The facilitative hexose transporter GLUT1 activity is blocked by tyrosine kinase inhibitors that include natural products such as flavones and isoflavones and synthetic compounds such as tyrphostins, molecules that are structurally unrelated to the transported substrates [Vera, et al. (2001) Biochemistry, 40, 777-790]. Here we analyzed the interaction of GLUT1 with quercetin (a flavone), genistein (an isoflavone), and tyrphostin A47 and B46 to evaluate if they share one common or have several binding sites on the protein. Kinetic assays showed that genistein, quercetin, and tyrphostin B46 behave as competitive inhibitors of equilibrium exchange and zero-trans uptake transport and noncompetitive inhibitors of net sugar exit out of human red cells, suggesting that they interact with the external surface of the GLUT1 molecule. In contrast, tyrphostin A47 was a competitive inhibitor of equilibrium exchange and zero-trans exit transport and a noncompetitive inhibitor of net sugar entry into red cells, suggesting that it interacts with the cytoplasmic surface of the transporter. Genistein protected GLUT1 against iodide-elicited fluorescence quenching and also decreased the affinity of d-glucose for its external binding site, while quercetin and tyrphostins B46 and A47 promoted fluorescence quenching and did not affect the external d-glucose binding site. These findings are explained by a carrier that presents at least three binding sites for tyrosine kinase inhibitors, in which (i) genistein interacts with the transporter in a conformation that binds glucose on the external surface (outward-facing conformation), in a site which overlaps with the external binding site for d-glucose, (ii) quercetin and tyrphostin B46 interact with the GLUT1 conformation which binds glucose by the internal side of the membrane (inward-facing conformation), but to a site accessible from the external surface of the protein, and (iii) the binding site for tyrphostin A47 is accessible from the inner surface of GLUT1 by binding to the inward-facing conformation of the transporter. These data provide groundwork for a molecular understanding of how the tyrosine kinase inhibitors directly affect glucose transport in animal cells.

Salas M, Obando P, Ojeda L, Ojeda P, Pérez A, VargasUribe M, Rivas CI, Vera JC, Reyes AM. Resolution of the direct interaction with and inhibition of the human GLUT1 hexose transporter by resveratrol from its effect on glucose accumulation. Am J Physiol Cell Physiol 305: C90 -C99, 2013. First published April 24, 2013; doi:10.1152/ajpcell.00387.2012.-Resveratrol acts as a chemopreventive agent for cancer and as a potential antiobesity and antidiabetic compound, by leading to reduced body fat and improved glucose homeostasis. The exact mechanisms involved in improving hyperglycemic state are not known, but most of the glucose uptake into mammalian cells is facilitated by the GLUT hexose transporters. Resveratrol is structurally similar to isoflavones such as genistein, which inhibit the glucose uptake facilitated by the GLUT1 hexose transporter. Here we examined the direct effects of resveratrol on glucose uptake and accumulation in HL-60 and U-937 leukemic cell lines, which express mainly GLUT1, under conditions that discriminate transport from the intracellular substrate phosphorylation/ accumulation. Resveratrol blocks GLUT1-mediated hexose uptake and thereby affects substrate accumulation on these cells. Consequently, we characterized the mechanism involved in inhibition of glucose uptake in human red cells. Resveratrol inhibits glucose exit in human red cells, and the displacement of previously bound cytochalasin B revealed the direct interaction of resveratrol with GLUT1. Resveratrol behaves as a competitive blocker of glucose uptake under zero-trans exit and exchange kinetic assays, but it becomes a mixed noncompetitive blocker when zero-trans entry transport was assayed, suggesting that the binding site for resveratrol lies on the endofacial face of the transporter. We propose that resveratrol interacts directly with the human GLUT1 hexose transporter by binding to an endofacial site and that this interaction inhibits the transport of hexoses across the plasma membrane. This inhibition is distinct from the effect of resveratrol on the intracellular phosphorylation/accumulation of glucose. glucose transport; GLUT1 transporter; hexose uptake; leukemic cells; resveratrol CANCER CELLS PRODUCE ENERGY predominantly via the anaerobic degradation of glucose. This process, which occurs even in the presence of high concentration of oxygen, assures the high rate of proliferation of cancer cells (67). Recent evidence indicates that the rapid breakdown of glucose through both the glycolytic and pentose phosphate pathways provides not only a quick source of energy but also an immediate supply of metabolites intermediates that will finally feed the biosynthetic pathways (13,24,61). Consistently, the consumption of glucose in cancer cells is significantly enhanced compared with normal cells, indicating that glucose plays a key role in the survival of cancer cells (12,20,25). Indeed, cancer cells are sensitive to glucose deprivation, as limiting the supply of glucose prevents cell proliferation and induces apoptosis (4,36,...

