Inverse relationship between GLUT-4 phosphorylation and its intrinsic activity.
In this study, we examined the effect of phosphorylation on GLUT-4 function in isolated rat adipocytes. Adipocytes labeled with 32P for 2 h were incubated with parathyroid hormone (PTH) (20 ng/ml) for 60 min and then exposed to insulin (25 ng/ml) for an additional 30 min. 32P-GLUT-4 was immunoprecipitated from the plasma membrane and low density microsomal fractions, and its degree of phosphorylation was determined by autoradiography and densitometry. Results were expressed as 32P-GLUT-4 specific activity (phosphorylation/unit of protein). GLUT-4 intrinsic activity was measured using [14C]2-deoxyglucose uptake in plasma membrane vesicles. PTH significantly increased GLUT-4 phosphorylation and eliminated the insulin-stimulated dephosphorylation of GLUT-4. Western blotting revealed normal distribution of GLUT-4 before and after insulin stimulation in control and PTH-treated cells, suggesting that phosphorylation of GLUT-4 does not interfere with its recruitment to the plasma membrane. In contrast, intrinsic activity of phosphorylated GLUT-4 was significantly reduced (p < 0.01). Preincubation of adipocytes with calcium channel blocker (nitrendipine) and cyclic AMP antagonist (RpcAMP) restored GLUT-4 intrinsic activity in the PTH-treated cells. In several experiments, GLUT-4 was phosphorylated in vitro in plasma membrane vesicles isolated from normal adipocytes exposed to insulin. This in vitro phosphorylation reduced GLUT-4 intrinsic activity by approximately 35% (p < 0.01). We conclude that phosphorylation of GLUT-4 significantly impairs the ability of insulin to stimulate its intrinsic activity.
Reusch
J E
JE
Department of Medicine, Veterans Affairs Medical Center, Denver, Colorado 80220.
Sussman
K E
KE
Draznin
B
B
D000273:Adipose Tissue / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D002250:Carbon Radioisotopes; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D003847:Deoxyglucose / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D010281:Parathyroid Hormone / Q000494:pharmacology; D010710:Phosphates / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley
D002250:Carbon Radioisotopes; D009004:Monosaccharide Transport Proteins; D010281:Parathyroid Hormone; D010710:Phosphates; D010761:Phosphorus Radioisotopes; D003847:Deoxyglucose