Glut 4 content in the plasma membrane of rat skeletal muscle: comparative studies of the subcellular fractionation method and the exofacial photolabelling technique using ATB-BMPA.
Employing subcellular membrane fractionation methods it has been shown that insulin induces a 2-fold increase in Glut 4 protein content in the plasma membrane of skeletal muscle from rats. Data based upon this technique are, however, impeded by poor plasma membrane recovery and cross-contamination with intracellular membrane vesicles. The present study was undertaken to compare the subcellular fractionation technique with the technique using [3H]ATB-BMPA exofacial photolabelling and immunoprecipitation of Glut 4 on soleus muscles from 3-week-old Wistar rats. Maximal insulin stimulation resulted in a 6-fold increase in 3-O-methylglucose uptake, and studies based on the subcellular fractionation method showed a 2-fold increase in Glut 4 content in the plasma membrane, whereas the exofacial photolabelling demonstrated a 6- to 7-fold rise in cell surface associated Glut 4 protein. Glucose transport activity was positively correlated with cell surface Glut 4 content as estimated by exofacial labelling.


CONCLUSIONS
(1) the increase in glucose uptake in muscle after insulin exposure is caused by an augmented concentration of Glut 4 protein on the cell surface membrane, (2) at maximal insulin stimulation (20 mU/ml) approximately 40% of the muscle cell content of Glut 4 is at the cell surface, and (3) the exofacial labelling technique is more sensitive than the subcellular fractionation technique in measuring the amount of glucose transporters on muscle cell surface.
Lund
S
S
Department of Endocrinology and Metabolism, Amtssygehuset, Aarhus University Hospital, Denmark.
Holman
G D
GD
Schmitz
O
O
Pedersen
O
O
D019325:3-O-Methylglucose; D000345:Affinity Labels; D000818:Animals; D001386:Azides; D001834:Body Water; D002458:Cell Fractionation; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D004187:Disaccharides; D051275:Glucose Transporter Type 4; D006027:Glycosides; D007328:Insulin / Q000494:pharmacology; D008297:Male; D008757:Methylglucosides / Q000378:metabolism; D009004:Monosaccharide Transport Proteins / Q000032:analysis*; D009124:Muscle Proteins; D009132:Muscles / Q000737:chemistry* / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D010777:Photochemistry; D011437:Propylamines; D051381:Rats; D017208:Rats, Wistar
D000345:Affinity Labels; D001386:Azides; D004187:Disaccharides; D051275:Glucose Transporter Type 4; D006027:Glycosides; D007328:Insulin; D008757:Methylglucosides; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D011437:Propylamines; C495694:Slc2a4 protein, rat; C065412:2-N-(4-(1-azitrifluoroethyl)benzoyl)-1,3-bis-(mannos-4-yloxy)-2-propylamine; D019325:3-O-Methylglucose