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.     

Resistance of the intact and reconstituted adipocyte hexose transport system to irreversible inhibition by sulfhydryl and amino reagents

Sensitivity of the adipocyte D-glucose transport system in intact plasma membranes or following solubilization and reconstitution into phospholipid vesicles to several protein-modifying reagents was investigated. When intact plasma membranes were incubated with N-ethylmaleimide (20 mM) or fluorodinitrobenzene (4 mM), D-glucose transport activity was virtually abolished. However, washing the membranes free of unreacted reagents restored transport activity, indicating that covalent interaction with the membranes did not mediate the transport inhibition. Reaction of [³H] N-ethylmaleimide with plasma membranes under similar conditions resulted in extensive labeling of all protein fractions resolved on dodecyl sulfate gels. Similarly, addition of N-ethyl-maleimide to cholate-solubilized membrane protein had no effect on transport activity in artifical phospholipid vesicles reconstituted under conditions where the membrane protein was free of unreacted N-ethylmaleimide. Transport activity in plasma membranes was also inhibited by both reduced and oxidized dithiothreitol or glutathione (15 mM) in a readily reversible manner, consistent with a noncovalent mode of inhibition. Thus, the insulin-responsive adipocyte D-glucose transport system differs from the red cell hexose transport system in its remarkable insensitivity to modulation by covalent blockade of sulfhydryal or amino groups by the reagents studied.     

Alcohol-induced versus anion-induced states of alpha-chymotrypsinogen A at low pH

Characterization of conformational transition and folding intermediates is central to the study of protein folding. We studied the effect of various alcohols (trifluoroethanol (TFE), butanol, propanol, ethanol and methanol) and salts (K(3)FeCN(6), Na(2)SO(4), KClO(4) and KCl) on the acid-induced state of alpha-chymotrypsinogen A, a predominantly beta-sheet protein, at pH 2.0 by near-UV circular dichroism (CD), far-UV CD and 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence measurements. Addition of alcohols led to an increase in ellipticity value at 222 nm indicating the formation of alpha-helical structure. The order of effectiveness of alcohols was shown to be TFE>butanol>propanol>ethanol>methanol. ANS fluorescence data showed a decrease in fluorescence intensity on alcohol addition, suggesting burial of hydrophobic patches. The near-UV CD spectra showed disruption of tertiary structure on alcohol addition. No change in ellipticity was observed on addition of salts at pH 2.0, whereas in the presence of 2 M urea, salts were found to induce a molten globule-like state as evident from the increases in ellipticity at 222 nm and ANS fluorescence indicating exposure of hydrophobic regions of the protein. The effectiveness in inducing the molten globule-like state, i.e. both increase in ellipticity at 222 nm and increase in ANS fluorescence, followed the order K(3)FeCN(6)>Na(2)SO(4)>KClO(4)>KCl. The loss of signal in the near-UV CD spectrum on addition of alcohols indicating disordering of tertiary structure results suggested that the decrease in ANS fluorescence intensity may be attributed to the unfolding of the ANS binding sites. The results imply that the alcohol-induced state had characteristics of an unfolded structure and lies between the molten globule and the unfolded state. Characterization of such partially folded states has important implications for protein folding.     

Hexose and amino acid transport by chicken embryo fibroblasts infected with temperature-sensitive mutant of Rous sarcoma virus. Comparison of transport properties of whole cells and membrane vesicles

The effect of transformation on hexose and amino acid transport has been studied using whole cells and membrane vesicles of chicken embryo fibroblasts infected with the temperature-sensitive mutant of the Rous sarcoma virus, TS-68. In whole cells, TS-68-infected chicken embryo fibroblasts cultured at the permissive temperature (37 degrees C) had a 2-fold higher rate of 2-deoxy-D-glucose uptake than the same cells cultured at the non-permissive temperature (41 degrees C). However, both the non-transformed and transformed cells had comparable rates of alpha-aminoisobutyric acid transport. Membrane vesicles, isolated from TS-68-infected chicken embryo fibroblasts cultured at 41 degrees C or 37 degrees C, displayed carrier-mediated, intravesicular uptake of D-glucose and alpha-aminoisobutyric acid. Membrane vesicles from TS-68-infected chicken embryo fibroblasts cultured at 37 degrees C had an approx. 50% greater initial rate of stereospecific hexose uptake than the membrane vesicles from fibroblasts cultured at 41 degrees C. The two types of membrane vesicle had similar uptake rates of alpha-aminoisobutyric acid. The results of hexose and amino acid uptake by the membrane vesicles correlated well with those observed with the whole cells. Km values for stereospecific D-glucose uptake by the membrane vesicles from TS-68-infected chicken embryo fibroblasts cultured at 41 and 37 degrees C were similar, but the V value was greater for the membrane vesicles from TS-68-infected cells cultured at 37 degrees C. Cytochalasin B competitively inhibited stereospecific hexose uptake in both types of membrane vesicle. These findings suggest that the membrane vesicles retained many of the features of hexose and amino acid transport observed in whole cells, and that the increased rate of hexose transport seen in the virally-transformed chicken embryo fibroblasts was due to an increase in the number or availability of hexose carriers.     

