To help resolve the question of
To help resolve the question of simple binding versus transport, we reasoned that if quercetin is transported by GLUT1, we should be able to block its uptake by other GLUT1 inhibitors. We initially investigated the effects of two newly identified GLUT1 inhibitors, BAY-876 and WZB-117. WZB-117 was first identified as a small molecule inhibitor of GLUT1 that had anticancer activity in a nude mouse model . Subsequently, the inhibitory and binding properties of WZB-117 were systematically investigated in erythrocytes and transformed HEK292 cells where it was shown to be an exofacial inhibitor binding preferentially to the central glucose channel in the outwardly open GLUT1 conformation . More recently, BAY-876 was identified as the first highly selective inhibitor of GLUT1 with a reported IC50 of 2 nM for GLUT1, which was 150–5000 times lower than its IC50 for GLUT2, GLUT3, or GLUT4 . There are no additional studies utilizing this inhibitor which delineate its mechanism of action. Our data reveal that both of these compounds are robust inhibitors of GLUT1-mediated 2DG uptake in L929 cells. The IC50 for WZB-117 inhibition of 2DG uptake in L929 cells is close to the values reported for its inhibition of 3-O-methylglucose uptake in erythrocytes (IC50 = 6.2 μM)  and its inhibition of cancer growth (IC50 = 10 μM) . In contrast, the IC50 for BAY-876 is about 10-fold higher than previously reported . This discrepancy can likely be attributed to the utilization of different assays for measuring glucose uptake and different cell types. In this study we utilized a 2DG uptake assay in L929 fibroblast cells, which is a more direct measure of GLUT1 transport activity while the previous study that utilized a luciferase assay for cellular ATP content in rotenone-poisoned CHO Azacyclonol as a proxy for GLUT transport activity. Somewhat unexpectedly, WZB-117 and BAY-876 differed in their effect on quercetin uptake. WZB-117 reduced quercetin uptake, consistent with inhibiting transport, but the more robust glucose uptake inhibitor, BAY-876, had no effect on quercetin uptake. Our findings with cytochalasin B are also inconsistent with quercetin transport. This classic potent inhibitor of GLUT1 activity actually increases the binding of quercetin to L929 cells. Unlike WZB-117, cytochalasin B is an endofacial binding inhibitor of the GLUT1 [37,38], which provides some information about the mode by which quercetin engages transporters. We also found that quercetin enhances cytochalasin B binding, which suggests that both inhibitors can simultaneously bind to GLUT1. A dual binding of quercetin and cytochalasin B is consistent with the observation that the combined inhibitory effects of these two compounds are greater than the inhibition of either alone. The most straightforward interpretation of these data is that cyochalasin B binds at an endofacial site on GLUT1, which stabilizes the external binding of quercetin, and conversely, the external binding of quercetin on GLUT1 stabilizes the endofacial binding of cytochalasin B. This model conflicts with a previous study that demonstrated that quercetin inhibited cytochalasin B binding to GLUT1 . That study, however, measured binding in unsealed erythrocyte ghosts where quercetin has access to both the internal and external surfaces of GLUT1. We propose that quercetin does not inhibit cytochalasin B binding in live L929 cells because it is not transported into the cells, and therefore does not have access to the endofacial cytochalasin B binding site. Our observation of the dual binding of quercetin and cytochalasin B is similar to the effects of WZB-117 on cytochalasin B binding. WZB-117 did not inhibit cytochalasin B binding in intact human erythrocytes, but did inhibit cytochalasin B binding in protein depleted membranes where WZB-117 has access to both surfaces of the erythrocyte membrane . The authors concluded from that observation that WZB-117 was not transported by GLUT1 into erythrocytes.