Possible CaMKK-dependent regulation of AMPK phosphorylation and glucose uptake at the onset of mild tetanic skeletal muscle contraction

The Ca(2+)/calmodulin (CaM) competitive inhibitor KN-93 has previously been used to evaluate 5'-AMP-activated protein kinase (AMPK)-independent Ca(2+)-signaling to contraction-stimulated glucose uptake in muscle during intense electrical stimulation ex vivo. With the use of low-intensity tetanic contraction of mouse soleus and extensor digitorum longus (EDL) muscles ex vivo, this study demonstrates that KN-93 can potently inhibit AMPK phosphorylation and activity after 2 min but not 10 min of contraction while strongly inhibiting contraction-stimulated 2-deoxyglucose uptake at both the 2- and 10-min time points. These data suggest inhibition of Ca(2+)/CaM-dependent signaling events upstream of AMPK, the most likely candidate being the novel AMPK kinase CaM-dependent protein kinase kinase (CaMKK). CaMKK protein expression was detected in mouse skeletal muscle. Similar to KN-93, the CaMKK inhibitor STO-609 strongly reduced AMPK phosphorylation and activity at 2 min and less potently at 10 min. Pretreatment with STO-609 inhibited contraction-stimulated glucose uptake at 2 min in soleus, but not EDL, and in both muscles after 10 min. Neither KN-93 nor STO-609 inhibited 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside-stimulated glucose uptake, AMPK phosphorylation, or recombinant LKB1 activity, suggestive of an LKB1-independent effect. Finally, neither KN-93 nor STO-609 had effects on the reductions in glucose uptake seen in mice overexpressing a kinase-dead AMPK construct, indicating that the effects of KN-93 and STO-609 on glucose uptake require inhibition of AMPK activity. We propose that CaMKKs act in mouse skeletal muscle regulating AMPK phosphorylation and glucose uptake at the onset of mild tetanic contraction and that an intensity- and/or time-dependent switch occurs in the relative importance of AMPKKs during contraction.     

Caffeine-induced Ca(2+) release increases AMPK-dependent glucose uptake in rodent soleus muscle

Previous studies have proposed that caffeine-induced activation of glucose transport in skeletal muscle is independent of AMP-activated protein kinase (AMPK) because alpha-AMPK Thr172 phosphorylation was not increased by caffeine. However, our previous studies, as well as the present, show that AMPK phosphorylation measured in whole muscle lysate is not a good indicator of AMPK activation in rodent skeletal muscle. In lysates from incubated rat soleus muscle, a predominant model in previous caffeine-studies, both acetyl-CoA carboxylase-beta (ACCbeta) Ser221 and immunoprecipitated alpha(1)-AMPK activity increased with caffeine incubation, without changes in AMPK phosphorylation or immunoprecipitated alpha(2)-AMPK activity. This pattern was also observed in mouse soleus muscle, where only ACCbeta and alpha(1)-AMPK phosphorylation were increased following caffeine treatment. Preincubation with the selective CaMKK inhibitor STO-609 (5 microM), the CaM-competitive inhibitor KN-93 (10 microM), or the SR Ca(2+) release blocking agent dantrolene (10 microM) all inhibited ACCbeta phosphorylation and alpha(1)-AMPK phosphorylation, suggesting that SR Ca(2+) release may work through a CaMKK-AMPK pathway. Caffeine-stimulated 2-deoxyglucose (2DG) uptake reflected the AMPK activation pattern, being increased with caffeine and inhibited by STO-609, KN-93, or dantrolene. The inhibition of 2DG uptake is likely causally linked to AMPK activation, since muscle-specific expression of a kinase-dead AMPK construct greatly reduced caffeine-stimulated 2DG uptake in mouse soleus. We conclude that a SR Ca(2+)-activated CaMKK may control alpha(1)-AMPK activation and be necessary for caffeine-stimulated glucose uptake in mouse soleus muscle.     

