GLUT-1 mediation of rapid glucose transport in dolphin (Tursiops truncatus) red blood cells.

D-Glucose entry into erythrocytes from adult dolphins (Tursiops truncatus) was rapid, showed saturation at high substrate concentrations, and demonstrated a marked stimulation by intracellular D-glucose. Kinetic parameters were estimated from the concentration dependence of initial rates of tracer entry at 6 degrees C: for zero-trans entry, Michaelis constant (K(m)) was 0.78 +/- 0.10 mM and maximal velocity (Vmax) was 300 +/- 9 mumol.l cell water-1.min-1; for equilibrium exchange entry, K(m) was 17.5 +/- 0.6 mM and Vmax was 8,675 +/- 96 mumol.l cell water-1.min-1. Glucose entry was inhibited by cytochalasin B, and mass law analysis of reversible, D-glucose-displaceable, cytochalasin B binding gave values of 0.37 +/- 0.03 nmol/mg membrane protein for maximal binding and 0.48 +/- 0.10 microM for the dissociation constant. Dolphin glucose transporter polypeptides were identified on sodium-dodecyl sulfate-polyacrylamide gel electrophoresis immunoblots [using antibodies that recognized human glucose transporter isoform (GLUT-1)] as two molecular species, apparent relative molecular weights of 53,000 and 47,000. Identity of these polypeptides was confirmed by D-glucose-sensitive photolabeling of membranes with [3H]cytochalasin B. Digestion of both dolphin and human red blood cell membranes with glycopeptidase F led to the generation of a sharp band of relative molecular weight 46,000 derived from GLUT-1. Trypsin treatment of human and dolphin erythrocyte membranes generated fragmentation patterns consistent with similar polypeptide structures for GLUT-1 in human and dolphin red blood cells.

Screening of potential transport systems for methyl mercury uptake in rat erythrocytes at 5 degrees by use of inhibitors and substrates.

The current study was designed to screen the potential transport systems for methyl mercury (MeHg) uptake by isolated erythrocytes from rats at 5 degrees. Several inhibitors and substrates were used to test which potential transport system might be involved in MeHg uptake. Probenecid was used to test the organic anion transport system, valinomycin was used to test the effect of the membrane potential, D-glucose and cytochalasin B were used to test the facilitated diffusive D-glucose transport system and colchicine and vinblastine were used to test the microtubule system. The effects of Ca++, Mg++ and Na+ on MeHg uptake have been examined. Ouabain, ATP and glucose were used to test the active transport system, cysteine for the cysteine-facilitated transport system, glycine for system Gly, DL-methionine for system L, and MeHgCl and 4',4-diisothiocyano-2',2-stilbenedisulfonic acid (DIDS) for the Cl- ion transport system. The results showed that MeHg uptake might be involved in the following transport systems at 5 degrees: 1) organic anion transport system; 2) facilitated diffusive D-glucose transport system; 3) cysteine-facilitated transport system; 4) Cl- ion transport system. Moreover, the transport systems for MeHg uptake were sensitive to the membrane potential. Although the mechanisms of interaction of transport systems have not been fully clarified, evidence has been presented which support the existence of several simultaneous transport systems for MeHg uptake.

Effects of ATP depletion on the mechanism of hexose transport in intact human erythrocytes.

Depletion of ATP is known to inhibit glucose transport in human erythrocytes, but the kinetic mechanism of this effect is controversial. Selective ATP depletion of human erythrocytes by 10 micrograms/ml A23187 in the presence of extracellular calcium inhibited 3-O-methylglucose influx noncompetitively and efflux competitively. ATP depletion also decreased the ability of either equilibrated 3-O-methylglucose or extracellular maltose to inhibit cytochalasin B binding in intact cells, whereas neither total high-affinity cytochalasin B binding nor its Kd was affected. Under the one-site model of hexose transport these data indicate that ATP depletion decreases both the affinity of the inward-facing glucose carrier for substrate and its ability to reorient outwardly in intact cells.

Reaction of an exofacial sulfhydryl group on the erythrocyte hexose carrier with an impermeant maleimide. Relevance to the mechanism of hexose transport.

