Insulin-stimulated Hexose Transport Research Articles

Insulin-induced activation of glycerol-3-phosphate acyltransferase by a chiro-inositol-containing insulin mediator is defective in adipocytes of insulin-resistant, type II diabetic, Goto-Kakizaki rats.

Type II diabetic Goto-Kakizaki (GK) rats were insulin-resistant in euglycemic-hyperinsulinemic clamp studies. We therefore examined insulin signaling systems in control Wistar and diabetic GK rats. Glycerol-3-phosphate acyltransferase (G3PAT), which is activated by headgroup mediators released from glycosyl-phosphatidylinositol (GPI), was activated by insulin in intact and cell-free adipocyte preparations of control, but not diabetic, rats. A specific chiro-inositol-containing inositol phosphoglycan (IPG) mediator, prepared from beef liver, bypassed this defect and comparably activated G3PAT in cell-free adipocyte preparations of both diabetic GK and control rats. A myo-inositol-containing IPG mediator did not activate G3PAT. Relative to control adipocytes, labeling of GPI by [3H]glucosamine was diminished by 50% and insulin failed to stimulate GPI hydrolysis in GK adipocytes. In contrast to GPI-dependent G3PAT activation, insulin-stimulated hexose transport was intact in adipocytes and soleus and gastrocnemius muscles of the GK rat, as was insulin-induced activation of mitogen-activated protein kinase and protein kinase C. We conclude that (i) chiro-inositol-containing IPG mediator activates G3PAT during insulin action, (ii) diabetic GK rats have a defect in synthesizing or releasing functional chiro-inositol-containing IPG, and (iii) defective IPG-regulated intracellular glucose metabolism contributes importantly to insulin resistance in diabetic GK rats.

Evidence that erythroid-type glucose transporter intrinsic activity is modulated by cadmium treatment of mouse 3T3-L1 cells.

Previous studies suggest that regulation of hexose uptake in Chinese hamster ovary fibroblasts can occur by alterations in glucose transporter intrinsic activity without changes in cell surface transporter number (Harrison, S. A., Buxton, J. M., Helgerson, A. L., MacDonald, R. G., Chlapowski, F. J., Carruthers, A., and Czech, M. P. (1990) J. Biol. Chem. 265, 5793-5801). We tested this hypothesis using 3T3-L1 fibroblasts and adipocytes which exhibit 5-6-fold increases in 2-deoxyglucose or 3-O-methylglucose uptake when exposed to low micromolar concentrations of cadmium for 18 h. Cadmium treatment decreased the apparent Km of 3T3-L1 fibroblasts for 3-O-methylglucose influx from approximately 28 to 9 mM and increased the apparent Vmax by 2-3-fold. These fibroblasts lack the skeletal muscle/adipocyte-type (GLUT4) transporter and showed only a small increase in total cellular immunoreactive HepG2 type (GLUT1) transporter in response to cadmium. Furthermore, cell surface GLUT1 levels did not change in 3T3-L1 fibroblasts exposed to cadmium, as assessed by the binding to intact cells of an antibody which recognizes an extracellular GLUT1 epitope. Insulin enhanced 2-deoxyglucose uptake 2-fold in 3T3-L1 fibroblasts, but did not further stimulate cadmium-activated transport rates. In contrast, insulin stimulated hexose transport 15-fold in 3T3-L1 adipocytes, which express both GLUT1 and GLUT4 proteins, and this effect was fully additive with the 5-fold effect of cadmium. Cadmium had little or no effect on immunoreactive GLUT1 or GLUT4 in isolated 3T3-L1 adipocyte plasma membranes. In contrast, insulin action led to marked recruitment (3-fold) of GLUT4 to the plasma membrane fraction in adipocytes treated with or without cadmium. Taken together, these data are consistent with the hypothesis that cadmium-activated sugar uptake is catalyzed by GLUT1, whereas insulin-stimulated sugar uptake is catalyzed predominantly by GLUT4 in 3T3-L1 adipocytes. Furthermore, the data suggest that the GLUT1 transporter can undergo significant increases in intrinsic catalytic activity in response to cadmium treatment of 3T3-L1 fibroblasts and adipocytes.

