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Adipocytes recognize and degrade oxidized low density lipoprotein through CD36
BBRC Biochemical and Biophysical Research Communications 295 (2002) 319–323 www.academicpress.com
Adipocytes recognize and degrade oxidized low density lipoprotein through CD36q Akihiko Kuniyasu,1 Shigeki Hayashi, and Hitoshi Nakayama* Department of Biofunctional Chemistry, Faculty of Pharmaceutical Sciences, Kumamoto University, 5-1 Ohe-Honmachi, Kumamoto 862-0973, Japan Received 28 May 2002
Abstract CD36 expressed on adipocytes is thought to function as a fatty acid transporter (FAT). Here we report that adipocytes can endocytose and lysosomally degrade OxLDL, mainly mediated by CD36. Mouse 3T3-L1 preadipocytes showed marked increase in uptake and degradation of 125 I-OxLDL during their differentiation to adipocytes. RT-PCR and immunoblot analysis indicated that expression of CD36 but not of scavenger receptor class A or macrosialin is required for the increase in uptake and degradation of 125 I-OxLDL in 3T3-L1 cells. An anti-CD36 antibody inhibited both uptake and degradation activities of 125 I-OxLDL up to 60%. These results strongly suggest that adipocytes may function as phagocytes like macrophages and that CD36 plays a novel role in adipose tissues. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: CD36; Scavenger receptor; Adipocytes; 3T3-L1 cells; Oxidized LDL; Endocytic uptake; Lysosomal degradation
Adipose tissues play an active role in a variety of physiological and pathological processes regulating energy metabolism. Triglycerides, a major energy source stored in adipose tissues, are synthesized from long chain fatty acids and glucose. Therefore, transport of the fatty acid and glucose is essential for the production of energy sources. CD36 is proposed to play a role as the fatty acid transporter (FAT) in adipocytes [1]. Marked decreases in FAT activity [2] and triglyceride synthesis [3], which are observed in CD36 null mutation mice, support this hypothesis. CD36 is a multifunctional receptor expressed in several cell types [4,5]. In macrophages, for example, CD36 functions as a scavenger receptor for oxidized low density lipoprotein (OxLDL) [6,7], while it functions as a receptor for thrombospondin-1 [8] and collagen type I/IV in platelets [9]. We recently showed that CD36 serves as a receptor for advanced glycation end products [10]. In addition, several studies show that CD36 can recognize anionic phospholipids to participate in phagocytotic q Abbreviations: FAT, fatty acid transporter; LDL, low density lipoprotein; OxLDL, oxidized LDL; SR-A, scavenger receptor class A. * Corresponding author. Fax: +81-96-372-7182. E-mail address: [email protected] (H. Nakayama). 1 Also corresponding author.
clearance by human monocytes [11,12] and dendritic cells [13], rat retinal pigment endothelium [14], and Drosophila hemocytes [15]. Recent observations indicate that adipose tissues are biologically active and dynamic and play important endocrine and possibly immunological roles in addition to the traditional function as energy storage depot [16–21]. Therefore, it is important to determine whether adipocytes have additional unknown functions. The 3T3-L1 cell line differentiates under the controlled conditions of cell culture from fibroblasts, or preadipocytes, to cells with the morphological and biochemical properties of adipocytes [22]. 3T3-L1 adipocytes are comparable with native adipocytes as they have the ability to accumulate lipid, respond to insulin, and secrete leptin [23]. In this study we determined whether adipocytes endocytosed and lysosomally degraded OxLDL. Here we demonstrate that 3T3-L1 adipocytes can recognize and degrade OxLDL, mainly mediated by CD36.
Materials and methods Materials. Na 125 I (3.7 GBq/ml) was purchased from Amersham Pharmacia Biotech (Buckinghamshire, UK). Goat anti-human CD36
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polyclonal antibody (L-17) and mouse anti-murine CD36 monoclonal antibody (clone 63) were purchased from Santa Cruz Biotech (Santa Cruz, CA, USA) and Cascade Bioscience (Winchester, MA, USA), respectively. Mouse IgA, kappa (TEPC 15) and porcine insulin were from Sigma (St. Louis, MO, USA). All other chemicals were of the analytical grade commercially available. Cell culture. Mouse 3T3-L1 cells (Health Science Research Resources Bank, Osaka, Japan) were plated and grown in a basal medium (Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, 20 U/ml penicillin, and 20 lg/ml streptomycin). Cells were then differentiated by changing to a medium composed of the basal medium plus 10 lg/ml insulin, 0.25 lM dexamethasone, and 0.5 mM isobutylmethylxanthine. After 48 h, the medium was replaced with basal medium containing 5 lg/ml insulin, and cells were maintained in this medium until use. Binding, cell association, and degradation of lipoproteins. Human low density lipoprotein (LDL, d ¼ 1:019–1:063 g/ml), acetylated LDL (AcLDL), OxLDL, and 125 I-labeled OxLDL (400–450 cpm/ng protein) were prepared essentially as described [24]. Binding, cell-association and degradation of 125 I-OxLDL were performed as described previously [10]. For the binding assay, cells were incubated with 1.0– 10 lg=ml 125 I-OxLDL at 4 °C for 1.5 h. Nonspecific binding was measured in the presence of a 20-fold excess of OxLDL and subtracted from the data. For cell-association and degradation assays, cells were incubated with 5 lg=ml 125 I-OxLDL at 37 °C for 16 h. For inhibition assays, cells were incubated with or without various compounds (100 lg/ml) or mouse antibodies (anti-CD36 antibody clone 63 or control IgA; 0.1-10 lg/ml). Results are represented as the means SD (n ¼ 3). RT-PCR amplification. Total RNAs were isolated from 3T3-L1 cells, J774 cells and mouse hearts using ISOGEN (Nippon Gene, Toyama, Japan) according to manufacturer’s recommendations. RNAs were reverse-transcribed, and the cDNA obtained was used for PCR amplification to estimate the expression of SR-A, CD36, and macrosialin. The primer sequences used were as follows. Scavenger receptor class A (SR-A) [25]: sense primer, 50 -ATGACAGAGAA TCAGAGGC-30 ; antisense primer, 50 -CATGTTCCTGGACTGAC GA-30 . CD36 [6]: sense primer, 50 -AGGTCCTTACACATACAGA GTTCG-30 ; antisense primer, 50 -GGACTTGCATGTAGGAAATGT GGA-30 . Macrosialin [26]: sense primer, 50 -ATGCGGCTCCC TGTGTGTC-30 ; antisense primer, 50 -TCAGAGGGGCTGGTAGG TTG-30 . The sizes of PCR products were 508 bp for SR-AI/II, 789 bp for CD36, and 981 bp for macrosialin. The amplified transcripts were analyzed by electrophoresis on 1.5% agarose gels. Immunoblotting analysis of CD36 in 3T3-L1 cells. Whole cell extracts from 3T3-L1 cells (50 lg) were separated in a 7.5% SDS–polyacrylamide gel and transferred to a nitrocellulose membrane. The membrane was incubated with 2 lg/ml of goat anti-human CD36 polyclonal antibody (L-17) for 2 h, followed by a horseradish peroxidase-conjugated secondary antibody, and visualized by enhanced chemiluminescence method.
