Mechanism of insulin resistance in the post receptor events in sheep: 3-O-methylglucose transport in ovine adipocytes

A severe resistance to the stimulatory action of insulin on glucose metabolism has been shown in ruminant adipose tissue or isolated adipocytes as compared to that of rats. To elucidate the mechanism of insulin resistance in ruminants, we measured the stimulatory effect of insulin on 3-O-methylgulose transport and on intracellular glucose metabolism in isolated adipocytes from sheep and rats. At a glucose concentration (0.1 mM) where transport is thought to be rate-limiting for metabolism, lipogenesis from [U-14C]glucose by ovine adipocytes was markedly less than by rat adipocytes in both the basal state and at all insulin concentrations. The responsiveness to insulin assessed by percent increase above basal was reduced to about 15% of that in rat adipocytes, but the insulin sensitivity was similar, because the insulin concentration giving half-maximal stimulation, ED50, did not differ significantly between ovine and rat adipocytes. The maximal insulin-stimulated 3-O-methylglucose transport in ovine adipocytes per cell was less than 20% of that in rat adipocytes, with a significant lowering in basal rates of transport. However, when data was expressed per 3-O-methylglucose equilibrium space no significant differences were found between ovine and rat in the basal transport rates, but a lowered ability of insulin to stimulate glucose transport was still seen in ovine adipocytes. The dose-response curve for glucose transport was slightly shifted to the right in ovine adipocytes compared to rat adipocytes, indicating a small decrease in insulin sensitivity. The decrease in glucose transport was due to 60% reduction in the maximum velocity in the insulin--stimulated state, with no change in the Km.     

The inhibition of hexose transport by permeant and impermeant sulfhydryl agents in rat adipocytes

The importance of exofacial sulfhydryl groups for hexose transport and its regulation was studied by comparing the effects of plasma membrane-permeant maleimide (N-ethylmaleimide) to an impermeant maleimide (glutathione-maleimide I) on 3-O-methylglucose transport into isolated rat adipocytes. The impermeant nature of glutathione-maleimide was confirmed by the finding that after a 15-min incubation, concentrations as high as 10 mM had no effect on intracellular glutathione content, while 1.7 mM N-ethylmaleimide decreased intracellular glutathione by 61%. Although N-ethylmaleimide appeared to be a more potent inhibitor of transport below 5 mM and at incubation times of less than 5 min, neither agent at concentrations which did not cause significant cell breakage inhibited basal transport rates more than 60-70%. The inhibition of transport by both agents was unaffected by extensive washing, suggesting a possible covalent interaction with the carrier. Preincubation with p-chloromercuribenzenesulfonic acid protected against the transport inhibition induced by both agents. However, only the transport inhibition induced by glutathione-maleimide was prevented by preincubation with D-glucose (50 mM) and maltose (50 mM). Transport in cells pretreated with insulin was inhibited by both agents to a similar extent as basal transport. However, treatment of cells with the maleimides before insulin caused a greater degree of inhibition. Thus, the insulin-induced increase in transport was inhibited half-maximally by 1 mM glutathione-maleimide. These results show that exofacial sulfhydryl groups, perhaps on the hexose-binding site of the carrier, are important for both the function and regulation of hexose transport.     

Exchange of 3-O-methylglucose in isolated fat cells. Concentration dependence and effect of insulin.

3-O-[14C]Methylglucose was used to study the insulin action on the sugar transport in white fat cells. The experiments comprised determinations of the 3-O-methylglucose space at stationary distribution, of the rate constants for 3-O-methylglucose equilibrium exchange under various conditions, and of the 3-O-methylglucose inhibition of the lipogenesis from glucose. The following was found. The intracellular distribution space for 3-O-methylglucose at equilibrium was unaffected by insulin and was identical with the intracellular 3H2O space. The half-time for the equilibrium exchange of 3-O-methylglucose at a concentration of 25 mM was about 240 s in the absence of insulin and about 15 s with insulin (0.7 muM) present. Addition of phloridzin (5 mM) decreased the rate of the exchange process about 25-fold in both cases. The self-exchange of 3-O-methylglucose (1 mM) was at least 50 times faster than the self-exchange of L-glucose (1 mM). The concentration dependence of the 3-O-methylglucose exchange rate was approximately hyperbolic both in the absence and the presence of insulin, although the saturation of the transport mechanism at high concentrations of sugar was not as complete as predicted. In the absence of insulin the estimate of the half-saturation constant (Kt) was about 5 mM; that of the maximal exchange rate (Vmax) varied from 0.07 mmol s-1/liter of intracellular water to 0.2 mmol s-1 liter-1. In the presence of insulin Kt remained about 5 mM, whereas Vmax was increased to about 1.7 mmol s-1 liter-1. The latter estimate was reproducible within about 20%. The incorporation of trace amounts of [U-14C]glucose into intracellular lipids was inhibited by unlabeled 3-O-methylglucose pre-equilibrated over the membrane. The inhibition constant estimated from such experiments was about 5 mM both in the absence and the presence of insulin, and the insulin-induced increase in the rate of glucose incorporation was similar to the increase in the rate of the 3-O-methylglucose exchange process. It is concluded that exchange of 3-O-methylglucose proceeds via a mechanism which shows stereospecificity and saturability and that insulin acts by increasing the maximal transport capacity without changing the half-saturation constant.     

