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.     

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.     

Regulation of hexose transport in BALB/c 3T3 preadipose cells: effects of glucose concentration and 12-O-tetradecanoylphorbol-13-acetate

Like many cell types in culture, both undifferentiated and differentiated BALB/c 3T3 preadipose cells respond to glucose deprivation with an increased uptake of 2-deoxy-D-glucose (deoxyglucose) and 3-O-methyl-D-glucose (methylglucose). Glucose readdition to glucose-deprived cultures resulted in a prompt fall in uptake activity; in undifferentiated cells, a half-maximally effective concentration of glucose was approximately 0.5 mM, while 0.1 mM was ineffective. Several hexoses differed in their efficacy of "deactivating" methylglucose transport in glucose-deprived cells; it appeared that a particular hexose must be metabolized beyond the 6-phosphate form to deactivate the transport system. Previous studies have shown that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulates hexose transport in undifferentiated and differentiated BALB/c 3T3 cells. In this study, it was found that TPA (and insulin in differentiated cells) prevented the glucose-induced deactivation of transport activity. Glucose-induced deactivation of transport activity was also prevented by cycloheximide or actinomycin D addition concomitantly with glucose. In glucose-starved cells, agents such as TPA and insulin appear to override a cellular control mechanism sensitive to the external concentration of glucose, so that elevated levels of transport activity are maintained under environmental conditions (i.e., a return to physiological glucose concentrations) that normally induce a fall in transport activity.     

Involvement of membrane sulfhydryls in the activation and maintenance of nutrient transport in chick embryo fibroblasts

At 5 μg/ml, insulin stimulates hexose, A-system amino acid, and nucleoside transport by serum-starved chick embryo fibroblasts (CEF). This stimulation, although variable, is comparable to that induced by 4% serum. The sulfhydryl oxidants diamide (1–20 μM). hydrogen peroxide (500 μM), and methylene blue (50 μM) mimic the effect of insulin in CEF. PCMB-S,¹ a sulfhydryl-reacting compound which penetrates the membrane slowly, has a complex effect on nutrient transport in serum- and glucose-starved CEF. Hexose uptake is inhibited by 0.1–1 mM PCMB-S in a time- and concentration-dependent manner, whereas A-system amino acid transport is inhibited maximally within 10 min of incubation and approaches control rates after 60 min. A differential sensitivity of CEF transport systems is also seen in cells exposed to membrane-impermeant glutathione-maleimide I, designated GS-Mal. At 2 mM GS-Mal reduces the rate of hexose uptake 80–100% in serum- and glucose-starved CEF; in contrast A-system amino acid uptake is unaffected. D-glucose, but not L-glucose or cytochalasin B, protects against GS-Mal inhibition. These results are consistent with the hypothesis that sulfhydryl groups are involved in nutrient transport and that those sulfhydryls associated with the hexose transport system and essential for its function are located near the exofacial surface of the membrane in CEF.     

Hexose transport after glucose refeeding of glucose-starved human fibroblasts: 1. The effects of tunicamycin and cycloheximide. 2. Insulin binding and action

Hexose transport in glucose-starved human fibroblasts was readily reversed by glucose refeeding. This hexose transport reversal was not inhibited by tunicamycin (1.5 microgram/ml) but was blocked by cycloheximide (20 micrograms/ml). The ability of insulin (100 mU/ml) to stimulate hexose transport was returned by glucose refeeding and this was not affected by tunicamycin. Cycloheximide which blocked the glucose refeeding effect on hexose transport, decreased the ability of insulin to stimulate hexose transport. Specific 125I-insulin binding was increased by glucose refeeding of glucose-starved cells and this change in binding was inhibited by tunicamycin and cycloheximide. Thus, it appears that under the conditions employed in human fibroblasts, the ability of insulin to stimulate hexose transport is differentially regulated more by factors affecting basal hexose transport than by those affecting changes in insulin binding.     

