IL-6 increases muscle insulin sensitivity only at superphysiological levels

Exercise induces an increase in glucose transport in muscle. As the acute increase in glucose transport reverses, it is replaced by an increase in insulin sensitivity. Interleukin-6 (IL-6) increases with exercise and has been reported to activate AMP-activated protein kinase (AMPK). Based on this information, we hypothesized that IL-6 would result in an increase in muscle insulin sensitivity. Rat epitrochlearis and soleus muscles were incubated with 120 ng/ml IL-6. Exposure to IL-6 induced a modest acute increase in glucose transport and was followed 3.5 h later by an increase in insulin sensitivity in epitrochlearis but not soleus muscles. IL-6 also brought about an increase in AMPK phosphorylation in epitrochlearis muscles. We conclude that exposure of fast-twitch muscle to 120 ng/ml IL-6 increases insulin sensitivity by activating AMPK. However, exposure of epitrochlearis muscles to 10 ng/ml IL-6, a concentration >100-fold higher than that attained in plasma during exercise, had no effect on glucose transport or insulin sensitivity. These findings provide evidence that the increases in glucose transport and insulin sensitivity induced by IL-6 are pharmacological rather than physiological effects. We interpret our results as evidence that the increase in IL-6 during exercise does not play a role in the exercise-induced increases in muscle glucose uptake and insulin sensitivity.     

Acute effects of troglitazone and nitric oxide on glucose uptake in L929 fibroblast cells

The thiazolidinedione class of antidiabetic drugs, including troglitazone, has an insulin-sensitizing effect for patients with type 2 diabetes. However, in some tissues, studies have shown that troglitazone also has an acute insulin-independent effect on glucose uptake. To determine the extent of this acute action of troglitazone, the effect of troglitazone on 2-deoxyglucose (2DG) uptake in L929 fibroblast cells was measured. Troglitazone stimulated 2DG uptake in a dose dependent manner with a maximum stimulation of >300% at 5-10 microM. In addition, nitric oxide has been shown to stimulate glucose uptake in peripheral muscle tissue. Therefore, the effect of nitric oxide on 2DG uptake in L929 cells was also investigated using the nitric oxide donor, sodium nitroprusside (SNP). SNP stimulated 2DG uptake by >200% with a maximally effective concentration of 5 mM. The combined effect of maximally effective concentrations of both stimulants (10 microM troglitazone + 5 mM SNP) was not additive suggesting a shared pathway for 2DG uptake. However, the nitric oxide synthase inhibitor, N(G)-monomethyl-L-arginine (L-NMMA, 50 microM) had no effect on troglitazone stimulated 2DG uptake, indicating that the troglitazone and nitric oxide pathways converge after nitric oxide production. In addition, 12.5 microM dantrolene was shown to have no effect on either troglitazone or SNP stimulated 2DG uptake suggesting that these stimulatory effects are independent of changes in calcium ion concentrations. These data provide important evidence for the acute regulation of glucose transport through GLUT 1 transporters.     

Increased submaximal insulin-stimulated glucose uptake in mouse skeletal muscle after treadmill exercise.

