Development of an intracellular pool of glucose transporters in 3T3-L1 cells.

The membrane-impermeant bis-mannose photolabel 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(D-mannos- 4-yloxy)-2- propylamine (ATB-BMPA) has been used to study the development of an intracellular pool of glucose transporters in 3T3-L1 cells. The subcellular distributions of the transporter isoforms GLUT1 and GLUT4 were determined by comparing the labeling obtained in cells in which the impermeant reagent only had access to the cell surface and the labeling obtained in digitonin-permeabilized cells. ATB-BMPA labeling showed that only GLUT1 was present in preconfluent fibroblasts and that most of the transporters were distributed to the cell surface. In preconfluent fibroblasts, the 2-deoxy-D-glucose transport activity was approximately 5 times higher than in confluent fibroblasts. ATB-BMPA labeling showed that the decrease in transport as cells reached confluence was associated with a decrease in the proportion of GLUT1 distributed to the cell surface. The sequestration of these transporters was associated with the development of an insulin-responsive transport activity which increased by approximately 2.5-fold compared with unstimulated confluent cells. ATB-BMPA labeling showed that insulin stimulation resulted in an approximately 2-fold increase in surface GLUT1 so that about one-half of the available transporters became recruited to the cell surface. Measurements of the changes in the distribution of both GLUT1 and GLUT4 throughout the differentiation of confluent fibroblasts into adipocytes showed that both transporters were sequestered in parallel. Basal levels of transport and photolabeling remained low throughout the differentiation period when the total pool of transporters (GLUT1 plus GLUT4) was increased by approximately 5-fold. These results suggest that the sequestration process was present before new transporters were synthesized. Thus, the sequestration mechanism develops in confluent growth-arrested fibroblasts although the capacity to sequester additional transporters may increase as differentiation proceeds.