Suppressed intrinsic catalytic activity of GLUT1 glucose transporters in insulin-sensitive 3T3-L1 adipocytes.

Previous studies indicated that the erythroidtype (GLUT1) glucose transporter isoform contributes to basal but not insulin-stimulated hexose transport in mouse 3T3-L1 adipocytes. In the present studies it was found that basal hexose uptake in 3T3-L1 adipocytes was about 50% lower than that in 3T3-L1 or CHO-K1 fibroblasts. Intrinsic catalytic activities of GLUT1 transporters in CHO-K1 and 3T3-L1 cells were compared by normalizing these hexose transport rates to GLUT1 content on the cell surface, as measured by two independent methods. Cell surface GLUT1 levels in 3T3-L1 fibroblasts and adipocytes were about 10- and 25-fold higher, respectively, than in CHO-K1 fibroblasts, as assessed with an anti-GLUT1 exofacial domain antiserum, delta. The large excess of cell surface GLUT1 transporters in 3T3-L1 adipocytes relative to CHO-K1 fibroblasts was confirmed by GLUT1 protein immunoblot analysis and by photoaffinity labelling (with 3-[125I]iodo-4-azidophenethylamido-7-O-succinyldeacetylforskoli n) of glucose transporters in isolated plasma membranes. Thus, GLUT1 intrinsic activity is markedly reduced in 3T3-L1 fibroblasts compared with the CHO-K1 fibroblasts, and further reduction occurs upon differentiation to adipocytes. Intrinsic catalytic activities specifically associated with heterologously expressed human GLUT1 protein in transfected CHO-K1 versus 3T3-L1 cells were determined by subtracting appropriate control cell values for hexose transport and delta-antibody binding from those determined in the transfected cells expressing high levels of human GLUT1. The results confirmed a greater than 90% inhibition of the intrinsic catalytic activity of human GLUT1 transporters on the surface of mouse 3T3-L1 adipocytes relative to CHO-K1 fibroblasts. We conclude that a mechanism that markedly suppresses basal hexose transport catalyzed by GLUT1 is a major contributor to the dramatic insulin sensitivity of glucose uptake in 3T3-L1 adipocytes.

Protein kinase C inhibitors block insulin and PMA-stimulated hexose transport in isolated rat adipocytes and BC3H-1 myocytes

Effects of protein kinase C (PKC) inhibitors and “down-regulation” on insulin and PMA-stimulated 2-deoxyglucose transport were determined in isolated rat adipocytes or BC3H-1 myocytes. In both model systems, H-7, sangivamycin, and staurosporine, inhibitors of the catalytic domain of PKC, each effectively blocked insulin and PMA-stimulated hexose uptake at similar concentrations. In the myocytes, staurosporine completely blocked the insulin effect retained post-chronic phorbol myristate acetate (PMA)-induced “down-regulation.” These findings indicate (1) that chronic pretreatment with PMA may not lead to a complete loss of PKC activity in the myocyte, and (2) that PKC is involved in insulin-stimulated hexose transport in both isolated rat adipocytes and BC3H-1 myocytes.

Antagonism by growth hormone of insulin-sensitive hexose transport in 3T3-F442A adipocytes.

We have studied the effects of GH on basal and insulin-stimulated hexose transport by 3T3-F442A adipocytes in a hormonally defined serum-free medium. Adipocytes preincubated in defined medium exhibit a low level of hexose transport which is acutely (15 min) stimulated (greater than 5-fold) by insulin (EC50, 0.1-0.2 nM). GH has acute (15-45 min) insulin-mimetic (greater than 2-fold) and chronic (4-48 h) diabetogenic (50-80%) effects on basal and insulin-stimulated hexose transport. The insulin-mimetic effect of GH has a higher EC50 (2 nM) than its diabetogenic effect (EC50, 0.2 nM). Chronic GH exposure decreases the maximal responsiveness (50-80%) and the acute sensitivity (approximately 2-fold) of hexose transport to insulin. Insulin-stimulated transport is more (approximately 5-fold) sensitive to the diabetogenic effect of GH than is basal transport. Insulin binding and degradation were not altered by chronic exposure to GH. The diabetogenic effect of GH may occur at a postinsulin binding level.

