Distinction of three types of D-glucose transport systems in animal cells

Immunoblotting of plasma membrane fractions from rat kidney cortex with antibody to human erythrocyte glucose transporter showed a single major cross-reacting material of 48K in basolateral membrane fractions possessing a facilitated diffusion system for D-glucose, but not in brush border membrane fractions which have a Na-dependent active transport system. Cytochalasin B inhibited D-glucose uptake in basolateral membrane vesicles but not in brush border vesicles. Cross-reacting materials of 44-55K were detected in several animal cells exhibiting facilitated diffusion systems, including a hormone dependent system. These results indicate molecular difference between glucose transporters of facilitated diffusion systems and active transport systems.     

6-Deoxy-D-glucose and D-xylose: Analogs for the study of D-glucose transport by mouse 3T3 cells

6-Deoxy-D-glucose and D-xylose, structural homomorphs of D-glucose that lack a 6-hydroxyl group or a 6-hydroxymethyl group, respectively, are transported efficiently by mouse 3T3 cells, with good affinity and high specificity for the D-glucose transport system. Since these analogs lack the 6-hydroxyl group, which is the site of phosphorylation of glucose by hexokinase, they are taken up and are recoverable from cells in an unchanged state. Thus, 6-deoxy-D-glucose and D-xylose offer advantages as transport substrates over 2-deoxy-D-glucose, which is phosphorylated by intercellular hexokinases, and 3-O-methyl-D-glucose, which shows a lower specificity for the D-glucose transport system.     

Facilitated Transport of Glucose from Blood into Peripheral Nerve

D-Glucose is the major substrate for energy metabolism in peripheral nerve. The mechanism of transfer of glucose across the blood-nerve barrier is unclarified. In this study an in situ perfusion technique was utilized, in anesthetized rats, to examine monosaccharide transport from blood into peripheral nerve. Unidirectional influxes of D-[14C]glucose, L-[14C]glucose, and [14C]3-O-methyl-D-glucose across capillaries of the tibial nerve were measured at different perfusate concentrations of unlabelled D-glucose. The permeability-surface area product (PA) for D-[14C]glucose and [14C]3-O-methyl-D-glucose decreased, whereas the PA for L-[14C]glucose remained constant, as the perfusate concentration of D-glucose was increased. In the presence of no added unlabelled D-glucose in the perfusate, the PA for L-[14C]glucose equaled one-fifth the PA for D-[14C]glucose. These results demonstrate self-saturation, competitive inhibition, and stereospecificity of glucose transfer, and for the first time show a unidirectional facilitated transport mechanism for D-monosaccharides at capillaries of mammalian peripheral nerve. The data were fit to a model for facilitated transport and passive diffusion. The half-saturation constant and maximal rate of transport for the saturable component of D-glucose influx equaled 23 +/- 11 mumol X ml-1 and 6.6 +/- 3.2 X 10(-3) mumol X s-1 X g-1, respectively. The constant of nonsaturable glucose influx equaled 0.5 +/- 0.1 X 10(-4) s-1. At normal plasma glucose concentrations, the saturable component comprises about 80% of total D-glucose influx into nerve.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of the glucose transport protein of the microvillous membrane of human placenta by photoaffinity labelling

The initial step in transfer of glucose from mother to fetus is facilitated diffusion transport across the microvillous membrane of the placental syncytium (1). We have used ³H-cytochalasin B as a photoaffinity label to identify the transport protein involved. Two binding proteins were present, one of which is apparently the glucose transport protein and one of which is actin. The two were identified by competition labeling with D-glucose, and cytochalasin E. They were separated by selective extraction with dimethyl maleic anhydride. The glucose transport protein is apparently a single molecular species of 52,000 molecular weight.     