Results Binding and endocytic uptake and degradation of OxLDL by differentiated 3T3-L1 cells
125
I-
We first examined endocytic uptake and degradation of 125 I-OxLDL using the mouse 3T3-L1 cell line. Specific levels of cell association and degradation markedly increased after differentiation of cells to adipocytes (day 5), as shown in Figs. 1A and B, respectively. The specific cell association of the adipocytes exhibited a dose-de-
Fig. 1. Endocytic uptake and degradation of 125 I-OxLDL by 3T3-L1 adipocytes. (A, B) 3T3-L1 cells of preadipocytes (day 0, open circle) and adipocytes (day 4, filled circle) were incubated with various concentrations of 125 I-OxLDL at 37 °C for 16 h. Specific amounts of cellassociated 125 I-OxLDL (A) and its degradation (B) were determined. (C) 3T3-L1 adipose cells (day 5) were incubated at 4 °C for 90 min with various concentrations of 125 I-OxLDL in the presence (open square) or absence (open circle) of excess OxLDL. Specific binding (filled circle) was determined by subtracting nonspecific binding from total binding. D: Scatchard analysis of the specific binding curve. B/F is bound/free. Values are means SD (n ¼ 3).
pendent saturation pattern with a plateau at 4 lg/mg of cell protein and apparent Kd of 2.5 lg/ml (Fig. 1A). Specific degradation of 125 I-OxLDL by adipocytes was similarly increased with a plateau at 2 lg/mg of cell protein and apparent Kd of 3.0 lg/ml (Fig. 1B). These results indicate that adipocytes have a novel function to uptake and degrade OxLDL. We next examined the cellular binding of 125 I-OxLDL to differentiated 3T3-L1 cells (day 5) at 4 °C. The total binding of 125 I-OxLDL was efficiently inhibited by excess unlabeled OxLDL. The specific binding was saturable (Fig. 1C). Scatchard analysis of the specific binding (Fig. 1D) revealed that cells have a binding site with an apparent Kd of 1.48 lg/ml and maximal surface binding of 460 ng/mg of cell protein, indicating that differentiated 3T3-L1 cells have a high affinity binding site for 125 I-OxLDL. Effect of modified LDL and ligands on endocytic uptake and degradation of 125 I-OxLDL in differentiated 3T3-L1 cells To determine the ligand specificity of a receptor recognizing OxLDL in differentiated 3T3-L1 cells, we examined the effects of LDL derivatives and other
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compounds on endocytic uptake and degradation of 125 I-OxLDL. The cell association of 125 I-OxLDL was effectively (90%) blocked by unlabeled OxLDL and AcLDL (Fig. 2A). Unmodified LDL, fucoidan and dextran sulfates, which are ligands for scavenger receptor class A (SR-A) [4,5], had negligible effect (<5%). Similarly, endocytic degradation of 125 I-OxLDL was effectively (88%) inhibited by unlabeled OxLDL (Fig. 2B). Inhibition by AcLDL was also effective (80%), but unmodified LDL, fucoidan and dextran sulfates had little effect (<13%). These results suggest that a scavenger receptor of class B could be responsible for endocytic uptake and degradation of OxLDL. Expression of OxLDL receptor in 3T3-L1 adipocytes To identify the scavenger receptor(s) expressed in 3T3-L1 adipocytes, we performed RT-PCR analysis using cDNA primers for the major mouse OxLDL receptors of macrophages, namely, SR-A, CD36, and macrosialin. As shown in Fig. 3A, mRNA for CD36 was strongly induced in 3T3-L1 adipocytes by differentia-
Fig. 3. Expression of mRNA and protein for CD36, and effects of antiCD36 antibody on endocytic uptake and degradation of 125 I-OxLDL in 3T3-L1 cells. (A) mRNA expression of scavenger receptors. mRNA levels were detected by RT-PCR. Positive controls (PC) are mRNAs from mouse J774.1 cells for SR-A and macrosialin and those from mouse heart for CD36. 3T3-L1 preadipocytes (day 0) and adipocytes (on day 2, 5) were used as samples. (B) Immunoblot of CD36 expression during adipogenic differentiation. 3T3-L1 cell proteins (50 lg) at day 0 and 2, 5, 8, 11, and 14 days of differentiation were separated on a 7.5% SDS–PAGE gel and subjected to immunoblot analysis using anti-CD36 polyclonal antibody (L-17). (C, D) Effect of anti-CD36 monoclonal antibody on endocytic uptake of 125 I-OxLDL. 3T3-L1 adipocytes (day 8) were incubated with 125 I-OxLDL (5 lg/ml) at 37 °C for 16 h in the presence of various concentrations of anti-murine CD36 monoclonal antibody (filled circle), control IgA (open circle), or 100 lg/ml of unlabeled OxLDL (filled triangle). The amounts of cellassociated 125 I-OxLDL (C) and its degradation products (D) were determined. The values for 100% association and degradation were 3900 and 510 lg/mg of cell protein, respectively.
tion. By contrast, neither SR-A nor macrosialin was detected in adipocytes and preadipocytes, although these messages were detected in macrophage-like J774 cells used as a positive control. In immunoblot analysis of 3T3-L1 cells during differentiation, CD36 protein was detected at day 5 and increased until day 8 (Fig. 3B), but decreased afterward. Anti-murine CD36 monoclonal antibody inhibits endocytic uptake of 125 I-OxLDL by 3T3-L1 adipocytes
Fig. 2. Effect of several ligands on cellular binding and endocytic uptake of 125 I-OxLDL by 3T3-L1 adipocytes. Cells were incubated at 37 °C for 16 h with 125 I-OxLDL (5 lg/ml) in the presence of unlabeled OxLDL, LDL, AcLDL, fucoidan, dextran sulfate. The amounts of cell- associated 125 I-OxLDL (A), and its degradation products (B) were determined. The values for 100% association and degradation were 3300 and 450 ng/mg of cell protein, respectively. Data represent the means of three separate experiments and error bars represent SD.