Glucose transporter (GLUT)1 has become an attractive target to block glucose uptake in malignant cells since most cancer cells overexpress GLUT1 and are sensitive to glucose deprivation. Methylxanthines are natural compounds that inhibit glucose uptake; however, the mechanism of inhibition remains unknown. Here, we used a combination of binding and glucose transport kinetic assays to analyze in detail the effects of caffeine, pentoxifylline, and theophylline on hexose transport in human erythrocytes. The displacement of previously bound cytochalasin B revealed a direct interaction between the methylxanthines and GLUT1. Methylxanthines behave as noncompetitive blockers (inhibition constant values of 2-3 mM) in exchange and zero-trans efflux assays, whereas mixed inhibition with a notable uncompetitive component is observed in zero-trans influx assays (inhibition constant values of 5-12 mM). These results indicate that methylxanthines do not bind to either exofacial or endofacial d-glucose-binding sites but instead interact at a different site accessible by the external face of the transporter. Additionally, infinite-cis exit assays (Sen-Widdas assays) showed that only pentoxifylline disturbed d-glucose for binding to the exofacial substrate site. Interestingly, coinhibition assays showed that methylxanthines bind to a common site on the transporter. We concluded that there is a methylxanthine regulatory site on the external surface of the transporter, which is close but distinguishable from the d-glucose external site. Therefore, the methylxanthine moiety may become an attractive framework for the design of novel specific noncompetitive facilitative GLUT inhibitors.

GLUT12 is a member of the facilitative family of glucose transporters. The goal of this study was to characterize the functional properties of GLUT12, expressed in Xenopus laevis oocytes, using radiotracer and electrophysiological methods. Our results showed that GLUT12 is a facilitative sugar transporter with substrate selectivity: d-glucose ≥ α-methyl-d-glucopyranoside (α-MG) > 2-deoxy-d-glucose(2-DOG) > d-fructose = d-galactose. α-MG is a characteristic substrate of the Na(+)/glucose (SGLT) family and has not been shown to be a substrate of any of the GLUTs. In the absence of sugar, (22)Na(+) was transported through GLUT12 at a higher rate (40%) than noninjected oocytes, indicating that there is a Na(+) leak through GLUT12. Genistein, an inhibitor of GLUT1, also inhibited sugar uptake by GLUT12. Glucose uptake was increased by the PKA activator 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) but not by the PKC activator phorbol-12-myristate-13-acetate (PMA). In high K(+) concentrations, glucose uptake was blocked. Addition of glucose to the external solution induced an inward current with a reversal potential of approximately -15 mV and was blocked by Cl(-) channel blockers, indicating the current was carried by Cl(-) ions. The sugar-activated Cl(-) currents were unaffected by genistein. In high external K(+) concentrations, sugar-activated Cl(-) currents were also blocked, indicating that GLUT12 activity is voltage dependent. Furthermore, glucose-induced current was increased by the PKA activator 8-Br-cAMP but not by the PKC activator PMA. These new features of GLUT12 are very different from those described for other GLUTs, indicating that GLUT12 must have a specific physiological role within glucose homeostasis, still to be discovered.

