective HDL CE uptake declined significantly in spleen, stomach, brain, and lungs compared with WT controls.
The liver is composed of distinct cell populations, including hepatocytes, KCs, and LSECs (40). Differences in HDL metabolism of specific hepatic cell fractions have been established (6). To investigate whether the regulation of HDL uptake by the CD36-deficient liver is due to a decrease of the respective pathways in distinct cell populations, HDL metabolism was explored in both primary hepatocytes and nonparenchymal liver cells (9). The cells were prepared from WT or CD36 KO mice (23, 26). After culturing, cells were incubated in medium containing 125I-TC-/[3H]CEt-HDL. The cellular tracer content was analyzed and expressed in terms of apparent HDL particle uptake (2, 25).
Murine hepatocytes were incubated in medium containing 125I-TC-/[3H]CEt-HDL (Fig. 4). In WT cells, apparent HDL particle uptake according to 125I-TC increased as the HDL concentration in the medium rose. Apparent HDL particle uptake due to [3H]CEt was in excess of that according to 125I-TC, and this lipid uptake increased in a dose-dependent manner throughout the entire concentration range. Apparent selective CE uptake ([3H]CEt − 125I-TC) rose in hepatocytes from WT mice as a function of the HDL concentration. Uptake of 125I-TC-/[3H]CEt-HDL was explored in CD36 KO hepatocytes in parallel. In CD36-deficient cells, apparent HDL particle uptake according to 125I-TC was dose-dependent, and this uptake decreased by 18-27% compared with WT hepatocytes (set as 100%). In mutant hepatocytes, apparent HDL particle uptake according to [3H]CEt was reduced by 21-26% throughout the HDL concentration range. Apparent selective HDL CE uptake ([3H]CEt − 125I-TC) was diminished by 21-30% in CD36-deficient hepatocytes compared with WT cells.
SR-BI KO-HDL yields a lower rate of selective CE uptake compared with WT-HDL in vitro (9). To address whether radiolabeled WT-HDL or CD36 KO-HDL yields similar or different rates of HDL uptake in primary murine liver cells, primary hepatocytes were isolated from WT mice. The cells were incubated (37°C, 2.0 h) in medium containing 125I-TC-/[3H]CEt-WT-HDL (40 µg HDL protein/ml) or 125I-TC-/[3H]CEt-CD36 KO-HDL (40 µg HDL protein/ml). Apparent HDL holo-particle uptake (125I-TC) was not significantly different between both HDL preparations (data not shown). Analogously, selective HDL CE uptake ([3H]CEt − 125I-TC) was not significantly different between both radiolabeled HDL preparations (data not shown).
The metabolism of 125I-TC-/[3H]CEt-HDL was investigated in primary nonparenchymal liver cells (Fig. 5). In cells from WT mice, apparent HDL particle uptake according to [3H]CEt was higher compared with 125I-TC, and the difference between both tracers yields apparent selective CE uptake ([3H]CEt − 125I-TC). In nonparenchymal hepatic cells from CD36 KO mice, apparent HDL particle uptake according to 125I-TC and [3H]CEt decreased by 8% and 25%, respectively, and this decline yielded a decrease of 31% for selective CE uptake (WT corresponds to 100%). Remarkably, based on cell protein, nonparenchymal hepatic cells quantitatively had a higher rate of HDL uptake compared with hepatocytes.
In summary, these experiments suggest a role for CD36 in both hepatocytes and in nonparenchymal hepatic cells for HDL metabolism.
In the experiments described above, mice or cells with a deficiency of CD36 were the model. Next the effect of a high CD36 expression on cellular HDL internalization was determined. CD36 expression was induced with a recombinant adenovirus which encodes murine CD36 (Ad-mCD36) (27, 28). H1299 cells were incubated in medium containing no virus (mock), Ad-GFP (control), or Ad-mCD36 (Fig. 6). After cell harvest, CD36 expression in postnuclear supernatants from these cells was explored by immunoblotting. Ad-mCD36 induced a substantial expression of CD36 in H1299 cells; in contrast, this scavenger receptor was not detected in cells incubated without virus (mock) or with Ad-GFP. Thus, cells with significant CD36 expression or with no immunodetectable CD36 were available.
