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FEBS Lett. 1993 Sep 20;330(3):312-8.

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.

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1
Department of Endocrinology and Metabolism, Amtssygehuset, Aarhus University Hospital, Denmark.

Abstract

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.

IN CONCLUSION:

(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.

PMID:
8375502
DOI:
10.1016/0014-5793(93)80895-2
[Indexed for MEDLINE]
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