GLUT4 trafficking in insulin-stimulated rat adipose cells: evidence that heterotrimeric GTP-binding proteins regulate the fusion of docked GLUT4-containing vesicles

Agents that activate the G-protein G(i) (e.g. adenosine) increase, and agen ts that activate G(s) [e.g. isoprenaline (isoproterenol)] decrease, steady- state insulin-stimulated glucose transport activity and cell-surface GLUT4 in isolated rat adipose cells without changing plasma membrane GLUT4 conten t. Here pile leave further examined the effects of R(s)G(s) and R(i)G(i) li gands (in which R-s and R-i are G(s)- and G(i)-coupled receptors respective ly) on insulin-stimulated cell-surface GLUT4 and the kinetics of GLUT4 traf ficking in these same cells. Rat adipose cells were preincubated for 2 min with or without isoprenaline (200 nM) and adenosine deaminase (1 unit/ml), to stimulate G(s) and decrease the stimulation of G(i) respectively, follow ed by 0-20 min with insulin (670 nM). Treatment with isoprenaline and adeno sine deaminase decreased insulin-stimulated glucose transport activity by 5 8%. Treatment with isoprenaline and adenosine deaminase also resulted in si milar decreases in insulin-stimulated cell-surface GLUT4 as assessed by bot h bis-mannose photolabelling of the substrate-binding site and biotinylatio n of the extracellular carbohydrate moiety when evaluated under similar exp erimental conditions. After stimulation with insulin in the absence of G(s) and the presence of G(i) agents, a distinct sequence of plasma membrane ev ents took place, starting with an increase in immunodetectable GLUT4, then an increase in the accessibility of GLUT4 to bis-mannose photolabel, and fi nally an increase in glucose transport activity. Pretreatment with isoprena line and adenosine deaminase before stimulation with insulin did not affect the time course of the increase in immunodetectable GLUT4 in the plasma me mbrane, but did delay both the increase in accessibility of GLUT4 to photol abel and the increase in glucose transport activity. These results suggest that R(s)G(s) and R(i)G(i) modulate insulin-stimulated glucose transport by influencing the extent to which GLUT4 is associated with occluded vesicles attached to the plasma membrane during exocytosis, perhaps by regulating t he fusion process through which the GLUT4 in docked vesicles becomes expose d on the cell surface.