Harrison S A, Buxton J M, Clancy B M, Czech M P
Program in Molecular Medicine, University of Massachusetts Medical Center, Worcester 01605.
J Biol Chem. 1990 Nov 25;265(33):20106-16.
Complementary DNA encoding a HepG2 cell-facilitated glucose transporter (GLUT1) was subcloned into a metal-inducible, mammalian expression vector, pLEN. Mouse 3T3-L1 fibroblasts transfected with this new construct, pLENGT, exhibited zinc-inducible expression of human glucose transporter mRNA, protein, and glucose transport activity, before and after differentiation into adipocytes. Both mouse host GLUT1 and expressed human GLUT1 proteins distributed about equally between 3T3-L1 adipocyte plasma membranes and low density microsomal membranes, while host skeletal muscle/adipocyte-type glucose transporter (GLUT4) was concentrated in the latter fraction. Mouse GLUT1 and GLUT4 proteins and the constitutively expressed human GLUT1 protein in pLENGT adipocytes were all redistributed from low density microsomal membrane to plasma membrane fractions in response to insulin. Insulin stimulated 2-deoxyglucose uptake in untransfected fibroblasts about 2-fold, while untransfected adipocytes displayed a 14-fold increase in deoxyglucose uptake in response to insulin. Both the expression of human GLUT1 protein and basal 2-deoxyglucose uptake by 75 microM zinc-treated pLENGT fibroblasts and adipocytes were increased approximately 3-fold over untransfected cells. In such pLENGT fibroblasts expressing human GLUT1 protein, however, the absolute values for insulin-stimulated increases in sugar uptake were no different than in control fibroblasts. As was observed in pLENGT fibroblasts, the increased basal sugar uptake by pLENGT adipocytes was additive with the insulin-stimulated increase in the rate of sugar uptake and, therefore, the -fold stimulation by insulin was markedly reduced. These data indicate that: 1) the membrane distributions of a glucose transporter protein, which is not responsive to insulin in HepG2 cells, and both mouse GLUT1 and GLUT4 glucose transporter isoforms are regulated by insulin in mouse 3T3-L1 adipocytes, and 2) the expressed human GLUT1 appears to contribute significantly to the rate of basal uptake but not to the insulin-stimulated increase in 2-deoxyglucose uptake by 3T3-L1 fibroblasts and adipocytes.
编码一种HepG2细胞促进型葡萄糖转运蛋白（GLUT1）的互补DNA被亚克隆到一种金属诱导型哺乳动物表达载体pLEN中。用这种新构建体pLENGT转染的小鼠3T3-L1成纤维细胞，在分化为脂肪细胞之前和之后，均表现出锌诱导的人葡萄糖转运蛋白mRNA、蛋白质及葡萄糖转运活性。小鼠宿主GLUT1和表达的人GLUT1蛋白在3T3-L1脂肪细胞质膜和低密度微粒体膜之间的分布大致相等，而宿主骨骼肌/脂肪细胞型葡萄糖转运蛋白（GLUT4）则集中在后一馏分中。pLENGT脂肪细胞中的小鼠GLUT1和GLUT4蛋白以及组成型表达的人GLUT1蛋白，在胰岛素作用下均从低密度微粒体膜重新分布到质膜馏分中。胰岛素刺激未转染的成纤维细胞对2-脱氧葡萄糖的摄取增加约2倍，而未转染的脂肪细胞对胰岛素的反应是脱氧葡萄糖摄取增加14倍。与未转染细胞相比，经75 microM锌处理的pLENGT成纤维细胞和脂肪细胞中人GLUT1蛋白的表达及基础2-脱氧葡萄糖摄取均增加约3倍。然而，在表达人GLUT1蛋白的此类pLENGT成纤维细胞中，胰岛素刺激的糖摄取增加的绝对值与对照成纤维细胞并无差异。正如在pLENGT成纤维细胞中观察到的那样，pLENGT脂肪细胞基础糖摄取的增加与胰岛素刺激的糖摄取速率增加是相加的，因此，胰岛素的刺激倍数显著降低。这些数据表明：1）一种在HepG2细胞中对胰岛素无反应的葡萄糖转运蛋白的膜分布，以及小鼠GLUT1和GLUT4葡萄糖转运蛋白同工型在小鼠3T3-L1脂肪细胞中均受胰岛素调节；2）表达的人GLUT1似乎对基础摄取速率有显著贡献，但对3T3-L1成纤维细胞和脂肪细胞中胰岛素刺激的2-脱氧葡萄糖摄取增加无贡献。
