Title: A Null Mutation in Murine CD36 Reveals an Important Role in Fatty Acid and Lipoprotein Metabolism
Abstract: A null mutation in the scavenger receptor gene CD36 was created in mice by targeted homologous recombination. These mice produced no detectable CD36 protein, were viable, and bred normally. A significant decrease in binding and uptake of oxidized low density lipoprotein was observed in peritoneal macrophages of null mice as compared with those from control mice. CD36 null animals had a significant increase in fasting levels of cholesterol, nonesterified free fatty acids, and triacylglycerol. The increase in cholesterol was mainly within the high density lipoprotein fraction, while the increase in triacylglycerol was within the very low density lipoprotein fraction. Null animals had lower fasting serum glucose levels when compared with wild type controls. Uptake of3H-labeled oleate was significantly reduced in adipocytes from null mice. However, the decrease was limited to the low ratios of fatty acid:bovine serum albumin, suggesting that CD36 was necessary for the high affinity component of the uptake process. The data provide evidence for a functional role for CD36 in lipoprotein/fatty acid metabolism that was previously underappreciated. A null mutation in the scavenger receptor gene CD36 was created in mice by targeted homologous recombination. These mice produced no detectable CD36 protein, were viable, and bred normally. A significant decrease in binding and uptake of oxidized low density lipoprotein was observed in peritoneal macrophages of null mice as compared with those from control mice. CD36 null animals had a significant increase in fasting levels of cholesterol, nonesterified free fatty acids, and triacylglycerol. The increase in cholesterol was mainly within the high density lipoprotein fraction, while the increase in triacylglycerol was within the very low density lipoprotein fraction. 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These animals had an increase in fasting serum/plasma cholesterol, an increase in nonesterified free fatty acid and triacylglycerol levels, and a decrease in fasting plasma glucose, revealing a more significant role for CD36 in lipoprotein and fatty acid metabolism than previously appreciated. Cell culture reagents including leukemia-inhibitory factor (Esgro) were obtained from Life Technologies, Inc.; fetal bovine serum was from Gemini Bio-Sciences, Inc.; total cholesterol, triacylglycerol, albumin, and glucose quantitative assay kits and calibrators and miscellaneous chemicals were from Sigma; standards and a quantitative assay kit for serum nonesterified fatty acids were from Wako; FITC-conjugated goat anti-rabbit IgG was from Kirkegaard and Perry Laboratories; rabbit anti-rat CD36 IgG was prepared as described by Ryeom et al.(74Ryeom S.W. Sparrow J.R. Silverstein R.L. J. 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Murine CD36 genomic sequences were obtained from a mouse strain 129/Sv genomic phage library (Stratagene) using a 551-base pair cDNA probe beginning at cDNA base pair 2. A phage containing a 13-kb insert was identified, and by restriction enzyme analysis, Southern blotting, and dideoxy sequencing (Sequenase, Amersham Pharmacia Biotech), it was proved to be genomic CD36 (data not shown). Construction of the CD36 targeting vector was carried out in the plasmid pPNT, which contains neo r and thymidine kinase for selection of homologous recombinants (75Tybulecwicz V.L.J. Crawford C.E. Jackson P.K. Bronson R.T. Mulligan R.C. Cell. 1991; 65: 1153-1163Abstract Full Text PDF PubMed Scopus (1159) Google Scholar). Two fragments flanking exon 3 of murine CD36, which contained the translation start site, were subcloned into pPNT in a transcription orientation opposite to the neo r cassette (Fig. 1 A). Exon 3, which encodes the first 40 amino acids of CD36 and contains the putative N-terminal transmembrane domain, was deleted entirely in the homologous recombined allele. The CJ7 ES cell line was cultured on growth-arrestedneo r embryonic fibroblasts as described (76Robertson E.J. Teratocarcinoma and Embryonic Stem Cells: A Practical Approach. IRL Press, Oxford1987Google Scholar). For electroporation, ES cells were resuspended at a concentration of 2.5 × 107/ml in phosphate-buffered saline (PBS) lacking calcium and magnesium. 