Monosaccharide uptake in common carp (Cyprinus carpio) EPC cells is mediated by a facilitative glucose carrier

In most animal cells, transport of monosaccharides across the plasma membrane is mediated by glucose transporters (GLUT). Mammals express at least five distinct transporters (GLUTs 1--5), which are well characterised both functionally and genetically. In contrast, the glucose transport system of fish remains poorly studied. Here we report studies of hexose uptake in carp EPC cells and cloning of a glucose transporter cDNA from these cells. Transport of radio-labelled methylglucose (3-OMG) followed Michaelis--Menten kinetics with a K(m) value (8.5 mM) similar to that of mammalian cells. The inhibition of transport by cytochalasin B and phloretin, but not by phloridzin or cyanide, strongly suggested the existence of a facilitative carrier. D-Glucose, 2-deoxyglucose, 3-OMG, D-mannose and D-xylose were competitive inhibitors of 3-OMG uptake, while L-glucose, mannitol, D-fructose, D-ribose and sucrose did not compete with 3-OMG. We cloned a carp glucose transporter (CyiGLUT1), using RT-PCR and RACE strategies. CyiGLUT1 was different from known carp and zebrafish EST sequences. The complete cDNA (3060 bp) contained one open reading frame encoding a predicted protein of 478 amino acids. The deduced amino acid sequence shared 78% identity with mammalian and avian GLUT1 proteins. Key amino acids involved in substrate selection and catalysis of mammalian GLUTs were conserved in the carp transporter.     

Isolation and characterization of hexose transport mutants in L6 rat myoblasts

A method for the selection and isolation of hexose transport mutants in undifferentiated rat myoblast L6 cells is reported; 2-deoxy-D-glucose (2-DOG)-and 2-deoxy-2-fluoro-D-glucose (2FG)-resistant mutants were selected after mutagenization of L6 cells with ethyl methanesulfonate. Of these, D18 and D23 (selected with 0.1 mM 2-DOG) and F72 and F76 (selected with 0.1 mM 2FG) exhibited the lowest hexose transport activity. Uptake of 0.06 mM 2-DOG, 2FG, or 3-O-methyl-D-glucose (3-OMG) by mutants grown in fructose medium supplemented with 0.05 mM 2FG was about four- to five-fold lower than the parental L6 cells. These mutants contain normal levels of ATP and glycolytic enzyme activities. They also exhibit normal transport activities for alpha-aminoisobutyric acid and fructose. Furthermore, hexose transport was observed to be decreased in plasma membrane vesicles prepared from these mutants. Kinetic analysis of 2-DOG and 3-OMG transport in mutant F72 demonstrated that the Vmax for 2-DOG uptake was significantly reduced, whereas the Vmax for 3-OMG transport was not affected. In all cases, the affinity for these hexose analogues was unaffected. In addition mutant F72 was found to be only slightly affected by treatment with various energy inhibitors and sulfhydryl reagents. The results suggest that this mutant is defective in, or has low levels of, a plasma membrane component(s) involved in the high-affinity hexose transport system.     

Regulation of hexose transport in rat myoblasts during growth and differentiation

We report here the effects of growth conditions and myogenic differentiation on rat myoblast hexose transport activities. We have previously shown that in undifferentiated myoblasts the preferred substrates for the high (HAHT)- and low (LAHT)-affinity hexose transport systems are 2-deoxyglucose (2-DG) and 3-O-methyl-D-glucose (3-OMG), respectively. The present study shows that at cell density higher than 4.4 x 10(4) cells/cm2, the activities of both transport processes decrease with increasing cell densities of the undifferentiated myoblasts. Since the transport affinities are not altered, the observed decrease is compatible with the notion that the number of functional hexose transporters may be decreased in the plasma membrane. Myogenic differentiation is found to alter the 2-DG, but not the 3-OMG, transport affinity. The Km values of 2-DG uptake are elevated upon the onset of fusion and are directly proportional to the extent of fusion. This relationship between myogenesis and hexose transport is further explored by using cultures impaired in myogenesis. Treatment of cells with 5-bromo-2'-deoxyuridine abolishes not only myogenesis but also the myogenesis-induced change in 2-DG transport affinity. Similarly, alteration in 2-DG transport affinity cannot be observed in a myogenesis-defective mutant, D1. However, under myogenesis-permissive condition, the myogenesis of this mutant is also accompanied by changes in its 2-DG transport affinity. The myotube 2-DG transport system also differs from its myoblast counterpart in its response to sulfhydryl reagents and in its turnover rate. It may be surmised from the above observations that myogenesis results in the alteration of the turnover rate or in the modification of the 2-DG transport system. Although glucose starvation has no effect on myogenesis, it is found to alter the substrate specificity and transport capacity of HAHT. In conclusion, the present study shows that hexose transport in rat myoblasts is very sensitive to the growth conditions and the stages of differentiation of the cultures. This may explain why different hexose transport properties have been observed with myoblasts grown under different conditions.     