The primary purpose of this study was to determine the effect of prior exercise on insulin-stimulated glucose uptake with physiological insulin in isolated muscles of mice. Male C57BL/6 mice completed a 60-min treadmill exercise protocol or were sedentary. Paired epitrochlearis, soleus, and extensor digitorum longus (EDL) muscles were incubated with [3H]-2-deoxyglucose without or with insulin (60 microU/ml) to measure glucose uptake. Insulin-stimulated glucose uptake for paired muscles was calculated by subtracting glucose uptake without insulin from glucose uptake with insulin. Muscles from other mice were assessed for glycogen and AMPK Thr172 phosphorylation. Exercised vs. sedentary mice had decreased glycogen in epitrochlearis (48%, P < 0.001), soleus (51%, P < 0.001), and EDL (41%, P < 0.01) and increased AMPK Thr172 phosphorylation (P < 0.05) in epitrochlearis (1.7-fold), soleus (2.0-fold), and EDL (1.4-fold). Insulin-independent glucose uptake was increased 30 min postexercise vs. sedentary in the epitrochlearis (1.2-fold, P < 0.001), soleus (1.4-fold, P < 0.05), and EDL (1.3-fold, P < 0.01). Insulin-stimulated glucose uptake was increased (P < 0.05) approximately 85 min after exercise in the epitrochlearis (sedentary: 0.266 +/- 0.045 micromol x g(-1) x 15 min(-1); exercised: 0.414 +/- 0.051) and soleus (sedentary: 0.102 +/- 0.049; exercised: 0.347 +/- 0.098) but not in the EDL. Akt Ser473 and Akt Thr308 phosphorylation for insulin-stimulated muscles did not differ in exercised vs. sedentary. These results demonstrate enhanced submaximal insulin-stimulated glucose uptake in the epitrochlearis and soleus of mice 85 min postexercise and suggest that it will be feasible to probe the mechanism of enhanced postexercise insulin sensitivity by using genetically modified mice.     

UCP3 gene expression does not correlate with muscle oxidation rates in troglitazone-treated Zucker fatty rats

Uncoupling protein-3 (UCP3), a mitochondrial carrier protein predominantly expressed in muscle, has been suggested to release stored energy as heat. The insulin-sensitizing thiazolidinediones enhance glucose disposal in skeletal muscle and have been reported to increase the expression of uncoupling proteins in various experimental systems. We therefore studied the effect of troglitazone treatment on UCP3 gene expression in muscles from lean and obese Zucker rats. In comparison with obese littermates, basal UCP3 mRNA levels in lean Zucker rats tended to be higher in white and red gastrocnemius muscles, but were lower in soleus (P<0.001) muscle and heart (P<0.01). In lean rats, troglitazone significantly increased UCP3 gene expression in white and red gastrocnemius and heart muscles (all P<0.01). In contrast, the drug reduced UCP3 mRNA expression in red gastrocnemius and soleus muscles of obese littermates (all P<0.001). The troglitazone-dependent decrease in UCP3 gene expression was accompanied by an increased weight gain in obese rats, while no such effect was observed in lean rats. In obese rats, improvement of insulin resistance by troglitazone was associated with increased rates of basal and insulin-stimulated CO(2) production from glucose measured in soleus muscle. These studies demonstrate that effects of troglitazone on UCP3 gene expression depend on the phenotype of Zucker rats and that troglitazone-induced metabolic improvements are not related to increased uncoupling resulting from upregulation of UCP3 mRNA expression in muscle.     

Nonradioisotope assay of glucose uptake activity in rat skeletal muscle using enzymatic measurement of 2-deoxyglucose 6-phosphate in vitro and in vivo

We investigated a nonradioisotope method for the evaluation of glucose uptake activity using enzymatic measurement of 2-deoxyglucose 6-phosphate (2DG6P) content in isolated rat soleus muscle in vitro and in vivo. The 2DG6P content in isolated rat soleus muscle after incubation with 2-deoxyglucose (2DG) was increased in a dose-dependent manner by insulin (ED(50) = 0.6 mU/ml), the maximum response being about 5 times that of the basal content in vitro. This increment was completely abolished by wortmannin (100 nM), with no effect on basal 2DG6P content. An insulin-mimetic compound, vanadium, also increased 2DG6P content in a dose-dependent manner. In isolated soleus muscle of Zucker fa/fa rats, well known as an insulin-resistant model, insulin did not increase 2DG6P content. The 2DG6P content in rat soleus muscle increased after 2DG (3 mmol/kg) injection in vivo, and conversely, the 2DG concentration in plasma was decreased in a dose-dependent manner by insulin (ED(50) = 0.11 U/kg). The maximum response of the accumulation of 2DG6P in soleus muscle was about 4 times that of the basal content. This method could be useful for evaluating glucose uptake (transport plus phosphorylation) activity in soleus muscle in vitro and in vivo without using radioactive materials.     

