Inhibition of glucose transport and direct interactions with type 1 facilitative glucose transporter (GLUT-1) by etomidate, ketamine, and propofol: a comparison with barbiturates.

Ketamine, etomidate, propofol, and pentobarbital were compared for effects on and interactions with the type 1 facilitative glucose transporter (GLUT-1). Fluxes of radiolabeled hexoses were used to determine the effects of anesthetics on GLUT-1 function. Hypotonic hemolysis of human erythrocytes was used to assess perturbations of membrane integrity. Quenching of intrinsic protein fluorescence was used to assess the direct interactions of anesthetics with purified GLUT-1. Pentobarbital, ketamine, etomidate, and propofol inhibited glucose transport in murine fibroblasts with IC(50) values of 0.8, 1. 6, 0.1, and 0.4 mM, respectively. Pentobarbital, ketamine, etomidate, and propofol also inhibited sugar transport in human erythrocytes. The IC(50) values for pentobarbital and ketamine exhibited substrate dependence for equilibrium exchange but not unidirectional effluxes. This was not observed for etomidate. Propofol did not inhibit equilibrium exchange but did inhibit unidirectional efflux with little substrate dependence. Pentobarbital protected against hemolysis, whereas etomidate and ketamine promoted hemolysis of erythrocytes. Propofol had no effect on membrane integrity. Pentobarbital, ketamine, and etomidate all interacted directly with GLUT-1, with apparent K(d) values of 2.2, 0.8, and 0.5 mM, respectively. Like barbiturates, ketamine, etomidate, and propofol inhibited GLUT-1 at concentrations near to those used pharmacologically. Inhibition of GLUT-1 by these intravenous general anesthetics was complex, exhibiting differential kinetic effects on equilibrium exchange versus unidirectional fluxes and contrasting substrate dependencies. Like barbiturates, ketamine and etomidate bound to GLUT-1 with affinities that paralleled inhibition of glucose transport. As a class, intravenous general anesthetics, in contrast to inhalation anesthetics, inhibit GLUT-1-mediated glucose transport.

Effects of barbiturates on facilitative glucose transporters are pharmacologically specific and isoform selective.

Barbiturates inhibit GLUT-1-mediated glucose transport across the blood-brain barrier, in cultured mammalian cells, and in human erythrocytes. Barbiturates also interact directly with GLUT-1. The hypotheses that this inhibition of glucose transport is (i) selective, preferring barbiturates over halogenated hydrocarbon inhalation anesthetics, and (ii) specific, favoring some GLUT-# isoforms over others were tested. Several oxy- and thio-barbiturates inhibited [3H]-2-deoxyglucose uptake by GLUT-1 expressing murine fibroblasts with IC50s of 0.2-2.9 mm. Inhibition of GLUT-1 by barbiturates correlates with their overall lipid solubility and pharmacology, and requires hydrophobic side chains on the core barbiturate structure. In contrast, several halogenated hydrocarbons and ethanol (all 10 mm). Thus, barbiturates selectively inhibit glucose transport by some, but not all, facilitative glucose transporter isoforms.

Barbiturate inhibition of GLUT-1 mediated hexose transport in human erythrocytes exhibits substrate dependence for equilibrium exchange but not unidirectional sugar flux.

Barbiturates inhibit GLUT-1 mediated hexose transport both in vivo [Gjedde & Rasmussen (1980) J. Neurochem. 35, 1382-1387; Otsuka et al. (1991) Am. J. Physiol. 261, R265-R275] and in vitro [Honkanen et al. (1995) Biochemistry 34, 535-544]. In the present study, the mechanism by which barbiturates inhibit GLUT-1 mediated hexose transport was examined by measuring both unidirectional zero trans and equilibrium exchange fluxes of hexoses in the functionally well-characterized, GLUT-1 rich human erythrocyte system. Unidirectional influx were both inhibited (> 80%) by 10 mM pentobarbital (PB). This symmetrical inhibition of unidirectional flux by PB was virtually independent of cis sugar concentration (2-130 mM) and exhibited an IC50 of approximately 2 mM. In contrast to unidirectional sugar flux, PB inhibition of equilibrium exchange sugar flux is attenuated by increased substrate concentration (e.g., 88% inhibition at 1 mM Glc versus 40% inhibition at 130 mM Glc in the presence of 10 mM PB) and exhibits an IC50 of approximately 10 mM at 100 mM Glc. Other barbiturates were found to inhibit sugar flux in human erythrocytes in this differential manner. These findings, when viewed with kinetic models proposed for GLUT-1 mediated transport [Carruthers (1990) Physiol. Rev. 70, 1135-1176], are consistent with barbiturates being noncompetitive inhibitors of Glc translocation and preferentially inhibiting the unoccupied form of the carrier protein. We propose, therefore, that barbiturates may prevent or alter the conformational changes associated with the reorientation of the carrier protein within the membrane. Overall, these results imply that barbiturates may more strongly inhibit GLUT-1 mediated Glc flux in vivo when the trans Glc is near zero as a result of either metabolism or another transport process.

