Exercise with low glycogen increases PGC-1α gene expression in human skeletal muscle

Recent studies suggest that carbohydrate restriction can improve the training-induced adaptation of muscle oxidative capacity. However, the importance of low muscle glycogen on the molecular signaling of mitochondrial biogenesis remains unclear. Here, we compare the effects of exercise with low (LG) and normal (NG) glycogen on different molecular factors involved in the regulation of mitochondrial biogenesis. Ten highly trained cyclists (VO_2max 65 ± 1 ml/kg/min, W _max 387 ± 8 W) exercised for 60 min at approximately 64 % VO_2max with either low [166 ± 21 mmol/kg dry weight (dw)] or normal (478 ± 33 mmol/kg dw) muscle glycogen levels achieved by prior exercise/diet intervention. Muscle biopsies were taken before, and 3 h after, exercise. The mRNA of peroxisome proliferator-activated receptor-γ coactivator-1 was enhanced to a greater extent when exercise was performed with low compared with normal glycogen levels (8.1-fold vs. 2.5-fold increase). Cytochrome c oxidase subunit I and pyruvate dehydrogenase kinase isozyme 4 mRNA were increased after LG (1.3- and 114-fold increase, respectively), but not after NG. Phosphorylation of AMP-activated protein kinase, p38 mitogen-activated protein kinases and acetyl-CoA carboxylase was not changed 3 h post-exercise. Mitochondrial reactive oxygen species production and glutathione oxidative status tended to be reduced 3 h post-exercise. We conclude that exercise with low glycogen levels amplifies the expression of the major genetic marker for mitochondrial biogenesis in highly trained cyclists. The results suggest that low glycogen exercise may be beneficial for improving muscle oxidative capacity.     

α-Actinin 4 and BAT1 interaction with the Cytochrome c promoter upon skeletal muscle differentiation

To identify common regulatory features of nuclear genes encoding mitochondrial proteins we searched for regulatory elements in the Cytochrome c promoter during skeletal muscle differentiation in cell culture. A consensus element with the sequence GCTGCCGCAC-(N4-20)-GGSCGYGGG was found in both rat Cyt c and coxIV promoters. This new sequence element with yet undescribed function, but high abundance in promoters of nuclear genes encoding mitochondrial proteins available from the databases, showed a striking change in protein binding in electromobility shift assays when myoblasts were compared to myotubes. Proteins involved in the observed protein-DNA complexes were isolated from myotubes and identified by MALDI-TOF as BAT1, a DEAD-box protein of yet unknown function, heat shock protein HSP84, and α-actinin 4, a non-muscle isoform of the structural protein α-actinin. α-actinin 4 was found to be preferentially localized in the nucleus upon induction of myogenesis, suggesting a signaling function during muscle differentiation. In conclusion, the analyzed sequence motif may be a new candidate for common regulatory elements specific for nuclear encoded mitochondrial genes, and α-actinin 4 may be involved in their regulation. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Contraction-induced changes in TNFα and Akt-mediated signalling are associated with increased myofibrillar protein in rat skeletal muscle

Resistance training results in skeletal muscle hypertrophy, but the molecular signalling mechanisms responsible for this altered phenotype are incompletely understood. We used a resistance training (RT) protocol consisting of three sessions [day 1 (d1), day 3 (d3), day 5 (d5)] separated by 48 h recovery (squat exercise, 4 sets × 10 repetitions, 3 min recovery) to determine early signalling responses to RT in rodent skeletal muscle. Six animals per group were killed 3 h after each resistance training session and 24 and 48 h after the last training session (d5). There was a robust increase in TNFα protein expression, and IKK^Ser180/181 and p38MAPK^{Thr180/Tyr182} phosphorylation on d1 (P < 0.05), which abated with subsequent RT, returning to control levels by d5 for TNFα and IKK^Ser180/181. There was a trend for a decrease in MuRF-1 protein expression, 48 h following d5 of training (P = 0.08). Notably, muscle myofibrillar protein concentration was elevated compared to control 24 and 48 h following RT (P < 0.05). Akt^Ser473 and mTOR^Ser2448 phosphorylation were unchanged throughout RT. Phosphorylation of p70S6k^Thr389 increased 3 h post-exercise on d1, d3 and d5 (P < 0.05), whilst phosphorylation of S6^Ser235/236 increased on d1 and d3 (P < 0.05). Our results show a rapid attenuation of inflammatory signalling with repeated bouts of resistance exercise, concomitant with summation in translation initiation signalling in skeletal muscle. Indeed, the cumulative effect of these signalling events was associated with myofibrillar protein accretion, which likely contributes to the early adaptations in response to resistance training overload in the skeletal muscle.     

