Heterogeneous Effects of Calorie Restriction on In Vivo Glucose Uptake and Insulin Signaling of Individual Rat Skeletal Muscles

Calorie restriction (CR) (consuming ∼60% of ad libitum, AL, intake) improves whole body insulin sensitivity and enhances insulin-stimulated glucose uptake by isolated skeletal muscles. However, little is known about CR-effects on in vivo glucose uptake and insulin signaling in muscle. Accordingly, 9-month-old male AL and CR (initiated when 3-months-old) Fischer 344xBrown Norway rats were studied using a euglycemic-hyperinsulinemic clamp with plasma insulin elevated to a similar level (∼140 µU/ml) in each diet group. Glucose uptake (assessed by infusion of [¹⁴C]-2-deoxyglucose, 2-DG), phosphorylation of key insulin signaling proteins (insulin receptor, Akt and Akt substrate of 160kDa, AS160), abundance of GLUT4 and hexokinase proteins, and muscle fiber type composition (myosin heavy chain, MHC, isoform percentages) were determined in four predominantly fast-twitch (epitrochlearis, gastrocnemius, tibialis anterior, plantaris) and two predominantly slow-twitch (soleus, adductor longus) muscles. CR did not result in greater GLUT4 or hexokinase abundance in any of the muscles, and there were no significant diet-related effects on percentages of MHC isoforms. Glucose infusion was greater for CR versus AL rats (P<0.05) concomitant with significantly (P<0.05) elevated 2-DG uptake in 3 of the 4 fast-twitch muscles (epitrochlearis, gastrocnemius, tibialis anterior), without a significant diet-effect on 2-DG uptake by the plantaris or either slow-twitch muscle. Each of the muscles with a CR-related increase in 2-DG uptake was also characterized by significant (P<0.05) increases in phosphorylation of both Akt and AS160. Among the 3 muscles without a CR-related increase in glucose uptake, only the soleus had significant (P<0.05) CR-related increases in Akt and AS160 phosphorylation. The current data revealed that CR leads to greater whole body glucose disposal in part attributable to elevated in vivo insulin-stimulated glucose uptake by fast-twitch muscles. The results also demonstrated that CR does not uniformly enhance either insulin signaling or insulin-stimulated glucose uptake in all muscles in vivo.     

Defective insulin signaling in skeletal muscle of the hypertensive TG(mREN2)27 rat

Essential hypertension is frequently associated with insulin resistance of skeletal muscle glucose transport, with a potential role of angiotensin II in the pathogenesis of both conditions. The male heterozygous TG(mREN2)27 rat harbors the mouse transgene for renin, exhibits local elevations in angiotensin II, and is an excellent model of both hypertension and insulin resistance. The present study was designed to investigate the potential cellular mechanisms for insulin resistance in this hypertensive animal model, including an assessment of elements of the insulin-signaling pathway. Compared with nontransgenic, normotensive Sprague-Dawley control rats, male heterozygous TG(mREN2)27 rats displayed elevated (P < 0.05) fasting plasma insulin (74%), an exaggerated insulin response (108%) during an oral glucose tolerance test, and reduced whole body insulin sensitivity. TG(mREN2)27 rats also exhibited decreased insulin-mediated glucose transport and glycogen synthase activation in both the type IIb epitrochlearis (30 and 46%) and type I soleus (22 and 64%) muscles. Importantly, there were significant reductions (approximately 30-50%) in insulin stimulation of tyrosine phosphorylation of the insulin receptor beta-subunit and insulin receptor substrate-1 (IRS-1), IRS-1 associated with the p85 subunit of phosphatidylinositol 3-kinase, Akt Ser473 phosphorylation, and Ser9 phosphorylation of glycogen synthase kinase-3beta in epitrochlearis and soleus muscles of TG(mREN2)27 rats. Soleus muscle triglyceride concentration was 25% greater in the transgenic group compared with nontransgenic animals. Collectively, these data provide the first evidence that the insulin resistance of the hypertensive male heterozygous TG(mREN2)27 rat can be attributed to specific defects in the insulin-signaling pathway in skeletal muscle.     

