Need for GLUT4 activation to reach maximum effect of insulin-mediated glucose uptake in brown adipocytes isolated from GLUT4myc-expressing mice.
There is a need to understand whether the amount of GLUT4 at the cell surface determines the extent of glucose uptake in response to insulin. Thus, we created a heterozygous mouse expressing modest levels of myc-tagged GLUT4 (GLUT4myc) in insulin-sensitive tissues under the control of the human GLUT4 promoter. Insulin stimulated 2-deoxyglucose uptake 6.5-fold in isolated brown adipocytes. GLUT1 did not contribute to the insulin response. The stimulation by insulin was completely blocked by wortmannin and partly (55 +/- 2%) by the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580. Insulin increased surface exposure of GLUT4myc twofold (determined by fluorescent or enzyme-linked myc immunodetection in intact adipocytes). Such increase was completely blocked by wortmannin but insensitive to SB203580. Insulin increased the kinase activity of the p38 MAPK beta-isoform 1.9-fold without affecting p38-alpha. In summary, the GLUT4myc mouse is a promising model for measuring GLUT4 translocation in intact primary cells. It affords direct comparison between GLUT4 translocation and glucose uptake in similar cell preparations, allowing one to study the regulation of GLUT4 activity. Using this animal model, we found that stimulation of glucose uptake into brown adipocytes involves both GLUT4 translocation and activation.
Programme in Cell Biology, The Hospital for Sick Children, Toronto, Ontario, Canada.
Daniel
D
Konrad
Philip J
PJ
Bilan
Zafar
Z
Nawaz
Gary
G
Sweeney
Wenyan
W
Niu
Zhi
Z
Liu
Costin N
CN
Antonescu
Assaf
A
Rudich
Amira
A
Klip
D017667:Adipocytes / Q000378:metabolism*; D002001:Adipose Tissue, Brown / Q000166:cytology / Q000378:metabolism*; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism; D003847:Deoxyglucose / Q000037:antagonists & inhibitors / Q000493:pharmacokinetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005947:Glucose / Q000493:pharmacokinetics*; D005951:Glucose Tolerance Test; D051275:Glucose Transporter Type 4; D017320:HIV Protease Inhibitors / Q000494:pharmacology; D006579:Heterozygote; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D019469:Indinavir / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology / Q000502:physiology*; D051379:Mice; D008822:Mice, Transgenic / Q000235:genetics; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000502:physiology*; D009124:Muscle Proteins; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D016324:Sequence Tagged Sites; D000077191:Wortmannin; D048051:p38 Mitogen-Activated Protein Kinases
D000730:Androstadienes; D004791:Enzyme Inhibitors; D051275:Glucose Transporter Type 4; D017320:HIV Protease Inhibitors; D007093:Imidazoles; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D016271:Proto-Oncogene Proteins c-myc; D011725:Pyridines; C495692:SLC2A4 protein, human; C495693:Slc2a4 protein, mouse; C495694:Slc2a4 protein, rat; D019469:Indinavir; D003847:Deoxyglucose; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D005947:Glucose; C093642:SB 203580; D000077191:Wortmannin