Scavenger receptor CD36 mediates uptake of high density lipoproteins in mice and by cultured cells.

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.


Materials
Primers were purchased from Metabion. Taq-DNA polymerase, a fl uorometric 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). 125 Iodine, [ 3 H]cholesteryl oleyl ether, [ 14 C]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, unesterifi ed cholesterol, and fatty acids were obtained from Wako. BSA, iodixanol gradient medium, and standard laboratory chemicals were purchased from Sigma Aldrich. Horseradish peroxidaseconjugated 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.

Mice
Male mice with a targeted mutation in the CD36 gene [CD36 2 / 2 , 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.

Lipoprotein preparation
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 ).

HDL metabolism in mice
For plasma decay analysis of 125 I-TC-/[ 3 H]CEt-HDL, the mice were fasted for 4 h before tracer injection ( 2 ). Then 125 I-TC-/ [ 3 H]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 125 I radioactivity, and [ 3 H] was analyzed after lipid extraction ( 21 ). Computer modeling was used to fi t (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. 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 defi ned ( 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 b , 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 specifi city 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)(13)(14). CD36 and SR-BI bind HDL with high affi nity, 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-defi cient mice, the function of CD36 for HDL metabolism was explored ( 15,16 ). Mice with a targeted mutation in the CD36 gene have a signifi cant 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 defi ciency of CD36 may promote HDL formation. A lack of CD36 may be associated with increased hepatic cholesterol and phospholipid effl ux 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 ).

Immunoblots
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-b -Actin (monoclonal anti-b -Actin, mouse, Sigma Aldrich) as primary antibodies. b -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 fi lms and quantifi ed using Image Quant software, version 5.2 (GE Healthcare).

Plasma lipase activity
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 2 80°C. Pre-and postheparin plasma lipase activity were assayed using an artifi cial glycerol tri-[1-14 C]oleatecontaining emulsion ( 33 ).

Adrenal cholesterol analysis
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 fl uorometric assay. Cholesterol content is expressed as µg cholesterol/mg protein.

Miscellaneous
Routinely, all mice were fasted for 4 h before blood was harvested for analytical or preparative purposes. Total cholesterol, Tissue sites of uptake of HDL-associated tracers were determined 24 h after injection of 125 I-TC-/[ 3 H]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 125 I radioactivity, and aliquots were analyzed for [ 3 H] 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 specifi c 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 specifi c 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. 125 I-TC represents the uptake of HDL holo-particles by tissues ( 19 ). Selective HDL CE uptake is calculated as the difference in organ FCR between [

Preparation of nonparenchymal liver cells
Non-parenchymal hepatic cells were prepared from murine liver as described ( 26 ). Briefl y, 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 125 I-TC-/[ 3 H]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 ).

Generation of recombinant adenoviral vectors and infection of H1299 cells
The recombinant, replication-defective adenoviruses Ad-mCD36 and Ad-GFP were generated as outlined ( 27 ). Briefl y, Ad-mCD36 contains an expression cassette encoding the murine murine HDL, which was radiolabeled in the protein ( 125 I-TC) as well as in the CE ([ 3 H]CEt) moiety ( 2,9 ). HDL apolipoprotein-associated 125 I-TC represents the metabolism of HDL holo-particles, and the difference between [  To investigate the function of CD36 in tissue HDL metabolism, mice were euthanized 24 h after 125 I-TC-/[ 3 H] 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 HDLassociated [ 3 H]CEt was higher than the respective rate for 125 I-TC ( Fig. 3A ). Selective HDL CE uptake ([ 3 H]CEt 2 125 I-TC) by the liver was substantial compared with hepatic HDL holo-particle internalization ( 125 I-TC) in WT animals ( 9 ). In contrast, in CD36 KO mice, the hepatic organ FCR for HDL-associated 125 I-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-defi cient liver, the organ FCR for [ 3 H]CEt was reduced by 32% and selective CE uptake from HDL ([ 3 H]CEt 2 125 I-TC) was diminished by 33% (WT corresponds to 100%). unesterifi ed 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.

Statistics and calculations
Values are means ± SEM. All statistical analyses were performed using Student's t -test. Probability values less than 0.05 were considered statistically signifi cant.

CD36, plasma lipids, and lipoproteins of mice
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 modifi ed by the CD36 defi ciency, 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 signifi cant 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 signifi cant differences between WT-HDL and CD36 KO-HDL.

CD36 and HDL metabolism in mice
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 ]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 ([ 3 H]CEt 2 125 I-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 signifi cantly in spleen, stomach, brain, and lungs compared with WT controls.