Reciprocal influence of glucose anomers upon their respective phosphorylation by hexokinase

The phosphorylation of D-glucose (1.0mM) was measured in homogenates of tumoral islet cells incubated at 7 degrees C in the presence of labelled alpha- and/or beta-D-glucose, with or without exogenous glucose 6-phosphate. The close-to-maximal reaction velocity of hexokinase was higher with beta- than alpha-D-glucose. The latter anomer inhibited beta-D-glucose phosphorylation more than the beta-anomer decreased the phosphorylation of alpha-D-glucose. This behaviour was accounted for by the higher affinity of hexokinase for alpha- than for beta-D-glucose. These direct measurements of the relative contribution of each anomer to the overall rate of glucose phosphorylation in the presence of mixed populations of alpha- and beta-D-glucose validate the concept that the phosphorylation of D-glucose displays anomeric specificity even when the hexose is used at anomeric equilibrium. Glucose 6-phosphate inhibited the phosphorylation of the two anomers more severely when alpha-D-glucose rather than beta-D-glucose was the most abundant anomer.     

Facilitated diffusion of monosaccharides in smooth muscle of rat vas deferens in vitro

The suitability of rat vas deferens for investigating sugar transport in smooth muscle was determined in vitro, with the nonmetabolized glucose analog 3-O-methyl-D-glucose as test sugar. Vas deferens smooth muscle contains a facilitated diffusion system for monosaccharides, as shown by saturation of the transport sites and by competition between 3-O-methyl-D-glucose and D-glucose. The activity of the facilitated diffusion system could be enhanced by hyperosmolarity and by contractile activity, but frequency dependency could not be established. A high concentration of insulin (100 mU/mL) was required to stimulate sugar transport. As smooth muscle is not a primary tissue for the storage of energy reserves, it does not require large numbers of insulin receptors.     

Topography and functions of sulfhydryl groups of the human erythrocyte glucose transport mechanism

Summary Membrane-impermeant and -permeant maleimides were applied to characterize the location and function of the sulfhydryl (SH) groups essential for the facilitated diffusion mediated by the human erythrocyte glucose transport protein. Three such classes have been identified. Type I SH is accessible to membrane-impermeant reagents at the outer (exofacial) surface of the intact erythrocyte. Alkylation of this class inhibits glucose transport; D-glucose and cytochalasin B protect against the alkylation. Type II SH is located at the inner (endofacial) surface of the membrane and is accessible to the membrane-impermeant reagent glutathione maleimide only after lysis of the erythrocyte. D-glucose enhances, while cytochalasin B reduces, the alkylation of Type II SH by maleimides. Reaction of Types I and II SH with an impermeant maleimide increases the half-saturation concentration for binding of D-glucose to erythrocyte membranes. By contrast, inactivation of Type III SH markedly decreases the half-saturation concentration for the binding of D-glucose and other transported sugars. Type III SH is inactivated by the relatively lipid-soluble reagents N-ethylmaleimide (NEM) and dipyridyl disulfide, but not by the impermeant glutathione maleimide. Type III SH is thus located in a hydrophobic membrane domain. A kinetic model constructed to explain these observations indicates that Type III SH is required for the translocation event in a hydrophobic membrane domain which leads to the dissociation of glucose bound to transport sites at the membrane surfaces.     

Decrease in glucose transport activity of Friend erythroleukemia cell caused by dimethylsulfoxide, a differentiation-inducing reagent

A transport system for D-glucose was found in a Friend erythroleukemia cell line, T-3-C1-2-O and was characterized as a facilitated diffusion system. D-Glucose transport activity showed a half-saturation concentration of 2.2 mM and was inhibited by mercuric ions, cytochalasin B, phloretin, and stilbestrol, but was not strongly inhibited by phloridzin. Transport of 3-O-methyl-D-glucose was faster than D-glucose and the intracellular concentration of the sugar was found to reach the concentration in the assay medium. The treatment of cells with a differentiation-inducing reagent, dimethylsulfoxide(Me2SO), for 24 h caused a marked decrease in glucose transport activity due to a decrease in Vmax. In an induction-insensitive Friend cell line, T-3-K-1, D-glucose transport activity was low in untreated cells and Me2SO treatment did not cause a significant decrease in transport activity. The results obtained in this study indicate that the decrease in glucose transport activity is not due to the direct effect of Me2SO on transport activity, but is associated with the induction of differentiation. By immunoblotting cell lysates of T-3-C1-2-O cells using antibody to human erythrocyte glucose transporter, a single major band having a molecular weight of 52,000 was detected, which may be a glucose transporter in Friend cells.     