To address whether CD36 mediates HDL uptake in vitro, H1299 cells were cultured in medium containing no virus (mock), Ad-GFP (control), or Ad-mCD36 (Fig. 7). Then the cells were incubated in the presence of 125I-TC-/[3H]CEt-HDL, and the HDL tracer uptake was analyzed. Compared with Ad-GFP, Ad-mCD36 induced an increase of 694% in apparent HDL particle uptake (125I-TC) in H1299 cells. Compared with Ad-GFP, Ad-mCD36 stimulated apparent HDL particle uptake according to [3H]CEt significantly (by 249%). In relation to Ad-GFP, Ad-mCD36 induced a 209% increase in selective CE uptake ([3H]CEt − 125I-TC) from HDL. Compared with control (mock, DMEM), virus Ad-GFP quantitatively had only a minor effect on HDL uptake by H1299 cells.
Next the effect of CD36 on the dose response curve for HDL uptake in H1299 cells was explored (supplementary Fig. I). Again, H1299 cells were kept in the medium containing no virus (mock), Ad-GFP, or Ad-mCD36. Following incubation of the cells in the presence of 125I-TC-/[3H]CEt-HDL, the respective concentration range was 10-100 µg HDL protein/ml. Compared with Ad-GFP, Ad-mCD36 induced an increase in apparent HDL particle uptake according to 125I-TC throughout the entire concentration range of HDL. Analogously, Ad-mCD36 induced an increase in apparent selective CE uptake ([3H]CEt − 125I-TC) throughout the entire concentration range of HDL compared with Ad-GFP.
In summary, the expression of CD36 substantially stimulates apparent HDL holo-particle internalization (125I-TC) as well as selective CE uptake from HDL under the experimental conditions of this study.
As described above, HDL uptake was diminished in the liver and adrenals of CD36 KO mice. SR-BI and, presumably, low density lipoprotein receptor-related protein1 (LRP1) mediate selective CE uptake by tissues (3, 41). With respect to molecular mechanism(s), we wondered whether the decrease in HDL uptake in CD36 KO mice was due to the deletion of this scavenger receptor per se or, alternatively, whether a CD36-mediated downregulation of SR-BI and/or of LRP1 was involved. To investigate this, the expression of SR-BI, LRP1, and CD36 was determined in membranes isolated from murine liver and adrenals. To ensure that these receptors were expressed by hepatocytes per se, postnuclear supernatants from primary hepatocytes were also prepared and probed for the respective receptors.
In liver membranes from WT and CD36 KO mice, SR-BI expression was identical in both membranes according to immunoblots (Fig. 8A). As expected, in membranes prepared from CD36 KO liver, no CD36 signal was observed, whereas the respective band was detected in WT proteins (Fig. 8B). CD36 expression was visible in these blots in murine peritoneal macrophages, which were included as reference for CD36 (5). To investigate SR-BI in liver parenchymal cells, postnuclear supernatants were prepared from hepatocytes isolated from WT and CD36 KO mice (Fig. 8C). SR-BI expression in these immunoblots was identical in hepatocyte proteins isolated from WT and CD36 KO mice. However, a signal for CD36 was detected only in postnuclear supernatants isolated from WT hepatocytes.
To address whether a downregulation of LRP1 was responsible for the reduced HDL uptake of CD36 KO mice, liver membranes and hepatocyte postnuclear supernatants were prepared from WT and CD36 KO mice (Fig. 9) (41). Immunoblots showed no difference in LRP1 expression in both groups of proteins from murine liver or hepatocytes.
In murine adrenals, immunoblots yielded an identical signal for SR-BI in membranes prepared from WT and CD36 KO mice (Fig. 10). In contrast, CD36 expression was detected only in adrenal membranes isolated from WT mice (data not shown).
