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.     

Effect of age and day time on the adenosine modulation of basal and insulin-stimulated glucose transport in rat adipocytes

1. The relationship between the activity of adenosine metabolizing enzymes 5'nucleotidase (5'N), adenosine kinase (A.K.) and adenosine deaminase (A.D.) with basal and insulin-stimulated glucose transport in isolated fat cells from young and old animals was studied at 08:00 and 16:00 hr. 2. In cells from young animals a larger insulin-stimulation of glucose transport was observed at 16:00 hr than at 08:00 hr. Also at 16:00 hr small changes in 5'N, A.K. and A.D. activities suggest a decrease in adenosine formation. 3. In the cells from old animals no effect of insulin was observed at any time, while a 3-5-fold increase in 5'N indicated a predominance of adenosine formation at both times studied. 4. An inverse relationship was observed in the changes of adenosine metabolism and insulin action.     

Interactions of insulin, catecholamines and adenosine in the regulation of glucose transport in isolated rat cardiac myocytes

The regulation of the glucose transport system by catecholamines and insulin has been studied in isolated rat cardiomyocytes. In the basal state, 1-isoproterenol exhibited a biphasic concentration-dependent regulation of 3-O-methylglucose transport. At low concentrations (less than 10 nM), isoproterenol induced a maximal inhibition of 65-70% of the basal rates, while at higher concentrations (greater than 10 nM) a 25-70% stimulation of transport was observed. In the presence of adenosine deaminase, the inhibition of isoproterenol at low doses was attenuated. No effect of adenosine deaminase was observed on the stimulation of transport at high doses of isoproterenol. The inhibitory effect of isoproterenol returned when N6-phenylisopropyladenosine (a non-metabolizable analog of adenosine) was included along with adenosine deaminase. Dibutyryl cAMP and forskolin both inhibited basal transport rates. In the presence of maximally stimulating concentrations of insulin, cardiomyocyte 3-O-methylglucose transport was generally elevated 200-300% above basal levels. In the presence of isoproterenol, insulin stimulation was inhibited at both high and low concentrations of catecholamine, with maximum inhibition occurring at the lowest concentrations tested. When cells were incubated with both adenosine deaminase and isoproterenol, the inhibition of the insulin response was greater at all concentrations of catecholamine and was almost completely blocked at isoproterenol concentrations of 10 nM or less. Dibutyryl cAMP inhibited the insulin response to within 10% of basal transport levels, while forskolin completely inhibited all transport activity in the presence of insulin. These results suggest that catecholamines regulate basal and insulin-stimulated glucose transport via both cAMP-dependent and cAMP-independent mechanisms and that this regulation is modulated in the presence of extracellular adenosine.     

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.     

Effects of adenosine deaminase on the sensitivity of glucose transport, glycolysis and glycogen synthesis to insulin in muscles of the rat

1. Soleus, extensor digitorum longus (EDL) or hemi-diaphragm muscles of the rat were incubated in the presence of insulin and rates of the processes of glycolysis and glycogen synthesis were measured. 2. The concentrations of insulin required to cause half-maximal stimulation of glycolysis in both soleus and EDL preparations were significantly decreased by the presence of adenosine deaminase in the medium. 3. Adenosine deaminase increased the sensitivity of the process of hexose transport to insulin (in an identical manner to the change in sensitivity of glycolysis) in the EDL preparation. 4. None of the adenosine mediated effects on insulin-stimulated rates of glycolysis were observed in the hemi-diaphragm preparation or on the rates of glycogen synthesis in any of the three muscle preparations. 5. Therefore, changes in the adenosine system in skeletal muscle influence insulin sensitivity regardless of fibre type composition of the muscle.     

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.     

In vivo and in vitro effect of p-chlorophenoxyisobutyrate on insulin binding and glucose transport in isolated rat adipocytes

We studied the in vivo and in vitro effect of p-chlorophenoxyisobutyrate (CPIB) on insulin binding and glucose transport in isolated rat adipocytes. In the in vitro study, adipocytes were incubated with 1mM of CPIB for 2 h at 37 degrees C, pH 7.4, and then insulin binding (37 degrees C, 60 min) and 3-0-methylglucose transport (37 degrees C, 2s) were measured. Incubation with CPIB did not affect either insulin binding or glucose transport in the cells. The addition of insulin (10 ng/ml) with CPIB to the incubation media also did not affect the following insulin binding and glucose transport. In the in vivo study, rats were fed a high sucrose-diet containing 0.25% CPIB for 7 days. Serum cholesterol, plasma free fatty acid, and insulin levels were significantly decreased in the CPIB-treated rats. The treated rats demonstrated an almost 2 fold increased maximal binding capacity for insulin (189,000 sites/cell for treated vs 123,000 sites/cell for control cells). Basal glucose transport (glucose transport in the absence of insulin) significantly decreased in the CPIB-treated rats, although insulin-stimulated glucose transport was comparable in treated and control cells. Thus, CPIB might have no direct effect on glucose transport and insulin binding, as determined by the in vitro studies. Furthermore, a relatively short-term in vivo treatment with CPIB, such as 7 days, did not stimulate glucose transport.     