Ion, water and neutral solute transport in Xenopus oocytes expressing frog lens MIP.

We have expressed frog (Rana pipiens) lens major intrinsic protein (MIP) in Xenopus oocytes and observed its effect on ion conductance, water permeability and neutral solute transport. SDS-PAGE and immunoblotting demonstrated oocytes injected with MIP mRNA expressed the protein at high levels. Immunolocalization indicated the expressed MIP migrated to the plasma membrane. MIP had no effect on the slope of oocyte I-V relations in the range -50 to +10 mV, although the averaged I-V curve was shifted 10 mV positive to control. MIP increased oocyte water permeability by a factor of 1.9 +/- 0.2, whereas the permeability to sucrose, 2-deoxyglucose, inositol, sorbitol, reduced glutathione or urea was unchanged. Glycerol permeability was enhanced in oocytes expressing MIP. In contrast to control oocytes, 3H-glycerol radioactivity accumulation did not follow first order kinetics. Radioactivity continued to accumulate even after 19 h of uptake and went beyond equilibrium with the bath. The time course of MIP-mediated glycerol uptake was modeled assuming metabolic trapping with good results. Based on this model, MIP increased oocyte glycerol permeability by a factor of 2.7.

Effect of lipid packing on the conformational states of purified GLUT-1 hexose transporter.

The purpose of this study was to determine the effect of increased lipid packing on the conformational states of the GLUT-1 hexose transporter purified in endogenous lipids. The binding of glucose results in a conformational change that can be followed by a decrease in fluorescence intensity. Lipid packing was increased by subjecting the samples to hydrostatic pressure. We have found that in the absence of ligand, the fluorescence intensity decreased approximately 20% in the 600 bar range studied. In the presence of either saturating or half-saturating amounts of D-glucose, a substantial loss in intensity (approximately 80%) was observed. Similar decreases were also seen the presence of a glucose analog, maltose, or a noncompetitive inhibitor, cytochalasin B. Changes in the accessibility of aqueous soluble quenchers (I- and acrylamide) to GLUT-1 Trp and Tyr residues suggested that ligand binding causes interfacial fluorophores to move closer to ionic groups in the lipid head group region of the membrane. This idea was substantiated by (1) increased static quenching of the GLUT-1 fluorophores in the presence of ligand, (2) increased energy transfer efficiency between GLUT-1 fluorophores and a fluorescent membrane probe located close to the head group region, and (3) reduced change in rotational motion with temperature in the presence of ligand. Since the application of pressure results in an increase in bilayer thickness, and ligand binding causes a population of fluorophores to move closer to the membrane surface, then these interfacial interactions can be more stabilized under pressure. Studies monitoring the change in quenching of membrane probes by GLUT-1 tryptophans and energy transfer of GLUT-1 tryptophans to membrane probes support this idea.

Barbiturates inhibit hexose transport in cultured mammalian cells and human erythrocytes and interact directly with purified GLUT-1.

