The volume changes associated with the operation of the 'simple' transporter.

The effects of hydrostatic pressure (0.1-50 MPa) on uridine transport mediated by the 'simple' facilitated nucleoside transporter of guinea-pig and human erythrocytes have been studied in an attempt to identify the volume changes which occur during transport. Pressure inhibited the zero-trans (influx or efflux) mode of uridine transport in guinea-pig cells significantly more (about 2.2- x) than equilibrium exchange. The equilibrium binding of 3H-nitrobenzylthioinosine, a potent specific inhibitor of nucleoside transport, to human red cells and ghosts, was not significantly altered by pressure suggesting that the permeation site was unperturbed. Thus pressure inhibited the transporter primarily by preventing the volume increase associated with the translocation step. Furthermore, the return of the 'empty' transporter was found to be rate-limiting because it required a larger increase in volume than when the transporter was loaded with substrate.     

Reconstitution of the glucose transporter from rat skeletal muscle

As a step in the purification and characterization of the glucose transporter from rat skeletal muscle, we have reconstituted glucose transport activity in liposomes. Plasma membranes were prepared from skeletal muscle which display D-glucose reversible binding of cytochalasin B (10 pmol sites/mg protein; KD = 0.3 microM). Older rats gave a slightly lower specific activity and much lower yield of sites per g muscle than young rats. Glucose transport activity was reconstituted into liposomes by the freeze-thaw procedure using either plasma membranes directly or cholate-extracted membrane proteins; the latter gave a 50% higher specific activity. The reconstituted transport activity was stereospecific, saturable, and inhibited by cytochalasin B, phloretin, and mercuric chloride. The optimum cholate concentration for extraction and reconstitution of transport activity was about 1.5%, and the highest specific activity of reconstituted transport was seen only at low ratios of protein to lipid in the reconstitution. Chromatography on agarose lentil lectin and agarose ethanethiol doubled both the specific activity of reconstituted transport and the fraction of glucose uptake which was stereospecific. In all of these respects the results were similar to our results with the bovine heart transporter (T. J. Wheeler and M. A. Hauck, Biochim. Biophys. Acta 818, 171-182 (1985)). Our findings suggest that further purification procedures developed for the heart transporter may be applicable to the skeletal muscle transporter as well.     

Comparison of glucose and fructose transport into adipocytes of the rat.

The purpose of these studies was to define the properties of the systems that transport hexoses into adipocytes. Glucose appears to enter adipocytes on a single transport system whose maximum velocity is stimulated by insulin and which is competitively inhibited by cytochalasin B, 5-thioglucose, fructose, mannose and 3-O-methylglucose. In contrast, fructose enters adipocytes by at least two separate mechanisms, one an insulin-sensitive transporter (probably the glucose transporter) and the other a mechanism that is insensitive to insulin. The fructose concentration required for half-maximal rates of transport is at least an order of magnitude higher than that for glucose and the maximum velocity of fructose transport is more than double that for glucose.     

Distributed control of the glycolytic flux in wild-type cells and catabolite repression mutants of Saccharomyces cerevisiae growing in carbon-limited chemostat cultures

The sensitivity of the control of glycolysis was studied in the wild-type (WT) strain CEN.PK122 and in isogenic catabolite-repression mutants growing in carbon-limited, aerobic chemostat cultures at different dilution rates, D. Based on a model of glycolysis in which the glucose transport step was considered reversible and inhibited by glucose 6-phosphate (G6P), the matrix method of metabolic control analysis was applied. In the present work, we report that the control of glycolysis was significantly distributed between the glucose uptake, hexokinase, and phosphofructokinase steps. The flux control properties were sensitive to the glucose gradient through the membrane and the extent of inhibition of the transport by G6P as parameters of the glucose-uptake kinetics in all strains tested. In the WT strain at low and high D, most of the control was exerted by the phosphofructokinase (PFK)-catalyzed step. In the cat1 mutant, the step catalyzed by PFK was the most rate controlling while in the cat3 strain, the control was shared between the PFK, hexokinase (HK), and glucose transport steps. On the other hand, the mig1 mutant exhibited high control by the glucose transporter depending on the glucose gradient across the membrane. The results obtained are discussed in terms of the dependence upon the type of metabolism displayed by yeast and the kinetics of the sugar transport step.     