Effect of insulin on the glucose utilization in isolated cardiac myocytes from adult rat

The acute effects of insulin on glucose utilization in isolated rat quiescent cardiac myocytes were studied. Insulin (80 nM) increased the rate of glucose clearance by 2-3 times in the presence of glucose ranging from 0.3 microM to 5.5 mM. Glucose transport, which was measured in terms of both D-glucose uptake in the presence of 0.3 microM D-glucose and initial rate of uptake of 3-O-methylglucose, was stimulated 3-fold in the presence of insulin. At higher glucose concentrations (greater than 100 microM), a decrease in glucose clearance rate due to a shift of the rate-limiting step from glucose transport to a post-transport step in the pathway of glucose metabolism was observed. At the physiological concentration of glucose (5.5 mM), about 73% of glucose was metabolized into lactate, about 10% was oxidized into CO2 and the rest (17%) remained inside the cells. The pentose phosphate pathway did not contribute to the glucose metabolism in these cells. Insulin (80 nM) significantly increased the uptake of glucose (112%), and the conversions of glucose into lactate (16%), glycogen (64%), and triglyceride (18%), but not into CO2 (3%). Insulin transiently increased the percentage of I-form of glycogen synthase by 16% above basal, but did not affect the percentage of a-form of glycogen phosphorylase. The content of glucose 6-phosphate in the cells was increased by 46% above the basal value in the presence of insulin. These results indicate that insulin has different acute stimulatory effects on various steps in the metabolic pathway of glucose in isolated quiescent cardiac myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amino acid regulation of insulin action in isolated adipocytes. Selective ability of amino acids to enhance both insulin sensitivity and maximal insulin responsiveness of the protein synthesis system

Using the number and concentration of amino acids in Dulbecco's modified Eagle's medium as reference (DMEM = 100%), we found that a maximally effective concentration of insulin (10 ng/ml) stimulated protein synthesis by 125% over basal rate in the presence of 50% amino acids (EC50 = 19%), but by only 48% in amino acid-free buffer. Moreover, time course experiments revealed that amino acid regulation of insulin action was very rapid (t1/2 of 9.5 min) and readily reversible (less than 30 min). This effect was specific in that basal rates of protein synthesis were unaltered by amino acids. A second effect of amino acids was to markedly enhance insulin sensitivity of the protein synthesis system in a dose-dependent manner. Thus, the half-maximally effective concentrations of insulin required to stimulate protein synthesis fell from 0.43 to 0.25 to 0.15 ng/ml in the presence of 0, 50, and 150% amino acids. Neither insulin sensitivity nor maximal insulin responsiveness of the glucose transport system was altered by amino acids, nor did amino acids affect the insulin binding capacity of cells. When we divided the 14 amino acids found in DMEM into two groups, we found that one group of 7 amino acids had little or no effect on insulin sensitivity or responsiveness, whereas the other group was fully active (a 157% increase in insulin responsiveness, ED50 of 0.21 ng/ml versus a 68% increase, ED50 of 0.51 ng/ml, with no amino acids). Isoleucine and serine together increased both insulin sensitivity and responsiveness to 60-70% of that seen with the full complement of amino acids. In conclusion: 1) amino acids modulate insulin action by enhancing maximal insulin responsiveness and insulin sensitivity of the protein synthesis system, and the regulatory site of amino acid action appears to be distal to the common signal pathway, within the insulin action-protein synthesis cascade, and 2) the effects of amino acids are specific, in that basal rates of protein synthesis are unaffected, only certain amino acids influence insulin action, and amino acids fail to alter insulin binding or the insulin-responsive glucose transport system. These studies, together with those in the companion paper, demonstrate that the pleiotropic actions of insulin on enhancing glucose uptake and protein synthesis are mediated through divergent pathways that can be independently regulated.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Suitability of 2-deoxyglucose for measuring initial rates of glucose uptake in isolated adipocytes