Involvement of hexose transport in myogenic differentiation

A high (HAHT) and a low (LAHT) affinity hexose transport system are present in undifferentiated rat L6 myoblasts; however, only the latter can be detected in multinucleated myotubes. This suggests that HAHT is either down-regulated or modified as a result of myogenesis. The present investigation examined the relationship between HAHT and myogenic differentiation. While myogenesis could be inhibited by the potent hexose transport inhibitor phloretin, it was not affected by phlorizin which had no effect on hexose transport. This relationship was further explored using six different HAHT-defective mutants. All six mutants, altered in either the HAHT transport affinity (Type I mutants) or capacity (Type II mutants), were impaired in myogenesis. Since these mutants were selected from both mutagenized and non-mutagenized cells with different reagents, or with different concentrations of the same reagent, the deficiency in myogenesis was likely due to changes in HAHT properties. This notion was confirmed by the observation that growth of Type I mutants in high D-glucose concentrations could rectify the defect in myogenesis. D-glucose was unlikely to rectify the defect in myogenesis, if this defect was due to a second unrelated mutation that may have arisen during isolation of the mutants. Since both types of mutants were not altered in LAHT, D-glucose should still be taken up into the cells. The fact that the glucose-mediated increase in fusion could not be observed in Type II mutants (deficient in the HAHT transporter) suggested that myogenesis was dependent on the presence of D-glucose or its metabolites in specific HAHT-accessible compartments. It is tempting to speculate that trans-acting regulators involved in myogenesis may be synthesized from the glucose metabolites in these specialized HAHT-accessible compartments.     

Conformational studies of NADPH cytochrome P-450 reductase by circular dichroism: interaction with phospholipids

Ultraviolet circular dichroism spectrum of purified NADPH cytochrome P-450 reductase was characterized by two negative bands centered at 208 and 222 nm. The approximation of the alpha-helical content from the value of the mean residue ellipticity at 222 nm indicated 28% of alpha-helical structures. Heat inactivation of the enzyme was associated to a drastic change in the secondary structure of the protein. Membrane reconstitution experiments by inclusion of the enzyme into liposomes revealed that the conformation of NADPH cytochrome P-450 reductase was sensitive to its phospholipid environment. Egg lecithin as well as synthetic phosphatidylcholines, at the optimal phospholipid-enzyme molar ratio 200, was able to increase up to 37% the mean residue ellipticity at 222 nm. Addition of phosphatidylserine or phosphatidylethanolamine produced no effect. Non-ionic detergent such as Emulgen 913 weakly enhanced the mean residue ellipticity.     

Anomalous asymmetric kinetics of human red cell hexose transfer: role of cytosolic adenosine 5'-triphosphate

Cytosolic adenosine 5'-triphosphate (ATP) modifies the properties of human red cell sugar transport. This interaction has been examined by analysis of substrate-induced sugar transporter intrinsic fluorescence quenching and by determination of Michaelis and velocity constants for D-glucose transport in red cell ghosts and inside-out vesicles lacking and containing ATP. When excited at 295 nm, human erythrocyte ghosts stripped of peripheral proteins display an emission spectrum characterized by a scattering peak and a single emission peak centered at about 333 nm. Addition of sugar transport substrate or cytochalasin B and phloretin (sugar transport inhibitors) reduces emission peak height by 10% and 5%, respectively. Cytochalasin B induced quenching is a simple saturable phenomenon with an apparent Kd (app Kd) of 60 nM and a capacity of 1.4 nmol of sites/mg of membrane protein. Quenching by D-glucose (and other transported sugars) is characterized by at least two (high and low) app Kd parameters. Inhibitor studies indicate that these sites correspond to sugar efflux and influx sites, respectively, and that both sites can exist simultaneously. ATP induces quenching of stripped ghost fluorescence with half-maximal effects at 20-30 microM ATP. ATP reduces the low app Kd and increases the high app Kd for sugar-induced fluorescence quenching. D-Glucose transport in intact red cells is asymmetric (Km and Vmax for influx less than Km and Vmax for efflux). In addition, two operational Km parameters for efflux are detected in zero- and infinite-trans efflux conditions. Protein-mediated sugar transport in ghosts and inside-out vesicles (IOVs) is symmetric with respect to Km and Vmax for entry and exit, and only one Km for exit is detected. Addition of millimolar levels of ATP to the interior of ghosts or to the exterior of IOVs restores both transport asymmetry and two operational Km parameters for native efflux. A model for red cell hexose transport is proposed in which ATP modifies the catalytic properties of the transport system. This model mimics the behavior of the sugar transport systems of intact cells, ghosts, and inside-out vesicles.     

