Retention of insulin-stimulated D-glucose transport activity by adipocyte plasma membranes following extraction of extrinsic proteins

Plasma membrane vesicles prepared from adipocytes incubated with insulin exhibited accelerated D-glucose transport activity characteristic of insulin action on intact fat cells. Both control and insulin-stimulated D-glucose transport activities were inhibited by cytochalasin B and thiol reagents. Extraction of plasma membranes with dimethylmaleic anhydride eluted 80% of the protein from plasma membrane vesicles. The two major glycoprotein bands (94,000 and 78,000 daltons) and small amounts of a 56,000-dalton band were retained in dodecyl sulfate gels of the extracted membranes. Both control and insulin-activated D-glucose transport activities were retained by plasma membrane vesicles extracted with dimethylmaleic anhydride. Cytochalasin B binding activity was also retained by extracted membrane vescles and D-glucose uptake into extracted vescles derived from untreated or insulin-treated fat cells was inhibited by cytochalasin B. These results suggest that the modification of the adipocyte hexose transport system elicited by insulin action is not altered by a major purification step which involves quantitative extraction of extrinsic membrane proteins.     

D-glucose binding increases secondary structure of human erythrocyte monosaccharide transport protein

Purified hexose transport protein ("band 4.5") from human erythrocytes, reconstituted in vesicles of its endogenous lipids, displays minima in its circular dichroism (CD) spectrum at 222 and 207 nm, a pattern diagnostic for alpha-helical content of proteins. Upon addition of D-glucose, a saturable increment of +10-12% in negative ellipticity at 222 nm is observed stereospecifically and reproducibly. Addition of L-glucose had no effect on the CD spectrum of the transport protein. Addition of cytochalasin B (CB), a reversible inhibitor of hexose transport, had no effect itself on transporter CD spectra, but restored the spectrum at 222 nm to its original value when added in the presence of D-glucose. The observed D-glucose-induced increase in ordered secondary structure is proposed to result from incorporation into the membrane of a segment of the transport protein originally at a membrane-water interface.     

Reconstitution of the glucose transporter from bovine heart

Reconstitution of the glucose transporter from heart should be useful as an assay in its purification and in the study of its regulation. We have prepared plasma membranes from bovine heart which display D-glucose reversible binding of cytochalasin B (33 pmol sites/mg protein; Kd = 0.2 muM). The membrane proteins were reconstituted into liposomes by the freeze-thaw procedure. Reconstituted liposomes showed D-glucose transport activity which was stereospecific, saturable and inhibited by cytochalasin B, phloretin, and mercuric chloride. Compared to membrane proteins reconstituted directly, proteins obtained by dispersal of the membranes with low concentrations of cholate or by cholate solubilization showed 1.2- or 2.3-fold higher specific activities for reconstituted transport, respectively. SDS-polyacrylamide gel electrophoresis followed by electrophoretic protein transfer and labeling with antisera prepared against the human erythrocyte transporter identified a single band of about 45 kDa in membranes from both dog and bovine hearts, a size similar to that reported for a number of other glucose transporters in various animals and tissues.     

Photolabeling of the human erythrocyte glucose carrier with androgenic steroids

Androgenic steroids, which are potent inhibitors of facilitated hexose transport in human erythrocytes, were tested as possible natural photolabels of the hexose carrier protein. Androstenedione, which inhibited 3-O-methylglucose uptake half-maximally at 30-50 microM (EC50), was the most potent inhibitor of the photolabile steroids tested. It appeared to interact directly with the carrier, since it (1) inhibited equilibrium [3H]cytochalasin B binding to high affinity D-glucose-sensitive sites in both intact cells (EC50 = 63 microM) and protein-depleted ghosts (EC50 = 61 microM), (2) inhibited cytochalasin B photolabeling of the band 4.5 carrier region in electrophoretic gels of protein-depleted ghosts (EC50 = 50 microM), and (3) underwent photoincorporation into the same gel region in a D-glucose- and cytochalasin B-sensitive fashion. However, Dixon plots for inhibition of both cytochalasin B binding and transport were upward-curving, indicating the binding of more than one molecule of androstenedione to the carrier. The photoincorporation of androstenedione into band 4.5 protein was both time- and concentration-dependent, and not associated with damage to unlabeled carrier. It probably occurred by activation of the alpha, beta-unsaturated ketone on the steroid rather than indirectly by photoactivation of a group on the carrier protein, as occurs with cytochalasin B. Although androstenedione may bind to more than one region of the carrier, as well as to other non-carrier proteins, tryptic digestion of photolabeled ghosts produced a labeled Mr = 18,000-20,000 fragment, the labeling of which was inhibited by cytochalasin B, and which had an electrophoretic mobility similar to the major labeled tryptic fragment in cytochalasin B-labeled ghosts. These data suggest that androstenedione interacts directly with the hexose carrier and that it or other similar naturally photolabile steroids may serve as useful probes for structural dissection of the carrier protein.     