Relationship between insulin stimulation and endogenous regulation of 2-deoxyglucose uptake in 3T3-L1 adipocytes

The occurrence of the endogenous regulatory response to high rates of 2-deoxyglucose (2-DG) uptake, as previously described for C6 glioma cells during incubation with 2 mM 2-DG (Lange et al.: J. Cell. Physiol., 1989), was studied in 3T3-L1 preadipocytes and adipocytes, and the influence of insulin on this endogenous uptake regulation was examined. In contrast to 3T3-L1 preadipocytes, insulin-sensitive differentiated 3T3-L1 adipocytes displayed the time-dependent cyclic pattern of 2-DG uptake rates characteristic of the membrane-limited and endogenously regulated cellular state of hexose utilization. Although insulin induced a threefold stimulation of 2-DG tracer uptake in adipocytes, the hormone did not additionally stimulate the uptake rates or affect the periodic response: maximum and minimum levels of uptake remained unchanged. Scanning electron microscopy (SEM) revealed that the acquirement of the differentiated state is accompanied by a conspicuous transformation of the smooth surface of undifferentiated 3T3-L1 cells into a surface covered by numerous microvilli of uniform size and appearance. Treatment with insulin (10 mU/ml; 10 minutes) converted these microvilli into voluminous saccular membrane protrusions of the same type as had been formed during incubation of 3T3-L1 adipocytes with 2 mM 2-DG, and which have previously been shown to be involved in the endogenous uptake regulation of C6 glioma cells (Lange et al.: J. Cell. Physiol., 1989). These insulin-induced saccated membrane areas appeared to become integrated into the cell surface. Accordingly, insulin treatment caused a twofold increase of the intracellular distribution space of 3-O-methylglucose (3-OMG) in 3T3-L1 adipocytes. This insulin-induced increase of the 3-OMG distribution space exhibited the same time (t1/2 = 2-2.5 minutes) and dose dependence (EC50 = 20 nM) as the insulin-induced stimulation of 3-OMG transport. Glucose deprivation during the differentiation period inhibited the outgrowth of microvilli from the cell surface. Glucose starvation (18 hours at less than 0.5 mM) induced a conspicuous reduction of the length of microvilli on differentiated 3T3-L1 cells. In this state, the stalks of the microvilli are almost invisible and the enlarged spherical tips of the microvilli (with an average diameter of 370 nm compared to 230 nm of fed cells) appeared to protrude directly out of the cell surface. Starvation-induced shortening of microvilli was accompanied by a threefold increase of the basal 3-OMG transport rate and a greater than twofold increase of the intracellular 3-OMG distribution space as compared to fed cells (10 mM; 18 hours).(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of the D-allose-mediated regulation of sugar transport in Chinese hamster fibroblasts

Exposure to D-allose has been demonstrated to lead to decreased 2-deoxy-D-glucose (2-DG) and 3-0-methyl-D-glucose transport in the V79 Chinese hamster lung fibroblast cell line. The effect of D-allose 1) was maximal after 4 hours exposure to the cells; 2) was optimal between 2.77 and 5.55 mM D-allose; and 3) led to a decreased Vmax for 2-DG transport with no change in the transport Km value. The decrease in 2-DG transport induced by D-allose was reversible and the reversal was differentially affected by cycloheximide, being blocked by a low concentration of cycloheximide (0.05 micrograms/ml) but not a high concentration of the inhibitor (5 micrograms/ml). D-allose did not competitively inhibit the transport of 2-DG while D-glucose under similar conditions yielded a Kl for 2-DG transport inhibition of 1.7 mM. Additionally, D-allose did not affect the phosphorylation of 2-DG by hexokinase in cell-free cytosol. The data indicate that D-allose has significant lowering effects on sugar transport activity. Additionally, while the sugar itself may be the active component in sugar transport regulation, the effect is not blocked by inhibition of protein synthesis but the synthesis of a regulatory protein(s) may be involved in the return of sugar transport following D-allose removal.     