Barbiturates reduce cerebral blood flow, metabolism, and Glc transfer across the blood-brain barrier. The effect of barbiturates on hexose transport in cultured mammalian cell lines and human erythrocytes was studied. Pentobarbital inhibits [3H]-2-dGlc uptake in 3T3-C2 murine fibroblasts by approximately 95% and approximately 50% at 10 and 0.5 mM, respectively. Uptake of [3H]-2-dGlc is linear with time in the presence or absence of pentobarbital, and the percent inhibition is constant. This suggests that hexose transport, not phosphorylation, is inhibited by barbiturates. Inhibition by pentobarbital of hexose transport in 3T3-C2 cells is rapid (< 1 min), is not readily reversible, is not altered by the presence of albumin [1% (w/v)], and is independent of temperature (4-37 degrees C) and the level of cell surface GLUT-1. The IC50's for inhibition of hexose transport in 3T3-C2 cells by pentobarbital, thiobutabarbital, and barbital are 0.8, 1.0, and 4 mM, respectively. This is consistent with both the Meyer-Overton rule and the pharmacology of barbiturates. Neither halothane (< or = 10 mM) nor ethanol [< or = 0.4% (v/v)] significantly inhibits hexose transport. Inhibition by pentobarbital (0.5 mM) of [3H]-2-dGlc uptake by 3T3-C2 cells decreases the apparent Vmax (approximately 50%) but does not alter the apparent Km (approximately 0.5 mM). Inhibition of hexose transport by barbiturates, but not ethanol [< or = 0.4% (v/v)], is also observed in human erythrocytes and four other cultured mammalian cell lines. Pentobarbital quenches (Qmax approximately 75%) the intrinsic fluorescence of purified and reconstituted GLUT-1 (Kd approximately 3 mM). Quenching is independent of Glc occupancy, is unchanged by mild proteolytic inactivation, and does not appear to directly involve perturbations of the lipid bilayer. We propose that barbiturates can interact directly with GLUT-1 and inhibit the intrinsic activity of the carrier. Glc crosses the blood-brain barrier primarily via the GLUT-1 of the endothelial cells of cerebral capillaries. Partial inhibition of this process by barbiturates may be of significance to cerebral protection.

Differential control of the functional cell surface expression and content of hexose transporter GLUT-1 by glucose and glucose metabolism in murine fibroblasts.

The present paper evaluates the contributions of glucose and its metabolites to the post-translational regulation of hexose transport and GLUT-1 content in murine fibroblasts. The effects of 3-O-methylglucose, a nearly non-metabolizable glucose analogue, on 2-deoxyglucose-uptake, cell-surface expression and content of GLUT-1, glucose 6-phosphate levels, and phosphoglucose isomerase (PGI) and hexokinase activities of murine fibroblasts were compared with those of glucose and fructose. Glucose (EC50 approximately 6 mM) or 3-O-methylglucose (EC50 approximately 12 mM), which are substrates of GLUT-1, but not fructose, which is not transported by GLUT-1, are able to prevent the glucose-deprivation-induced increases in both hexose transport and cell-surface expression of GLUT-1. In contrast, glucose (EC50 approximately 6 mM), but not 3-O-methylglucose or fructose, prevents the glucose-deprivation-induced accumulation of total GLUT-1 polypeptides. Glucose (> or = 5 mM), but not fructose or 3-O-methylglucose, leads to significant glucose 6-phosphate accumulation. Although 3-O-methylglucose is weakly phosphorylated by fibroblasts, accumulation of phosphorylated product does not correlate with hexose-transport regulation. The activities of hexokinase and PGI are not altered by glucose, fructose or 3-O-methylglucose. We suggest that, in murine fibroblasts: (i) hexose transport and GLUT-1 content are differentially regulated; (ii) substrates of GLUT-1 and/or their immediate metabolites regulate the cell-surface expression of functional GLUT-1; and (iii) glucose metabolism is required for the regulation of GLUT-1 content.

The cDNA sequence encoding the major intrinsic protein of frog lens.

A cDNA clone encoding the frog lens major intrinsic protein (MIP) has been isolated and sequenced. The predicted protein of 28 kDa has high sequence identity and similarity to mammalian and avian lens MIP sequences. Frog lens MIP is encoded by a transcript of 4.4 kb.

Expression and characterization of a canine hippocampal inwardly rectifying K+ current in Xenopus oocytes.

1. An inwardly rectifying potassium current expressed in Xenopus laevis oocytes injected with canine hippocampal poly(A)+ RNA was investigated with the two-microelectrode voltage clamp technique. 2. Xenopus oocytes injected with canine hippocampal poly(A)+ RNA expressed a current activated by hyperpolarization. This current contained an instantaneous and a time-dependent component. Both components were inwardly rectifying and could be blocked by extracellular Cs+ or Ba2+. 3. The expressed current was carried mainly by K+. Its reversal potential measured in different [K+]os could be fitted by the Nernst equation with a slope of -50.7 per tenfold change in [K+]o. Extracellular Cl- and Na+ made minimal contributions to the current. 4. The activation of the expressed current depended on both voltage and [K+]o. Activation started near EK and the activation curve shifted along the voltage axis in parallel with EK when [K+]o was altered. 5. The activation time constants of the expressed current also depended on both voltage and [K+]o. The voltage dependence of the time constants was bell-shaped and the peak value was at a potential 30-50 mV more negative than EK. The voltage dependence of the time constants shifted along the voltage axis when EK was changed. 6. The poly(A)+ RNA extracted from canine hippocampus was fractionated in a 10-31% linear sucrose gradient. The size of the mRNA required to express the inwardly rectifying current was estimated to be around 4 kb. 7. In conclusion, the expressed current is an inwardly rectifying potassium current. The canine hippocampal mRNA should be an excellent source for expression-cloning of the inward rectifier channel.