Direct interaction of phenylarsine oxide with hexose transporters in isolated rat adipocytes

It has previously been shown that phenylarsine oxide (PhAsO), an inhibitor of protein internalization, also inhibits stereospecific uptake of D-glucose and 2-deoxyglucose in both basal and insulin-stimulated rat adipocytes. This inhibition of hexose uptake was found to be dose-dependent. PhAsO rapidly inhibited sugar transport into insulin-stimulated adipocytes, but at low concentrations inhibition was transient. Low doses of PhAsO (1 microM) transiently inhibit stereospecific hexose uptake and near total (approx. 90%) recovery of transport activity occurs within 20 min. Interestingly, once recovered, the adipocytes can again undergo rapid inhibition and recovery of transport activity upon further treatment with PhAsO (1 microM). In addition, PhAsO is shown to inhibit cytochalasin B binding to plasma membranes from insulin-stimulated adipocytes in a concentration-dependent manner which parallels the dose-response inhibition of hexose transport by PhAsO. The data presented suggest a direct interaction between the D-glucose transporter and PhAsO, resulting in inhibition of transport. The results are consistent with the current recruitment hypothesis of insulin activation of sugar transport and indicate that a considerable reserve of intracellular glucose carriers exists within fat cells.     

Regulation of hexose transporters in chicken embryo fibroblasts: stimulation by the phorbol ester TPA leads to increased numbers of functioning transporters

As has been observed with many types of cultured cells, chicken embryo fibroblasts (CEF) when exposed to the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) develop a 3- to 4-fold increase in hexose transport activity in 4 h. This increase in transport activity occurred despite a modest decline of 20% in [3H]leucine incorporation into acid insoluble fractions. Cycloheximide largely, but not completely, blocked the increase in transport activity during TPA exposure. The effects of TPA were somewhat similar to those of glucose starvation induced enhancement of hexose transport activity. Furthermore, with TPA there was no additive effect to that produced by glucose starvation. Plasma membrane enriched fractions were prepared from CEF treated with or without TPA. Membranes prepared from TPA exposed cells had a two-fold enhancement of stereospecific D-glucose transport activity as well as D-glucose inhibitable [3H]cytochalasin B binding as compared to the membranes from control CEF. There was no effect on transport when membranes were exposed to TPA in vitro. These results provide strong evidence that TPA exposure leads to an increase in the number of functioning transporters, an effect largely requiring protein synthesis.     

Insulin regulation of glucose metabolism in HT29 colonic adenocarcinoma cells: activation of glycolysis without augmentation of glucose transport

The effects of insulin on glucose transport and metabolism were examined in cultured HT29 human colonic adenocarcinoma cells. The presence of glucose transporters was verified by D-glucose displaceable [3H]cytochalasin B binding. The Kd and Bmax values from cytochalasin B binding studies were 190 +/- 30 nM and 8.4 +/- 1.4 pmol/mg protein, respectively. Glucose transport determined with 3-O-methylglucose showed saturable kinetics with a Km of 5.8 +/- 0.4 mM and a Vmax of 0.047 +/- 0.003 mumol/mg protein per min at 25 degrees C. Moreover, in HT29 cells, two classes of insulin binding sites were detected in radioligand binding experiments. Although insulin failed to stimulate glucose transport, it was found to activate glycolysis in HT29 cells. Glucose consumption increased from 0.33 +/- 0.03 mumol/mg protein per h to 0.49 +/- 0.05 mumol/mg protein per h and lactate production was augmented from 0.67 +/- 0.04 mumol/mg protein per h to 0.87 +/- 0.06 mumol/mg protein per h in response to 10(-7) to 10(-5) M insulin. Insulin also enhanced mannose metabolism. Apart from these two hexoses, HT29 cells exhibited a surprisingly narrow substrate specificity. With the possible exception of glyceraldehyde, little lactate was produced from alternative substrates, including adenosine, inosine, ribose, deoxyribose, dihydroxyacetone, galactose and fructose either with or without insulin. Despite its limited utilization by the glycolytic pathway, adenosine was readily salvaged for de novo synthesis of adenine nucleotides. These findings suggest that insulin directly influences substrate utilization through the glycolytic pathway in HT29 cells without activating the glucose transport pathway.     