Effect of adrenaline on glucose transport in red cells of Rana balcanica

The characteristics of 3-O-methyl-D-glucose (3-OMG) uptake by frog erythrocytes were studied. 3-OMG transport was increased by adrenaline. Although the transport is inhibited by phloretin, the lack of saturation kinetics suggests that a glucose transporter doesn't exist or that its affinity for glucose is extremely low. Frog Rana balcanica red cells suspended in an isotonic medium containing adrenaline enlarge rapidly to reach a new pH-dependent steady state volume. At pH 8.0, the cells swell less than at pH 7.3. This is explained by a differential pH effect on the two pathways controlling the movement of the cations: as pH becomes more acidic K+ loss decreases. On the contrary as pH becomes more acidic Na+ uptake increases. The increase in glucose transport after osmotic swelling and the inhibition of swelling-induced glucose transport by phloretin suggest that the glucose transport pathway in Rana balcanica erythrocytes may is a volume-activated channel.     

Acute activation of glucose uptake by glucose deprivation in L929 fibroblast cells

Glucose is a very important energy source for a wide variety of cells, and the ability of cells to respond to changes in glucose availability or other cell stresses is of critical importance. Many mammalian cells respond to acute stress by increasing the V(max) of transport through GLUT1; the most ubiquitously expressed glucose transporter isoform. This study investigated the acute response of glucose uptake to glucose deprivation in L929 fibroblast cells--a cell line that expresses only the GLUT1 transporter. Results indicated that glucose deprivation of only a minute activated glucose uptake 10-fold and reached a maximum of 20-fold within 10 min. The activation was dose dependent and only partially muted by addition of up to 20mM pyruvate as an alternate energy source. In contrast to the kinetics of acute metabolic stress, glucose deprivation decreased the K(m) of transport, but did not alter the V(max). Maximal activation of glucose transport by glucose deprivation was completely additive to activation of transport by methylene blue--a stimulant that increased the V(max) of transport without a change in the K(m). Glucose-deprived activation of glucose transport was not inhibited by wortmannin or herbimycin A, but was completely inhibited by phenylarsine oxide. Altogether, the data indicate that L929 fibroblast cells respond quickly and robustly to the cell stress of glucose deprivation and methylene blue treatment by two distinct activation pathways.     

Interaction between facilitated diffusion of glucose across the plasma membrane and its metabolism in Trichomonas vaginalis

Abstract The parasitic protist Trichomonas vaginalis transport glucose across the plasma membrane by facilitated diffusion. The K _m of the transporter for glucose was 1.6 mM. The uptake of labelled glucose in a minimal medium not allowing growth reached saturation only after 2.5 h, indicating the turnover of storage carbohydrate. Organisms grown on glucose showed higher activities both of the transporter and of the subsequent metabolic pathway than organisms grown on maltose. At low external glucose concentrations the transport step was rate limiting, at higher levels a subsequent enzymatic step. The uptake mechanism for glucose of T. vaginalis resembled that of parasitic kinetoplastid protist and Entamoeba histolytica .     

Effects of three proposed inhibitors of adipocyte glucose transport on the reconstituted transporter