The suitability of [3H]-2-deoxyglucose from measuring initial rates of glucose uptake in isolated rat adipocytes was assessed using three approaches. Basal and insulin-stimulated rates of glucose uptake were directly compared in 2 sec and 5 min assays using [14C]-3-O-methylglucose, [3H]-2-deoxyglucose, and [3H]-D-glucose. Equilibrium kinetics of 2-deoxyglucose uptake were compared with those of 3-O-methylglucose through impairment of hexokinase activity by depleting cellular energy with 2,4-dinitrophenol. The equivalence of these glucose analogues in a dynamic system was assessed by measuring the lag time preceding insulin stimulation of glucose uptake, insulin activation rates, and the T 1/2 of insulin activation. Our results demonstrate that no fundamental difference exists in the initial transport of 3-O-methylglucose, 2-deoxyglucose, and D-glucose.     

The insulin-like effect of sodium vanadate on adipocyte glucose transport is mediated at a post-insulin-receptor level.

Sodium vanadate has several insulin-like effects. To determine whether vanadate acts via the insulin receptor, I investigated the effect of vanadate on glucose transport (2-deoxyglucose uptake) in adipocytes that had been treated to decrease the number of insulin receptors. Trypsin (100 micrograms/ml) caused greater than 95% loss of 125I-insulin binding and rendered glucose transport resistant to both insulin and an anti-insulin-receptor antibody. However, vanadate caused an 8-fold increase in the transport rate [EC50 (concn. giving 50% of maximum effect) 0.2 mM] in both control and trypsin-treated cells, demonstrating that the insulin receptor does not have to be intact for vanadate to stimulate glucose transport. Insulin receptors were depleted by treatment of adipocytes with insulin (100 ng/ml) in the presence of Tris (which blocks receptor recycling). A 2 h treatment caused 60% loss of receptors, and a shift to the right in the dose-response curve for insulin stimulation of glucose transport (EC50 0.3 ng of insulin/ml in controls, 1.2 ng/ml in treated cells). The response to vanadate was again unaffected. Treatment with insulin for 4 h caused a 67% decrease in insulin binding and, in addition to the rightward shift in the insulin dose-response curve, a decrease in basal and maximal transport rates (which cannot be explained by decreased insulin receptor number). The EC50 of vanadate was again equal in control and treated cells, but glucose transport in the presence of a maximally effective concentration of vanadate (1 mM) was decreased. I conclude that the effect of vanadate on glucose transport is independent of the insulin receptor. Induction of a post-receptor defect (which may be a decrease in the total number of cellular glucose transporters) by prolonged exposure to insulin decreases the potency of a maximally effective concentration of vanadate. The findings demonstrate that vanadate stimulates glucose transport by an effect at a level distal to the insulin receptor.     

Relationship between insulin stimulation and endogenous regulation of 2-deoxyglucose uptake in 3T3-L1 adipocytes

