Efflux Of D-glucose Research Articles

Neutral-sugar transport by rat liver lysosomes

Transport of D-glucose was studied in Percoll-gradient-purified rat liver lysosomes. D-Glucose uptake had a Km of 22 mM and a t1/2 of approx. 30 s. D-Fucose, 2-deoxyglucose and methyl alpha-glucoside were the most effective competitors for uptake of D-glucose, although D-galactose, D-mannose, D-xylose and L-fucose also appeared to compete for uptake. L-Glucose was a poor competitor for uptake. No competition was observed with N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, D-glucuronic acid, N-acetylneuraminic acid, D-glucosamine or the amino acids L-glycine, L-lysine and L-proline. Uptake was unaffected by N-ethylmaleimide, dithiothreitol, KCl, NaCl, ATP/Mg or alteration of buffer pH. D-Glucose efflux from lysosomes was temperature-dependent, with a Q10 of 2.3, and was inhibited by cytochalasin B. Counter-transport could not be demonstrated. In contrast, L-fucose uptake had a Km of 65 mM and was largely unaffected by 5 M excess of neutral D-sugars. Both uptake and efflux of L-fucose were inhibited by cytochalasin B. It appears that lysosomes possess a facilitated transport system for D-glucose and perhaps other neutral D-sugars that is discrete from transport systems for acetylated and acidic sugars.

Effect of insulin on d-glucose transport by human placental brush border membranes

We studied the effect of insulin on the uptake of d-glucose by human placental brush border membranes (BBM) in vitro. d-glucose transport through placental BBM is a Na +-independent transport, inhibited by 0.5 mM phloretin. Increasing the substrate concentration from 1 to 50 mM resulted in an increase in glucose uptake according to an S-shaped relationship. Hill plot analysis suggests that at least two molecules of d-glucose are transported at the same time by the carrier. Preincubation of the placental tissue with insulin for 45 min at 22 °C significantly enhanced the d-glucose influx into the membrane vesicles, without influencing the slope of the Hill plot. A dose-response curve of the effect of insulin revealed that although the effect was already significant at 10 −9 M, the maximal activity was reached at 10 −8 M. The influence of insulin on d-glucose uptake was present only when preincubation of the placental tissue with the hormone was performed in the presence of Mn 2+. Incubation of placental tissue with 10 -8 M insulin did not influence d-glucose efflux from the BBM vesicles. Finally, direct incubation of the membranes with insulin had no effect on the glucose influx into these membrane vesicles. We conclude that insulin, at physiological concentrations, enhances glucose uptake by the BBM, and that such a regulation might contribute to the glucose homeostasis in the fetal circulation, independent of the maternal variations in glycemia.

Comparison of adrenergic agonist and insulin effects on 3-0-methyl- d-glucose efflux and sarcolemmal cytochalasin B binding by perfused rat heart

1. 1. α- and β-Adrenergic agonists as well as insulin stimulate 3-0-methyl- d-glucose efflux by the perfused rat heart and increase d-glucose inhibitable cytochalasin B binding by isolated sarcolemma. 2. 2. α- and β-Agonists like insulin increase V max for 3-0-methyl- d-glucose efflux and increase B max for cytochalasin B binding. 3. 3. The effects of α- and β-agonists are totally Ca 2+-dependent whilst those of insulin appear to be only partly Ca 2+-dependent.

ATP regulation of the human red cell sugar transporter.

Purified human red blood cell sugar transport protein intrinsic tryptophan fluorescence is quenched by D-glucose and 4,6-ethylidene glucose (sugars that bind to the transport), phloretin and cytochalasin B (transport inhibitors), and ATP. Cytochalasin B-induced quenching is a simple saturable phenomenon with Kd app of 0.15 microM and maximum capacity of 0.85 cytochalasin B binding sites per transporter. Sugar-induced quenching consists of two saturable components characterized by low and high Kd app binding parameters. These binding sites appear to correspond to influx and efflux transport sites, respectively, and coexist within the transporter molecule. ATP-induced quenching is also a simple saturable process with Kd app of 50 microM. Indirect estimates suggest that the ratio of ATP-binding sites per transporter is 0.87:1. ATP reduces the low Kd app and increases the high Kd app for sugar-induced fluorescence quenching. This effect is half-maximal at 45 microM ATP. ATP produces a 4-fold reduction in Km and 2.4-fold reduction in Vmax for cytochalasin B-inhibitable D-glucose efflux from inside-out red cell membrane vesicles (IOVs). This effect on transport is half-maximal at 45 microM ATP. AMP, ADP, alpha, beta-methyleneadenosine 5'-triphosphate, and beta, gamma-methyleneadenosine 5'-triphosphate at 1 mM are without effect on efflux of D-glucose from IOVs. ATP modulation of Km for D-glucose efflux from IOVs is immediate in onset and recovery. ATP inhibition of Vmax for D-glucose exit is complete within 5-15 min and is only partly reversed following 30-min incubation in ATP-free medium. These findings suggest that the human red cell sugar transport protein contains a nucleotide-binding site(s) through which ATP modifies the catalytic properties of the transporter.

