Agents that activate the G-protein Gi (e.g. adenosine) increase, and agents that activate Gs [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 RsGs and RiGi ligands (in which Rs and Ri are Gs- and Gi-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 Gs and decrease the stimulation of Gi 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 Gs and the presence of Gi 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 RsGs and RiGi 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.
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Research Article|
October 25 1999
GLUT4 trafficking in insulin-stimulated rat adipose cells: evidence that heterotrimeric GTP-binding proteins regulate the fusion of docked GLUT4-containing vesicles
Cynthia M. FERRARA;
Cynthia M. FERRARA
1
1Experimental Diabetes, Metabolism, and Nutrition Section, Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 10 Center Drive MSC 1420 (10/5N102), Bethesda, MD 20892-1420, U.S.A.
1To whom correspondence should be addressed (cindy@grecc.ab.umd.edu). University of Maryland, Baltimore Veterans Administration Medical Center, 10 North Greene Street, GRECC (BT/18/GR), Baltimore, MD 21201-1524, U.S.A.
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Samuel W. CUSHMAN
Samuel W. CUSHMAN
1Experimental Diabetes, Metabolism, and Nutrition Section, Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 10 Center Drive MSC 1420 (10/5N102), Bethesda, MD 20892-1420, U.S.A.
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Publisher: Portland Press Ltd
Received:
April 30 1999
Revision Received:
July 26 1999
Accepted:
August 17 1999
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1999
1999
Biochem J (1999) 343 (3): 571–577.
Article history
Received:
April 30 1999
Revision Received:
July 26 1999
Accepted:
August 17 1999
Citation
Cynthia M. FERRARA, Samuel W. CUSHMAN; GLUT4 trafficking in insulin-stimulated rat adipose cells: evidence that heterotrimeric GTP-binding proteins regulate the fusion of docked GLUT4-containing vesicles. Biochem J 1 November 1999; 343 (3): 571–577. doi: https://doi.org/10.1042/bj3430571
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