Isolated Mouse Soleus Research Articles

Effect of high-fat diet on isometric, concentric and eccentric contractile performance of skeletal muscle isolated from female CD-1 mice.

Despite evidence inferring muscle and contractile mode-specific effects of high-fat diet (HFD), no study has yet considered the impact of HFD directly on eccentric muscle function. The present work uniquely examined the effect of 20-week HFD on the isometric, concentric and eccentric muscle function of isolated mouse soleus (SOL) and extensor digitorum longus (EDL) muscles. CD-1 female mice were randomly split into a control (n=16) or HFD (n=17) group and for 20 weeks consumed standard lab chow or HFD. Following this period, SOL and EDL muscles were isolated and assessments of maximal isometric force and concentric work loop (WL) power were performed. Each muscle was then subjected to either multiple concentric or eccentric WL activations. Post-fatigue recovery, as an indicator of incurred damage, was measured via assessment of concentric WL power. In the EDL, absolute concentric power and concentric power normalised to muscle mass were reduced in the HFD group (P<0.038). HFD resulted in faster concentric fatigue and reduced eccentric activity-induced muscle damage (P<0.05). For the SOL, maximal isometric force was increased, and maximal eccentric power normalised to muscle mass and concentric fatigue were reduced in the HFD group (P<0.05). HFD effects on eccentric muscle function are muscle-specific and have little relationship with changes in isometric or concentric function. HFD has the potential to negatively affect the intrinsic concentric and eccentric power-producing capacity of skeletal muscle, but a lack of a within-muscle uniform response indicates disparate mechanisms of action which require further investigation.

The effects of muscle starting length on work loop power output of isolated mouse soleus and extensor digitorum longus muscle.

Force-length relationships derived from isometric activations may not directly apply to muscle force production during dynamic contractions. As such, different muscle starting lengths between isometric and dynamic conditions could be required to achieve maximal force and power. Therefore, this study examined the effects of starting length [±5-10% of length corresponding to maximal twitch force (L0)] on work loop (WL) power output (PO), across a range of cycle frequencies, of the soleus (SOL) and extensor digitorum longus muscle (EDL; N=8-10) isolated from ∼8week old C57 mice. Furthermore, passive work was examined at a fixed cycle frequency to determine the association of passive work and active net work. Starting length affected maximal WL PO of the SOL and EDL across evaluated cycle frequencies (P<0.030, ηp2>0.494). For the SOL, PO produced at -5% L0 was greater than that at most starting lengths (P<0.015, Cohen's d>0.6), except -10% L0 (P=0.135, d<0.4). However, PO produced at -10% L0 versus L0 did not differ (P=0.138, d=0.35-0.49), indicating -5% L0 is optimal for maximal SOL WL PO. For the EDL, WL PO produced at -10% L0 was lower than that at most starting lengths (P<0.032, d>1.08), except versus -5% L0 (P=0.124, d<0.97). PO produced at other starting lengths did not differ (P>0.163, d<1.04). For the SOL, higher passive work was associated with reduced PO (Spearman's r=0.709, P<0.001), but no relationship was observed between passive work and PO of the EDL (Pearson's r=0.191, r2=0.04, P=0.184). This study suggests that starting length should be optimised for both static and dynamic contractions and confirms that the force-length curve during dynamic contractions is muscle specific.

High-fat diet effects on contractile performance of isolated mouse soleus and extensor digitorum longus when supplemented with high dose vitamin D.

