ATP does not regulate the reconstituted glucose transporter

ATP has been reported to affect glucose transport in human erythrocytes and resealed erythrocyte ghosts [Jacquez, J. A. (1983) Biochim. Biophys. Acta 727, 367-378; Jensen, M. R., & Brahm, J. (1987) Biochim. Biophys. Acta 900, 282-290]. In more detailed studies, effects of micromolar levels of ATP on transport in ghosts and inside-out vesicles, and on the fluorescence of ghosts and the purified glucose transporter [Carruthers, A. (1986) Biochemistry 25, 3592-3602; Hebert, D. N., & Carruthers, A. (1986) J. Biol. Chem. 261, 10093-10099; Carruthers, A. (1986) J. Biol. Chem. 261, 11028-11037], have been interpreted as supporting a model in which ATP regulates the catalytic properties of the transporter. Both allosteric and covalent effects of ATP were proposed; among the allosteric effects was a 60% reduction in the Km for zero-trans uptake. In order to test whether allosteric ATP regulation of the transporter occurs, we reconstituted glucose transport activity into liposomes using erythrocyte membranes without detergent treatment. The effects of ATP, present either outside, inside, or both inside and outside the liposomes, on the transport activity were examined. Effects of ATP on trypsin-treated liposomes, which have only a single orientation of active transporters, were also tested. While the model predicts activation by ATP, only inhibition was observed. This was significant only at millimolar concentrations of ATP, in contrast to the previously reported effects at micromolar levels, and was primarily on the extracellular surface of the transporter. In addition, the ATP effects on reconstituted transport were nonspecific, with similar effects produced by tripolyphosphate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reconstitution of the glucose transporter from rat skeletal muscle

As a step in the purification and characterization of the glucose transporter from rat skeletal muscle, we have reconstituted glucose transport activity in liposomes. Plasma membranes were prepared from skeletal muscle which display D-glucose reversible binding of cytochalasin B (10 pmol sites/mg protein; KD = 0.3 microM). Older rats gave a slightly lower specific activity and much lower yield of sites per g muscle than young rats. Glucose transport activity was reconstituted into liposomes by the freeze-thaw procedure using either plasma membranes directly or cholate-extracted membrane proteins; the latter gave a 50% higher specific activity. The reconstituted transport activity was stereospecific, saturable, and inhibited by cytochalasin B, phloretin, and mercuric chloride. The optimum cholate concentration for extraction and reconstitution of transport activity was about 1.5%, and the highest specific activity of reconstituted transport was seen only at low ratios of protein to lipid in the reconstitution. Chromatography on agarose lentil lectin and agarose ethanethiol doubled both the specific activity of reconstituted transport and the fraction of glucose uptake which was stereospecific. In all of these respects the results were similar to our results with the bovine heart transporter (T. J. Wheeler and M. A. Hauck, Biochim. Biophys. Acta 818, 171-182 (1985)). Our findings suggest that further purification procedures developed for the heart transporter may be applicable to the skeletal muscle transporter as well.     

Reconstitution of the glucose transport activity of rat adipocytes

Rat epididymal fat cell membrane proteins were extracted from adipocyte ghosts with octylglucoside and incorporated by detergent dialysis into unilamellar phosphatidylcholine vesicles approx. 200 nm in diameter. The rate of glucose transport into the vesicles under zero-trans conditions was substrate dependent, saturable and inhibited by phloretin and cytochalasin B. Their maximum specific transport activity was 35.6 mumol/min per mg protein, and their half saturation constant for glucose was 15 mM. Glucose transport into the reconstituted vesicles was inhibited by only those sugars which competitively inhibited glucose transport into intact adipocytes. A major protein component of the vesicles was a 100 kDa protein which we had previously found to react with the affinity label maltosyl isothiocyanate (Malchoff, D.M., Olansky, L., Pohl, S. and Langdon, R.G. (1981) Fed. Proc. 40, 1893). Removal of adipocyte ghost membrane extrinsic proteins with dimethylmaleic anhydride followed by extraction of the resulting membrane pellet with octylglucoside yielded a solution which contained two major proteins, of Mr 100 000 and 85 000, with very small quantities of lower Mr proteins. Vesicles into which these proteins were incorporated had average specific transport activities of 624 mumol/min per mg protein and half saturation constants of 22 mM. Our results strongly indicate that the native glucose transporter of the rat adipocyte, like that of the human erythrocyte (Shelton, R.L. and Langdon, R.G. (1983) Biochim. Biophys. Acta 733, 25-33), is a 100 kDa protein.     