Sulfonylurea Binding to Adipocyte Membranes and Potentiation of Insulin-stimulated Hexose Transport

We have previously shown that the sulfonylureas increase insulin-stimulated glucose transport in adipocytes mainly by enhancing the insulin-induced recruitment of glucose transporter from its intracellular storage pool to the plasma membrane (Jacobs, D. B., and Jung, C. Y. (1985) J. Biol. Chem. 260, 2593-2596). In order to determine if this sulfonylurea effect is mediated by a specific membrane-associated sulfonylurea-binding protein, in the present report we measured exact dose dependence of the transport enhancement activities of different sulfonylureas in adipocytes in primary culture and equilibrium binding affinities of these agents to various adipocyte membrane fractions. Glycuride was found to increase the insulin-stimulated, 3-O-methyl-D-glucose equilibrium exchange in cultured rat adipocytes by up to 60% with little effect in the absence of insulin. The effect developed gradually reaching the maximum level at 24 h of incubation. The effect was concentration dependent showing a simple, one-to-one stoichiometry and an apparent activation constant (Ka) of approximately 1 microM. Glypizide, tolazamide, and tolbutamide also enhanced the insulin-stimulated hexose transport by up to 60%, but with Ka of approximately 2, 11, and 25 microM, respectively. HB-699 and ciglitazone, non-sulfonylureas, were without effect under the same condition. In equilibrium binding experiments, [3H]glyburide was found to bind to adipocyte membranes at two or more protein-specific, saturable sites, with similar apparent dissociation constants (KD) ranging 1-3 microM. These protein-specific glyburide bindings were displaced not only by tolazamide and tolbutamide, but also by ciglitazone and HB-699, with indicated KD of 11-16, 80-85, 20-25, and 85-95 microM, respectively. However, with the plasma membrane fraction, the displacements by ciglitazone and HB-699 were partial and did not exceed 56-61% at maximum. Based on these findings, we propose that there is a sulfonylurea-specific-binding protein in the plasma membrane of adipocytes, and that this sulfonylurea-binding protein may play a key role in the enhancement of insulin-stimulated hexose transport by sulfonylureas, probably via potentiation of the insulin-induced recruitment of glucose transporter.

Failure of fenfluramine to affect basal and insulin-stimulated hexose transport in rat skeletal muscle

Fenfluramine is an effective appetite supressant that mediates its action via serotoninergic neurons. We studied the effect of pure d- and l-fenfluramine on in vitro hexose transport in isolated rat soleus muscles and skeletal muscle cells in culture. We found no evidence to suggest that the fenfluramine enantiomers affect the basal transport activity. Furthermore, the drugs did not interfere with the ability of glucose to regulate its own transport. Muscle responsiveness to insulin was not altered by the enantiomers, nor did insulin unmask any effect of fenfluramine on muscle hexose transport. These conclusions are based on experiments performed with a wide concentration range of drug and insulin, from the therapeutic to suprapharmacological levels. We discuss our results in view of published data on the effects of fenfluramine on peripheral glucose metabolism.

Insulin-stimulated Hexose Transport and Glucose Oxidation in rat Adipocytes Is Inhibited by Sphingosine at a Step after Insulin Binding

Spingosine, a naturally occurring inhibitor of protein kinase C, has recently been shown to have potent bioregulatory effects on a variety of cellular processes involving signal transduction mechanisms. In the present studies, we have investigated its effects on activation by insulin of hexose transport and glucose oxidation in isolated rat adipocytes. Preincubation of cells with this long-chain base blocked both the marked activation of these processes by insulin and the smaller activation by phorbol myristate acetate. Inhibition of both insulin and phorbol 12-myristate 13-acetate activation showed the same sphingosine concentration dependence, suggesting a common locus of action. The effectiveness of sphingosine was inversely proportional to the lipid content in the incubation (which was a function of both the age of the animal and the number of cells used) presumably due to dilution of the lipophilic long-chain base into the cellular triglycerides. Sphingosine did not affect either insulin binding to its receptor or the half-maximal concentration of the hormone required to activate hexose transport, but reduced the maximal responses. Thus, the inhibition was at a step distal to the binding of insulin to its receptor. Basal transport activity was not inhibited, suggesting a locus of action prior to the glucose transporter. The inhibitor was also effective when added following activation by insulin of hexose transport and resulted in a rapid reversal of activation (t 1/2 for inhibition was 2-4 min.). Sphingosine and its analogs showed a parallel potency for inhibition both of isolated protein kinase C and of insulin activation in adipocytes, consistent with an essential role for protein kinase C in the activation of hexose transport by insulin.