The occurrence of the endogenous regulatory response to high rates of 2-deoxyglucose (2-DG) uptake, as previously described for C6 glioma cells during incubation with 2 mM 2-DG (Lange et al.: J. Cell. Physiol., 1989), was studied in 3T3-L1 preadipocytes and adipocytes, and the influence of insulin on this endogenous uptake regulation was examined. In contrast to 3T3-L1 preadipocytes, insulin-sensitive differentiated 3T3-L1 adipocytes displayed the time-dependent cyclic pattern of 2-DG uptake rates characteristic of the membrane-limited and endogenously regulated cellular state of hexose utilization. Although insulin induced a threefold stimulation of 2-DG tracer uptake in adipocytes, the hormone did not additionally stimulate the uptake rates or affect the periodic response: maximum and minimum levels of uptake remained unchanged. Scanning electron microscopy (SEM) revealed that the acquirement of the differentiated state is accompanied by a conspicuous transformation of the smooth surface of undifferentiated 3T3-L1 cells into a surface covered by numerous microvilli of uniform size and appearance. Treatment with insulin (10 mU/ml; 10 minutes) converted these microvilli into voluminous saccular membrane protrusions of the same type as had been formed during incubation of 3T3-L1 adipocytes with 2 mM 2-DG, and which have previously been shown to be involved in the endogenous uptake regulation of C6 glioma cells (Lange et al.: J. Cell. Physiol., 1989). These insulin-induced saccated membrane areas appeared to become integrated into the cell surface. Accordingly, insulin treatment caused a twofold increase of the intracellular distribution space of 3-O-methylglucose (3-OMG) in 3T3-L1 adipocytes. This insulin-induced increase of the 3-OMG distribution space exhibited the same time (t1/2 = 2-2.5 minutes) and dose dependence (EC50 = 20 nM) as the insulin-induced stimulation of 3-OMG transport. Glucose deprivation during the differentiation period inhibited the outgrowth of microvilli from the cell surface. Glucose starvation (18 hours at less than 0.5 mM) induced a conspicuous reduction of the length of microvilli on differentiated 3T3-L1 cells. In this state, the stalks of the microvilli are almost invisible and the enlarged spherical tips of the microvilli (with an average diameter of 370 nm compared to 230 nm of fed cells) appeared to protrude directly out of the cell surface. Starvation-induced shortening of microvilli was accompanied by a threefold increase of the basal 3-OMG transport rate and a greater than twofold increase of the intracellular 3-OMG distribution space as compared to fed cells (10 mM; 18 hours).(ABSTRACT TRUNCATED AT 400 WORDS)     

Transport and metabolism of D-glucose in human adipocytes. Studies of the dependence on medium glucose and insulin concentrations

Uptake and metabolism of the physiologically labelled D-glucose (D-[U-14C]glucose) has been characterized in human adipocytes at several unlabelled D-glucose concentrations in the absence and presence of insulin. Following a 90 min incubation, about 80% of the intracellular radioactivity was incorporated into total lipids at tracer glucose concentration, as well as at higher glucose concentrations in basal and insulin-stimulated cells, whereas 20% was recovered as hydrophilic metabolites. The only 14C-labelled metabolite escaping the cells in detectable amounts was CO2, which accounted about 4%. At trace glucose concentrations (5 mumol/l), the rate of glucose uptake was linear with time. Comparative studies of initial glucose uptake after 10 s and tracer D-glucose conversion to total lipids after 90 min showed high coefficients of correlation between basal rates (r = 0.87), maximal response above basal level to insulin (r = 0.92) and insulin sensitivity (r = 0.78). Thus, under these conditions glucose transport is rate-limiting for net glucose uptake, and measurements over long time intervals of rates for total cell-associated radioactivity or lipogenesis may serve as reliable estimates of initial glucose influx rates. However, the conversion rate of tracer glucose to metabolites decreased progressively with the glucose concentration and with an apparent Km of about 0.2 mmol/l. The three metabolic pathways exhibited similar percentage decreases in their activities, suggesting that a common enzymatic step is rate-limiting. In comparison, the Km for initial D-glucose uptake rate was about 7 mmol/l. Hence, the capacity for total glucose metabolism comprised only a small fraction of the glucose transport capacity at medium glucose concentrations above tracer concentrations. Both basal, half-maximal and maximal insulin-stimulated rates of adipocyte glucose utilization were dependent on the glucose concentration. Thus, comparing lipogenesis at tracer and at 0.5 mmol/l medium glucose concentration, it was shown that the higher medium glucose concentration was associated with a 60% lowering of the basal rate, a 35% reduction in the percentage response above baseline to maximal insulin stimulation and a 4-fold increase in the insulin sensitivity. Obviously, these findings reflect some intracellular step(s) being rate-limiting at medium glucose levels above tracer values.     