A role for calcium/calmodulin kinase in insulin stimulated glucose transport

Previous research has shown that the CAMK (calcium/calmodulin dependent protein kinase) inhibitor, KN62, can lead to reductions in insulin stimulated glucose transport. Although controversial, an L-type calcium channel mechanism has also been hypothesized to be involved in insulin stimulated glucose transport. The purpose of this report was to determine if 1) L-type calcium channels and CAMK are involved in a similar signaling pathway in the control of insulin stimulated glucose transport and 2) determine if insulin induces an increase in CAMKII phosphorylation through an L-type calcium channel dependent mechanism. Insulin stimulated glucose transport was significantly (p<0.05) inhibited to a similar extent ( approximately 30%) by both KN62 and nifedipine in rat soleus and epitrochelaris muscles. The new finding of these experiments was that the combined inhibitory effect of these two compounds was not greater than the effect of either inhibitor alone. To more accurately determine the interaction between CAMK and L-type calcium channels, we measured insulin induced changes in CAMKII phosphorylation using Western blot analysis. The novel finding of this set of experiments was that insulin induced an increase in phosphorylated CAMKII ( approximately 40%) in rat soleus muscle that was reversed in the presence of KN62 but not nifedipine. Taken together these results suggest that a CAMK signaling mechanism may be involved in insulin stimulated glucose transport in skeletal muscle through an L-type calcium channel independent mechanism.     

Are tyrosine kinases involved in mediating contraction-stimulated muscle glucose transport?

Muscle contractions and insulin stimulate glucose transport into muscle by separate pathways. The contraction-mediated increase in glucose transport is mediated by two mechanisms, one involves the activation of 5'-AMP-activated protein kinase (AMPK) and the other involves the activation of calcium/calmodulin-dependent protein kinase II (CAMKII). The steps leading from the activation of AMPK and CAMKII to the translocation of GLUT4 to the cell surface have not been identified. Studies with the use of the tyrosine kinase inhibitor genistein suggest that one or more tyrosine kinases could be involved in contraction-stimulated glucose transport. The purpose of the present study was to determine the involvement of tyrosine kinases in contraction-stimulated glucose transport in rat soleus and epitrochlearis muscles. Contraction-stimulated glucose transport was completely prevented by pretreatment with genistein (100 microM) and the related compound butein (100 microM). However, the structurally distinct tyrosine kinase inhibitors 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyridine and herbimycin did not reduce contraction-stimulated glucose transport. Furthermore, genistein and butein inhibited glucose transport even when muscles were exposed to these compounds after being stimulated to contract. Muscle contractions did not result in increases in tyrosine phosphorylation of proteins such as proline-rich tyrosine kinase and SRC. These results provide evidence that tyrosine kinases do not mediate contraction-stimulated glucose transport and that the inhibitory effects of genistein on glucose transport result from direct inhibition of the glucose transporters at the cell surface.     

A role for AMP-activated protein kinase in contraction- and hypoxia-regulated glucose transport in skeletal muscle

Eukaryotic cells possess systems for sensing nutritional stress and inducing compensatory mechanisms that minimize the consumption of ATP while utilizing alternative energy sources. Such stress can also be imposed by increased energy needs, such as in skeletal muscle of exercising animals. In these studies, we consider the role of the metabolic sensor, AMP-activated protein kinase (AMPK), in the regulation of glucose transport in skeletal muscle. Expression in mouse muscle of a dominant inhibitory mutant of AMPK completely blocked the ability of hypoxia or AICAR to activate hexose uptake, while only partially reducing contraction-stimulated hexose uptake. These data indicate that AMPK transmits a portion of the signal by which muscle contraction increases glucose uptake, but other AMPK-independent pathways also contribute to the response.     