The effect of membrane composition and alcohols on the insulin-sensitive reconstituted monosaccharide-transport system of rat adipocyte plasma membranes.

The monosaccharide transporter from the plasma membranes of rat adipocytes and insulin-stimulated adipocytes has been reconstituted in sonicated liposomes. The stereospecific D-glucose uptake by liposomes made from a range of phospholipids and incorporating fatty acids has been investigated. D-Glucose uptake is correlated with an increase in lipid fluidity as a consequence of the addition of fluidizing fatty acids, changes in phospholipid acyl chain length and temperature. Benzyl alcohol and ethyl alcohol, which are generally considered to increase bilayer fluidity, decrease stereo-specific D-glucose uptake in both whole adipocytes and reconstituted liposomes. It is suggested that, although these alcohols may affect D-glucose transport by lipid-mediated fluidity changes, they also interact directly with the transporter resulting in inhibition of transport.     

Reconstitution of the glucose transporter from bovine heart

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

Long term regulation of glucose transporters by insulin in mature 3T3-F442A adipose cells. Differential effects on two glucose transporter subtypes

The question of a long term regulatory role of insulin on adipocyte glucose transporter content was addressed using the differentiating or fully mature 3T3-F442A adipocytes. Glucose transport was measured in intact cells. Glucose transporter content in plasma membranes and low density microsomes (LDM) was assessed by cytochalasin B binding and Western analysis. In insulin- versus spontaneously differentiated adipocytes, glucose transport and glucose transporters content of plasma membranes and LDM were increased 5-, 4-, and 2-fold, respectively. Insulin deprivation for 24 h induced a redistribution of glucose transporters in those cells which then displayed 2-fold higher glucose transport and glucose transporter content in plasma membranes than spontaneously differentiated cells and 3-fold more glucose transporters in LDM. When fully insulin-differentiated adipocytes were insulin-deprived for 4 days, there was a marked decrease in glucose transporters in both membrane fractions that was fully reversible by reexposing the cells to insulin for 4 days. Glucose uptake changes were closely proportionate to changes in glucose transporter content of plasma membranes as assessed by an antiserum to the C-terminal peptide of the erythrocyte/HepG2/brain-type glucose transporter. When Western blots were immunoblotted with 1F8 monoclonal antibody, specific for glucose transporter in insulin responsive tissues, an abundant immunoreactive protein was detected in both plasma membranes and LDM but the amount of this glucose transporter did not change with insulin exposure in any membrane fractions. In conclusion, insulin plays a long term regulatory role on cultured adipocyte glucose transporter content through a selective effect on the erythrocyte/HepG2/brain-type glucose transporter.     

Glycosylation of the human erythrocyte glucose transporter: a minimum structure is required for glucose transport activity

The involvement of the carbohydrate moiety of the human erythrocyte glucose transporter in glucose transport activity was previously demonstrated (Feugeas et al. (1990) Biochim. Biophys. Acta 1030, 60-64): N-glycanase treatment of the transport glycoprotein reconstituted in proteoliposomes resulted in a dramatic decrease of the Vmax. In this study, kinetic measurements of glucose equilibrium influx confirm our previous results. In order to investigate that a minimum glycosidic structure is required to maintain glucose transport activity, proteoliposomes were respectively treated with either sialidase, or sialidase and endo-beta-galactosidase, or a pool of exo-glycosidases which allows the release of all the sugar residues, except the proximal N-acetylglucosamine. Kinetic measurements of zero-trans influx made on sialidase- and (sialidase + endo-beta-galactosidase)-treated proteoliposomes did not reveal any significant changes in the glucose transport activity. On the contrary, treatment of the same proteoliposomes by a pool of exoglycosidases led to a complete abolition of activity, suggesting that a minimum glycosidic structure is required for glucose transport activity.     

