Evidence that nitric oxide increases glucose transport in skeletal muscle

Balon, Thomas W., and Jerry L. Nadler. Evidence thatnitric oxide increases glucose transport in skeletal muscle.J. Appl. Physiol. 82(1): 359-363, 1997.Nitric oxide synthase (NOS) is expressed in skeletal muscle.However, the role of nitric oxide (NO) in glucose transport in thistissue remains unclear. To determine the role of NO in modulatingglucose transport, 2-deoxyglucose (2-DG) transport was measured in ratextensor digitorum longus (EDL) muscles that were exposed to either amaximally stimulating concentration of insulin or to an electricalstimulation protocol, in the presence ofN^G-monomethyl-L-arginine,a NOS inhibitor. In addition, EDL preparations were exposed to sodiumnitroprusside (SNP), an NO donor, in the presence of submaximal andmaximally stimulating concentrations of insulin. NOS inhibition reducedboth basal and exercise-enhanced 2-DG transport but had no effect oninsulin-stimulated 2-DG transport. Furthermore, SNP increased 2-DGtransport in a dose-responsive manner. The effects of SNP and insulinon 2-DG transport were additive when insulin was present inphysiological but not in pharmacological concentrations. Chronictreadmill training increased protein expression of both type I and typeIII NOS in soleus muscle homogenates. Our results suggest that NO maybe a potential mediator of exercise-induced glucose transport.

     

Effect of passive stretch on intracellular nitric oxide and superoxide activities in single skeletal muscle fibres: influence of ageing

Skeletal muscle is repeatedly exposed to passive stretches due to the activation of antagonist muscles and to external forces. Stretch has multiple effects on muscle mass and function, but the initiating mechanisms and intracellular signals that modulate those processes are not well understood. Mechanical stretch applied to some cell types induces production of reactive oxygen species (ROS) and nitric oxide that modulate various cellular signalling pathways. The aim of this study was to assess whether intracellular activities of ROS and nitric oxide were modulated by passive stretches applied to single mature muscle fibres isolated from young and old mice. We developed a novel approach to apply passive stretch to single mature fibres from the flexor digitorum brevis muscle in culture and to monitor the activities of ROS and nitric oxide in situ by fluorescence microscopy. Passive stretch applied to single skeletal muscle fibres from young mice induced an increase in dihydroethidium oxidation (reflecting intracellular superoxide) with no increase in intracellular DAF-FM oxidation (reflecting nitric oxide activity) or CM-DCFH oxidation. In contrast, in fibres isolated from muscles of old mice passive stretch was found to induce an increase in intracellular nitric oxide activities with no change in DHE oxidation.     

Regulation of nitric oxide production in response to skeletal muscle activation

Nitric oxide(NO) is synthesized in normal muscle fibers by the neuronal (nNOS) andthe endothelial (ecNOS) isoforms of nitric oxide synthase (NOS). NOcontributes to the regulation of several processes such asexcitation-contraction coupling and mitochondrial respiration. Weassessed in this study whether NO production is regulated in responseto an acute increase in muscle activation. Three groups ofanesthetized, tracheostomized, spontaneously breathing rats wereexamined after an experimental period of 3 h. Group 1 served as a control (no loading), whereasgroups 2 and3 were exposed to moderate and severeinspiratory resistive loads, respectively, which elicited trachealpressures of 30 and 70% of maximum, respectively. Ventilatory(diaphragm, intercostal, and transverse abdominis) and limb(gastrocnemius) muscles were excised at the end of the experimentalperiod and examined for NOS activity and NOS protein expression.Neither submaximal nor maximum tracheal pressures were altered after 3 h of resistive loading. Diaphragmatic and intercostal muscle NOSactivities declined significantly in response to moderate and severeloading, whereas those of transverse abdominis and gastrocnemiusmuscles remained unchanged. On the other hand, resistive loading had nosignificant effect on ventilatory and limb muscle NOS isoformexpression. We propose that a contraction-induced decline in muscle NOSactivity represents a compensatory mechanism through which musclecontractility and mitochondrial function are protected from theinhibitory influence of NO.

