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.     

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.     

Regulation of sugar transport in cultured diploid human skin fibroblasts

The regulation of hexose transport under glucose-starvation conditions was studied in cultured human skin fibroblasts. Glucose starvation enhanced the transport of 2-DG and 3-O-methyl-D-glucose (3-OMG) but not of L-glucose. Glucose-starvation enhanced transport was inhibited by cytochalasin B (10 μM). The starvation-induced change in 2-DG transport was due to an increase in the V_max of both the high and low affinity transport sites (2.8- and 2.4-fold, respectively) with no effect on their K_ms. The presence of 5.55 mM galactose, fructose, or L-glucose in the medium resulted in transport increases similar to those seen in glucose-starved cells, while the presence of 5.55 mM glucose, mannose, or 3-OMG repressed 2-DG transport. Glucose-starvation enhancement of 2-DG transport was blocked by cycloheximide (20 μg/ml) but not by actinomycin D (0.03 μg/ml) or α-amanitin (3.5 μM). Readdition of glucose (5.55 mM) for six hours to glucose-starved cells led to a rapid decrease in hexose transport that could be blocked by cycloheximide but not actinomycin D. Although readdition of 3-OMG to glucose-starved cells had little effect on reversing the transport increases, glucose plus 3-OMG were more effective than glucose alone. Serum containing cultures (10% v/v) of glucose-fed or glucose-starved cells exhibited rapid decreases in 2-DG transport when exposed to glucose-containing serum-free medium. These decreases were prevented by employing glucose-free, serum-free medium. The data indicate that hexose transport regulation in cultured human fibrob asts involves protein synthesis of hexose carriers balanced by interactions of glucose with a regulatory protein(s) and glucose metabolism as they affect the regulation and/or turnover of the carrier molecules.     

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.     

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.     

Properties of hexose-transport regulatory mutants isolated from L6 rat myoblasts.

A hexose-transport regulatory mutant (D1/S4) was isolated from L6 rat myoblasts on the basis of its resistance to detachment and cell lysis in the presence of antibody and complement. Growth studies indicated that D1/S4 cells had a slower doubling time (29 h) compared with the parental L6 cells (22 h). Furthermore, after 9 days growth, less than 1% cell fusion was observed with D1/S4 cells, whereas 95% cell fusion was observed with the L6 cells. When the parental L6 cells were starved of glucose or treated with anti-L6 antibody, a significant increase in the Vmax, of 2-deoxy-D-glucose (dGlc) and 3-O-methyl-D-glucose (MeGlc) transport was observed. Although glucose-grown D1/S4 cells possessed normal hexose-transport activity, the above treatments had no effect on dGlc and MeGlc transport in these cells. Electrophoresis and immunoblotting studies revealed that D1/S4 cells possessed decreased amounts of a 112 kDa plasma-membrane protein. It is conceivable that this protein may play a role in triggering the antibody- and glucose-starvation-mediated activation of hexose transport and in myogenic differentiation. Unlike D1/S4, mutant F72, a mutant defective in the high-affinity hexose-transport system, was found to possess normal amounts of the 112 kDa protein. Although glucose starvation has no effect on the hexose-transport activity in this mutant, its hexose transport activity can be increased by antibody treatment. These studies with mutants suggest the involvement of regulatory components in the activation of hexose transport.     

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.     

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.     

The hexose transporters at the plasma membrane and the tonoplast of transformed plant cells: kinetic characterization of two distinct carriers.