Hexose metabolism in pancreatic islets: preferential utilization of mitochondrial ATP for glucose phosphorylation

The respective contribution of exogenous and intramitochondrially formed ATP to D-glucose phosphorylation by mitochondria-bound hexokinase was examined in both rat liver and pancreatic islet mitochondria by comparing the generation of D-glucose 6-[32P]phosphate from exogenous [gamma-32P]ATP to the total rate of D-[U-14C]glucose phosphorylation. In liver mitochondria, the fractional contribution of exogenous ATP to D-glucose phosphorylation ranged from 4 to 74%, depending on the availability of endogenous ATP formed by either oxidative phosphorylation or in the reaction catalyzed by adenylate kinase. Likewise, in islet mitochondria exposed to exogenous ATP but deprived of exogenous nutrient, about 60% of D-glucose phosphorylation was supported by mitochondrial ATP. Such a fractional contribution was further increased in the presence of ADP and succinate, and suppressed by mitochondrial poisons. It is concluded that, in islet like in liver mitochondria, mitochondrial ATP is used preferentially to exogenous ATP as a substrate for D-glucose phosphorylation by mitochondria-bound hexokinase. This may favour the maintenance of a high cytosolic ATP concentration in glucose-stimulated islet cells.     

Rapid kinetics of the glucose transporter from human erythrocytes. Detection and measurement of a half-turnover of the purified transporter

The stopped flow method combined with fluorescence detection has been employed to study the rapid kinetics of the glucose transporter from human erythrocytes. Upon mixing the purified transporter reconstituted into unsealed membranes of erythrocyte lipids with 4,6-ethylidene D-glucose, a derivative that binds preferentially to the substrate site on the outer domain of the transporter, there was a rapid, first-order decrease in the intrinsic fluorescence of the protein. Three properties of this transient indicate that it represents a half-turnover of the transporter from a conformation with the substrate site facing inward to one with this site facing outward. The first-order rate constant decreased as the concentration of ethylidene glucose was increased; the value of the rate constant for the process is similar to that expected from steady-state kinetic studies of transport in the erythrocyte; and D-glucose at low concentration increased the rate of reaction. This study is the first determination of the kinetics of a half-turnover for a transport system of the facilitated diffusion type. The identification of this step provides direct evidence for the alternating conformation mechanism of transport.     

Kinetic characterization of N-acetyl-D-glucosamine kinase from rat liver and kidney.

1. Under normal assay conditions the N-acetyl-D-glucosamine kinases from rat liver and kidney show a pH-dependent lag phase before reaching a steady state, which is probably due to reversible dissociation of the dimeric enzyme. 2. The enzyme catalyses the phosphorylation of N-acetyl-D-glucosamine, N-acetyl-D-mannosamine and D-glucose at pH 7.5, with apparent Km values of 0.06, 0.95 and 600 mM respectively for the enzyme from liver and 0.04, 1.0 and 410 mM respectively for the kidney enzyme. It is strongly inhibited by ADP. 3. The interaction between the enzymes and acceptor substrates shows non-Michaelian kinetics with respect to N-acetyl-D-glucosamine but normal behaviour towards N-acetyl-D-mannosamine and D-glucose. 4. Both N-acetyl-D-glucosamine and N-acetyl-D-mannosamine inhibit the phosphorylation of D-glucose; this inhibition appears to be mixed in character. 5. The facts that the enzymes catalyse the phosphorylation of N-acetyl-D-mannosamine and D-glucose do not detract from the designation of the enzymes as N-acetyl-D-glucosamine kinase. Phosphorylation of glucose in vivo by these kinases is unlikely.     

