Exercise In Rat Skeletal Muscle Research Articles

Exercise increases the expression of glucose transport and lipid metabolism genes at optimum level time point 6 hours post exercise in rat skeletal muscle

AimType 2 diabetes constitutes more than 90% of all diabetic cases and is characterized by persistent increase in glucose level in the blood (hyperglycemia) as well as lipid and protein metabolic disorders that may induce insulin resistance. Increased lipid levels induce type 2 diabetes by interfering with the insulin‐signaling pathway responsible for glucose transport. Individuals who suffer from type 2 diabetes are usually characterized in part by down‐regulation of glucose transport genes such as glut4 and down‐regulation of mitochondrial lipid oxidizing genes such as carnitine palmitoyltransferase‐1 (Cpt‐1). Nuclear respiratory factor (NRF)‐1 is a mitochondrial transcriptional factor which is shown to be involved in glucose transport and is set as a potential therapeutic modality in the treatment and management of type 2 diabetes. In this study, we sought to access NRF‐1 and its target genes expression during exercise which is crucial in glucose transport and lipid oxidation.MethodFive to six weeks old male Wistar rats were exercised to identify the time point for an optimum increase in the levels of NRF‐1 and its target genes. Gastrocnemius muscles were harvested after 0, 2, 4, 6, 8, 10, 12, and 15 h post‐exercise and non‐exercise rats (control). Primers were used to amplify the region of following genes; Nrf‐1, glut 4, carnitine palmitoyl transferase (Cpt‐1 & Cpt‐2), peroxisome proliferator‐activated receptor gamma co‐activator 1 (Pgc‐1), mef2a, and acetyl‐CoA carboxylase‐1 (Acc‐1). Relative mRNA expression was normalized to the Actin reference gene.ResultsFrom the result of this study, cpt‐1, Nrf‐1, mef2a, glut4, cpt2, and Pgc‐1 showed 2.5, 8, 1.2, 4.1, 4.6, 3.5‐folds increases respectively after 8h post‐exercise compared with control whereas Acc‐1 showed a 3.1‐fold decrease in gene expression ratio after 6 h post‐exercise compared with control. Nrf‐1 binding to cpt‐1 and mef2a increased with 3 and 3.5‐folds respectively 6h post‐exercise compared with control.ConclusionConclusively, nrf‐1 was increased by exercise and also, it's binding to target genes which has huge implications in ameliorating type 2 diabetes and insulin resistance.

Dietary Aronia melanocarpa extract enhances mTORC1 signaling, but has no effect on protein synthesis and protein breakdown-related signaling, in response to resistance exercise in rat skeletal muscle

BackgroundUrsolic acid altered muscle protein metabolism in normal and resting conditions after acute resistance exercise, suggesting that eating fruits rich in ursolic acid could enhance muscle protein synthesis and decrease muscle degradation. Aronia melanocarpa, a member of the family Rosaceae and native to North America and Eastern Canada, is rich in ursolic acid. In this study, we examined the effects of A. melanocarpa extract (AME) supplementation on the mTORC1 signaling pathway and muscle degradation-related factors in rats, both alone and in combination with resistance exercise.MethodsMale Sprague-Dawley rats were divided into AME and normal chow (NOR) groups. AME group was fed chow providing a dose of 3 g/kg of AME and 115 mg/kg of ursolic acid for 7 days, whereas NOR rats were fed normal powder chow. The right gastrocnemius muscle of each animal was isometrically exercised (5 sets of ten 3-s contractions, with a 7-s interval between contractions and 3-min rest intervals between sets), while the left gastrocnemius muscle served as an internal control. Western blotting and real-time polymerase chain reaction were used to assess expression of factors involved in the mTORC1 signaling pathway and muscle degradation.ResultsAt 1 h after resistance exercise, phosphorylation of ERK1/2 was significantly increased by AME consumption. At 6 h after resistance exercise, AME consumption significantly increased the phosphorylation of Akt, p70S6K, rpS6, and AMPK. It also increased MAFbx expression. Furthermore, AME significantly increased the phosphorylation of p70S6K and rpS6 in response to resistance exercise. However, AME did not increase muscle protein synthesis (MPS) after resistance exercise. AME did not affect the expression of any of the mediators of protein degradation, with the exception of MAFbx.ConclusionsDietary AME enhanced mTORC1 activation in response to resistance exercise without increasing MPS. Moreover, it neither accelerated muscle protein degradation nor otherwise negatively affected protein metabolism. Further study is needed to clarify the effect of the combination of AME and chronic resistance training on muscle hypertrophy.

Diclofenac pretreatment modulates exercise-induced inflammation in skeletal muscle of rats through the TLR4/NF-κB pathway.