The plasma membrane hexose transporter and the tonoplast hexose transporter from heterotrophically grown transformed Nicotiana tabacum cells have been studied in vitro using membrane vesicles for trans-zero transport studies. In highly purified phase-partitioned outside-out plasma membrane vesicles (PMV) the hexose transporter showed an apparent Km value of 230 microM (substrate: 3-O-methyl-D-glucose (3-OMG); pHi 7.2/pHo 7.2), which was reduced to 120 microM when a pH gradient was imposed (pHo 5.7/pHi 7.2). However, the Vmax value was not affected indicating that no stable pH gradient was formed. Uptake experiments with 14C-labelled acetate supported this interpretation. Transport was insensitive to N-ethylmaleimide (NEM; up to 1 mM concentration) and p-chloromercuribenzene sulfonate (PCMBS; up to 500 microM), whereas the tonoplast hexose transporter (in mixed inside / out and outside / out vesicles) was inhibited by NEM in a substrate-protectable manner, and PCMBS was also inhibitory. Kinetically two components with apparent Km values of 6 and 20 mM could be distinguished for the tonoplast hexose transporter. Substrate specificities of both transporters were similar except for D-galactose and D-fructose. The results indicate structural differences between the tonoplast and plasma membrane hexose transporters in plants.     

Evidence that activation of 2-deoxy-D-glucose transport in rat thymocyte suspensions results from enhanced coupling between transport and hexokinase activity.

1. Suspensions of rat thymocytes accumulate free 2-deoxy-D-glucose (2-dGlc) within the cytosol to a concentration approx. 25-fold above the external concentration. This active accumulation was enhanced by 40 nM-phorbol 12-myristate 13-acetate (phorbol). 2. The Km for zero-trans uptake in control cells was 2.3 +/- 0.14 mM and Vmax. was 0.41 +/- 0.08 mumol/min per 10(10) cells (n = 6). In cells treated with phorbol (40 nM) the Km for zero-trans uptake was 1.2 +/- 0.13 mM and Vmax. 0.46 +/- 0.03 mumol/min per 10(10) cells (n = 6). The Km was decreased significantly by phorbol (P less than 0.01). 3. Phorbol-dependent activation of thymocytes delayed exit of free 2-dGlc into sugar-free solution and prevented exchange exit. Activation had no effect on 3-O-methyl D-glucoside (3-OMG) exit. 4. Coupling of 2-dGlc transport to hexokinase activity was determined by observing the effects of various concentrations of unlabelled cytosolic 2-dGlc on influx of labelled 2-dGlc into the hexose phosphate pool. In control cells this coupling was 0.81 +/- 0.02 and in phorbol-activated cells it was 0.92 +/- 0.01 (P less than 0.01). 5. The high-affinity inhibitor of hexokinase, mannoheptulose, inhibited uptake of 2-dGlc in both control and phorbol-treated cells. These data are consistent with a model for activation of sugar transport in which hexokinase activity is integrated with the sugar transporter at the endofacial surface. The results suggest that phorbol increases the degree of coupling transport with hexokinase activity, thereby leading to an increase in the rate of uptake of 2-dGlc, a decrease in exit of free 2-dGlc from the cytosol and an increase in free 2-dGlc accumulation.     

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.     

How hexoses and inhibitors influence the malate transport system in Zygosaccharomyces bailii

When grown in fructose or glucose the cells of Zygosaccharomyces bailii were physiologically different. Only the glucose grown cells (glucose cells) possessed an additional transport system for glucose and malate. Experiments with transport mutants had lead to the assumption that malate and glucose were transported by one carrier, but further experiments proved the existence of two separate carrier systems. Glucose was taken up by carriers with high and low affinity. Malate was only transported by an uptake system and it was not liberated by starved malate-loaded cells, probably due to the low affinity of the intracellular anion to the carrier. The uptake of malate was inhibited by fructose, glucose, mannose, and 2-DOG but not by non metabolisable analogues of glucose. The interference of malate transport by glucose, mannose or 2-DOG was prevented by 2,4-dinitrophenol, probably by inhibiting the sugar phosphorylation by hexokinase. Preincubation of glucose-cells with metabolisable hexoses promoted the subsequent malate transport in a sugar free environment. Preincubation of glucose-cells with 2-DOG, but not with 2-DOG/2,4-DNP, decreased the subsequent malate transport. The existence of two separate transport systems for glucose and malate was demonstrated with specific inhibitors: malate transport was inhibited by sodium fluoride and glucose transport by uranylnitrate. A model has been discussed that might explain the interference of hexoses with malate uptake in Z. bailii.Abbreviations 2,4-DNP 2,4-dinitrophenol - 2-DOG 2-deoxyglucose - 6-DOG 6-deoxyglucose - pCMB para-hydroxymercuribenzoate     