Binding of [3H]cytochalasin B to tumoral islet cells

Tumoral pancreatic islet cells of the RINm5F line are equipped with two classes of [3H]cytochalasin B binding sites with respective Kd of 0.4 and 7 microM. The binding of the fungal metabolite and its dissociation from the binding sites display rapid time courses. The binding is inhibited by D-glucose, more than by L-glucose, by phlorizin and by cytochalasin E. These findings are considered in the light of the dual action of cytochalasin B upon hexose transport and motile activity in islet cells.     

Inhibitors of protein synthesis cause increased hexose transport in cultured human fibroblasts by a mechanism other than transporter translocation.

We have investigated the effect of various inhibitors of protein synthesis on hexose transport in human skin fibroblasts using 2-deoxy-D-glucose (2-DG) and 3-0-methyl-D-glucose (3-OMG) to measure hexose transport. Exposure of glucose-fed, serum-free cultures to cycloheximide (CHX) (50 micrograms/ml) for 6 h resulted in increased 2-DG transport (3.81 +/- .53 vs. 6.62 +/- .88 nmoles/mg protein/2 min; n = 9) and 3-OMG transport (1.36 +/- .66 vs. 3.18 +/- .83 nmoles/mg protein/30 sec; n = 4) in the CHX exposed group. Under these conditions inhibition of protein synthesis was greater than 90%. This CHX induced transport increase was time dependent (approaching maximum within 1 h of exposure to CHX) and related to an increase in the Vmax of hexose transport in the CHX exposed group (18.4 +/- 2.4 vs. 4.8 +/- 1.1 nmoles 2-DG/mg protein/min) with no difference in the transport Km (1.55 +/- .63 vs. 2.92 +/- .59 mM). Further, the CHX induced increase in hexose transport was reversible. Exposure of human fibroblasts to inhibitors of protein synthesis with different mechanisms of action (e.g., puromycin, pactamycin, or CHX) all generated hexose transport increases in a concentration-dependent fashion correlating with their increasing inhibitory effects on protein synthesis. Nucleotidase enriched (i.e., plasma membrane) fractions of control and CHX-exposed cells showed no differences in D-glucose inhibitable cytochalasin B binding activity. Further, quantitative Western analysis of nucleotidase enriched fractions indicated CHX exposure resulted in no significant increase in glucose transporter mass compared with control plasma membrane fractions. Glucose deprived cells, however, which exhibited increased sugar transport comparable to the CHX-exposed group, did show increased glucose transporter mass in the plasma membrane fraction. The data indicate that inhibitors of protein synthesis can cause a significant elevation in hexose transport and that the hexose transporter mass in the isolated plasma membrane fractions did not reflect the whole cell transport change. It is suggested that a mechanism other than glucose transporter translocation to the plasma membrane may be involved in causing this sugar transport increase.     

Ligand-dependent quenching of tryptophan fluorescence in human erythrocyte hexose transport protein