Nonsteroidal anti-inflammatory drugs, such as diclofenac, are widely used to treat inflammation and pain in several conditions, including sports injuries. This study analyzes the influence of diclofenac on the toll-like receptor-nuclear factor kappa B (TLR-NF-κB) pathway in skeletal muscle of rats submitted to acute eccentric exercise. Twenty male Wistar rats were divided into 4 groups: control-saline, control-diclofenac, exercise-saline, and exercise-diclofenac. Diclofenac or saline were administered for 7 days prior to an acute eccentric exercise bout. The inflammatory status was evaluated through mRNA levels of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), IL-1β, and tumor necrosis factor alpha (TNF-α), and protein content of COX-2, IL-6, and TNF-α in vastus lateralis muscle. Data obtained showed that a single bout of eccentric exercise significantly increased COX-2 gene expression. Similarly, mRNA expression and protein content of other inflammation-related genes also increased after the acute exercise. However, these effects were attenuated in the exercise + diclofenac group. TLR4, myeloid differentiation primary response gene 88 (MyD88), and p65 were also upregulated after the acute eccentric bout and the effect was blunted by the anti-inflammatory drug. These findings suggest that pretreatment with diclofenac may represent an effective tool to ameliorate the pro-inflammatory status induced by acute exercise in rat skeletal muscle possibly through an attenuation of the TLR4-NF-κB signaling pathway.

Hydrogen Peroxide Does Not Potentiate Exercise-induced Mtorc1 In Rat Skeletal Muscle

Our previous study showed that administration of an antioxidant attenuated the phosphorylation of p70S6K and skeletal muscle hypertrophy induced by mechanical overload (Makanae et al. 2013), suggesting that reactive oxygen species (ROS) could be an activator of mammalian target of rapamycin complex 1(mTORC1), a key regulator of muscle protein synthesis. However, no study has investigated the effects of exogenous injection of ROS into skeletal muscle on the mTORC1 activity. PURPOSE: The purpose of this sturdy was to investigate the effect of ROS injection on mTORC1 activity at baseline and after a bout of exercise in rat skeletal muscle. METHODS: Experiment 1: The male Sprague-Dawley rats, at 11 weeks of age, were injected with hydrogen peroxide (1 mM, 0.1 mL) or placebo (PBS, 0.1 mL) into the right gastrocnemius muscle under isoflurane anesthesia, and killed by blood removal at 1 h after the injection. Experiment 2: The right gastrocnemius muscle of the rats was isometrically exercised (stimulating 3 sec × 10 contractions, with a 7 sec interval between contractions, per set for 5 sets, with 3 min rest intervals) via percutaneous electrical stimulation under isoflurane anesthesia. The hydrogen peroxide or placebo were injected into the right gastrocnemius muscle immediately after the resistance exercise. RESULTS: The injection of hydrogen peroxide increased significantly the phosphorylation of p70S6K (Thr389) and rspS6 (Ser235/Ser236) at 1 h after the injection (P < 0.05) as compared to the placebo injection. Resistance exercise increased the phosphorylation of p70S6K (Thr389) and prS6 (Ser235/Ser236) at 3 h after exercise in the both hydrogen peroxide and placebo injected groups. However, there was no difference in the level of either p70S6K (Thr389) or prS6 (Ser235/Ser236) phosphorylation between groups. CONCLUSIONS: These results suggest that ROS is one of the activator of mTORC1 signaling in the skeletal muscle, but the exogenous increase in ROS does not further stimulate resistance exercise-induced mTORC1 activation.

The calcineurin antagonist RCAN1-4 is induced by exhaustive exercise in rat skeletal muscle

The aim of this work was to study the regulation of the calcineurin antagonist regulator of calcineurin 1 (RCAN1) in rat skeletal muscles after exhaustive physical exercise, which is a physiological modulator of oxidative stress. Three skeletal muscles, namely extensor digitorum longus (EDL), gastrocnemius, and soleus, were investigated. Exhaustive exercise increased RCAN1-4 protein levels in EDL and gastrocnemius, but not in soleus. Protein oxidation as an index of oxidative stress was increased in EDL and gastrocnemius, but remained unchanged in soleus. However, lipid peroxidation was increased in all three muscles. CuZnSOD and catalase protein levels were increased at 3 h postexercise in soleus, whereas they remained unchanged in EDL and gastrocnemius. Calcineurin enzymatic activity declined in EDL and gastrocnemius but not in soleus, and its protein expression was decreased in all three muscles. The level of PGC1-α protein remained unchanged, whereas the protein expression of the transcription factor NFATc4 was decreased in all three muscles. Adiponectin expression was increased in all three muscles. RCAN1-4 expression in EDL and gastrocnemius muscles was augmented by the oxidative stress generated from exhaustive exercise. We propose that increased RCAN1-4 expression and the signal transduction pathways it regulates represent important components of the physiological adaptation to exercise-induced oxidative stress.

