The structure and activity of GLUT has been most intensively

The structure and activity of GLUT1 has been most intensively studied in erythrocytes, in which this transporter makes up 10–20% of membrane protein content. Carruthers has shown that, while GLUT1 can exist and transport glucose as monomers, dimers and tetramers, in erythrocytes GLUT1 exists predominately as homotetrameric complexes. These tetramers are stabilized by an internal disulfide bond within each GLUT1 monomer and have greater transport activity than the monomeric or dimeric forms [1], [21]. The self-association of GLUT1 has also been demonstrated in kidney Mycophenolate Mofetil where it is dependent, at least in part, on GLUT1 concentration [22]. Thus, stimulated association of GLUT1 into tetramers is an appealing mechanism to explain the acute activation of glucose uptake in L929 cells, but this dynamic multimerization has yet to be directly demonstrated. Given the sensitivity of acute GLUT1 activation to cysteine chemistry, however, it remains possible that the activation of glucose uptake in L929 cells involves an increase in tetramer formation driven by a dynamic regulation of disulfide bond chemistry within individual GLUT1 proteins [23], [24], [25], [26], [27]. One indirect means of demonstrating the presence of tetramers within a given cell line is to test its response to an inhibitor of glucose transport that preferentially binds to and inhibits the tetramer form of GLUT1. One such inhibitor is the nucleotide triphosphate ATP, which acts as an uncompetitive inhibitor of GLUT1. The nucleotide-binding site maps to an endofacial site that is only available in tetramers [28], [29], and is lost when tetrameric structure is disrupted [30], [31]. Recently, it was shown that caffeine mimics ATP by binding to this nucleotide-binding site and eliciting a similar conformational change in GLUT1 that is induced by ATP [32], [33].
Materials and methods
Results
Discussion Caffeine binds to the ATP binding site located on the endofacial side of GLUT1, and like ATP, is a potent uncompetitive inhibitor of GLUT1 activity [32], [33]. However, this work was done in erythrocytes where the concentration of GLUT1 is particularly high (10–20% of membrane protein) and thus the effects of caffeine may be unique to these cells. In erythrocytes the dominant structure of GLUT1 is a homotetramer, which has greater transport activity than the dimer and monomer forms of GLUT1 [1], [40]. The tetramer is stabilized by the formation of an internal disulfide bond between Cys347 and Cys421 within each subunit [21], [40]. ATP binds to the tetramer form, and thus exposure of erythrocytes to a reducing agent leads to the transition of GLUT1 tetramers to dimers and the subsequent loss of ATP binding and inhibition [30], [31]. Caffeine binds to the same site as ATP and induces a similar conformational change in GLUT1. Specifically, both ATP and caffeine appear to trigger a conformational change that brings the C-terminus of GLUT1 closer to the intracellular loop connecting transmembrane helices 6–7, creating a ‘cage-like’ structure around the endofacial exit site for glucose transport that likely impedes the release of glucose into the cytoplasmic space [32]. This does not affect glucose exchange through the transporter but does lead to uncompetitive inhibition of net glucose uptake. These data suggest that caffeine inhibition may be an indirect probe for the tetrameric structure of GLUT1. Therefore, the purpose of this study was to determine if caffeine inhibits glucose uptake in other GLUT1 expressing cells and if the inhibition correlates with the content level and/or activity level of the transporter. The L929 fibroblast cell line, which we have used to characterize glucose uptake by GLUT1, presents a unique cellular model in contrast to erythrocytes. These cells express exclusively GLUT1, but at a much lower level than in erythrocytes [37]. We show that caffeine inhibits 2DG uptake in L929 cells in a dose-dependent manner with uncompetitive kinetics, which mirrors its effects in erythrocytes. The effects of caffeine are immediate and reversible, and are also additive to the competitive inhibitory effects of glucose, which is consistent with previous work showing that caffeine does not interfere with glucose binding sites [32]. We also show that the inhibition by caffeine is not additive to the effects of cytochalasin B. This is again consistent to the results from erythrocytes, where the high affinity binding sites for caffeine and cytochalasin B map to overlapping sites and caffeine can inhibit specific 3H-cytochalasin B binding [32]. Finally, it had been suggested by an earlier study that curcumin might share a binding site with caffeine based on the observation that both inhibitors compete with cytochalasin B binding but not with glucose [38]. However, contrary to that suggestion, we show that the inhibitory effects of the maximally effective concentrations of caffeine and curcumin are additive, indicating that these inhibitors do not share binding sites.