Abstract
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.
Highlights
Exposure of Clone 9 cells to inhibitors of oxidative [3,4,5,6,7,8,9] and hasbeen the subject of renewed interest in the past phosphorylation results in a rapid and striking stimu- few years [10,11,12,13,14,15,16]
We focussed on the early phase of the response because the 7.5fold stimulation of glucose transport that occurs over this interval takes place in the absence of any change in whole cell GLUT-1 mRNA or GLUT-1 content (Ref. 16 and Figs. 1 and 3), suggesting that the stimulation is mediated entirely by post-translational mechanisms
Further studieswere carried out to differentiate between mechanisms that result in a redistribution of GLUT-1 sites leading to an increased number of glucose transporter molecules in the plasma membrane and mechanisms that lead to an activation of transporter molecules pre-existing in theplasma membrane
Summary
Exposure of Clone 9 cells to inhibitors of oxidative [3,4,5,6,7,8,9] and hasbeen the subject of renewed interest in the past phosphorylation results in a rapid and striking stimu- few years [10,11,12,13,14,15,16]. The enrichmentin a1 subunit is evident azide, there was a significant increase inthe GLUT-1 content in the lanes containing theplasma membranes (lanes 9-12); of the cell homogenate (1.6 f 0.1-fold), the postnuclear fracin repeated experiments the enrichment averaged 5.3 f 0.6- tion (2.0 f 0.3-fold), the supernatant following reaction with fold and was the same in controland azide-treated cells.
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