Effects of insulin receptor down-regulation on hexose transport in human erythrocytes.

D-Glucose and D-galactose influx and efflux rates in human erythrocytes were studied using infinite-cis and zero-trans assay methods. It was found that insulin decreased the infinite-cis Km for both D-glucose and D-galactose influx by 44 and 56%, respectively, while the Vmax was unchanged. The Km for D-glucose efflux in the presence of insulin decreased by 47% when compared to controls, and the change in Vmax was statistically insignificant. If insulin receptors were first down regulated, and then influx and efflux assays were performed, decreases in the infinite-cis and zero-trans Km values were also observed in the absence of exogenous insulin. These affinity changes were not due to persistent surface insulin receptor occupation by the insulin which was used to induce down-regulation. These affinity changes were comparable to those observed in non-down-regulated cells in the presence of insulin.

A rapid method of reconstituting human erythrocyte sugar transport proteins.

A rapid reconstitution procedure for human erythrocyte hexose transfer activity is described. The procedure (reverse-phase evaporation) avoids exposure of the isolated proteins to detergent, organic solvent, sonication, or freeze-thaw steps during insertion into synthetic membranes and may be effected within 15 min. The so-formed vesicles are unilamellar structures with a large encapsulated volume, narrow size range, and low passive permeabilities. Contamination by carry-through of endogenous (red cell) lipids is less than 1%. Reconstituted hexose transfer activity was examined by using unfractionated proteins (bands 3, 4.5, and 6) and purified proteins (bands 4.5 and 3). With unfractionated proteins, hexose transport activity is low [0.34 mumol X (mg of protein)-1 X min-1], is inhibited by cytochalasin B, and increases monotonically with protein concentration. Kinetic analysis indicates that Vmax values for both influx and efflux of D-glucose are identical. Reconstitution of the cytochalasin B binding protein (band 4.5) results in hexose transport with high specific activity [5 mumol X (mg of protein)-1 X min-1] and symmetry in transfer kinetics. Band 3 proteins also appear to mediate cytochalasin B sensitive D-glucose transport activity.

Demonstration of sodium-dependent, electrogenic substrate transport in rat small intestinal brush border membrane vesicles by a cyanine dye.

The cyanine dye DiS-C2(5) was tested as an indicator for changes in membrane potential of subfractionated rat jejunal brush border membrane vesicles. The fluorescence of this dye increased with inside positive and decreased with inside negative potentials. The sensitivity to inside negative potentials was greater than to inside positive potentials. The addition of L-alanine, L-phenylalanine, L-methionine, D-galactose and D-glucose in the presence of sodium provoked a transient fluorescence increase indicating an inside positive membrane potential due to electrogenic, sodium-coupled transport. Besides the sodium-dependence, the dye reflected stereo-specificity and saturability of D-glucose transport. When D-glucose loaded vesicles were incubated in D-glucose-free medium, a decrease in fluorescence was observed indicating that D-glucose efflux is also electrogenic.

Reconstitution of D-glucose transporter from human placental microvillous plasma membranes.

Proteins from microvillous plasma membrane vesicles of the maternal surface of human trophoblast were solubilized with octyl beta-D-glucoside. After incorporation of the soluble protein into phospholipid liposomes D-glucose uptake exceeded that of L-glucose. The reconstituted system showed that D-glucose uptake was not sodium dependent and was inhibited by cytochalasin B. Efflux of D-glucose from the proteoliposomes was retarded by cytochalasin B. D-Glucose uptake, but not L-glucose, was proportional to the amount of protein used in the reconstitution procedure. Membrane protein was also solubilized with octylglucoside from vesicles that had been extracted previously by dimethylmaleic anhydride. Proteoliposomes prepared from these latter proteins showed D-glucose uptake threefold greater than that from octylglucoside solubilization alone, but sodium dodecyl sulfate polyacrylamide gel electrophoresis of the extracted protein showed no clear difference between the double extraction procedure and the pattern obtained with the single detergent.

