Smith Jill, Su Xiong, El-Maghrabi Raafat, Stahl Philip D, Abumrad Nada A
Department of Medicine, Center for Human Nutrition, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
J Biol Chem. 2008 May 16;283(20):13578-85. doi: 10.1074/jbc.M800008200. Epub 2008 Mar 18.
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 microm 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.
FAT/CD36是一种膜清道夫受体，可促进肌肉对长链脂肪酸的摄取。据报道，胰岛素和收缩可使膜CD36和脂肪酸摄取急性增加。在本研究中，我们探讨了蛋白质泛素化作为调节CD36水平的一种潜在机制。在中国仓鼠卵巢（CHO）或HEK 293细胞中表达的CD36被发现通过涉及泛素赖氨酸48和63的过程进行多聚泛素化。使用表达胰岛素受体（CHO/hIR）和CD36的CHO细胞，结果显示添加胰岛素（100 nM，10和30分钟）可显著降低CD36的泛素化。相反，脂肪酸（200 μM棕榈酸或油酸，2小时）可强烈增强泛素化。同样，C2C12肌管中的内源性CD36被泛素化，油酸处理可增强这种泛素化，同时也降低了细胞裂解物中总的CD36蛋白。胰岛素降低了CD36的泛素化，增加了CD36蛋白，并抑制了脂肪酸对这两个参数的相反作用。这些变化与脂肪酸摄取的变化平行，脂肪酸摄取可被CD36抑制剂油酰基磺基琥珀酰亚胺阻断。CD36羧基末端尾巴中的两个赖氨酸残基发生突变，显著减弱了CHO细胞中表达的蛋白质的泛素化，并与CD36水平升高、油酸摄取增加以及甘油三酯掺入增加有关。总之，脂肪酸和胰岛素在CD36泛素化方面诱导相反的改变，调节CD36水平和脂肪酸摄取。CD36周转的改变可能导致胰岛素抵抗肌肉中脂肪酸摄取异常。
