title: GLUT4 trafficking in insulin-stimulated rat adipose cells: evidence that heterotrimeric GTP-binding proteins regulate the fusion of docked GLUT4-containing vesicles.
abstract: Agents that activate the G-protein G(i) (e.g. adenosine) increase, and agents 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 content. Here we have further examined the effects of R(s)G(s) and R(i)G(i) ligands (in which R(s) and R(i) are G(s)- and G(i)-coupled receptors respectively) on insulin-stimulated cell-surface GLUT4 and the kinetics of GLUT4 trafficking 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, followed by 0-20 min with insulin (670 nM). Treatment with isoprenaline and adenosine deaminase decreased insulin-stimulated glucose transport activity by 58%. Treatment with isoprenaline and adenosine deaminase also resulted in similar decreases in insulin-stimulated cell-surface GLUT4 as assessed by both bis-mannose photolabelling of the substrate-binding site and biotinylation of the extracellular carbohydrate moiety when evaluated under similar experimental conditions. After stimulation with insulin in the absence of G(s) and the presence of G(i) agents, a distinct sequence of plasma membrane events took place, starting with an increase in immunodetectable GLUT4, then an increase in the accessibility of GLUT4 to bis-mannose photolabel, and finally an increase in glucose transport activity. Pretreatment with isoprenaline and adenosine deaminase before stimulation with insulin did not affect the time course of the increase in immunodetectable GLUT4 in the plasma membrane, but did delay both the increase in accessibility of GLUT4 to photolabel 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 the fusion process through which the GLUT4 in docked vesicles becomes exposed on the cell surface.
authors.affiliation: Experimental Diabetes, Metabolism, and Nutrition Section, Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-1420, USA. cindy@grecc.ab.umd.edu
authors.forename: C M
authors.initials: CM
authors.lastname: Ferrara
authors.forename: S W
authors.initials: SW
authors.lastname: Cushman
mesh_terms: D019325:3-O-Methylglucose / Q000493:pharmacokinetics; D000241:Adenosine / Q000502:physiology; D000243:Adenosine Deaminase / Q000378:metabolism; D000273:Adipose Tissue / Q000187:drug effects / Q000502:physiology*; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D004822:Epididymis; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism; D051275:Glucose Transporter Type 4; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism*; D007328:Insulin / Q000494:pharmacology*; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D008561:Membrane Fusion / Q000187:drug effects; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D009124:Muscle Proteins; D051381:Rats
chemical_list: D051275:Glucose Transporter Type 4; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; C495694:Slc2a4 protein, rat; D019325:3-O-Methylglucose; D000243:Adenosine Deaminase; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D019205:GTP-Binding Protein alpha Subunits, Gs; D020962:Heterotrimeric GTP-Binding Proteins; D000241:Adenosine; D007545:Isoproterenol