To assess the relative contribution of CD36 to uptake of 125 I-OxLDL by 3T3-L1 adipocytes, we examined the effects of anti-murine CD36 monoclonal antibody (clone 63) on these activities. As shown in Fig. 3C, the cell association of 125 I-OxLDL was inhibited in a dose-dependent manner by addition of antibody and the inhibition level reached 63% of controls with 5 lg/ml of antibody. The antibody was equally effective against the endocytic degradation of 125 I-OxLDL (Fig. 3D). Control antibody was not effective on either activity. These results indicate that CD36 plays a major role in both cell association and subsequent endocytic degradation of OxLDL in 3T3-L1 adipocytes.
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Discussion Adipose tissue provides a primary energy reservoir for the body. In addition, adipose tissue is now recognized to be a biologically active and dynamic tissue playing major endocrine and possibly immunological roles [16–21]. In this paper, we show that adipocytes both recognize and degrade OxLDL. Endocytic uptake and degradation activity of OxLDL was promoted by differentiation of preadipocytes to adipocytes and correlated with induction of CD36. CD36 in adipocytes has been generally considered to function as a long chain fatty acid transporter (FAT) [1]. CD36 functions as different receptors expressed in several cell types [4,5]: e.g., receptors for thrombospondin-1 [8] and collagen type I/IV in platelets [9], receptors for OxLDL in macrophage [6,7], and receptors for anionic phospholipids in retinal pigment epithelium [14]. However, ours is the first report to show that CD36 in adipocytes functions as a receptor for OxLDL. It is noteworthy that adipocytes can not only recognize OxLDL, but degrade it. This finding suggests that adipocytes may function as phagocytes, like macrophages. There is accumulating evidence that blood levels of OxLDL positively correlate with severity of acute coronary syndromes [27,28]. The hepatic Kupffer cell has been considered a major site for clearance of OxLDL in blood [29]. It is possible that adipocytic phagocytosis of OxLDL by CD36 is also a physiological mechanism that reduces the severity and risk of atherogenesis, since adipose tissues are abundant in the body. Alternatively, apoptosis of adipose cells may reduce tissue mass, as recent reports claim [30,31]. Little is known, however, about the removal of apoptotic cells. Interestingly, CD36 mediates phagocytosis of apoptotic cells in monocytes [11,12], dendritic cells [13], and the retinal pigment epithelium [14]. Therefore, it is intriguing to hypothesize that CD36 in adipocytes may participate in the recognition and clearance of nearby damaged, senescent, or apoptotic adipocytes. Further study is required to elucidate the physiological significance of phagocytosis in adipocytes. CD36 and SR-A expressed in macrophages are important for the pathogenesis of atherosclerosis, since evidence suggests that uptake of large amounts of OxLDL by monocytes/macrophages is a key early event in lesion development [4,5,32–35]. Adipose tissue, on the other hand, secretes various cytokines, including tumor necrosis factor-a [18], resistin [19], and plasminogen activator inhibitor-1 [20], which are associated with occurrence of insulin resistance and/or atherosclerosis. Obesity markedly increases the circulating levels of cytokines [17–20]. Therefore, it is likely that uptake of large amounts of OxLDL by adipocytes leads to over-accumulation of lipids, which favors obesity and may, in turn, provide a link to insulin resistance and atherosclerosis.
In summary, we have demonstrated that 3T3-L1-derived adipocytes have a novel function to uptake and degrade OxLDL and that CD36 plays a major role in this process. Further studies are required to determine how uptake of OxLDL through CD36 contributes physiologically and/or correlates pathophysiologically to common diseases, such as obesity, insulin resistance, and atherosclerosis.
Acknowledgments We thank Professors S. Horiuchi and A. Miyazaki for discussions and Dr. N. Ohgami for technical advice. This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to A.K. and H.N.).
References [1] N.A. Abumrad, M.R. el-Maghrabi, E.Z. Amri, E. Lopez, P.A. Grimaldi, Cloning of a rat adipocyte membrane protein implicated in binding or transport of long-chain fatty acids that is induced during preadipocyte differentiation, homology with human CD36, J. Biol. Chem. 268 (1993) 17665–176658. [2] C.T. Coburn, F.F. Knapp Jr., M. Febbraio, A.L. Beets, R.L. Silverstein, N.A. Abumrad, Defective uptake and utilization of long chain fatty acids in muscle and adipose tissues of CD36 knockout mice, J. Biol. Chem. 275 (2000) 32523–32529. [3] M. Febbraio, N.A. Abumrad, D.P. Hajjar, K. Sharma, W. Cheng, S.F. Pearce, R.L. Silverstein, A null mutation in murine CD36 reveals an important role in fatty acid and lipoprotein metabolism, J. Biol. Chem. 274 (1999) 19055–19062. [4] M. Krieger, The other side of scavenger receptors: pattern recognition for host defense, Curr. Opin. Lipidol. (1997) 275–280. [5] U.P. Steinbrecher, Receptors for oxidized low density lipoprotein, Biochim. Biophys. Acta 1436 (1999) 279–298. [6] G. Endemann, L.W. Stanton, K.S. Madden, C.M. Bryant, R.T. White, A.A. Protter, CD36 is a receptor for oxidized low density lipoprotein, J. Biol. Chem. 268 (1993) 11811–11816. [7] A.C. Nicholson, S. Frieda, A. Pearce, R.L. Silverstein, Oxidized LDL binds to CD36 on human monocyte-derived macrophages and transfected cell lines, evidence implicating the lipid moiety of the lipoprotein as the binding site, Arterioscler. Thromb. Vasc. Biol. 15 (1995) 269–275. [8] R.L. Silverstein, A.S. Asch, R.L. Nachman, Glycoprotein IV mediates thrombospondin-dependent platelet-monocyte and platelet-U937 cell adhesion, J. Clin. Invest. 84 (1989) 546–552. [9] N.N. Tandon, U. Kralisz, G.A. Jamieson, Identification of glycoprotein IV (CD36) as a primary receptor for platelet-collagen adhesion, J. Biol. Chem. 264 (1989) 7576–7583. [10] N. Ohgami, R. Nagai, M. Ikemoto, H. Arai, A. Kuniyasu, S. Horiuchi, H. Nakayama, CD 36, a member of the class B scavenger receptor family as a receptor for advanced glycation end products, J. Biol. Chem. 276 (2001) 3195–3202. [11] J. Savill, N. Hogg, Y. Ren, C. Haslett, Thrombospondin cooperates with CD36 and the vitronectin receptor in macrophage recognition of neutrophils undergoing apoptosis, J. Clin. Invest. 90 (1992) 1513–1522. [12] V.A. Fadok, M.L. Warner, D.L. Bratton, P.M. Henson, CD36 is required for phagocytosis of apoptotic cells by human macrophages that use either a phosphatidylserine receptor or the vitronectin receptor (avb3), J. Immunol. 161 (1998) 6250–6257.