Evidence suggests vitamin D3 (VD) supplementation can reduce accumulation of adipose tissue and inflammation and promote myogenesis in obese individuals, and thus could mitigate obesity-induced reductions in skeletal muscle (SkM) contractility. However, this is yet to be directly investigated. This study, using the work-loop technique, examined effects of VD (cholecalciferol) supplementation on isolated SkM contractility. Female mice (n = 37) consumed standard low-fat diet (SLD) or high-fat diet (HFD), with or without VD (20,000IU/kg-1 ) for 12weeks. Soleus and EDL (n = 8-10 per muscle per group) were isolated and absolute and normalized (to muscle size and body mass) isometric force and power output (PO) were measured, and fatigue resistance determined. Absolute and normalized isometric force and PO of soleus were unaffected by diet (P>0.087). However, PO normalized to body mass was reduced in HFD groups (P<0.001). Isometric force of extensor digitorum longus (EDL) was unaffected by diet (P>0.588). HFD reduced EDL isometric stress (P=0.048) and absolute and normalized PO (P<0.031), but there was no effect of VD (P>0.493). Cumulative work during fatiguing contractions was lower in HFD groups (P<0.043), but rate of fatigue was unaffected (P>0.060). This study uniquely demonstrated that high-dose VD had limited effects on SkM contractility and did not offset demonstrated adverse effects of HFD. However, small and moderate effect sizes suggest improvement in EDL muscle performance and animal morphology in HFD VD groups. Given effect sizes observed, coupled with proposed inverted U-shaped dose-effect curve, future investigations are needed to determine dose/duration specific responses to VD, which may culminate in improved function of HFD SkM.

The potassium-glycogen interaction on force and excitability in mouse skeletal muscle: implications for fatigue.

A reduced muscle glycogen content and potassium (K+ ) disturbances across muscle membranes occur concomitantly during repeated intense exercise and together may contribute to skeletal muscle fatigue. Therefore, we examined whether raised extracellular K+ concentration ([K+ ]o ) (4 to 11 mM) interacts with lowered glycogen to reduce force production. Isometric contractions were evoked in isolated mouse soleus muscles (37°C) using direct supramaximal field stimulation. (1) Glycogen declined markedly in non-fatigued muscle with >2h exposure in glucose-free physiological saline compared with control solutions (11 mM glucose), i.e. to <45% control. (2) Severe glycogen depletion was associated with increased 5'-AMP-activated protein kinase activity, indicative of metabolic stress. (3) The decline of peak tetanic force at 11mM [K+ ]o was exacerbated from 67% initial at normal glycogen to 22% initial at lowered glycogen. This was due to a higher percentage of inexcitable fibres (71% vs. 43%), yet without greater sarcolemmal depolarisation or smaller amplitude action potentials. (4) Returning glucose while at 11mM [K+ ]o increased both glycogen and force. (5) Exposure to 4mM [K+ ]o glucose-free solutions (15 min) did not increase fatiguability during repeated tetani; however, after recovery there was a greater force decline at 11mM [K+ ]o at lower than normal glycogen. (6) An important exponential relationship was established between relative peak tetanic force at 11mM [K+ ]o and muscle glycogen content. These findings provide direct evidence of a synergistic interaction between raised [K+ ]o and lowered muscle glycogen as the latter shifts the peak tetanic force-resting EM relationship towards more negative resting EM due to lowered sarcolemmal excitability, which hence may contribute to muscle fatigue. KEY POINTS: Diminished muscle glycogen levels and raised extracellular potassium concentrations ([K+ ]o ) occur simultaneously during intense exercise and together may contribute to muscle fatigue. Prolonged exposure of isolated non-fatigued soleus muscles of mice to glucose-free physiological saline solutions markedly lowered muscle glycogen levels, as does fatigue then recovery in glucose-free solutions. For both approaches, the subsequent decline of maximal force at 11mM[K+ ]o , which mimics interstitial [K+ ] levels during intense exercise, was exacerbated at lowered compared with normal glycogen. This was mainly due to many more muscle fibres becoming inexcitable. We established an important relationship that provides evidence of a synergistic interaction between raised [K+ ]o and lowered glycogen content to reduce force production. This paper indicates that partially lowered muscle glycogen (and/or metabolic stress) together with elevated interstitial [K+ ] interactively lowers muscle force, and hence may diminish performance especially during repeated high-intensity exercise.

Ergogenic effect of ischemic preconditioning is not directly conferred to isolated skeletal muscle via blood.

