Determination of the rates of appearance and loss of glucose transporters at the cell surface of rat adipose cells.
We have used an impermeant bis-mannose compound (2-N-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]-1,3-bis-(D-mannos+ ++- 4-yloxy)-2- propylamine; ATB-BMPA) to photolabel the glucose transporter isoforms GLUT4 and GLUT1 that are present in rat adipose cells. Plasma-membrane fractions and light-microsome membrane fractions were both labelled by ATB-BMPA. The labelling of GLUT4 in the plasma membrane fraction from insulin-treated cells was approximately 3-fold higher than that of basal cells and corresponded with a decrease in the labelling of the light-microsome fraction. In contrast with this, the cell-surface labelling of GLUT4 from insulin-treated intact adipose cells was increased approximately 15-fold above basal levels. In these adipose cell preparations, insulin stimulated glucose transport activity approximately 30-fold. Thus the cell-surface labelling, but not the labelling of membrane fractions, closely corresponded with the stimulation of transport. The remaining discrepancy may be due to an approx. 2-fold activation of GLUT4 intrinsic transport activity. We have studied the kinetics of trafficking of transporters and found the following. (1) Lowering the temperature to 18 degrees C increased basal glucose transport and levels of cell-surface glucose transporters by approximately 3-fold. This net increase in transporters probably occurs because the process of recruitment of transporters is less temperature-sensitive than the process involved in internalization of cell-surface transporters. (2) The time course for insulin stimulation of glucose transport activity occurred with a slight lag period of 47 s and a t 1/2 3.2 min. The time course of GLUT4 and GLUT1 appearance at the cell surface showed no lag and a t 1/2 of approximately 2.3 min for both isoforms. Thus at early times after insulin stimulation there was a discrepancy between transporter abundance and transport activity. The lag period in the stimulation of transport activity may represent the time required for the approximately 2-fold stimulation of transporter intrinsic activity. (3) The decrease in transport activity after insulin removal occurred with a very high activation energy of 159 kJ.mol-1. There was thus no significant decrease in transport or less of cell-surface transporters over 60 min at 18 degrees C. The decrease in transport activity occurred with a t1/2 of 9-11 min at 37 degrees C.(ABSTRACT TRUNCATED AT 400 WORDS)
Clark
A E
AE
Department of Biochemistry, University of Bath, U.K.
Holman
G D
GD
Kozka
I J
IJ
D019325:3-O-Methylglucose; D000273:Adipose Tissue / Q000187:drug effects / Q000378:metabolism*; D000345:Affinity Labels; D000818:Animals; D001386:Azides; D001692:Biological Transport / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism; D004187:Disaccharides; D006027:Glycosides; D007328:Insulin / Q000494:pharmacology; D007425:Intracellular Membranes / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008757:Methylglucosides / Q000378:metabolism; D008861:Microsomes / Q000648:ultrastructure; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D010777:Photochemistry; D011190:Potassium Cyanide / Q000494:pharmacology; D011437:Propylamines; D051381:Rats; D011919:Rats, Inbred Strains
D000345:Affinity Labels; D001386:Azides; D004187:Disaccharides; D006027:Glycosides; D007328:Insulin; D008757:Methylglucosides; D009004:Monosaccharide Transport Proteins; D011437:Propylamines; C065412:2-N-(4-(1-azitrifluoroethyl)benzoyl)-1,3-bis-(mannos-4-yloxy)-2-propylamine; D019325:3-O-Methylglucose; D011190:Potassium Cyanide