     

Obesity, insulin resistance, and skeletal muscle nitric oxide synthase

The molecular mechanisms responsible for impaired insulin action have yet to be fully identified. Rodent models demonstrate a strong relationship between insulin resistance and an elevation in skeletal muscle inducible nitric oxide synthase (iNOS) expression; the purpose of this investigation was to explore this potential relationship in humans. Sedentary men and women were recruited to participate (means ± SE: nonobese, body mass index = 25.5 ± 0.3 kg/m(2), n = 13; obese, body mass index = 36.6 ± 0.4 kg/m(2), n = 14). Insulin sensitivity was measured using an intravenous glucose tolerance test with the subsequent modeling of an insulin sensitivity index (S(I)). Skeletal muscle was obtained from the vastus lateralis, and iNOS, endothelial nitric oxide synthase (eNOS), and neuronal nitric oxide synthase (nNOS) content were determined by Western blot. S(I) was significantly lower in the obese compared with the nonobese group (～43%; P < 0.05), yet skeletal muscle iNOS protein expression was not different between nonobese and obese groups. Skeletal muscle eNOS protein was significantly higher in the nonobese than the obese group, and skeletal muscle nNOS protein tended to be higher (P = 0.054) in the obese compared with the nonobese group. Alternative analysis based on S(I) (high and low tertile) indicated that the most insulin-resistant group did not have significantly more skeletal muscle iNOS protein than the most insulin-sensitive group. In conclusion, human insulin resistance does not appear to be associated with an elevation in skeletal muscle iNOS protein in middle-aged individuals under fasting conditions.     

Stretch-induced nitric oxide modulates mechanical properties of skeletal muscle cells

In this study, we examined the hypothesis that stretch-induced (nitric oxide) NO modulates the mechanical properties of skeletal muscles by increasing accumulation of protein levels of talin and vinculin and by inhibiting calpain-induced proteolysis, thereby stabilizing the focal contacts and the cytoskeleton. Differentiating C₂C₁₂ myotubes were subjected to a single 10% step stretch for 0–4 days. The apparent elastic modulus of the cells, E_app, was subsequently determined by atomic force microscopy. Static stretch led to significant increases (P < 0.01) in E_app beginning at 2 days. These increases were correlated with increases in NO activity and neuronal NO synthase (nNOS) protein expression. Expression of talin was upregulated throughout, whereas expression of vinculin was significantly increased only on days 3 and 4. Addition of the NO donor L-arginine onto stretched cells further enhanced E_app, NOS activity, and nNOS expression, whereas the presence of the NO inhibitor N-nitro-L-arginine methyl ester (L-NAME) reversed the effects of mechanical stimulation and of L-arginine. Overall, viscous dissipation, as determined by the value of hysteresis, was not significantly altered. For assessment of the role of vinculin and talin stability, cells treated with L-NAME showed a significant decrease in E_app, whereas addition of a calpain inhibitor abolished the effect. Thus our results show that NO inhibition of calpain-initiated cleavage of cytoskeleton proteins was correlated with the changes in E_app. Together, our data suggest that NO modulates the mechanical behavior of skeletal muscle cells through the combined action of increased talin and vinculin levels and a decrease in calpain-mediated talin proteolysis. mechanical stimulation; apparent elastic modulus; skeletal muscle cells; nitric oxide; stretch     

Nitric oxide synthase-independent generation of nitric oxide in rat skeletal muscle ischemia-reperfusion injury.

We have used electron paramagnetic resonance to investigate the time course of nitric oxide (NO) generation and its susceptibility to inhibitors of nitric oxide synthase (NOS) in ischemia-reperfusion (IR) injury to rat skeletal muscle in vivo. Significant levels of muscle nitroso-heme complexes were detected 24 h postreperfusion, but not after at 0.05, 3, and 8 h of reperfusion. The levels of muscle nitroso-heme complexes were not decreased by the NOS inhibitor N-nitro-L-arginine methyl ester as a single dose (30 mg/kg) prior to reperfusion or as multiple doses continued throughout the reperfusion (total administered, 120 mg/kg) or by the potent NOS inhibitor S-methylisothiourea (3 mg/kg). In contrast, nitroso-heme levels were reduced by the glucocorticoid dexamethasone (2.5 mg/kg). Muscle necrosis in vitro did not result in the formation of nitroso-heme complexes. The finding that reperfusion after ischemia is necessary for NO formation suggests that an inflammatory pathway is responsible for NOS-independent NO formation in IR injury to skeletal muscle.     