In summary, SR-BI and LRP1 expression were identical in liver of WT and of CD36 KO mice, and the same was true for SR-BI in adrenal glands. These results imply that in the liver a deficiency of CD36 is responsible for the decreased HDL uptake observed in this organ in vivo.
Lipoprotein lipase can stimulate selective HDL CE uptake independent from lipolysis (42). In CD36-deficient mice, LPL-mediated hydrolysis of lipoproteins was decreased compared with WT animals (37). Therefore, it could be that differences in HDL metabolism between WT and CD36 KO mice were due to variations in LPL activity. To investigate this, lipase activity was analyzed in WT and CD36 KO mice (Table 3). No difference in pre- and postheparin plasma lipase activity was detected between mice with and without CD36 expression.
HDL delivers cholesterol to adrenals as substrate for steroid hormone synthesis (38, 43). Analyzed with an isotope approach, in adrenals from CD36 KO mice, HDL uptake decreased compared with the respective HDL internalization of WT glands (Fig. 3B). To address the role of CD36 for HDL-mediated adrenal lipid accumulation in more detail, cholesterol mass was analyzed in glands from WT and mutant mice (Table 3). Total cholesterol mass decreased by 22% in glands isolated from CD36 KO mice compared with WT adrenals (corresponding to 100%). This result reinforces a physiological role of CD36 for HDL-mediated cholesterol delivery to tissues.
A major effect of CD36 deficiency in mice is an increase in plasma cholesterol; this change in phenotype is primarily due to a rise in HDL cholesterol. In this study, HDL cholesterol increased by 37% in CD36 KO mice. In previous investigations, a 33% increase in this lipoprotein fraction (15) and a 34% increase in total plasma cholesterol (16) were observed. This consistent change in HDL steady-state level is in agreement with a physiologic function of CD36 in HDL metabolism in vivo. Plasma triacylglycerol and fatty acids were not different between CD36 KO and WT animals in this study; this observation is in contrast to a previous report (15) but in agreement with another study (11). However, mouse strain-related genetic differences and specific experimental conditions (like diet) presumably explain the discrepancy.
Both HDL biosynthesis and HDL catabolism yield the steady state-level of this lipoprotein fraction in plasma. A recent investigation suggested that a deficiency of CD36 in mice is associated with an increase in HDL formation that finally yields an increase in HDL cholesterol (16). CD36 and SR-BI have a similar structure (4, 13), and the latter scavenger receptor has a major role in cellular HDL uptake and catabolism in vivo (8, 9). A function of CD36 in HDL uptake has been proposed on the basis of in vitro experiments; however, the lipid transfer rate of this protein may be lower compared with SR-BI (12, 13). In view of these observations, the hypothesis emerged that CD36 may have a role in HDL uptake and catabolism in vivo, analogous to SR-BI. We examined this hypothesis in mice and in liver and adrenals, the tissues of primary interest.
To explore the role of CD36 in HDL catabolism, a technique was employed that tracks the fate of distinct HDL components independently (2, 19). Using protein- and lipid-labeled murine HDL, HDL holo-particle (125I-TC) catabolism from the HDL plasma pool by tissues was detected in WT mice. Substantial selective CE removal from the HDL plasma pool by murine WT tissues was observed (9). As a major result of this study, a lack of CD36 reduced HDL holo-particle catabolism as well as selective CE clearance from HDL from the intravascular space. These data are in contrast to a recent study by Yue et al. (16) in CD36-deficient mice in which no effect of CD36 on HDL catabolism was suggested. However, several methodological differences between this and the other investigation (16) have to be considered. For example, Yue et al. (16) injected a huge mass of labeled HDL protein (180 µg HDL protein per mouse), whereas here only a trace amount of this lipoprotein fraction was given (30 µg HDL protein per mouse). Such a large mass of HDL increases the endogenous plasma pool of this lipoprotein fraction and thus may decrease the rate of HDL catabolism. This technical difference raises the question whether the experimental conditions of the other study (16) were physiologically appropriate.