A. Kuniyasu et al. / Biochemical and Biophysical Research Communications 295 (2002) 319–323 [13] M.L. Albert, S.F. Pearce, L.M. Francisco, B. Sauter, P. Roy, R.L. Silverstein, N. Bhardwaj, Immature dendritic cells phagocytose apoptotic cells via avb5 and CD36 and cross-present antigens to cytotoxic T lymphocytes, J. Exp. Med. 188 (1998) 1359–1368. [14] S.W. Ryeom, R.L. Silverstein, A. Scotto, J.R. Sparrow, Binding of anionic phospholipids to retinal pigment epithelium may be mediated by the scavenger receptor CD36, J. Biol. Chem. 271 (1996) 20536–205359. [15] N.C. Franc, P. Heitzler, R.A. Ezekowitz, K. White, Requirement for croquemort in phagocytosis of apoptotic cells in Drosophila, Science 284 (1999) 1991–1994. [16] V. Mohamed-Ali, J.H. Pinkney, S.W. Coppack, Adipose tissue as an endocrine and paracrine organ, Int. J. Obes. Relat. Metab. Disord. 22 (1998) 1145–1158. [17] J.M. Friedman, J.L. Halaas, Leptin and the regulation of body weight in mammals, Nature 395 (1998) 763–770. [18] G.S. Hotamisligil, N.S. Shargill, B.M. Spiegelman, Adipose expression of tumor necrosis factor-a: direct role in obesity-linked insulin resistance, Science 259 (1993) 87–91. [19] C.M. Steppan, S.T. Bailey, S. Bhat, E.J. Brown, R.R. Banerjee, C.M. Wright, H.R. Patel, R.S. Ahima, M.A. Lazar, The hormone resistin links obesity to diabetes, Nature 409 (2001) 307–312. [20] I. Shimomura, T. Funahashi, M. Takahashi, K. Maeda, K. Kotani, T. Nakamura, S. Yamashita, M. Miura, Y. Fukuda, K. Takemura, K. Tokunaga, Y. Matsuzawa, Enhanced expression of PAI-1 in visceral fat: possible contributor to vascular disease in obesity, Nat. Med. 2 (1996) 800–803. [21] K. Maeda, K. Okubo, I. Shimomura, T. Funahashi, Y. Matsuzawa, K. Matsubara, cDNA cloning and expression of a novel adipose specific collagen-like factor, apM1 (AdiPose Most abundant Gene transcript 1), Biochem. Biophys. Res. Commun. 221 (1996) 286–289. [22] H. Green, O. Kehinde, An established preadipose cell line and its differentiation in culture. II. Factors affecting the adipose conversion, Cell 1 (1975) 19–27. [23] R.M. Cowherd, R.E. Lyle, R.E. McGehee Jr., Molecular regulation of adipocyte differentiation, Semin. Cell Dev. Biol. 10 (1999) 3–10. [24] M. Sakai, A. Miyazaki, H. Hakamata, T. Kodama, H. Suzuki, S. Kobori, M. Shichiri, S. Horiuchi, The scavenger receptor serves as a route for internalization of lysophosphatidylcholine in oxidized low density lipoprotein-induced macrophage proliferation, J. Biol. Chem. 271 (1996) 27346–27352.
323
[25] J. Ashkenas, M. Penman, E. Vasile, S. Acton, M. Freeman, M. Krieger, Structures and high and low affinity ligand binding properties of murine type I and type II macrophage scavenger receptors, J. Lipid Res. 34 (1993) 983–1000. [26] C.L. Holness, R.P. da Silva, J. Fawcett, S. Gordon, D.L. Simmons, Macrosialin, a mouse macrophage-restricted glycoprotein is a member of the lamp/lgp family, J. Biol. Chem. 268 (1993) 9661–9666. [27] P. Holvoet, A. Mertens, P. Verhamme, K. Bogaerts, G. Beyens, R. Verhaeghe, D. Collen, E. Muls, F. Van de Werf, Circulating oxidized LDL is a useful marker for identifying patients with coronary artery disease, Arterioscler. Thromb. Vasc. Biol. 21 (2001) 844–848. [28] S. Ehara, M. Ueda, T. Naruko, K. Haze, A. Itoh, M. Otsuka, R. Komatsu, T. Matsuo, H. Itabe, T. Takano, Y. Tsukamoto, M. Yoshiyama, K. Takeuchi, J. Yoshikawa, A.E. Becker, Elevated levels of oxidized low density lipoprotein show a positive relationship with the severity of acute coronary syndromes, Circulation 103 (2001) 1955–1960. [29] T.J. Van Berkel, Y.B. De Rijke, J.K. Kruijt, Different fate in vivo of oxidatively modified low density lipoprotein and acetylated low density lipoprotein in rats. Recognition by various scavenger receptors on Kupffer and endothelial liver cells, J. Biol. Chem. 266 (1991) 2282–2289. [30] J.B. Prins, S. O’Rahilly, Regulation of adipose cell number in man, Clin. Sci. 92 (1997) 3–11. [31] A. Sorisky, R. Magun, A.M. Gagnon, Adipose cell apoptosis: death in the energy depot, Int. J. Obes. Relat. Metab. Disord. 24 (Suppl. 4) (2000) S3–S7. [32] D. Steinberg, S. Parthasarathy, T.E. Carew, J.C. Khoo, J.L. Witztum, Beyond cholesterol, modifications of low-density lipoprotein that increase its atherogenicity, N. Engl. J. Med. 320 (1989) 915–924. [33] G.M. Chisolm, D. Steinberg, The oxidative modification hypothesis of atherogenesis: an overview, Free Radic. Biol. Med. 28 (2000) 1815–1826. [34] P. Tontonoz, L. Nagy, J.G. Alvarez, V.A. Thomazy, R.M. Evans, PPARc promotes monocyte/macrophage differentiation and uptake of oxidized LDL, Cell 93 (1998) 241–252. [35] L. Nagy, P. Tontonoz, J.G. Alvarez, H. Chen, R.M. Evans, Oxidized LDL regulates macrophage gene expression through ligand activation of PPARc, Cell 93 (1998) 229–240.