Immunological evidence that band 3 is the major glucose transporter of the human erythrocyte membrane

We have previously reported that human erythrocyte band 3 contains 90-95% of the reconstitutable glucose transport activity of the erythrocyte membrane (Shelton, R.L. and Langdon, R.G. (1983) Biochim. Biophys. Acta 733, 25-33). We have now found that monoclonal and polyclonal antibodies to epitopes on band 3 specifically removed band 3 and more than 90% of the reconstitutable glucose transport activity from unfractionated octylglucoside extracts of erythrocyte membranes; nonimmune serum removed neither. Western blots of whole membrane extracts revealed that the polyclonal antibody to band 4.5 used to isolate cDNA clones presumed to code for the transporter (Mueckler, M., Caruso, C., Baldwin, C.A., Pancio, M., Blench, J., Morris, H.B., Allard, W.J., Lienhard, G.E. and Lodish, H.F. (1985) Science 229, 941-945) reacts strongly with six discrete bands in the 4.5 region. A monoclonal antibody to band 3 also reacts with a Mr 55,000 component of band 4.5. We conclude that band 3 contains the major glucose transporter of human erythrocytes, and that the transport activity in band 4.5 might be attributable to a band 3 fragment. Band 3 is probably a multifunctional transport protein responsible for transport of glucose, anions, and water.     

Functional reconstitution of the malate carrier of barley mesophyll vacuoles in liposomes

The malate carrier of barley (Hordeum vulgare L.) mesophyll vacuoles was highly purified by chromatography on hydroxyapatite followed by affinity-chromatography using 5-amino-1,2,3-benzenetricarboxylic acid as ligand. The carrier, reconstituted in asolectin liposomes, had properties similar to those described previously for the carrier in intact vacuoles (Martinoia, E., Flügge, U.I., Kaiser, G., Heber, U. and Heldt, H.W. (1985) Biochim. Biophys. Acta 806, 311-319). The apparent Km for malate uptake was 2-3 mM, and the uptake was inhibited by other carboxylic acids (preferentially tricarboxylic). The sulfhydryl reagent, p-chloromercuribenzenesulfonate, as well as the anion transport inhibitor 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid, also inhibited malate uptake. The transport was dependent on the membrane potential with an optimum at about 35 mV.     

Sulfonylurea binding to adipocyte membranes and potentiation of insulin-stimulated hexose transport

We have previously shown that the sulfonylureas increase insulin-stimulated glucose transport in adipocytes mainly by enhancing the insulin-induced recruitment of glucose transporter from its intracellular storage pool to the plasma membrane (Jacobs, D. B., and Jung, C. Y. (1985) J. Biol. Chem. 260, 2593-2596). In order to determine if this sulfonylurea effect is mediated by a specific membrane-associated sulfonylurea-binding protein, in the present report we measured exact dose dependence of the transport enhancement activities of different sulfonylureas in adipocytes in primary culture and equilibrium binding affinities of these agents to various adipocyte membrane fractions. Glycuride was found to increase the insulin-stimulated, 3-O-methyl-D-glucose equilibrium exchange in cultured rat adipocytes by up to 60% with little effect in the absence of insulin. The effect developed gradually reaching the maximum level at 24 h of incubation. The effect was concentration dependent showing a simple, one-to-one stoichiometry and an apparent activation constant (Ka) of approximately 1 microM. Glypizide, tolazamide, and tolbutamide also enhanced the insulin-stimulated hexose transport by up to 60%, but with Ka of approximately 2, 11, and 25 microM, respectively. HB-699 and ciglitazone, non-sulfonylureas, were without effect under the same condition. In equilibrium binding experiments, [3H]glyburide was found to bind to adipocyte membranes at two or more protein-specific, saturable sites, with similar apparent dissociation constants (KD) ranging 1-3 microM. These protein-specific glyburide bindings were displaced not only by tolazamide and tolbutamide, but also by ciglitazone and HB-699, with indicated KD of 11-16, 80-85, 20-25, and 85-95 microM, respectively. However, with the plasma membrane fraction, the displacements by ciglitazone and HB-699 were partial and did not exceed 56-61% at maximum. Based on these findings, we propose that there is a sulfonylurea-specific-binding protein in the plasma membrane of adipocytes, and that this sulfonylurea-binding protein may play a key role in the enhancement of insulin-stimulated hexose transport by sulfonylureas, probably via potentiation of the insulin-induced recruitment of glucose transporter.     