Modulation of glucose uptake in animal cells. Studies using plasma membrane vesicles isolated from nontransformed and simian virus 40-transformed mouse fibroblast cultures.

Plasma membrane vesicles isolated from nontransformed and Simian virus 40-transformed mouse fibroblast cultures catalyzed carrier-mediated D-glucose transport without detectable metabolic conversion to glucose 6-phosphate. Glucose transport activity was stereospecific, temperature-dependent, sensitive to inactivation by p-chloromercuriphenylsulfonate, and accompanied plasma membrane material during subcellular fractionation. D-Glucose efflux from vesicles was inhibited by phloretin, an inhibitor of glucose uptake in intact cells. Cytochalasin B, a potent inhibitor of glucose uptake when tested with the intact cells used for vesicle isolation did not inhibit glucose transport in vesicles despite the presence of high affinity cytochalasin binding sites in isolated membranes. The enhanced glucose uptake observed in intact cells after viral transformation was not expressed in vesicles: no significant differences in glucose transport specific activity could be detected in vesicle preparations from nontransformed and transformed mouse fibroblast cultures. These findings indicate that cellular components distinct from glucose carriers can mediate changes in glucose uptake in mouse fibroblast cultures in at least two cases: sensitivity to inhibition by cytochalasin B and the enhanced cellular sugar uptake observed after viral transformation.

The effect of the strongly bound protein fraction on sugar transport in human erythrocyte ghosts

The protein fraction released from human erythrocyte membranes with 90% acetic acid enhanced the transport of several sugar species when enclosed in erythrocyte ghosts. Both the influx and the efflux of d-glucose were increased so that permeation rather than sugar binding was involved. The permeation increase was selective, being found with d-glucose, d-galactose and d-xylose but not with l-glucose or lactose. The protein-dependent sugar transport was saturable and the incorporation of proteins into the ghost membrane brought J max to the level corresponding to intact erythrocytes, leaving K m unchanged.

Reconstitution of D-glucose transport in vesicles composed of lipid and a partially purified protein from the human erythrocyte membrane

The stereospecific efflux of D-glucose from liposomes formed upon sonication of erythrocyte lipid with various membrane fractions was measured in order to assess their D-glucose transport activity. Extrinsic and intrinsic membrane proteins were separated into soluble and membrane-bound fractions, respectively by extraction of ghosts with 2,3-dimethylamleic anhydride. Reconstitution of D-glucose transport was catalyzed only by the intrinsic membrane proteins. Upon subsequent Triton X-100 extraction of these proteins, reconstitution of D-glucose transport was associated with both the extract and membrane residue separated by high-speed centrifugation. The membrane residue did not contain any periodic acid-Schiff-sensitive glycoprotein and consisted chiefly of a protein component of 95,000 molecular weight (Band 3), a portion of which was present in the Triton X-100 extract. These results support the proposal that Band 3 proteins are directly involved in D-glucose transport.

A method using 3-O-methyl-D-glucose and phloretin for the determination of intracellular water space of cells in monolayer culture

A technique is described by which the intracellular water space can be determined in cells which are attached to a culture dish. A major advantage of this procedure is that the cells maintain their normal morphology. This technique takes advantage of the transport of the nonmetabolizable hexose, 3- O-methyl- d-glucose, to an intracellular concentration equal to the extracellular concentration, and the inhibition of 3- O-methyl- d-glucose efflux by phloretin. It requires only a determination of the nanomoles of hexose taken up at equilibrium plus a protein or cell number determination.

