Molecular and cellular regulation of glucose transporter (GLUT) proteins in cancer

Malignant cells are known to have accelerated metabolism, high glucose requirements, and increased glucose uptake. Transport of glucose across the plasma membrane of mammalian cells is the first rate-limiting step for glucose metabolism and is mediated by facilitative glucose transporter (GLUT) proteins. Increased glucose transport in malignant cells has been associated with increased and deregulated expression of glucose transporter proteins, with overexpression of GLUT1 and/or GLUT3 a characteristic feature. Oncogenic transformation of cultured mammalian cells causes a rapid increase of glucose transport and GLUT1 expression via interaction with GLUT1 promoter enhancer elements. In human studies, high levels of GLUT1 expression in tumors have been associated with poor survival. Studies indicate that glucose transport in breast cancer is not fully explained by GLUT1 or GLUT3 expression, suggesting involvement of another glucose transporter. Recently, a novel glucose transporter protein, GLUT12, has been found in breast and prostate cancers. In human breast and prostate tumors and cultured cells, GLUT12 is located intracellularly and at the cell surface. Trafficking of GLUT12 to the plasma membrane could therefore contribute to glucose uptake. Several factors have been implicated in the regulation of glucose transporter expression in breast cancer. Hypoxia can increase GLUT1 levels and glucose uptake. Estradiol and epidermal growth factor, both of which can play a role in breast cancer cell growth, increase glucose consumption. Estradiol and epidermal growth factor also increase GLUT12 protein levels in cultured breast cancer cells. Targeting GLUT12 could provide novel methods for detection and treatment of breast and prostate cancer.     

Glucose transporter isoform-3 mutations cause early pregnancy loss and fetal growth restriction

Glucose transporter isoform-3 (GLUT3) is the trophoblastic facilitative glucose transporter. To investigate the role of this isoform in embryonic development, we created a novel GLUT3-null mouse and observed arrested early embryonic development and loss at neurulation stage when both alleles were mutated. This loss occurred despite the presence of other related isoforms, particularly GLUT1. In contrast, when a single allele was mutated, despite increased embryonic cell apoptosis, adaptive changes in the subcellular localization of GLUT3 and GLUT1 in the preimplantation embryo led to postimplantation survival. This survival was compromised by decreased GLUT3-mediated transplacental glucose transport, causing late-gestation fetal growth restriction. This yielded young male and female adults demonstrating catch-up growth, with normal basal glucose, insulin, insulin-like growth factor-I and IGF-binding protein-3 concentrations, fat and lean mass, and glucose and insulin tolerance. We conclude that GLUT3 mutations cause a gene dose-dependent early pregnancy loss or late-gestation fetal growth restriction despite the presence of embryonic and placental GLUT1 and a compensatory increase in system A amino acid placental transport. This critical life-sustaining functional role for GLUT3 in embryonic development provides the basis for investigating the existence of human GLUT3 mutations with similar consequences during early pregnancy.     

Differential expression of glucose transporter isoforms during embryonic stem cell differentiation

In mouse blastocysts six facilitative glucose transporter isoforms (GLUT)1-4, 8 and 9 are expressed. We have used the mouse embryonic stem (ES) cell line D3 and spontaneously differentiating embryoid bodies (EB) to investigate GLUT expression and the influence of glucose during differentiation of early embryonic cells. Both ES cells and EBs (2d-20d) expressed GLUT1, 3, and 8, whereas the isoforms 2 and 4 were detectable exclusively in EBs. Differentiation-associated expression of GLUT was analyzed by double staining with stage-specific embryonic antigen (SSEA-1), cytokeratins (CK18, 19), nestin, and desmin. Similar to trophoblast cells in mouse blastocysts the outer cell layer of endoderm-like cells showed a high GLUT3 expression in early EBs. In 20-day-old EBs no GLUT3 protein and only minor GLUT3 mRNA amounts could be detected. A minimal glucose concentration of 5 mM applied during 2 and 8 days of EB culture resulted in up-regulated GLUT4, Oct-4 and SSEA-1 levels and a delay in EB differentiation. We conclude that GLUT expression depends on cellular differentiation and that the expression is modulated by glucose concentration. The developmental and glucose-dependent regulation of GLUT strongly suggests a functional role of glucose and glucose transporters in ES cell differentiation and embryonic development.     

