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.     

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.     

Regulation of glucose uptake by stressed cells.

Lactate production by BHK cells is stimulated by arsenite, azide, or by infection with Semliki Forest virus (SFV). In the case of arsenite or SFV infection, the increase correlates approximately with the increase in glucose transport as measured by uptake of [3H] deoxy glucose (dGlc); in the case of azide, the increase in lactate production exceeds that of glucose transport. Hence glucose utilization by BHK cells and its stimulation by anaerobic and other types of cellular stress is controlled at least in part at the level of glucose transport. The glucose uptake by BHK cells is also stimulated by serum and by glucose deprivation. In these circumstances, as with arsenite, stimulation is reversible, with t1/2 of 1-2 hours; stimulation is compatible with a translocation of the glucose transporter protein between an intracellular site and the plasma membrane (shown here for serum and previously for arsenite). The surface binding and rate of internalization of [125I]-labelled transferrin and [125I] alpha 2-macroglobulin was studied to determine whether changes in glucose transport are accompanied by changes in the surface concentration or rate of internalization of membrane proteins. The findings indicate that changes in glucose transport do not reflect a consistent and general redistribution of membrane receptors. Taken together, the results are compatible with the proposal that BHK cells exposed to stimuli like insulin or serum, or to stresses like arsenite, azide, SFV infection, or deprivation of glucose, respond in the same manner: namely, by an increased capacity to transport glucose brought about by reversible and specific translocation of the transporter protein from an (inactive) intracellular site to the plasma membrane.     

Glucose deprivation induces Akt-dependent synthesis and incorporation of GLUT1, but not of GLUT4, into the plasma membrane of 3T3-L1 adipocytes

Reduction of the glucose concentration in the culture medium of 3T3-L1 adipose cells below 1.25 mM produces a 4-8-fold stimulation of 2-deoxyglucose uptake which starts after a lag phase of 2 h and is maximal after 10-16 h. In the present study, we employed the 'membrane sheet assay' in order to re-assess the contribution of the transporter isoforms GLUT1 and GLUT4 to this effect. Immunochemical assay of glucose transporters in membranes prepared with the 'sheet assay' revealed that the effect reflected a marked increase of GLUT1 in the plasma membrane with no effect on GLUT4. Glucose deprivation increased the total cellular GLUT1 protein in parallel with the transport activity, whereas GLUT4 was unaltered. The specific PI 3-kinase inhibitor wortmannin inhibited the effect of glucose deprivation on transport activity and also on GLUT1 synthesis. Glucose deprivation produced a moderate, biphasic increase in the activity of the protein kinase Akt/PKB that was inhibitable by wortmannin. When wortmannin was added after stimulation of cells in order to assess the internalization rate of transporters, the effect of insulin was reversed considerably faster (T1/2 = 18 min) than that of glucose deprivation (T1/2 > 60 min). These data are consistent with the conclusion that the effect of glucose deprivation reflects a specific, Akt-dependent de-novo synthesis of GLUT1, and not of GLUT4, and its insertion into a plasma membrane compartment which is distinct from that of the insulin-sensitive GLUT1.     

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.     

Regulation of the functional expression of hexose transporter GLUT-1 by glucose in murine fibroblasts: role of lysosomal degradation.

The nature of the membrane compartments involved in the regulation by glucose of hexose transport is not well defined. The effect of inhibitors of lysosomal protein degradation on hexose transport (i.e., uptake of [3H]-2-deoxy-D-glucose) and hexose transporter protein GLUT-1 (i.e., immunoblotting with antipeptide serum) in glucose-fed and -deprived cultured murine fibroblasts (3T3-C2 cells) was studied. The acidotropic amines chloroquine (20 microM) and ammonium chloride (10 mM) cause accumulation (both approximately 4-fold) of GLUT-1 protein and a small increase (both approximately 25%) in hexose transport in glucose-fed fibroblasts (24 h). The endopeptidase inhibitor, leupeptin (100 microM) causes accumulation (approximately 4-fold) of GLUT-1 protein in glucose-fed fibroblasts (24 h) without changing hexose transport (less than or equal to 5%). These agents do not greatly alter the electrophoretic mobility of GLUT-1. Neither chloroquine nor leupeptin augment the glucose deprivation (24 h) induced increases in hexose transport (approximately 4-fold) and GLUT-1 content (approximately 7-fold). In contrast, chloroquine or leupeptin diminish the reversal by glucose refeeding of the glucose deprivation induced accumulation of GLUT-1 protein but fail to alter the return of hexose transport to control levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of cell surface glucose transporters measured by photoaffinity labeling of insulin-sensitive 3T3-L1 adipocytes.

