title: Mechanism of stimulation of glucose transport in response to inhibition of oxidative phosphorylation: analysis with myc-tagged Glut1.
abstract: To determine the role of 'translocation' vs. 'activation' of Glut1 in the stimulation of glucose transport in response to inhibition of oxidative phosphorylation, we measured the abundance of myc-tagged Glut1 in plasma membrane of stably transfected Clone 9 cells, a rat liver cell line expressing only the Glut1 isoform. The myc epitope-tag is located between Ile56 and Pro57 in the putative first extracellular loop of Glut1. Under basal conditions, transfected cells expressed approximately 3 fold higher levels of Glut1 and exhibited a approximately 3 fold higher rate of glucose transport than non-transfected cells. To delineate the mechanism mediating the stimulation of glucose transport by a azide we employed two strategies: (1) mild cell surface biotinylation followed by isolation of plasma membranes and quantitation of Glut1 sites in Western blots employing anti-Glut1 and anti-myc antibodies, and (2) quantitative immunofluorescence of myc epitopes in plasma membrane sheets. The rate of glucose transport increased 2.9 +/- 0.5 fold in transfected cells exposed to 5 mM azide for 1 h. Exposure to azide, however, resulted in no significant increase in Glut1 content of plasma membranes using anti-Glut1 or anti-myc antibodies in Western blots (1.0 +/- 0.1 and 0.9 +/- 0.2 fold, respectively; azide/control), and was associated with no detectable increase in immunofluorescence using either anti-Glut1 or anti-myc antibodies (p > 0.1 for both measurements). Treatment of cells with cobalt chloride (employed as a positive control) resulted in marked increases in glucose transport, cell and plasma membrane Glut1 content, and immunofluorescence of plasma membrane sheets (8-10 fold increase in each parameter). We conclude that the stimulation of glucose transport by azide results mainly from activation of Glut1 transporters pre-existing in the plasma membrane.
authors.affiliation: Department of Medicine, Case Western Reserve University, Cleveland, OH 44106-4951, USA.
authors.forename: M H
authors.initials: MH
authors.lastname: Koseoglu
authors.forename: F I
authors.initials: FI
authors.lastname: Beigi
mesh_terms: D000818:Animals; D001386:Azides / Q000494:pharmacology; D001692:Biological Transport; D015153:Blotting, Western; D002999:Clone Cells; D003035:Cobalt / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D016259:Genes, myc; D005947:Glucose / Q000378:metabolism*; D051272:Glucose Transporter Type 1; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D010085:Oxidative Phosphorylation; D051381:Rats
chemical_list: D001386:Azides; D051272:Glucose Transporter Type 1; D009004:Monosaccharide Transport Proteins; C495679:Slc2a1 protein, rat; D003035:Cobalt; C018021:cobaltous chloride; D005947:Glucose