DETERMINATION OF THE RATES OF APPEARANCE AND LOSS OF GLUCOSE TRANSPORTERS AT THE CELL-SURFACE OF RAT ADIPOSE-CELLS

We have used an impermeant bis-mannose compound {2-N-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]-1.3-bis-(D-mannos-4-yloxy)-2-propylamine; ATB-BMPA} to photolabel the glucose transporter isoforms GLUT4 and GLUT1 that are present in rat adipose cells. Plasma-membrane fractions and light-microsome membrane fractions were both labelled by ATB-BMPA. The labelling of GLUT4 in the plasma membrane fraction from insulin-treated cells was approximately 3-fold higher than that of basal cells and corresponded with a decrease in the labelling of the light-microsome fraction. In contrast with this, the cell-surface labelling of GLUT4 from insulin-treated intact adipose cells was increased approximately 15-fold above basal levels. In these adipose cell preparations, insulin stimulated glucose transport activity approximately 30-fold. Thus the cell-surface labelling, but not the labelling of membrane fractions, closely corresponded with the stimulation of transport. The remaining discrepancy may be due to an approx. 2-fold activation of GLUT4 intrinsic transport activity. We have studied the kinetics of trafficking of transporters and found the following. (1) Lowering the temperature to 18-degrees-C increased basal glucose transport and levels of cell-surface glucose transporters by approximately 3-fold. This net increase in transporters probably occurs because the process of recruitment of transporters is less temperature-sensitive than the process involved in internalization of cell-surface transporters. (2) The time course for insulin stimulation of glucose transport activity occurred with a slight lag period of 47 s and a t1/2 of 3.2 min. The time course of GLUT4 and GLUT1 appearance at the cell surface showed no lag and a t1/2 of approximately 2.3 min for both isoforms. Thus at early times after insulin stimulation there was a discrepancy between transporter abundance and transport activity. The lag period in the stimulation of transport activity may represent the time required for the approximately 2-fold stimulation of transporter intrinsic activity. (3) The decrease in transport activity after insulin removal occurred with a very high activation energy of 159 kJ.mol-1. There was thus no significant decrease in transport or loss of cell-surface transporters over 60 min at 18-degrees-C. The decrease in transport activity occurred with a t1/2 of 9-11 min at 37-degrees-C. Using the ATB-BMPA photolabel to monitor remaining cell-surface transporters, we have shown that the loss of GLUT4 and GLUT1 occurred with t1/2 values of approximately 12 min. Thus the decrease in transport activity corresponds with the decrease in cell-surface transporters.