Adipocytes recognize and degrade oxidized low density lipoprotein through CD36q Akihiko Kuniyasu,1 Shigeki Hayashi, and Hitoshi Nakayama* Department of Biofunctional Chemistry, Faculty of Pharmaceutical Sciences, Kumamoto University, 5-1 Ohe-Honmachi, Kumamoto 862-0973, Japan Received 28 May 2002
Abstract CD36 expressed on adipocytes is thought to function as a fatty acid transporter (FAT). Here we report that adipocytes can endocytose and lysosomally degrade OxLDL, mainly mediated by CD36. Mouse 3T3-L1 preadipocytes showed marked increase in uptake and degradation of 125 I-OxLDL during their differentiation to adipocytes. RT-PCR and immunoblot analysis indicated that expression of CD36 but not of scavenger receptor class A or macrosialin is required for the increase in uptake and degradation of 125 I-OxLDL in 3T3-L1 cells. An anti-CD36 antibody inhibited both uptake and degradation activities of 125 I-OxLDL up to 60%. These results strongly suggest that adipocytes may function as phagocytes like macrophages and that CD36 plays a novel role in adipose tissues. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: CD36; Scavenger receptor; Adipocytes; 3T3-L1 cells; Oxidized LDL; Endocytic uptake; Lysosomal degradation
Adipose tissues play an active role in a variety of physiological and pathological processes regulating energy metabolism. Triglycerides, a major energy source stored in adipose tissues, are synthesized from long chain fatty acids and glucose. Therefore, transport of the fatty acid and glucose is essential for the production of energy sources. CD36 is proposed to play a role as the fatty acid transporter (FAT) in adipocytes [1]. Marked decreases in FAT activity [2] and triglyceride synthesis [3], which are observed in CD36 null mutation mice, support this hypothesis. CD36 is a multifunctional receptor expressed in several cell types [4,5]. In macrophages, for example, CD36 functions as a scavenger receptor for oxidized low density lipoprotein (OxLDL) [6,7], while it functions as a receptor for thrombospondin-1 [8] and collagen type I/IV in platelets [9]. We recently showed that CD36 serves as a receptor for advanced glycation end products [10]. In addition, several studies show that CD36 can recognize anionic phospholipids to participate in phagocytotic q Abbreviations: FAT, fatty acid transporter; LDL, low density lipoprotein; OxLDL, oxidized LDL; SR-A, scavenger receptor class A. * Corresponding author. Fax: +81-96-372-7182. E-mail address: [email protected] (H. Nakayama). 1 Also corresponding author.
clearance by human monocytes [11,12] and dendritic cells [13], rat retinal pigment endothelium [14], and Drosophila hemocytes [15]. Recent observations indicate that adipose tissues are biologically active and dynamic and play important endocrine and possibly immunological roles in addition to the traditional function as energy storage depot [16–21]. Therefore, it is important to determine whether adipocytes have additional unknown functions. The 3T3-L1 cell line differentiates under the controlled conditions of cell culture from fibroblasts, or preadipocytes, to cells with the morphological and biochemical properties of adipocytes [22]. 3T3-L1 adipocytes are comparable with native adipocytes as they have the ability to accumulate lipid, respond to insulin, and secrete leptin [23]. In this study we determined whether adipocytes endocytosed and lysosomally degraded OxLDL. Here we demonstrate that 3T3-L1 adipocytes can recognize and degrade OxLDL, mainly mediated by CD36.
Materials and methods Materials. Na 125 I (3.7 GBq/ml) was purchased from Amersham Pharmacia Biotech (Buckinghamshire, UK). Goat anti-human CD36
0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 6 - 2 9 1 X ( 0 2 ) 0 0 6 6 6 - 6
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polyclonal antibody (L-17) and mouse anti-murine CD36 monoclonal antibody (clone 63) were purchased from Santa Cruz Biotech (Santa Cruz, CA, USA) and Cascade Bioscience (Winchester, MA, USA), respectively. Mouse IgA, kappa (TEPC 15) and porcine insulin were from Sigma (St. Louis, MO, USA). All other chemicals were of the analytical grade commercially available. Cell culture. Mouse 3T3-L1 cells (Health Science Research Resources Bank, Osaka, Japan) were plated and grown in a basal medium (Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, 20 U/ml penicillin, and 20 lg/ml streptomycin). Cells were then differentiated by changing to a medium composed of the basal medium plus 10 lg/ml insulin, 0.25 lM dexamethasone, and 0.5 mM isobutylmethylxanthine. After 48 h, the medium was replaced with basal medium containing 5 lg/ml insulin, and cells were maintained in this medium until use. Binding, cell association, and degradation of lipoproteins. Human low density lipoprotein (LDL, d ¼ 1:019–1:063 g/ml), acetylated LDL (AcLDL), OxLDL, and 125 I-labeled OxLDL (400–450 cpm/ng protein) were prepared essentially as described [24]. Binding, cell-association and degradation of 125 I-OxLDL were performed as described previously [10]. For the binding assay, cells were incubated with 1.0– 10 lg=ml 125 I-OxLDL at 4 °C for 1.5 h. Nonspecific binding was measured in the presence of a 20-fold excess of OxLDL and subtracted from the data. For cell-association and degradation assays, cells were incubated with 5 lg=ml 125 I-OxLDL at 37 °C for 16 h. For inhibition assays, cells were incubated with or without various compounds (100 lg/ml) or mouse antibodies (anti-CD36 antibody clone 63 or control IgA; 0.1-10 lg/ml). Results are represented as the means SD (n ¼ 3). RT-PCR amplification. Total RNAs were isolated from 3T3-L1 cells, J774 cells and mouse hearts using ISOGEN (Nippon Gene, Toyama, Japan) according to manufacturer’s recommendations. RNAs were reverse-transcribed, and the cDNA obtained was used for PCR amplification to estimate the expression of SR-A, CD36, and macrosialin. The primer sequences used were as follows. Scavenger receptor class A (SR-A) [25]: sense primer, 50 -ATGACAGAGAA TCAGAGGC-30 ; antisense primer, 50 -CATGTTCCTGGACTGAC GA-30 . CD36 [6]: sense primer, 50 -AGGTCCTTACACATACAGA GTTCG-30 ; antisense primer, 50 -GGACTTGCATGTAGGAAATGT GGA-30 . Macrosialin [26]: sense primer, 50 -ATGCGGCTCCC TGTGTGTC-30 ; antisense primer, 50 -TCAGAGGGGCTGGTAGG TTG-30 . The sizes of PCR products were 508 bp for SR-AI/II, 789 bp for CD36, and 981 bp for macrosialin. The amplified transcripts were analyzed by electrophoresis on 1.5% agarose gels. Immunoblotting analysis of CD36 in 3T3-L1 cells. Whole cell extracts from 3T3-L1 cells (50 lg) were separated in a 7.5% SDS–polyacrylamide gel and transferred to a nitrocellulose membrane. The membrane was incubated with 2 lg/ml of goat anti-human CD36 polyclonal antibody (L-17) for 2 h, followed by a horseradish peroxidase-conjugated secondary antibody, and visualized by enhanced chemiluminescence method.