Besides plasma, the role of individual tissues for HDL catabolism was explored in this study. In WT mice, the liver quantitatively had the highest uptake rates for HDL holo-particles (125I-TC) and for selective CE internalization. This result is consistent with a dominant role of this organ for HDL catabolism in vivo (2, 9). Adrenals showed the highest ratio of HDL lipid compared with protein uptake in WT rodents. HDL internalization by WT kidneys demonstrated a higher uptake of 125I-TC compared with [3H]CEt. This observation is in line with a preferential renal catabolism of HDL-associated apolipoproteins (39).
For tissue HDL metabolism of CD36-deficient mice, a really novel result from this investigation is a significant decline in selective HDL CE uptake by liver and adrenal glands compared with WT littermates. HDL holo-particle (125I-TC) internalization by liver and adrenals was also significantly diminished in mutant rodents. This decreased HDL catabolism by organs of CD36-deficient mice provides evidence that CD36 mediates hepatic and adrenal HDL uptake and thus catabolism of this lipoprotein fraction. This diminished HDL uptake by tissues in CD36-deficient mice represents a mechanism that contributes to the increase of plasma HDL cholesterol of these mutant rodents. Remarkably, a recent study in CD36 mutant mice did not address the role of CD36 for HDL uptake by liver and adrenals in vivo (16). The physiologic relevance of CD36 for organ cholesterol homeostasis is underscored here by adrenal gland lipid mass analysis. In adrenals from CD36 KO mice, cholesterol mass declined significantly compared with WT. This observation also provides evidence for a function of CD36 in HDL-mediated cholesterol delivery to tissues.
The liver is composed of distinct cell populations, and only ~70% of all liver cells are parenchymal cells (i.e., hepatocytes) (40). HDL metabolism by defined hepatic cell types is heterogeneous (6). Due to this heterogeneity, the decreased hepatic HDL uptake in CD36 KO mice did not necessarily represent a decline in HDL catabolism by liver parenchymal cells per se. Alternatively, a decrease in HDL uptake by nonparenchymal hepatic cells could be the mechanism of the impairment of HDL uptake by the whole liver in CD36 KO animals. In this investigation, a CD36 deficiency significantly diminished selective HDL CE uptake and HDL holo-particle (125I-TC) internalization by hepatocytes and nonparenchymal liver cells, and both results are in qualitative agreement with those obtained for the liver in vivo. Therefore, the decrease in HDL uptake observed in vivo must be attributed to a decline of these pathways in both hepatocytes and nonparenchymal liver cells. This observation is in contrast to results from Yue et al. (16) which suggested that a CD36 deficiency of murine hepatocytes had no role for hepatic HDL internalization. Again, substantial methodological differences have to be considered between that study (16) and this investigation. For example, in the other study, human HDL3 (d = 1.125-1.21 g/ml) was radiolabeled with [3H]cholesteryl oleyl ether, whereas here for HDL uptake analysis doubly-radiolabeled murine HDL (d = 1.063-1.21 g/ml) was applied. Only total HDL lipid uptake could be estimated by using [3H]cholesteryl oleyl ether-labeled HDL3 in the other study (16); a distinction between selective CE uptake and HDL particle internalization is not possible with this single isotope tracer. Besides, Yue et al. (16) used very high concentrations of radiolabeled HDL (100-300 µg HDL protein/ml) compared with this investigation, and such high HDL concentrations may be above saturation of the pathway. These technical differences presumably explain the discrepancy in results.
A possible role of CD36 for cellular HDL metabolism was explored in this study with a model in which this scavenger receptor was expressed using an adenovirus-mediated gene transfer. Ad-mCD36 induced a substantial expression of CD36 in cultured cells, and in parallel, Ad-mCD36 mediated an increase in HDL selective uptake and in HDL particle internalization. In contrast to these results, CD36 previously had a low efficiency for HDL uptake in cultured cells compared with SR-BI (12–14). A likely explanation for this discrepancy in results may be technical. Whereas in a previous study a plasmid vector was used for gene transfer, here an adenovirus was applied (13). Different methods for gene transfer may yield variations in the CD36 protein expression level. In addition, minor differences in the CD36 molecule structure may contribute to quantitative variations in HDL uptake efficiency of this scavenger receptor.