Uniaxial cyclic stretch increases glucose uptake into C2C12 myotubes through a signaling pathway independent of insulin-like growth factor I

Insulin-like growth factor I (IGF-I), an autocrine/paracrine growth factor involved in myogenesis, has rapid effects on muscle metabolism. In a manner analogous to insulin and mechanical stimuli such as stretch, IGF-I stimulates glucose transport through recruitment of glucose transporters to surface membranes in skeletal muscles. It is known that IGF-I is secreted from skeletal muscle cells in response to stretch. Therefore, we examined whether IGF-I is involved in the mechanism by which mechanical stretch regulates glucose transport using cultured C2C12 myotubes. IGF-I increased 2-deoxy- D-glucose (2-DG) uptake, and this created an additive effect with mechanical stretch, suggesting that these stimuli enhance glucose transport through different mechanisms. In fact, IGF-I-stimulated 2-DG uptake was not blocked by dantrolene (an inhibitor of Ca (2+)release from sarcoplasmic reticulum), whereas the stretch-stimulated effect was abolished. Conversely, the IGF-I-stimulated 2-DG uptake was prevented by phosphatidylinositol 3-kinase inhibitor wortmannin, which did not prevent the stretch-stimulated 2-DG uptake. In addition, experiments using media conditioned by stretched myotubes indicated that a mechanically induced release of locally acting autocrine/paracrine growth factors was not sufficient for induction of 2-DG uptake. Thus, our results demonstrate that mechanical stretch signaling for glucose transport is independent of the mechanism through which IGF-I increases this transport.     

Evidence for the involvement of CaMKII and AMPK in Ca2+-dependent signaling pathways regulating FA uptake and oxidation in contracting rodent muscle.

Calcium-calmodulin/dependent protein kinase II (CaMKII), AMP-activated protein kinase (AMPK), and extracellular signal-regulated kinase (ERK1/2) have each been implicated in the regulation of substrate metabolism during exercise. The purpose of this study was to determine whether CaMKII is involved in the regulation of FA uptake and oxidation and, if it is involved, whether it does so independently of AMPK and ERK1/2. Rat hindquarters were perfused at rest with (n = 16) or without (n = 10) 3 mM caffeine, or during electrical stimulation (n = 14). For each condition, rats were subdivided and treated with 10 muM of either KN92 or KN93, inactive and active CaMKII inhibitors, respectively. Both caffeine treatment and electrical stimulation significantly increased FA uptake and oxidation. KN93 abolished caffeine-induced FA uptake, decreased contraction-induced FA uptake by 33%, and abolished both caffeine- and contraction-induced FA oxidation (P < 0.05). Caffeine had no effect on ERK1/2 phosphorylation (P > 0.05) and increased alpha(2)-AMPK activity by 68% (P < 0.05). Electrical stimulation increased ERK1/2 phosphorylation and alpha(2)-AMPK activity by 51% and 3.4-fold, respectively (P < 0.05). KN93 had no effect on caffeine-induced alpha(2)-AMPK activity, ERK1/2 phosphorylation, or contraction-induced ERK1/2 phosphorylation (P > 0.05). Alternatively, it decreased contraction-induced alpha(2)-AMPK activity by 51% (P < 0.05), suggesting that CaMKII lies upstream of AMPK. These results demonstrate that regulation of contraction-induced FA uptake and oxidation occurs in part via Ca(2+)-independent activation of ERK1/2 as well as Ca(2+)-dependent activation of CaMKII and AMPK.     

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.     

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.     

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.     

Effect of fiber type and nutritional state on AICAR- and contraction-stimulated glucose transport in rat muscle

AMP-activated protein kinase (AMPK) may mediate the stimulatory effect of contraction and 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) on glucose transport in skeletal muscle. In muscles with different fiber type composition from fasted rats, AICAR increased 2-deoxyglucose transport and total AMPK activity approximately twofold in epitrochlearis (EPI), less in flexor digitorum brevis, and not at all in soleus muscles. Contraction increased both transport and AMPK activity more than AICAR did. In EPI muscles, the effects of AICAR and contractions on glucose transport were partially additive despite a lower AMPK activity with AICAR compared with contraction alone. In EPI from fed rats, glucose transport responses were smaller than what was seen in fasted rats, and AICAR did not increase transport despite an increase in AMPK activity. AICAR and contraction activated both alpha(1)- and alpha(2)-isoforms of AMPK. Expression of both isoforms varied with fiber types, and alpha(2) was highly expressed in nuclei. In conclusion, AICAR-stimulated glucose transport varies with muscle fiber type and nutritional state. AMPK is unlikely to be the sole mediator of contraction-stimulated glucose transport.     