Results Binding and endocytic uptake and degradation of OxLDL by differentiated 3T3-L1 cells
125
I-
We first examined endocytic uptake and degradation of 125 I-OxLDL using the mouse 3T3-L1 cell line. Specific levels of cell association and degradation markedly increased after differentiation of cells to adipocytes (day 5), as shown in Figs. 1A and B, respectively. The specific cell association of the adipocytes exhibited a dose-de-
Fig. 1. Endocytic uptake and degradation of 125 I-OxLDL by 3T3-L1 adipocytes. (A, B) 3T3-L1 cells of preadipocytes (day 0, open circle) and adipocytes (day 4, filled circle) were incubated with various concentrations of 125 I-OxLDL at 37 °C for 16 h. Specific amounts of cellassociated 125 I-OxLDL (A) and its degradation (B) were determined. (C) 3T3-L1 adipose cells (day 5) were incubated at 4 °C for 90 min with various concentrations of 125 I-OxLDL in the presence (open square) or absence (open circle) of excess OxLDL. Specific binding (filled circle) was determined by subtracting nonspecific binding from total binding. D: Scatchard analysis of the specific binding curve. B/F is bound/free. Values are means SD (n ¼ 3).
pendent saturation pattern with a plateau at 4 lg/mg of cell protein and apparent Kd of 2.5 lg/ml (Fig. 1A). Specific degradation of 125 I-OxLDL by adipocytes was similarly increased with a plateau at 2 lg/mg of cell protein and apparent Kd of 3.0 lg/ml (Fig. 1B). These results indicate that adipocytes have a novel function to uptake and degrade OxLDL. We next examined the cellular binding of 125 I-OxLDL to differentiated 3T3-L1 cells (day 5) at 4 °C. The total binding of 125 I-OxLDL was efficiently inhibited by excess unlabeled OxLDL. The specific binding was saturable (Fig. 1C). Scatchard analysis of the specific binding (Fig. 1D) revealed that cells have a binding site with an apparent Kd of 1.48 lg/ml and maximal surface binding of 460 ng/mg of cell protein, indicating that differentiated 3T3-L1 cells have a high affinity binding site for 125 I-OxLDL. Effect of modified LDL and ligands on endocytic uptake and degradation of 125 I-OxLDL in differentiated 3T3-L1 cells To determine the ligand specificity of a receptor recognizing OxLDL in differentiated 3T3-L1 cells, we examined the effects of LDL derivatives and other
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compounds on endocytic uptake and degradation of 125 I-OxLDL. The cell association of 125 I-OxLDL was effectively (90%) blocked by unlabeled OxLDL and AcLDL (Fig. 2A). Unmodified LDL, fucoidan and dextran sulfates, which are ligands for scavenger receptor class A (SR-A) [4,5], had negligible effect (<5%). Similarly, endocytic degradation of 125 I-OxLDL was effectively (88%) inhibited by unlabeled OxLDL (Fig. 2B). Inhibition by AcLDL was also effective (80%), but unmodified LDL, fucoidan and dextran sulfates had little effect (<13%). These results suggest that a scavenger receptor of class B could be responsible for endocytic uptake and degradation of OxLDL. Expression of OxLDL receptor in 3T3-L1 adipocytes To identify the scavenger receptor(s) expressed in 3T3-L1 adipocytes, we performed RT-PCR analysis using cDNA primers for the major mouse OxLDL receptors of macrophages, namely, SR-A, CD36, and macrosialin. As shown in Fig. 3A, mRNA for CD36 was strongly induced in 3T3-L1 adipocytes by differentia-
Fig. 3. Expression of mRNA and protein for CD36, and effects of antiCD36 antibody on endocytic uptake and degradation of 125 I-OxLDL in 3T3-L1 cells. (A) mRNA expression of scavenger receptors. mRNA levels were detected by RT-PCR. Positive controls (PC) are mRNAs from mouse J774.1 cells for SR-A and macrosialin and those from mouse heart for CD36. 3T3-L1 preadipocytes (day 0) and adipocytes (on day 2, 5) were used as samples. (B) Immunoblot of CD36 expression during adipogenic differentiation. 3T3-L1 cell proteins (50 lg) at day 0 and 2, 5, 8, 11, and 14 days of differentiation were separated on a 7.5% SDS–PAGE gel and subjected to immunoblot analysis using anti-CD36 polyclonal antibody (L-17). (C, D) Effect of anti-CD36 monoclonal antibody on endocytic uptake of 125 I-OxLDL. 3T3-L1 adipocytes (day 8) were incubated with 125 I-OxLDL (5 lg/ml) at 37 °C for 16 h in the presence of various concentrations of anti-murine CD36 monoclonal antibody (filled circle), control IgA (open circle), or 100 lg/ml of unlabeled OxLDL (filled triangle). The amounts of cellassociated 125 I-OxLDL (C) and its degradation products (D) were determined. The values for 100% association and degradation were 3900 and 510 lg/mg of cell protein, respectively.
tion. By contrast, neither SR-A nor macrosialin was detected in adipocytes and preadipocytes, although these messages were detected in macrophage-like J774 cells used as a positive control. In immunoblot analysis of 3T3-L1 cells during differentiation, CD36 protein was detected at day 5 and increased until day 8 (Fig. 3B), but decreased afterward. Anti-murine CD36 monoclonal antibody inhibits endocytic uptake of 125 I-OxLDL by 3T3-L1 adipocytes
Fig. 2. Effect of several ligands on cellular binding and endocytic uptake of 125 I-OxLDL by 3T3-L1 adipocytes. Cells were incubated at 37 °C for 16 h with 125 I-OxLDL (5 lg/ml) in the presence of unlabeled OxLDL, LDL, AcLDL, fucoidan, dextran sulfate. The amounts of cell- associated 125 I-OxLDL (A), and its degradation products (B) were determined. The values for 100% association and degradation were 3300 and 450 ng/mg of cell protein, respectively. Data represent the means of three separate experiments and error bars represent SD.