Local effect of vanadate on interstitial glucose and lactate concentrations in human skeletal muscle

The aim of this study was to investigate the local effect of the insulin-mimetic agent vanadate on glucose metabolism in human skeletal muscle in vivo. Interstitial concentrations of glucose and lactate were determined by microdialysis at a low flow rate in the quadriceps femoris muscle of 18 men. In the same leg two microdialysis catheters were inserted. In one catheter, the perfusion medium was supplemented with sodium metavanadate (10-100 mM) after a basal period, the other catheter served as control. In the catheter perfused with metavanadate, the interstitial glucose concentration was decreased by 13-50% compared to the control catheter (p<0.05). The lactate concentration was higher in the 50 mM and 100 mM metavanadate catheters compared to control (39-89%, p<0.05). There was no difference between control and metavanadate catheters in urea concentrations. Five of the subjects were insulin-resistant and for them the results were similar, although the effect was somewhat smaller. The decreased interstitial glucose concentration, and the increased lactate concentration, in the vicinity of the microdialysis catheter most likely reflects an increased cellular glucose uptake. The present study thus indicates that vanadate mimics the effect of insulin in human skeletal muscle in vivo.     

Expression of the inducible nitric oxide synthase gene in diaphragm and skeletal muscle

Thompson, Marita, Lisa Becker, Debbie Bryant, Gary Williams,Daniel Levin, Linda Margraf, and Brett P. Giroir. Expression ofthe inducible nitric oxide synthase gene in diaphragm and skeletal muscle. J. Appl. Physiol. 81(6):2415-2420, 1996.Nitric oxide (NO) is a pluripotent molecule thatcan be secreted by skeletal muscle through the activity of the neuronalconstitutive isoform of NO synthase. To determine whether skeletalmuscle and diaphragm might also express the macrophage-inducible formof NO synthase (iNOS) during provocative states, we examined tissuefrom mice at serial times after intravenous administration ofEscherichia coli endotoxin. In thesestudies, iNOS mRNA was strongly expressed in the diaphragm and skeletalmuscle of mice 4 h after intravenous endotoxin and was significantlydiminished by 8 h after challenge. Induction of iNOS mRNA was followedby expression of iNOS immunoreactive protein on Western immunoblots.Increased iNOS activity was demonstrated by conversion of arginine tocitrulline. Immunochemical analysis of diaphragmatic explants exposedto endotoxin in vitro revealed specific iNOS staining in myocytes, inaddition to macrophages and endothelium. These results may be importantin understanding the pathogenesis of respiratory pump failure duringseptic shock, as well as skeletal muscle injury during inflammation ormetabolic stress.

     

Upregulation of neuronal nitric oxide synthase in skeletal muscle by swim training

Exercise enhances cardiac output and blood flow to working skeletal muscles but decreases visceral perfusion. The alterations in nitric oxide synthase (NOS) activity and/or expression of the cardiopulmonary, skeletal muscle, and visceral organs induced by swim training are unknown. In sedentary and swim-trained rats (60 min twice/day for 3-4 wk), we studied the alterations in NOS in different tissues along with hindquarter vasoreactivity in vivo during rest and mesenteric vascular bed reactivity in vitro. Hindquarter blood flow and conductance were reduced by norepinephrine in both groups to a similar degree, whereas N(G)-nitro-L-arginine methyl ester reduced both indexes to a greater extent in swim-trained rats. Vasodilator responses to ACh, but not bradykinin or S-nitroso-N-acetyl-penicillamine, were increased in swim-trained rats. Ca(2+)-dependent NOS activity was enhanced in the hindquarter skeletal muscle, lung, aorta, and atria of swim-trained rats together with increased expression of neuronal NOS in the hindquarter skeletal muscle and endothelial NOS in the cardiopulmonary organs. Mesenteric arterial bed vasoreactivity was unaltered by swim training. Physiological adaptations to swim training are characterized by enhanced hindquarter ACh-induced vasodilation with upregulation of neuronal NOS in skeletal muscle and endothelial NOS in the lung, atria, and aorta.     

