GLUT-4 phosphorylation and its intrinsic activity. Mechanism of Ca(2+)-induced inhibition of insulin-stimulated glucose transport.
In this study, we examined the influence of high levels of cytosolic calcium on phosphorylation status and function of GLUT-4 in isolated rat adipocytes. Intracellular calcium was elevated by exposing adipocytes to either extracellular ATP (1.6 mM) or thapsigargin (100 nM). Both agents increased cytosolic calcium 2-3 fold. While basal glucose uptake was unaffected, both ATP and thapsigargin reduced insulin-stimulated glucose transport by 40-70% (p < 0.05). Neither ATP nor thapsigargin affected GLUT-4 content or its translocation from the low density microsomes to the plasma membrane (PM). In contrast, GLUT-4 immunoprecipitated from the PM of adipocytes exposed to either ATP or thapsigargin was phosphorylated to a greater extent than the GLUT-4 isolated from control cells. ATP and thapsigargin also abolished insulin-stimulated dephosphorylation of GLUT-4. At the same time, GLUT-4 intrinsic activity was significantly reduced in adipocytes with high levels of cytosolic calcium (p < 0.05). Preincubation of adipocytes with cAMP antagonist, RpcAMP (10(-4) M), and calcium channel blocker, nitrendipine (30 microM), improved the ability of insulin to dephosphorylate GLUT-4 and restored insulin-stimulated GLUT-4 intrinsic activity. We conclude that elevated levels of cytosolic calcium interfere with insulin's ability to dephosphorylate GLUT-4, thus reducing its intrinsic activity.
Department of Medicine, Veterans Affairs Medical Center, Denver, Colorado 80220.
N
N
Begum
W
W
Leitner
J E
JE
Reusch
K E
KE
Sussman
B
B
Draznin
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000273:Adipose Tissue / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D003847:Deoxyglucose / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism*; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D008297:Male; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D009568:Nitrendipine / Q000494:pharmacology; D010710:Phosphates / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin; D013873:Thionucleotides / Q000494:pharmacology
D007328:Insulin; D009004:Monosaccharide Transport Proteins; D010710:Phosphates; D010761:Phosphorus Radioisotopes; D013729:Terpenes; D013873:Thionucleotides; C016957:adenosine-3',5'-cyclic phosphorothioate; D019284:Thapsigargin; D000255:Adenosine Triphosphate; D009568:Nitrendipine; D003847:Deoxyglucose; D000242:Cyclic AMP; D000252:Calcium-Transporting ATPases; D005947:Glucose; D002118:Calcium