Glucose uptake by Trypanosoma brucei. Rate-limiting steps in glycolysis and regulation of the glycolytic flux

Glucose uptake and metabolism in the bloodstream form of the glycosome-containing protozoan parasite Trypanosoma brucei was measured using 14C-labeled glucose in combination with the silicone oil centrifugation technique in short term (5-60 s) incubations. Glucose rather than glucose analogues was used to study the interrelation between the uptake process and the subsequent metabolic steps. Glucose uptake over the plasma membrane occurred by facilitated diffusion, which limited the overall glycolytic rate at external glucose concentrations (glcout) below 5 mM. At higher glcout another step, either transport over the glycosomal membrane or phosphorylation by hexokinase became rate-limiting. Mathematical modeling assuming that glucose uptake occurs by facilitated diffusion followed by an enzymatic step accurately predicts the experimental data. As predicted by the model, the internal concentration of non-metabolized glucose remains low till glcout = 5 mM and increases at higher external concentrations. In contrast to glucose, glycerol entered the cell by simple diffusion. Externally supplied glycerol did not affect glucose metabolism but externally added glucose interfered with glycerol metabolism in a way that suggests that the rate-limiting step is at the level of glycerol kinase. Our observations suggest that the bloodstream form of T. brucei adapts its glucose transport in a way that gives maximum yield at minimum expense.     

Transport of glucose by Bifidobacterium animalis subsp. lactis occurs via facilitated diffusion

Two strains of Bifidobacterium animalis subsp. lactis were indistinguishable by several nucleic acid-based techniques; however, the type strain DSMZ 10140 was glucose utilization positive, while RB 4825, an industrially employed strain, was unable to grow rapidly on glucose as the principal carbon source. This difference was attributed to the presence of a low-affinity facilitated-diffusion glucose transporter identified in DSMZ 10140 but lacking in RB 4825. Uptake of D-[U-(14)C]glucose in DSMZ 10140 was stimulated by monovalent cations (ammonium, sodium, potassium, and lithium) and inhibited by divalent cations (calcium and magnesium). When competitor carbohydrates were included in the uptake assays, stereospecific inhibition was exhibited, with greater competition by methyl-beta-glucoside than methyl-alpha-glucoside. Significant inhibition (>30%) was observed with phloretin, an inhibitor of facilitated diffusion of glucose, whereas there was no inhibition by sodium fluoride, iodoacetate, sodium arsenate, sodium azide, 2,4-dinitrophenol, monensin, or valinomycin, which typically reduce energy-driven transport. Based on kinetic analyses, the mean values for K(t) and V(max) were 14.8 +/- 3.4 mM D-glucose and 0.13 +/- 0.03 micromol glucose/min/mg cell protein, respectively. Glucose uptake by several glucose-utilizing commercial strains of B. animalis subsp. lactis was also inhibited by phloretin, indicating the presence of facilitated diffusion glucose transporters in those strains. Since DSMZ 10140 has been previously reported to lack a functional glucose phosphoenolpyruvate phosphotransferase system, the glucose transporter identified here is responsible for much of the organism's glucose uptake.     

Inhibition of glucose-induced insulin release by 3-O-methyl-D-glucose: Enzymatic,metabolic and cationic determinants

