Cadmium Increases GLUT1 Substrate Binding Affinity in Vitro While Reducing Its Cytochalasin B Binding Affinity
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Cadmium stimulates glucose transport in fibroblasts, apparently by increasing the intrinsic activity of GLUT1 [Harrison, S. A., Buxton, J. M., Clancy, B. M., & Czech, M. P. (1991) J. Biol. Chem. 266, 19438−19449]. In the present study, we examined whether cadmium affects the binding in vitro of purified GLUT1 to glucose and cytochalasin B. Cadmium inhibited cytochalasin B binding to GLUT1 competitively by reducing its binding affinity with an apparent inhibition constant of approximately 0.2 mM. However, d-glucose displaced cytochalasin B bound to GLUT1 as effectively in the presence of cadmium as in its absence, and detailed analysis of this displacement revealed that cadmium in fact increases the substrate binding affinity significantly. These findings suggest that cadmium induces a specific conformational change in GLUT1 that interferes with cytochalasin B binding but enhances substrate binding. This is the first clear demonstration in which the substrate and cytochalasin B binding activities of GLUT1 are differentially affected, which may offer insight into the workings of the glucose transporter.Keywords:
Cytochalasin B
Cytochalasin
Cadmium stimulates glucose transport in fibroblasts, apparently by increasing the intrinsic activity of GLUT1 [Harrison, S. A., Buxton, J. M., Clancy, B. M., & Czech, M. P. (1991) J. Biol. Chem. 266, 19438−19449]. In the present study, we examined whether cadmium affects the binding in vitro of purified GLUT1 to glucose and cytochalasin B. Cadmium inhibited cytochalasin B binding to GLUT1 competitively by reducing its binding affinity with an apparent inhibition constant of approximately 0.2 mM. However, d-glucose displaced cytochalasin B bound to GLUT1 as effectively in the presence of cadmium as in its absence, and detailed analysis of this displacement revealed that cadmium in fact increases the substrate binding affinity significantly. These findings suggest that cadmium induces a specific conformational change in GLUT1 that interferes with cytochalasin B binding but enhances substrate binding. This is the first clear demonstration in which the substrate and cytochalasin B binding activities of GLUT1 are differentially affected, which may offer insight into the workings of the glucose transporter.
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At any instant, the human erythrocyte sugar transporter presents at least one sugar export site but multiple sugar import sites. The present study asks whether the transporter also presents more than one sugar exit site. We approached this question by analysis of binding of [3H]cytochalasin B (an export conformer ligand) to the human erythrocyte sugar transporter and by analysis of cytochalasin B modulation of human red blood cell sugar uptake. Phloretin-inhibitable cytochalasin B binding to human red blood cells, to human red blood cell integral membrane proteins, and to purified human red blood cell glucose transport protein (GluT1) displays positive cooperativity at very low cytochalasin B levels. Cooperativity between sites and Kd(app) for cytochalasin B binding are reduced in the presence of intracellular ATP. Red cell sugar uptake at subsaturating sugar levels is inhibited by high concentrations of cytochalasin B but is stimulated by lower (<20 nM) concentrations. Increasing concentrations of the e1 ligand forskolin also first stimulate then inhibit sugar uptake. Cytochalasin D (a cytochalasin B analogue that does not interact with GluT1) is without effect on sugar transport over the same concentration range. Cytochalasin B and ATP binding are synergistic. ATP (but not AMP) enhances [3H]cytochalasin B photoincorporation into GluT1 while cytochalasin B (but not cytochalasin D) enhances [γ-32P]azidoATP photoincorporation into GluT1. We propose that the red blood cell glucose transporter is a cooperative tetramer of GluT1 proteins in which each protein presents a translocation pathway that alternates between uptake (e2) and export (e1) states but where, at any instant, two subunits must present uptake (e2) and two subunits must present exit (e1) states.