To assess the relative contribution of CD36 to uptake of 125 I-OxLDL by 3T3-L1 adipocytes, we examined the effects of anti-murine CD36 monoclonal antibody (clone 63) on these activities. As shown in Fig. 3C, the cell association of 125 I-OxLDL was inhibited in a dose-dependent manner by addition of antibody and the inhibition level reached 63% of controls with 5 lg/ml of antibody. The antibody was equally effective against the endocytic degradation of 125 I-OxLDL (Fig. 3D). Control antibody was not effective on either activity. These results indicate that CD36 plays a major role in both cell association and subsequent endocytic degradation of OxLDL in 3T3-L1 adipocytes.
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Discussion Adipose tissue provides a primary energy reservoir for the body. In addition, adipose tissue is now recognized to be a biologically active and dynamic tissue playing major endocrine and possibly immunological roles [16–21]. In this paper, we show that adipocytes both recognize and degrade OxLDL. Endocytic uptake and degradation activity of OxLDL was promoted by differentiation of preadipocytes to adipocytes and correlated with induction of CD36. CD36 in adipocytes has been generally considered to function as a long chain fatty acid transporter (FAT) [1]. CD36 functions as different receptors expressed in several cell types [4,5]: e.g., receptors for thrombospondin-1 [8] and collagen type I/IV in platelets [9], receptors for OxLDL in macrophage [6,7], and receptors for anionic phospholipids in retinal pigment epithelium [14]. However, ours is the first report to show that CD36 in adipocytes functions as a receptor for OxLDL. It is noteworthy that adipocytes can not only recognize OxLDL, but degrade it. This finding suggests that adipocytes may function as phagocytes, like macrophages. There is accumulating evidence that blood levels of OxLDL positively correlate with severity of acute coronary syndromes [27,28]. The hepatic Kupffer cell has been considered a major site for clearance of OxLDL in blood [29]. It is possible that adipocytic phagocytosis of OxLDL by CD36 is also a physiological mechanism that reduces the severity and risk of atherogenesis, since adipose tissues are abundant in the body. Alternatively, apoptosis of adipose cells may reduce tissue mass, as recent reports claim [30,31]. Little is known, however, about the removal of apoptotic cells. Interestingly, CD36 mediates phagocytosis of apoptotic cells in monocytes [11,12], dendritic cells [13], and the retinal pigment epithelium [14]. Therefore, it is intriguing to hypothesize that CD36 in adipocytes may participate in the recognition and clearance of nearby damaged, senescent, or apoptotic adipocytes. Further study is required to elucidate the physiological significance of phagocytosis in adipocytes. CD36 and SR-A expressed in macrophages are important for the pathogenesis of atherosclerosis, since evidence suggests that uptake of large amounts of OxLDL by monocytes/macrophages is a key early event in lesion development [4,5,32–35]. Adipose tissue, on the other hand, secretes various cytokines, including tumor necrosis factor-a [18], resistin [19], and plasminogen activator inhibitor-1 [20], which are associated with occurrence of insulin resistance and/or atherosclerosis. Obesity markedly increases the circulating levels of cytokines [17–20]. Therefore, it is likely that uptake of large amounts of OxLDL by adipocytes leads to over-accumulation of lipids, which favors obesity and may, in turn, provide a link to insulin resistance and atherosclerosis.
In summary, we have demonstrated that 3T3-L1-derived adipocytes have a novel function to uptake and degrade OxLDL and that CD36 plays a major role in this process. Further studies are required to determine how uptake of OxLDL through CD36 contributes physiologically and/or correlates pathophysiologically to common diseases, such as obesity, insulin resistance, and atherosclerosis.
Acknowledgments We thank Professors S. Horiuchi and A. Miyazaki for discussions and Dr. N. Ohgami for technical advice. This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to A.K. and H.N.).
References [1] N.A. Abumrad, M.R. el-Maghrabi, E.Z. Amri, E. Lopez, P.A. Grimaldi, Cloning of a rat adipocyte membrane protein implicated in binding or transport of long-chain fatty acids that is induced during preadipocyte differentiation, homology with human CD36, J. Biol. Chem. 268 (1993) 17665–176658. [2] C.T. Coburn, F.F. Knapp Jr., M. Febbraio, A.L. Beets, R.L. Silverstein, N.A. Abumrad, Defective uptake and utilization of long chain fatty acids in muscle and adipose tissues of CD36 knockout mice, J. Biol. Chem. 275 (2000) 32523–32529. [3] M. Febbraio, N.A. Abumrad, D.P. Hajjar, K. Sharma, W. Cheng, S.F. Pearce, R.L. Silverstein, A null mutation in murine CD36 reveals an important role in fatty acid and lipoprotein metabolism, J. Biol. Chem. 274 (1999) 19055–19062. [4] M. Krieger, The other side of scavenger receptors: pattern recognition for host defense, Curr. Opin. Lipidol. (1997) 275–280. [5] U.P. Steinbrecher, Receptors for oxidized low density lipoprotein, Biochim. Biophys. Acta 1436 (1999) 279–298. [6] G. Endemann, L.W. Stanton, K.S. Madden, C.M. Bryant, R.T. White, A.A. Protter, CD36 is a receptor for oxidized low density lipoprotein, J. Biol. Chem. 268 (1993) 11811–11816. [7] A.C. Nicholson, S. Frieda, A. Pearce, R.L. Silverstein, Oxidized LDL binds to CD36 on human monocyte-derived macrophages and transfected cell lines, evidence implicating the lipid moiety of the lipoprotein as the binding site, Arterioscler. Thromb. Vasc. Biol. 15 (1995) 269–275. [8] R.L. Silverstein, A.S. Asch, R.L. Nachman, Glycoprotein IV mediates thrombospondin-dependent platelet-monocyte and platelet-U937 cell adhesion, J. Clin. Invest. 84 (1989) 546–552. [9] N.N. Tandon, U. Kralisz, G.A. Jamieson, Identification of glycoprotein IV (CD36) as a primary receptor for platelet-collagen adhesion, J. Biol. Chem. 264 (1989) 7576–7583. [10] N. Ohgami, R. Nagai, M. Ikemoto, H. Arai, A. Kuniyasu, S. Horiuchi, H. Nakayama, CD 36, a member of the class B scavenger receptor family as a receptor for advanced glycation end products, J. Biol. Chem. 276 (2001) 3195–3202. [11] J. Savill, N. Hogg, Y. Ren, C. Haslett, Thrombospondin cooperates with CD36 and the vitronectin receptor in macrophage recognition of neutrophils undergoing apoptosis, J. Clin. Invest. 90 (1992) 1513–1522. [12] V.A. Fadok, M.L. Warner, D.L. Bratton, P.M. Henson, CD36 is required for phagocytosis of apoptotic cells by human macrophages that use either a phosphatidylserine receptor or the vitronectin receptor (avb3), J. Immunol. 161 (1998) 6250–6257.