D-Glucose transport by the 492-residue human erythrocyte hexose transport protein may involve ligand-mediated conformational/positional changes. To examine this possibility, hydrophilic quencher molecules [potassium iodide and acrylamide (ACR)] were used to monitor the quenching of the total protein intrinsic fluorescence exhibited by the six protein tryptophan (Trp) residues in the presence and absence of substrate D-glucose, and in the presence of the inhibitors maltose and cytochalasin B. Protein fluorescence was found to be quenched under various conditions, ca. 14-24% by KI and ca. 25-33% by ACR, indicating that the bulk of the Trp residue population occurs in normally inaccessible hydrophobic regions of the erythrocyte membrane. However, in the presence of D-glucose, quenching by KI and ACR decreased an average of -3.4% and -4.4%, respectively; Stern-Volmer plots displayed decreased slopes in the presence of D-glucose, confirming the relatively reduced quenching. In contrast, quenching efficiency increased in the presence of maltose (+5.9%, +3.3%), while addition of cytochalasin B had no effect on fluorescence quenching. The overall results are interpreted in terms of ligand-activated movement of an initially aqueous-located protein segment containing a Trp residue into, or toward, the cellular membrane. Relocation of this segment, in effect, opens the D-glucose channel; maltose and cytochalasin B would thus inhibit transport by mechanisms which block this positional change. Conformational and hydropathy analyses suggested that the region surrounding Trp-388 is an optimal "dynamic segment" which, in response to ligand activation, could undergo the experimentally deduced aqueous/membrane domain transfer.     

Photoaffinity labeling of insulin-sensitive hexose transporters in intact rat adipocytes. Direct evidence that latent transporters become exposed to the extracellular space in response to insulin

Irradiation of intact rat adipocytes with high intensity ultraviolet light in the presence of 0.5 microM [3H] cytochalasin B results in the labeling of Mr 43,000 and 46,000 proteins that reside in the plasma membrane fraction. In contrast to the Mr 46,000 protein, the Mr 43,000 component is not observed in the microsome fraction and exhibits lower affinity for [3H]cytochalasin B. Photolabeling of the Mr 43,000 protein is inhibited by cytochalasin D, indicating it is not a hexose transporter component. The Mr 46,000 protein exhibits characteristics expected for the glucose transporter such that D-glucose or 3-O-methylglucose but not cytochalasin D inhibits its photolabeling with [3H] cytochalasin B. Furthermore, insulin addition to intact cells either prior to or after photoaffinity labeling of the Mr 46,000 protein causes a redistribution of this component from the low density microsomes to the plasma membrane fraction, as expected for the hexose transporter. Photolabeling of transporters in both the low density microsome and plasma membrane fractions is inhibited when intact cells are equilibrated with 50 mM ethylidene glucose prior to irradiation with [3H]cytochalasin B. Incubation of intact cells with 50 mM ethylidene glucose for 1 min at 15 degrees C leads to an intracellular concentration of only 2 mM. Under these conditions, the photoaffinity labeling in intact cells of hexose transporters that fractionate with the low density microsomes is unaffected, indicating these transporters are not exposed to the extracellular medium. In contrast, photolabeling in intact insulin-treated cells of hexose transporters that fractionate with the plasma membrane is inhibited under these incubation conditions. The results demonstrate that insulin action results in the exposure to the extracellular medium of previously sequestered hexose transporters.     

Transport of alpha- and beta-D-glucose by the intact human red cell

The kinetics of alpha- and beta-D-glucose mutarotation and the transport of these anomers by intact human red cells were determined at 0.6 and 36.6 degrees C. The mutarotation coefficients for alpha- and beta-D-glucose in cell-free tris(hydroxymethyl)aminomethane medium (pH 7.4) at 0.6 degrees C are (2.25 +/- 0.2) and (1.73 +/- 0.42) X 10(-3) min-1, respectively, and at 36.6 degrees C are (69 +/- 12) and (75 +/- 5) X 10(-3) min-1, respectively. These values are in good agreement with previous estimates. At 0.6 degrees C, the red cell contains no detectable mutarotase activity. Initial rates of sugar uptake were measured by using radiolabeled D-glucose and time courses of uptake by turbidimetry. The time courses of alpha- and beta-D-glucose and an equilibrium mixture of alpha- and beta-D-glucose infinite-cis entry are identical at 0.66 degrees C (n = 41) where negligible mutarotation is observed. The apparent Ki values for inhibition of radiolabeled D-glucose initial uptake by unlabeled alpha- or beta-D-glucose at 0.6 degrees C are identical (1.6 mM). The calculated Vmax parameters for uptake of the radiolabeled anomers at this temperature are also indistinguishable. The time courses of infinite-cis alpha- and beta-D-glucose uptake at 36.66 degrees C are identical (n = 40). While D-glucose mutarotation is more rapid at this temperature, the anomers of D-glucose are not transported differently by the red cell hexose transfer system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic characteristics and regulation of hexose transport in a galactokinase-negative Chinese hamster fibroblast cell line: a good model for studies on sugar transport in cultured mammalian cells