Ischemic preconditioning (IPC) in humans has been demonstrated to confer ergogenic benefit to aerobic exercise performance, with an improvement in the response rate when the IPC stimulus is combined with concurrent exercise. Despite potential performance improvements, the nature of the neuronal and humoral mechanisms of conferral and their respective contributions to ergogenic benefit remain unclear. We sought to examine the effects of the humoral component of ischemic preconditioning on skeletal muscle tissue using preconditioned human serum and isolated mouse soleus. Isolated mouse soleus was electrically stimulated to contract while in human serum preconditioned with either traditional (IPC) or augmented (AUG) ischemic preconditioning compared to control (CON) and exercise (ERG) preconditioning. Force frequency (FF) curves, twitch responses, and a fatigue-recovery protocol were performed on muscles before and after the addition of serum. After preconditioning, human participants performed a 4km cycling time trial in order to identify responders and non-responders to IPC. No differences in indices of contractile function, fatiguability, nor recovery were observed between conditions in mouse soleus muscles. Further, no human participants improved performance in a 4-km cycling time trial in response to traditional nor augmented ischemic preconditioning compared to control or exercise conditions (CON 407.7 ± 41.1s, IPC 411.6 ± 41.9s, ERG 408.8 ± 41.4s, AUG 414.1 ± 41.9s). Our findings do not support the conferral of ergogenic benefit via a humoral component of IPC at the intracellular level. Ischemic preconditioning may not manifest prominently at submaximal exercise intensities, and augmented ischemic preconditioning may have a hormetic relationship with performance improvements.

High-fat diet affects measures of skeletal muscle contractile performance in a temperature-specific manner but does not influence regional thermal sensitivity.

The present study examined whether high-fat diet (HFD) consumption for 20weeks had a temperature-specific effect on the contractile performance and regional thermal sensitivity of isolated mouse soleus and diaphragm muscle. Four-week-old female CD-1 mice were randomly selected to consume either a standard laboratory diet or a standard laboratory diet in conjunction with a HFD for 20weeks. Peripheral soleus and core diaphragm were isolated from each animal and maximal isometric force and work loop power were assessed at 20, 28, 35 and 40°C. Increasing temperature to 35°C resulted in greater isometric stress, lower activation and relaxation time, and higher work loop power in both muscles. A further increase in temperature to 40°C did not affect isometric force but increased work loop power output of the soleus. Conversely, isometric force of the diaphragm was reduced and work loop power maintained when temperature was increased to 40°C. HFD consumption resulted in greater isometric force and absolute work loop power of the soleus and reduced isometric stress of the diaphragm, effects that were less apparent at lower temperatures. When the relationship between temperature and each measure of contractile function was examined by linear regression, there was no difference in slope between the control or HFD groups for either the soleus or diaphragm. These results indicate that whilst contractile function initially increases with temperature, the temperature to elicit maximal performance is muscle and contractile mode specific. Furthermore, HFD effects on contractile function are temperature specific, but HFD does not influence the relationship between temperature and performance.

Insulin-stimulated glucose uptake partly relies on p21-activated kinase (PAK)2, but not PAK1, in mouse skeletal muscle.

Muscle-specific genetic ablation of p21-activated kinase (PAK)2, but not whole-body PAK1 knockout, impairs glucose tolerance in mice. Insulin-stimulated glucose uptake partly relies on PAK2 in glycolytic extensor digitorum longus muscle By contrast to previous reports, PAK1 is dispensable for insulin-stimulated glucose uptake in mouse muscle. The group I p21-activated kinase (PAK) isoforms PAK1 and PAK2 are activated in response to insulin in skeletal muscle and PAK1/2signalling is impaired in insulin-resistant mouse and human skeletal muscle. Interestingly, PAK1has been suggested to be required for insulin-stimulated glucose transporter 4 translocation in mouse skeletal muscle. Therefore, the present study aimed to examine the role of PAK1 in insulin-stimulated muscle glucose uptake. The pharmacological inhibitor of group I PAKs, IPA-3 partially reduced (-20%) insulin-stimulated glucose uptake in isolated mouse soleus muscle (P<0.001). However, because there was no phenotype with genetic ablation of PAK1 alone, consequently, the relative requirement for PAK1 and PAK2 in whole-body glucose homeostasis and insulin-stimulated muscle glucose uptake was investigated. Whole-body respiratory exchange ratio was largely unaffected in whole-body PAK1 knockout (KO), muscle-specific PAK2 KO and in mice with combined whole-body PAK1 KO and muscle-specific PAK2 KO. By contrast, glucose tolerance was mildly impaired in mice lacking PAK2specifically in muscle, but not PAK1 KO mice. Moreover, while PAK1 KO muscles displayed normal insulin-stimulated glucose uptake in vivo and in isolated muscle, insulin-stimulated glucose uptake was slightly reduced in isolated glycolytic extensor digitorum longus muscle lacking PAK2 alone (-18%) or in combination with PAK1 KO (-12%) (P<0.05). In conclusion, glucose tolerance and insulin-stimulated glucose uptake partly rely on PAK2 in glycolytic mouse muscle, whereas PAK1 is dispensable for whole-body glucose homeostasis and insulin-stimulated muscle glucose uptake.