CD36 and HDL metabolism of hepatocytes and of nonparenchymal liver cells
The liver is composed of distinct cell populations, including hepatocytes, KCs, and LSECs ( 40 ). Differences in HDL metabolism of specifi c hepatic cell fractions have been established ( 6 ). To investigate whether the regulation of HDL uptake by the CD36-defi cient 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 125 I-TC-/[ 3 H]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 125 I-TC-/[ 3 H]CEt-HDL ( Fig. 4 ). In WT cells, apparent HDL particle uptake according to 125 I-TC increased as the HDL concentration in the medium rose. Apparent HDL particle uptake due to [ 3 H]CEt was in excess of that according to 125 I-TC, and this lipid uptake increased in a dose-dependent manner throughout the entire concentration range. Apparent selective CE uptake ([ 3 H]CEt 2 125 I-TC) rose in hepatocytes from WT mice as a function of the HDL concentration. Uptake of 125 I-TC-/[ 3 H]CEt-HDL was explored in CD36 KO hepatocytes in parallel. In CD36defi cient cells, apparent HDL particle uptake according to 125 I-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 [

Adenovirus-mediated CD36 expression and HDL metabolism in vitro
In the experiments described above, mice or cells with a defi ciency 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 signifi cant CD36 expression or with no immunodetectable CD36 were available.

The metabolism of 125 I-TC-/[
3 H]CEt-HDL was investigated in primary nonparenchymal liver cells ( Fig. 5 ). In cells from WT mice, apparent HDL particle uptake according to [ 3 H]CEt was higher compared with 125 I-TC, and the difference between both tracers yields apparent selective CE uptake ([ 3 H]CEt 2 125 I-TC). In nonparenchymal hepatic cells from CD36 KO mice, apparent HDL particle uptake according to 125 I-TC and [ 3 H]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.   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 125 I-TC-/[ 3 H] 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 125 I-TC throughout the entire concentration range of HDL. Analogously, Ad-mCD36 induced an increase in apparent selective CE uptake ([ 3 H]CEt 2 125 I-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 ( 125 I-TC) 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 125 I-TC-/[ 3 H]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 ( 125 I-TC) in H1299 cells. Compared with Ad-GFP, Ad-mCD36 stimulated apparent HDL particle uptake according to [   (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 de termined 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 as well as selective CE uptake from HDL under the experimental conditions of this study.

SR-BI, LRP1, and CD36 expression in murine liver, hepatocytes, and adrenal glands
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 Then postnuclear supernatants were prepared from these cells. The proteins were subjected to electrophoresis followed by transfer to a membrane. Then the proteins were immunoblotted using CD36-or b -actin-specifi c antibodies. Shown is a typical blot; three independent experiments yielded qualitatively identical results. CD36, cluster of differentiation 36 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 defi ciency of CD36 is responsible for the decreased HDL uptake observed in this organ in vivo.

Lipase activity and HDL metabolism
Lipoprotein lipase can stimulate selective HDL CE uptake independent from lipolysis ( 42 ). In CD36-defi cient 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.

CD36 and adrenal gland cholesterol
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.

DISCUSSION
A major effect of CD36 defi ciency 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 steadystate 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 specifi c 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. Fig. 8. SR-BI and CD36 expression in liver membranes and postnuclear supernatants prepared from hepatocytes isolated from WT or from CD36 KO mice. A, B: Membrane fractions were isolated from livers of WT and CD36 KO male mice. As reference for CD36, a postnuclear supernatant was prepared from WT murine peritoneal macrophages (B). C: Hepatocytes were isolated from WT and CD36 KO male mice. After culture (37°C, 2.0 h), postnuclear supernatants were prepared from these cells. The indicated mass of protein was subjected to electrophoresis and transferred to a membrane. The proteins were immunoblotted using SR-BI-, CD36-, or b -actin-specifi c antibodies. b -actin was used as loading control. Typical blots are shown; four (A), three (B), or three (C) independent blots yielded qualitatively identical results. CD36, cluster of differentiation 36; KO, knockout; SR-BI, scavenger receptor class B type I; WT, wildtype. lipid-labeled murine HDL, HDL holo-particle ( 125 I-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-defi cient 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.
A recent investigation suggested that a defi ciency of CD36 in mice is associated with an increase in HDL formation that fi nally 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    ( 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 effi ciency for HDL uptake in cultured cells compared with SR-BI (12)(13)(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 effi ciency of this scavenger receptor.
With respect to the molecular mechanisms responsible for the decrease in tissue HDL uptake in CD36 KO mice and CD36-defi cient 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-defi cient liver and cells that fi nally 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 specifi c 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 defi ciency in mice signifi cantly 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 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 ( 125 I-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 125 I-TC compared with [ 3 H]CEt. This observation is in line with a preferential renal catabolism of HDL-associated apolipoproteins ( 39 ).
For tissue HDL metabolism of CD36-defi cient mice, a really novel result from this investigation is a signifi cant decline in selective HDL CE uptake by liver and adrenal glands compared with WT littermates. HDL holo-particle ( 125 I-TC) internalization by liver and adrenals was also signifi cantly diminished in mutant rodents. This decreased HDL catabolism by organs of CD36-defi cient 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 signifi cantly 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 ‫ف‬ 70% of all liver cells are parenchymal cells (i.e., hepatocytes) ( 40 ). HDL metabolism by defi ned 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 defi ciency signifi cantly diminished selective HDL CE uptake and HDL holo-particle ( 125 I-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 defi ciency 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 HDL 3 (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 es-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-defi cient mice, an alternative mechanism has been suggested as well ( 16 ). Accordingly, a CD36 defi ciency 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-defi cient 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 modifi ed 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 identifi ed. 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 modifi ed particles, a role of CD36 in the metabolism of native lipoprotein fractions presumably contributes to the relationship between CD36 and atherosclerosis.