Extremely low-frequency electromagnetic fields differentially regulate estrogen receptor-α and -β expression in the rat olfactory bulb

Recently, the effects of extremely low-frequency electromagnetic fields (ELF EMF) on biological systems have been extensively investigated. In this report, the influence of ELF EMF on olfactory bulb (OB) estrogen receptor-α (ERα) mRNA and -β (ERβ) mRNA expression was studied by RT-PCR in adult female and male rats. Results reveal for the first time that ELF EMF exerted a biphasic effect on female OB ERβ mRNA gene expression, which increased during diestrous and decreased during estrous. We did not observe any influence of ELF EMF on female OB ERα mRNA expression. Our data demonstrate a fluctuating pattern of ER-α and -β mRNA expression in the female OB throughout the phases of the estrous cycle in non-ELF EMF-exposed animals. Thus the highest ERα expression was observed in diestrous and the lowest in proestrous. The pattern of ERβ mRNA was less variable, the lowest expression was observed in diestrous. ER-α mRNA and -β mRNA expression level in the male OB did not exhibit any variation either in ELF EMF-exposed or non-ELF EMF-exposed animals. In summary, ELF EMF modulate ERβ gene expression in the OB of female adult rats but not in males.     

Na+–K+ pump location and translocation during muscle contraction in rat skeletal muscle

Muscle contraction may up-regulate the number of Na(+)-K(+) pumps in the plasma membrane by translocation of subunits. Since there is still controversy about where this translocation takes place from and if it takes place at all, the present study used different techniques to characterize the translocation. Electrical stimulation and biotin labeling of rat muscle revealed a 40% and 18% increase in the amounts of the Na(+)-K(+) pump alpha(2) subunit and caveolin-3 (Cav-3), respectively, in the sarcolemma. Exercise induced a 36% and 19% increase in the relative amounts of the alpha(2) subunit and Cav-3, respectively, in an outer-membrane-enriched fraction and a 41% and 17% increase, respectively, in sarcolemma giant vesicles. The Na(+)-K(+) pump activity measured with the 3-O-MFPase assay was increased by 37% in giant vesicles from exercised rats. Immunoprecipitation with Cav-3 antibody showed that 17%, 11% and 14% of the alpha(1) subunits were associated with Cav-3 in soleus, extensor digitorum longus, and mixed muscles, respectively. For the alpha(2), the corresponding values were 17%, 5% and 16%. In conclusion; muscle contraction induces translocation of the alpha subunits, which is suggested to be caused partly by structural changes in caveolae and partly by translocation from an intracellular pool.     

The PPARα agonists fenofibrate and CP-778875 cause increased β-oxidation, leading to oxidative injury in skeletal and cardiac muscle in the rat

Weak peroxisome proliferator-activated receptor (PPAR) α agonists (fibrates) are used to treat dyslipidemia. This study compared the effects of the potent and selective PPARα agonist CP-778875 on peroxisomal β-oxidation and cardiac and/or skeletal muscle injury with those of the weak PPARα agonist fenofibrate. We hypothesized that these muscle effects are mediated through the PPARα receptor, leading to increased β-oxidation and consequent oxidative stress. CP-778875 (5 or 500 mg/kg) and fenofibrate (600 or 2,000→1,200 mg/kg, dose lowered because of intolerance) were administered to rats for six weeks. Standard end points, serum troponin I, heart and skeletal muscle β-oxidation of palmitoyl-CoA, and acyl co-oxidase (AOX) mRNA were assessed. Both compounds dose-dependently increased the incidence and/or severity of cardiomyocyte degeneration and necrosis, heart weight, troponin I, and skeletal muscle degeneration. Mean heart β-oxidation (3.4- to 5.1-fold control) and AOX mRNA (2.4- to 3.2-fold control) were increased with CP-778875 500 mg/kg and both doses of fenofibrate. β-Oxidation of skeletal muscle was not affected by either compound; however, a significant increase in AOX mRNA (1.6- to 2.1-fold control) was observed with CP-778875 500 mg/kg and both doses of fenofibrate. Taken together, these findings were consistent with PPARα agonism and support the link between increased cardiac and skeletal muscle β-oxidation and resultant muscle injury in the rat.     