Regulation of the functional expression of hexose transporter GLUT-1 by glucose in murine fibroblasts: role of lysosomal degradation.

The nature of the membrane compartments involved in the regulation by glucose of hexose transport is not well defined. The effect of inhibitors of lysosomal protein degradation on hexose transport (i.e., uptake of [3H]-2-deoxy-D-glucose) and hexose transporter protein GLUT-1 (i.e., immunoblotting with antipeptide serum) in glucose-fed and -deprived cultured murine fibroblasts (3T3-C2 cells) was studied. The acidotropic amines chloroquine (20 microM) and ammonium chloride (10 mM) cause accumulation (both approximately 4-fold) of GLUT-1 protein and a small increase (both approximately 25%) in hexose transport in glucose-fed fibroblasts (24 h). The endopeptidase inhibitor, leupeptin (100 microM) causes accumulation (approximately 4-fold) of GLUT-1 protein in glucose-fed fibroblasts (24 h) without changing hexose transport (less than or equal to 5%). These agents do not greatly alter the electrophoretic mobility of GLUT-1. Neither chloroquine nor leupeptin augment the glucose deprivation (24 h) induced increases in hexose transport (approximately 4-fold) and GLUT-1 content (approximately 7-fold). In contrast, chloroquine or leupeptin diminish the reversal by glucose refeeding of the glucose deprivation induced accumulation of GLUT-1 protein but fail to alter the return of hexose transport to control levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucose transport is reduced in the blood-brain barrier of aged rats.

To determine the biochemical basis of decreased brain uptake of glucose with age, the brain influx of 3-O-methylglucose (3-O-MG) was measured in male Fischer 344 rats at various ages using the arterial injection-tissue sampling technique of Oldendorf. The Vmax of 3-O-MG transport in the 24-month-old rats (0.22 +/- 0.14 mumol/min/g) was significantly lower than that in 3-month-old rats (0.88 +/- 0.18 mumol/min/g) (P less than 0.05). The Km of transport in aged rats (10.1 +/- 4.8 mM) was not different from that in young rats (8.1 +/- 2.5 mM). The cytochalasin B binding sites in cerebral microvessels isolated from aged rats (13.9 +/- 0.9 pmol/mg) compared to the binding sites in cerebral microvessels of young rats (21.9 +/- 1.4 pmol/mg) were significantly reduced (P less than 0.001). However, the immunoreactive mass of glucose transporter of cerebral microvessels was not altered with age. The enrichment of capillary preparations with gamma-glutamyl transpeptidase activity, a marker of endothelial cells, was not altered in aged rats, suggesting that the reduced blood-brain barrier transport of glucose is due to specific reduction in glucose binding sites of the transporter rather than secondary to a non-specific age-related effect of endothelial cell drop-out.

Glucose deprivation induces the selective accumulation of hexose transporter protein GLUT-1 in the plasma membrane of normal rat kidney cells.