Inhibition by forskolin of insulin-stimulated glucose transport in L6 muscle cells

The cardioactive diterpene forskolin is a known activator of adenylate cyclase, but recently a specific interaction of this compound with the glucose transporter has been identified that results in the inhibition of glucose transport in several human and rat cell types. We have compared the sensitivity of basal and insulin-stimulated hexose transport to inhibition by forskolin in skeletal muscle cells of the L6 line. Forskolin completely inhibited both basal and insulin-stimulated hexose transport when present during the transport assay. The inhibition of basal transport was completely reversible upon removal of the diterpene. In contrast, insulin-stimulated hexose transport did not recover, and basal transport levels were attained instead. This effect of inhibiting (or reversing) the insulin-stimulated fraction of transport is a novel effect of the diterpene. Forskolin treatment also inhibited the stimulated fraction of transport when the stimulus was by 4 beta-phorbol 12,13-dibutyrate, reversing back to basal levels. Half-maximal inhibition of the above-basal insulin-stimulated transport was achieved with 35-50 microM-forskolin, and maximal inhibition with 100 microM. Forskolin did not inhibit 125I-insulin binding under conditions where it caused significant inhibition of insulin-stimulated hexose transport. Forskolin significantly elevated the cyclic AMP levels in the cells; however its inhibitory effect on the above basal, insulin-stimulated fraction of hexose transport was not mediated by cyclic AMP since: (i) 8-bromo cyclic AMP and cholera toxin did not mimic this effect of the diterpene, (ii) significant decreases in cyclic AMP levels caused by 2',3'-dideoxyadenosine in the presence of forskolin did not prevent inhibition of insulin-stimulated hexose transport, (iii) isobutylmethylxanthine did not potentiate forskolin effects on glucose transport but did potentiate the elevation in cyclic AMP, and (iv) 1,9-dideoxyforskolin, which does not activate adenylate cyclase, inhibited hexose transport analogously to forskolin. We conclude that forskolin can selectively inhibit the insulin- and phorbol ester-stimulated fraction of hexose transport under conditions where basal transport is unimpaired. The results are compatible with the suggestions that glucose transporters operating in the stimulated state (insulin or phorbol ester-stimulated) differ in their sensitivity to forskolin from transporters operating in the basal state, or, alternatively, that a forskolin-sensitive signal maintains the stimulated transport rate.

Effect of chronic isoproterenol exposure on insulin binding and insulin-stimulated hexose transport in isolated rat adipocytes

The effect of chronic exposure of isolated rat adipocytes to the beta-adrenergic agonist isoproterenol has been studied with respect to insulin binding and insulin-stimulated hexose uptake. Isoproterenol exposure led to a progressive decrease in both the number of surface insulin receptors and the stimulation of hexose uptake. The effect on insulin binding was reversible by removal of the beta-agonist within an hour of its addition. Later exposures of adipocytes to isoproterenol resulted in an irreversible cellular defect by leading to a progressive inability of the cells to regain their normal level of insulin-stimulated hexose uptake and insulin binding.

Insulin elicits a redistribution of transferrin receptors in 3T3-L1 adipocytes through an increase in the rate constant for receptor externalization.