Improved insulin sensitivity with calorie restriction does not require reduced JNK1/2, p38, or ERK1/2 phosphorylation in skeletal muscle of 9-month-old rats

Calorie restriction [CR; ～40% below ad libitum (AL) intake] improves the health of many species, including rats, by mechanisms that may be partly related to enhanced insulin sensitivity for glucose disposal by skeletal muscle. Excessive activation of several mitogen-activated protein kinases (MAPKs), including JNK1/2, p38, and ERK1/2 has been linked to insulin resistance. Although insulin can activate ERK1/2, this effect is not required for insulin-mediated glucose uptake. We hypothesized that skeletal muscle from male 9-mo-old Fischer 344/Brown Norway rats CR (35-40% beginning at 3 mo old) versus AL rats would have 1) attenuated activation of JNK1/2, p38, and ERK1/2 under basal conditions; and 2) no difference for insulin-induced ERK1/2 activation. In contrast to our hypothesis, there were significant CR-related increases in the phosphorylation of p38 (epitrochlearis, soleus, and gastrocnemius), JNK1 (epitrochlearis and soleus), and JNK2 (gastrocnemius). Consistent with our hypothesis, CR did not alter insulin-mediated ERK1/2 activation. The greater JNK1/2 and p38 phosphorylation with CR was not attributable to diet effects on muscle oxidative stress (assessed by protein carbonyls and 4-hydroxynonenal protein conjugates). In muscles from the same rats used for the present study, we previously reported a CR-related increase in insulin-mediated glucose uptake by the epitrochlearis and the soleus (Sharma N, Arias EB, Bhat AD, Sequea DA, Ho S, Croff KK, Sajan MP, Farese RV, Cartee GD. Am J Physiol Endocrinol Metab 300: E966-E978, 2011). The present results indicate that the improved insulin sensitivity with CR is not attributable to attenuated MAPK phosphorylation in skeletal muscle.     

Epinephrine inhibits insulin-stimulated muscle glucose transport.

We recently demonstrated that epinephrine could inhibit the activation by insulin of insulin receptor substrate-1 (IRS-1)-associated phosphatidylinositol 3-kinase (PI3-kinase) in skeletal muscle (Hunt DG, Zhenping D, and Ivy JL. J Appl Physiol 92: 1285-1292, 2002). Activation of PI3-kinase is recognized as an essential step in the activation of muscle glucose transport by insulin. We therefore investigated the effect of epinephrine on insulin-stimulated glucose transport in both fast-twitch (epitrochlearis) and slow-twitch (soleus) muscle of the rat by using an isolated muscle preparation. Glucose transport was significantly increased in the epitrochlearis and soleus when incubated in 50 and 100 microU/ml insulin, respectively. Activation of glucose transport by 50 microU/ml insulin was inhibited by 24 nM epinephrine in both muscle types. This inhibition of glucose transport by epinephrine was accompanied by suppression of IRS-1-associated PI3-kinase activation. However, when muscles were incubated in 100 microU/ml insulin, 24 nM epinephrine was unable to inhibit IRS-1-associated PI3-kinase activation or glucose transport. Even when epinephrine concentration was increased to 500 nM, no attenuating effect was observed on glucose transport. Results of this study indicate that epinephrine is capable of inhibiting glucose transport activated by a moderate, but not a high, physiological insulin concentration. The inhibition of glucose transport by epinephrine appears to involve the inhibition of IRS-1-associated PI3-kinase activation.     