Effects of chlorpromazine and other calmodulin antagonists on phosphatidylcholine-induced vesiculation of platelet plasma membranes

Dilauroylglycerophosphocholine (C12:0PC)-induced vesiculation of platelet plasma membranes (Kobayashi, T., Okamoto, H., Yamada, J.-I., Setaka, M. and Kwan, T. (1984) Biochim. Biophys. Acta 778, 210-218; Kobayashi, T., Yamada, J.-I., Satoh, N., Setaka, M. and Kwan, T. (1985) Biochim. Biophys. Acta 817, 307-312) was inhibited by chlorpromazine. Preincubation of platelets with chlorpromazine was required for inhibition but incorporation of chlorpromazine into C12:0PC liposomes was not necessary for it, indicating that the observed inhibition of vesiculation was mainly due to the effect of chlorpromazine on platelets and not that on liposomes. The change in platelet membrane fluidity caused by chlorpromazine was not the cause of inhibition of vesiculation. The inhibition of vesiculation by various other calmodulin antagonists was also observed. The inhibitory activities of these calmodulin antagonists and chlorpromazine correspond very well to their abilities to bind to calmodulin. N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) inhibited vesiculation but a structural analogue of it, N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5), had no inhibitory activity. These results suggest the involvement of calmodulin in membrane vesiculation.     

Identification and partial purification of the insulin-responsive glucose transporter from 3T3-L1 adipocytes

The glucose transporter in 3T3-L1 adipocytes has been identified as a polypeptide of average Mr 51000 by means of its reaction with antibodies raised against the purified human erythrocyte glucose transporter and by photolabeling with [3H]cytochalasin B. The finding that the antibodies immunoprecipitated the photolabeled polypeptide demonstrated that both methods detected the same polypeptide. The 3T3-L1 adipocyte glucose transporter has been partially purified. The main steps in the purification procedure were the preparation of salt-washed cellular membranes, Triton X-100 solubilization, and immunoaffinity chromatography on affinity-purified antibodies against the human erythrocyte transporter. A simple method of affinity purification of these antibodies, which consists of adsorption from serum onto protein-depleted erythrocyte membranes and release with acid, and an assay for the 3T3-L1 adipocyte transporter polypeptide, which employs immunoblotting, have been developed.     

Dissociation of insulin-stimulated glucose transport from the translocation of glucose carriers in rat adipose cells

Cycloheximide, a potent inhibitor of protein synthesis, has been used to examine the relationship between recruitment of hexose carriers and the activation of glucose transport by insulin in rat adipocytes. Adipocytes were preincubated +/- cycloheximide for 90 min then +/- insulin for a further 30 min. We measured 3-O-methylglucose uptake in intact cells and in isolated plasma membrane vesicles. The concentration of glucose transporters in plasma membranes and low density microsomes was measured using a cytochalasin B binding assay. Cycloheximide had no affect on basal or insulin-stimulated 3-O-methylglucose uptake in intact cells or in plasma membrane vesicles. However, the number of glucose carriers in plasma membranes prepared from cells incubated with cycloheximide and insulin was markedly reduced compared to that from cells incubated with insulin alone (14 and 34 pmol/mg protein, respectively). Incubation of cells with cycloheximide alone did not change the concentration of glucose carriers in either plasma membranes or in low density microsomes compared to control cells. When isolated membranes were analyzed with an antiserum prepared against human erythrocyte glucose transporter, decreased cross-reactivity was observed in plasma membranes prepared from cycloheximide/insulin-treated cells compared to those from insulin cells. The present findings indicate that incubation of adipocytes with cycloheximide greatly reduces the number of hexose carriers in the plasma membrane of insulin-stimulated cells. Despite this reduction, insulin is still able to maximally stimulate glucose uptake. Thus, these data suggest an apparent dissociation between insulin stimulation of glucose transport activity and the recruitment of glucose carriers by the hormone.     

Inhibition of the binding of cytochrome b5 to phosphatidylcholine vesicles by cholesterol

The binding of cytochrome b₅ to single-walled liposomes of egg phosphatidylcholine was inhibited by the presence of cholesterol in the lipid bilayer under conditions where a limited amount of liposomes was incubated with the cytochrome. Since similar conditions seem to apply for the binding of cytochrome b₅ to erythrocyte ghosts, this observation supports the conclusion of Enomoto and Sato (Enomoto, K. and Sato, R. (1977) Biochim. Biophys. Acta 466, 136–147) that the localization of cholesterol on the outer surface of the ghost membrane prevents the binding of cytochrome b₅ to this surface. The finding reported by Roseman et al. (Roseman, M.A., Holloway, P.W. and Calabro, M.A. (1978) Biochim. Biophys. Acta 507, 552–556) that cholesterol did not prevent the cytochrome binding to phosphatidylcholine liposomes in the presence of a large excess of liposomes could be confirmed in the present study, but this does not contradict the abovementioned conclusion.     