Incubation of 3T3-L1 adipocytes with insulin at 37 degrees C resulted in a 2-fold increase in specific binding of transferrin to cell-surface receptors, as measured by a subsequent incubation of cells at 4 degrees C with 125I-transferrin. The insulin concentration required for half-maximal effect was 10 nM, and the half-time for insulin action was 40 s. By comparison, insulin stimulated hexose transport in 3T3-L1 adipocytes with a half-maximal effect at 8 nM and a half-time of 105 s. Scatchard analysis of 125I-transferrin binding to cells at 4 degrees C showed that the insulin-induced increase in transferrin receptor binding was due to an increase in the number of surface transferrin receptors. When cells were incubated for 2 h at 37 degrees C with 125I-transferrin to achieve steady-state binding and then exposed to insulin, there was a 1.7-fold increase in surface-bound transferrin (acid-sensitive) and a corresponding decrease in intracellularly bound transferrin (acid-insensitive). Thus, insulin elicits translocation of intracellular transferrin receptors to the plasma membrane. Concomitant with the 2-fold increase in surface receptors in response to insulin, there was a 2-fold increase in the rate of 59Fe3+ uptake from 59Fe3+-loaded transferrin. The rate of externalization of the intracellular 125I-transferrin-receptor complex at 37 degrees C was determined for basal and insulin-treated cells. Insulin increased the first-order rate constant for this process 1.7-fold. The effect of insulin on the rate of externalization is sufficient to account for the increase in surface transferrin receptors.

Forskolin inhibition of hexose transport in cardiomyocytes.

The effects of insulin, forskolin, isoproterenol, and epinephrine on 3-O-methylglucose (hexose) transport and cell cyclic AMP levels were determined in adult rat cardiomyocytes. Insulin stimulated hexose transport in these cells an average of 2.5-fold. Initial hexose transport rates at 1 mM hexose were 3.75 X 10(-2) nmol/mg cell protein/second in the absence of insulin, and 8.25 X 10(-2) nmol/mg cell protein/second in the presence of 12.3 microM insulin. Forskolin at 5 microM nearly abolished hexose transport within 3 s of exposure, but did not increase cell cyclic AMP concentrations within 9 s. The apparent Ki for hexose transport inhibition was about 0.3 microM forskolin. Epinephrine and isoproterenol at 50 microM increased cell cyclic AMP 4-fold during 9 s exposure, but did not affect hexose transport. Treatment of cells with these catecholamines of forskolin for up to 99 s increased cell cyclic AMP, but only forskolin inhibited hexose transport. We conclude from these results that forskolin acts on hexose transport independent of its action on adenyl cyclase, and that cyclic AMP does not inhibit or stimulate hexose transport.

The glucose transporter in 3T3-L1 adipocytes is phosphorylated in response to phorbol ester but not in response to insulin.

At maximally active concentrations with 20-min exposure, insulin and phorbol myristate acetate (PMA) stimulated hexose transport in 3T3-L1 adipocytes by 11- and 2-fold, respectively. The potential role of phosphorylation of the glucose transporter (GT) in these stimulations was investigated by the isolation of GT through immunoprecipitation from ortho[32P]phosphate-labeled 3T3-L1 adipocytes. It was found that there was no significant 32P incorporation into GT from basal adipocytes after 2- or 18 h-labeling in the presence of 0.5 mCi of 32Pi/ml. Furthermore, under these labeling conditions, insulin treatment for 1, 4, or 30 min failed to stimulate the phosphorylation of GT. Also, there was no detectable phosphate incorporation into GT upon reversal of insulin-stimulated hexose transport by the removal of insulin (half-time for reversal approximately 8 min). In contrast to these results, exposure of adipocytes to PMA (1 microM) for 20 min elicited a phosphorylation of GT to the extent of about 0.1 phosphate/GT molecule. Exposure of cells to both insulin and PMA resulted in a 3-fold increase in the level of phosphate in GT compared to that seen with PMA alone. Possibly this increase is due to the translocation of GT to the plasma membrane where it is a better substrate for activated protein kinase C. Stimulation of hexose transport was the same with the combined treatment of insulin and PMA compared to that seen with insulin alone. These results indicate that neither a change in the phosphorylation state of the GT nor activation of protein kinase C is involved in the mechanism by which the insulin receptor stimulates glucose transport.

Postreceptor myocardial metabolic defect in a rat model of non-insulin-dependent diabetes mellitus.

