REGULATION OF THE GLUT1 GLUCOSE-TRANSPORTER IN CULTURED MYOCYTES - TOTAL NUMBER AND SUBCELLULAR-DISTRIBUTION AS DETERMINED BY PHOTOAFFINITY-LABELING

We have used the impermeant photoaffinity label yl-[2-H-3]1,3-bis-(D-m annos-4-yloxy)-2-propylamine (ATB-[2-H-3]BMPA) to identify and quantif y the glucose transporters on the surface of BC3H-1 cells, a continuou sly cultured skeletal-muscle cell line lacking the MyoD transcription factor required for cell fusion. ATB-[2-H-3]BMPA was used in combinati on with immunoprecipitation of the GLUT1 glucose transporter, the only isoform expressed in these cells. The total cellular GLUT1 content wa s also determined by photolabelling and immunoprecipitation after cell permeabilization with digitonin (0.025 %). In glucose-starved cells, 85 % of the glucose transporters were present at the cell surface in t he basal state, with little change in response to insulin (200 nM), co rrelating with lack of additional 2-deoxyglucose uptake in response to insulin. Feeding the cells with glucose (25 mM) for 24 h resulted in an 80 % decrease in the total GLUT1 content relative to starved cells, of which only 25 % were present on the cell surface. This was associa ted with an 85 % decrease in 2-deoxyglucose uptake. In addition, acute stimulation of the fed cells with insulin or phorbol 12-myristate 13- acetate (PMA) led to an increase in GLUT1 at the cell surface, and, in correspondence, an increase in 2-deoxyglucose uptake by approx. 2- an d 4-fold respectively. We conclude that exofacial photoaffinity labell ing of glucose transporters with ATB-[2-H-3]BMPA in the presence and a bsence of digitonin, followed by specific immunoprecipitation, provide s an accurate measure of total and cell-surface glucose transporters i n differentiated BC3H-1 muscle cells. This technique demonstrates that glucose pre-feeding (1) decreases the total number of GLUT1 and (2) r edistributes the majority of the remaining transporters to an intracel lular site, where they can now be translocated to the cell surface in response to insulin and PMA.