The presence of a reactive exofacial sulfhydryl on the human erythrocyte hexose carrier was used to test several predictions of the alternating conformation or one-site model of transport. The cell-impermeant glutathione-maleimide-I (GS-Mal) irreversibly inhibited hexose entry by decreasing the transport Vmax. This effect was potentiated by phloretin and maltose but decreased by cytochalasin B, indicating that under the one-site model the external sulfhydryl is on the outward-facing carrier but that it does not overlap with the exofacial substrate-binding site. Incubation of erythrocytes with maltose competitively inhibited the binding of [3H]cytochalasin B to the inward-facing carrier (Ki = 40 mM). Furthermore, both equilibrium cytochalasin B binding and its photolabeling of the band 4.5 carrier protein were decreased in ghosts prepared from GS-Mal-treated cells. Thus induction of an outward-facing carrier conformation with either maltose or GS-Mal caused the endofacial substrate-binding site to disappear. Dose-response studies of GS-Mal treatment of intact cells suggested that some functional carriers lack a reactive external sulfhydryl, which can be partially regenerated by pretreatment with excess cysteine. These data provide direct support for the one-site model of transport and further define the role of the external sulfhydryl in the transport mechanism.

Inhibition of hexose transport by adenosine derivatives in human erythrocytes.

The human erythrocyte membrane carriers for hexoses and nucleosides have several structural features in common. In order to assess functional similarities, the effects of adenosine derivatives on hexose transport and cytochalasin B binding sites were studied. Adenosine inhibited zero-trans uptake of 3-O-methylglucose half-maximally at 5 mM, while more hydrophobic adenosine deaminase-resistant derivatives were ten- to 20-fold more potent transport inhibitors. However, degradation of adenosine accounted for very little of this difference in potency. Hexose transport was rapidly inhibited by N6-(L-2-phenylisopropyl)adenosine at 5 degrees C in a dose-dependent fashion (EC50 = 240 microM), to lower the transport Vmax without affecting the Km. A direct interaction with the carrier protein was further indicated by the finding that N6-(L-2-phenylisopropyl)adenosine competitively inhibited [3H]cytochalasin B binding to erythrocytes (Ki = 143 microM) and decreased [3H]cytochalasin B photolabeling of hexose carriers in erythrocyte ghosts. The cross-reactivity of adenosine and several of its derivatives with the hexose carrier suggests further homologies between the carriers for hexoses and nucleosides, possibly related to their ability to transport hydrophilic molecules through the lipid core of the plasma membrane.

Inhibition by nucleosides of glucose-transport activity in human erythrocytes.

The interaction of nucleosides with the glucose carrier of human erythrocytes was examined by studying the effect of nucleosides on reversible cytochalasin B-binding activity and glucose transport. Adenosine, inosine and thymidine were more potent inhibitors of cytochalasin B binding to human erythrocyte membranes than was D-glucose [IC50 (concentration causing 50% inhibition) values of 10, 24, 28 and 38 mM respectively]. Moreover, low concentrations of thymidine and adenosine inhibited D-glucose-sensitive cytochalasin B binding in an apparently competitive manner. Thymidine, a nucleoside not metabolized by human erythrocytes, inhibited glucose influx by intact cells with an IC50 value of 9 mM when preincubated with the erythrocytes. In contrast, thymidine was an order of magnitude less potent as an inhibitor of glucose influx when added simultaneously with the radioactive glucose. Consistent with this finding was the demonstration that glucose influx by inside-out vesicles prepared from human erythrocytes was more susceptible to thymidine inhibition than glucose influx by right-side-out vesicles. These data, together with previous suggestions that cytochalasin B binds to the glucose carrier at the inner face of the membrane, indicate that nucleosides are capable of inhibiting glucose-transport activity by interacting at the cytoplasmic surface of the glucose transporter. Nucleosides may also exhibit a low-affinity interaction at the extracellular face of the glucose transporter.

Direct evidence for ATP modulation of sugar transport in human erythrocyte ghosts.