Differential regulation of three glucose transporter genes in a renal epithelial cell line

Three hexose transporter genes, the Na(+)/glucose cotransporters SGLT1 and SGLT3 (formerly SAAT1/pSGLT2) and the facilitative transporter GLUT1, are expressed in a renal epithelial cell line with proximal tubule characteristics. A number of studies have demonstrated that SGLT1 expression is coupled to the cellular differentiation state and is also negatively regulated by its substrate glucose. In the present study, we demonstrate that SGLT3 mRNA expression is relatively unaffected by conditions promoting dedifferentiation (reseeding to a subconfluent density, activation of protein kinase C) or differentiation (confluent cell density, activation of protein kinase A) nor was expression sensitive to hyperglycemic glucose levels in the medium. We further demonstrate that protein kinase A and protein kinase C exert opposing effects on GLUT1 and SGLT1 mRNA levels in polarized cell monolayers, indicating that GLUT1 mRNA is also highly regulated in polarized epithelial cells by agents affecting cell differentiation. The relatively constitutive expression of SGLT3 mRNA suggests a novel role for this low-affinity Na(+)/glucose cotransporter, to provide concentrative glucose uptake under hyperglycemic conditions where expression of high-affinity glucose cotransporter SGLT1 mRNA is significantly downregulated.     

Detection of the GLUT3 facilitative glucose transporter in rat L6 muscle cells: regulation by cellular differentiation, insulin and insulin-like growth factor-I.

The GLUT3 facilitative glucose transporter protein was found to be expressed in rat L6 muscle cells. It was detected at both the myoblast and myotube stage. GLUT3 protein content per mg of total membrane protein increased significantly during L6 cell differentiation. Subcellular fractionation demonstrated that the GLUT3 protein was predominantly localized in plasma membrane-enriched fractions of either myoblasts or myotubes. Short-term exposure of L6 myotubes to IGF-I or insulin caused a redistribution of GLUT3 protein from an intracellular membrane fraction to the plasma membrane, without affecting total membrane GLUT3 protein content. Long-term exposure of L6 myotubes to IGF-I produced an increase of GLUT3 protein in total membranes and all subcellular membrane fractions, especially the plasma membrane. We propose that the GLUT3 glucose transporter may play an important role in glucose metabolism in developing muscle.     

Growth factors, mitogens, oncogenes and the regulation of glucose transport

The erythrocyte (or HepG2/brain) type glucose transporter (GLUT 1) was the first of the family of facilitative glucose transporter proteins to be cloned [M. Mueckler et al., Science 229, 941–945, 1985]. GLUT 1 is expressed in most tissue types, all cells lines, transformed cells and tumour cells. It is thought to be responsible for ‘housekeeping’ levels of glucose transport, i.e. the uptake of glucose required for oxidative phosphorylation. The rate of glucose transport via GLUT 1 can be regulated under conditions in which the metabolic rate must be adjusted such as cell division (mitosis and meiosis), differentiation, transformation and nutrient starvation. Here we review the recent literature on the control of glucose transport of mitogens, growth factors and oncogenes, and discuss some of the implications for the integration of cellular signalling pathways and cell growth.     

