Rapid activation of GLUT-1 glucose transporter following inhibition of oxidative phosphorylation in clone 9 cells.
Exposure of Clone 9 cells to inhibitors of oxidative phosphorylation results in a rapid and striking stimulation of facilitated glucose transport (7.5-fold at 2 h) that is mediated by the GLUT-1 transporter. We have previously shown that this rapid stimulation of glucose transport occurs in the absence of any detectable increase in cell GLUT-1 or GLUT-1 mRNA content. To determine whether this early enhancement of transport is attributable to a translocation of glucose transporters to the plasma membrane, or instead to an activation of transporters already present in the plasma membrane, we have employed four different approaches to determine whether the stimulation of transport is accompanied by a corresponding increase in plasma membrane GLUT-1 sites: 1) immunofluorescence microscopy; 2) quantitation of GLUT-1 sites in plasma membrane fractions isolated by differential centrifugation and subsequent Western blotting; 3) cell surface biotinylated followed by isolation of plasma membranes and quantitation of GLUT-1 sites by Western blotting; and 4) quantitation of GLUT-1 sites in plasma membrane fractions by [3H]cytochalasin B binding. Each of these experimental approaches led to the same conclusion, namely that the large stimulation of glucose transport observed during the early phase of the response to azide is associated with only a slight increase in the abundance of GLUT-1 sites in the plasma membrane. These results strongly suggest that activation of GLUT-1 sites pre-existing in the plasma membrane is the dominant mechanism mediating the early glucose transport response to inhibition of oxidative phosphorylation.
Shetty
M
M
Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, New York 10032.
Loeb
J N
JN
Vikstrom
K
K
Ismail-Beigi
F
F
D000818:Animals; D001386:Azides / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D001710:Biotin; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003571:Cytochalasin B / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D005947:Glucose / Q000378:metabolism*; D051272:Glucose Transporter Type 1; D007700:Kinetics; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D010085:Oxidative Phosphorylation; D051381:Rats
D001386:Azides; D051272:Glucose Transporter Type 1; D009004:Monosaccharide Transport Proteins; C495679:Slc2a1 protein, rat; D003571:Cytochalasin B; D001710:Biotin; D005947:Glucose