The Relationship Between the Metal Clusters and the Conformation of Molybdenum-Iron Protein from Azotobacter vinelandii

The ultraviolet CD spectrum of nitrogenase MoFe protein from Azotobacter vinelandii had a negative trough with double peaks at 208 nm and 222 nm, respectively, and the shape of the trough was similar to those of other proteins with a-helix structure. After treatment with o-phenanthroline under an aerobic or anaerobic condition, the height of the peak at 222 nm (h222 nm) decreased with the decrease of the C2H2-reduction activity, Fe content and CD spectra at both 450 nm and 660 nm, or at 450 nm of the treated proteins. However, after reconstituting with a reconstituent solution containing Na2MoO4, Na2S, dithiothreitol and either ferric homocitrate or ferric citrate, the h222 nm Of the reconstituted proteins could be restored as well as the activity, Fe content and CD spectra at both of 450 nm and 660 nm. The results show that there is a significant relationship between the metal clusters (FeMoco and P-cluster) and the conformation of MoFe protein.     

Control of sugar transport in human fibroblasts independent of glucose metabolism or carrier-substrate interaction

Transport regulation by different metabolizable and nonmetabolizable sugars was studied in human fibroblasts. Sugars were classed as glucose-like (D-mannose, 3-0-methyl-D-glucose, thio-D-glucose, and D-allose) and starvation-like (D-galactose, D-fructose, L-glucose, D-xylose, 6-deoxy-D-glucose and 2-deoxy-D-glucose) based on their competence in curbing glucose starvation enhanced transport. No significant correlation existed between the ability of a sugar to curb hexose transport and the KI of that sugar in inhibiting hexose transport. Independence of the transport curb from glucose metabolism was observed since nonmetabolizable analogs of D-glucose when substituted for D-glucose in the culture medium effected glucose [i.e. 3-0-methyl-D-glucose (3-OMG)] and starvation-like (i.e. 6- and 2-deoxy-D-glucose) effects. The KI of inhibition pf 2-deoxy-D-glucose transport for 3-OMG was 8.5 mM, similar to those obtained for 6-deoxyglucose and 2-deoxyglucose on 2-deoxyglycose transport (7.5 and 3.5 mM, respectively) and on 3-0-methylglucose transport (3.5 and 2.5 mM, respectively). An equimolar mixture of D-glucose and 3-OMG (5.55 mM each) was more effective than 11.1 mM D-glucose or 3-OMG alone in curbing hexose transport or reversing hexose starvation induced increases in transport. The effect of 3-OMG may be independent of glucose metabolism but it is possible that 3-OMG structurally mimics a metabolite of glucose that may interact with intracellular regulators of carrier degradation and or expression.     

Effects of the anticancer agent VM-26 on hexose uptake in Ehrlich cells

The chemotherapeutic agent VM-26 is a membrane-interactive drug which we have previously demonstrated to be a potent inhibitor of nucleoside transport. Since the carriers mediating nucleoside and hexose transport are structurally and functionally similar, we have further characterized the membrane related properties of this agent by examining its effect on the transport and phosphorylation of hexoses in Ehrlich ascites cells. Under conditions in which only the transport component of hexose uptake was measured, VM-26 had no effect on the influx of 2-deoxyglucose, 3-0-methylglucose, or D-glucose. Glucose-sensitive cytochalasin B binding was only weakly inhibited by the drug. However, VM-26 was an apparent non-competitive inhibitor of the net uptake of 2-deoxyglucose (transport and phosphorylation). Measurement of hexokinase activity in cell extracts failed to demonstrate any significant effect of VM-26 on enzyme activity. In summary, although VM-26 is a potent inhibitor of the transport of nucleosides, it has no apparent effect on the transmembrane flux of hexoses indicating a differential effect on nucleoside and hexose transporters. The ability of the drug to decrease the net accumulation of hexoses in the absence of any detectable effect on hexokinase activity warrants further investigation.     