Isoproterenol enhances force production in mouse glycolytic and oxidative muscle via separate mechanisms

Fight or flight is a biologic phenomenon that involves activation of β-adrenoceptors in skeletal muscle. However, how force generation is enhanced through adrenergic activation in different muscle types is not fully understood. We studied the effects of isoproterenol (ISO, β-receptor agonist) on force generation and energy metabolism in isolated mouse soleus (SOL, oxidative) and extensor digitorum longus (EDL, glycolytic) muscles. Muscles were stimulated with isometric tetanic contractions and analyzed for metabolites and phosphorylase activity. Under conditions of maximal force production, ISO enhanced force generation markedly more in SOL (22%) than in EDL (8%). Similarly, during a prolonged tetanic contraction (30 s for SOL and 10 s for EDL), ISO-enhanced the force × time integral more in SOL (25%) than in EDL (3%). ISO induced marked activation of phosphorylase in both muscles in the basal state, which was associated with glycogenolysis (less in SOL than in EDL), and in EDL only, a significant decrease (16%) in inorganic phosphate (Pi). ATP turnover during sustained contractions (1 s EDL, 5 s SOL) was not affected by ISO in EDL, but essentially doubled in SOL. Under conditions of maximal stimulation, ISO has a minor effect on force generation in EDL that is associated with a decrease in Pi, whereas ISO has a marked effect on force generation in SOL that is associated with an increase in ATP turnover. Thus, phosphorylase functions as a phosphate trap in ISO-mediated force enhancement in EDL and as a catalyzer of ATP supply in SOL.

Does Dietary-Induced Obesity in Old Age Impair the Contractile Performance of Isolated Mouse Soleus, Extensor Digitorum Longus and Diaphragm Skeletal Muscles?

Ageing and obesity independently have been shown to significantly impair isolated muscle contractile properties, though their synergistic effects are poorly understood. We uniquely examined the effects of 9 weeks of a high-fat diet (HFD) on isometric force, work loop power output (PO) across a range of contractile velocities, and fatigability of 79-week-old soleus, extensor digitorum longus (EDL) and diaphragm compared with age-matched lean controls. The dietary intervention resulted in a significant increase in body mass and gonadal fat pad mass compared to the control group. Despite increased muscle mass for HFD soleus and EDL, absolute isometric force, isometric stress (force/CSA), PO normalised to muscle mass and fatigability was unchanged, although absolute PO was significantly greater. Obesity did not cause an alteration in the contractile velocity that elicited maximal PO. In the obese group, normalised diaphragm PO was significantly reduced, with a tendency for reduced isometric stress and fatigability was unchanged. HFD soleus isolated from larger animals produced lower maximal PO which may relate to impaired balance in older, larger adults. The increase in absolute PO is smaller than the magnitude of weight gain, meaning in vivo locomotor function is likely to be impaired in old obese adults, with an association between greater body mass and poorer normalised power output for the soleus. An obesity-induced reduction in diaphragm contractility will likely impair in vivo respiratory function and consequently contribute further to the negative cycle of obesity.

Effect of postexercise temperature elevation on postexercise glycogen metabolism of isolated mouse soleus muscle.

