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 of 3H-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.
Highlights
A null mutation in the scavenger receptor gene CD36 was created in mice by targeted homologous recombination
Monocytes/macrophages isolated from a null mouse carrying a mutation in the class A receptors showed partial loss in the ability to bind and internalize oxidized low density lipoprotein (LDL) (ϳ30%) [36], and a lack of murine SR-AI/II receptors in the context of an atherogenic environment was partially protective against the formation of atherogenic lesions, decreasing lesion size by 40% [15]
Materials—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 [74]; FITC-conjugated rat anti-mouse SR-A antibody (2F8) was from Serotec, Ltd.; carrier-free 125I-NaCl was from Amersham Pharmacia Biotech; [␣-32P]dCTP, 14C-labeled palmitate, and 3H-labeled oleate were from NEN Life Science Products; the targeting vector, pPNT, was a gift from H.-C
Summary
Materials—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 [74]; FITC-conjugated rat anti-mouse SR-A antibody (2F8) was from Serotec, Ltd.; carrier-free 125I-NaCl was from Amersham Pharmacia Biotech; [␣-32P]dCTP, 14C-labeled palmitate, and 3H-labeled oleate were from NEN Life Science Products; the targeting vector, pPNT, was a gift from H.-C. Long Chain Fatty Acid Transport and Lipid Incorporation into Adipocytes—Adipocytes were isolated from epididymal fat pads of nonfasted (normal chow) age- and sex-matched 6 –16-week-old mice by collagenase (type 1) digestion (1 mg/ml, Worthington) in Krebs-Ringer medium (pH 7.4) lacking phosphate, buffered with HEPES (KRH) and containing 2% BSA (Fraction V, fatty acid-free), 2 mM glucose [55, 82,83,84], and 200 nM adenosine to inhibit lipolysis [85]. DNA was measured using aliquots of cell suspension identical to those used for the transport assays, and the DNA content/packed cell volume was obtained This was found to be similar for suspensions from wild type and null mice, indicating that cell size and number were not different. Cell-associated radioactivity was determined in quadruplicate, and the experiment was repeated twice
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