Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.
Biochem J. 2010 Mar 15;427(1):143-50. doi: 10.1042/BJ20091529.
Insulin stimulates glucose transport in fat and skeletal muscle cells primarily by inducing the translocation of GLUT4 (glucose transporter isoform 4) to the PM (plasma membrane) from specialized GSVs (GLUT4 storage vesicles). Glycosphingolipids are components of membrane microdomains and are involved in insulin-regulated glucose transport. Cellular glycosphingolipids decrease during adipocyte differentiation and have been suggested to be involved in adipocyte function. In the present study, we investigated the role of glycosphingolipids in regulating GLUT4 translocation. We decreased glycosphingolipids in 3T3-L1 adipocytes using glycosphingolipid synthesis inhibitors and investigated the effects on GLUT4 translocation using immunocytochemistry, preparation of PM sheets, isolation of GSVs and FRAP (fluorescence recovery after photobleaching) of GLUT4-GFP (green fluorescent protein) in intracellular structures. Glycosphingolipids were located in endosomal vesicles in pre-adipocytes and redistributed to the PM with decreased expression at day 2 after initiation of differentiation. In fully differentiated adipocytes, depletion of glycosphingolipids dramatically accelerated insulin-stimulated GLUT4 translocation. Although insulin-induced phosphorylation of IRS (insulin receptor substrate) and Akt remained intact in glycosphingolipid-depleted cells, both in vitro budding of GLUT4 vesicles and FRAP of GLUT4-GFP on GSVs were stimulated. Glycosphingolipid depletion also enhanced the insulin-induced translocation of VAMP2 (vesicle-associated membrane protein 2), but not the transferrin receptor or cellubrevin, indicating that the effect of glycosphingolipids was specific to VAMP2-positive GSVs. Our results strongly suggest that decreasing glycosphingolipid levels promotes the formation of GSVs and, thus, GLUT4 translocation. These studies provide a mechanistic basis for recent studies showing that inhibition of glycosphingolipid synthesis improves glycaemic control and enhances insulin sensitivity in animal models of Type 2 diabetes.
胰岛素主要通过诱导 GLUT4（葡萄糖转运蛋白同工型 4）从特殊的 GSVs（GLUT4 储存小泡）向 PM（质膜）易位，来刺激脂肪和骨骼肌细胞中的葡萄糖转运。糖脂是膜微区的组成部分，参与胰岛素调节的葡萄糖转运。细胞糖脂在脂肪细胞分化过程中减少，并被认为参与脂肪细胞功能。在本研究中，我们研究了糖脂在调节 GLUT4 易位中的作用。我们使用糖脂合成抑制剂降低 3T3-L1 脂肪细胞中的糖脂，并通过免疫细胞化学、PM 片制备、GSV 分离和 GLUT4-GFP（绿色荧光蛋白）在细胞内结构中的 FRAP（光漂白后荧光恢复）来研究其对 GLUT4 易位的影响。糖脂在预脂肪细胞中位于内体小泡中，并在分化开始后第 2 天随着表达的减少而重新分布到 PM。在完全分化的脂肪细胞中，糖脂耗竭显著加速了胰岛素刺激的 GLUT4 易位。尽管在糖脂耗尽的细胞中，胰岛素诱导的 IRS（胰岛素受体底物）和 Akt 的磷酸化仍然完整，但 GLUT4 小泡的体外出芽和 GLUT4-GFP 在 GSV 上的 FRAP 都被刺激。糖脂耗竭也增强了胰岛素诱导的 VAMP2（囊泡相关膜蛋白 2）易位，但不增强转铁蛋白受体或细胞分裂素，表明糖脂的作用是针对 VAMP2 阳性 GSV 的。我们的结果强烈表明，降低糖脂水平促进了 GSV 的形成，从而促进了 GLUT4 的易位。这些研究为最近的研究提供了机制基础，这些研究表明抑制糖脂合成可改善 2 型糖尿病动物模型的血糖控制和增强胰岛素敏感性。