The analog of D-glucose, 3-O-methyl-D-glucose, is thought to delay the equilibration of D-glucose concentration across the plasma membrane of pancreatic islet B-cells, but not to exert any marked inhibitory action upon the late phase of glucose-stimulated insulin release. In this study, however, 3-O-methyl-D-glucose, when tested in high concentrations (30-80 mM) was found to cause a rapid, sustained and not rapidly reversible inhibition of glucose-induced insulin release in rat pancreatic islets. In relative terms, the inhibitory action of 3-O-methyl-D-glucose was more marked at low than high concentrations of D-glucose. It could not be attributed to hyperosmolarity and appeared specific for the insulinotropic action of D-glucose, as distinct from non-glucidic nutrient secretagogues. Although 3-O-methyl-D-glucose and D-glucose failed to exert any reciprocal effect upon the steady-state value for the net uptake of these monosaccharides by the islets, the glucose analog inhibited D-[5-3H]glucose utilization and D-[U-14C]glucose oxidation. This coincided with increased 86Rb outflow and decreased 45Ca outflow from prelabelled islets, as well as decreased 45Ca net uptake. A preferential effect of 3-O-methyl-D-glucose upon the first phase of glucose-stimulated insulin release was judged compatible with an altered initial rate of D-glucose entry into islet B-cells. The long-term inhibitory action of the glucose analog upon the metabolic and secretory response to D-glucose, however, may be due, in part at least, to an impaired rate of D-glucose phosphorylation. The phosphorylation of the hexose by beef heart hexokinase and human B-cell glucokinase, as well as by parotid and islet homogenates, was indeed inhibited by 3-O-methyl-D-glucose. The relationship between insulin release and D-glucose utilization or oxidation in the presence of 3-O-methyl-D-glucose was not different from that otherwise observed at increasing concentrations of either D-glucose or D-mannoheptulose. It is concluded, therefore, that 3-O-methyl-D-glucose adversely affects the metabolism and insulinotropic action of D-glucose by a mechanism largely unrelated to changes in the intracellular concentration of the latter hexose.     

D-Glucose uptake in fish hepatocytes: mediated by transporter in rainbow trout (Oncorhynchus mykiss), but only by diffusion in prespawning lamprey (Lampetra fluviatilis) and in RTH-149 cell line

The transport of D-glucose into rainbow trout (Oncorhynchus mykiss) and river lamprey (Lampetra fluviatilis) hepatocytes, as well as into rainbow trout hepatoblastoma cell line RTH-149 was studied using tracer methods. The half-time for D-glucose equilibration was 15 s for rainbow trout. The half-times for the non-metabolizable D-glucose analog, 3-O-methyl-D-glucose equilibration were 8 s, 37 s and 38 s for rainbow trout, lamprey and RTH-149 cells, respectively. The 3-O-methyl-D-glucose was taken up by rainbow trout hepatocytes by facilitated diffusion in addition to simple diffusion. The uptake showed saturation kinetics with the K(m) of 37 mM and V(max) of 62 mmol kg(-1) cells min(-1). The uptake was sensitive to phloretin and cytochalasin B, but not affected by ouabain. The 3-O-methyl-D-glucose uptake by lamprey hepatocytes and RTH-149 cells showed no indication of saturation up to 160 mM, and was not affected by phloretin, cytochalasin B or ouabain, which suggests the mode of transport to be by passive diffusion. However, immunocytochemical stainings revealed the existence of mammalian type GLUT1 and GLUT2 transporters in all cells studied. The lack of a functioning carrier-mediated glucose uptake in lamprey hepatocytes might be due to its physiological state (prespawning starvation). The minor 3-O-methyl-D-glucose uptake into RTH-149 cells compared to freshly isolated rainbow trout hepatocytes might reflect low metabolic activity of the cell lines. Under the conditions applied the RTH-149 cell line is no suitable in vitro model for glucose transport in fish cells.     

Glucose transport across plasma membrane in human platelets

Studies have been carried out in the presence of 2-deoxyglucose, by utilizing a technique of platelet rapid filtration. Kinetic data suggest that glucose uptake across plasma membrane is the rate limiting step in its utilization. 2-deoxyglucose is transported by facilitated diffusion. L-glucose is transferred at only 1/1200 of the rate of glucose. Transport system shows high affinity for substrate. Transport is inhibited by cytochalasin B, phloretin and N-ethylmaleimide. Cytochalasin E does not affect 2-deoxyglucose uptake. Diamide can have activating or inhibitory effect. t-Butyl hydroperoxide is always activating. Insulin has no effect on rate transport. D-glucose, 3-O-methylglucose, non radioactive 2-deoxyglucose and D-mannose are strong competitors, whereas D-galactose and D-fructose compete weakly with 2-deoxyglucose transport.     

