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

FEBS Letters

Lund, S.²  Schmitz, O.²  Holman, G.D.³  Pedersen, O.¹ 

[1] Steno Diabetes Center, Copenhagen, Denmark;Department of Endocrinology and Metabolism, Amtssygehuset, Aarhus University Hospital, Aarhus, and Medical Department M, (Endocrinology and Diabetes) Kommunehospitalet, Aarhus, Denmark;Department of Biochemistry, University of Bath, Claverton Down, Bath, UK

关键词: Glucose transporter;    Exofacial photolabelling;    Subcellular fractionation;    Skeletal muscle;    ATB-BMPA;    ATB-BMPA;    2-N-4-(1-azi-2;    2;    2-trifluoroethyl) benzoyl-1;    3-bis-(D-mannose-4-yloxy)-2-propylamine;    C12E9;    nonaethyleneglycol dodecyl;    DOC;    deoxycholic acid;    GLUT 4;    insulin-sensitive muscle/fat glucose transporter;   

DOI  :  10.1016/0014-5793(93)80895-2

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Employing subcellular membrane fractionation methods it has been shown that insulin induces a 2-fold increase in the 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.

【 授权许可】

Unknown