Evidence for glucose-6-phosphate transport in rat liver microsomes

The existence of glucose-6-phosphate transport across the liver microsomal membrane is still controversial. In this paper, we show that S3483, a chlorogenic acid derivative known to inhibit glucose-6-phosphatase in intact microsomes, caused the intravesicular accumulation of glucose-6-phosphate when the latter was produced by glucose-6-phosphatase from glucose and carbamoyl-phosphate. S3483 also inhibited the conversion of glucose-6-phosphate to 6-phosphogluconate occurring inside microsomes in the presence of electron acceptors (NADP or metyrapone). These data indicate that liver microsomal membranes contain a reversible glucose-6-phosphate transporter, which furnishes substrate not only to glucose-6-phosphatase, but also to hexose-6-phosphate dehydrogenase.     

Spatial requirements for insulin-sensitive sugar transport in rat adipocytes

(1) Alkyl sugar inhibition of d-allose uptake into adipocytes has been used to explore the spatial requirements of the external sugar transport site in insulin-treated cells. α-methyl and β-methyl glucosides show low affinity indicating very little space around C-1. The high affinity of d-glucosamine (K_i = 9.05 ± 0.66 mM) is lost by N-acetylation. $\text{N-}\text{Acetyl-}\text{d}\text{-glucosamine}$ shows no detectable affinity, indicating that a bulky group at C-2 is not accepted. Similarly $\text{2,3-}\text{di}\text{-O-}\text{methyl-}\text{d}\text{-glucose}$ (K_i = 42.1 ± 7.5 mM) has lower affinity than $\text{3-O-}\text{methyl-}\text{d}\text{-glucose}$ (K_i = 5.14 ± 0.32 mM) indicating very little space around C-2 but much more around C-3. A reduction in affinity does occur if a propyl group is introduced into the C-3 position. The K_i for $\text{3-O-}\text{propyl-}\text{d}\text{-glucose}$ is 11.26 ± 2.12 mM. $\text{6-O-}\text{Methyl-}\text{d}\text{-galactose}$ (K_i = 87.2 ± 17.9 mM) and $\text{6-O-}\text{propyl-}\text{d}\text{-glucose}$ (K_i = 78.07 ± 12.6 mM) show low affinity compared with d-galactose and d-glucose, indicating steric constraints around C-6. High affinity is restored in $\text{6-O-}\text{pentyl-}\text{d}\text{-galactose}$ (K_i = 4.66 ± 0.23 mM) possibly indicating a hydrophobic binding site around C-6). (2) In insulin treated cells $\text{4,6-O-}\text{ethylidene-}\text{d}\text{-glucose}$ (K_i = 6.11 ± 0.5 mM) and maltose (K_i = 23.5 ± 2.1 mM) are well accommodated by the site but trehalose shows no detectable inhibition. These results indicate that the site requires a specific orientation of the sugar as it approaches the transporter from the external solution. C-1 faces the inside while C-4 faces the external solution. (3) To determine the spatial and hydrogen bonding requirements for basal cells 40 μM $\text{3-O-}\text{methyl-}\text{d}\text{-glucose}$ was used as the substrate. Poor hydrogen bonding analogues and analogues with sterically hindering alkyl groups showed similar K_i values to those determined for insulin-treated cells. These results indicate that insulin does not change the specificity of the adipocyte transport system.     

Effect of puromycin on sugar transport in isolated rat adipocytes

This study was performed to determine whether puromycin effects sugar transport into isolated rat adipocytes. Time-course and kinetic studies demonstrated that puromycin competitively inhibited 3-O-methylglucose transport. The data indicate that there is an acute effect of puromycin on sugar transport via competitive inhibition that is independent of the inhibition of protein synthesis.     

Stimulation of glucose transport by guanine nucleotides in permeabilized rat adipocytes.

Effects of guanine nucleotides on glucose transport were studied in permeabilized rat epididymal fat cells. GTP gamma S and Gpp(NH)p, but not App(NH)p, stimulated 3-O-methylglucose transport. Effect of GTP gamma S was dose-dependent, being detectable at 0.1 mM, and 1.0 mM GTP gamma S stimulated glucose transport to the same extent as insulin. GTP gamma S (0.3 mM) enhanced insulin-stimulated glucose transport while 1 mM GTP gamma S did not affect insulin-mediated transport. GDP beta S had no effect on glucose transport by itself but rather enhanced insulin action. NaF, which is known to activate trimeric G proteins, increased glucose transport to the same extent as insulin. Likewise, mastoparan augmented glucose transport. These results indicate that a certain type of trimeric G protein(s) is involved in the regulation of glucose transport.     