A GSK-3/TSC2/mTOR pathway regulates glucose uptake and GLUT1 glucose transporter expression

Glucose transport is a highly regulated process and is dependent on a variety of signaling events. Glycogen synthase kinase-3 (GSK-3) has been implicated in various aspects of the regulation of glucose transport, but the mechanisms by which GSK-3 activity affects glucose uptake have not been well defined. We report that basal glycogen synthase kinase-3 (GSK-3) activity regulates glucose transport in several cell types. Chronic inhibition of basal GSK-3 activity (8-24 h) in several cell types, including vascular smooth muscle cells, resulted in an approximately twofold increase in glucose uptake due to a similar increase in protein expression of the facilitative glucose transporter 1 (GLUT1). Conversely, expression of a constitutively active form of GSK-3beta resulted in at least a twofold decrease in GLUT1 expression and glucose uptake. Since GSK-3 can inhibit mammalian target of rapamycin (mTOR) signaling via phosphorylation of the tuberous sclerosis complex subunit 2 (TSC2) tumor suppressor, we investigated whether chronic GSK-3 effects on glucose uptake and GLUT1 expression depended on TSC2 phosphorylation and TSC inhibition of mTOR. We found that absence of functional TSC2 resulted in a 1.5-to 3-fold increase in glucose uptake and GLUT1 expression in multiple cell types. These increases in glucose uptake and GLUT1 levels were prevented by inhibition of mTOR with rapamycin. GSK-3 inhibition had no effect on glucose uptake or GLUT1 expression in TSC2 mutant cells, indicating that GSK-3 effects on GLUT1 and glucose uptake were mediated by a TSC2/mTOR-dependent pathway. The effect of GSK-3 inhibition on GLUT1 expression and glucose uptake was restored in TSC2 mutant cells by transfection of a wild-type TSC2 vector, but not by a TSC2 construct with mutated GSK-3 phosphorylation sites. Thus, TSC2 and rapamycin-sensitive mTOR function downstream of GSK-3 to modulate effects of GSK-3 on glucose uptake and GLUT1 expression. GSK-3 therefore suppresses glucose uptake via TSC2 and mTOR and may serve to match energy substrate utilization to cellular growth.     

The glucose transport activity of GLUT1 is markedly decreased by substitution of a single amino acid with a different charge at residue 415

GLUT1 glucose transporter cDNA was modified to introduce a single amino acid substitution of aspartic acid for asparagine 415, which is conserved among all facilitative glucose transporter isoforms. Although a significant amount of the mutated transporter was expressed into plasma membranes of Chinese hamster ovary cells by transfection with expression vector, almost no increase in glucose transport activity was observed. Analysis of glucose uptake with Lineweaver-Burk plot depicts that the mutation induced a marked decrease (more than 5-fold) in turnover number and a slight increase (1.5-fold) in Km compared with the wild-type GLUT1. Results obtained with cytochalasin B and ethylidene glucose suggested that the inner but not outer glucose binding site was modulated. These results suggest that asparagine 415 is located close to the inner glucose binding site and the putative inner gate of GLUT1 glucose transporter and that an ionic charge in this domain might play an important role in the rate of conformational change between an inward-facing form and an outward-facing form of glucose transporter.     

The predicted ATP-binding domains in the hexose transporter GLUT1 critically affect transporter activity.

The glucose transporter GLUT1 has three short amino acid sequences (domains I-III) with homology to typical ATP-binding domains. GLUT1 is a facilitative transporter, however, and transports its substrates down a concentration gradient without a specific requirement for energy or hydrolysis of ATP. Therefore, we assessed the functional role of the predicted ATP-binding domains in GLUT1 by site-directed mutagenesis and expression in Xenopus oocytes. For each mutant, we determined the level of protein expression and the kinetics of transport under zero-trans influx, zero-trans efflux, and equilibrium exchange conditions. Although all five mutants were expressed at levels similar to that of the wild-type GLUT1, each single amino acid change in domains I or III profoundly affected GLUT1 function. The mutants Gly116-->Ala in domain I and Gly332-->Ala in domain III exhibited only 10-20% of the transport activity of the wild-type GLUT1. The mutants Gly111-->Ala in domain I and Leu336-->Ala in domain III showed altered kinetic properties; neither the apparent Km nor the Vmax for 3-methylglucose transport were increased under equilibrium exchange conditions, and they did not show the expected level of countertransport acceleration. The mutant Lys117-->Arg in domain I showed a marked increase in the apparent Km for 3-methylglucose transport under zero-trans efflux and equilibrium exchange conditions while maintaining countertransport acceleration. These results indicate that the predicted ATP-binding domains I and III in GLUT1 are important components of the region in GLUT1 involved in transport of the substrate and that their integrity is critical for maintaining the activity and kinetic properties of the transporter.     

