Activation of Glut1 glucose transporter in response to inhibition of oxidative phosphorylation - Role of sites of mitochondrial inhibition and mechanism of Glut1 activation

We have previously shown that exposure of Clone 9 cells to hypoxia, cyanide , or azide results in an acute stimulation of glucose transport that is lar gely mediated by "activation" of glucose transporter (Glut1) sites preexist ing in the plasma membrane. However, it is not known whether inhibition of oxidative phosphorylation only at its terminal step, or at any of its steps , leads to the glucose transport response. Hence, the effect of azide (5 mM ), rotenone (1 mu M), rotenone (1 mu M) plus thenoyltrifluoroacetone (TTFA) (5 mu M), antimycin A (0.3 mu M), dinitrophenol (0.25 mM), carbonyl cyanid e m-chlorophenylhydrazone (CCCP) (2.5 mu M), and, oligomycin B (0.15 mu M) on glucose transport was determined. All of the above agents elicited a sim ilar similar to 4-fold stimulation of cytochalasin B (CB)-inhibitable 3-O-m ethyl glucose (3-OMG) uptake in Clone 9 cells. The stimulatory effect of az ide on 3-OMG uptake was not inhibited by antioxidants 2-mercaptopropionyl g lycine (1.2 mM) and 1,10-phenanthroline (40 mu M), while, in contrast, the antioxidants attenuated the stimulation of glucose transport in response to 250 mu M H2O2 by similar to 50%. To differentiate between an increase in t he number of functional Glut1 sites in the plasma membrane (in the absence of "translocation") versus an increase in the "intrinsic activity" of Glut1 , the effect of azide on the energy of activation (E-a) of glucose transpor t was measured. The E-a was determined by measuring the rate of CB-inhibita ble 3-OMG uptake at 24.0, 28.0, 35.0, and 40 degrees C. The E-a of control Clone 9 cells and of cells exposed to 10 mM azide for 2 h was 32,530 +/- 18 30 and 31,220 +/- 600 J/mol, respectively (P > 0.1), while the rate of CB-i nhibitable 3-OMG uptake was 9.3 +/- 0.7-fold higher in azide-treated cells. It is concluded that (i) inhibition of oxidative phosphorylation, at any o f its steps, leads to a stimulation of glucose transport, and (ii) the mech anism of stimulation of glucose transport in response to azide appears to b e predominately mediated by an apparent increase in the number of functiona l Glut1 sites in the plasma membrane (instead of an increase in their "intr insic activity"), suggesting an "unmasking" mechanism, (C) 1999 Academic Pr ess.