0.9-ml aliquots were mixed with 20 μg of the linearized targeting vector and pulsed at 250 V, 500 microfarads (Bio-Rad Gene Pulser). The cells were then diluted in culture medium and plated at 5 × 106 cells/100-mm dish. After 24 h, G418 (Life Technologies) selection (250 μg/ml) was begun, and selection with ganciclovir (2 μm; Cytovene from Syntex Laboratories, Inc.) began on day 3. Resistant colonies were picked on days 8–10, dissociated with trypsin, and divided into two wells of a 96-well plate. Upon confluence, one well of ES cells was frozen and the other was expanded for DNA isolation and Southern blot analysis. Genomic DNA was prepared from ES cells and tail biopsies by digestion at 55 °C with proteinase K. Approximately 10 μg of genomic DNA were digested with the appropriate restriction endonuclease, electrophoresed through a 1% agarose gel, transferred to ZetaProbe GT (Bio-Rad), and hybridized with the random primed32P-radiolabeled probe indicated in Fig. 1. The probe/enzyme strategy was such that a different size restriction fragment would result from the endogenous locus, integration of the plasmid nonspecifically, and homologous recombination. A second probe/restriction enzyme strategy and probes to both the thymidine kinase and neo r genes were used to ensure that homologous recombination and not random integration of the targeting vector had occurred (data not shown). Primers specific to exon 3 were designed, and these failed to give rise to product from genomic DNA of null mice in polymerase chain reactions while yielding a fragment of the correct size in wild type animals (data not shown). Two separate ES cell clones were injected into C57BL/6 blastocysts as described (76Robertson E.J. Teratocarcinoma and Embryonic Stem Cells: A Practical Approach. IRL Press, Oxford1987Google Scholar) and resulted in chimeras with 75–99% agouti coats. Male chimeras were bred with C57BL/6 females, and agouti offspring were screened for the presence of the mutated CD36 gene by Southern blot hybridization. Offspring heterozygous for the mutation were interbred, and mice homozygous for the CD36 disrupted allele were identified by Southern blot hybridization. Tissues were excised from euthanized mice after perfusion with PBS and resuspended in radioimmune precipitation buffer (150 mm NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 50 mm Tris, pH 7.4) containing protease inhibitors (1 mmphenylmethylsulfonyl fluoride, 0.5 μg/ml leupeptin, 1 μg/ml pepstatin, and 2 μg/ml aprotinin). The tissues were Dounce-homogenized, insoluble material was removed by centrifugation, and the extracts were stored at −20 °C. Protein concentration was determined using the BCA reagent (Pierce). Immunoprecipitation was carried out using a polyclonal rabbit anti-rat CD36 antiserum (74Ryeom S.W. Sparrow J.R. Silverstein R.L. J. Cell Sci. 1996; 109: 387-395Crossref PubMed Google Scholar), followed by incubation with protein A-Sepharose. After immunoprecipitation for 2 h at 4 °C, beads were thoroughly washed, and bound proteins were analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting with the same antiserum. Proteins were visualized with an ECL detection system (Bio-Rad). Human and mouse platelets served as positive controls. To demonstrate specificity, an identical blot was incubated with normal rabbit serum. LDL (density 1.019–1.063 g/ml) was prepared from human plasma and isolated by sequential ultracentrifugation, extensively dialyzed against HEPES-buffered saline containing 0.3 mm EDTA, sterilized by filtration (0.22 μm), and stored under nitrogen gas at 4 °C. Protein concentration was determined using the BCA reagent. LDL was iodinated by the method of Goldstein et al. (77Goldstein J.L. Basu S. Brown M.S. Methods Enzymol. 1983; 98: 241-260Crossref PubMed Scopus (1284) Google Scholar), using carrier-free 125I-NaCl. LDL and 125I-LDL were oxidized by dialyzing against 5 μmol/liter CuSO4 in PBS at 37 °C for 16 h. The purity and charge of the lipoproteins were evaluated by examining electrophoretic mobility in an agarose gel. The degree of oxidation was determined by measuring the amount of thiobarbituric acid-reactive substances. LDL had thiobarbituric acid-reactive substances of <1 nmol/mg. Oxidized LDL had thiobarbituric acid-reactive substances of >10 and <30 nmol/mg. The specific activity of the 125I-oxidized LDL was approximately 0.05–0.1 μCi/μg. Resident peritoneal macrophages and thioglycolate-elicited macrophages (4 days after intraperitoneal injection of 2–4 ml of 4% sterile thioglycolate solution) were collected by lavage into ice-cold PBS and cultured in Dulbecco's modified Eagle's medium containing 5% heat-inactivated fetal bovine serum, 1% l-glutamine, and 1% penicillin/streptomycin for 1–2 days. Blood was collected into heparinized syringes by heart puncture. Buffy coat cells were collected after centrifugation and in