Schürmann A, Monden I, Joost H G, Keller K
Institut für Pharmakologie und Toxikologie RWTH, Aachen, Germany.
Biochim Biophys Acta. 1992 Jul 15;1131(3):245-52. doi: 10.1016/0167-4781(92)90022-r.
In adipose and muscle cells, the glucose transporter isoform GLUT4 is mainly located in an intracellular, vesicular compartment from which it is translocated to the plasma membrane in response to insulin. In order to test the hypothesis that this preferential targeting of a glucose transporter to an intracellular storage site is conferred only by its primary sequence, we compared the subcellular distribution of the fat/muscle glucose transporter GLUT4 with that of the erythrocyte/brain-type glucose transporter GLUT1 after transient expression in COS-7 cells. Full-length cDNA was ligated into the expression vector pCMV that is driven by the cytomegalovirus promoter, and introduced into COS cells by the DEAE-dextran method. Cells were homogenized and fractionated by differential centrifugation to yield plasma membranes and a Golgi-enriched fraction of intracellular membranes (low-density microsomes). In these membrane fractions, the abundance of glucose transporters was assessed by immunoblotting with specific antibodies against GLUT1 and GLUT4, and their transport activity was assayed after solubilization and reconstitution into lecithin liposomes. Uptake rates of 2-deoxyglucose assayed in parallel samples were higher in cells expressing GLUT1 or GLUT4 as compared with control cells (transfection of pCMV without transporter cDNA). Reconstituted glucose transport activity in plasma membranes was about 5-fold higher after expression of GLUT1 and GLUT4 as compared with control cells. The relative amount of GLUT4 in the low-density microsomes as detected by reconstitution and immunoblotting exceeded that of the GLUT1, but was much lower than that observed in typical insulin-sensitive cells, e.g., rat fat cells or 3T3-L1 adipocytes. These data indicate that COS-7 cells transfected with glucose transporter cDNA express the active transport proteins and can be used for functional studies.
在脂肪细胞和肌肉细胞中，葡萄糖转运体异构体GLUT4主要位于细胞内的囊泡区室，在胰岛素的作用下，它会从该区室转运至质膜。为了验证葡萄糖转运体优先靶向细胞内储存位点仅由其一级序列决定这一假说，我们在COS-7细胞中瞬时表达后，比较了脂肪/肌肉葡萄糖转运体GLUT4与红细胞/脑型葡萄糖转运体GLUT1的亚细胞分布。将全长cDNA连接到由巨细胞病毒启动子驱动的表达载体pCMV中，并通过DEAE-葡聚糖法导入COS细胞。细胞经匀浆后通过差速离心进行分级分离，以获得质膜和富含高尔基体的细胞内膜组分（低密度微粒体）。在这些膜组分中，通过用针对GLUT1和GLUT4的特异性抗体进行免疫印迹来评估葡萄糖转运体的丰度，并在溶解并重新组装到卵磷脂脂质体后测定其转运活性。与对照细胞（转染无转运体cDNA的pCMV）相比，表达GLUT1或GLUT4的细胞中平行样本检测的2-脱氧葡萄糖摄取率更高。与对照细胞相比，表达GLUT1和GLUT4后质膜中重组的葡萄糖转运活性约高5倍。通过重组和免疫印迹检测，低密度微粒体中GLUT4的相对含量超过GLUT1，但远低于在典型的胰岛素敏感细胞（如大鼠脂肪细胞或3T3-L1脂肪细胞）中观察到的含量。这些数据表明，用葡萄糖转运体cDNA转染的COS-7细胞表达有活性的转运蛋白，可用于功能研究。
