M. Shetty et al., RAPID ACTIVATION OF GLUT-1 GLUCOSE-TRANSPORTER FOLLOWING INHIBITION OF OXIDATIVE-PHOSPHORYLATION IN CLONE-9 CELLS, The Journal of biological chemistry, 268(23), 1993, pp. 17225-17232
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.