Caffeine inhibition of GLUT1 is dependent on the activation state of the transporter

Abstract Caffeine has been shown to be a robust uncompetitive inhibitor of glucose uptake in erythrocytes. It preferentially binds to the nucleotide-binding site on GLUT1 in its tetrameric form and mimics the inhibitory action of ATP. Here we demonstrate that caffeine is also a dose-dependent, uncompetitive inhibitor of 2-deoxyglucose (2DG) uptake in L929 fibroblasts. The inhibitory effect on 2DG uptake in these cells was reversible with a rapid onset and was additive to the competitive inhibitory effects of glucose itself, confirming that caffeine does not interfere with glucose binding. We also report for the first time that caffeine inhibition was additive to inhibition by curcumin, suggesting distinct binding sites for curcumin and caffeine. In contrast, caffeine inhibition was not additive to that of cytochalasin B, consistent with previous data that reported that these two inhibitors have overlapping binding sites. More importantly, we show that the magnitude of maximal caffeine inhibition in L929 cells is much lower than in erythrocytes (35% compared to 90%). Two epithelial cell lines, HCLE and HK2, have both higher concentrations of GLUT1 and increased basal 2DG uptake (3–4 fold) compared to L929 cells, and subsequently display greater maximal inhibition by caffeine (66–70%). Interestingly, activation of 2DG uptake (3-fold) in L929 cells by glucose deprivation shifted the responsiveness of these cells to caffeine inhibition (35%–70%) without a change in total GLUT1 concentration. These data indicate that the inhibition of caffeine is dependent on the activity state of GLUT1, not merely on the concentration.