Nocodazole inhibits insulin-stimulated glucose transport in 3T3-L1 adipocytes via a microtubule-independent mechanism

Insulin stimulates glucose transport in adipocytes and muscle cells by triggering redistribution of the GLUT4 glucose transporter from an intracellular perinuclear location to the cell surface. Recent reports have shown that the microtubule-depolymerizing agent nocodazole inhibits insulin-stimulated glucose transport, implicating an important role for microtubules in this process. In the present study we show that 2 microm nocodazole completely depolymerized microtubules in 3T3-L1 adipocytes, as determined morphologically and biochemically, resulting in dispersal of the perinuclear GLUT4 compartment and the Golgi apparatus. However, 2 microm nocodazole did not significantly effect either the kinetics or magnitude of insulin-stimulated glucose transport. Consistent with previous studies, higher concentrations of nocodazole (10-33 microm) significantly inhibited basal and insulin-stimulated glucose uptake in adipocytes. This effect was not likely the result of microtubule depolymerization because in the presence of taxol, which blocked nocodazole-induced depolymerization of microtubules as well as the dispersal of the perinuclear GLUT4 compartment, the inhibitory effect of 10-33 microm nocodazole on insulin-stimulated glucose uptake prevailed. Despite the decrease in insulin-stimulated glucose transport with 33 microm nocodazole we did not observe inhibition of insulin-stimulated GLUT4 translocation to the cell surface under these conditions. Consistent with a direct effect of nocodazole on glucose transporter function we observed a rapid inhibitory effect of nocodazole on glucose transport activity when added to either 3T3-L1 adipocytes or to Chinese hamster ovary cells at 4 degrees C. These studies reveal a new and unexpected effect of nocodazole in mammalian cells which appears to occur independently of its microtubule-depolymerizing effects.     

Kinetic properties of the reconstituted glucose transporter from human erythrocytes

The kinetic parameters of D-glucose transport in liposomes reconstituted with the purified glucose transporter were determined. Net uptake and efflux both had Km values of 0.7 to 1.2 mM and Vmax values of 1.6 mumol/mg of protein/min. Equilibrium exchange had a Km of 35 mM and a Vmax of 50 mumol/mg of protein/min. By separating the liposomes from unreconstituted protein using density centrifugation, the Vmax of exchange was increased to 86 mumol/mg of protein/min, about 3 times that of the erythrocyte membrane. Trypsin, which inhibits erythrocyte glucose transport only from the cytoplasmic side, inhibited reconstituted transport activity about 40% when added externally. With internal treatment as well, the inhibition was about 80%. This suggests that the reconstituted transporter is oriented about equally in both directions. Antibody prepared against the purified transporter inhibits transport to a maximum of about 50%, also indicating a scrambled orientation. External trypsin treatment decreased the Km for uptake and increased the Km for efflux, consistent with asymmetric kinetic parameters for the two faces of the transporter. However, the calculated Km values are lower than those reported for erythrocytes. Phloretin and diethylstilbestrol inhibit the reconstituted transporter. However, they bind to liposomes, producing anomalous results under some experimental conditions. When this binding is taken into account, phloretin inhibits completely and symmetrically. The binding accounts for the apparent asymmetric effects of phloretin reported by others. The inhibitory effects of mercuric ions are consistent with action at two classes of binding sites. Treatment with trypsin increases the sensitivity to Hg2+, indicating that the more sensitive site is on the external face of the transporter.     

Location of the maltosyl isothiocyanate binding site on the human erythrocyte glucose transporter

The covalent affinity probe maltosyl isothiocyanate (MITC) has been used previously to identify the glucose transporter of human erythrocytes as a component of band 3. By use of limited proteolysis, the site on the Mr 100 000 protein to which MITC attaches has been localized to a 17 000-dalton region near the center of the polypeptide chain which is intimately associated with the membrane. The erythrocyte anion transporter, which is probably homologous to the glucose carrier, has a corresponding segment which is known to bind the covalent affinity label 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid [Ramjeesingh, M., Gaarn, A., & Rothstein, A. (1980) Biochim. Biophys. Acta 559, 127-139]. These results suggest that, in addition to having structural features in common, the two carrier proteins may be quite similar with regard to functional organization.