Hexose transport in L6 rat myoblasts. I. Rate-limiting step, kinetic properties, and evidence for two systems

The hexose transport system of undifferentiated L6 rat myoblasts was investigated. 2-Deoxy-D-glucose (2-DOG) and 2-deoxy-2-fluoro-D-glucose (2FG) were used as analogues to investigate the rate-limiting step of hexose uptake into the cell. Virtually all of the 2-DOG or 2FG taken up into the cell was found to be in the phosphorylated form. No significant pool of intracellular free sugar could be detected. This demonstrates that hexose transport, not phosphorylation, is the rate-limiting step. The inhibitory effect of various glucose analogues on 2-DOG and 3-O-methyl-D-glucose (3-OMG) uptake revealed that these two sugars may be taken up into the cell by different carriers. In addition, kinetics analysis of the transport of both sugars also indicates that two hexose transport systems may be present in L6 cells. 2-DOG is transported by high and low affinity transport systems (Km 0.6 mM and 2.9 mM, respectively), whereas 3-OMG is transported by a low affinity system (Km 3.5 mM). Treatment of cells with ionophores or energy uncouplers results in inactivation of the high affinity system, but not the low affinity system.     

Regulation of insulin binding and stimulation of sugar transport in cultured human fibroblasts by sugar levels in the culture medium

Studies were carried out on cultures of human skin fibroblasts to explore the effects of culture medium glucose levels on insulin binding and action. Cell cultures in 5.55 mm glucose-containing medium depleted their medium glucose within 3 days, and at that time exhibited elevated deoxy-d-glucose (2-DG) transport (84% greater than control cultures fed 22.2 mm glucose) and failure of insulin to stimulate 2-DG transport (an insulin:control transport ratio of 1.02). There was also a significant negative correlation between basal 2-DG transport and insulin binding (r = ?0.621; n = 29; P < 0.01), while insulin binding exhibited a significant positive correlation with insulin action (r = 0.816; n = 12; P < 0.01). Glucose starvation of cultures for 18 h resulted in several changes: (i) a 49% decrease in specific ¹²⁵I-insulin binding due to a reduction in binding capacity; (ii) elevated basal 2-DG transport; and (iii) an absence of insulin stimulation of 2-DG transport. Exposure to increasing concentrations of glucose for 18 h led to a glucose concentration-dependent increase in specific insulin binding. Additionally, the various changes in the glucose-starved group were reversed after as little as 6 h of glucose refeeding. The results indicate that basal sugar transport, and insulin binding and action can be regulated by the amount of glucose in the medium.     

Derepression and carrier turnover: evidence for two distinct mechanisms of hexose transport regulation in animal cells.

Hexose uptake by hamster cells was increased five to ten fold by either substituting D-fructose for glucose or by completely omitting D-glucose from the culture medium for 24 to 48 hours. Conversely, when cycloheximide was present for 24 hours in media containing glucose, up to 20-fold decreases in hexose uptake were observed. However, these decreases in uptake activity were only observed over a narrow range of cycloheximide concentrations. After extended exposure to low concentrations of cycloheximide (0.05 to 10 mug/ml), the uptake by the fed cells decreased parallel with inhibition of protein synthesis whereas at high concentrations (greater than 50 mug/ml) uptake was increased. Cells deprived of glucose and maintained in the presence of cycloheximide did not show decreases in uptake activity. In separate experiments the high uptake rates of glucose-starved cells could be decreased by addition of glucose-free medium. The reversal was complete in 6 to 8 hours. The analog of glucose, 2-deoxy-D-glucose, did not promote the time-dependent decrease suggesting that the 6-phosphoester of glucose is not an inhibitor of transport. In addition, when cycloheximide is added at the same time as glucose, there is no decrease in uptake for at least 12 hours. We propose that turnover of components of hexose uptake systems could account for part of the control of hexose transport. Moreover, the results indicate that the turnover mechanism becomes inactive during glucose starvation and must be resynthetized following refeeding of the starved cells with glucose.     