Several studies have demonstrated that the intrinsic catalytic activity of cell surface glucose transporters is highly regulated in 3T3-L1 adipocytes expressing GLUT1 (erythrocyte/brain) and GLUT4 (adipocyte/skeletal muscle) glucose transporter isoforms. For example, inhibition of protein synthesis in these cells by anisomycin or cycloheximide leads to marked increases in hexose transport without a change in the levels of cell surface glucose transporter proteins (Clancy, B. M., Harrison, S. A., Buxton, J. M., and Czech, M. P. (1991) J. Biol. Chem. 266, 10122-10130). In the present work the exofacial hexose binding sites on GLUT1 and GLUT4 in anisomycin-treated 3T3-L1 adipocytes were labeled with the cell-impermeant photoaffinity reagent [2-3H]2-N-[4-(1-azitrifluoroethyl)benzoyl]-1,3-bis- (D-mannos-4-yloxy)-2-propylamine [( 2-3H] ATB-BMPA) to determine which isoform is activated by protein synthetic blockade. As expected, a 15-fold increase in 2-deoxyglucose uptake in response to insulin was associated with 1.7- and 2.6-fold elevations in plasma membrane GLUT1 and GLUT4 protein levels, respectively. Anisomycin treatment of cultured adipocytes for 5 h produced an 8-fold stimulation of hexose transport but no increase in the content of glucose transporters in the plasma membrane fraction as measured by protein immunoblot analysis. Cell surface GLUT1 levels were also shown to be unaffected on 3T3-L1 adipocytes in response to anisomycin using an independent method, the binding of an antiexofacial GLUT1 antibody to intact cells. In contrast, anisomycin fully mimicked the action of insulin to stimulate (about 4-fold) the radiolabeling of GLUT1 transporters specifically immunoprecipitated from intact 3T3-L1 adipocytes irradiated after incubation with [2-3H] ATB-BMPA. Photolabeling of GLUT4 under these conditions was also significantly enhanced (1.8-fold) by anisomycin treatment, but this effect was only 15% of that caused by insulin. These results suggest that: 1) the photoaffinity reagent [2-3H]ATB-BMPA labels those cell surface glucose transporters present in a catalytically active state rather than total cell surface transporters as assumed previously and 2) inhibition of protein synthesis in 3T3-L1 adipocytes stimulates sugar transport primarily by enhancing the intrinsic catalytic activity of cell surface GLUT1, and to a lesser extent, GLUT4 proteins.     

Stimulation of hexose transport in L6 rat myoblasts by antibody and by glucose starvation.

Treatment of glucose-grown L6 rat myoblasts with rabbit or sheep anti-(L6-rat myoblast) antibody for 35 min or glucose starvation for at least 8 h results in a 2-fold increase in the Vmax. of 2-deoxy-D-glucose (dGlc) and 3-O-methyl-D-glucose uptake. In both cases, apparent transport affinities were not affected. Furthermore, once stimulation has occurred, further increases in hexose uptake could not be produced. Assays of antibody binding to whole cells suggested that the antibody is not internalized but remains bound on the cell surface. To elucidate the site and mechanism of antibody action, plasma-membrane vesicles from L6 cells were prepared. Anti-L6 antibody was found to cause a time- and dosage-dependent stimulation of dGlc transport in these vesicles. Maximum activation was achieved after 30 min exposure. This antibody-mediated activation could be inhibited by treatment of vesicles with various proteinase inhibitors. Treatment of vesicles with trypsin was also found to activate dGlc transport to levels observed with antibody. These results are virtually identical with those obtained with whole cells and suggest that antibody-mediated activation of hexose transport results from interaction of antibody with a specific membrane component(s).     

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.     

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.     

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.     

Differential regulation of the HepG2 and adipocyte/muscle glucose transporters in 3T3L1 adipocytes. Effect of chronic glucose deprivation.