Effects of prostaglandin E2, indomethacin and adenosine deaminase on basal and insulin-stimulated glucose metabolism in human adipocytes

The effects of prostaglandin E2 were studied on glucose metabolism (3-O-methylglucose transport, CO2 production and lipogenesis) in human adipocytes. Initially, the effects of endogenously produced adenosine and prostaglandins were indirectly demonstrated by using adenosine deaminase and indomethacin in the incubations. From these studies it was found that adenosine deaminase (5 micrograms/ml) had a pronounced effect on adipocyte glucose metabolism in vitro. In the basal (nonhormonal-stimulated) state, glucose transport, CO2 production and lipogenesis were inhibited by about 30% (P less than 0.05). Furthermore, adenosine deaminase significantly inhibited the isoproterenol- and insulin-stimulated CO2 production and lipogenesis (P less than 0.01). Indomethacin (50 microM) had a consistently inhibitory effect on the insulin-stimulated CO2 production (P less than 0.05), whereas indomethacin had no significant effects on basal or isoproterenol-stimulated glucose metabolism. In contrast to the relatively minor effect of endogenous prostaglandins, the addition of exogenous prostaglandin E2 significantly stimulated the glucose transport, glucose oxidation and lipogenesis in human adipocytes, especially in the presence of adenosine deaminase. Half-maximal stimulation was obtained at prostaglandin E2 concentrations of 2.2, 0.8 and 0.8 nM, respectively. The effect of prostaglandin E2 was specific, since the structurally related prostaglandin, prostaglandin F2 alpha, had practically no effect on glucose metabolism. The maximal effect of prostaglandin E2 (1 microM) on glucose metabolism was 30-35% of the maximal insulin (1 nM) effect. When insulin and prostaglandin E2 were added together, the effect of prostaglandin E2 on glucose metabolism was additive at all insulin concentrations tested.     

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.     

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.     

PKB inhibition prevents the stimulatory effect of insulin on glucose transport and protein translocation but not the antilipolytic effect in rat adipocytes

We identified 1-(5 chloronaphthalenesulfonyl)-1H-hexahydro-1, 4-diazepine, also known as ML-9, as a powerful inhibitor of PKB activity in different cells as well as of recombinant PKB. It also inhibits other downstream serine/threonine kinases, such as PKA and p90 S6 kinase, but not upstream tyrosine phosphorylation or PI3-kinase activation in response to insulin. We compared the effects of ML-9 and wortmannin on several insulin-stimulated effects in isolated rat fat cells. Both ML-9 and wortmannin inhibited glucose transport and GLUT4/IGF II receptor translocation to the plasma membrane. In contrast, only wortmannin inhibited the antilipolytic effect and PDE3B activation by insulin. Thus, ML-9 inhibits PKB but not PI3-kinase activation in response to insulin and is useful to differentiate between these effects. Both PI3-kinase and PKB are important for glucose transport and intracellular protein translocation while PKB does not appear to play an important role for the antilipolytic effect or activation of PDE3B in response to insulin.     

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.     

Glucagon inhibition of insulin-stimulated 2-deoxyglucose uptake by rat adipocytes in the presence of adenosine deaminase.

Effects of adenosine deaminase and glucagon on insulin-stimulated 2-deoxyglucose uptake by rat adipocytes are reported. (1) Adenosine deaminase (10 micrograms/ml) caused a rightward shift in the dose-response curve for the stimulation by insulin of 2-deoxyglucose uptake, but the enzyme did not alter either the basal or the maximally insulin-stimulated uptake rate. (2) In adipocytes obtained from 24 h-starved rats, glucagon inhibited the effect of insulin on 2-deoxyglucose uptake in the presence (but not in the absence) of adenosine deaminase. Basal uptake rates were unaffected. (3) Glucagon inhibited insulin-stimulated 2-deoxyglucose uptake to a greater extent in cells isolated from starved rats than in cells from fed rats. (4) Adipocytes isolated from fed and from starved rats did not differ in their capacity for degradation of 125I-labelled glucagon. The results suggest that adenosine and glucagon are regulators of insulin action in adipose tissue.     

Effects of islet-activating protein on insulin- and isoprenaline-stimulated glucose transport in isolated rat adipocytes

The effects of islet-activating protein (IAP), a Bordetella pertussis toxin, on insulin- and isoprenaline-stimulated glucose transport were studied in isolated rat adipocytes. Basal as well as insulin-stimulated glucose transport were not affected when cells were pretreated with IAP. In contrast, IAP pretreatment abolished the stimulatory effect of isoprenaline. When IAP-pretreated cells were exposed to a combination of insulin and isoprenaline, the catecholamine significantly reduced the stimulatory effect of insulin. Since IAP is supposed to specifically block the inhibitory component Ni of adenylate cyclase, the results suggest that: (a) the effect of insulin is unrelated to the regulation of adenylate cyclase; (b) isoprenaline may exert both stimulatory and inhibitory effects depending on activation of Ni. The inhibitory regulation of adenylate cyclase may thus be a pivotal link in the regulation of glucose transport.     