Sugar transport in human erythrocyte ghosts is modulated by low molecular weight factors present in red cell cytosol that induce an asymmetry in Michaelis and velocity constants for sugar entry and exit (Carruthers, A., and Melchior, D. L. (1983) Biochim. Biophys. Acta 728, 254-266). This study examines the possibility that ATP is the transport-modulating factor. The intracellular factor must satisfy at least three criteria. It must reduce Km and Vmax for sugar efflux from inside-out red cell membrane vesicles. It should increase Km for efflux from red cell ghosts. It should have a molecular weight of less than 10 kDa. These criteria are satisfied by ATP. AMP, ADP, GTP, UTP, and ITP are without effect on sugar transport. The following results support the view that the cytosolic factor is ATP. Red cell lysate (obtained by hypotonic lysis of red cells) is unable to modify transport following dialysis against ATP-free medium. The ability of lysate to modify transport is retained following acid extraction. ATP depletion of acid-extracted lysate by treatment with apyrase results in the loss of transport-modulating potency. Myokinase partly restores both the ATP content and the ability of ATP-depleted (apyrase-treated) lysate to modify transport. Addition of ATP to ATP-depleted lysate mimics the ability to myokinase to restore the transport-modulating potency of lysate. ATP is without effect on the number and molecular size of D-glucose-sensitive cytochalasin B-binding proteins in the red cell membrane. These findings demonstrate that the transport-modulating potency of red cell cytosol is quantitatively accounted for by intracellular ATP which acts to modify the catalytic activity of plasmalemmal transporters.

Characterisation of monoclonal antibodies which specifically recognise the human erythrocyte glucose transport protein.

Two monoclonal antibodies (mabs) of subclass IgG1 have been raised against the human erythrocyte glucose transport protein. The mabs bound to the purified glucose transporter in both its membrane-bound and detergent-solubilised forms. However, they exhibited little or no binding to the detergent-solubilised nucleoside transport protein, which is present as a minor contaminant in the glucose transport protein preparation. Both mabs inhibited the binding of cytochalasin B to the glucose transport protein, reducing the affinity of this binding by greater than 2-fold. Each mab labelled the transporter polypeptide on Western blots both before and after treatment of the protein with endoglycosidase F, indicating that the epitopes recognised were located on the protein moiety of the glycoprotein. However, the mabs did not bind to the large fragments produced by tryptic or chymotryptic digestion of the native protein, although both mabs were shown to bind to sites on the cytoplasmic surface of the erythrocyte membrane.

Inhibition of 3-O-methylglucose transport in human erythrocytes by forskolin.

The effect of forskolin, an activator of adenylate cyclase, was investigated on glucose transport in human erythrocytes. Forskolin was found to be a potent inhibitor of 3-O-methylglucose (3-O-MG) influx in human erythrocytes. The inhibition of 3-O-MG transport was instantaneous and reversible. The inhibitory effect of forskolin was concentration-dependent, having an IC50 value of 7.5 microM. Forskolin caused a decrease in Vmax of carrier-mediated 3-O-MG transport from 35.32 to 1.56 mumol/ml of cell X min in the presence of 50 microM forskolin. Inhibition of influx was not reversed at high concentrations of 3-O-MG. In addition, forskolin inhibited the influx of other carbohydrates including galactose, ribose, and fructose. In contrast, forskolin was without effect on adenosine transport. To unravel the underlying mechanism responsible for the inhibitory action of forskolin, the possible involvement of cyclic AMP in controlling glucose transport was examined. Erythrocytes treated with 50 microM forskolin exhibited an increase in cyclic AMP content from the basal levels of 258 fmol/ml of cell to 334 fmol/ml of cell within 10 s after forskolin exposure. However, erythrocytes in which cyclic AMP was allowed to accumulate in excess of 10,000 times the basal level, by means of preincubation with exogenous cyclic AMP, displayed 3-O-MG transport indistinguishable from that of cyclic AMP-poor control cells. In view of the finding that cyclic AMP plays no discernible role in the erythrocyte 3-O-MG transport, it is suggested that the forskolin inhibition is mediated by a mechanism other than by stimulating adenylate cyclase activity. Moreover, forskolin appears to directly inactivate the 3-O-MG transport system since glucose-sensitive cytochalasin B binding to erythrocyte membranes is virtually abolished by 50 microM forskolin.

Familial hyperinsulinemia complicated by extreme insulin resistance during pregnancy: a probable postreceptor defect.