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.     

Glucose transporter content and glucose uptake in skeletal muscle constructs engineered in vitro

Engineered muscle may eventually be used as a treatment option for patients suffering from loss of muscle function. The metabolic and contractile function of engineered muscle has not been well described; therefore, the purpose of this experiment was to study glucose transporter content and glucose uptake in engineered skeletal muscle constructs called myooids. Glucose uptake by way of 2-deoxyglucose and GLUT-1 and GLUT-4 transporter protein content was measured in basal and insulin-stimulated myooids that were engineered from soleus muscles of female Sprague-Dawley rats. There was a significant increase in the basal 2-deoxyglucose uptake of myooids compared with adult control (fivefold), contraction-stimulated (3.4-fold), and insulin-stimulated (threefold) soleus muscles (P = 0.0001, 0.0001, and 0.0001, respectively). In addition, there was a significant increase in the insulin-stimulated 2-deoxyglucose uptake of myooids compared with adult control soleus muscles in basal conditions (6.5-fold) and adult contraction-stimulated (4.5-fold) and insulin- stimulated (3.9-fold) soleus muscles (P = 0.0001, 0.0001, and 0.0001, respectively). There was a significant 30% increase in insulin-stimulated compared with basal 2-deoxyglucose uptake in the myooids. The myooid GLUT-1 protein content was 820% of the adult control soleus muscle, whereas the GLUT-4 protein content was 130% of the control soleus muscle. Myooid GLUT-1 protein content was 6.3-fold greater than GLUT-4 protein content, suggesting that the glucose transport of the engineered myooids is similar in several respects to that observed in both fetal and denervated skeletal muscle tissue.     

The alpha-subunit of AMPK is essential for submaximal contraction-mediated glucose transport in skeletal muscle in vitro

AMP-activated protein kinase (AMPK) is a key signaling protein in the regulation of skeletal muscle glucose uptake, but its role in mediating contraction-induced glucose transport is still debated. The effect of contraction on glucose transport is impaired in EDL muscle of transgenic mice expressing a kinase-dead, dominant negative form of the AMPKalpha(2) subunit (KD-AMPKalpha(2) mice). However, maximal force production is reduced in this muscle, raising the possibility that the defect in glucose transport was due to a secondary decrease in force production and not impaired AMPKalpha(2) activity. Generation of force-frequency curves revealed that muscle force production is matched between wild-type (WT) and KD-AMPKalpha(2) mice at frequencies < or =50 Hz. Moreover, AMPK activation is already maximal at 50 Hz in muscles of WT mice. When EDL muscles from WT mice were stimulated at a frequency of 50 Hz for 2 min (200-ms train, 1/s, 30 volts), contraction caused an approximately 3.5-fold activation of AMPKalpha(2) activity and an approximately 2-fold stimulation of glucose uptake. Conversely, whereas force production was similar in EDL of KD-AMPKalpha(2) animals, no effect of contraction was observed on AMPKalpha(2) activity, and glucose uptake stimulation was reduced by 50% (P < 0.01) As expected, 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranosyl 5'-monophosphate (AICAR) caused a 2.3-fold stimulation of AMPKalpha(2) activity and a 1.7-fold increase in glucose uptake in EDL from WT mice, whereas no effect was detected in muscle from KD-AMPKalpha(2) mice. These data demonstrate that AMPK activation is essential for both AICAR and submaximal contraction-induced glucose transport in skeletal muscle but that AMPK-independent mechanisms are also involved.     