Three compounds which inhibit glucose transport in rat adipocytes have been proposed to act directly on the glucose transporter protein. We tested these proposals by examining the effects of the compounds on the stereospecific glucose uptake catalyzed by adipocyte membrane proteins after reconstitution into liposomes. Effects on the transport activity reconstituted from human erythrocyte membranes were also examined. Glucose 6-phosphate, which was suggested to inhibit the transporter noncompetitively (Foley, J.E. and Huecksteadt, T.P. (1984) Biochim. Biophys. Acta 805, 313-316), had no effect on either type of reconstituted transporter, even when present at 5 mM on both sides of the liposomal membranes. Thus, it is unlikely to act directly on the transporter. The metalloendoproteinase substrate dipeptide Cbz-Gly-Phe-NH2, which inhibited insulin-stimulated but not basal glucose uptake in adipocytes (Aiello, L.P., Wessling-Resnick, M. and Pilch, P.F. (1986) Biochemistry 25, 3944-3950), inhibited the reconstituted erythrocyte transporter noncompetitively with a Ki of 1.5-2 mM. The inhibition of the erythrocyte transporter was identical in liposomes of soybean and egg lipid. Transport reconstituted using adipocyte membrane fractions was also inhibited by the dipeptide, with the activity from basal microsomes more sensitive than that from insulin-stimulated plasma membranes. These results indicate that the dipeptide interacts directly with the transporter, and may be a potentially useful probe for changes in transporter structure accompanying insulin action. Phenylarsine oxide, which was suggested to act directly on the adipocyte transporter (Douen, A.G., and Jones, M.N. (1988) Biochim. Biophys. Acta 968, 109-118), produced only slight (about 10%) inhibition of the reconstituted adipocyte and erythrocyte transporters, even when present at 100-200 microM and after 30 min of pretreatment. These results suggest that the major actions of phenylarsine oxide observed in adipocytes are not direct effects on the transporter, but rather effects on the pathways by which insulin regulates glucose transport activity (Frost, S.C. and Lane, M.D. (1985) J. Biol. Chem. 260, 2646-2652).     

Redefining the facilitated transport of mannose in human cells: absence of a glucose-insensitive, high-affinity facilitated mannose transport system

Current evidence suggests that extracellular mannose can be transported intracellularly and utilized for glycoprotein synthesis; however, the identity and the functional characteristics of the transporters of mannose are controversial. Although the glucose transporters are capable of transporting mannose, it has been postulated that the entry of mannose in mammalian cells is mediated by a transporter that is insensitive to glucose [Panneerselvam, K., and Freeze, H. (1996) J. Biol. Chem. 271, 9417-9421] or by a transporter induced by cell treatment with metformin [Shang, J., and Lehrman, M. A. (2004) J. Biol. Chem. 279, 9703-9712]. We performed a detailed analysis of the uptake of mannose in normal human erythrocytes and in leukemia cell line HL-60. Short uptake assays allowed the identification of a single functional activity involved in mannose uptake in both cell types, with a K(m) for transport of 6 mM. Transport was inhibited in a competitive manner by classical glucose transporter substrates. Similarly, the glucose transporter inhibitors cytochalasin B, genistein, and myricetin inhibited mannose transport by 100%. Using long uptake experiments, we identified a second, high-affinity component associated with the intracellular trapping of mannose in the HL-60 cells that is not directly involved in the transport of mannose via the glucose transporters. Thus, the transport of mannose via glucose transporters is a process which is kinetically and biologically separable from its intracellular trapping. A general survey of human cells revealed that mannose uptake was entirely blocked by concentrations of cytochalasin B that obliterates the activity of the glucose transporters. The transport and inhibition data demonstrate that extracellular mannose, whose physiological concentration is in the micromolar range, enters cells in the presence of physiological concentrations of glucose. Overall, our data indicate that transport through the glucose transporter is the main mechanism by which human cells acquire mannose.     

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.     

The impact of N- and O-glycosylation on the functions of Glut-1 transporter in human thyroid anaplastic cells