Glucose transport in 3T3L1 adipocytes is mediated by two facilitated diffusion transport systems. We examined the effect of chronic glucose deprivation on transport activity and on the expression of the HepG2 (GLUT 1) and adipocyte/muscle (GLUT 4) glucose transporter gene products in this insulin-sensitive cell line. Glucose deprivation resulted in a maximal increase in 2-deoxyglucose uptake of 3.6-fold by 24 h. Transport activity declined thereafter but was still 2.4-fold greater than the control by 72 h. GLUT 1 mRNA and protein increased progressively during starvation to values respectively 2.4- and 7.0-fold greater than the control by 72 h. Much of the increase in total immunoreactive GLUT 1 protein observed later in starvation was the result of the accumulation of a non-functional or mistargeted 38 kDa polypeptide. Immunofluorescence microscopy indicated that increases in GLUT 1 protein occurred in presumptive plasma membrane (PM) and Golgi-like compartments during prolonged starvation. The steady-state level of GLUT 4 protein did not change during 72 h of glucose deprivation despite a greater than 10-fold decrease in the mRNA. Subcellular fractionation experiments indicated that the increased transport activity observed after 24 h of starvation was principally the result of an increase in the 45-50 kDa GLUT 1 transporter protein in the PM. The level of the GLUT 1 transporter in the PM and low-density microsomes (LDM) was increased by 3.9- and 1.4-fold respectively, and the GLUT 4 transporter content of the PM and LDM was 1.7- and 0.6-fold respectively greater than that of the control after 24 h of glucose deprivation. These data indicate that newly synthesized GLUT 1 transporters are selectively shuttled to the PM and that GLUT 4 transporters undergo translocation from an intracellular compartment to the PM during 24 h of glucose starvation. Thus glucose starvation results in an increase in glucose transport in 3T3L1 adipocytes via a complex series of events involving increased biosynthesis, decreased turnover and subcellular redistribution of transporter proteins.     

Effect of Glucose Starvation on Glucose Transport in Neuronal Cells in Primary Culture from Rat Brain

The regulation of glucose transport into cultured brain cells during glucose starvation was studied. On glucose deprivation for 40 h, 2-deoxy-D-glucose (2-DG) uptake was stimulated twofold in neuronal cells but was not changed significantly in astrocytes. On refeeding, the increased activity of neuronal cells rapidly returned to the basal level, an observation indicating that the effect of glucose starvation was reversible. The increase was due solely to change in the Vmax, a finding suggesting that the number of glucose transporters on the plasma membrane is increased in starved cells. Cycloheximide inhibited this increase. In the presence of cycloheximide, the activity of 2-DG uptake of starved cells remained constant for 12 h and then slowly decreased, whereas that of fed cells decreased rapidly. These findings suggest that glucose starvation regulates glucose transport by changing the rate of net synthesis of the transporter in neuronal cells in culture.     

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.     

Reduction of glucose uptake through inhibition of hexose transporters and enhancement of their endocytosis by methylglyoxal in Saccharomyces cerevisiae

Diabetes mellitus is characterized by an impairment of glucose uptake even though blood glucose levels are increased. Methylglyoxal is derived from glycolysis and has been implicated in the development of diabetes mellitus, because methylglyoxal levels in blood and tissues are higher in diabetic patients than in healthy individuals. However, it remains to be elucidated whether such factors are a cause, or consequence, of diabetes. Here, we show that methylglyoxal inhibits the activity of mammalian glucose transporters using recombinant Saccharomyces cerevisiae cells genetically lacking all hexose transporters but carrying cDNA for human GLUT1 or rat GLUT4. We found that methylglyoxal inhibits yeast hexose transporters also. Glucose uptake was reduced in a stepwise manner following treatment with methylglyoxal, i.e. a rapid reduction within 5 min, followed by a slow and gradual reduction. The rapid reduction was due to the inhibitory effect of methylglyoxal on hexose transporters, whereas the slow and gradual reduction seemed due to endocytosis, which leads to a decrease in the amount of hexose transporters on the plasma membrane. We found that Rsp5, a HECT-type ubiquitin ligase, is responsible for the ubiquitination of hexose transporters. Intriguingly, Plc1 (phospholipase C) negatively regulated the endocytosis of hexose transporters in an Rsp5-dependent manner, although the methylglyoxal-induced endocytosis of hexose transporters occurred irrespective of Plc1. Meanwhile, the internalization of hexose transporters following treatment with methylglyoxal was delayed in a mutant defective in protein kinase C.     

Effect of hypertonicity on hexose transporter regulation in chicken embryo fibroblasts

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

Functional expression of mammalian glucose transporters in Xenopus laevis oocytes: evidence for cell-dependent insulin sensitivity.

