Samih N, Hovsepian S, Aouani A, Lombardo D, Fayet G
INSERM Unité 260, Faculté de Médecine, Université de la Mediterranée, Marseille, France.
Endocrinology. 2000 Nov;141(11):4146-55. doi: 10.1210/endo.141.11.7793.
It was previously demonstrated that insulin or TSH treatment of FRTL-5 cells resulted in an elevation of glucose transport and in an increase of cell surface expression of the glucose transporter Glut-1. However, the signaling mechanisms leading to the insulin or TSH-induced increase in the cell surface expression of Glut-1 were not investigated. In the present study, we demonstrated that wortmannin and LY294002, two specific inhibitors of phosphatidylinositol 3-kinase (PI3-kinase), interfere both in the signaling pathways of insulin and TSH leading to glucose consumption enhancement and Glut-1 translocation. Two hours after insulin treatment, TSH or cAMP analog (Bu)2cAMP stimulation, glucose transport was increased and most of the intracellular Glut-1 pool was translocated to plasma membranes. Wortmannin or LY294002 blocked the insulin, (Bu)2cAMP, and the TSH-induced translocation of Glut-1. Wortmannin or LY294002 alone did not alter the basal ratio between intracellular and cell surface Glut-1 molecules. These results suggest that in FRTL-5 cells wortmannin and LY294002 inhibited the insulin, (Bu)2cAMP and TSH events leading to Glut-1 translocation from an intracellular compartment to the plasma membrane. Likewise, (Bu)2cAMP effects on glucose transport and Glut-1 translocation to plasma membrane were repressed by PI3-kinase inhibitors but not by the protein kinase A (PKA) inhibitor H89. We suggest that (Bu)2cAMP stimulates Glut-1 translocation to plasma membrane through PI3-kinase-dependent and PKA-independent signaling pathways. To further elucidate mechanisms that regulate the translocation of Glut-1 to cell membrane, we extended this study to the role played by the N-glycosylation in the translocation and in the biological activity of Glut-1 in FRTL-5 cells. For this purpose we used tunicamycin, an inhibitor of the N-glycosylation. Our experiments with tunicamycin clearly showed that both the glycosylated and unglycosylated forms of the transporter reached the cell surface. Likewise, a decrease in glucose consumption (-50%) after treatment of cells with tunicamycin was accompanied by a decrease (-70% vs. control) in the membrane expression of a 50-kDa form of Glut-1 and an increase in its unglycosylated 41-kDa form. These results suggest that carbohydrate moiety is essential for the biological activity of glucose transport but is not required for the translocation of Glut-1 from the intracellular membrane pool to the plasma membrane.
先前的研究表明，用胰岛素或促甲状腺激素（TSH）处理FRTL-5细胞会导致葡萄糖转运增加以及葡萄糖转运蛋白Glut-1的细胞表面表达增加。然而，导致胰岛素或TSH诱导Glut-1细胞表面表达增加的信号传导机制尚未得到研究。在本研究中，我们证明了渥曼青霉素和LY294002这两种磷脂酰肌醇3激酶（PI3激酶）的特异性抑制剂，会干扰胰岛素和TSH的信号传导途径，导致葡萄糖消耗增加和Glut-1易位。胰岛素处理、TSH或环磷酸腺苷（cAMP）类似物（Bu）2cAMP刺激两小时后，葡萄糖转运增加，并且大部分细胞内Glut-1库易位到质膜。渥曼青霉素或LY294002阻断了胰岛素、（Bu）2cAMP和TSH诱导的Glut-1易位。单独使用渥曼青霉素或LY294002不会改变细胞内和细胞表面Glut-1分子之间的基础比例。这些结果表明，在FRTL-5细胞中，渥曼青霉素和LY294002抑制了导致Glut-1从细胞内区室易位到质膜的胰岛素、（Bu）2cAMP和TSH事件。同样，PI3激酶抑制剂可抑制（Bu）2cAMP对葡萄糖转运和Glut-1易位到质膜的作用，但蛋白激酶A（PKA）抑制剂H89则不能。我们认为，（Bu）2cAMP通过PI3激酶依赖性和PKA非依赖性信号传导途径刺激Glut-1易位到质膜。为了进一步阐明调节Glut-1易位到细胞膜的机制，我们将这项研究扩展到N-糖基化在FRTL-5细胞中Glut-1易位及其生物学活性中所起的作用。为此，我们使用了衣霉素，一种N-糖基化抑制剂。我们用衣霉素进行的实验清楚地表明，转运蛋白的糖基化和非糖基化形式都能到达细胞表面。同样，用衣霉素处理细胞后，葡萄糖消耗减少（-50%），同时50 kDa形式的Glut-1膜表达减少（与对照相比-70%），其非糖基化41 kDa形式增加。这些结果表明，碳水化合物部分对于葡萄糖转运的生物学活性至关重要，但对于Glut-1从细胞内膜库易位到质膜并非必需。