Insulin regulates GLUT1-mediated glucose transport in MG-63 human osteosarcoma cells

Osteosarcoma is the most common type of malignant bone cancer, accounting for 35% of primary bone malignancies. Because cancer cells utilize glucose as their primary energy substrate, the expression and regulation of glucose transporters (GLUT) may be important in tumor development and progression. GLUT expression has not been studied previously in human osteosarcoma cell lines. Furthermore, although insulin and insulin-like growth factor (IGF-I) play an important role in cell proliferation and tumor progression, the role of these hormones on GLUT expression and glucose uptake, and their possible relation to osteosarcoma, have also not been studied. We determined the effect of insulin and IGF-I on GLUT expression and glucose transport in three well-characterized human osteosarcoma cell lines (MG-63, SaOs-2, and U2-Os) using immunocytochemical, RT-PCR and functional kinetic analyses. Furthermore we also studied GLUT isoform expression in osteosarcoma primary tumors and metastases by in situ hybridization and immunohistochemical analyses. RT-PCR and immunostaining show that GLUT1 is the main isoform expressed in the cell lines and tissues studied, respectively. Immunocytochemical analysis shows that although insulin does not affect levels of GLUT1 expression it does induce a translocation of the transporter to the plasma membrane. This translocation is associated with increased transport of glucose into the cell. GLUT1 is the main glucose transporter expressed in osteosarcoma, furthermore, this transporter is regulated by insulin in human MG-63 cells. One possible mechanism through which insulin is involved in cancer progression is by increasing the amount of glucose available to the cancer cell.     

Replacement of intracellular C-terminal domain of GLUT1 glucose transporter with that of GLUT2 increases Vmax and Km of transport activity.

The intracellular C-terminal domain is diverse in size and amino acid sequence among facilitative glucose transporter isoforms. The characteristics of glucose transport are also divergent, and GLUT2 has far higher Km and Vmax values compared with GLUT1. To investigate the role of the intracellular C-terminal domain in glucose transport, we expressed in Chinese hamster ovary cells the mutated GLUT1 protein whose intracellular C-terminal domain was replaced with that of GLUT2 by means of engineering the chimeric cDNA. Cytochalasin B, for which GLUT2 protein has much lower affinity, bound to this chimeric protein in a fashion similar to GLUT1. In contrast, greater transport activity was observed in this chimeric glucose transporter compared with the wild-type GLUT1 at 10 mM 2-deoxy-D-glucose concentration. The kinetic studies on 2-deoxy-D-glucose uptake revealed a 3.8-fold increase in Km and a 4.3-fold increase in Vmax in this chimeric glucose transporter compared with the wild-type GLUT1. Thus, replacement of the intracellular C-terminal domain confers the GLUT2-like property on the glucose transporter. These results strongly suggest that the diversity of intracellular C-terminal domain contributes to the diversity of glucose transport characteristics among isoforms.     

Differential regulation of the HepG2 and adipocyte/muscle glucose transporters in 3T3L1 adipocytes. Effect of chronic glucose deprivation.