Dissociation of 5' AMP-activated protein kinase activation and glucose uptake stimulation by mitochondrial uncoupling and hyperosmolar stress: differential sensitivities to intracellular Ca2+ and protein kinase C inhibition

2,4-dinitrophenol (DNP) compromises ATP production within the cell by disrupting the mitochondrial electron transport chain. The resulting loss of ATP leads to an increase in glucose uptake for anaerobic generation of ATP. In L6 skeletal muscle cells, DNP increases the rate of glucose uptake by twofold. We previously showed that DNP increases cell surface levels of glucose transporter 4 (GLUT4) and hexose uptake via a Ca2+-sensitive and conventional protein kinase C (cPKC)-dependent mechanism. Recently, 5' AMP-activated protein kinase (AMPK) has been proposed to mediate the stimulation of glucose uptake by energy stressors such as exercise and hypoxia. Changes in Ca2+ and cPKC have also been invoked in the stimulation of glucose uptake by exercise and hypoxia. Here we examine whether changes in cytosolic Ca2+ or cPKC lead to activation of AMPK. We show that treatment of L6 cells with DNP (0.5 mM) or hyperosmolar stress (mannitol, 0.6 M) increased AMPK activity by 3.5-fold. AMPK activation peaked by 10-15 min prior to maximal stimulation of glucose uptake. Intracellular Ca2+ chelation and cPKC inhibition prior to treatment with DNP and hyperosmolarity significantly reduced cell surface GLUT4 levels and hexose uptake but had no effect on AMPK activation. These results illustrate a break in the relationship between AMPK activation and glucose uptake in skeletal muscle cells. Activation of AMPK does not suffice to stimulate glucose uptake in response to DNP and hyperosmolarity.     

Contraction-activated glucose uptake is normal in insulin-resistant muscle of the obese Zucker rat.

The rates of muscle glucose uptake of lean and obese Zucker rats were assessed via hindlimb perfusion under basal conditions (no insulin), in the presence of a maximal insulin concentration (10 mU/ml), and after electrically stimulated muscle contraction in the absence of insulin. The perfusate contained 28 mM glucose and 7.5 microCi/mmol of 2-deoxy-D-[3H-(G)]glucose. Glucose uptake rates in the soleus (slow-twitch oxidative fibers), red gastrocnemius (fast-twitch oxidative-glycolytic fibers), and white gastrocnemius (fast-twitch glycolytic fibers) under basal conditions and after electrically stimulated muscle contraction were not significantly different between the lean and obese rats. However, the rate of glucose uptake during insulin stimulation was significantly lower for obese than for lean rats in all three fiber types. Significant correlations were found for insulin-stimulated glucose uptake and glucose transporter protein isoform (GLUT-4) content of soleus, red gastrocnemius, and white gastrocnemius of lean (r = 0.79) and obese (r = 0.65) rats. In contrast, the relationships between contraction-stimulated glucose uptake and muscle GLUT-4 content of lean and obese rats were negligible because of inordinately low contraction-stimulated glucose uptakes by the solei. These results suggest that maximal skeletal muscle glucose uptake of obese Zucker rats is resistant to stimulation by insulin but not to contractile activity. In addition, the relationship between contraction-stimulated glucose uptake and GLUT-4 content appears to be fiber-type specific.     

Salicylate acutely stimulates 5′-AMP-activated protein kinase and insulin-independent glucose transport in rat skeletal muscles

Salicylate (SAL) has been recently implicated in the antidiabetic effect in humans. We assessed whether 5′-AMP-activated protein kinase (AMPK) in skeletal muscle is involved in the effect of SAL on glucose homeostasis. Rat fast-twitch epitrochlearis and slow-twitch soleus muscles were incubated in buffer containing SAL. Intracellular concentrations of SAL increased rapidly (<5 min) in both skeletal muscles, and the Thr¹⁷² phosphorylation of the α subunit of AMPK increased in a dose- and time-dependent manner. SAL increased both AMPKα1 and AMPKα2 activities. These increases in enzyme activity were accompanied by an increase in the activity of 3-O-methyl-d-glucose transport, and decreases in ATP, phosphocreatine, and glycogen contents. SAL did not change the phosphorylation of insulin receptor signaling including insulin receptor substrate 1, Akt, and p70 ribosomal protein S6 kinase. These results suggest that SAL may be transported into skeletal muscle and may stimulate AMPK and glucose transport via energy deprivation in multiple muscle types. Skeletal muscle AMPK might be part of the mechanism responsible for the metabolic improvement induced by SAL.     