Antibody to the carboxyl-terminal of hexose transporter protein GLUT-1 was used to localize this carrier in normal rat kidney (NRK) cells during D-glucose (Glc) deprivation. Glc-deprivation of NRK cells induces increased hexose transport, inhibits the glycosylation of GLUT-1, and increases the content of both native, 55,000 apparent mol wt (Mr) and aglyco, 38,000 Mr GLUT-1 polypeptides. The distribution of GLUT-1 protein in subcellular fractions isolated from Glc-fed NRK cells shows that the 55,000 Mr polypeptide is most abundant in intracellular membrane fractions. Glc-fed cells that have been tunicamycin treated contain principally the 38,000 Mr GLUT-1 polypeptide, which is found predominantly in intracellular membrane fractions. In Glc-deprived cells the 55,000 Mr GLUT-1 polypeptide localizes predominantly in the Golgi and plasma membrane fractions, whereas the more abundant 38,000 Mr GLUT-1 polypeptide is distributed throughout all membrane fractions. In Glc-deprived but fructose-fed cells only the 55,000 Mr GLUT-1 polypeptide is detected, and it is found predominantly in the plasma membrane and Golgi fractions. The localization of GLUT-1 protein was directly and specifically visualized in NRK cells by immunofluorescence microscopy. Glc-fed cells show little labeling of cell borders and a small punctate juxtanuclear pattern suggestive of localization to the Golgi and, perhaps, endoplasmic reticulum. Glc-fed cells that have been tunicamycin treated show large punctate intracellular accumulations suggestive of localization to distended Golgi and perhaps endoplasmic reticulum. Glc-deprived cells exhibited intense labeling of cell borders as well as intracellular accumulations. Glc-deprived but fructose-fed cells show fewer intracellular accumulations, and the labeling is, in general, limited to the cell borders. Our results suggest that Glc deprivation induces the selective accumulation of GLUT-1 in the plasma membrane of NRK cells.

Regulation of rat brain/HepG2 glucose transporter gene expression by phorbol esters in primary cultures of neuronal and astrocytic glial cells.

We have demonstrated regulation of the rat brain/Hep G2 glucose transporter gene (GT1) by Northern blot analysis with a rat brain glucose transporter cDNA probe. Incubation of both neuronal and glial cells derived from neonatal rats with 12-O-tetradecanoyl-phorbol-13-acetate induced a time- and dose-dependent increase in the steady state levels of GT1 mRNA. In glial cells, this corresponded to an increase in both the level of GT1 protein and glucose transporter activity, as demonstrated by Western blot analysis and [3H]2-deoxyglucose (dGlc) uptake studies. In contrast, in neuronal cells 12-O-tetradecanoyl-phorbol-13-acetate had no effect on either the concentration/level of the GT or [3H]dGlc uptake. These results suggest that phorbol esters regulate dGlc uptake at the transcriptional level in both neuronal and glial cells, but that the increase in expression of the GT1 gene is dissociated from posttranscriptional events involved in dGlc uptake in neuronal cells.

Antagonism by growth hormone of insulin-sensitive hexose transport in 3T3-F442A adipocytes.

We have studied the effects of GH on basal and insulin-stimulated hexose transport by 3T3-F442A adipocytes in a hormonally defined serum-free medium. Adipocytes preincubated in defined medium exhibit a low level of hexose transport which is acutely (15 min) stimulated (greater than 5-fold) by insulin (EC50, 0.1-0.2 nM). GH has acute (15-45 min) insulin-mimetic (greater than 2-fold) and chronic (4-48 h) diabetogenic (50-80%) effects on basal and insulin-stimulated hexose transport. The insulin-mimetic effect of GH has a higher EC50 (2 nM) than its diabetogenic effect (EC50, 0.2 nM). Chronic GH exposure decreases the maximal responsiveness (50-80%) and the acute sensitivity (approximately 2-fold) of hexose transport to insulin. Insulin-stimulated transport is more (approximately 5-fold) sensitive to the diabetogenic effect of GH than is basal transport. Insulin binding and degradation were not altered by chronic exposure to GH. The diabetogenic effect of GH may occur at a postinsulin binding level.

Structure, biosynthesis, and function of the hexose transporter in Chinese hamster ovary cells deficient in N-acetylglucosaminyltransferase 1 activity.

We have used a Chinese hamster ovary cell line deficient in N-acetylglucosaminyltransferase 1 activity (Lec1) to study the effects of altered asparagine-linked oligosaccharides on the structure, biosynthesis, and function of glucose transporter protein. Immunoblots of membranes of Lec1 cells show a glucose transporter protein of Mr 40,000, whereas membranes of wild-type (WT) cells contain a broadly migrating Mr 55,000 form similar to that observed in several other mammalian tissues. The total content of immunoreactive glucose transporters in Lec1 cells is 3.5-fold greater than that of WT cells. Digestion with endoglycosidases, treatment with inhibitors of glycosylation, and interactions with agarose-bound lectins demonstrate that glucose transporters of both cell lines derive from a similar Mr 38,000 core polypeptide and that both contain asparagine-linked oligosaccharide. Transporters in Lec1 cells contain primarily "undecorated" but "trimmed" mannose-type asparagine-linked oligosaccharides, while the protein in WT cells contains a mixture of "decorated" and "trimmed" asparagine-linked oligosaccharides. Biosynthetic and turnover studies demonstrate that Lec1 cells, in contrast to WT cells, are unable fully to process the core asparagine-linked oligosaccharides of maturing glucose transporters. When radiolabeled in methionine-deficient medium both Lec1 and WT cells show similar rates of synthesis and turnover of glucose transporter proteins. It should be noted, however, that starvation for a critical amino acid may alter the ability of the cell to synthesize or degrade proteins. The abilities of Lec1 and WT cells to transport hexoses and to interact with the inhibitor cytochalasin B are very similar. The results indicate that, although altered asparagine-linked glycosylation can affect the content and biogenesis of glucose transporters, these changes do not greatly modify cellular hexose uptake. The possibility that alterations in asparagine-linked glycosylation may change the cell surface localization or acquisition of a "functional conformation" of the glucose transporter is also suggested.