Hexose transport in plasma membrane vesicles prepared from L6 rat myoblasts

Hexose transport in plasma membrane vesicles prepared from L6 rat myoblasts was shown to be stereospecific, activated by glucose starvation and occurred by both high and low affinity systems. Transport by the high affinity system was shown to occur by an active transport process. Furthermore, the high affinity system was shown to be defective in vesicles prepared from F72 cells (hexose transport mutant). These results indicate that the high affinity hexose transport system is retained in the plasma membrane vesicles. Thus plasma membrane vesicles could be of value in further characterization of the L6 high affinity hexose transport system, without interference from the various metabolic events occurring in whole cells.     

Transport and accumulation of 2-deoxy-D-glucose in wild-type and hexokinase-deficient cultured Chinese-hamster ovary (CHO) cells.

Hexokinase-deficient mutants and wild-type Chinese-hamster ovary cells have been used to investigate the role of hexokinase in uptake and accumulation of 2-D-deoxyglucose (2-dGlc). The evidence for a specific sugar transport system in both types of cells is that there is similar saturable phloretin-sensitive uptake of 2-dGlc and 3-O-methyl-D-glucose (3-OMG) in both types of cell. In wild-type cells, 2-dGlc is accumulated to a tissue:medium ratio of 10- and in the mutant only 3-fold; 3-OMG is not accumulated by either mutant or wild-type cells. The evidence that hexokinase affects the membrane transport process is that the rate of exit of free 2-dGlc from wild-type cells is 5-fold less than from mutant cells, whereas there is no difference in the rate of loss of 3-OMG between mutant and wild-type cells.     

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)     

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.     

Specificity of the Glucose Transport System in Leishmania tropica Promastigotes*

SYNOPSIS. The glucose transport system in Leishmania tropica promastigotes was characterized by the use of labeled 2-deoxy-D-glucose (2-DOG), a nonmetabolizable glucose analog. The uptake system has a Q₁₀ of 2 and a heat of activation of 10.2 kcal/mole. The glucose transport system is subject to competitive inhibition by 2-DOG, glucosamine, N-acetyl glucosamine, mannose, galactose, and fructose which suggests that substitutions in the hexose chain at carbons 2 and 4 do not affect carrier specificity. In contrast, changes at carbon 1 (α-methyl-D-glucoside, 1,5-anhydroglucitol) and carbon 3 (3–0-methyl glucose) lead to loss of carrier affinity since these sugars do not compete for the glucose carrier. Sugars that compete with the glucose carrier have one common feature—they all exist in the pyranose form in solution. The carrier for D-glucose does not interact with L-glucose or any of the pentose sugars tested. Uptake of 2-DOG is inhibited by glycerol. This inhibition, however, is noncompetitive; it is evident, therefore, that glucose and glycerol do not compete for the same carrier. Glycerol does not repress the glucose carrier since cells grown in presence of glycerol transport the sugar normally.     

Sugar uptake in a 2-deoxy-d-glucose resistant mutant ofSaccharomyces cerevisiae

Summary The non-metabolizable and toxic glucose analogue 2-deoxy-d-glucose (2-DOG) has been widely employed to screen for regulatory mutants which lack catabolite repression. A number of yeast mutants resistant to 2-DOG have recently been isolated in this laboratory. One such mutant, derived from aSaccharomyces cerevisiae haploid strain, was demonstrated to be derepressed for maltose, galactose and sucrose uptake. Furthermore, kinetic analysis of glucose transport suggested that the high affinity glucose transport system was also derepressed in the mutant strain. In addition, the mutant had an increased intracellular concentration of trehalose relative to the parental strain. These results indicate that the 2-DOG resistant mutant is defective in general glucose repression.