Non-insulin dependent diabetic patients have increased glucose uptake in red blood cells

The kinetic characteristics of 3-O-methyl glucose (3-OMG) uptake were examined in red blood cells (RBC) from seven normal individuals (controls) and nine patients with non-insulin-dependent diabetes mellitus (NIDDM) treated with diet and oral hypoglycemic medication. Comparison of rates of 3-OMG uptake at 5 different substrate concentrations revealed significantly higher overall 3-OMG uptake in the diabetic group (P less than 0.0001). Kinetic parameters obtained for individual subjects showed there was not a significant difference in the Km between the diabetic (3.17 +/- 0.45 mM; mean +/- SE) and the control (2.46 +/- 0.25 mM) groups. However, Vmax was significantly increased (61%; P less than 0.025) in the diabetics (217.8 +/- 28.9 pmol/2 sec per 10(6) cells) compared to controls (135.2 +/- 15.6 pmol/2 sec per 10(6) cells). There was no correlation between HbA1C levels in the diabetic patients and Vmax values for 3-OMG uptake, suggesting that the increased hexose uptake was not accounted for simply by increased glycosylation in these cells. Glucose transport in RBC in hyperglycemic states may be a useful model for delineating the regulation of the non-insulin-mediated disposal of glucose in diabetes.     

Hexose transport in L6 rat myoblasts. II. The effects of sulfhydryl reagents

The importance of sulfhydryl groups for hexose transport in undifferentiated L6 rat myoblasts was investigated. N-ethylmaleimide (NEM) and p-chloromer-curibenzenesulfonic acid (pCMBS) inhibited 2-deoxy-D-glucose (2-DOG) transport in a time and concentration-dependent manner. The inhibition produced by both reagents was virtually complete within 5 min, although neither reagent inhibited transport more than 70–80% regardless of the concentrations or incubation times used. Furthermore, the inhibition of 2-DOG transport by pCMBS or NEM could not be prevented by simultaneous preincubation of cells with 20 mM D-glucose or 20 mM 2-DOG. This suggests that sulfhydryl groups required for transport are separate from the hexose binding and transport site. By comparing the effects of the membrane impermeant pCMBS to those of the membrane permeant NEM, cell surface sulfhydryl groups were shown to be essential for hexose binding and transport. In contrast to the inhibition of 2-DOG transport, pCMBS and NEM had much less of an effect on 3-O-methyl-D-glucose (3-OMG) transport. For example, 1 mM NEM inhibited 2-DOG transport by 66%, whereas 3-OMG transport was inhibited by only 7%. This supports the suggestion that these hexose analogues may be transported by different carriers. Kinetic analysis of transport shows that treatment of cells with 1 mM NEM or 1 pCMBS results in inactivation of the high affinity 2-DOG transport system, whereas the low affinity transport system is unaffected. 3-OMG is preferentially transported by the low affinity system.     

Endogenous regulation of 2-deoxyglucose uptake in C6 glioma cells correlates with cytoskeleton-mediated changes of surface morphology

The cellular basis of the membrane-limited state of glucose utilization and the mechanism of the endogenous regulation of hexose uptake in dense monolayers of C6 glioma cells were investigated. In an earlier study, it was shown that at high rates of glucose transport and phosphorylation combined with the inhibition of glycolytic adenosine triphosphate (ATP) production by iodoacetate, an endogenous regulatory response occurred that resulted in rapid, periodic variations of the glucose uptake rates (Lange et al., 1982). Similar time-dependent periodic changes of uptake rates also occurred during incubation of C6 glioma cells with 2 mM 2-deoxyglucose (2-DG) without pretreatment of the cells with iodoacetate. These changes were accompanied by variations of the intracellular ATP content, by distinct alterations of the shape and arrangement of microvilli and lamellae (lamellipodia) on the cell surface, and by changes of the cytoskeletal F-actin content. Because the changes of 2-DG uptake rates occurred independent of the intracellular 2-DG concentration, the bulk of this 2-DG pool was assumed to be localized apart from the membranal transport sites. Downregulation of 2-DG uptake appeared to be triggered by a rapid decrease of a small pool of the cellular ATP involved in the phosphorylation of transported hexose. Scanning and transmission electron microscopic observations of cells fixed in different states of the endogenous uptake regulation supported the assumption that the interior of lamellae and microvilli may represent a small entrance compartment for transported hexoses in which occurred the observed close coupling between hexose transport and phosphorylation as well as the rapid variations of ATP content. Hexose uptake is supposed to be regulated by cytoskeleton-mediated changes of volume and diffusional accessibility of this compartment, modulating the degree of its metabolic coupling with the cytoplasmic main compartment.     