PPARdelta activator GW-501516 has no acute effect on glucose transport in skeletal muscle

It has been reported that treatment of cultured human skeletal muscle myotubes with the peroxisome proliferator-activated receptor-delta (PPARdelta) activator GW-501516 directly stimulates glucose transport and enhances insulin action. Cultured myotubes are minimally responsive to insulin stimulation of glucose transport and are not a good model for studying skeletal muscle glucose transport. The purpose of this study was to evaluate the effect of GW-501516 on glucose transport to determine whether the findings on cultured myotubes have relevance to skeletal muscle. Rat epitrochlearis and soleus muscles were treated for 6 h with 10, 100, or 500 nM GW-501516, followed by measurement of 2-deoxyglucose uptake. GW-501516 had no effect on glucose uptake. There was no effect on insulin sensitivity or responsiveness. Also, in contrast to findings on myotubes, treatment of muscles with GW-501516 did not result in increased phosphorylation or increased expression of AMP-activated protein kinase (AMPK) or p38 mitogen-activated protein kinase (MAPK). Treatment of epitrochlearis muscles with GW-501516 for 24 h induced a threefold increase in uncoupling protein-3 mRNA, providing evidence that the GW-501516 compound that we used gets into and is active in skeletal muscle. In conclusion, our results show that, in contrast to myotubes in culture, skeletal muscle does not respond to GW-501516 with 1) an increase in AMPK or p38 MAPK phosphorylation or expression or 2) direct stimulation of glucose transport or enhanced insulin action.     

Glucose transport into rat skeletal muscle: interaction between exercise and insulin

This study was done to evaluate the effect of insulin on sugar transport into skeletal muscle after exercise. The permeability of rat epitrochlearis muscle to 3-O-methylglucose (3-MG) was measured after exposure to a range of insulin concentrations 30, 60, and 180 min after a bout of exercise. Thirty and 60 min after exercise, the effects of exercise and insulin on 3-MG transport were additive over a wide range of insulin concentrations, with no increase in sensitivity or responsiveness to insulin. After 180 min, when approximately 66% of the exercise-induced increase in sugar transport had worn off, both the responsiveness and sensitivity of the glucose transport process to insulin were increased. These findings appear compatible with the hypothesis that the actions of exercise and insulin result in activation and/or translocation into the plasma membrane of two separate pools of glucose transporters in mammalian skeletal muscle.     

Propranolol prevents epinephrine from limiting insulin-stimulated muscle glucose uptake during contraction.

Beta-blockade results in rapid glucose clearance and premature fatigue during exercise. To investigate the cause of this increased glucose clearance, we studied the acute effects of propranolol on insulin-stimulated muscle glucose uptake during contraction in the presence of epinephrine with an isolated rat muscle preparation. Glucose uptake increased in both fast- (epitrochlearis) and slow-twitch (soleus) muscle during insulin or contraction stimulation. In the presence of 24 nM epinephrine, glucose uptake during contraction was completely suppressed when insulin was present. This suppression of glucose uptake by epinephrine was accompanied by a decrease in insulin receptor substrate (IRS)-1-phosphatidylinositol 3 (PI3)-kinase activity. Propranolol had no direct effect on insulin-stimulated glucose uptake during contraction. However, epinephrine was ineffective in attenuating insulin-stimulated glucose uptake during contraction in the presence of propranolol. This ineffectiveness of epinephrine to suppress insulin-stimulated glucose uptake during contraction occurred in conjunction with its inability to completely suppress IRS-1-PI3-kinase activity. Results of this study indicate that the effectiveness of epinephrine to inhibit insulin-stimulated glucose uptake during contraction is severely diminished in muscle exposed to propranolol. Thus the increase in glucose clearance and premature fatigue associated with beta-blockade could result from the inability of epinephrine to attenuate insulin-stimulated muscle glucose uptake.     