Hearts isolated from non-insulin-dependent diabetic rats were found to exhibit reduced rates of basal and insulin-stimulated glucose metabolism. Since tissue levels of fructose 1,6-bisphosphate are significantly reduced in the diabetic heart, it was concluded that phosphofructokinase may be inhibited. However, neither glycogen nor glucose 6-phosphate accumulated in the myocyte, indicating that the phosphofructokinase reaction was not a bottleneck diverting substrate away from glycolysis. The other major factor contributing to decreased glycolytic flux in the diabetic heart is the impairment in glucose transport. Both basal and insulin-stimulated transport of 3-O-methyl-D-glucose was 30% less in the diabetic heart. While insulin sensitivity was unaltered in the diabetic rat, insulin responsiveness was decreased, indicating that the impairment in insulin-stimulated hexose transport was caused by a post-receptor defect. The net result of these abnormalities in glucose metabolism is a significant reduction in the rate of ATP synthesis by the diabetic heart.

Sensitivity of adipocyte basal and insulin-stimulated hexose transport to the membrane lipid structure

A series of anesthetic alcohols inhibited basal and insulin-stimulated 2-deoxy- d-[1- 14C]glucose transport in adipocytes over total alcohol concentration ranges that cause local anesthesia of rat sciatic nerve. The relative potencies of the inhibition caused by the alcohols increased in the following order: methanol < ethanol < propanol < butanol < benzyl alcohol < hexanol < octanol. The inhibition was reversible and correlated well with the known partitioning of the alcohols into lipids of biological membranes. Adipocyte membranes were labeled with the 5-nitroxide stearate spin probe to investigate the effects of the alcohols on the dynamic structure of membrane lipids of the adipocyte. The alcohols increased the membrane “fluidity”, and the relative concentration dependence of the effects closely paralleled that noted from methanol to octanol in transport studies. Alcohols from methanol to hexanol caused inhibition of hexose transport at molar potencies comparable to that observed for membrane disordering. This suggests that hydrophobic regions of the transporter and its lipid environment are perturbed by a comparable mechanism for each alcohol. The cholesterol-complexing polyene antibiotic filipin inhibited hexose transport and influenced the mobility of lipid domains sampled with the nitroxide cholestane, cholesterol-like spin probe. The data are consistent with the consistent with the concept that the membrane structural/functional effects are mediated by formation of 1:1 cholesterol:filipin complexes. Alcohols and filipin inhibited inherent transporter activity and perturbed the membrane lipid structure without dramatically diminishing transport stimulation by insulin above basal. The specific organization of membrane lipids (particularly cholesterol) may provide an essential environment for optimal transport system activity.

Effects of sulfonylureas on the actions of insulin and insulin-mimickers: Potentiation of stimulated hexose transport in adipocytes

The sulfonylurea glyburide, a ‘second-generation’ oral hypoglycemic compound, was studied in vitro in order to determine its cellular mechanism of action in adipocytes prepared from cultured rat epididymal fat tissue. Glyburide treatment (1 μg/ml) for 20 h did not alter insulin receptor number or affinity, or down-regulation by insulin. Biologic responses of these cells were measured in the presence of insulin or the oxidants Vitamin K 5 and H 2O 2, which have insulin-like activity, but do not act through the binding portion of the receptor. 2-Deoxyglucose uptake was not significantly changed by exposure to glyburide alone. However, the sulfonylurea increased the insulin-stimulated or insulin-mimicker-activated uptake by approximately 30%. Insulin-stimulated glucose oxidation was also potentiated when glucose transport was rate limiting for metabolism. These findings extend our earlier observation that in adipose tissue the primary cellular mechanism of action of sulfonylureas is to potentiate insulin-stimulated hexose transport, and that this process may account for their hypoglycemic activity.

The effect of membrane phospholipid modification on insulin action in adipocytes from normal and streptozotocin-induced diabetic rats.

The effects of membrane phospholipid modification on insulin-stimulated hexose transport and antilipolysis was studied in isolated adipocytes derived from normal and streptozotocin (STZ)-diabetic rats. The marked inhibition of glucose transport observed in normal adipocytes pretreated with phospholipid vesicles composed of dioleoylphosphatidylcholine and phosphatidylserine (DOPC-PC) was not apparent in cells from diabetic animals. Inhibition of glucose oxidation in cells from non-diabetic, but not diabetic rats, was also detected under conditions where transport rate was insufficient to saturate the intracellular processes leading to oxidation. The antilipolytic effect of insulin, on the other hand, was not significantly impaired by vesicle pretreatment of cells from either the control or diabetic animals. These studies suggest that vesicles may have a specific effect on the glucose transport system and that changes in responsiveness of insulin-stimulated glucose transport may be related to alterations of membrane phospholipids.