Kinetics of insulin action on protein synthesis in isolated adipocytes. Ability of glucose to selectively desensitize the glucose transport system without altering insulin stimulation of protein synthesis

When adipocytes were exposed to [3H]leucine for times ranging from 5 to 180 s, leucine was found to enter cells rapidly and equilibrate with the cell interior within 5 s. After an additional 15-30 s [3H]leucine was incorporated into nascent protein, and the rate of incorporation was linear for up to 6 h in both control and insulin-treated cells. Since treatment of adipocytes with 10 ng/ml insulin enhanced the rate of leucine incorporation 2-3-fold with minimal or no effect on the rate of protein degradation or leucine uptake, we conclude that the predominant effect of insulin is on enhancement of protein synthesis. To assess the time required for insulin to stimulate protein synthesis, we preincubated cells with 10 ng/ml of insulin for various times from 2 to 30 min and then measured [3H]leucine incorporation into protein during a 4-min assay. These results revealed that the insulin stimulation of protein synthesis is rapid (t 1/2 of 4.4 min), but 9-fold slower than insulin activation of glucose transport (t 1/2 less than 0.5 min under identical conditions). In contrast to the rapidity of insulin activation, we found that deactivation proceeded at much slower rates (t 1/2 of 32 and 21 min for protein synthesis and glucose transport, respectively). Desensitization of the glucose transport system has previously been shown to occur after adipocytes are exposed to high glucose and insulin. To examine the specificity of desensitization, we treated cells for 6 h with 20 mM glucose and 25 ng/ml insulin and then examined insulin sensitivity and maximal insulin responsiveness of both the glucose transport and protein synthesis systems. After treatment, the glucose transport was markedly insulin-resistant (60% loss in maximal insulin responsiveness and a marked loss in insulin sensitivity), whereas the protein synthesis system exhibited neither diminished insulin responsiveness nor loss of insulin sensitivity. In fact, insulin sensitivity actually increased, as indicated by the finding that less insulin was required to stimulate protein synthesis (insulin ED50 values of 0.25 and 18 ng/ml at 0 and 6 h of treatment). From these studies we conclude that desensitization of the glucose transport system by glucose and insulin treatment appears to be specific for this particular effector system and does not reflect a state of generalized cellular insulin resistance.     

Phorbol esters imitate in rat fat-cells the full effect of insulin on glucose-carrier translocation, but not on 3-O-methylglucose-transport activity.

Tumour-promoting phorbol esters have insulin-like effects on glucose transport and lipogenesis in adipocytes and myocytes. It is believed that insulin activates the glucose-transport system through translocation of glucose transporters from subcellular membranes to the plasma membrane. The aim of the present study was to investigate if phorbol esters act through the same mechanism as insulin on glucose-transport activity of rat adipocytes. We compared the effects of the tumour-promoting phorbol ester tetradecanoylphorbol acetate (TPA) and of insulin on 3-O-methylglucose transport and on the distribution of D-glucose-inhibitable cytochalasin-B binding sites in isolated rat adipocytes. Insulin (100 mu units/ml) stimulated 3-O-methylglucose uptake 9-fold, whereas TPA (1 nM) stimulated the uptake only 3-fold (mean values of five experiments, given as percentage of equilibrium reached after 4 s: basal 7 +/- 1.3%, insulin 60 +/- 3.1%, TPA 22 +/- 2.3%). In contrast, both agents stimulated glucose-transporter translocation to the same extent [cytochalasin B-binding sites (pmol/mg of protein; n = 7): plasma membranes, basal 6.2 +/- 1.0, insulin 13.4 +/- 2.0, TPA 12.7 +/- 2.7; low-density membranes, basal 12.8 +/- 2.1, insulin 6.3 +/- 0.9, TPA 8.9 +/- 0.7; high-density membranes, 6.9 +/- 1.1; insulin 12.5 +/- 1.0, TPA 8.1 +/- 0.9]. We conclude from these data: (1) TPA stimulates glucose transport in fat-cells by stimulation of glucose-carrier translocation; (2) insulin and TPA stimulate the carrier translocation to the same extent, whereas the stimulation of glucose uptake is 3-fold higher with insulin, suggesting that the stimulatory effect of insulin on glucose-transport activity involves other mechanisms in addition to carrier translocation.     

Influence of exogenous sugars and polyols on C1-influx and efflux by the ascomycete Neocosmospora vasinfecta.