Endotoxin-induced skeletal muscle contractile dysfunction: contribution of nitric oxide synthases

The aims of thisstudy were to assess the role of nitric oxide (NO) and the contributionof different NO synthase (NOS) isoforms in skeletal muscle contractiledysfunction in septic shock. Four groups of conscious rats wereexamined. Group 1 served as control; groups 2, 3, and4 were injected withEscherichia coli endotoxin [lipopolysaccharide (LPS), 20 mg/kg ip] and killed after 6, 12, and 24 h, respectively. Protein expression was assessed byimmunoblotting and immunostaining. LPS injection elicited a transientexpression of the inducible NOS isoform, which peaked 12 h after LPSinjection and disappeared within 24 h. This expression coincided with a significant increase in nitrotyrosine formation (peroxynitrite footprint). Muscle expression of the endothelial and neuronal NOSisoforms, by comparison, rose significantly and remained higher thancontrol levels 24 h after LPS injection. In vitro measurement of musclecontractility 24 h after LPS injection showed that incubation with NOSinhibitor (S-methyliosothiourea)restored the decline in submaximal force generation, whereas maximalmuscle force remained unaffected. We conclude that NO plays asignificant role in muscle contractile dysfunction in septic animalsand that increased NO production is due to induction of the inducibleNOS isoform and upregulation of constitutive NOS isoforms.

     

Formation of hydrogen peroxide and nitric oxide in rat skeletal muscle cells during contractions

We examined intra- and extracellular H(2)O(2) and NO formation during contractions in primary rat skeletal muscle cell culture. The fluorescent probes DCFH-DA/DCFH (2,7-dichlorofluorescein-diacetate/2,7-dichlorofluorescein) and DAF-2-DA/DAF-2 (4,5-diaminofluorescein-diacetate/4,5-diaminofluorescein) were used to detect H(2)O(2) and NO, respectively. Intense electrical stimulation of muscle cells increased the intra- and extracellular DCF fluorescence by 171% and 105%, respectively, compared with control nonstimulated cells (p <.05). The addition of glutathione (GSH) or Tiron prior to electrical stimulation inhibited the intracellular DCFH oxidation (p <.05), whereas the addition of GSH-PX + GSH inhibited the extracellular DCFH oxidation (p <.05). Intense electrical stimulation also increased (p <.05) the intra- and extracellular DAF-2 fluorescence signal by 56% and 20%, respectively. The addition of N(G)-nitro-L-arginine (L-NA) completely removed the intra- and extracellular DAF-2 fluorescent signal. Our results show that H(2)O(2) and NO are formed in skeletal muscle cells during contractions and suggest that a rapid release of H(2)O(2) and NO may constitute an important defense mechanism against the formation of intracellular (*)OH and (*)ONOO. Furthermore, our data show that DCFH and DAF-2 are suitable probes for the detection of ROS and NO both intra- and extracellularly in skeletal muscle cell cultures.     

Agonist-dependent variablity of contributions of nitric oxide and prostaglandins in human skeletal muscle.