Inhibition of insulin-stimulated glucose transport in rat adipocytes by nucleoside transport inhibitors

In isolated rat adipocytes, basal as well as insulin-stimulated 3-O-methylglucose transport was inhibited nearly completely (maximal inhibition: 95%) by the nucleoside transport inhibitors dipyridamole (IC50 = 5 microM), nitrobenzylthioguanosine (20 microM), nitrobenzylthioinosine (35 microM) and papaverine (130 microM). Transport kinetics in the presence of 10 microM dipyridamole revealed a significant increase in the transport Km value of 3-O-methylglucose (3.45 +/- 0.6 vs 2.36 +/- 0.29 mM in the controls) as well as a decrease in the Vmax value (4.84 +/- 0.95 vs 9.03 +/- 1.19 pmol/s per microliter lipid in the controls). Half-maximally inhibiting concentrations of dipyridamole were one order of magnitude higher than those inhibiting nucleoside (thymidine) uptake (0.48 microM). The inhibitory effect of dipyridamole (5 microM) reached its maximum within 30 s. The agent failed to affect insulin's half-maximally stimulating concentration (0.075 nM) indicating that it did not interfere with the mechanism by which insulin stimulates glucose transport. Further, dipyridamole fully suppressed the glucose-inhibitable cytochalasin B binding (IC50 = 1.65 +/- 0.05 microM). The data indicate that nucleoside transport inhibitors reduce glucose transport by a direct interaction with the transporter or a closely related protein. It is suggested that glucose and nucleoside transporters share structural, and possibly functional, features.     

Glucose oxidation,glucose transport and insulin binding in isolated rat epididymal adipocytes in the presence of anesthetics

Insulin binding and glucose oxidation were measured in isolated rat adipocytes in the presence of several anesthetics; ethanol, n-octanol, pentobarbital, chlorpromazine and tetracaine. Ethanol and chlorpromazine, at anesthetic and pentobarbitol at sub-anesthetic concentrations are inhibitory to both basal and insulin stimulated rates of glucose oxidation. At all concentrations of ethanol, pentobarbital or chlorpromazine tested binding of insulin is not affected. Since anesthetics may alter membrane fluidity, it is suggested that an anesthetic-induced increase in membrane fluidity beyond that which occurs at 37°C is detrimental to glucose oxidation. Of the 5 anesthetics examined, only chlorpromazine (10 μM or less) and tetracaine (500 μM) stimulate glucose oxidation. These two agents are known to bind to a cell's cytoskeletal system; the binding of chlorpromazine to microtubules is entropy driven. The temperature and concentration dependence of chlorpromazine stimulation of glucose oxidation (transport) are consistent with this form of binding. It is proposed that chlorpromazine binds to the cytoskeletal system of the adipocyte and that this system is normally restrictive to the motion of membrane proteins. Disruption of the cytoskeletal system by chlorpromazine or tetracaine would increase the frequency of insulin-receptor and glucose-carrier contact. Activation of glucose transport could ensue.     

Stimulation of glucose transport and glucose transporter phosphorylation by okadaic acid in rat adipocytes

