FAT/CD36 is a membrane scavenger receptor that facilitates long chain fatty acid uptake by muscle. Acute increases in membrane CD36 and fatty acid uptake have been reported in response to insulin and contraction. In this study we have explored protein ubiquitination as one potential mechanism for the regulation of CD36 level. CD36 expressed in Chinese hamster ovary (CHO) or HEK 293 cells was found to be polyubiquitinated via a process involving both lysines 48 and 63 of ubiquitin. Using CHO cells expressing the insulin receptor (CHO/hIR) and CD36, it is shown that addition of insulin (100 nM, 10 and 30 min) significantly reduced CD36 ubiquitination. In contrast, ubiquitination was strongly enhanced by fatty acids (200 M palmitate or oleate, 2 h). Similarly, endogenous CD36 in C2C12 myotubes was ubiquitinated, and this was enhanced by oleic acid treatment, which also reduced total CD36 protein in cell lysates. Insulin reduced CD36 ubiquitination, increased CD36 protein, and inhibited the opposite effects of fatty acids on both parameters. These changes were paralleled by changes in fatty acid uptake, which could be blocked by the CD36 inhibitor sulfosuccinimidyl oleate. Mutation of the two lysine residues in the carboxyl-terminal tail of CD36 markedly attenuated ubiquitination of the protein expressed in CHO cells and was associated with increased CD36 level and enhanced oleate uptake and incorporation into triglycerides. In conclusion, fatty acids and insulin induce opposite alterations in CD36 ubiquitination, modulating CD36 level and fatty acid uptake. Altered CD36 turnover may contribute to abnormal fatty acid uptake in the insulin-resistant muscle.Long chain fatty acids (FA) 3 are the predominant fuel used by oxidative muscle and heart. FA uptake into muscle was shown to involve, in addition to passive diffusion, proteinfacilitated transfer with a major role for the FA translocase FAT/CD36 (1-3). CD36 was identified as a FA receptor based on its binding to inhibitors of FA uptake in isolated adipocytes. The most specific of these inhibitors is membrane-impermeable sulfosuccinimidyl oleic acid (SSO), which forms a covalent bond with free amino groups on the protein (4). Transgenic mice that overexpress CD36 in heart and muscle tissues exhibit increased FA utilization and glucose sparing (5). In contrast, deficiency in CD36 is associated in mice (3, 6) and humans (7) with a defect in FA uptake that is most pronounced in the heart and that is compensated for by increased glucose utilization.Muscle CD36 content is highly regulated both at chronic and acute levels (8). Up-regulation of protein levels has been shown to associate with fasting (9) and insulin resistance in mice (10) and humans (8). Chronic leptin down-regulates the protein (11), whereas resistin (12) has the opposite effect. Acute up-regulation of muscle CD36 has been documented in response to insulin and to contraction and has been postulated to involve recruitment of the protein from intracellular stores to the plasma membrane (2). This recruitm...

Abstract-Mice deficient in both macrophage-colony-stimulating factor (M-CSF, op) and apolipoprotein E (apoE) have elevated cholesterol levels but are protected from atherosclerosis. To assess the contribution of macrophage (M) phenotypic heterogeneity and scavenger receptor (SR-A) expression to this seeming paradox, we characterized the M phenotype by immunohistochemistry in these animals. Lesion size was determined in animals fed a chow or Western-type diet, and lipoprotein clearance studies were performed in vivo. Op0/E0 mice have fourfold smaller aortic root lesions than op2/E0 animals despite 2.5-fold higher total plasma cholesterol levels. Ms in atherosclerotic lesions of op2/E0 mice constitute a predominantly recruited and M-CSF-dependent population.

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