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Specific, anti-peptide antibodies against the five known mammalian facilitative D-glucose transporter isoforms (GLUT1-5) were used to investigate the transporter content of lactating rat mammary gland. Western blots of gland homogenate showed the apparent presence of GLUT1 and GLUT4. However, in isolated epithelial cells only GLUT1 was present, a result which indicated the mammary GLUT4 is present within adipocytes. The putative GLUT1 glucose transporter was recognised by antibodies against several hydrophilic regions of human erythrocyte GLUT1 and endoglycosidase F digestion decreased its apparent Mr from 50,000 to 42,000, a value essentially identical to that of the deglycosylated human protein. Its identity as a glucose transporter was confirmed by the ability of anti-GLUT1 antibodies to immunoprecipitate a mammary protein of apparent 50,000, which could be photolabelled in a D-glucose-sensitive fashion by the transport inhibitor [4-3H]cytochalasin B. Immunocytochemical staining of sections confirmed that the primary location of GLUT1 in the mammary gland is the epithelial cell. Sub-cellular fractionation experiments showed that the transporter is not only located at the cell surface, but also within the Golgi. However, quantitative Western blotting indicated that GLUT1 could only account for about half of the D-glucose-sensitive cytochalasin B binding sites in Golgi vesicles, suggesting the presence of a second, so far unidentified, glucose transporter isoform. Developmental changes in transporter expression in the gland were investigated both at the level of protein and mRNA. GLUT1 protein levels became detectable in the epithelial cells during the final 24-48hr of pregnancy, rose to a peak immediately following parturition, then fell slightly at the time of peak lactation. No major changes in GLUT1 mRNA were observed, implying a post-transcriptional control of GLUT1 expression. Removal of the litter for 24hr at peak lactation resulted in a total loss of GLUT1, suggesting feed-back inhibition influencing GLUT1 expression. The hormonal basis of these changes was investigated using explant culture of alveoli from mid-pregnant mammary gland. These experiments indicated that GLUT1 expression was dependent upon a lactogenic hormone, prolactin. Insulin and cortisol were also required for explant viability, but neither alone could stimulate synthesis of GLUT1.
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Facilitative glucose transporter-1 (GLUT1) is expressed abundantly and has an important role in glucose transfer in placentas. However, little is known about the regulation of GLUT1 expression in placental cells. We studied the changes in placental GLUT1 levels in relation to changes in glucose concentration in vitro and in vivo. In in vitro experiments, dispersed mouse placental cells were incubated under control (5.5 mM) and moderately high (22 mM) glucose concentrations, and 2-deoxyglucose uptake into cells was studied on days 1-5 of culture. After 4 days of incubation under both conditions, GLUT1 mRNA and proten levels were examined by Northern and immunoblot analyses. Treatment of cells with 22 mM glucose resulted in a significant decrease in 2-deoxyglucose uptake compared with control, from day 2 to day 5 of culture. Moreover, GLUT1 mRNA and protein levels on day 4 of culture were significantly reduced in cells incubated with 22 mM glucose compared with control. Next, we rendered mice diabetic by administering 200 micrograms/g body weight streptozotocin (STZ) on day 8 of pregnancy. Animals were killed on day 12 of pregnancy and placental tissues were obtained. [3H]Cytochalasin B binding study was carried out to assess total GLUTs, and GLUT1 mRNA and protein were measured as above. [3H]Cytochalasin B binding sites in placentas from STZ-treated mice were significantly less than those in control mice. Northern and immunoblot analyses revealed a significant decrease in GLUT1 mRNA and protein levels in diabetic mice compared with the controls. These findings suggest that the glucose concentration may regulate the expression of placental GLUT1.
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Significance This paper reports the first structure of WT-human glucose transporter 1 (hGLUT1), to our knowledge, cocrystallized with inhibitors. The structures provide a template to develop therapeutic inhibitors applicable to cancers, because cancer cells become dependent on greatly increased glucose consumption. This dependence results in up-regulation of glucose transporter expression, especially hGLUT1. The bound inhibitors include the natural compound cytochalasin B and two of a series of previously undescribed organic compounds that bind in the submicromolar range. Our results emphasize that modulation of glucose import by hGLUTs should focus on making good interaction points for compounds and that the actual chemical backbone of the inhibitor is of less importance.
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The human erythrocyte-type glucose transporter (GLUT1) has been abundantly expressed in insect cells by using a recombinant baculovirus. At 4 days after infection with the virus, the insect cell-surface and intracellular membranes were found to contain greater than 200 pmol of D-glucose-sensitive binding sites for the transport inhibitor cytochalasin B per mg of protein. The characteristics of binding were identical with those of the erythrocyte transporter, although the two proteins differed substantially in apparent Mr, probably as a result of glycosylation differences.
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