The effects of temperature elevation after intense repeated contractions on glycogen and energy metabolism as well as contractile function of isolated mouse soleus muscle (slow twitch, oxidative) were investigated. Muscles were stimulated electrically to perform repeated tetanic contractions for 10 min at 25°C, which reduced tetanic force by ~85% and glycogen by 50%. After 120-min recovery at 25°C glycogen was fully restored (~125% of basal), whereas after recovery at 35°C glycogen decreased further (~25% of basal). Glycogen synthase fractional activity averaged 31.8 ± 3.1% (baseline = 33.8 ± 3.4%) after 120-min recovery at 25°C but was increased after recovery at 35°C (63.8 ± 4.8%; P < 0.001 vs. 25°C). Phosphorylase fractional and total activities were not affected by the higher temperature. However, recovery at 35°C resulted in a significantly higher content of the phosphorylase substrate inorganic phosphate (~20%; P < 0.01 vs. 25°C). Finally, fatigue development during a subsequent bout of repeated contractions at 25°C was similar after 120-min recovery at 25°C and 35°C. These data demonstrate that after intense contractions elevated temperature inhibits glycogen accumulation, likely by increasing the availability of the phosphorylase substrate inorganic phosphate, but has no effect on fatigue development. Thus after heat exposure phosphorylase plays a significant role in glycogen accumulation, and glycogen does not limit muscle performance in isolated mouse soleus muscle after recovery from elevated temperature. NEW & NOTEWORTHY Whether elevated temperature affects glycogen biogenesis and contractile performance of isolated slow-twitch muscle is not known. Here we show that after a bout of repeated contractions in isolated mouse soleus muscle at 25°C, increasing muscle temperature during recovery to 35°C blocked glycogen accumulation compared with recovery at 25°C. Surprisingly, during a subsequent bout of repeated contractions at 25°C, the rate of fatigue was not different between groups after recovery at the two temperatures.

Investigating a dose-response relationship between high-fat diet consumption and the contractile performance of isolated mouse soleus, EDL and diaphragm muscles.

Recent evidence has demonstrated an obesity-induced, skeletal muscle-specific reduction in contractile performance. The extent and magnitude of these changes in relation to total dose of high-fat diet consumption remains unclear. This study aimed to examine the dose-response relationship between a high-fat diet and isolated skeletal muscle contractility. 120 female CD1 mice were randomly assigned to either control group or groups receiving 2, 4, 8 or 12 weeks of a high-calorie diet (N = 24). At 20weeks, soleus, EDL or diaphragm muscle was isolated (n = 8 in each case) and isometric force, work loop power output and fatigue resistance were measured. When analysed with respect to feeding duration, there was no effect of diet on the measured parameters prior to 8weeks of feeding. Compared to controls, 8-week feeding caused a reduction in normalised power of the soleus, and 8- and 12-week feeding caused reduced normalised isometric force, power and fatigue resistance of the EDL. Diaphragm from the 12-week group produced lower normalised power, whereas 8- and 12-week groups produced significantly lower normalised isometric force. Correlation statistics indicated that body fat accumulation and decline in contractility will be specific to the individual and independent of the feeding duration. The data indicate that a high-fat diet causes a decline in muscle quality with specific contractile parameters being affected in each muscle. We also uniquely demonstrate that the amount of fat gain, irrespective of feeding duration, may be the main factor in reducing contractile performance.

The Effect of Increasing Age on the Concentric and Eccentric Contractile Properties of Isolated Mouse Soleus and Extensor Digitorum Longus Muscles.

There is currently a limited amount of literature investigating the age-related changes in eccentric muscle function in vitro. The present study uniquely uses the work loop (WL) technique, to better replicate in vivo muscle function, in the assessment of the age- and muscle-specific changes in acute and sustained concentric and eccentric power and recovery. Whole soleus or extensor digitorum longus (EDL) muscles were isolated from 10-week and 78-week-old mice and acute and sustained concentric and eccentric WL power assessed. Despite an age-related increase in body and muscle mass, peak absolute power for both muscles was unaffected by age. Peak concentric power normalized to muscle mass declined significantly for each muscle, while peak normalized eccentric power declined only for soleus. Fatigue resistance and recovery for the soleus did not differ between age or contraction type. Older EDL was less resistant to concentric fatigue, but was better able to withstand sustained eccentric activity than young EDL. We have shown that age-related changes in muscle quality are more limited for eccentric function than concentric function. A greater bodily inertia is likely to further reduce in vivo locomotor performance in older animals.

