Harrison S A, Clancy B M, Pessino A, Czech M P
Program in Molecular Medicine, University of Massachusetts Medical Center, Worcester 01605.
J Biol Chem. 1992 Feb 25;267(6):3783-8.
Several studies have demonstrated that the intrinsic catalytic activity of cell surface glucose transporters is highly regulated in 3T3-L1 adipocytes expressing GLUT1 (erythrocyte/brain) and GLUT4 (adipocyte/skeletal muscle) glucose transporter isoforms. For example, inhibition of protein synthesis in these cells by anisomycin or cycloheximide leads to marked increases in hexose transport without a change in the levels of cell surface glucose transporter proteins (Clancy, B. M., Harrison, S. A., Buxton, J. M., and Czech, M. P. (1991) J. Biol. Chem. 266, 10122-10130). In the present work the exofacial hexose binding sites on GLUT1 and GLUT4 in anisomycin-treated 3T3-L1 adipocytes were labeled with the cell-impermeant photoaffinity reagent [2-3H]2-N-[4-(1-azitrifluoroethyl)benzoyl]-1,3-bis- (D-mannos-4-yloxy)-2-propylamine [( 2-3H] ATB-BMPA) to determine which isoform is activated by protein synthetic blockade. As expected, a 15-fold increase in 2-deoxyglucose uptake in response to insulin was associated with 1.7- and 2.6-fold elevations in plasma membrane GLUT1 and GLUT4 protein levels, respectively. Anisomycin treatment of cultured adipocytes for 5 h produced an 8-fold stimulation of hexose transport but no increase in the content of glucose transporters in the plasma membrane fraction as measured by protein immunoblot analysis. Cell surface GLUT1 levels were also shown to be unaffected on 3T3-L1 adipocytes in response to anisomycin using an independent method, the binding of an antiexofacial GLUT1 antibody to intact cells. In contrast, anisomycin fully mimicked the action of insulin to stimulate (about 4-fold) the radiolabeling of GLUT1 transporters specifically immunoprecipitated from intact 3T3-L1 adipocytes irradiated after incubation with [2-3H] ATB-BMPA. Photolabeling of GLUT4 under these conditions was also significantly enhanced (1.8-fold) by anisomycin treatment, but this effect was only 15% of that caused by insulin. These results suggest that: 1) the photoaffinity reagent [2-3H]ATB-BMPA labels those cell surface glucose transporters present in a catalytically active state rather than total cell surface transporters as assumed previously and 2) inhibition of protein synthesis in 3T3-L1 adipocytes stimulates sugar transport primarily by enhancing the intrinsic catalytic activity of cell surface GLUT1, and to a lesser extent, GLUT4 proteins.
多项研究表明，在表达GLUT1（红细胞/脑型）和GLUT4（脂肪细胞/骨骼肌型）葡萄糖转运体异构体的3T3-L1脂肪细胞中，细胞表面葡萄糖转运体的内在催化活性受到高度调控。例如，茴香霉素或环己酰亚胺抑制这些细胞中的蛋白质合成会导致己糖转运显著增加，而细胞表面葡萄糖转运体蛋白水平却没有变化（克兰西，B.M.，哈里森，S.A.，巴克斯顿，J.M.，以及捷克，M.P.（1991年）《生物化学杂志》266卷，10122 - 10130页）。在本研究中，用细胞不可渗透的光亲和试剂[2 - 3H]2 - N - [4 - (1 - 叠氮三氟乙基)苯甲酰基]-1,3 - 双 - （D - 甘露糖 - 4 - 氧基）-2 - 丙胺[（2 - 3H]ATB - BMPA）标记茴香霉素处理的3T3-L1脂肪细胞中GLUT1和GLUT4的胞外己糖结合位点，以确定哪种异构体被蛋白质合成阻断激活。正如预期的那样，响应胰岛素时2 - 脱氧葡萄糖摄取增加15倍，分别伴随着质膜GLUT1和GLUT4蛋白水平升高1.7倍和2.6倍。用茴香霉素处理培养的脂肪细胞5小时，己糖转运受到8倍的刺激，但通过蛋白质免疫印迹分析测定，质膜部分葡萄糖转运体的含量没有增加。使用一种独立方法，即抗胞外GLUT1抗体与完整细胞的结合，也表明茴香霉素处理对3T3-L1脂肪细胞的细胞表面GLUT1水平没有影响。相反，茴香霉素完全模拟了胰岛素的作用，刺激（约4倍）从用[2 - 3H]ATB - BMPA孵育后照射的完整3T3-L1脂肪细胞中特异性免疫沉淀的GLUT1转运体的放射性标记。在这些条件下，茴香霉素处理也显著增强（1.8倍）了GLUT4的光标记，但这种效应仅为胰岛素引起的效应的15%。这些结果表明：1）光亲和试剂[2 - 3H]ATB - BMPA标记处于催化活性状态的那些细胞表面葡萄糖转运体，而不是如先前假设的总细胞表面转运体；2）3T3-L1脂肪细胞中蛋白质合成的抑制主要通过增强细胞表面GLUT1的内在催化活性来刺激糖转运，在较小程度上也增强GLUT4蛋白的内在催化活性。