Anti-fertility and other actions of gossypol analogues.

From a series of gossypol derivatives studied, we conclude that the carbonyl groups of gossypol are needed for inhibition of erythrocyte anion transport and the hydroxy groups affect but are not essential to that inhibition. In an in vitro mouse erythroleukemia cytocidal assay, the most active compounds were gossypol and apogossypol. The latter was not active in the inhibition of erythrocyte anion transport or in a spermicidal assay. Of the more simple structures related to gossypol, those that were active in the cytocidal and spermicidal assays were bi-aromatic, linked by a 1- and not a 4-carbon chain and had free phenolic hydroxyl groups. These results are included in a discussion of the specificity and mechanism of action of gossypol.

PIP: The analysis of multiple biological assays of gossypol and its derivatives suggests that the carbonyl groups of gossypol are required for inhibition of erythrocyte anion transport and the hydroxy groups affect but are not essential to that inhibition. In an in vitro mouse erythroleukemia cytocidal assay, the most active compounds were gossypol and apogossypol. The latter was not active in the inhibition of erythrocyte anion transport or in a spermicidal assay. Of the more simple structures related to gossypol, those that were active in the cytocidal and spermicidal assays were biaromatic, linked by a 1- and not a 4-carbon chain, and had free phenolic hydroxyl groups. When gossypol inhibits the anion transporter, the carbonyl group does not seem to form a Schiff base. Gossypol is a unique compound since it alone, but not any of its derivatives, has in vivo as well as in vitro antifertility activity. It remains unknown, however, whether similar mechanisms are involved in gossypol's in vivo and in vitro effects. In whatever manner gossypol exerts its toxic effects, the selectivity for testicular tissue must be explained.

Characterization of antisera to a synthetic carboxyl-terminal peptide of the glucose transporter protein.

Peptides corresponding to amino acid residues 1-12 of the amino terminal and 480-492 of the carboxyl terminal of the deduced sequence of the glucose transporter were synthesized and used to produce site-specific polyclonal antipeptide sera. In a solid-phase radioimmunoassay, antiserum to the carboxyl terminal recognizes peptide 480-492 and purified human erythrocyte glucose transporter, but not peptide 1-12. Antiserum to the amino terminal recognizes peptide 1-12 but neither peptide 480-492 nor the erythrocyte transporter. The antiserum to the carboxyl terminal specifically immunoblots the Mr 55,000 glucose transporter in erythrocyte membranes and the purified erythrocyte transporter. It also recognizes a Mr 40,000-60,000 polypeptide in membranes of cells derived from different mammalian species and tissues including insulin-sensitive rat adipocytes as well as a Mr 20,000 tryptic fragment of the transporter which contains the site for photolabeling by cytochalasin B. Antiserum to the carboxyl terminal of the transporter binds specifically to leaky erythrocyte membranes but not to intact erythrocytes. This binding is saturable and competitively inhibited by peptide 480-492. Using immunofluorescence microscopy, this antiserum detects glucose transporter protein in permeabilized erythrocytes, but not in intact erythrocytes. These studies provide immunochemical evidence in support of the predicted cytoplasmic orientation of the carboxyl terminus of the glucose transporter, allow us to suggest a spatial relationship of the cytochalasin B binding site to the carboxyl terminal of the glucose transporter and suggest that antisera directed to the carboxyl terminal domain of the protein may be useful for the immunocytochemical localization of the glucose transporter.

Transformation of rat fibroblasts by FSV rapidly increases glucose transporter gene transcription.