We report the kinetic characteristics for D-galactose, 2-deoxy-D-glucose and 3-O-methyl-D-glucose transport in a galactokinase null-allele mutant of a Chinese hamster V79 cell line. GalKl cells exhibited a Km and Vmax for D-galactose, 2-deoxy-D-glucose, and 3-O-methyl-D-glucose transport of 8.6 +/- 2.6 mM and 26.1 +/- 7.2 nmol/mg p/min, 4.1 +/- 1.2 mM and 40.3 +/- 9.5 nmol/mg p/min, and 7.01 +/- .85 mM and 11.6 +/- 4.8 nmol/mg p/30 s, respectively. Nonsaturable hexose uptake was determined using cytochalasin B inhibition of galactose uptake (89.6 +/- 3.7% of galactose uptake was cytochalasin B inhibitable) and L-glucose uptake (7.5% of the galactose uptake). D-Galactose was not metabolized and effluxed rapidly from preloaded cells. The Kls for the inhibition of D-galactose transport were 4.5 +/- 2.5 mM for D-glucose, 7.0 +/- 2.0 mM for 2-deoxy-D-glucose, 6 mM for 2-deoxy-D-galactose and 6.0 +/- 0.6 mM for 3-O-methyl-D-glucose. This indicates the operation of a single common carrier. The hexose transport rate decreased 50-60% after 24 h serum deprivation. Addition of insulin was shown to increase hexose transport (more than twofold) in serum-deprived cells. Hexose transport rates increased substantially in glucose-deprived, D-fructose- or D-galactose-fed cells as compared to glucose-fed cells. Since GalKl does not metabolize galactose, the hexose transport increases induced by feeding cells galactose suggest that carrier interaction with ligand is not a significant factor in transport regulation in GalKl. The kinetic and regulatory characteristics of D-galactose transport in the GalKl cell line indicate that this system is a good model to study sugar transport from a mechanistic and regulatory point of view.     

Studies on avian erythrocyte metabolism. XVII. Kinetics and transport properties of myo-inositol in chicken reticulocytes

The uptake of myo-inositol was determined in a reticulocyte-enriched fraction prepared from chicken blood and compared with uptake in mature erythrocytes. While reticulocytes accumulated inositol at levels more than threefold that of the plasma concentration, erythrocyte levels were only slightly higher than that of the plasma concentration. The rate of uptake in reticulocytes was approximately 66 mumol/ml rbc/h compared to 5 mumol/ml rbc/h in mature erythrocytes when measured at an inositol medium concentration of 250 microM. The kinetic analysis of inositol influx by reticulocytes reveals a two component system: saturable and nonsaturable. The saturable component, which has a Km for inositol of approximately 222 microM, is Na-dependent. This Na-dependent saturable component, which presumably reflects active transport of inositol, accounts for 30-35% of the transport process. The saturable component is completely inhibited by amiloride but to a lesser extent by ouabain and bumetanide. Moreover, in the course of reticulocyte maturation, the saturable component is lost concomitantly with the completion of the synthesis of myo-inositol pentakisphosphate and the drastic decrease in the membrane permeability to inositol. In addition, phloretin and cytochalasin B, which bind to hexose carriers and inhibit hexose sugar transport, also inhibited inositol transport. The uptake of inositol was not affected by excesses of 3-O-methylglucose (100 mM) or by physiological concentrations of D-glucose. Thus, the transport mechanism of myo-inositol appears distinct from that of D-glucose.     

Genetic evidence indicating the identity of the cytochalasin B photolabelled components in rat myoblasts

While photolabelling with cytochalasin B (CB) has been widely used in the identification of eukaryotic glucose transporters, there is presently no unequivocal evidence indicating that the CB-labelled components are indeed the glucose transporters. A combination of biochemical, physiological and genetic manipulations was used in the present investigation to demonstrate that the plasma membrane hexose transporters can indeed by photolabelled by CB. In this study, plasma membranes from glucose-grown and glucose-starved hexose transport mutant D23 and its parental L6 cells were photolyzed in the presence of 3H-CB. The amount of CB bound to the 40-60 kDa region (CB50) was found to be differentially inhibited by D-glucose, 2-deoxy-D-glucose (dGlc) and 3-O-methyl-glucose (MeGlc). Mutant D23 exhibited not only reduced hexose transport activity but also significantly lower level of CB50. Glucose-starvation resulted not only in elevated hexose transport activity but also increased level of CB50. It should be noted glucose-starvation did not have much effect on the hexose transport activity and on the level of CB50 in mutant D23. The present study provides the first genetic evidence indicating that the CB-labelled component(s) are indeed associated with the hexose transport systems.     

