Yang J, Clark A E, Harrison R, Kozka I J, Holman G D
Department of Biochemistry, University of Bath, Avon, U.K.
Biochem J. 1992 Feb 1;281 ( Pt 3)(Pt 3):809-17. doi: 10.1042/bj2810809.
We have compared the rates of insulin stimulation of cell-surface availability of glucose-transporter isoforms (GLUT1 and GLUT4) and the stimulation of 2-deoxy-D-glucose transport in 3T3-L1 cells. The levels of cell-surface transporters have been assessed by using the bismannose compound 2-N-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]-1,3-bis(D-mannos -4-yloxy) propyl-2-amine (ATB-BMPA). At 27 degrees C the half-times for the appearance of GLUT1 and GLUT4 at the cell surface were 5.7 and 5.4 min respectively and were slightly shorter than that for the observed stimulation of transport activity (t 1/2 8.6 min). This lag may be due to a slow dissociation of surface transporters from trafficking proteins responsible for translocation. When fully-insulin-stimulated cells were subjected to a low-pH washing procedure to remove insulin at 37 degrees C, the cell-surface levels of GLUT1 and GLUT4 decreased, with half-times of 9.2 and 6.8 min respectively. These times correlated well with decrease in 2-deoxy-D-glucose transport activity that occurred during this washing procedure (t1/2 6.5 min). When fully-insulin-stimulated cells were treated with phenylarsine oxide (PAO), a similar decrease in transport activity occurred (t1/2 9.8 min). However, surface labelling showed that this corresponded with a decrease in GLUT4 only (t1/2 7.8 min). The cell-surface level of GLUT1 remained high throughout the PAO treatment. Light-microsome membranes were isolated from cells which had been cell-surface-labelled with ATB-BMPA. Internalization of both transporter isoforms to this pool occurred when cells were maintained in the presence of insulin for 60 min. In contrast with the surface-labelling results, we have shown that the transfer to the light-microsome pool of both transporters occurred in cells treated with insulin and PAO. These results suggest that both transporters are recycled by fluid-phase endocytosis and exocytosis. PAO may inhibit this recycling at a stage which involves the re-emergence of internalized transporters at the plasma membrane. The GLUT1 transporters that are recycled to the surface in insulin- and PAO-treated cells appear to have low transport activity. This may be because of a failure to dissociate fully from trafficking proteins at the cell surface. GLUT4 transporters appear to have a greater tendency to remain internalized if the normal mechanisms that commit transporters to the cell surface, such as dissociation from trafficking proteins, are uncoupled.
我们比较了胰岛素刺激3T3-L1细胞中葡萄糖转运体亚型（GLUT1和GLUT4）的细胞表面可用性的速率以及对2-脱氧-D-葡萄糖转运的刺激作用。通过使用双甘露糖化合物2-N-[4-(1-叠氮基-2,2,2-三氟乙基)苯甲酰基]-1,3-双（D-甘露糖-4-氧基）丙基-2-胺（ATB-BMPA）来评估细胞表面转运体的水平。在27℃时，GLUT1和GLUT4出现在细胞表面的半衰期分别为5.7分钟和5.4分钟，略短于观察到的转运活性刺激的半衰期（t1/2 8.6分钟）。这种延迟可能是由于表面转运体与负责转运的运输蛋白缓慢解离所致。当在37℃下对完全胰岛素刺激的细胞进行低pH洗涤程序以去除胰岛素时，GLUT1和GLUT4的细胞表面水平下降，半衰期分别为9.2分钟和6.8分钟。这些时间与在此洗涤过程中发生的2-脱氧-D-葡萄糖转运活性的下降密切相关（t1/2 6.5分钟）。当用苯砷酸氧化物（PAO）处理完全胰岛素刺激的细胞时，转运活性也出现类似的下降（t1/2 9.8分钟）。然而，表面标记显示这仅对应于GLUT4的下降（t1/2 7.8分钟）。在整个PAO处理过程中，GLUT1的细胞表面水平保持较高。从用ATB-BMPA进行细胞表面标记的细胞中分离出轻微粒体膜。当细胞在胰岛素存在下维持60分钟时，两种转运体亚型都内化到这个池中。与表面标记结果相反，我们已经表明在用胰岛素和PAO处理的细胞中，两种转运体都转移到了轻微粒体池中。这些结果表明，两种转运体都是通过液相内吞作用和胞吐作用进行循环利用的。PAO可能在涉及内化转运体重新出现在质膜的阶段抑制这种循环利用。在胰岛素和PAO处理的细胞中循环回到表面的GLUT1转运体似乎具有较低的转运活性。这可能是因为未能在细胞表面与运输蛋白完全解离。如果使转运体定位于细胞表面的正常机制，如与运输蛋白解离，被解除偶联，GLUT4转运体似乎更倾向于保持内化状态。