Hexose metabolism in pancreatic islets. Insignificance of D-glucose futile cycling in rat islets.

When rat pancreatic islets were incubated in the presence of unlabelled D-glucose (16.7 mM) and 3HOH, the production of 3H-labelled material susceptible to be phosphorylated by yeast hexokinase and then detritiated by yeast phosphoglucoisomerase did not exceed 2.66 +/- 0.21 pmol/islet per 180 min, i.e. about 1% of the rate of exogenous D-[5-3H]glucose utilization. Such a material accounted for 43 +/- 4% of the total radioactivity, associated with tritiated hexose(s). It is proposed, therefore, that the futile cycling of D-glucose in the reactions catalyzed in the islet cells by the hexokinase isoenzymes and glucose-6-phosphatase represents a negligible fraction of the total rate of D-glucose phosphorylation.     

Menadione- and cyclic AMP-induced alteration of the ratio between D-[2-3H]glucose detritiation and phosphorylation in rat erythrocytes

When rat erythrocytes are exposed to both dibutyryl-cyclic AMP and isobutylmethylxanthine, the rates of D-glucose phosphorylation and glycolysis are both markedly increased. On the contrary, menadione by increasing the flow rate through the pentose phosphate pathway, decreases the generation rate of triose phosphates from exogenous D-glucose. Yet, both procedures lead to a decrease in the production of³HOH from D-[2-³H]glucose relative to its phosphorylation rate. It is proposed, therefore, that such a radioactive measurement should no more be considered as a reliable index of D-glucose phosphorylation, at variance with current practice.     

Effect of glucose uptake on growth rate of mouse 3T3 cells

The objective of this investigation was to determine whether the rate of glucose uptake by mouse 3T3 cells was a primary determinant of growth rate. The experimental approach was to control the rate of glucose uptake into intracellular pools by supplying this sugar at varying concentration in minimal Eagle's medium with dialyzed serum in the absence and presence of 6-deoxy-D-glucose, a metabolically inert homomorphic analog of D-glucose that competitively inhibits the uptake of D-glucose. Total hexose (D-glucose and 6-deoxy-D-glucose) concentration was maintained at the physiological concentration of 5.5 mM, in order to maintain saturation and maximum activity of the D-glucose transport system; thus the flux of D-glucose into the cell was controlled by adjusting its concentration relative to its competing nonmetabolizable analog. It was found that even when the concentration of D-glucose was reduced to 0.7 mM, one eighth of the “normal” level of 5.5 mM. and 6-deoxy-D-glucose was present in sevenfold excess (4.8 mM), conditions under which glucose uptake was reduced to 20% of that shown by cells in the presence of 5.5 mM D-glucose, and intracellular pools of glucose and phosphorylated sugars derived from glucose were reduced to approximately 14% of normal, there was not a significant decrease in growth rate. These data support the view that the rate of glucose uptake is not a primary determinant of growth rate under the usual conditions of cell culture.     

Anomeric specificity of D-glucose metabolism in rat adipocytes

The anomeric specificity of D-glucose metabolism was investigated in rat adipocytes exposed for 60 min at 8 degrees C to pure alpha- or beta-D-glucose or to equilibrated D-glucose. The rate of D-[5-3H]glucose utilization was higher with alpha- than beta-D-glucose. However, as judged from the oxidation of D-[1-14C]glucose and D-[6-14C]glucose anomers, the fraction of D-glucose catabolism occurring via the pentose cycle was higher with beta- than alpha-D-glucose. In the presence of equilibrated D-glucose, the utilization of alpha-D-[5-3H]glucose and the oxidation of both alpha-D-[1-14C]glucose and alpha-D-[6-14C]glucose were higher, relative to the anomer concentration, than the corresponding values for beta-D-glucose. It is concluded that the anomeric specificity of D-glucose metabolism is operative in adipocytes, even when they are exposed to equilibrated D-glucose.