Gossypol is a natural disesquiterpene that blocks the activity of the mammalian facilitative hexose transporter GLUT1. In human HL-60 cells, which express GLUT1, Chinese hamster ovary cells overexpressing GLUT1, and human erythrocytes, gossypol inhibited hexose transport in a concentration-dependent fashion, indicating that blocking of GLUT1 activity is independent of cellular context. With the exception of red blood cells, the inhibition of cellular transport was instantaneous. Gossypol effect was specific for the GLUT1 transporter since it did not alter the uptake of nicotinamide by human erythrocytes. Gossypol affects the glucose-displaceable binding of cytochalasin B to GLUT1 in human erythrocyte ghost in a mixed noncompetitive way, with a K(i) value of 20 microM. Likewise, GLUT1 fluorescence was quenched approximately 80% by gossypol, while Stern-Volmer plots for quenching by iodide displayed increased slopes by gossypol addition. These effects on protein fluorescence were saturable and unaffected by the presence of D-glucose. Gossypol did not alter the affinity of D-glucose for the external substrate site on GLUT1. Kinetic analysis of transport revealed that gossypol behaves as a noncompetitive inhibitor of zero-trans (substrate outside but not inside) transport, but it acts as a competitive inhibitor of equilibrium-exchange (substrate inside and outside) transport, which is consistent with interaction at the endofacial surface, but not at the exofacial surface of the transporter. Thus, gossypol behaves as a quasi-competitive inhibitor of GLUT1 transport activity by binding to a site accessible through the internal face of the transporter, but it does not, in fact, compete with cytochalasin B binding. Our observations suggest that some effects of gossypol on cellular physiology may be related to its ability to disrupt the normal hexose flux through GLUT1, a transporter expressed in almost every kind of mammalian cell and responsible for the basal uptake of glucose.

The polyphenol nordihydroguaiaretic acid ( NDGA ) has antineoplastic properties, hence it is critical to understand its action at the molecular level. Here, we establish that NDGA inhibits glucose uptake and cell viability in leukemic HL ‐60 and U‐937 cell lines. We monitored hexose uptake using radio‐labeled 2‐deoxyglucose (2 DG ) and found that the inhibition by NDGA followed a noncompetitive mechanism. In addition, NDGA blocked hexose transport in human red blood cells and displaced prebound cytochalasin B from erythrocyte ghosts, suggesting a direct interaction with the glucose transporter GLUT 1. We propose a model for the mechanism of action of NDGA on glucose uptake. Our study shows for the first time that NDGA can act as inhibitor of the glucose transporter GLUT 1.

Facilitative glucose transport through plasma membrane is facilitated by GLUT1, transporter protein expressed in most cells. To identify regions on the protein involved on sensitivity towards inhibitors of transport, we identify three short sequences that show homology with typical ATP‐binding sites, as putative targets for inhibitor interaction. We probe the functional role of these regions by site directed mutagenesis and expression on Xenopus oocytes. For each mutant we analyze their kinetic properties for transport and their abilities to be affected by cytochalasin B, quercetin and tyrphostin A47. We identify mutants of exofacial orientation that suffers complete loss of sensitivity towards quercetin. Besides, we detected two groups of mutants of endofacial orientation that exhibit a decreased sensitivity to tyrphostin A47 or cytochalasin B. These data suggests that there are at least two regulatory regions, one accessible from the external side and another located on the internal surface of the protein and identify amino acid residues implied on the interaction with these ligands (Financed by FONDECYT 1060198 and FONDEF D07I1117).

The GLUT2 carrier is the major glucose transporter isoform expressed in hepatocytes, insulin‐secreting pancreatic beta cells, and absorptive epithelial cells of the intestinal mucosa and kidney. GLUT2 is suggested to play a critical role in maintaining glucose homeostasis because it functions as a low affinity, high‐turnover transport machinery. However, little is known regarding the mechanism of transport at the structural level. Here, we designed a cysteine‐less isoform of GLUT2 (C‐less GLUT2) to serve as platform for functional and structural studies, i.e. cysteine‐scanning mutagenesis in combination with thiol chemical modification. The kinetic properties of the C‐less GLUT2 expressed into Xenopus oocytes were like those of the native GLUT2, except that the overall activity was lower than that of the wild‐type transporter. Like the native protein, the C‐less GLUT2 transported methylglucose and deoxyglucose, and the activity was reduced in the presence of other sugars such as fructose, sorbitol, 3‐O‐methyl‐D‐glucose, galactose and ribose. Similarly, the activity of the C‐less isoform was inhibited by the classical GLUT2 inhibitors cytochalasin B, quercetin and resveratrol. This C‐less protein was refractory to MTSET, a membrane‐impermeable thiol‐alkylating reagent that obliterated the activity of native GLUT2. Our data suggest that the C‐less isoform of GLUT2 retains native‐like function and therefore is a suitable platform to perform structure‐function analysis by cysteine‐scanning mutagenesis.Support or Funding InformationSupported by grants from FONDECYT 1130386, and FONDEF D11I1131