Mechanisms for increased insulin-stimulated Akt phosphorylation and glucose uptake in fast- and slow-twitch skeletal muscles of calorie-restricted rats

Calorie restriction [CR; ~65% of ad libitum (AL) intake] improves insulin-stimulated glucose uptake (GU) and Akt phosphorylation in skeletal muscle. We aimed to elucidate the effects of CR on 1) processes that regulate Akt phosphorylation [insulin receptor (IR) tyrosine phosphorylation, IR substrate 1-phosphatidylinositol 3-kinase (IRS-PI3K) activity, and Akt binding to regulatory proteins (heat shock protein 90, Appl1, protein phosphatase 2A)]; 2) Akt substrate of 160-kDa (AS160) phosphorylation on key phosphorylation sites; and 3) atypical PKC (aPKC) activity. Isolated epitrochlearis (fast-twitch) and soleus (slow-twitch) muscles from AL or CR (6 mo duration) 9-mo-old male F344BN rats were incubated with 0, 1.2, or 30 nM insulin and 2-deoxy-[(3)H]glucose. Some CR effects were independent of insulin dose or muscle type: CR caused activation of Akt (Thr(308) and Ser(473)) and GU in both muscles at both insulin doses without CR effects on IRS1-PI3K, Akt-PP2A, or Akt-Appl1. Several muscle- and insulin dose-specific CR effects were revealed. Akt-HSP90 binding was increased in the epitrochlearis; AS160 phosphorylation (Ser(588) and Thr(642)) was greater for CR epitrochlearis at 1.2 nM insulin; and IR phosphorylation and aPKC activity were greater for CR in both muscles with 30 nM insulin. On the basis of these data, our working hypothesis for improved insulin-stimulated GU with CR is as follows: 1) elevated Akt phosphorylation is fundamental, regardless of muscle or insulin dose; 2) altered Akt binding to regulatory proteins (HSP90 and unidentified Akt partners) is involved in the effects of CR on Akt phosphorylation; 3) Akt effects on GU depend on muscle- and insulin dose-specific elevation in phosphorylation of Akt substrates, including, but not limited to, AS160; and 4) greater IR phosphorylation and aPKC activity may contribute at higher insulin doses.     

Preventing the calorie restriction-induced increase in insulin-stimulated Akt2 phosphorylation eliminates calorie restriction's effect on glucose uptake in skeletal muscle

Calorie restriction (CR; ~60% of ad libitum, AL, consumption) improves insulin-stimulated glucose uptake in skeletal muscle. The precise cellular mechanism for this healthful outcome is unknown, but it is accompanied by enhanced insulin-stimulated activation of Akt. Previous research using Akt2-null mice demonstrated that Akt2 is essential for the full CR-effect on insulin-stimulated glucose uptake by muscle. However, because Akt2-null mice were completely deficient in Akt2 in every cell throughout life, it would be valuable to assess the efficacy of transient, muscle-specific Akt inhibition for attenuation of CR-effects on glucose uptake. Accordingly, we used a selective Akt inhibitor (MK-2206) to eliminate the CR-induced elevation in insulin-stimulated Akt2 phosphorylation and determined the effects on Akt substrates and glucose uptake. We incubated isolated epitrochlearis muscles from 9-month-old AL and CR (~60-65% of AL intake for 6months) rats with or without MK-2206 and measured insulin-stimulated (1.2nM) glucose uptake and phosphorylation of the insulin receptor (Tyr1162/1163), pan-Akt (Thr308 and Ser473), Akt2 (Thr308 and Ser473), AS160/TBC1D4 (Thr642), and Filamin C (Ser2213). Incubation of isolated skeletal muscles with a dose of a selective Akt inhibitor that eliminated the CR-induced increases in Akt2 phosphorylation prevented CR's effects on insulin-stimulated glucose uptake, pAS160(Thr642) and pFilamin C(Ser2213) without altering pIR(Tyr1162/1163). These data provide compelling new evidence linking the CR-induced increase in insulin-stimulated Akt2 phosphorylation to CR's effects on insulin-mediated phosphorylation of Akt substrates and glucose uptake in skeletal muscle.     

Vasoconstrictor effects of insulin in skeletal muscle arterioles are mediated by ERK1/2 activation in endothelium