Transport of d-glucose by brush border membranes isolated from the renal cortex

The transport of d-glucose by brush border membranes isolated from the rabbit renal cortex was studied. At concentrations less than 2 mM, the rate of d-glucose uptake increased linearly with the concentration of the sugar. No evidence was found for a “high-affinity” (μM) saturable site. Saturation was indicated at concentrations of d-glucose greater than 5 mM. The uptake of d-glucose was stereospecific and selectively inhibited by d-galactose and other sugars. Phlorizin inhibited the uptake of d-glucose in the presence and absence of Na +. The glycoside was a potent inhibitor of the efflux of d-glucose. Preloading the brush border membrane vesicles with d-glucose, but not with l-glucose, accelerated exchange diffusion of d-glucose. These results demonstrate that the uptake of d-glucose by renal brush borders represents transport into an intravesicular space rather than solely binding. The rate of d-glucose uptake was increased when the Na + in the extravesicular medium was high and the membranes were preloaded with a Na +-free medium. The rate of d-glucose uptake was inhibited by preloading the brush border membranes with Na +. These results are consistent with the Na + gradient hypothesis for d-glucose transport in the kidney. Thus, the presence of a Na +-dependent facilitated transport of d-glucose in isolated renal brush border membranes is indicated. This finding is consistent with what is known of the transport of the sugar in more physiologically intact preparations and suggests that the membranes serve as an effective model system in examining the mechanism of d-glucose transport in the kidney.

An alternative to the carrier model for sugar transport across red cell membranes.

The quantitative predictions of the carrier model diverge from the experimental data for sugar transport across red cell membranes in several ways. It has been found that labelled D-glucose efflux from red cells is accelerated more when concentrations of D-galactose sufficient to saturate the transport system replace similar concentrations of D-glucose in the external solution (1, 2). It has been suggested that this finding requires that the mobility of the galactose-carrier complex should exceed that of the glucose-carrier complex (1). Thus the mobile carrier model predicts that labelled galactose efflux should be accelerated more by external galactose than is glucose efflux accelerated by external glucose. In fact the experimental data show that at 20°C labelled galactose efflux is accelerated less by external galactose than is labelled glucose efflux accelerated by external glucose and that at 30°C no acceleration of labelled galactose efflux, following addition of galactose to the bulk solution, occurs (2).

Kinetics of glucose transfer in adult and fetal human erythrocytes.

Extract: Efflux of D-glucose at 4° was different in erythrocytes of healthy adults than it was in erythrocytes of term human infants. The calculated Ks. was 30.2 mM for the adult cells and 18.0 mM for the infant cells. The likeliest explanation of these results is the ontogeny of the sugar-carrier apparatus in the human erythrocyte. Speculation: Evidence is presented that in the early stages of mammalian development transfer rates for certain solutes across the plasma membrane change substantially and that three of four possible transfer mechanisms participate in this change. This change may affect infant nutritional requirements and should be considered in establishing normal values of certain nutrients (e.g., glucose) in the premature and newly born. In addition, response of infants to drugs may be related to drug access to the target site. Whether other functions of the plasma membrane undergo developmental change in early postnatal life remains to be determined.

Glucose Transport in Fat Cell Membranes

Abstract The properties of d-glucose transport by isolated fat cell membrane preparations have been studied by measuring directly the rates of sugar accumulation and efflux. Observations are made under conditions in which zero order kinetics prevails and during which no detectable metabolic alteration of glucose occurs. The initial velocities measured are proportional to the amount of membrane material present. This is a relatively simple model system which permits direct study of glucose transport and insulin action. The rate of d-glucose accumulation is a temperature-dependent process. In contrast to l-glucose accumulation, d-glucose transport rapidly reaches a steady state, displays saturation kinetics with respect to substrate, is inhibited by 3-O-methyl glucose but not by l-glucose, is markedly suppressed by low concentrations of phloretin, and is enhanced by insulin. The transport process is not coupled to energy-producing metabolic processes, and no accumulation occurs against a chemical gradient. The unidirectional efflux of d-glucose from fat cell membranes has been studied separately. This process is also temperature-dependent, energy (metabolic)-independent, stereospecific, saturable with respect to the concentration of glucose inside the membrane, and sensitive to the presence of insulin or phloretin in the medium external to the membrane. The kinetic rate constants for efflux (Kt, 0.2 mm; Vmax, 3.8) differ from those calculated from the influx data (Kt, 3.3 mm; Vmax, 12.3). Although these differences may reflect differences in the biological function of the membrane surfaces, they do not constitute definitive proof of asymmetry of the transport structures or processes. Evidence for the operation of a mobile carrier mechanism of facilitated diffusion was obtained by detecting accelerated exchange diffusion. The unidirectional d-glucose flux from the inside of the membranes into a medium containing negligible glucose is accelerated by the presence of 3-O-methyl glucose in the medium. This suggests that the rate of transfer of free carrier from the outside to the inside phases of the membrane is a limiting process in the outward movement of the d-glucose-carrier complex.