Okadaic acid, an inhibitor of Type I and IIa protein phosphatases, was recently found to stimulate 2-deoxyglucose uptake in rat adipocytes (Haystead, T. A. J., Sim, A. T. R., Carling, D., Honnor, R. C., Tsukitani, Y., Cohen, P., and Hardie, D. G. (1989) Nature 337, 78-81). In the present experiments the effect of okadaic acid on the phosphorylation and subcellular distribution of the insulin-regulatable glucose transporter (IRGT) was investigated. At maximally effective concentrations, insulin and okadaic acid increased the amount of IRGT in the plasma membrane by 10- and 4-fold, respectively. Thus, the stimulation of glucose transport by okadaic acid was apparently due to an increase in the surface concentration of the IRGT. However, despite its stimulatory actions, okadaic acid partially inhibited the ability of insulin to enhance glucose transport and translocation of the transporter. When cells were incubated with okadaic acid alone or in combination with insulin, phosphorylation of the IRGT in the plasma membrane was increased by approximately 3-fold relative to the intracellular pool of transporters in control cells. Phosphorylation of the IRGT was confined to the presumed cytoplasmic domain at the COOH terminus of the protein. Glucose transporters were dephosphorylated in vitro by Type I or Type IIa protein phosphatases, indicating that inhibition of one or both of these phosphatases could account for the increased phosphorylation produced by okadaic acid. The observation that okadaic acid stimulated translocation of the IRGT implicated a serine/threonine phosphorylation event in triggering movement of the intracellular IRGT-containing vesicles (GTV) to the cell surface. Immunoadsorption of GTV from 32P-labeled adipocytes revealed that the IRGT was the major phosphoprotein in these vesicles. The phosphorylation of at least three other GTV proteins was increased by okadaic acid, and these species would appear to be candidates for regulators of GTV movement to the plasma membrane. It is unlikely that phosphorylation of the IRGT is the signal for translocation because insulin did not increase phosphorylation of the protein. Rather, the inhibitory effect of okadaic acid on insulin-stimulated translocation is consistent with the hypothesis that phosphorylation of the IRGT promotes its internalization.     

Catecholamine-induced insulin resistance of glucose transport in isolated rat adipocytes.

The effects of pre-incubation with isoprenaline and noradrenaline on insulin binding and insulin stimulation of D-glucose transport in isolated rat adipocytes are reported. (1) Pre-incubation of the cells with isoprenaline (0.1-10 microM) in Krebs-Ringer-Hepes [4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid] buffer (30 min, 37 degrees C) at D-glucose concentrations of 16 mM, in which normal ATP levels were maintained, caused a rightward-shift in sensitivity of D-glucose transport to insulin stimulation by 50% and a decrease in maximal responsiveness by 30% (2) [A14-125I]insulin binding was reduced significantly by 35% at insulin concentrations less than 100 mu-units/ml and Scatchard analysis showed that this consisted mainly of a decrease in high-affinity binding. (3) Pre-incubation with catecholamines under the same conditions but at low glucose concentrations (0-5 mM) caused a fall in intracellular ATP levels of 65 and 45% respectively. (4) The fall in ATP additionally lowered insulin binding by 50% at all insulin concentrations and a parallel shift of the binding curves in the Scatchard plot showed that this was due to a decrease in the number of receptors. (5) At low and high ATP concentrations the insulin stimulation of D-glucose transport was inhibited to a similar extent. (6) Pre-incubation with catecholamines thus inhibited insulin stimulation of D-glucose transport in rat adipocytes mainly by a decrease in high-affinity binding of insulin, which was not mediated by low ATP levels. This mechanism may play a role in the pathogenesis of catecholamine-induced insulin resistance in vivo.     

Insulin-stimulated glucose transport regulated by adenylate cyclase system in rat adipocytes

1. In rat adipocytes, there was an inverse correlation between insulin-stimulated 2-deoxyglucose (2-DG) uptake and cAMP levels, indicating that cAMP suppressed the 2-DG uptake stimulated by insulin. 2. This inhibitory effect of cAMP was due to suppression of translocation of glucose transporters rather than that of insulin-binding to its receptors. 3. Phosphodiesterase inhibitors (IBMX, Ro 20-1724, and cilostamide) inhibited the 2-DG uptake, which was brought about by direct interaction with glucose transporters in the plasma membranes.     

The mechanism of glucose 6-phosphate transport by Escherichia coli

To evaluate anion exchange as the mechanistic basis of sugar phosphate transport, natural and artificial membranes were used in studies of glucose 6-phosphate (Glc-6-P) and inorganic phosphate (Pi) accumulation by the uhpT-encoded protein (UhpT) of Escherichia coli. Experiments with intact cells demonstrated that UhpT catalyzed the neutral exchange of internal and external Pi, and work with everted as well as right-side-out membrane vesicles showed further that UhpT mediated the heterologous exchange of Pi and Glc-6-P. When loaded with Pi, but not when loaded with morpholinopropanesulfonate (MOPS), everted vesicles took up Glc-6-P to levels 100-fold above medium concentration in a reaction unaffected by the ionophores valinomycin, valinomycin plus nigericin, and carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Similarly, right-side-out vesicles were capable of Glc-6-P transport, but only if a suitable internal countersubstrate was available. Thus, in MOPS-loaded vesicles, oxidative metabolism established a proton-motive force that supported proline or Pi accumulation, but transport of Glc-6-P was found only if vesicles could accumulate Pi during a preincubation. After reconstitution of UhpT into proteoliposomes it was possible to show as well that the level of accumulation of Glc-6-P (17 to 560 nmol/mg of protein) was related directly to the internal concentration of Pi. These results are most easily understood if the transport of glucose 6-phosphate in E. coli occurs by anion exchange rather than by nH+/anion support.     