Insulin exerts both NO-dependent vasodilator and endothelin-dependent vasoconstrictor effects on skeletal muscle arterioles. The intracellular enzymes 1-phosphatidylinositol 3-kinase (PI3-kinase) and Akt have been shown to mediate the vasodilator effects of insulin, but the signaling molecules involved in the vasoconstrictor effects of insulin in these arterioles are unknown. Our objective was to identify intracellular mediators of acute vasoconstrictor effects of insulin on skeletal muscle arterioles. Rat cremaster first-order arterioles (n=40) were isolated, and vasoreactivity to insulin was studied using a pressure myograph. Insulin induced dose-dependent vasoconstriction of skeletal muscle arterioles (up to -22 +/- 3% of basal diameter; P <0.05) during PI3-kinase inhibition with wortmannin (50 nmol/l). Insulin-induced vasoconstriction was abolished by inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) with PD-98059 (40 micromol/l). In addition, inhibition of ERK1/2 without PI3-kinase inhibition uncovered insulin-mediated vasodilatation in skeletal muscle arterioles (up to 37 +/- 10% of baseline diameter; P <0.05). Effects of insulin on ERK1/2 activation in arterioles were then investigated by Western blot analysis. Insulin induced a transient 2.4-fold increase in ERK1/2 phosphorylation (maximal at approximately 15 min) in skeletal muscle arterioles (P <0.05). Removal of the arteriolar endothelium abolished insulin-induced vasoconstriction, which suggests that activation of ERK1/2 in endothelial cells is involved in acute insulin-mediated vasoconstriction. To investigate this, acute effects of insulin on ERK1/2 phosphorylation were studied in human microvascular endothelial cells. In support of the findings in skeletal muscle arterioles, insulin induced a 1.9-fold increase in ERK1/2 phosphorylation (maximal at approximately 15 min) in microvascular endothelial cells (P <0.05). We conclude that acute vasoconstrictor effects of insulin in skeletal muscle arterioles are mediated by activation of ERK1/2 in endothelium. This ERK1/2-mediated vasoconstrictor effect antagonizes insulin-induced, PI3-kinase-dependent vasodilatation in skeletal muscle arterioles. These findings provide a novel mechanism by which insulin may determine blood flow and glucose disposal in skeletal muscle.     

NKCC activity restores muscle water during hyperosmotic challenge independent of insulin,ERK, and p38 MAPK

In isosmotic conditions, insulin stimulation of PI 3-K/Akt and p38 MAPK pathways in skeletal muscle inhibits Na(+)-K(+)-2Cl(-) cotransporter (NKCC) activity induced by the ERK1,2 MAPK pathway. Whether these signaling cascades contribute to NKCC regulation during osmotic challenge is unknown. Increasing osmolarity by 20 mosM with either glucose or mannitol induced NKCC-mediated (86)Rb uptake and water transport into rat soleus and plantaris skeletal muscle in vitro. This NKCC activity restored intracellular water. In contrast to mannitol, hyperosmolar glucose increased ERK1,2 and p38 MAPK phosphorylation. Glucose, but not mannitol, impaired insulin-stimulated phosphorylation of Akt and p38 MAPK in the plantaris and soleus muscles, respectively. Hyperosmolarity-induced NKCC activation was insensitive to insulin action and pharmacological inhibition of ERK1,2 and p38 MAPK pathways. Paradoxically, cAMP-producing agents, which stimulate NKCC activity in isosmotic conditions, suppressed hyperosmolar glucose- and mannitol-induced NKCC activity and prevented restoration of muscle cell volume in hyperosmotic media. These results indicate that NKCC activity helps restore muscle cell volume during hyperglycemia. Moreover, hyperosmolarity activates NKCC regulatory pathways that are insensitive to insulin inhibition.     

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.     

Insulin-independent pathways mediating glucose uptake in hindlimb-suspended skeletal muscle.

Insulin resistance accompanies atrophy in slow-twitch skeletal muscles such as the soleus. Using a rat hindlimb suspension model of atrophy, we have previously shown that an upregulation of JNK occurs in atrophic muscles and correlates with the degradation of insulin receptor substrate-1 (IRS-1) (Hilder TL, Tou JC, Grindeland RF, Wade CE, and Graves LM. FEBS Lett 553: 63-67, 2003), suggesting that insulin-dependent glucose uptake may be impaired. However, during atrophy, these muscles preferentially use carbohydrates as a fuel source. To investigate this apparent dichotomy, we examined insulin-independent pathways involved in glucose uptake following a 2- to 13-wk hindlimb suspension regimen. JNK activity was elevated throughout the time course, and IRS-1 was degraded as early as 2 wk. AMP-activated protein kinase (AMPK) activity was significantly higher in atrophic soleus muscle, as were the activities of the ERK1/2 and p38 MAPKs. As a comparison, we examined the kinase activity in solei of rats exposed to hypergravity conditions (2 G). IRS-1 phosphorylation, protein, and AMPK activity were not affected by 2 G, demonstrating that these changes were only observed in soleus muscle from hindlimb-suspended animals. To further examine the effect of AMPK activation on glucose uptake, C2C12 myotubes were treated with the AMPK activator metformin and then challenged with the JNK activator anisomycin. While anisomycin reduced insulin-stimulated glucose uptake to control levels, metformin significantly increased glucose uptake in the presence of anisomycin and was independent of insulin. Taken together, these results suggest that AMPK may be an important mediator of insulin-independent glucose uptake in soleus during skeletal muscle atrophy.     