Acute exposure of L6 myotubes to cis-9, trans-11 and trans-10, cis-12 conjugated linoleic acid isomers stimulates glucose uptake by modulating Ca2+/calmodulin-dependent protein kinase II

Conjugated linoleic acid (CLA), a dietary fat, has been considered beneficial in metabolic syndrome. Despite several findings indicating that CLA improves glucose clearance, little information is available regarding the cellular dynamics of CLA on skeletal muscle. We sought to investigate the role of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in cis-9, trans-11(c9,t11) and trans-10, cis-12 (t10,c12) CLA isomer-mediated glucose transport by L6 myotubes. t10,c12-CLA stimulated both intracellular Ca(2+) release (Ca(i)(2+)) and CaMKII phosphorylation, whereas c9,t11-CLA showed only modest effects on both. Sequestering Ca(i)(2+) with BAPTA/AM abrogated the effect of both CLA isomers on Akt substrate-160 kDa (AS160) phosphorylation and glucose uptake by myotubes. Exposing myotubes to KN-93 or autocamtide 2-related inhibitory peptide to block CaMKII activity prevented both CLA isomers from inducing AS160 phosphorylation and glucose transport. Likewise, genetic knockdown of CaMKII in myotubes using siRNA completely abolished CLA isomer-mediated glucose uptake. These results indicate that CLA isomers require Ca(i)(2+)-CaMKII to mediate glucose uptake. Evidence that CaMKII blockers inhibit t10,c12-CLA-mediated AMP-activated protein kinase (AMPK) activation indicated that CaMKII acts upstream of AMPK in response to t10,c12-CLA. Lastly, CLA isomers stimulated the formation of reactive oxygen species but had no effect on stress-activated protein kinase/c-jun NH(2)-terminal kinase. These data establish that t10,c12-CLA acts via Ca(i)(2+)-CaMKII-AMPK-AS160 to stimulate skeletal muscle glucose transport, whereas the mechanism of c9,t11-CLA remains unclear. Given that impairments in muscle glucose utilisation are apparent in metabolic syndrome, delineating the molecular mechanisms by which CLA isomers mediate muscle glucose uptake may identify new approaches to manage this condition.     

Defining the contribution of AMP-activated protein kinase (AMPK) and protein kinase C (PKC) in regulation of glucose uptake by metformin in skeletal muscle cells

The importance of AMP-activated protein kinase (AMPK) and protein kinase C (PKC) as effectors of metformin (Met) action on glucose uptake (GU) in skeletal muscle cells was investigated. GU in L6 myotubes was stimulated 2-fold following 16 h of Met treatment and acutely enhanced by insulin in an additive fashion. Insulin-stimulated GU was sensitive to PI3K inhibition, whereas that induced by Met was not. Met and its related biguanide, phenformin, stimulated AMPK activation/phosphorylation to a level comparable with that induced by the AMPK activator, 5-amino-1-β-d-ribofuranosyl-imidazole-4-carboxamide (AICAR). However, the increase in GU elicited by AICAR was significantly lower than that induced by either biguanide. Expression of a constitutively active AMPK mimicked the effects of AICAR on GU, whereas a dominant interfering AMPK or shRNA silencing of AMPK prevented AICAR-stimulated GU and Met-induced AMPK signaling but only repressed biguanide-stimulated GU by ～20%. Consistent with this, analysis of GU in muscle cells from α1(-/-)/α2(-/-) AMPK-deficient mice revealed a significant retention of Met-stimulated GU, being reduced by ～35% compared with that of wild type cells. Atypical PKCs (aPKCs) have been implicated in Met-stimulated GU, and in line with this, Met and phenformin induced activation/phosphorylation of aPKC in L6 myotubes. However, although cellular depletion of aPKC (>90%) led to loss in biguanide-induced aPKC phosphorylation, it had no effect on Met-stimulated GU, whereas inhibitors targeting novel/conventional PKCs caused a significant reduction in biguanide-induced GU. Our findings indicate that although Met activates AMPK, a significant component of Met-stimulated GU in muscle cells is mediated via an AMPK-independent mechanism that involves novel/conventional PKCs.