Identification of the glucose transport protein of the microvillous membrane of human placenta by photoaffinity labelling

The initial step in transfer of glucose from mother to fetus is facilitated diffusion transport across the microvillous membrane of the placental syncytium (1). We have used ³H-cytochalasin B as a photoaffinity label to identify the transport protein involved. Two binding proteins were present, one of which is apparently the glucose transport protein and one of which is actin. The two were identified by competition labeling with D-glucose, and cytochalasin E. They were separated by selective extraction with dimethyl maleic anhydride. The glucose transport protein is apparently a single molecular species of 52,000 molecular weight.     

Effect of insulin, prostaglandin E1 and uptake inhibitors on glucose transport in the perfused guinea-pig placenta

The effects of insulin, prostaglandin E1 (PGE1) and uptake inhibitors on unidirectional D-glucose influx at brush border (maternal) and basal (fetal) sides of the guinea-pig syncytotrophoblast were investigated in the intact, perfused guinea-pig placenta by rapid, paired-tracer dilution. Experiments were performed in either an in situ preparation artificially perfused through the umbilical vessels (intact maternal circulation) or in the fully isolated dually-perfused placenta in which both interfaces were studied simultaneously. Kinetic characterization of unidirectional D-glucose influx gave apparent Km values (mean +/- SEM) at maternal and fetal sides of 70 +/- 6 and 87 +/- 16 mM respectively; corresponding Vmax values were 53 +/- 3 and 82 +/- 6 mumol min-1g-1. At the fetal side (singly-perfused placenta) cytochalasin B (50 microM), ethylidene-D-glucose (100 mM) and PGE1 (1 microM) partially inhibited D-glucose uptake whereas cortisol (50 microM) and progesterone (100 microM) had no effect. Abolition of the sodium gradient across the fetal interface did not modulate the kinetics of influx. In the presence of 150 mu units ml-1 insulin (dually-perfused placenta), unidirectional uptake into the trophoblast and transplacental D-[3H]glucose transfer were unaltered. In contrast, prostaglandin E1 (1 microM) markedly reduced the Km and Vmax for D-glucose at both interfaces and the inhibitory effect was reflected in a reduction in specific transplacental D-glucose transfer. Further experiments showed that the isolated placenta releases prostaglandins (PGE; PGF2 alpha) into both circulations. Bilateral insulin perfusion did not affect either lactate release by the placenta or rapid metabolism of D-[14C]glucose to [3H]lactate (usually less than 10% effluent [14C]lactate in 5 min). An asymmetric degradation of exogenous insulin was observed in the dually-perfused placenta: uterine venous samples contained 24 +/- 7 microunits ml-1 immunoreactive insulin when compared to the arterial concentration (151 +/- 3 microU ml-1 perfusate) while no change was measureable in the fetal circulation within the same time period (152 +/- 5 microU ml-1). This asymmetry was confirmed in experiments employing [125I]insulin. These results demonstrate that glucose transport in the intact guinea-pig placenta occurs by a sodium-independent, cytochalasin B-inhibitable system which is insulin-insensitive. Prostaglandin E1 appeared to be a potent transport inhibitor which suggests that prostaglandins may be involved in the 'down' regulation of placental glucose transport in vivo.     