Contraction activates glucose uptake and glycogen synthase normally in muscles from dexamethasone-treated rats

Glucocorticoids cause insulin resistance in skeletal muscle. The aims of the present study were to investigate the effects of contraction on glucose uptake, insulin signaling, and regulation of glycogen synthesis in skeletal muscles from rats treated with the glucocorticoid analog dexamethasone (1 mg x kg(-1) x day(-1) ip for 12 days). Insulin resistance in dexamethasone-treated rats was confirmed by reduced insulin-stimulated glucose uptake (approximately 35%), glycogen synthesis (approximately 70%), glycogen synthase activation (approximately 80%), and PKB Ser(473) phosphorylation (approximately 40%). Chronic dexamethasone treatment did not impair glucose uptake during contraction in soleus or epitrochlearis muscles. In epitrochlearis (but not in soleus), the presence of insulin during contraction enhanced glucose uptake to similar levels in control and dexamethasone-treated rats. Contraction also increased glycogen synthase fractional activity and dephosphorylated glycogen synthase at Ser(645), Ser(649), Ser(653), and Ser(657) normally in muscles from dexamethasone-treated rats. After contraction, insulin-stimulated glycogen synthesis was completely restored in epitrochlearis and improved in soleus from dexamethasone-treated rats. Contraction did not increase insulin-stimulated PKB Ser(473) or glycogen synthase kinase-3 (GSK-3) phosphorylation. Instead, contraction increased GSK-3beta Ser(9) phosphorylation in epitrochlearis (but not in soleus) in muscles from control and dexamethasone-treated rats. In conclusion, contraction stimulates glucose uptake normally in dexamethasone-induced insulin resistant muscles. After contraction, insulin's ability to stimulate glycogen synthesis was completely restored in epitrochlearis and improved in soleus from dexamethasone-treated rats.     

Oxidant stress and skeletal muscle glucose transport: roles of insulin signaling and p38 MAPK

Oxidative stress can impact the regulation of glucose transport activity in a variety of cell lines. In the present study, we assessed the direct effects of an oxidant stress on the glucose transport system in intact mammalian skeletal muscle preparations. Type IIb (epitrochlearis) and type I (soleus) muscles from insulin-sensitive lean Zucker rats were incubated in 8 mM glucose for 2 h in the absence or presence of 100 mU/ml glucose oxidase to produce the oxidant hydrogen peroxide (H(2)O(2)) (60-90 microM). Glucose transport, glycogen synthase activity, and metabolic signaling factors were then assessed. H(2)O(2) significantly (p < 0.05) activated basal glucose transport and glycogen synthase activities and increased insulin receptor tyrosine phosphorylation, insulin receptor substrate-1 associated with the p85 subunit of phosphatidylinositol-3' kinase (PI3-kinase), and Ser(473) phosphorylation of Akt in both muscle types. This induction of glucose transport by the oxidant stress was prevented by the PI3-kinase inhibitor wortmannin. The oxidant stress also significantly increased phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) and 5'-AMP-dependent protein kinase. Interestingly, selective inhibition of p38 MAPK using A304000 substantially reduced the activation of glucose transport induced by the oxidant stress. These results support a direct role for oxidative stress in the activation of the glucose transport system in mammalian skeletal muscle and indicate that this process involves engagement of and possible interactions between the PI3-kinase-dependent signaling pathway and activation of p38 MAPK.     

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.     

Effects of a unique conjugate of alpha-lipoic acid and gamma-linolenic acid on insulin action in obese Zucker rats