Glucose as a regulator of insulin-sensitive hexose uptake in 3T3 adipocytes

In the present study we examined the role of glucose in the regulation of its own transport activity in the cultured 3T3 fat cell. A regulatory control of glucose became apparent after these cells were cultured in the absence of glucose. Glucose deprivation of the cells was accompanied by a specific time and protein synthesis-dependent increase in dGlc (2-deoxyglucose) uptake (up to 5-fold), which was due to an increase in the apparent Vmax of the transport system. Concomitantly, the stimulatory effect of insulin on hexose uptake almost completely disappeared. Addition of glucose to the glucose-deprived cells rapidly reversed the deprivation effects. Cycloheximide experiments revealed that the glucose deprivation-induced increase in hexose uptake required protein synthesis as well as a protein synthesis-independent response to glucose deprivation that retarded the turnover of hexose transport activity. Taken together, these data indicate that glucose deprivation is accompanied by retardation of the rate of degradation, internalization, or inactivation of hexose transporters while the increase in dGlc uptake requires at least the continuation of protein synthesis-dependent de novo synthesis, insertion, or activation of hexose transporters. Hexose competitively taken up with dGlc, including the nonmetabolizable glucose analogue 3-O-methylglucose, could replace glucose in the process of prevention and reversal of the deprivation effects, indicating that competitive transport but not the metabolism of hexose is a prerequisite for the regulatory effect of glucose on the activity of its own transport system. In conclusion, our results indicate that in cultured 3T3 fat cells glucose itself is involved in the regulation of the activity of its own transport system by influencing the rate of degradation, internalization, or inactivation of hexose transporters by a protein synthesis-independent mechanism.     

Differential effects of follicle-stimulating hormone, insulin, and insulin-like growth factor I on hexose uptake and lactate production by rat Sertoli cells

The stimulatory effects of follicle-stimulating hormone (FSH), insulin, and insulin-like growth factor I (IGF-I) on lactate production and hexose uptake by Sertoli cells from immature rats were studied. The time-courses and the maximal stimulatory effects of FSH, insulin, and IGF-I on lactate production were virtually identical. When Sertoli cells were incubated in the presence of FSH in combination with insulin or IGF-I (submaximal doses), additive but no pronounced synergistic effects were observed. The stimulatory effects of FSH and insulin were not dependent on the presence of extracellular calcium. 2-Deoxy-D-glucose (2-DOG), an analogue of D-glucose, was used to investigate the hexose transport system of Sertoli cells. Uptake of 2-DOG was linear in time and virtually all of the intracellular 2-DOG was phosphorylated up to 30 min of incubation; 2-DOG uptake was inhibited by cytochalasin B, but not by cytochalasin E. D-glucose, but not D-galactose, appeared to be an effective competitor of 2-DOG uptake. The Km of 2-DOG uptake was not influenced by FSH, insulin, and IGF-I. FSH had no effect on the Vmax of 2-DOG uptake, whereas insulin and IGF-I caused a 30% stimulation of the Vmax. It is concluded that FSH, insulin, and IGF-I stimulate lactate production by cultured Sertoli cells, but that only insulin and IGF-I stimulate hexose transport. The insulin-like effect of FSH on Sertoli cells may principally involve stimulation of glycolytic enzyme activities.     

Characterization of a glucose transport system in Vibrio parahaemolyticus.

Cells of a glucose-PTS (phosphoenolpyruvate:carbohydrate phosphotransferase system)-negative mutant of Vibrio parahaemolyticus transport D-glucose in the presence of Na+. Maximum stimulation of D-glucose transport was observed at 40 mM NaCl, and Na+ could be replaced partially with Li+. Addition of D-glucose to the cell suspension under anaerobic conditions elicited Na+ uptake. Thus, we conclude that glucose is transported by a Na+/glucose symport mechanism. Calculated Vmax and Km values for the Na(+)-dependent D-glucose transport were 15 nmol/min/mg of protein and 0.57 mM, respectively, when NaCl was added at 40 mM. Na+ lowered the Km value without affecting the Vmax value. D-Glucose was the best substrate for this transport system, followed by galactose, alpha-D-fucose, and methyl-alpha-glucoside, judging from the inhibition pattern of the glucose transport. D-Glucose itself partly repressed the transport system when cells were grown in its presence.     