The effect of gonadotropin on glucose transport and apoptosis in rat ovary

Although the effects of Gonadotropin on ovarian physiology have been known for many decades, its action on glucose uptake in the rat ovary remained poorly understood. Evidence also suggests that glucose uptake is mediated by a number of glucose transporter proteins (Glut). Therefore, we examined the rat ovary for the presence of Glut1-4 and blood glucose level after eCG (equine chorionic gonadotropin) and anti-eCG antiserum treatment. All of the glucose transports were present in the ovarian oocyte, granulosa cells and theca cells in different stage follicles. The expression of Glut in ovary was up-regulated by eCG, however, anti-eCG antiserum reversed eCG action. Western blot analysis also demonstrated the content of Glut1 was higher in eCG treatment group compared with anti-eCG antiserum and control group. The same tendency was shown in other glut isoforms. Moreover, there were no significant difference between the anti-eCG antiserum and control group. In additional, the level of serum glucose in eCG treatment group was significantly higher than others, which is similar with glut expression pattern. High glucose level in blood is correlated with increased expression of glucose transporter proteins in rat ovary. Meanwhile, anti-eCG antiserum increased granulosa cell apoptosis in antral follicle compared with those in eCG group. Our observations provide potential explanation for the effects of Glut on follicular development in rat ovary and a role for eCG in the regulation of ovarian glucose uptake.     

Counter-regulation of insulin-stimulated glucose transport by catecholamines in the isolated rat adipose cell

The interaction between catecholamines and insulin in regulating glucose transport in isolated rat adipose cells has been evaluated. In the absence of insulin, 1 microM isoproterenol stimulates 3-O-methylglucose transport approximately 2-fold. However, isoproterenol in combination with adenosine deaminase inhibits glucose transport activity approximately 60%. N6-Phenylisopropyladenosine, a nonmetabolizable adenosine analogue, substantially reverses this inhibitory effect and actually stimulates glucose transport activity approximately 2-fold in the absence of isoproterenol. Dibutyryl cAMP inhibits glucose transport activity approximately 75% regardless of adenosine deaminase. While none of these agents significantly influences the basal concentration of plasma membrane glucose transporters, as assessed by specific D-glucose-inhibitable cytochalasin B binding, isoproterenol or dibutyryl cAMP in combination with adenosine deaminase reduces that in the low density microsomes 19 and 58%, respectively. In the presence of insulin, both isoproterenol and adenosine deaminase alone inhibit glucose transport activity approximately 25%. However, only the latter is accompanied by a corresponding decrease in the insulin-stimulated concentration of plasma membrane glucose transporters. Together, isoproterenol and adenosine deaminase inhibit insulin-stimulated glucose transport activity approximately 75%, even in the presence of 5 mM glucose to maintain cellular ATP levels. A similar inhibition is observed with dibutyryl cAMP. However, these agents decrease the insulin-stimulated concentration of plasma membrane glucose transporters only approximately 45%. Nevertheless, all of these inhibitory effects occur through decreases in the transport Vmax. In addition, N6-phenylisopropyladenosine partially reverses the inhibitory effects induced by the presence of adenosine deaminase. These results suggest that catecholamines counter-regulate basal and insulin-stimulated glucose transport in rat adipose cells through a cAMP-mediated mechanism, but only in part by modulating the translocation of glucose transporters.     

The effect of experimental diabetes on the circadian pattern of adenosine deaminase and myeloperoxidase activities in rat liver

This study investigated time-dependent variations in the activities of adenosine deaminase (ADA), an adenosine-metabolizing enzyme, and myeloperoxidase (MPO), an oxidation reaction-catalyzing enzyme, in control and streptozotocin (STZ)-induced diabetic rat liver. The animals were sacrificed at six different times of day (1, 5, 9, 13, 17 and 21 hours after lights on - HALO). The hepatic activity of ADA did not change depending on the STZ treatment whereas MPO activity was significantly higher in the diabetics than in the controls. Hepatic ADA activity was dependent on the time of sacrifice with the lowest activity at 21 HALO and the highest activity at 5 HALO. Both enzyme activities failed to show any significant interaction between STZ treatment and time of sacrifice, which means that diabetes does not influence the 24 h pattern of these activities. Since MPO, a heme protein localized in the leukocytes, is involved in the killing of microorganisms, increased MPO activity in diabetic rat liver may reflect leukocyte infiltration secondary to diabetes. A reduction in ADA activity during the dark (activity/feeding) period will presumably lead to high concentrations of adenosine in the liver, possibly contributing to changes in some metabolic processes, such as glycogen turnover and oxygen supply.