Effects of exercise on insulin binding and glucose metabolism in muscle

To elucidate the mechanism of enhanced insulin sensitivity by muscle after exercise, we studied insulin binding, 2-deoxy-D-[1-14C]glucose (2-DOG) uptake and [5-3H]glucose utilization in glycolysis and glycogenesis in soleus and extensor digitorum longus (EDL) muscles of mice after 60 min of treadmill exercise. In the soleus, glycogenesis was increased after exercise (P less than 0.05) and remained sensitive to the action of insulin. Postexercise insulin-stimulated glycolysis was also increased in the soleus (P less than 0.05). In the EDL, glycogenesis was increased after exercise (P less than 0.05). However, this was already maximal in the absence of insulin and was not further stimulated by insulin (0.1-4 nM). The disposal of glucose occurred primarily via the glycolytic pathway (greater than 60%) in the soleus and EDL at rest and after exercise. The uptake of 2-DOG uptake was not altered in the soleus after exercise (4 h incubation at 18 degrees C). However, with 1-h incubations at 37 degrees C, a marked increase in 2-DOG uptake after exercise was observed in the soleus (P less than 0.05) in the absence (0 nM) and presence of insulin (0.2-4 nM) (P less than 0.05). A similar postexercise increase in 2-DOG uptake occurred in EDL. Despite the marked increase in glucose uptake and metabolism, no changes in insulin binding were apparent in either EDL or soleus at 37 degrees C or 18 degrees C. This study shows that the postexercise increase of glucose disposal does not appear to be directly attributable to increments in insulin binding to slow-twitch and fast-twitch muscles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of endurance and sprint exercise on the sensitivity of glucose metabolism to insulin in the epitrochlearis muscle of sedentary and trained rats

The effects of two types of acute exercise (1 h treadmill running at 20 m.min-1, or 6 x 10-s periods at 43 m.min-1, 0 degree inclination), as well as two training regimes (endurance and sprint) on the sensitivity of epitrochlearis muscle [fast twitch (FT) fibres] to insulin were measured in vitro in rats. The hormone concentration in the incubation medium producing the half maximal stimulation of lactate (la) production and glycogen synthesis was determined and used as an index of the muscle insulin sensitivity. A single period of moderate endurance as well as the sprint-type exercise increased the sensitivity of la production to insulin although the rate of la production enhanced markedly only after sprint exercise at 10 and 100 microU.ml-1 of insulin. These effects persisted for up to 2 h after the termination of exercise. Both types of exercise significantly decreased the muscle glycogen content, causing a moderate enhancement in the insulin-stimulated rates of glycogen synthesis in vitro for up to 2 h after exercise. However, a significant increase in the sensitivity of this process to insulin was found only in the muscle removed 0.25 h after the sprint effort. Training of the sprint and endurance types increased insulin-stimulated rates of glycolysis 24 h after the last period of exercise. The sensitivity of this process to insulin was also increased at this instant. Both types of training increased the basal and maximal rates of glycogen synthesis, as well as the sensitivity of this process to insulin at the 24th h following the last training session.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metformin, but not exercise training, increases insulin responsiveness in skeletal muscle of Sprague-Dawley rats

We assessed the effects of combined metformin treatment and exercise training on body composition, on insulin concentration following glucose loading, on insulin-stimulated glucose transport in skeletal muscle, and on muscle glycogen content. Male Sprague-Dawley rats were treated for 35 days with or without metformin (320 mg/kg/day) and/or treadmill exercise training (20 min at 20 m/min, 5 days/wk). Because metformin reduces food intake, pair-fed controls were included. Metformin, training, and pair-feeding all decreased food intake, body weight, and insulin concentration following glucose loading. Metformin and training reduced intra-abdominal fat, but pair feeding did not. In isolated strips derived from soleus, epitrochlearis and extensor carpi ulnaris muscles, metformin increased insulin-stimulated transport of [3H]-2-deoxyglucose by 90%, 89% and 125%, respectively (P < 0.02) and training increased [3H]-2-deoxyglucose transport in the extensor carpi ulnaris muscle only (66%, P < 0.05). Pair-feeding did not alter [3H]-2-deoxyglucose transport. Training increased gastrocnemius muscle glycogen by 100% (P < 0.001). Metformin and pair-feeding did not alter muscle glycogen. We conclude that metformin reverses the maturation-induced impairment of insulin responsiveness in Sprague-Dawley rats by increasing insulin-stimulated glucose transport in skeletal muscle and that this effect is not secondary to reduced food intake. We also conclude that metformin and exercise training may increase insulin sensitivity by different mechanisms, with training causing increased glucose transport only in some muscles and also causing increased muscle glycogen storage.     