Detailed studies of a family with hyperinsulinemia are reported. The index patient, a 30-yr-old woman with polycystic ovary syndrome, presented with gestational diabetes which was completely resistant to insulin in the presence of severe endogenous hyperinsulinemia. Sensitivity to insulin was regained after delivery. Therapy with cyproterone acetate and ethinyl estradiol for hirsutism exacerbated the hyperinsulinemia toward the levels occurring in pregnancy, with a concomitant deterioration of glucose tolerance. Five other members of her family also were found to have hyperinsulinemia together with high concentrations of circulating C-peptide. Antibodies to insulin and to insulin receptors were not detected, insulin antagonists were not increased, and insulin degradation in the circulation was normal. Insulin extracted from the patient's serum was identical to normal insulin by the criteria of Sephadex chromatography, placental membrane insulin receptor binding, and stimulation of 2-deoxyglucose uptake in isolated rat adipocytes. Although [125I]insulin binding to erythrocytes of all family members and to the patient's placental membranes was markedly reduced, binding to fibroblast cultures from the patient was normal. Insulin-stimulated glucose transport in these fibroblasts also was normal, but there was a mild (20%) reduction in the concentration of cytochalasin B-binding sites in erythrocyte ghosts. Insulin resistance in this family may be due to a partial defect distal to the insulin receptor. This is asymptomatic unless metabolic stresses (pregnancy or steroid administration) are superimposed.

The monosaccharide transport system of the human erythrocyte. Orientation upon reconstitution.

Treatment of intact human erythrocytes with trypsin had no effect upon either the rate of hexose transport or the binding of cytochalasin B to the transport system. In contrast, proteolysis of inside-out vesicles prepared from human erythrocyte membranes inactivated both hexose transport and cytochalasin B binding. When purified hexose transporter, reconstituted into phospholipid vesicles of undetermined size, was treated with trypsin, approx. 50% of the cytochalasin B binding activity was lost. This loss correlated with a decrease in the amount of the transporter polypeptide, as assayed by gel electrophoresis. These results show that the orientation of the transporter can be established through trypsin treatment in conjunction with cytochalasin B binding. Small unilamellar vesicles containing transporter were prepared by sonication of larger species and by a cycle of cholate solubilization and removal of the detergent. In the former case, the transporter orients almost randomly, whereas in the latter approx. 75% of the transporters have the cytoplasmic domain external.

Cytochalasin B-binding proteins in rabbit erythrocyte membranes and their post-natal change in relation to the glucose carrier activity.

Two distinct, carrier-mediated glucose uptake processes, a fast, cytochalasin B-sensitive and a slow, cytochalasin B-insensitive flux are identified in parallel in newborn rabbit erythrocytes. The fast, cytochalasin B-sensitive carrier function disappears as rabbits age, and only the slow cytochalasin B-insensitive carrier function is observed with adult rabbit erythrocytes. Three different cytochalasin B binding sites are distinguished in newborn rabbit erythrocytes; a glucose-sensitive site (site I), a cytochalasin E-sensitive site (site II), and a site insensitive to both glucose and cytochalasin E. With adult rabbit erythrocytes, only a cytochalasin E-sensitive site is detected. With glucose-sensitive site disappears as rabbits age, with a time course which is comparable to that of the disappearance of the cytochalasin B-sensitive glucose carrier function. The cytochalasin E-sensitive cytochalasin B binding site does not increase during this change, thus the disappearance of the glucose-sensitive site is not due to its conversion to a cytochalasin E-sensitive site. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of rabbit erythrocyte ghosts revealed a partial decrease in each of the membrane polypeptides of approximate molecular weights of 240 000, 160 000 and 50 000 as rabbits aged. It is concluded that the cytochalasin B-sensitive glucose carrier of fetal rabbit erythrocytes, like that of the human erythrocyte, is tightly associated with the site I cytochalasin B-binding protein, while the cytochalasin B-insensitive glucose carrier, operative in adult rabbit erythrocytes, is not.

Purification of the cytochalasin B binding component of the human erythrocyte monosaccharide transport system.

The cytochalasin B binding component of the human erythrocyte monosaccharide transport system has been purified. The preparation appears to contain one major protein with an apparent polypeptide chain molecular weight of 55,000 and about 0.4 binding sites per chain. Cytochalasin B binds to the reconstituted preparation with a dissociation constant of 1.3.10(-7) M, a value which is similar to that reported for the transport system in the intact erythrocyte.