Evaluation of the bioconversion of genetically modified switchgrass using simultaneous saccharification and fermentation and a consolidated bioprocessing approach

Background

In mixed sugar fermentations with recombinant Saccharomyces cerevisiae strains able to ferment D-xylose and L-arabinose the pentose sugars are normally only utilized after depletion of D-glucose. This has been attributed to competitive inhibition of pentose uptake by D-glucose as pentose sugars are taken up into yeast cells by individual members of the yeast hexose transporter family. We wanted to investigate whether D-glucose inhibits pentose utilization only by blocking its uptake or also by interfering with its further metabolism.

Results

To distinguish between inhibitory effects of D-glucose on pentose uptake and pentose catabolism, maltose was used as an alternative carbon source in maltose-pentose co-consumption experiments. Maltose is taken up by a specific maltose transport system and hydrolyzed only intracellularly into two D-glucose molecules. Pentose consumption decreased by about 20 - 30% during the simultaneous utilization of maltose indicating that hexose catabolism can impede pentose utilization. To test whether intracellular D-glucose might impair pentose utilization, hexo-/glucokinase deletion mutants were constructed. Those mutants are known to accumulate intracellular D-glucose when incubated with maltose. However, pentose utilization was not effected in the presence of maltose. Addition of increasing concentrations of D-glucose to the hexo-/glucokinase mutants finally completely blocked D-xylose as well as L-arabinose consumption, indicating a pronounced inhibitory effect of D-glucose on pentose uptake. Nevertheless, constitutive overexpression of pentose-transporting hexose transporters like Hxt7 and Gal2 could improve pentose consumption in the presence of D-glucose.

Conclusion

Our results confirm that D-glucose impairs the simultaneous utilization of pentoses mainly due to inhibition of pentose uptake. Whereas intracellular D-glucose does not seem to have an inhibitory effect on pentose utilization, further catabolism of D-glucose can also impede pentose utilization. Nevertheless, the results suggest that co-fermentation of pentoses in the presence of D-glucose can significantly be improved by the overexpression of pentose transporters, especially if they are not inhibited by D-glucose.     

Conformational changes of type II regulatory subunit of cAMP-dependent protein kinase on cAMP binding

The effect of cAMP on the conformation of the regulatory subunit of type II cAMP-dependent protein kinase (RII) from bovine heart was investigated by UV-difference and circular dichroism (CD) spectroscopy. The UV-difference spectrum of RII with and without cAMP showed a positive band around 278 nm and a negative band at 256 nm. Similarly, cAMP enhanced the ellipticity of RII in the region between 280 and 300 nm and decreased that between 250 and 280 nm. In addition, cAMP transformed the far-UV CD spectrum of RII from that of a negative band minimally at 209 nm with a shoulder at 223 nm to one with two minima at 222 and 211 nm. These data show that cAMP induces conformational changes of RII upon binding. Such structural changes may be the basis of activation of cAMP-dependent protein kinases by cAMP.     

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.     

Characterization of a photosensitive glucose derivative. A photoaffinity reagent for the erythrocyte hexose transporter

The photosensitive reagent 6-N-(4-azido-2-hydroxy-3,5-diiodobenzoyl)-D-glucosamine has been assessed as a potential photoaffinity label for the hexose transporter. Under zero-trans conditions, transport experiments performed in the dark reveal that the reagent inhibits the uptake of D-glucose in resealed human erythrocyte ghosts. Increasing the concentration of glucose in the transport medium has a protective effect, reducing the inhibition. Kinetic analysis indicates that the probe acts as a competitive inhibitor with high affinity for the erythrocyte hexose transporter (Ki between 0.07 and 0.2 microM). Exposure to a 280 nm filtered high intensity mercury-vapor lamp results in a rapid and efficient photolysis. At low concentrations of the probe, specific labeling of membrane preparations was observed. Autoradiograms of 10% SDS gels revealed the specific labeling of bands 4.51 and 6. This labeling was concentration-dependent and protected by D-glucose (not the L-isomer) and phloretin in the medium. When subjected to multiple exposures of low concentration of the photoaffinity reagent, apparent saturation was achieved.     

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.     

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.     

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.