Elevation of glucose transport is an alteration common to most virally induced tumors. Rat fibroblasts transformed with wild-type or a temperature-sensitive Fujinami sarcoma virus (FSV) were studied in order to determine the mechanisms underlying the increased transport. Five- to tenfold increases in total cellular glucose transporter protein in response to transformation were accompanied by similar increases in transporter messenger RNA levels. This, in turn, was preceded by an absolute increase in the rate of glucose transporter gene transcription within 30 minutes after shift of the temperature-sensitive FSV-transformed cells to the permissive temperature. The transporter messenger RNA levels in transformed fibroblasts were higher than those found in proliferating cells maintained at the nonpermissive temperature. The activation of transporter gene transcription by transformation represents one of the earliest known effects of oncogenesis on the expression of a gene encoding a protein of well-defined function.

Cloning and characterization of a cDNA encoding the rat brain glucose-transporter protein.

Antibody raised against the human erythrocyte glucose transporter identified a recombinant lambda gt11 bacteriophage in a cDNA library prepared from immunoselected polysomal RNA from adult rat brain. The cDNA predicts a 492-amino acid protein that demonstrates 97.6% identity to the human hepatoma hexose carrier. The tissue distribution of the transporter mRNA is identical to that of immunologically identifiable protein and transport activity, except in liver in which high levels of transport are associated with little or no transporter mRNA or protein. As assayed by blot-hybridization analysis, mRNA from insulin-responsive and nonresponsive tissues are indistinguishable. These data suggest that a genetically unrelated protein is responsible for hexose transport in normal liver.

Glucose deprivation and hexose transporter polypeptides of murine fibroblasts.

The effect of Glc deprivation (starvation) on hexose transporter (GT) polypeptide(s) (pp) was studied in 3T3-C2 murine fibroblasts. Cells deprived of Glc exhibit 5-fold increases in hexose transport and Glc-displaceable cytochalasin B binding. Immunoblots of membranes reveal a Mr 55,000 GT pp in fed (4 g of Glc/liter) cells and Mr 55,000 and Mr 42,000 GT pp in starved cells. A 10-40-fold increase in total GT pp occurs upon Glc deprivation; part of this accumulation (2-5-fold) is in the Mr 55,000 GT pp, and the remaining increase is in the Mr 42,000 GT pp. During the first 12 h of Glc deprivation only the Mr 55,000 GT pp accumulates. At later times (24-72 h) the Mr 42,000 GT pp appears and constitutes a larger fraction of the total accumulation. Similarly, the Glc concentration dependence of these phenomena reveals that the Mr 55,000 GT pp accumulates at higher concentrations of Glc (less than or equal to g/liter) than the Mr 42,000 GT pp (less than or equal to 0.5 g/liter). Using alternative nutrients, sugar analogs, and inhibitors we observed that the accumulation of total GT pp is dependent upon both hexose phosphate metabolism and the interaction of substrate with the GT. The role(s) of oligosaccharide biosynthesis, protein synthesis, and the transport process itself in the Glc deprivation-induced accumulation of GT pp were examined. The appearance of the Mr 42,000 GT pp but not the Mr 55,000 GT pp was dependent upon protein synthesis. The Glc deprivation-induced accumulation of GT pp is reversible upon refeeding with Glc (4 g/liter, 12 h). This reversal was dependent upon protein synthesis. The electrophoretic mobility of the Mr 42,000 GT pp is similar to the GT pp observed after tunicamycin treatment. The Mr 55,000 but not the Mr 42,000 GT pp binds specifically to agarose-bound wheat germ agglutinin and is sensitive to endoglycosidase F digestion. Oligosaccharide-stripped GT pp and the Mr 42,000 GT pp have the same Mr. The results suggest that the accumulation of total GT pp induced by Glc deprivation is partially independent of the effect of Glc deprivation on glycoprotein biogenesis. The appearance of the Mr 42,000 GT pp with aglyco characteristics is the result of the latter. The accumulation of total GT pp, however, is the result of a specialized and sensitive adaptation of the cell to Glc deprivation. The GT pp synthesized during chronic Glc deprivation has an Mr of 42,000; fed cells synthesize the Mr 55,000 GT pp. Neither the level of in vitro translatable GT mRNA nor the rate of GT pp synthesis are increased by Glc deprivation.(ABSTRACT TRUNCATED AT 400 WORDS)