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.
Department of Endocrinology and Metabolism, Amtssygehuset, Aarhus University Hospital, Denmark.
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Lund
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Holman
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Schmitz
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Pedersen
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