Glucose and other transportable sugars and polyols inhibited Cl- influx very soon after addition to mycelium in the process of Cl- accumulation. Under the usual experimental conditions (0.1 mM KCl, glucose greater than or equal to 2 mM) the mean percentage of inhibition of Cl- influx by glucose was 54.1 +/- 8.0 (+/- standard error; N = 26). Transport of the exogenous carbohydrate was necessary for inhibition of Cl- influx. Thus, the estimated Ki for glucose inhibition of Cl- influx (28 muM) was close to the Km for glucose transport; glycerol did not inhibit Cl- influx unless it was itself transported, and the degree of inhibition exerted by various carbohydrates correlated with their uptake rates. Inhibition was not caused by the accumulated sugar itself, as high levels (ca. 60 mM) of intramycelial 3-O-methylglucose gave rise to a stimulation of Cl- influx when the exogenous sugar was removed. It is suggested that interaction of Cl- and carbohydrate transport arises from competition for a common energy-coupling mechanism in the cell membrane. Both glucose and 3-O-methylglucose elicited Cl- efflux, but the maximal Cl- efflux rates were observed only after 40 min of incubation and only in the presence of the readily metabolizable glucose. Removal of the exogenous glucose, even after maximal Cl- efflux had been established, resulted in the rapid cessation of efflux. Studies under anaerobic conditions gave further evidence that glucose uptake was necessary and that efflux was not due to temporary depletion of energy reserves. It is proposed that glucose-induced leakage of Cl- is due to reversal of the Cl- uptake system, even though the Km for efflux is much greater than that for influx.     

High affinity insulin binding and insulin receptor-effector coupling: modulation by Ca2+.

Insulin binding and insulin stimulated amino acid and glucose uptake were determined in cultured HTC hepatoma cells in the presence of Ca2+ and ruthenium red (RR) in order to further characterise the putative calcium binding site on the receptor. These ions increased insulin receptor high affinity binding and the sensitivity of these responses to insulin. The insulin concentration required to half-maximally stimulate amino acid uptake decreased significantly from 26.9 +/- 5.8 ng/ml to 6.0 +/- 1.3 ng/ml in the presence of 10 mM Ca2+ and to 1.3 +/- 0.5 ng/ml in the presence of RR. The effect of Ca2+ and RR was more pronounced on insulin stimulated glucose uptake. These agents also increased receptor-effector coupling, reducing the percentage of occupied receptors required for maximal insulin stimulation of amino acid uptake from 10.8% in control cells to 3.4 and 1.4% in the presence of Ca2+ and RR respectively. The receptor occupancy required to produce maximal insulin responses on glucose uptake decreased from 20% (control) to 3.8% (Ca2+ and RR). We hypothesize that since Ca2+ and RR have similar effects, that occupation of Ca2+ binding sites on the receptor produces a conformational change in the insulin receptor which increases insulin receptor affinity, insulin sensitivity and acts on an early post-receptor event responsible for coupling binding to 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.     

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.     

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-induced insulin resistance of skeletal-muscle glucose transport and uptake.

The ability of glucose and insulin to modify insulin-stimulated glucose transport and uptake was investigated in perfused skeletal muscle. Here we report that perfusion of isolated rat hindlimbs for 5 h with 12 mM-glucose and 20,000 microunits of insulin/ml leads to marked, rapidly developing, impairment of insulin action on muscle glucose transport and uptake. Thus maximal insulin-stimulated glucose uptake at 12 mM-glucose decreased from 34.8 +/- 1.9 to 11.5 +/- 1.1 mumol/h per g (mean +/- S.E.M., n = 10) during 5 h perfusion. This decrease in glucose uptake was accompanied by a similar change in muscle glucose transport as measured by uptake of 3-O-[14C]-methylglucose. Simultaneously, muscle glycogen stores increased to 2-3.5 times initial values, depending on fibre type. Perfusion for 5 h in the presence of glucose but in the absence of insulin decreased subsequent insulin action on glucose uptake by 80% of the effect of glucose with insulin, but without an increase in muscle glycogen concentration. Perfusion for 5 h with insulin but without glucose, and with subsequent addition of glucose back to the perfusate, revealed glucose uptake and transport similar to initial values obtained in the presence of glucose and insulin. The data indicate that exposure to a moderately increased glucose concentration (12 mM) leads to rapidly developing resistance of skeletal-muscle glucose transport and uptake to maximal insulin stimulation. The effect of glucose is enhanced by simultaneous insulin exposure, whereas exposure for 5 h to insulin itself does not cause measurable resistance to maximal insulin stimulation.     