We report the functional expression of two different mammalian facilitative glucose transporters in Xenopus oocytes. The RNAs encoding the rat brain and liver glucose transporters were transcribed in vitro and microinjected into Xenopus oocytes. Microinjected cells showed a marked increase in 2-deoxy-D-glucose uptake as compared with controls injected with water. 2-Deoxy-D-glucose uptake increased during the 5 days after microinjection of the RNAs, and the microinjected RNAs were stable for at least 3 days. The expression of functional glucose transporters was dependent on the amount of RNA injected. The oocyte-expressed transporters could be immunoprecipitated with anti-brain and anti-liver glucose transporter-specific antibodies. Uninjected oocytes expressed an endogenous transporter that appeared to be stereospecific and inhibitable by cytochalasin B. This transporter was kinetically and immunologically distinguishable from both rat brain and liver glucose transporters. The uniqueness of this transporter was confirmed by Northern (RNA) blot analysis. The endogenous oocyte transporter was responsive to insulin and to insulinlike growth factor I. Most interestingly, both the rat brain and liver glucose transporters, which were not insulin sensitive in the tissues from which they were cloned, responded to insulin in the oocyte similarly to the endogenous oocyte transporter. These data suggest that the insulin responsiveness of a given glucose transporter depends on the type of cell in which the protein is expressed. The expression of hexose transporters in the microinjected oocytes may help to identify tissue-specific molecules involved in hormonal alterations in hexose transport activity.     

The hexose carrier from Chlorella. cDNA cloning of a eucaryotic H+-cotransporter

The cDNA coding for the inducible H+/hexose cotransporter of Chlorella kessleri has been cloned and sequenced. It was isolated by differential screening of a cDNA library prepared from glucose-induced cells. The increase in expression of the gene correlates quantitatively with the increase in uptake activity due to induction; it is not expressed in a hexose transport mutant. An open reading frame allows for a membrane protein of 533 amino acids with a relative molecular mass of 57 kDa. This protein is highly homologous to the human and rat glucose transporters catalyzing facilitated diffusion and to the bacterial H+/pentose cotransporters. It is not related to the H+/lactose cotransporter of E. colli and to the mammalian Na+/glucose cotransporter.     

Hexose transport stimulation and membrane redistribution of glucose transporter isoforms in response to cholera toxin, dibutyryl cyclic AMP, and insulin in 3T3-L1 adipocytes

Exposure of 3T3-L1 adipocytes to 100 ng/ml of cholera toxin or 1 mM dibutyryl cyclic AMP caused a marked stimulation of deoxyglucose transport. A maximal increase of 10- to 15-fold was observed after 12-24 h of exposure, while 100 nM insulin elicited an increase of similar magnitude within 30 min. A short term exposure (4 h) of cells to cholera toxin or dibutyryl cyclic AMP resulted in a 3- to 4-fold increase in deoxyglucose transport which was associated with significant redistribution of both the HepG2/erythrocyte (GLUT1) and muscle/adipocyte (GLUT4) glucose transporters from low density microsomes to the plasma membrane fraction. Total cellular amounts of both transporter proteins remained constant. In contrast, cells exposed to cholera toxin or dibutyryl cyclic AMP for 12 h exhibited elevations in total cellular contents of GLUT1 (but not GLUT4) protein to about 1.5- and 2.5-fold above controls, respectively. Although such treatments of cells with cholera toxin (12 h) versus insulin (30 min) caused similar 10-fold enhancements of deoxyglucose transport, a striking discrepancy was observed with respect to the content of glucose transporter proteins in the plasma membrane fraction. While insulin elicited a 2.6-fold increase in the levels of GLUT4 protein in the plasma membrane fraction, cholera toxin increased the amount of this transporter by only 30%. Insulin or cholera toxin increased the levels of GLUT1 protein in the plasma membrane fraction equally (1.6-fold). Thus, a greater number of glucose transporters in the plasma membrane fraction is associated with transport stimulation by insulin compared to cholera toxin. We conclude that: 1) at early times (4 h) after the addition of cholera toxin or dibutyryl cyclic AMP to 3T3-L1 adipocytes, redistribution of glucose transporters to the plasma membrane appears to contribute to elevated deoxyglucose uptake rates, and 2) the stimulation of hexose uptake after prolonged treatment (12-18 h) of cells with cholera toxin may involve an additional increase in the intrinsic activity of one or both glucose transporter isoforms.