Comparison of glucose and fructose transport into adipocytes of the rat.

The purpose of these studies was to define the properties of the systems that transport hexoses into adipocytes. Glucose appears to enter adipocytes on a single transport system whose maximum velocity is stimulated by insulin and which is competitively inhibited by cytochalasin B, 5-thioglucose, fructose, mannose and 3-O-methylglucose. In contrast, fructose enters adipocytes by at least two separate mechanisms, one an insulin-sensitive transporter (probably the glucose transporter) and the other a mechanism that is insensitive to insulin. The fructose concentration required for half-maximal rates of transport is at least an order of magnitude higher than that for glucose and the maximum velocity of fructose transport is more than double that for glucose.     

Evidence for surface glycoprotein involvement in the intracellular bioactivity of insulin in rat adipocytes.

Lectins specific for D-mannose (concanavalin A), N-acetyl-D-glucosamine (wheat-germ agglutinin) or D-galactose (Ricinus communis agglutinin I) inhibited insulin binding and activated glucose transport in rat adipocytes [Cherqui, Caron, Capeau & Picard (1982) Mol. Cell. Endocrinol. 28, 627-643]. In the present investigation, the intracellular activities of insulin and lectins on lipogenesis and protein synthesis were studied under conditions where neither agent had an effect on membrane transport processes. (1) When glucose transport was rate-limiting (0.5 mM-glucose), insulin (0.8 ng/ml) and lectins (20 micrograms/ml) increased lipogenesis by 2.4-3-fold. (2) When passive diffusion of glucose was amplified (10 mM-glucose), insulin (0.8 ng/ml) and lectins (20 micrograms/ml) increased lipogenesis by 1.6-1.8-fold even in the presence of 50 microM-cytochalasin B, which completely blocked glucose transport. (3) Insulin (6 ng/ml), concanavalin A and wheat-germ agglutinin (40 micrograms/ml) stimulated the incorporation of L-[U-14C]leucine into fat-cell protein 1.5-fold but did not modify alpha-aminoisobutyric acid uptake or 14C-labelled protein degradation. (4) Peanut and soya-bean agglutinins (specific for O-glycosidically-linked oligosaccharides), known not to alter insulin binding, were ineffective. (5) Lectin effects were dose-dependent and were markedly inhibited by specific monosaccharides (50 mM). (6) Insulin and lectin maximal effects were not additive and were completely abolished by neuraminidase treatment of fat-cells (0.05 unit/ml). These data indicate involvement of surface sialylated glycoproteins of the complex N-linked type in the insulin stimulation of glucose and amino acid intracellular metabolic processes. They suggest, together with our previous results, that the transmission of the insulin signal for both membrane and intracellular effects occurs via glycosylated effector entities of, or closely linked to, the insulin-receptor complex.     

Dehydrogenation of the phosphonate analogue of glucose 6-phosphate by glucose 6-phosphate dehydrogenase.

6,7 -Dideoxy-alpha-D-gluco-heptose 7-phosphonic acid, the isosteric phosphonate analogue of glucose 6-phosphate, was synthesized in six steps from the readily available precursor benzyl 4,6-O-benzylidene-alpha-D-glucopyranoside. The analogue is a substrate for yeast glucose 6-phosphate dehydrogenase, showing Michaelis-Menten kinetics at pH7.5 and 8.0. At both pH values the Km values of the analogue are 4-5 fold higher and the values approx. 50% lower than those of the natural substrate. The product of enzymic dehydrogenation of the phosphonate analogue at pH8.5 is itself a substrate for gluconate 6-phosphate dehydrogenase.     