PGC-1α mRNA level and oxidative capacity of the plantaris muscle in rats with metabolic syndrome, hypertension, and type 2 diabetes

We examined the fiber profiles and the mRNA levels of peroxisome proliferator-activated receptors (PPARα and PPARδ/β) and of the PPARγ coactivator-1α (PGC-1α) in the plantaris muscles of 15-week-old control (WR), metabolic syndrome (CP), hypertensive (SHR), and type 2 diabetic (GK) rats. The deep regions in the muscles of SHR and GK rats exhibited lower percentages of high-oxidative type I and IIA fibers and higher percentages of low-oxidative type IIB fibers compared with WR and CP rats. The surface regions in the muscles of CP, SHR, and GK rats exhibited lower percentages of high-oxidative type IIA fibers and higher percentages of low-oxidative type IIB fibers compared with WR rats. The muscles of SHR and GK rats had lower oxidative enzyme activity compared with WR rats. The muscles of SHR rats had the lowest PPARδ/β mRNA level. In addition, the muscles of SHR and GK rats had lower PGC-1α mRNA level compared with WR and CP rats. We concluded that the plantaris muscles of rats with hypertension and type 2 diabetes have lower oxidative capacity, which is associated with the decreased level of PGC-1α mRNA.     

The effects of β-adrenergic stimulation and exercise on NR4A3 protein expression in rat skeletal muscle

β-Adrenergic stimulation and exercise up-regulate the mRNA expression of nuclear receptor NR4A3, which is involved in the regulation of glucose and fatty acid utilization genes in skeletal muscle. The objective of our study was to examine the effects of β-adrenergic stimulation and exercise on the expression of NR4A3 protein in rat skeletal muscle. A single subcutaneous injection of clenbuterol, which is a β2-adrenergic receptor (β2-AR) agonist, increased NR4A3 mRNA and protein expression in the fast-twitch glycolytic triceps muscle. On the other hand, an acute 3-h session of either treadmill running or swimming did not increase the NR4A3 protein level in the exercised muscle, although both treadmill running and swimming increased NR4A3 mRNA. Finally, loss of postural contractile activity because of hindlimb immobilization reduced NR4A3 mRNA and protein in the slow-twitch oxidative soleus muscle. These results suggest that: β-adrenergic stimulation up-regulates not only NR4A3 mRNA but also NR4A3 protein in fast-twitch glycolytic muscle; exercise may increase NR4A3 mRNA but not NR4A3 protein in skeletal muscle; and local postural contractile activity plays a crucial role in maintaining NR4A3 protein expression level in postural muscle.     

Effects of myostatin deficiency on PGC-1α and FNDC5 expression in three different murine muscle types

Myostatin (MSTN) is a key negative regulator of muscle growth and development. Skeletal, cardiac, and smooth muscles were isolated from MSTN knockout (MSTN?∕?) and control mice to investigate the effect of knocking out MSTN on peroxisome proliferator-activated receptor 1 coactivator (PGC-1α)-III and fibronectin domain 5 (FNDC5) expression. Various molecular biology techniques were used to analyze the changes in PGC-1α-FNDC5 in different muscle types from MSTN?∕? mice. The expression levels of PGC-1α and FNDC5 in the skeletal, cardiac, and smooth muscles of MSTN?∕? mice differed from those in the skeletal, cardiac, and smooth muscles of normal mice. This study revealed that knocking out MSTN resulted in inconsistent PGC-1α and FNDC5 expression in specific muscles. It proved for the first time that MSTN deletion attenuated the expression of PGC-1α and FNDC5 in three different murine muscle types. MSTN deletion may have additional effects on the status ofFNDC5 expression. Further research, however, is needed to confirm this conclusion.     

Caffeine activates preferentially α1-isoform of 5'AMP-activated protein kinase in rat skeletal muscle