Phlorizin inhibits hexose transport across the plasmalemma of Riccia fluitans

The effect of phlorizin has been tested on hexose transport and hexose-induced changes of electrical potential (_m) and conductance (g _m) across the plasmalemma of rhizoid and thallus cells of the aquatic liverwort Riccia fluitans. The decrease of _m (depolarization) and g _m induced by 1 mM 3-oxymethyl-D-glucose (3-OMG) is substantially inhibited by simultaneous addition of 2 mM phlorizin, whereas no significant response was observed when phlorizin was added alone or several minutes after the sugar. Current-voltage data show that the 3-OMG-generated electrical inward current of 0.016 A m^-2 drops to 0.010 A m^-2 when phlorizin is present. Uptake as well as efflux of [¹⁴C]-3-OMG is strongly and reversibly inhibited by phlorizin between 0.2 and 5 mM. The results are consistent with our hypothesis that the hexose carrier has one binding site with competitive inhibition of glucose uptake by phlorizin (k _i=0.08 mM). The electrical data indicate that phlorizin affects an _m step of the carrier transport cycle.Abbreviation 3-OMG 3-oxymethyl-D-glucose     

Counter-transport in chick embryo fibroblasts. A significant factor in measurement of glucose entry.

Enhanced rates of carrier-mediated 3-O-methyl-D-glucose (0.1 mM) transport were observed in primary cell cultures of chicken embryo fibroblasts deprived of glucose for 1 day. The addition of 5.5 mM-glucose, glucosamine or 2-deoxy-D-glucose for 15 min (37 degrees C) to glucose-starved cultures followed by washing and immediate measurement of 3-O-methyl-D-glucose transport resulted in an apparent further stimulation of transport. Transport stimulation increased with increasing concentrations of the added preincubation sugar and was observed at test concentrations ranging from 0.1 mM- to 10 mM-3-O-methyl-D-glucose. This enhancement occurred when the preloaded sugar was rapidly effluxing from cells and was eliminated by allowing cultures to incubate in buffer without sugar for 30 min (37 degrees C) after the removal of hexose and before measuring transport. A transient overshoot in the cumulative uptake of 3-O-methyl-D-glucose was observed in glucose-starved cultures that were pre-incubated in the presence of 55 mM-glucose or -glucosamine for 15 min (37 degrees C). These data suggest that counter-transport accounts for the apparent enhancement of glucose-transport capability observed in glucose-starved cells when they are briefly re-exposed to hexose.     

Regulation of hexose transport in L8 myocytes by glucose: possible sites of interaction

Previous work demonstrated that glucose controls its own transport rate in rat skeletal muscle: exposure to high glucose levels down-regulates muscle hexose transport, while glucose withdrawal results in elevated transport rates (J. Biol. Chem. 261:16827-16833, 1986). The present study investigates the mechanism of this autoregulatory system. Preincubation of L8 myocytes at 16 mM glucose reduced subsequent 2-deoxy-D-glucose (dGlc) uptake by 40% within 3 h. Cycloheximide (1 microM) mimicked the action of glucose; the effects of glucose and cycloheximide were not additive. At 50 microM, cycloheximide prevented the modulations of glucose transport induced by exposure of muscle cells to high or low glucose concentrations. Inhibition of glycosylation with tunicamycin A1 reduced the basal dGlc uptake, but did not prevent its up-regulation following glucose withdrawal. Inhibition of RNA synthesis by actinomycin D prevented the down-regulatory effect of glucose. These results indicate that continuous protein synthesis and protein glycosylation are required for the maintenance of the steady-state dGlc uptake. We suggest that glucose exerts its autoregulatory effect on hexose transport by modifying the incorporation of active glucose transporters into the plasma membrane rather than changing their rate of degradation. It is hypothesized that this effect is mediated by a non-glycosylated protein involved in the translocation or activation of glucose transporters.