The relative contributions of endothelium-dependent dilators [nitric oxide (NO), prostaglandins (PGs), and endothelium-derived hyperpolarizing factor (EDHF)] in human limbs are poorly understood. We tested the hypothesis that relative contributions of NO and PGs differ between endothelial agonists acetylcholine (ACh; 1, 2, and 4 microg.dl(-1).min(-1)) and bradykinin (BK; 6.25, 25, and 50 ng.dl(-1).min(-1)). We measured forearm blood flow (FBF) using venous occlusion plethysmography in 50 healthy volunteers (27 +/- 1 yr) in response to brachial artery infusion of ACh or BK in the absence and presence of inhibitors of NO synthase [NOS; with NG-monomethyl-L-arginine (L-NMMA)] and cyclooxygenase (COX; with ketorolac). Furthermore, we tested the idea that the NOS + COX-independent dilation (in the presence of L-NMMA + ketorolac, presumably EDHF) could be inhibited by exogenous NO administration, as reported in animal studies. FBF increased approximately 10-fold in the ACh control; L-NMMA reduced baseline FBF and ACh dilation, whereas addition of ketorolac had no further effect. Ketorolac alone did not alter ACh dilation, but addition of L-NMMA reduced ACh dilation significantly. For BK infusion, FBF increased approximately 10-fold in the control condition; L-NMMA tended to reduce BK dilation (P < 0.1), and addition of ketorolac significantly reduced BK dilation. Similar to ACh, ketorolac alone did not alter BK dilation, but addition of L-NMMA reduced BK dilation. To test the idea that NO can inhibit the NOS + COX-independent portion of dilation, we infused a dose of sodium nitroprusside (NO-clamp technique) during ACh or BK that restored the reduction in baseline blood flow due to L-NMMA. Regardless of treatment order, the NO clamp restored baseline FBF but did not reduce the NOS + COX-independent dilation to ACh or BK. We conclude that the contribution of NO and PGs differs between ACh and BK, with ACh being more dependent on NO and BK being mostly dependent on a NOS + COX-independent mechanism (EDHF) in healthy young adults. The NOS + COX-independent dilation does not appear sensitive to feedback inhibition from NO in the human forearm.     

Deficiency of inducible nitric oxide synthase attenuates immobilization-induced skeletal muscle atrophy in mice

The present study examined the effects of inducible nitric oxide synthase (iNOS) deficiency on skeletal muscle atrophy in single leg-immobilized iNOS knockout (KO) and wild-type (WT) mice. The left leg was immobilized for 1 wk, and the right leg was used as the control. Muscle weight and contraction-stimulated glucose uptake were reduced by immobilization in WT mice, which was accompanied with increased iNOS expression in skeletal muscle. Deficiency of iNOS attenuated muscle weight loss and the reduction in contraction-stimulated glucose uptake by immobilization. Phosphorylation of Akt, mTOR, and p70S6K was reduced to a similar extent by immobilization in both WT and iNOS KO mice. Immobilization decreased FoxO1 phosphorylation and increased mRNA and protein levels of MuRF1 and atrogin-1 in WT mice, which were attenuated in iNOS KO mice. Aconitase and superoxide dismutase activities were reduced by immobilization in WT mice, and deficiency of iNOS normalized these enzyme activities. Increased nitrotyrosine and carbonylated protein levels by immobilization in WT mice were reversed in iNOS KO mice. Phosphorylation of ERK and p38 was increased by immobilization in WT mice, which was reduced in iNOS KO mice. Immobilization-induced muscle atrophy was also attenuated by an iNOS-specific inhibitor N(6)-(1-iminoethyl)-l-lysine, and this finding was accompanied by increased FoxO1 phosphorylation and reduced MuRF1 and atrogin-1 levels. These results suggest that deficiency of iNOS attenuates immobilization-induced skeletal muscle atrophy through reduced oxidative stress, and iNOS-induced oxidative stress may be required for immobilization-induced skeletal muscle atrophy.     

Involvement of nitric oxide synthase in skeletal muscle adaptation to chronic overload.