The purpose of this study was to assess the individual and interactive effects of the antioxidant alpha-lipoic acid (LPA) and the n-6 essential fatty acid gamma-linolenic acid (GLA) on insulin action in insulin-resistant obese Zucker rats. LPA, GLA, and a unique conjugate consisting of equimolar parts of LPA and GLA (LPA-GLA) were administered for 14 days at 10, 30, or 50 mg. kg body wt(-1). day(-1). Whereas LPA was without effect at 10 mg/kg, at 30 and 50 mg/kg it elicited 23% reductions (P < 0.05) in the glucose-insulin index (the product of glucose and insulin areas under the curve during an oral glucose tolerance test and an index of peripheral insulin action) that were associated with significant increases in insulin-mediated (2 mU/ml) glucose transport activity in isolated epitrochlearis (63-65%) and soleus (33-41%) muscles. GLA at 10 and 30 mg/kg caused 21-25% reductions in the glucose-insulin index and 23-35% improvements in insulin-mediated glucose transport in epitrochlearis muscle. The beneficial effects of GLA disappeared at 50 mg/kg. At 10 and 30 mg/kg, the LPA-GLA conjugate elicited 29 and 38% reductions in the glucose-insulin index. These LPA-GLA-induced improvements in whole body insulin action were accompanied by 28-63 and 38-57% increases in insulin-mediated glucose transport in epitrochlearis and soleus muscles and resulted from the additive effects of LPA and GLA. At 50 mg/kg, the metabolic improvements due to LPA-GLA were substantially reduced. In summary, these results indicate that the conjugate of the antioxidant LPA and the n-6 essential fatty acid GLA elicits significant dose-dependent improvements in whole body and skeletal muscle insulin action on glucose disposal in insulin-resistant obese Zucker rats. Moreover, these actions of LPA-GLA are due to the additive effects of its individual components.     

Restoration of impaired p38 activation by insulin in insulin resistant skeletal muscle cells treated with thiazolidinediones

We have previously reported that thiazolidinediones (TZDs) are able to restore the tyrosine phosphorylation of insulin receptor and insulin receptor substrate-1, activation of phosphatidyl inositol 3-kinase and glucose uptake in insulin resistant skeletal muscle cells. In this study, we investigated the effects of insulin stimulation and TZDs on the role of mitogen-activated protein kinase (MAPK) in insulin resistant skeletal muscle cells. All the three MAPKs [extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAPK] were activated by insulin in the sensitive skeletal muscle cells. In contrast, activation of p38 MAPK was impaired in insulin resistant cells, where as ERK and JNK were activated by insulin. Treatment with TZDs resulted in the restoration of p38 MAPK activity in insulin resistant cells. The treatment of cells with p38 MAPK inhibitor, SB203580, blocked the insulin stimulated glucose uptake in sensitive as well as resistant cells and it also prevented the activation of p38 by insulin. These results suggest the potential involvement of p38 as well as the mechanistic role of TZDs in insulin resistance.     

Acute selective glycogen synthase kinase-3 inhibition enhances insulin signaling in prediabetic insulin-resistant rat skeletal muscle

Glycogen synthase kinase-3 (GSK3) has been implicated in the multifactorial etiology of skeletal muscle insulin resistance in animal models and in human type 2 diabetic subjects. However, the potential molecular mechanisms involved are not yet fully understood. Therefore, we determined if selective GSK3 inhibition in vitro leads to an improvement in insulin action on glucose transport activity in isolated skeletal muscle of insulin-resistant, prediabetic obese Zucker rats and if these effects of GSK3 inhibition are associated with enhanced insulin signaling. Type I soleus and type IIb epitrochlearis muscles from female obese Zucker rats were incubated in the absence or presence of a selective, small organic GSK3 inhibitor (1 microM CT118637, Ki < 10 nM for GSK3alpha and GSK3beta). Maximal insulin stimulation (5 mU/ml) of glucose transport activity, glycogen synthase activity, and selected insulin-signaling factors [tyrosine phosphorylation of insulin receptor (IR) and IRS-1, IRS-1 associated with p85 subunit of phosphatidylinositol 3-kinase, and serine phosphorylation of Akt and GSK3] were assessed. GSK3 inhibition enhanced (P <0.05) basal glycogen synthase activity and insulin-stimulated glucose transport in obese epitrochlearis (81 and 24%) and soleus (108 and 20%) muscles. GSK3 inhibition did not modify insulin-stimulated tyrosine phosphorylation of IR beta-subunit in either muscle type. However, in obese soleus, GSK3 inhibition enhanced (all P < 0.05) insulin-stimulated IRS-1 tyrosine phosphorylation (45%), IRS-1-associated p85 (72%), Akt1/2 serine phosphorylation (30%), and GSK3beta serine phosphorylation (39%). Substantially smaller GSK3 inhibitor-mediated enhancements of insulin action on these insulin signaling factors were observed in obese epitrochlearis. These results indicate that selective GSK3 inhibition enhances insulin action in insulin-resistant skeletal muscle of the prediabetic obese Zucker rat, at least in part by relieving the deleterious effects of GSK3 action on post-IR insulin signaling. These effects of GSK3 inhibition on insulin action are greater in type I muscle than in type IIb muscle from these insulin-resistant animals.     