A. Kuniyasu et al. / Biochemical and Biophysical Research Communications 295 (2002) 319–323 [13] M.L. Albert, S.F. Pearce, L.M. Francisco, B. Sauter, P. Roy, R.L. Silverstein, N. Bhardwaj, Immature dendritic cells phagocytose apoptotic cells via avb5 and CD36 and cross-present antigens to cytotoxic T lymphocytes, J. Exp. Med. 188 (1998) 1359–1368. [14] S.W. Ryeom, R.L. Silverstein, A. Scotto, J.R. Sparrow, Binding of anionic phospholipids to retinal pigment epithelium may be mediated by the scavenger receptor CD36, J. Biol. Chem. 271 (1996) 20536–205359. [15] N.C. Franc, P. Heitzler, R.A. Ezekowitz, K. White, Requirement for croquemort in phagocytosis of apoptotic cells in Drosophila, Science 284 (1999) 1991–1994. [16] V. Mohamed-Ali, J.H. Pinkney, S.W. Coppack, Adipose tissue as an endocrine and paracrine organ, Int. J. Obes. Relat. Metab. Disord. 22 (1998) 1145–1158. [17] J.M. Friedman, J.L. Halaas, Leptin and the regulation of body weight in mammals, Nature 395 (1998) 763–770. [18] G.S. Hotamisligil, N.S. Shargill, B.M. Spiegelman, Adipose expression of tumor necrosis factor-a: direct role in obesity-linked insulin resistance, Science 259 (1993) 87–91. [19] C.M. Steppan, S.T. Bailey, S. Bhat, E.J. Brown, R.R. Banerjee, C.M. Wright, H.R. Patel, R.S. Ahima, M.A. Lazar, The hormone resistin links obesity to diabetes, Nature 409 (2001) 307–312. [20] I. Shimomura, T. Funahashi, M. Takahashi, K. Maeda, K. Kotani, T. Nakamura, S. Yamashita, M. Miura, Y. Fukuda, K. Takemura, K. Tokunaga, Y. Matsuzawa, Enhanced expression of PAI-1 in visceral fat: possible contributor to vascular disease in obesity, Nat. Med. 2 (1996) 800–803. [21] K. Maeda, K. Okubo, I. Shimomura, T. Funahashi, Y. Matsuzawa, K. Matsubara, cDNA cloning and expression of a novel adipose specific collagen-like factor, apM1 (AdiPose Most abundant Gene transcript 1), Biochem. Biophys. Res. Commun. 221 (1996) 286–289. [22] H. Green, O. Kehinde, An established preadipose cell line and its differentiation in culture. II. Factors affecting the adipose conversion, Cell 1 (1975) 19–27. [23] R.M. Cowherd, R.E. Lyle, R.E. McGehee Jr., Molecular regulation of adipocyte differentiation, Semin. Cell Dev. Biol. 10 (1999) 3–10. [24] M. Sakai, A. Miyazaki, H. Hakamata, T. Kodama, H. Suzuki, S. Kobori, M. Shichiri, S. Horiuchi, The scavenger receptor serves as a route for internalization of lysophosphatidylcholine in oxidized low density lipoprotein-induced macrophage proliferation, J. Biol. Chem. 271 (1996) 27346–27352.
323
[25] J. Ashkenas, M. Penman, E. Vasile, S. Acton, M. Freeman, M. Krieger, Structures and high and low affinity ligand binding properties of murine type I and type II macrophage scavenger receptors, J. Lipid Res. 34 (1993) 983–1000. [26] C.L. Holness, R.P. da Silva, J. Fawcett, S. Gordon, D.L. Simmons, Macrosialin, a mouse macrophage-restricted glycoprotein is a member of the lamp/lgp family, J. Biol. Chem. 268 (1993) 9661–9666. [27] P. Holvoet, A. Mertens, P. Verhamme, K. Bogaerts, G. Beyens, R. Verhaeghe, D. Collen, E. Muls, F. Van de Werf, Circulating oxidized LDL is a useful marker for identifying patients with coronary artery disease, Arterioscler. Thromb. Vasc. Biol. 21 (2001) 844–848. [28] S. Ehara, M. Ueda, T. Naruko, K. Haze, A. Itoh, M. Otsuka, R. Komatsu, T. Matsuo, H. Itabe, T. Takano, Y. Tsukamoto, M. Yoshiyama, K. Takeuchi, J. Yoshikawa, A.E. Becker, Elevated levels of oxidized low density lipoprotein show a positive relationship with the severity of acute coronary syndromes, Circulation 103 (2001) 1955–1960. [29] T.J. Van Berkel, Y.B. De Rijke, J.K. Kruijt, Different fate in vivo of oxidatively modified low density lipoprotein and acetylated low density lipoprotein in rats. Recognition by various scavenger receptors on Kupffer and endothelial liver cells, J. Biol. Chem. 266 (1991) 2282–2289. [30] J.B. Prins, S. O’Rahilly, Regulation of adipose cell number in man, Clin. Sci. 92 (1997) 3–11. [31] A. Sorisky, R. Magun, A.M. Gagnon, Adipose cell apoptosis: death in the energy depot, Int. J. Obes. Relat. Metab. Disord. 24 (Suppl. 4) (2000) S3–S7. [32] D. Steinberg, S. Parthasarathy, T.E. Carew, J.C. Khoo, J.L. Witztum, Beyond cholesterol, modifications of low-density lipoprotein that increase its atherogenicity, N. Engl. J. Med. 320 (1989) 915–924. [33] G.M. Chisolm, D. Steinberg, The oxidative modification hypothesis of atherogenesis: an overview, Free Radic. Biol. Med. 28 (2000) 1815–1826. [34] P. Tontonoz, L. Nagy, J.G. Alvarez, V.A. Thomazy, R.M. Evans, PPARc promotes monocyte/macrophage differentiation and uptake of oxidized LDL, Cell 93 (1998) 241–252. [35] L. Nagy, P. Tontonoz, J.G. Alvarez, H. Chen, R.M. Evans, Oxidized LDL regulates macrophage gene expression through ligand activation of PPARc, Cell 93 (1998) 229–240.