Glucose transporter recycling in rat adipose cells. Effects of potassium depletion.
Depletion of intracellular potassium (K+) induced a 4-fold increase in basal and 1 microM phorbol-12-myristate-13-acetate (PMA)-stimulated 3-O-methylglucose transport in rat adipose cells. K+ depletion had no effect on the maximum insulin (0.7 microM)-stimulated transport rate but enhanced the sensitivity to insulin 3-fold (EC50 = 0.05 versus 0.15 nM) by a mechanism that did not result from changes in the insulin receptor binding, autophosphorylation, or tyrosine kinase activity. Western blotting analysis revealed that K+ depletion induced a 2.2-fold increase in GLUT4 in plasma membranes from basal cells, enhanced the PMA-stimulated GLUT4 translocation by 4-fold, and increased the 5-fold insulin-stimulated GLUT4 translocation by 15%, indicating the presence of an inactive GLUT4 intermediate. The time course for insulin's stimulation of transport activity was accelerated by K+ depletion (t1/2 = 3 versus 1.5 min). Conversely, the reversal of transport activity, on removal of insulin, was delayed (t1/2 = 11 versus 22 min). The corresponding t1/2 values for the loss of GLUT4 were 22 min in control cells and 40 min in K(+)-depleted cells, again indicating the existence of an inactive intermediate. Photolabeling intact cells with the impermeant, exofacial photolabel 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannos-4 - yloxy)-2-propylamine in the continuous presence of insulin revealed that K+ depletion had no effect on the GLUT4 externalization rate but halved the rate of internalization. K+ depletion elicited entirely analogous effects on the recycling of insulin-like growth factor II/mannose 6-phosphate receptor, strongly supporting the involvement of a coated pit mechanism in the recycling of GLUT4 transporters. An inactive conformation of GLUT4 has been detected in plasma membranes from insulin-stimulated cells, which is enhanced by K+ depletion, suggesting a limitation in the adipose cells' capacity to express active GLUT4 transporters.
Nishimura
H
H
Experimental Diabetes, Metabolism and Nutrition Section, National Institute of Diabetes and Digestive and Kidney Disease, National Institutes of Health, Bethesda, Maryland 20892.
Zarnowski
M J
MJ
Simpson
I A
IA
D019325:3-O-Methylglucose; D000273:Adipose Tissue / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D066298:In Vitro Techniques; D007328:Insulin / Q000494:pharmacology; D007425:Intracellular Membranes / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008757:Methylglucosides / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins / Q000302:isolation & purification / Q000378:metabolism*; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine; D011188:Potassium / Q000494:pharmacology*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D051381:Rats; D017527:Receptor, IGF Type 2 / Q000302:isolation & purification / Q000378:metabolism; D011972:Receptor, Insulin / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis
D007328:Insulin; D008757:Methylglucosides; D009004:Monosaccharide Transport Proteins; D010750:Phosphoproteins; D017527:Receptor, IGF Type 2; D019325:3-O-Methylglucose; D019000:Phosphotyrosine; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D013755:Tetradecanoylphorbol Acetate; D011188:Potassium