Glucose transport in 3T3L1 adipocytes is mediated by two facilitated diffusion transport systems. We examined the effect of chronic glucose deprivation on transport activity and on the expression of the HepG2 (GLUT 1) and adipocyte/muscle (GLUT 4) glucose transporter gene products in this insulin-sensitive cell line. Glucose deprivation resulted in a maximal increase in 2-deoxyglucose uptake of 3.6-fold by 24 h. Transport activity declined thereafter but was still 2.4-fold greater than the control by 72 h. GLUT 1 mRNA and protein increased progressively during starvation to values respectively 2.4- and 7.0-fold greater than the control by 72 h. Much of the increase in total immunoreactive GLUT 1 protein observed later in starvation was the result of the accumulation of a non-functional or mistargeted 38 kDa polypeptide. Immunofluorescence microscopy indicated that increases in GLUT 1 protein occurred in presumptive plasma membrane (PM) and Golgi-like compartments during prolonged starvation. The steady-state level of GLUT 4 protein did not change during 72 h of glucose deprivation despite a greater than 10-fold decrease in the mRNA. Subcellular fractionation experiments indicated that the increased transport activity observed after 24 h of starvation was principally the result of an increase in the 45-50 kDa GLUT 1 transporter protein in the PM. The level of the GLUT 1 transporter in the PM and low-density microsomes (LDM) was increased by 3.9- and 1.4-fold respectively, and the GLUT 4 transporter content of the PM and LDM was 1.7- and 0.6-fold respectively greater than that of the control after 24 h of glucose deprivation. These data indicate that newly synthesized GLUT 1 transporters are selectively shuttled to the PM and that GLUT 4 transporters undergo translocation from an intracellular compartment to the PM during 24 h of glucose starvation. Thus glucose starvation results in an increase in glucose transport in 3T3L1 adipocytes via a complex series of events involving increased biosynthesis, decreased turnover and subcellular redistribution of transporter proteins.     

A possible role for a mammalian facilitative hexose transporter in the development of resistance to drugs.

We show that D- but not L-hexoses modulate the accumulation of radioactive vinblastine in injected Xenopus laevis oocytes expressing the murine Mdr1b P-glycoprotein. We also show that X. laevis oocytes injected with RNA encoding the rat erythroid/brain glucose transport protein (GLUT1) and expressing the corresponding functional transporter exhibit a lower accumulation of [3H]vinblastine and show a greater capacity to extrude the drug than do control oocytes not expressing the rat GLUT1 protein. Cytochalasin B and phloretin, two inhibitors of the mammalian facilitative glucose transporters, can overcome the reduced drug accumulation conferred by expression of the rat GLUT1 protein in Xenopus oocytes but have no significant effect on the accumulation of drug by Xenopus oocytes expressing the mouse Mdr1b P-glycoprotein. These drugs also increase the accumulation of [3H]vinblastine in multidrug-resistant Chinese hamster ovary cells. Cytochalasin E, an analog of cytochalasin B that does not affect the activity of the facilitative glucose transporter, has no effect on the accumulation of vinblastine by multidrug-resistant Chinese hamster cells or by oocytes expressing either the mouse Mdr1b P-glycoprotein or the GLUT1 protein. In all three cases, the drug verapamil produces a profound effect on the cellular accumulation of vinblastine. Interestingly, although immunological analysis indicated the presence of massive amounts of P-glycoprotein in the multidrug-resistant cells, immunological and functional studies revealed only a minor increase in the expression of a hexose transporter-like protein in resistant versus drug-sensitive cells. Taken together, these results suggest the participation of the mammalian facilitative glucose transporter in the development of drug resistance.     

Regulation of Human Trophoblast GLUT1 Glucose Transporter by Insulin-Like Growth Factor I (IGF-I)

Glucose transport to the fetus across the placenta takes place via glucose transporters in the opposing faces of the barrier layer, the microvillous and basal membranes of the syncytiotrophoblast. While basal membrane content of the GLUT1 glucose transporter appears to be the rate-limiting step in transplacental transport, the factors regulating transporter expression and activity are largely unknown. In view of the many studies showing an association between IGF-I and fetal growth, we investigated the effects of IGF-I on placental glucose transport and GLUT1 transporter expression. Treatment of BeWo choriocarcinoma cells with IGF-I increased cellular GLUT1 protein. There was increased basolateral (but not microvillous) uptake of glucose and increased transepithelial transport of glucose across the BeWo monolayer. Primary syncytial cells treated with IGF-I also demonstrated an increase in GLUT1 protein. Term placental explants treated with IGF-I showed an increase in syncytial basal membrane GLUT1 but microvillous membrane GLUT1 was not affected. The placental dual perfusion model was used to assess the effects of fetally perfused IGF-I on transplacental glucose transport and syncytial GLUT1 content. In control perfusions there was a decrease in transplacental glucose transport over the course of the perfusion, whereas in tissues perfused with IGF-I through the fetal circulation there was no change. Syncytial basal membranes from IGF-I perfused tissues showed an increase in GLUT1 content. These results demonstrate that IGF-I, whether acting via microvillous or basal membrane receptors, increases the basal membrane content of GLUT1 and up-regulates basal membrane transport of glucose, leading to increased transepithelial glucose transport. These observations provide a partial explanation for the mechanism by which IGF-I controls nutrient supply in the regulation of fetal growth.     