It has been previously shown that glucose transporter Glut-1 expression was detectable by immunostaining in tissue sections from anaplastic carcinoma, but not in normal thyroid tissue. Using human thyroid anaplastic carcinoma cells, we studied the mechanism by which Glut-1 molecules are translocated from the endoplasmic reticulum to the cell surface. The contribution of N- and O-linked glycans for the translocation and activity of Glut-1 transporter is emphasized. The inhibition of N-glycosylation with tunicamycin (TM) led to a 50% decrease in glucose transport while glycosylated and unglycosylated forms of Glut-1 were found at the cell surface. However, the inhibition of N-linked oligosaccharide processing with deoxymannojirimycin (dMJ) and swainsonine (SW) influenced neither the intracellular trafficking nor the activity of the transporter. On the other hand, Glut-1 bound to the O-linked glycan-specific lectin jacalin and the O-glycosylation inhibitor benzyl-N-acetylgalactosamine dramatically inhibited glucose transport. These results show that O- and N-linked oligosaccharides arbored by Glut-1 are essential for glucose transport in anaplastic carcinoma cells. The quantitative and qualitative alterations of Glut-1 glycosylation and the increase in glucose transport are associated with the anaplastic phenotype of human thyroid cells.     

Glucose transport byAspergillus niger: the low-affinity carrier is only formed during growth on high glucose concentrations

The filamentous fungusAspergillus niger accumulates large levels of citric acid in the medium when grown under conditions favouring a high rate of sugar catabolism. With the aim of understanding the mechanisms involved in this process we investigated glucose transport in this fungus. To this end a medium was designed that enables growth of the fungus into a fine, hairy filamentous mycelium, suitable for transport studies. It was found thatA. niger contains a single, high-affinity glucose transporter when grown on a low (1% w/v) glucose concentration, but forms an additional low-affinity transporter when grown on a high (15% w/v) glucose concentration. Both glucose transporters exhibit decreased activities at low pH and are inhibited by citric acid. However, the activity of the low-affinity transporter is much less affected by these conditions. Two 2-deoxyglucose-resistant (dgr) mutants ofA. niger, which produce citric acid at a much lower rate than the parent strain, are impaired in the formation of the low-affinity transporter, but form the high-affinity transporter with higher activities. We conclude that the low-affinity glucose transporter takes part in the mechanism by whichA. niger responds to high extracellular glucose concentrations leading to citric acid accumulation.     

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.     

Regulation of glucose transport in Clone 9 cells by thyroid hormone.

Triiodothyronine (T3) is found to stimulate cytochalasin B-inhibitable glucose transport in Clone 9 cells, a 'non-transformed' rat liver cell line. After an initial lag period of more than 3 h, glucose transport rate is significantly increased at 6 h and reaches more than 3-times the control rate at 24 h. The enhancement of glucose transport by T3 is due to an increase in transport Vmax and occurs in the absence of a change in either the Km for glucose transport (approximately 3 mM) or the Ki for inhibition of transport by cytochalasin B ((1-2).10(-7) M). Consistent with the observed Ki for cytochalasin B, Northern blot analysis of RNA from control and T3-treated cells employing cDNA probes encoding GTs of the human erythrocyte/rat brain/HepG2 cell transporter (GLUT-1), rat muscle/fat cell transporter (GLUT-4), and rat liver transporter (GLUT-2) types indicates expression of only the GLUT-1 mRNA isoform in these cells. The abundance of GLUT-1 mRNA increases approx. 1.9-fold after 24 h of T3 treatment and is accompanied by an approx. 1.3-fold increase in the abundance of GLUT-1 in whole-cell extracts as demonstrated by Western blot analysis employing a polyclonal antibody directed against the 13 amino acid C-terminal peptide of GLUT-1. The more than 3-fold stimulation of glucose transport at 24 h substantially exceeds the fractional increment in transporter abundance suggesting that, in addition to increasing total GLUT-1 abundance, exposure to T3 may result in a translocation of transporters to the plasma membrane or an activation of pre-existing membrane transporter sites.     

Degradation and biosynthesis of the glucose transporter protein in chicken embryo fibroblasts transformed by the src oncogene.