Influence of dexamethasone on insulin action: inhibition of basal and insulin-stimulated hexose transport is dependent on length of exposure in vitro.

Rat adipose tissue was exposed to 0.1 μ M dexamethasone in a biochemically defined environment for up to 48 h in order to characterize the cellular alterations in insulin action resulting from long term glucocorticoid treatment. After treatment, adipocytes were isolated by collagenase digestion, and insulin action was determined by acute experiments that examined insulin binding, hexose transport, and glucose metabolism. Neither 2-h nor longer 10- to 48-h exposures to dexamethasone influenced insulin receptor number or affinity. Conversely, steroid treatment directly reduced both hexose transport and glucose metabolism, and the extent of the effects was dependent on the length of exposure. Two-hour treatment with dexamethasone reduced glucose transport and metabolism in the absence but not the presence of insulin. However, after 10- to 48-h exposures to dexamethasone, both basal and insulin-stimulated hexose uptake and glucose metabolism were significantly inhibited. For example, after 24-h treatment, -de...

Modulation by adrenalectomy and fasting of insulin effects in perfused hindlimb muscle.

Perfused hindlimb muscle from fed adrenalectomized rats accumulated more 2-deoxyglucose at submaximal concentrations of insulin in comparison to muscle from fed normal rats. However, in the fasted state, insulin-stimulated 2-deoxyglucose uptake was largely inhibited by adrenalectomy. Basal 2-deoxyglucose uptake did not differ between fed and fasted normal or adrenalectomized rats. The changes in insulin effects caused by adrenalectomy were due to altered hexose transport as shown by measurements of 3-O-methylglucose uptake and of intracellular free and phosphorylated 2-deoxyglucose. Muscles of fasted normal and fed or fasted adrenalectomized rats showed higher basal glycogen synthase --glucose-6-P/+glucose-6-P activity ratios than those of fed normal rats probably because of decreased glycogen content. However, muscles from fed or fasted adrenalectomized rats did not show any alterations in insulin effects on the activity ratio and half-maximal activation constant (A0.5) for glucose-6-P of glycogen synthase. Because of the dissociation of the effects of insulin on hexose transport and glycogen synthase in muscle of fasted adrenalectomized rats, it is concluded that the impairment in insulin-stimulated hexose transport in these animals is due to a defect lying beyond the interaction of insulin with its receptor.

In vitro effects of a sulfonylurea on insulin action in adipocytes. Potentiation of insulin-stimulated hexose transport.

The mechanism(s) by which the oral sulfonylurea, tolazamide, exerts its extrapancreatic hypoglycemic effects was studied using rat epididymal adipose tissue maintained 20-44 h in the presence or absence of the drug. Insulin binding, hexose transport and glucose metabolism were compared in adipocytes isolated from the cultured tissue. In contrast to earlier reports that suggested that sulfonylureas alter the binding of insulin, neither receptor number nor affinity were changed by tolazamide treatment. The uptake of the glucose analogs 2-deoxyglucose and 3-0-methylglucose in the absence of insulin (i.e., basal) was also unchanged. However, exposure to tolazamide resulted in a potentiation of the stimulatory effects of insulin by approximately 30% at each hormone concentration assayed (0.4-40 ng/ml). This potentiation was dependent on the tolazamide concentration (0.003-0.30 mg/ml), with a maximal effect observed at therapeutic levels. A tolazamide analog hypoglycemic activity in vivo was found not to enhance either basal or insulin-stimulated uptake in vitro. Conversion of 0.1-5.0 mM glucose to CO2 and total lipids in the presence of insulin was also potentiated by tolazamide treatment. The inability of the drug to directly stimulate basal glucose uptake was paralleled by its lack of effect on glucose metabolism. At 50 mM glucose, where transport is no longer rate-limiting, tolazamide did not potentiate metabolism in the absence or the presence of insulin. These studies demonstrate that tolazamide in vitro alters postreceptor insulin action without influencing the receptor, and suggests insulin-stimulated hexose transport as the cellular process responsible for the hypoglycemic effect of sulfonyureas in adipose tissue.