Liu Ping, Leffler Brian J, Weeks Lara K, Chen Guoli, Bouchard Christine M, Strawbridge Andrew B, Elmendorf Jeffrey S
Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Center for Diabetes Research, Indianapolis, IN 46202, USA.
Am J Physiol Cell Physiol. 2004 Feb;286(2):C317-29. doi: 10.1152/ajpcell.00073.2003. Epub 2003 Oct 1.
A basis for the insulin mimetic effect of sphingomyelinase on glucose transporter isoform GLUT4 translocation remains unclear. Because sphingomyelin serves as a major determinant of plasma membrane cholesterol and a relationship between plasma membrane cholesterol and GLUT4 levels has recently become apparent, we assessed whether GLUT4 translocation induced by sphingomyelinase resulted from changes in membrane cholesterol content. Exposure of 3T3-L1 adipocytes to sphingomyelinase resulted in a time-dependent loss of sphingomyelin from the plasma membrane and a concomitant time-dependent accumulation of plasma membrane GLUT4. Degradation products of sphingomyelin did not mimic this stimulatory action. Plasma membrane cholesterol amount was diminished in cells exposed to sphingomyelinase. Restoration of membrane cholesterol blocked the stimulatory effect of sphingomyelinase. Increasing concentrations of methyl-beta-cyclodextrin, which resulted in a dose-dependent reversible decrease in membrane cholesterol, led to a dose-dependent reversible increase in GLUT4 incorporation into the plasma membrane. Although increased plasma membrane GLUT4 content by cholesterol extraction with concentrations of methyl-beta-cyclodextrin above 5 mM most likely reflected decreased GLUT4 endocytosis, translocation stimulated by sphingomyelinase or concentrations of methyl-beta-cyclodextrin below 2.5 mM occurred without any visible changes in the endocytic retrieval of GLUT4. Furthermore, moderate loss of cholesterol induced by sphingomyelinase or low concentrations of methyl-beta-cyclodextrin did not alter membrane integrity or increase the abundance of other plasma membrane proteins such as the GLUT1 glucose transporter or the transferrin receptor. Regulation of GLUT4 translocation by moderate cholesterol loss did not involve known insulin-signaling proteins. These data reveal that sphingomyelinase enhances GLUT4 exocytosis via a novel cholesterol-dependent mechanism.
鞘磷脂酶对葡萄糖转运蛋白异构体GLUT4易位的胰岛素模拟作用的基础仍不清楚。由于鞘磷脂是质膜胆固醇的主要决定因素，且质膜胆固醇与GLUT4水平之间的关系最近已变得明显，我们评估了鞘磷脂酶诱导的GLUT4易位是否源于膜胆固醇含量的变化。将3T3-L1脂肪细胞暴露于鞘磷脂酶会导致质膜中鞘磷脂随时间依赖性丢失，同时质膜GLUT4随时间依赖性积累。鞘磷脂的降解产物不会模拟这种刺激作用。暴露于鞘磷脂酶的细胞中质膜胆固醇量减少。膜胆固醇的恢复阻断了鞘磷脂酶的刺激作用。增加甲基-β-环糊精的浓度会导致膜胆固醇呈剂量依赖性可逆性降低，从而导致GLUT4掺入质膜的量呈剂量依赖性可逆性增加。尽管用高于5 mM的甲基-β-环糊精浓度提取胆固醇导致质膜GLUT4含量增加很可能反映了GLUT4内吞作用的降低，但鞘磷脂酶或低于2.5 mM的甲基-β-环糊精浓度刺激的易位发生时，GLUT4的内吞回收没有任何明显变化。此外，鞘磷脂酶或低浓度甲基-β-环糊精诱导的适度胆固醇丢失不会改变膜完整性，也不会增加其他质膜蛋白如GLUT1葡萄糖转运蛋白或转铁蛋白受体的丰度。适度胆固醇丢失对GLUT4易位的调节不涉及已知的胰岛素信号蛋白。这些数据表明，鞘磷脂酶通过一种新的胆固醇依赖性机制增强GLUT4的胞吐作用。