Recovery of maximal insulin responsiveness and insulin sensitivity after induction of insulin resistance in primary cultured adipocytes

Treatment of primary cultured adipocytes with 50 ng/ml insulin and 20 mM glucose for 0-6 h resulted in a loss of maximal insulin responsiveness (MIR) which was immediate (no lag period), rapid (t1/2 of 3 h), linear, and extensive (80% of that seen at 24 h), whereas loss of insulin sensitivity from 0-24 h was slow (t1/2 = 8 h), extensive (insulin ED50 of 0.3 and 1.45 ng/ml at 2 and 24 h, respectively), and was preceded by an initial 2-h lag. Recovery of MIR and insulin sensitivity was assessed by inducing desensitization for various times from 2-24 h, removing insulin and glucose, and then measuring MIR and insulin sensitivity over a subsequent 1-6-h period. After 2 h, recovery of MIR in desensitized cells was rapid (251 pmol of glucose/3 min/h), whereas after 24 h, recovery was much slower (35 pmol/3 min/h). In contrast, the opposite trend was seen for recovery of insulin sensitivity: at early times recovery of insulin sensitivity was slow (0.05 ng/ml/h) but was rapid after 24 h (0.12 ng/ml/h). Thus, it appears that MIR and insulin sensitivity can be independently regulated since recovery rates for MIR and insulin sensitivity diverged with the progression of insulin resistance. When the effects of insulin and glucose on recovery were examined, we found that insulin alone was unable to block recovery of MIR or insulin sensitivity. Glucose alone, however, was effective in preventing recovery of insulin sensitivity but not recovery of MIR. In the presence of 20 mM glucose, low doses of insulin (treatment EC50 = 0.22-0.46 ng/ml) effectively prevented recovery of both MIR and insulin sensitivity. De novo protein synthesis apparently is not involved in the development of insulin resistance or the reversal of desensitization since inhibition of protein synthesis by cycloheximide had no effect on the loss of MIR and insulin sensitivity or recovery.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of insulin, prostaglandin E1 and uptake inhibitors on glucose transport in the perfused guinea-pig placenta

The effects of insulin, prostaglandin E1 (PGE1) and uptake inhibitors on unidirectional D-glucose influx at brush border (maternal) and basal (fetal) sides of the guinea-pig syncytotrophoblast were investigated in the intact, perfused guinea-pig placenta by rapid, paired-tracer dilution. Experiments were performed in either an in situ preparation artificially perfused through the umbilical vessels (intact maternal circulation) or in the fully isolated dually-perfused placenta in which both interfaces were studied simultaneously. Kinetic characterization of unidirectional D-glucose influx gave apparent Km values (mean +/- SEM) at maternal and fetal sides of 70 +/- 6 and 87 +/- 16 mM respectively; corresponding Vmax values were 53 +/- 3 and 82 +/- 6 mumol min-1g-1. At the fetal side (singly-perfused placenta) cytochalasin B (50 microM), ethylidene-D-glucose (100 mM) and PGE1 (1 microM) partially inhibited D-glucose uptake whereas cortisol (50 microM) and progesterone (100 microM) had no effect. Abolition of the sodium gradient across the fetal interface did not modulate the kinetics of influx. In the presence of 150 mu units ml-1 insulin (dually-perfused placenta), unidirectional uptake into the trophoblast and transplacental D-[3H]glucose transfer were unaltered. In contrast, prostaglandin E1 (1 microM) markedly reduced the Km and Vmax for D-glucose at both interfaces and the inhibitory effect was reflected in a reduction in specific transplacental D-glucose transfer. Further experiments showed that the isolated placenta releases prostaglandins (PGE; PGF2 alpha) into both circulations. Bilateral insulin perfusion did not affect either lactate release by the placenta or rapid metabolism of D-[14C]glucose to [3H]lactate (usually less than 10% effluent [14C]lactate in 5 min). An asymmetric degradation of exogenous insulin was observed in the dually-perfused placenta: uterine venous samples contained 24 +/- 7 microunits ml-1 immunoreactive insulin when compared to the arterial concentration (151 +/- 3 microU ml-1 perfusate) while no change was measureable in the fetal circulation within the same time period (152 +/- 5 microU ml-1). This asymmetry was confirmed in experiments employing [125I]insulin. These results demonstrate that glucose transport in the intact guinea-pig placenta occurs by a sodium-independent, cytochalasin B-inhibitable system which is insulin-insensitive. Prostaglandin E1 appeared to be a potent transport inhibitor which suggests that prostaglandins may be involved in the 'down' regulation of placental glucose transport in vivo.