Aim: Caffeine activates 5′AMP‐activated protein kinase (AMPK), a signalling intermediary implicated in the regulation of glucose, lipid and energy metabolism in skeletal muscle. Skeletal muscle expresses two catalytic α subunits of AMPK, α1 and α2, but the isoform specificity of caffeine‐induced AMPK activation is unclear. The aim of this study was to determine which α isoform is preferentially activated by caffeine in vitro and in vivo using rat skeletal muscle. Methods: Rat epitrochlearis muscle was isolated and incubated in vitro in the absence or presence of caffeine. In another experiment, the muscle was dissected after intravenous injection of caffeine. Isoform‐specific AMPK activity, the phosphorylation status of AMPKα Thr¹⁷² and acetyl‐CoA carboxylase (ACC) Ser⁷⁹, the concentrations of ATP, phosphocreatine (PCr) and glycogen, and 3‐O‐methyl‐d ‐glucose (3MG) transport activity were estimated. Results: Incubation of isolated epitrochlearis muscle with 1 mm of caffeine for 15 min increased AMPKα1 activity, but not AMPKα2 activity; concentrations of ATP, PCr and glycogen were not affected. Incubation with 3 mm of caffeine activated AMPKα2 and reduced PCr and glycogen concentrations. Incubation with 1 mm of caffeine increased the phosphorylation of AMPK and ACC and enhanced 3MG transport. Intravenous injection of caffeine (5 mg kg^?1) predominantly activated AMPKα1 and increased 3MG transport without affecting energy status. Conclusion: Our results suggest that of the two α isoforms of AMPK, AMPKα1 is predominantly activated by caffeine via an energy‐independent mechanism and that the activation of AMPKα1 increases glucose transport and ACC phosphorylation in skeletal muscle.     

CL 316,243, a selective β3-adrenergic agonist, inhibits protein breakdown in rat skeletal muscle

The in vitro effect of CL 316,243 (CL), a selective ₃-adrenoceptor agonist in the rate of overall proteolysis, the activity of proteolytic systems (lysosomal, Ca²⁺-dependent, ATP-dependent, and ATP-independent) and in the process of protein synthesis was investigated in rat skeletal muscles. The rate of overall proteolysis in soleus muscle from rats incubated with CL (10^–4 and 10^–5 M) or epinephrine (10^–5 M) was significantly decreased. In vitro rates of maximal activity of Ca²⁺-dependent proteolysis in soleus muscles were decreased by about 41% in the presence of 10^–5 M CL. No change was observed in the activities of the lysosomal, ATP-dependent or ATP-independent proteolytic systems. The anti-proteolytic effect of CL or epinephrine was partially prevented by 10^–5 M SR 59230A, a selective ₃-adrenoceptor antagonist. The increase of proteolysis induced by food deprivation in soleus was abolished by in vitro addition of 10^–5 M CL. No change in proteolysis was observed in extensor digitorum longus (EDL) muscles incubated with any concentration of the ₃-adrenoceptor agonist tested. Rates of protein synthesis were not affected by 10^–4 M CL neither in soleus nor EDL. The data suggest that a ₃-adrenoceptor-mediated inhibition of Ca²⁺-dependent proteolysis participates of the antiproteolytic effect of catecholamines in oxidative muscles.     

Resistance exercise, but not endurance exercise, induces IKKβ phosphorylation in human skeletal muscle of training-accustomed individuals

The mammalian target of rapamycin complex 1 (mTORC1) is considered an important role in the muscular adaptations to exercise. It has been proposed that exercise-induced signaling to mTORC1 do not require classic growth factor PI3K/Akt signaling. Activation of IKKβ and the mitogen-activated protein kinases (MAPKs) Erk1/2 and p38 has been suggested to link inflammation and cellular stress to activation of mTORC1 through the tuberous sclerosis 1 (TSC1)/tuberous sclerosis 2 (TSC2) complex. Consequently, activation of these proteins constitutes potential alternative mechanisms of mTORC1 activation following exercise. Previously, we demonstrated that mTOR is preferentially activated in response to resistance exercise compared to endurance exercise in trained individuals without concomitant activation of Akt. In the present study, we extended this investigation by examining IκB kinase complex (IKK), TSC1, MAPK, and upstream Akt activators, along with gene expression of selected cytokines, in skeletal muscles from these subjects. Biopsies were sampled prior to, immediately after, and in the recovery period following resistance exercise, endurance exercise, and control interventions. The major finding was that IKKβ phosphorylation increased exclusively after resistance exercise. No changes in TSC1, Erk1/2, insulin receptor, or insulin receptor substrate 1 phosphorylation were observed in any of the groups, while p38 phosphorylation was higher in the resistance exercise group compared to both other groups immediately after the intervention. Resistance and endurance exercise increased IL6, IL8, and TNFα gene expression immediately after exercise. The non-exercise control group demonstrated that cytokine gene expression is also sensitive to repeated biopsy sampling, whereas no effect of repeated biopsy sampling on protein expression and phosphorylation was observed. In conclusion, resistance exercise, but not endurance exercise, increases IKKβ phosphorylation in trained human subjects, which support the idea that IKKβ can influence the activation of mTORC1 in human skeletal muscle.