Restoration of impaired p38 activation by insulin in insulin resistant skeletal muscle cells treated with thiazolidinediones

We have previously reported that thiazolidinediones (TZDs) are able to restore the tyrosine phosphorylation of insulin receptor and insulin receptor substrate-1, activation of phosphatidyl inositol 3-kinase and glucose uptake in insulin resistant skeletal muscle cells [21]. In this study, we investigated the effects of insulin stimulation and TZDs on the role of mitogen-activated protein kinase (MAPK) in insulin resistant skeletal muscle cells. All the three MAPKs [extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAPK] were activated by insulin in the sensitive skeletal muscle cells. In contrast, activation of p38 MAPK was impaired in insulin resistant cells, where as ERK and JNK were activated by insulin. Treatment with TZDs resulted in the restoration of p38 MAPK activity in insulin resistant cells. The treatment of cells with p38 MAPK inhibitor, SB203580, blocked the insulin stimulated glucose uptake in sensitive as well as resistant cells and it also prevented the activation of p38 by insulin. These results suggest the potential involvement of p38 as well as the mechanistic role of TZDs in insulin resistance.     

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.     

Age-related differences in skeletal muscle insulin signaling: the role of stress kinases and heat shock proteins

Aging is associated with an increase in insulin resistance in skeletal muscle, yet the underlying mechanism is not well established. We hypothesize that with aging, a chronic increase in stress kinase activation, coupled with a decrease in oxidative capacity, leads to insulin resistance in skeletal muscle. In aged (24 mo old) and young (3 mo old) Fischer 344 rats, 2-deoxyglucose uptake and insulin signaling [as measured by phosphorylation of insulin receptor substrate-1 (IRS-1), Akt (protein kinase B), and Akt substrate of 160 kDa (AS160)] decreased significantly with age. Activation of, c-Jun NH(2)-terminal kinase (JNK), glycogen serine kinase-3beta (GSK-3beta), and degradation of IkappaBalpha by the upstream inhibitor of kappa B kinase (IKKbeta), as measured by Western blot analysis, were increased with age in both soleus and epitrochlearis (Epi) muscles. However, much higher activation of these kinases in Epi muscles from young rats compared with soleus results in a greater effect of these kinases on insulin signaling in fast-twitch muscle with age. Heat shock protein (HSP) 72 expression and phosphorylation of HSP25 were higher in soleus compared with Epi muscles, and both parameters decreased with age. Age and fiber type differences in cytochrome oxidase activity are consistent with observed changes in HSP expression and activation. Our results demonstrate a significant difference in the ability of slow-twitch and fast-twitch muscles to respond to insulin and regulate glucose with age. A greater constitutive HSP expression and lower stress kinase activation may account for the ability of slow-twitch muscles to preserve the capacity to respond to insulin and maintain glucose homeostasis with age.     

Static stretch increases c-Jun NH2-terminal kinase activity and p38 phosphorylation in rat skeletal muscle

Physicalexercise and contraction increase c-Jun NH₂-terminal kinase(JNK) activity in rat and human skeletal muscle, and eccentriccontractions activate JNK to a greater extent than concentric contractions in human skeletal muscle. Because eccentric contractions include a lengthening or stretch component, we compared the effects ofisometric contraction and static stretch on JNK and p38, the stress-activated protein kinases. Soleus and extensor digitorum longus(EDL) muscles dissected from 50- to 90-g male Sprague-Dawley rats weresubjected to 10 min of electrical stimulation that produced contractions and/or to 10 min of stretch (0.24 N tension, 20-25% increase in length) in vitro. In the soleus muscle, contraction resulted in a small, but significant, increase in JNK activity (1.8-fold above basal) and p38 phosphorylation (4-fold). Static stretchhad a much more profound effect on the stress-activated proteinkinases, increasing JNK activity 19-fold and p38 phosphorylation 21-fold. Increases in JNK activation and p38 phosphorylation in response to static stretch were fiber-type dependent, with greater increases occurring in the soleus than in the EDL. Immunohistochemistry performed with a phosphospecific antibody revealed that activation ofJNK occurred within the muscle fibers. These studies suggest that thestretch component of a muscle contraction may be a major contributor tothe increases in JNK activity and p38 phosphorylation observed afterexercise in vivo.