Hydrogen bonding requirements for the insulin-sensitive sugar transport system of rat adipocytes

(1) The $\text{t}\text{1}\text{2}$ for 1.3 mM D-allose uptake and efflux in insulin-stimulated adipocytes is 1.7 ± 0.1 min. In the absence of insulin mediated uptake of D-allose is virtually eliminated and the uptake rate ($\text{t}\text{1}\text{2}\text{= 75.8 ± 4.99}\text{min}$) is near that calculated for nonmediated transport. The kinetic parameters for D-allose zero-trans uptake in insulin-treated cells are K_zt^oi = 271.3 ± 34.2 mM, V_zt^oi = 1.15 ± 0.12 mM · s^?1. (2) A kinetic analysis of the single-gate transporter (carrier) model interacting with two substrates (or substrate plus inhibitor) is presented. The analysis shows that the heteroexchange rates for two substrates interacting with the transporter are not unique and can be calculated from the kinetic parameters for each sugar acting alone with the transporter. This means that the equations for substrate analogue inhibition of the transport of a low affinity substrate such as D-allose can be simplified. It is shown that for the single gate transporter the K_i for a substrate analogue inhibitor should equal the equilibrium exchange K_m for this analogue. (3) Analogues substituted at C-1 show a fused pyranose ring is accepted by the transporter. 1-Deoxy-D-glucose is transported but has low affinity for the transporter. High affinity can be restored by replacing a fluorine in the β-position at C-1. The K_i for $\text{d}\text{-glucose}\text{= 8.62}\text{mM}$; the K_i for $\text{β-}\text{fluoro-}\text{d}\text{-glucose}\text{= 6.87}\text{mM}$. Replacing the ring oxygen also results in a marked reduction in affinity. The K_i for $\text{5-}\text{thio-}\text{d}\text{-glucose}\text{= 42.1}\text{mM}$. (4) A hydroxyl in the gluco configuration at C-2 is not required as 2-deoxy-d-galactose (K_i = 20.75 mM) has a slightly higher affinity than d-galactose (K_i = 24.49 mM). A hydroxyl in the manno configuration at C-2 interferes with transport as d-talose (K_i = 35.4 mM) has a lower affinity than d-galactose. (5) d-Allose (K_m = 271.3 mM) and 3-deoxy-d-glucose (K_i = 40.31 mM) have low affinity but high affinity is restored by substituting a fluorine in the gluco configuration at C-3. The K_i for $\text{3-}\text{fluoro-}\text{d}\text{-glucose}\text{= 7.97}\text{mM}$. (6) Analogues modified at C-4 and C-6 do not show large losses in affinity. However, 6-deoxy-d-glucose (K_i = 11.08 mM) has lower affinity than d-glucose and 6-deoxy-d-galactose K_i = 33.97 mM) has lower affinity than d-galactose. Fluorine substitution at C-6 of d-galactose restores high affinity. The K_i for $\text{6-}\text{fluoro-}\text{d}\text{-galactose}\text{= 6.67}\text{mM}$. Removal of the C-5 hydroxymethyl group results in a large affinity loss. The K_i$\text{d}\text{-xylose}\text{= 45.5}\text{mM}$. The K_i for $\text{l}\text{-arabinose}\text{= 49.69}\text{mM}$. (7) These results indicate that the important hydrogen bonding positions involved in sugar interaction with the insulin-stimulated adipocytes transporter are the ring oxygen, C-1 and C-3. There may be a weaker hydrogen bond to C-6. Sugar hydroxyls in non-gluco configurations may sterically hinder transport.     

Regulation of glucose transport in isolated adipocytes by levamisole.

When isolated rat adipocytes were incubated with increasing concentrations of levamisole (0.5-5 mM), basal glucose oxidation decreased by almost 50% and insulin-stimulated glucose oxidation decreased by 90%. The decrease in glucose oxidation correlated with an inhibition of glucose transport, since levamisole at 5.0 mM decreased basal 3-O-methylglucose transport by 60% and insulin-stimulated transport by 80%. Diamide-stimulated glucose transport was also inhibited approximately 80% by 5.0 mM levamisole. Levamisole at concentrations up to 5.0 mM had no effect on phosphofructokinase activity. The present results suggest that levamisole inhibits glucose utilization by inhibiting glucose transport in a concentration-dependent manner.