Abstract
The amounts of the brain type and muscle type glucose transporters (designated Glut 1 and 4, respectively) in 3T3-L1 adipocytes have been determined by quantitative immunoblotting with antibodies against their carboxyl-terminal peptides. There are about 950,000 and 280,000 copies of Glut 1 and 4, respectively, per cell. Insulin caused the translocation of both types of transporters from an intracellular location to the plasma membrane. The insulin-elicited increase in cell surface transporters was assessed by labeling the surface transporters with a newly developed, membrane-impermeant, photoaffinity labeling reagent for glucose transporters. The increases in Glut 1 and 4 averaged 6.5- and 17-fold, respectively, whereas there was a 21-fold in hexose transport. These results indicate that the translocation of Glut 4 could largely account for the insulin effect on transport rate, but only if the intrinsic activity of Glut 4 is much higher than that of Glut 1. The two transporters are colocalized intracellularly: vesicles (average diameter 72 nm) isolated from the intracellular membranes by immunoadsorption with antibodies against Glut 1 contained 95% of the Glut 4 and, conversely, vesicles isolated with antibodies against Glut 4 contained 85% of the Glut 1.
Highlights
From the Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03756 and the SDepartment of Biochemistry, The University of Bath, Bath BA2 7A Y, United Kingdom
The insulin-elicited increase in cell surface transporters was assessed by labeling the surface transporters with a newly developed, membrane-impermeant, photoaffinity labeling reagent for glucose transporters
Ll adipocytes is located in intracellular membranes and that insulin causes the translocation of a portion of this intracellular Glut 1 to the plasma membrane
Summary
4 in insulin-stimulated hexose transport in 3T3-Ll adipocytes. Insulin stimulates hexose transport in 3T3-Ll adipocytes rapidly and typically by lo-fold or more (1,Z). Because of this large effect, this cultured ceil line has served as a model for investigations of the basis for insulin action on transport. It has been known for a number of years that 3T3-Ll adipocytes contain a glucose transporter of the type found in brain and human erythrocytes (hereafter referred to as Glut 1)’ [3]. ‘The abbreviations used are: Glut 1, brain-type glucose transporter; Glut 4, fat-muscle type glucose transporter; ATB-BMPA, photoaffinity labeling reagent for the glucose transporters; C,,E,, octaethyleneglycol dodecyl ether; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid, KRP, Krebs-Ringer-phosphate buffer; PBS, phosphate-buffered saline; Staph A, Staphylococcus aureus; SDS, sodium dodecyl sulfate
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