Effect of hypertonicity on hexose transporter regulation in chicken embryo fibroblasts

The regulation of hexose transporters of cultured fibroblasts was investigated by exposing chicken embryo fibroblasts (CEF) to hypertonic culture medium, a condition known to enhance hexose transport activity. The effects of hypertonicity and the role of protein synthesis were examined with CEF in the basal (glucose fed) and transport enhanced (glucose starved) states. Glucose-fed CEF exposed to hypertonic conditions developed four-fold enhancement of hexose transport activity within 4 hrs; this declined in the following 20 hrs to a level slightly higher than the fed control. Protein synthesis was required in part for this effect, since the presence of cycloheximide during hypertonic exposure of fed CEF blocked the increase in of transport by almost 50%. Although the increased transport produced by glucose starvation was not further enhanced by hypertonicity, hypertonic treatment of starved CEF during glucose refeeding largely prevented the loss of transport activity to the basal, fed state. The hypertonic effects were concentration dependent (240mOsm optimal) and could be elicited with NaCl, KCl, or sucrose. Hypertonic treatment typically led to a greater than 50% decline in the incorporation of [3H]leucine into acid-insoluble fractions. The changes in transport were evident at the plasma membrane level, and studies of membrane vesicles prepared from hypertonically treated fed CEF showed a doubling of both [3H]cytochalasin B binding and the Vmax of D-glucose transport. These findings indicate that exposure of CEF to hypertonic conditions has some effects similar to those produced by glucose starvation and suggest that protein synthesis is to some extent involved in the regulation of hexose transporters in CEF.     

Use of a genetic variant to study the hexose transport properties of human skin fibroblasts.

Human skin fibroblasts from 'normal' subjects were found to possess at least two hexose transport systems. One system was responsible for the uptake of 2-deoxy-D-glucose (dGlc), D-glucose and D-galactose, whereas the other was responsible primarily for the uptake of 3-O-methyl-D-glucose (MeGlc). The transport of dGlc was the rate-limiting step in the uptake process; over 97% of the internalized dGlc was phosphorylated and the specific activity of hexokinase was several times higher than that for dGlc transport. The dGlc transport system was activated by glucose starvation, and was very sensitive to inhibition by cytochalasin B and energy uncouplers. Fibroblasts isolated from a patient with symptoms of hypoglycaemia were found to differ from their normal counterparts in the dGlc transport system. They exhibited a much higher transport affinity for dGlc, D-glucose and D-galactose, with no change in the respective transport capacity. Transport was not the rate-limiting step in dGlc uptake by these cells. Moreover, the patient's dGlc transport system was no longer sensitive to inhibition by cytochalasin B and energy uncouplers. This suggested that the intrinsic properties of the patient's dGlc transport system were altered. It should be noted that the patient's dGlc transport system could still be activated by glucose starvation. Despite the changes in the dGlc transport system, the MeGlc transport system in the patient's fibroblasts remained unaltered. The observed difference in the properties of the two hexose transport systems in the 'normal' and the patient's fibroblasts strongly suggests that the two transport systems may be coded or regulated by different genes. The present finding provides the first genetic evidence from naturally occurring fibroblasts indicating the presence of two different hexose transport systems.     

Reconstitution studies of the human erythrocyte nucleoside transporter

The human erythrocyte nucleoside transporter has been identified as a band 4.5 polypeptide (Mr 45,000-66,000) on the basis of reversible binding and photoaffinity labeling experiments with the nucleoside transport inhibitor, nitrobenzylthioinosine (NBMPR). In the present study, the NBMPR-binding protein was extracted from protein-depleted human erythrocyte "ghosts" with Triton X-100 and reconstituted into soybean phospholipid vesicles by a freeze-thaw-sonication procedure. The reconstituted proteoliposomes exhibited nitrobenzylthioguanosine (NBTGR)-sensitive [14C]uridine transport. A partially purified preparation of the NBMPR-binding protein, consisting largely of band 4.5 polypeptides, was also shown to have nucleoside transport activity. This band 4.5 preparation exhibited a 10-fold increase in uridine transport activity and a 7-fold increase in NBMPR-binding activity relative to the crude membrane extract. Uridine transport by the reconstituted band 4.5 preparation was saturable (apparent Km = 0.21 mM; Vmax = 9 nmol/mg of protein/5 s) and was inhibited by dipyridamole, dilazep, adenosine, and inosine. The vesicles reconstituted with the band 4.5 preparation also exhibited stereospecific glucose transport which was inhibited by cytochalasin B, but unaffected by NBTGR. In contrast, cytochalasin B was a poor inhibitor of NBTGR-sensitive uridine transport. These experiments implicate band 4.5 polypeptides in both nucleoside and sugar permeation.