The purpose of this study was to determine the necessity of nitric oxide (NO) for hypertrophy and fiber-type transition in overloaded (OL) skeletal muscle. Endogenous NO production was blocked by administering N(G)-nitro-L-arginine methyl ester (L-NAME; 0.75 mg/ml; approximately 100 mg x kg-1 x day-1) in drinking water. Thirty-eight female Sprague-Dawley rats (approximately 250 g) were randomly divided into four groups: control-nonoverloaded (Non-OL), control-OL, L-NAME-Non-OL, and L-NAME-OL. Chronic overload of the plantaris was induced bilaterally by surgical removal of the gastrocnemius and soleus. Rats in the Non-OL groups received sham surgeries. L-NAME treatment began 24 h before surgery and continued until the rats were killed 14 days postsurgery. Although OL induced hypertrophy in both control (+76%) and L-NAME (+39%) conditions (P < 0.05), mean plantaris-to-body mass ratio in the L-NAME-OL group was significantly lower (P < 0.05) than that in the control-OL group. Microphotometric analysis of histochemically determined fiber types revealed increases in cross-sectional area (P < 0.05) for all fiber types (types I, IIA, and IIB/X) in the OL plantaris from control rats, whereas L-NAME-OL rats exhibited increases only in type I and IIB/X fibers. SDS-PAGE analysis of myosin heavy chain (MHC) composition in the plantaris indicated a significant (P < 0.05) OL effect in the control rats. Specifically, the mean proportion of type I MHC increased 6% (P < 0.05), whereas the proportion of type IIb MHC decreased approximately 9% (P < 0.05). No significant OL effects on MHC profile were observed in the L-NAME rats. These data support a role of NO in overload-induced skeletal muscle hypertrophy and fiber-type transition.     

A fluorescence-based method for measuring nitric oxide in extracts of skeletal muscle.

We describe here a fluorescence assay for nitric oxide synthase activity in skeletal muscle based on a new indicator, 4,5-diaminofluorescein (DAF-2). The rapid and irreversible binding of DAF-2 to oxidized NO allows real-time measurement of NO production. The method is safer and more convenient than the usual citrulline radioassay and can be used with crude muscle extracts. Rabbit fast tibialis anterior (TA) muscle had a nitric oxide synthase (NOS) activity of 44.3 +/- 3.5 pmol/min/mg muscle. Addition of NOS blocker N(G)-allyl-L-arginine reduced this activity by 43%. Slow soleus muscle displayed NOS activity of 7.3 +/- 2.5 pmol/min/mg muscle, 16% that of the TA muscle. Continuous stimulation of TA muscle at 10 Hz for 3 weeks reduced NOS activity by 47% to an intermediate value consistent with the associated conversion of the muscle phenotype from fast to slow.     

Nitric oxide in skeletal muscle: inhibition of nitric oxide synthase inhibits walking speed in rats.

Nitric oxide (NO*) is a multifunctional messenger molecule generated by a family of enzymes called the nitric oxide synthases (NOSs). Although NOSs have been identified in skeletal muscle, specifically brain NOS (bNOS) and endothelial NOS (eNOS), their role has not been well clarified. The goals of this investigation were to (1) characterize the immunoreactivity, Ca(2+) dependence, and activity of NOS in human and rat skeletal muscle and (2) using a rat model, investigate the effect of chronic blockade of NOS on skeletal muscle structure and function. Our results showed that both human and rodent skeletal muscle had NOS activity. This NOS activity was similar to that of the endothelial and brain NOS isoforms in that it was calcium-dependent. However, Western blot analysis consistently showed that a polyclonal antibody raised against a peptide sequence of human inducible NOS (iNOS) reacted with a protein with a molecular weight (95 kDa) that was different from that of other NOS isoforms. RT-PCR analysis identified the mRNA expression of not only eNOS and bNOS but also iNOS in human and rat muscle. Inhibition of nitric oxide synthase in rats with N(omega)-nitro-L-arginine methyl ester (L-NAME) resulted in a progressive, severe reduction in walking speed (30-fold reduction in walking velocity at day 22, P < 0.001), muscle fiber cross-sectional area (40% reduction at day 22, P < 0.001), and muscle mass (40% reduction in dry weight at day 22, P < 0.01). Rats fed the same regimen of the enantiomer of L-NAME (d-NAME) had normal motor function, muscle fiber morphology, and muscle mass. Taken together, these results imply that there may be a novel nitric oxide synthase in muscle and that NO. generated from muscle may be important in muscle function.