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 r esults in a rapid and striking stimulation of facilitated glucose tran sport (7.5-fold at 2 h) that is mediated by the GLUT-1 transporter. We have previouslY shown that this rapid stimulation of glucose transpor t occurs in the absence of any detectable increase in cell GLUT-1 or G LUT-1 mRNA content. To determine whether this early enhancement of tra nsport is attributable to a translocation of glucose transporters to t he plasma membrane, or instead to an activation of transporters alread y present in the plasma membrane, we have employed four different appr oaches to determine whether the stimulation of transport is accompanie d by a corresponding increase in plasma membrane GLUT-1 sites: 1) immu nofluorescence microscopy; 2) quantitation of GLUT-1 sites in plasma m embrane fractions isolated by differential centrifugation and subseque nt Western blotting; 3) cell surface biotinylation followed by isolati on of plasma membranes and quantitation of GLUT-1 sites by Western blo tting; and 4) quantitation of GLUT-1 sites in plasma membrane fraction s by H-3!cytochalasin B binding. Each of these experimental approache s led to the same conclusion, namely that the large stimulation of glu cose transport observed during the early phase of the response to azid e is associated with only a slight increase in the abundance of GLUT-1 sites in the plasma membrane. These results strongly suggest that act ivation of GLUT-1 sites pre-existing in the plasma membrane is the dom inant mechanism mediating the early glucose transport response to inhi bition of oxidative phosphorylation.