The effect of obesity on the contractile performance of isolated mouse soleus, EDL, and diaphragm muscles.

The effect of obesity on isolated muscle function is surprisingly underresearched. The present study is the first to examine the effects of obesity on isolated muscle performance using a method that more closely represents real-world muscle function. This work uniquely establishes a muscle-specific profile of mechanical changes in relation to underpinning mechanisms. These findings may be important to understanding the negative cycle of obesity and in designing interventions for improving weight status.

The Impact of Hyperthermia on Receptor-Mediated Interleukin-6 Regulation in Mouse Skeletal Muscle.

In inflammatory cells, hyperthermia inhibits lipopolysaccharide (LPS)-induced interleukin-6 (IL-6) gene expression and protein secretion. Since hyperthermia alone stimulates IL-6 in skeletal muscle, we hypothesized that it would amplify responses to other receptor-mediated stimuli. IL-6 regulation was tested in C2C12 myotubes and in soleus during treatment with epinephrine (EPI) or LPS. In EPI-treated myotubes (100 ng/ml), 1 h exposure at 40.5°C-42°C transiently increased IL-6 mRNA compared to EPI treatment alone at 37°C. In LPS-treated myotubes (1 μg/ml), exposure to 41°C-42°C also increased IL-6 mRNA. In isolated mouse soleus, similar amplifications of IL-6 gene expression were observed in 41°C, during both low (1 ng/ml) and high dose (100 ng/ml) EPI, but only in high dose LPS (1 μg/ml). In myotubes, heat increased IL-6 secretion during EPI exposure but had no effect or inhibited secretion with LPS. In soleus there were no effects of heat on IL-6 secretion during either EPI or LPS treatment. Mechanisms for the effects of heat on IL-6 mRNA were explored using a luciferase-reporter in C2C12 myotubes. Overexpression of heat shock factor-1 (HSF-1) had no impact on IL-6 promoter activity during EPI stimulation, but elevated IL-6 promoter activity during LPS stimulation. In contrast, when the activator protein-1 (AP-1) element was mutated, responses to both LPS and EPI were suppressed in heat. Using siRNA against activating transcription factor-3 (ATF-3), a heat-stress-induced inhibitor of IL-6, no ATF-3-dependent effects were observed. The results demonstrate that, unlike inflammatory cells, hyperthermia in muscle fibers amplifies IL-6 gene expression to EPI and LPS. The effect appears to reflect differential engagement of HSF-1 and AP-1 sensitive elements on the IL-6 gene, with no evidence for involvement of ATF-3. The functional significance of increased IL-6 mRNA expression during heat may serve to overcome the well-known suppression of protein synthetic pathways occurring during heat shock.

Does a physiological concentration of taurine increase acute muscle power output, time to fatigue, and recovery in isolated mouse soleus (slow) muscle with or without the presence of caffeine?

High concentrations of caffeine and taurine are key constituents of many ergogenic supplements ingested acutely to provide legal enhancements in athlete performance. Despite this, there is little evidence supporting the claims for the performance-enhancing effects of acute taurine supplementation. In-vitro models have demonstrated that a caffeine-induced muscle contracture can be further potentiated when combined with a high concentration of taurine. However, the high concentrations of caffeine used in previous research would be toxic for human consumption. Therefore, this study aimed to investigate whether a physiological dose of caffeine and taurine would directly potentiate skeletal muscle performance. Isolated mouse soleus muscle was used to examine the effects of physiological taurine (TAU), caffeine (CAF), and taurine-caffeine combined (TC) on (i) acute muscle power output; (ii) time to fatigue; and (iii) recovery from fatigue, compared with the untreated controls (CON). Treatment with TAU failed to elicit any significant difference in the measured parameters. Treatment with TC resulted in a significant increase in acute muscle power output and faster time to fatigue. The ergogenic benefit posed by TC was not different from the effects of caffeine alone, suggesting no acute ergogenic benefit of taurine.