The rate of glucose transport in cultured fibroblasts is regulated to a number of physiological variables, including malignant transformation by src, glucose starvation, and stimulation with mitogens. Much of this transport regulation can be accounted for by variations in the amount of transporter protein in the cells. To determine the mechanisms by which levels of the transporter are regulated, we measured the rates of synthesis and degradation of the transporter by pulse-chase experiments and immunoprecipitation of the transporter. We found that transformation by the src oncogene results in a large decrease in the rate at which the transporter protein is degraded but that it does not appreciably increase the rate of transporter biosynthesis. On the other hand, glucose starvation and mitogen stimulation increase the rate of transporter biosynthesis, although a role for control of degradation is possible in these circumstances also. Variations in the rate of glucose transport or the amount of the transporter are not associated with phosphorylation of the transporter protein.     

Differential sensitivity to guanine nucleotides of basal and insulin-stimulated glucose transporter activity reconstituted from adipocyte membrane fractions

The effects of GTP gamma S on glucose transport activity reconstituted from adipocyte membrane fractions were studied in order to test the hypothesis that intrinsic activity changes of the insulin-sensitive glucose transporter may be mediated by guanine nucleotide-dependent mechanisms. GTP gamma S and GTP inhibited reconstituted glucose transport activity by 50% in membrane fractions from insulin-treated cells in a concentration-dependent manner; no inhibitory effect was observed in membrane fractions obtained from basal cells. GDP, GMP and guanosine were less effective than GTP, whereas the adenine nucleotides ATP gamma S and AMP failed to reduce the reconstituted transport activity. The data indicate that guanine nucleotides may modulate the activity of the adipocyte glucose transporter. Since the effect is dependent on treatment of cells with insulin, the hormone appears to induce a specific functional alteration of the glucose transporter.     

Effect of erythropoietin on the glucose transport of rat erythrocytes and bone marrow cells

The effect of Ep on radioactive glucose and methyl-alpha-D-glucoside transport by rat erythrocytes and bone marrow cells were studied. There is initial linearity followed by saturation kinetics of [14C]glucose transport by the erythrocytes of starved and starved plus Ep-treated rats at different concentrations of glucose. Starvation caused slight inhibition of glucose transport which increased markedly on Ep administration to starved rats. Normal animals failed to show any significant change in glucose transport after Ep treatment. Methyl-alpha-D-glucoside inhibited the Ep-stimulated glucose transport significantly. Ep also stimulated the transport of radioactive methyl-alpha-D-glucoside which was competitively inhibited in presence of D-glucose. Glucose transport in erythrocytes was found to be sensitive to metabolic inhibitors like azide and DNP. A sulfhydryl reagent and ouabain also inhibited the transport process. Ep stimulated glucose and methyl-alpha-D-glucoside transport in the bone marrow cells of starved rats. The sugar analog competitively inhibited the glucose transport in bone marrow cells and vice versa.     

Expression of a functional glucose transporter in Xenopus oocytes

A cDNA encoding the rat brain glucose transporter was inserted between the 5' and 3' untranslated regions from the Xenopus globin gene and downstream of an SP6 RNA polymerase start site. RNA synthesized from this vector was microinjected into oocytes from Xenopus laevis; this resulted in expression of the glucose transporter, as determined by both immunoblotting and the appearance of transport activity. The properties of the transporter were those expected from previous studies: it was glycosylated, and its activity, measured by 3-O-methylglucose transport, was inhibited by D-glucose and cytochalasin B, but not by L-glucose. The low level of endogenous glucose transport activity found in water-injected oocytes makes this a useful system in which to determine the kinetic parameters of transport. The Km for 3-O-methylglucose was found to be 20 mM under equilibrium exchange conditions. Despite the fact that oocytes exhibit insulin-dependent responses, insulin did not stimulate 3-O-methylglucose transport by injected oocytes.     

The GLUT4 glucose transporter

Few physiological parameters are more tightly and acutely regulated in humans than blood glucose concentration. The major cellular mechanism that diminishes blood glucose when carbohydrates are ingested is insulin-stimulated glucose transport into skeletal muscle. Skeletal muscle both stores glucose as glycogen and oxidizes it to produce energy following the transport step. The principal glucose transporter protein that mediates this uptake is GLUT4, which plays a key role in regulating whole body glucose homeostasis. This review focuses on recent advances on the biology of GLUT4.