With respect to the molecular mechanisms responsible for the decrease in tissue HDL uptake in CD36 KO mice and CD36-deficient cells, several molecules, including SR-BI, LRP1 and LPL, may play a role (3, 41, 42). We considered whether regulation of these proteins occurred in CD36-deficient liver and cells that finally yielded the decrease in HDL uptake. Expression of SR-BI and LRP1 in liver and hepatocytes from WT and CD36 KO mice were identical in immunoblots. In adrenal glands, SR-BI expression in plasma membranes was identical in the presence and absence of CD36. Analogously, lipase activity in plasma was not different between both groups of animals. These results are in line with another study (16) and reinforce the concept that the inactivation of CD36 represents the mechanism responsible for the decrease in HDL uptake by tissues and the increase in plasma HDL cholesterol in CD36 KO mice.
SR-BI KO mice had an increase of 96% in plasma HDL cholesterol compared with WT littermates in a previous investigation (9). CD36 KO mice showed a rise of 37% in HDL cholesterol in this study. This comparison suggests that both SR-BI and CD36 have a function in HDL-mediated cholesterol delivery to tissues. However, on a quantitative basis, SR-BI has a major role in HDL metabolism and in tissue HDL uptake. With respect to specific organs, the expression of both SR-BI and CD36 in liver and adrenals are relevant for the regulation of plasma HDL cholesterol in mice.
In summary, a CD36 deficiency in mice significantly increases plasma HDL cholesterol (15, 16). One mechanism that contributes to this lipid increase is a decrease in HDL uptake by the liver and adrenals. In line with this observation in vivo, a lack of CD36 in hepatocytes and in nonparenchymal liver cells in vitro diminished cellular HDL uptake. On the other hand, an adenovirus-mediated expression of CD36 stimulated HDL uptake by cells in vitro. Thus several lines of evidence show that CD36 has a role for cellular HDL uptake and catabolism. With respect to the increase in plasma HDL in CD36-deficient mice, an alternative mechanism has been suggested as well (16). Accordingly, a CD36 deficiency is associated with an increase in HDL formation in mice. However, results presented in both investigations are in line with the conclusion that presumably two mechanisms (i.e., increased HDL biosynthesis (16) and reduced HDL catabolism) contribute to the increase in steady-state plasma HDL cholesterol level in CD36-deficient mice.
CD36 has been implicated in the pathogenesis of atherosclerosis (4, 5). Initially this effect was attributed to a function of this scavenger receptor in the internalization of oxidatively modified lipoproteins by macrophages and subsequent lipid accumulation and foam cell formation in the arterial wall. However, recent studies point to a role of CD36 in the metabolism of native lipoproteins. This study and several others present evidence for a function of CD36 in HDL metabolism in mice (15, 16). Variations in the CD36 gene were implicated in the etiology of the metabolic syndrome in humans (44), and associations between variants in the gene of this receptor, HDL cholesterol levels, and the metabolic syndrome were identified. Evidence for a role of CD36 in the catabolism of native LDL in mice has been suggested as well (11). A function of CD36 in the clearance of fatty acid-enriched, triglyceride-rich particles (45) from plasma has been proposed, and intestinal CD36 has been involved in chylomicron formation (46). These results are in line with physiologic functions of CD36 in the metabolism of native lipoproteins. Thus, in addition to effects on the catabolism of modified particles, a role of CD36 in the metabolism of native lipoprotein fractions presumably contributes to the relationship between CD36 and atherosclerosis.
The authors appreciate S. Ehret, B. Schulz, M. Thiel, and D. Evans for their assistance. R. E. Morton and D. J. Greene donated CETP, and U. J. F. Tietge supplied mice.
This work was supported by Grant Ri 436/8-1 from Deutsche Forschungsgemeinschaft (DFG), Bonn, Germany, and by Werner Otto Stiftung, Hamburg, Germany.
[S]The online version of this article (available at http://www.jlr.org) contains supplementary data in the form of one figure and one table.