High-fat diet-induced muscle insulin resistance: relationship to visceral fat mass

It has been variously hypothesized that the insulin resistance induced in rodents by a high-fat diet is due to increased visceral fat accumulation, to an increase in muscle triglyceride (TG) content, or to an effect of diet composition. In this study we used a number of interventions: fish oil, leptin, caloric restriction, and shorter duration of fat feeding, to try to disassociate an increase in visceral fat from muscle insulin resistance. Substituting fish oil (18% of calories) for corn oil in the high-fat diet partially protected against both the increase in visceral fat and muscle insulin resistance without affecting muscle TG content. Injections of leptin during the last 4 days of a 4-wk period on the high-fat diet partially reversed the increase in visceral fat and the muscle insulin resistance, while completely normalizing muscle TG. Restricting intake of the high-fat diet to 75% of ad libitum completely prevented the increase in visceral fat and muscle insulin resistance. Maximally insulin-stimulated glucose transport was negatively correlated with visceral fat mass (P < 0.001) in both the soleus and epitrochlearis muscles and with muscle TG concentration in the soleus (P < 0.05) but not in the epitrochlearis. Thus we were unable to dissociate the increase in visceral fat from muscle insulin resistance using a variety of approaches. These results support the hypothesis that an increase in visceral fat is associated with development of muscle insulin resistance.     

Insulin sensitivity and responsiveness of epitrochlearis and soleus muscles from fed and starved rats. Recognition of differential changes in insulin sensitivities of protein synthesis and glucose incorporation into glycogen.

The insulin sensitivity of protein synthesis and glucose incorporation into glycogen by the soleus and epitrochlearis muscles from fed rats and 24 h-starved rats was determined in vitro during the first and second hours of incubation after isolation of the muscles. Rates of protein synthesis by both muscles from fed rats in the first hour of incubation were 2-fold higher than in the second hour and were not increased by insulin. Rates of protein synthesis during the first hour in the presence of 6000 microunits of insulin/ml were increased in soleus, but not in epitrochlearis, muscles from starved rats. Rates of protein synthesis in both muscles from fed and starved rats were increased significantly by insulin during the second hour. High concentrations of insulin caused a marked stimulation of the rates of glucose incorporation by both muscles from fed and starved rats in both the first and second hours of incubation. The insulin sensitivity of glucose incorporation during the second hour, defined as the concentration of insulin causing half-maximal stimulation, was increased 10-fold for both muscle types from starved rats (soleus, 65 microunits/ml; epitrochlearis, 45 microunits/ml) relative to muscles from fed rats (soleus, 600 microunits/ml; epitrochlearis, 500 microunits/m). The insulin sensitivity of protein synthesis in the second hour was greater for soleus muscles from starved rats (65 microunits/ml) than from fed rats (500 microunits/ml). In contrast, the insulin sensitivity of protein synthesis in epitrochlearis muscles from starved rats was significantly decreased (225 microunits/ml) compared with fed rats (25 microunits/ml Maximal rates achieved by high concentrations of insulin were not different from those in the same muscle from fed rats. It is suggested that protein synthesis, in distinction to glucose utilization, may be resistant to insulin stimulation during periods of acute starvation in muscles with fibre compositions similar to the epitrochlearis, but not in muscles with fibre compositions similar to the soleus. Partial reversal of the resistance observed in vitro for epitrochlearis muscles from starved rats may be due to the loss of factors which suppress the effect of insulin in vivo.     

Modulation of insulin resistance and hypertension by voluntary exercise training in the TG(mREN2)27 rat.