The effects of elevated levels of sodium bicarbonate (NaHCO3) on the acute power output and time to fatigue of maximally stimulated mouse soleus and EDL muscles

This study examined the effects of elevated buffer capacity [~32 mM HCO₃(-)] through administration of sodium bicarbonate (NaHCO₃) on maximally stimulated isolated mouse soleus (SOL) and extensor digitorum longus (EDL) muscles undergoing cyclical length changes at 37 °C. The elevated buffering capacity was of an equivalent level to that achieved in humans with acute oral supplementation. We evaluated the acute effects of elevated [HCO₃(-)] on (1) maximal acute power output (PO) and (2) time to fatigue to 60 % of maximum control PO (TLIM60), the level of decline in muscle PO observed in humans undertaking similar exercise, using the work loop technique. Acute PO was on average 7.0 ± 4.8 % greater for NaHCO₃-treated EDL muscles (P < 0.001; ES = 2.0) and 3.6 ± 1.8 % greater for NaHCO₃-treated SOL muscles (P < 0.001; ES = 2.3) compared to CON. Increases in PO were likely due to greater force production throughout shortening. The acute effects of NaHCO₃ on EDL were significantly greater (P < 0.001; ES = 0.9) than on SOL. Treatment of EDL (P = 0.22; ES = 0.6) and SOL (P = 0.19; ES = 0.9) with NaHCO₃ did not alter the pattern of fatigue. Although significant differences were not observed in whole group data, the fatigability of muscle performance was variable, suggesting that there might be inter-individual differences in response to NaHCO₃ supplementation. These results present the best indication to date that NaHCO₃ has direct peripheral effects on mammalian skeletal muscle resulting in increased acute power output.

AICAR reverses ketone body mediated insulin resistance in isolated oxidative muscle

Recently it was demonstrated that the ketone body β-hydroxybutyrate (BOH) inhibits insulin-mediated glucose transport in isolated oxidative muscle, which was associated with decreased phosphorylation of Akt/protein kinase B. The purpose of the present study was to determine if activation of AMP-dependent protein kinase by the pharmacological activator AICAR could reverse the insulin resistance induced by BOH. Isolated mouse soleus muscle was incubated in vitro in the absence or presence of 5mM BOH for ∼20h. Following prolonged incubation, insulin increased 2-deoxyglucose glucose (2-DG) uptake 3-fold, but in the presence of BOH most of the insulin response was lost (only ∼30% remained). Addition of 2mM AICAR during the last 2h of prolonged incubation increased the insulin response in the presence of BOH to ∼80% of the normal insulin effect on 2-DG uptake. The AICAR-mediated reversal of the insulin resistance was not associated with a restoration of the insulin effect on Akt/protein kinase B phosphorylation. However, AICAR enhanced the insulin-induced phosphorylation of the Akt substrate, AS160. In conclusion, these data demonstrate that AICAR reverses the negative effect of BOH on insulin-mediated glucose uptake and this is attributed to activation of a late step in insulin signaling.

Clenbuterol-Stimulated Glucose Uptake Activates both GS and GI Pathways through Β2-Adrenoceptor in Mouse Isolated Soleus Muscle