Expression of the glucose transporter isoform GLUT 4 is insufficient to confer insulin-regulatable hexose uptake to cultured muscle cells.

Glucose transporter isoform expression was studied in the skeletal muscle-like cell line, C2C12. Northern and Western blot analysis showed that the insulin-responsive muscle/fat glucose transporter isoform, GLUT 4, was expressed in these cells at very low levels, whereas the erythrocyte isoform, GLUT 1, was expressed at readily detectable levels. Insulin did not stimulate glucose transport in this cultured muscle cell line. The C2C12 cells were then transfected separately with either GLUT 1 or GLUT 4, and stable cell lines expressing high levels of mRNA and protein were isolated. GLUT 1-transfected cells exhibited a 3-fold increase in the amount of the GLUT 1 transporter protein which was accompanied by a 2- to 3-fold increase in the glucose uptake rate. However, despite at least a 10-fold increase in GLUT 4 mRNA and protein detected after GLUT 4 cDNA transfection, the glucose uptake of these cells was unchanged and remained insulin-insensitive. By laser confocal immunofluorescence imaging, it was established that the transfected GLUT 4 protein was localized almost entirely in cytoplasmic compartments. In contrast, the GLUT 1 isoform was detected both at the plasma membrane as well as in intracellular compartments. These results suggest that acute insulin stimulation of glucose transport is not solely dependent on the presence of the insulin receptor and the GLUT 4 protein, and that the presence of some additional protein(s) must be required.     

GLUT1 deficiency links nutrient availability and apoptosis during embryonic development

GLUT1 is essential for human brain development and function, as evidenced by the severe epileptic encephalopathy observed in children with GLUT1 deficiency syndrome resulting from inherited loss-of-function mutations in the gene encoding this facilitative glucose transporter. To further elucidate the pathophysiology of this disorder, the zebrafish orthologue of human GLUT1 was identified, and expression of this gene was abrogated during early embryonic development, resulting in a phenotype of aberrant brain organogenesis consistent with the observed expression of Glut1 in the embryonic tectum and specifically rescued by human GLUT1 mRNA. Affected embryos displayed impaired glucose uptake concomitant with increased neural cell apoptosis and subsequent ventricle enlargement, trigeminal ganglion cell loss, and abnormal hindbrain architecture. Strikingly, inhibiting expression of the zebrafish orthologue of the proapoptotic protein Bad resulted in complete rescue of this phenotype, and this occurred even in the absence of restoration of apparent glucose uptake. Taken together, these studies describe a tractable system for elucidating the cellular and molecular mechanisms of Glut1 deficiency and provide compelling in vivo genetic evidence directly linking nutrient availability and activation of mitochondria-dependent apoptotic mechanisms during embryonic brain development.     

Human articular chondrocytes express three facilitative glucose transporter isoforms: GLUT1, GLUT3 and GLUT9

Glucose is an important metabolite and a structural precursor for articular cartilage and its transport has significant consequences for cartilage development and functional integrity. In this study the expression of facilitative glucose transporters (GLUTs) in human chondrocytes was investigated. Results showed that at least three GLUT isoforms (GLUT1, GLUT3 and GLUT9) are expressed by normal chondrocytes. Given the central role of glucose in chondrocyte physiology and metabolism, its regular provision via GLUTs will influence the metabolic activity and survival of chondrocytes in cartilage matrices.