Hypertension is often accompanied by insulin resistance of skeletal muscle glucose transport. The male heterozygous TG(mREN2)27 rat, which harbors a mouse transgene for renin, displays local elevations in the renin-angiotensin system and exhibits markedly elevated systolic blood pressure (SBP). The present study was undertaken to characterize insulin-stimulated skeletal muscle glucose transport in male heterozygous TG(mREN2)27 rats and to evaluate the effect of voluntary exercise training on SBP and skeletal muscle glucose transport. Compared with normotensive Sprague-Dawley rats, TG(mREN2)27 rats displayed a 53% elevation (P < 0.05) in SBP, a twofold increase in plasma free fatty acid levels, and an exaggerated insulin response during an oral glucose tolerance test. Moreover, insulin-mediated glucose transport (2-deoxyglucose uptake) in isolated epitrochlearis and soleus muscles of TG(mREN2)27 animals was 33 and 43% less, respectively, than in Sprague-Dawley controls. TG(mREN2)27 rats ran voluntarily for 6 wk and achieved daily running distances of 6-7 km over the final 3 wk. Training caused a 36% increase in peak aerobic capacity and a 16% reduction in resting SBP. Fasting plasma insulin (21%) and free fatty acid (34%) levels were reduced in the trained TG(mREN2)27 rats. Whole body glucose tolerance was improved in the trained TG(mREN2)27 rats and was associated with increases of 39 and 50% in insulin-mediated glucose transport in epitrochlearis and soleus muscles, respectively. Whole muscle GLUT-4 protein was increased in the soleus (23%), but not in the epitrochlearis, of trained TG(mREN2)27 rats. These data indicate that the male heterozygous TG(mREN2)27 rat is a model of both hypertension and insulin resistance. Importantly, both of these defects can be beneficially modified by voluntary exercise training.     

Phorbol esters affect skeletal muscle glucose transport in a fiber type-specific manner

Recent evidence has shown that activation of lipid-sensitive protein kinase C (PKC) isoforms leads to skeletal muscle insulin resistance. However, earlier studies demonstrated that phorbol esters increase glucose transport in skeletal muscle. The purpose of the present study was to try to resolve this discrepancy. Treatment with the phorbol ester 12-deoxyphorbol-13-phenylacetate 20-acetate (dPPA) led to an approximately 3.5-fold increase in glucose transport in isolated fast-twitch epitrochlearis and flexor digitorum brevis muscles. Phorbol ester treatment was additive to a maximally effective concentration of insulin in fast-twitch skeletal muscles. Treatment with dPPA did not affect insulin signaling in the epitrochlearis. In contrast, phorbol esters had no effect on basal glucose transport and inhibited maximally insulin-stimulated glucose transport approximately 50% in isolated slow-twitch soleus muscle. Furthermore, dPPA treatment inhibited the insulin-stimulated tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and the threonine and serine phosphorylation of PKB by approximately 50% in the soleus. dPPA treatment also caused serine phosphorylation of IRS-1 in the slow-twitch soleus muscle. In conclusion, our results show that phorbol esters stimulate glucose transport in fast-twitch skeletal muscles and inhibit insulin signaling in slow-twitch soleus muscle of rats. These findings suggest that mechanisms other than PKC activation mediate lipotoxicity-induced whole body insulin resistance.     

Effect of exercise training on insulin binding and glucose metabolism in mouse soleus muscle

Training stimulates glucose uptake and metabolism by muscles independent of a rise in serum glucose. Whether this increased insulin action is associated with enhanced insulin binding in muscles is unknown. We studied the effect of 6 weeks of treadmill running on insulin binding, uptake of 2-deoxy-D-glucose, glycolysis, and glycogenesis by the soleus muscle of Swiss Webster mice. Training was progressively increased. The in vitro studies using intact soleus preparations were done 48 h after the last exercise bout. Training increased insulin binding, insulin-stimulated uptake of 2-deoxy-D-glucose, and glycogenesis but not glycolysis in the soleus. Our data suggest that the enhanced glucose uptake and metabolism in muscles induced by exercise training are associated with an increase in insulin binding.     