&beta;2-adrenoceptors activated by adrenaline can also couple to both Gs and Gi proteins. The former is associated with an increase in cAMP to illicit the effect of the catecholamine. In the later, &beta;2-AR induces PKA-catalysed phosphorylation of the receptor, which intends couples to Gi, at high concentration. We proposed that, clenbuterol which stimulates glucose uptake at low concentra-tion and inhibits it at high concentration might have identical signalling pathway as adrenaline. Mouse isolated muscles were pre-incubated in flasks containing 3 ml of Krebs-Henseleit Bicar-bonate buffer. After 120 min of pre-incubation, with the appropriate concentration of PTX the muscles were transferred to another incubation flask containing 3 ml of the same buffer and 0.3mCi 2-deoxy[1-14C]glucose containing varying concentrations of adrenaline, clenbuterol or 1nM insulin with or without PTX concentrations of 100ng/ml for adrenaline and clenbuterol and 0.1 or 1.0ng/ml for insulin, or 1M acetylcholine for 45 minutes. Adrenaline stimulated glu-cose uptake in isolated mouse soleus muscle at low concentration (10-10M) and inhibited it at high concentration (10-5M). The effect of the lower concentration was mediated through the &beta;2-AR coupling to the Gs protein and to the Gi protein at high concentration. Similarly, clenbuterol stimulated glucose uptake at lower concentration (10-11M) is mediated through the &beta;2-AR cou-pling to the Gs protein and to the Gi protein at the higher concentration (10-7M). These effects of high concentration of adrenaline and clenbuterol were supported by the fact that 100ng/ml PTX relieved their inhibitory effects. The higher concentration effect of clenbuterol was additionally supported by the fact that, 1M acetylcholine, relieved the inhibitory effect. Keywords: PTX, Pertusis toxin, G-proteins, Guanine nucleotide binding proteins, &beta;-AR, beta adrenoceptor, M2, muscarinic receptors

β-Hydroxybutyrate inhibits insulin-mediated glucose transport in mouse oxidative muscle

Blood ketone body levels increase during starvation and untreated diabetes. Here we tested the hypothesis that ketone bodies directly inhibit insulin action in skeletal muscle. We investigated the effect of d,l-beta-hydroxybutyrate (BOH; the major ketone body in vivo) on insulin-mediated glucose uptake (2-deoxyglucose) in isolated mouse soleus (oxidative) and extensor digitorum longus (EDL; glycolytic) muscle. BOH inhibited insulin-mediated glucose uptake in soleus (but not in EDL) muscle in a time- and concentration-dependent manner. Following 19.5 h of exposure to 5 mM BOH, insulin-mediated (20 mU/ml) glucose uptake was inhibited by approximately 90% (substantial inhibition was also observed in 3-O-methylglucose transport). The inhibitory effect of BOH was reproduced with d- but not l-BOH. BOH did not significantly affect hypoxia- or AICAR-mediated (activates AMP-dependent protein kinase) glucose uptake. The BOH effect did not require the presence/utilization of glucose since it was also seen when glucose in the medium was substituted with pyruvate. To determine whether the BOH effect was mediated by oxidative stress, an exogenous antioxidant (1 mM tempol) was used; however, tempol did not reverse the BOH effect on insulin action. BOH did not alter the levels of total tissue GLUT4 protein or insulin-mediated tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 but blocked insulin-mediated phosphorylation of protein kinase B by approximately 50%. These data demonstrate that BOH inhibits insulin-mediated glucose transport in oxidative muscle by inhibiting insulin signaling. Thus ketone bodies may be potent diabetogenic agents in vivo.

Electrical interval stimulation improves calcium handling rapidly but transiently in isolated mouse soleus muscle

Running wheel training in mice involves burst activity and boosts cellular capacity for cation homeostasis in hindlimb muscles (de Snoo, FASEB J 19:A132, 2005). Here, we investigated what biochemical mechanism underlies improved calcium handling in soleus muscle. Soleus (SOL) of 6-wks trained and control C57Bl/6 mice were ligated at the tendons, tied to a force transducer, and set at resting length. Baseline force (BLF) was used as index of free calcium. First, a 300 s bout of 6 Hz stimulation was recorded. BLF increased in control SOL to 5-fold higher levels than in trained SOL. Next, a short-term training protocol mimicking mouse running activity was executed consisting of four sets, separated by 60 s rest, of ten 10 s trains (duty cycle 0.5) of 6 Hz stimulation. In set I, train 1, BLF steeply increased in control SOL as before. During trains 2–10, BLF remained higher in control though markedly less pronounced. In set II, the same result was found. In set III, however, changes in peak- and baseline force were identical for control and trained SOL from train 6 on. In set IV, this result was found from train 3 on. During a next 150 s bout of 6 Hz stimulation control and trained SOL mechanically performed identically. This acquired ‘short-term training’ property was lost after 600 s of rest in control, but not trained SOL. These results may be explained by transient activation of the cAMP-PKA pathway transiently boosting calcium pumping via disinhibition of SERCA2A through phospholamban phosphorylation.