Insulin action and signalling in fat and muscle from dexamethasone-treated rats

Glucocorticoids initiate whole body insulin resistance and the aim of the present study was to investigate effects of dexamethasone on protein expression and insulin signalling in muscle and fat tissue. Rats were injected with dexamethasone (1 mg/kg/day, i.p.) or placebo for 11 days before insulin sensitivity was evaluated in vitro in soleus and epitrochlearis muscles and in isolated epididymal adipocytes. Dexamethasone treatment reduced insulin-stimulated glucose uptake and glycogen synthesis by 30-70% in epitrochlearis and soleus, and insulin-stimulated glucose uptake by ∼40% in adipocytes. 8-bromo-cAMP-stimulated lipolysis was ∼2-fold higher in adipocytes from dexamethasone-treated rats and insulin was less effective to inhibit cAMP-stimulated lipolysis. A main finding was that dexamethasone decreased expression of PKB and insulin-stimulated Ser⁴⁷³ and Thr³⁰⁸ phosphorylation in both muscles and adipocytes. Expression of GSK-3 was not influenced by dexamethasone treatment in muscles or adipocytes and insulin-stimulated GSK-3β Ser⁹ phosphorylation was reduced in muscles only. A novel finding was that glycogen synthase (GS) Ser⁷ phosphorylation was higher in both muscles from dexamethasone-treated rats. GS expression decreased (by 50%) in adipocytes only. Basal and insulin-stimulated GS Ser⁶⁴¹ and GS Ser^{645,649,653,657} phosphorylation was elevated in epitrochlearis and soleus muscles and GS fractional activity was reduced correspondingly. In conclusion, dexamethasone treatment (1) decreases PKB expression and insulin-stimulated phosphorylation in both muscles and adipocytes, and (2) increases GS phosphorylation (reduces GS fractional activity) in muscles and decreases GS expression in adipocytes. We suggest PKB and GS as major targets for dexamethasone-induced insulin resistance.     

Insulin receptor substrate-2 is not necessary for insulin- and exercise-stimulated glucose transport in skeletal muscle.

Insulin receptor substrate-2-deficient (IRS2(-/-)) mice develop type 2 diabetes. The purpose of this study was to determine whether there is a defect in basal, insulin-, and exercise-stimulated glucose transport in the skeletal muscle of these animals. IRS2(-/-) and wild-type (WT) mice (male, 8-10 weeks) exercised on a treadmill for 1 h or remained sedentary. 2-Deoxyglucose (2DG) uptake was measured in isolated soleus muscles incubated in vitro in the presence or absence of insulin. Resting blood glucose concentration in IRS2(-/-) mice (10.3 mM) was higher than WT animals (4.1 mM), but there was a wide range among the IRS2(-/-) mice (3-25 mM). Therefore, IRS2(-/-) mice were divided into two subgroups based on blood glucose concentrations (IRS2(-/-)L < 7.2 mM, IRS2(-/-)H > 7.2 mM). Only IRS2(-/-)H had lower basal, exercise-, and submaximally insulin-stimulated 2DG uptake, while maximal insulin-stimulated 2DG uptake was similar among the three groups. The ED(50) for insulin to stimulate 2DG uptake above basal in IRS2(-/-)H was higher than WT and IRS2(-/-)L mice, suggesting insulin resistance in the skeletal muscle from the IRS2(-/-) mice with high blood glucose concentrations. Furthermore, resting blood glucose concentrations from all groups were negatively correlated to submaximally insulin-stimulated 2DG uptake (r(2) = 0.33, p < 0.01). Muscle GLUT4 content was significantly lower in IRS2(-/-)H mice compared with WT and IRS2(-/-)L mice. These results demonstrate that the IRS2 protein in muscle is not necessary for insulin- or exercise-stimulated glucose transport, suggesting that the onset of diabetes in the IRS2(-/-) mice is not due to a defect in skeletal muscle glucose transport; hyperglycemia may cause insulin resistance in the muscle of IRS2(-/-) mice.