Mitochondrial Biosynthesis Factors of Offspring before and during Maternal Training Exercise in Rat Skeletal Muscle

Mitochondrial Biosynthesis Factors of Offspring before and during Maternal Training Exercise in Rat Skeletal Muscle

Ursolic acid stimulates mTORC1 signaling after resistance exercise in rat skeletal muscle

A recent study identified ursolic acid (UA) as a potent stimulator of muscle protein anabolism via PI3K/Akt signaling, thereby suggesting that UA can increase Akt-independent mTOR complex 1 (mTORC1) activation induced by resistance exercise via Akt signaling. The purpose of the present study was to investigate the effect of UA on resistance exercise-induced mTORC1 activation. The right gastrocnemius muscle of male Sprague-Dawley rats aged 11 wk was isometrically exercised via percutaneous electrical stimulation (stimulating ten 3-s contractions per set for 5 sets), while the left gastrocnemius muscle served as the control. UA or placebo (PLA; corn oil only) was injected intraperitoneally immediately after exercise. The rats were killed 1 or 6 h after the completion of exercise and the target tissues removed immediately. With placebo injection, the phosphorylation of p70(S6K) at Thr(389) increased 1 h after resistance exercise but attenuated to the control levels 6 h after the exercise. On the other hand, the augmented phosphorylation of p70(S6K) was maintained even 6 h after exercise when UA was injected immediately after exercise. A similar trend of prolonged phosphorylation was observed in PRAS40 Thr(246), whereas UA alone or resistance exercise alone did not alter its phosphorylation level at 6 h after intervention. These results indicate that UA is able to sustain resistance exercise-induced mTORC1 activity.

Induction of amino acid transporters expression by endurance exercise in rat skeletal muscle

We here investigated whether an acute bout of endurance exercise would induce the expression of amino acid transporters that regulate leucine transport across plasma and lysosomal membranes in rat skeletal muscle. Rats ran on a motor-driven treadmill at a speed of 28m/min for 90min. Immediately after the exercise, we observed that expression of mRNAs encoding l-type amino acid transporter 1 (LAT1) and CD98 was induced in the gastrocnemius, soleus, and extensor digitorum longus (EDL) muscles. Sodium-coupled neutral amino acid transporter 2 (SNAT2) mRNA was also induced by the exercise in those three muscles. Expression of proton-assisted amino acid transporter 1 (PAT1) mRNA was slightly but not significantly induced by a single bout of exercise in soleus and EDL muscles. Exercise-induced mRNA expression of these amino acid transporters appeared to be attenuated by repeated bouts of the exercise. These results suggested that the expression of amino acid transporters for leucine may be induced in response to an increase in the requirement for this amino acid in the cells of working skeletal muscles.

Effects of dietary extra-virgin olive oil on oxidative stress resulting from exhaustive exercise in rat skeletal muscle: A morphological study

Physical exercise induces oxidative stress through production of reactive oxygen species and can cause damage to muscle tissue. Oxidative stress, resulting from exhaustive exercise is high and improvement of antioxidant defenses of the body may ameliorate damage caused by free radicals. Extra-virgin olive oil is widely considered to possess anti-oxidative properties. The aim of this study was to determine if extra-virgin olive oil improved the adaptive responses in conditions of oxidative stress. Twenty-four 12-week-old male Sprague-Dawley rats were divided in three groups: (1) rats fed with standard chow and not subjected to physical exercise; (2) rats fed with standard chow and subjected to exhaustive exercise; (3) rats fed with a diet rich in oleic acid, the major component of extra-virgin olive oil, and subjected to exhaustive exercise. Exhaustive exercise consisted of forced running in a five-lane 10° inclined treadmill at a speed of 30m/min for 70–75min. We studied some biomarkers of oxidative stress and of antioxidant defenses, histology and ultrastructure of the Quadriceps femoris muscle (Rectus femoris). We observed that, in rats of group 3, parameters indicating oxidative stress such as hydroperoxides and thiobarbituric acid-reactive substances decreased, parameters indicating antioxidant defenses of the body such as non-enzymatic antioxidant capacity and Hsp70 expression increased, and R. femoris muscle did not show histological and ultrastructural alterations. Results of this study support the view that extra-virgin olive oil can improve the adaptive response of the body in conditions of oxidative stress.

Inactivity Suppresses the Expression of Amino Acid Transporters in Rat Skeletal Muscle

Leucine is one of the building blocks of protein but also stimulates the translation in protein synthesis through the activation of mTORC1 signaling. Furthermore, leucine is oxidized as a fuel to synthesize ATP during exercise in the muscle. These features of leucine let us hypothesized that the expression of leucine transporters would be modified in proportion to its cellular demand induced by physical activity. This study investigated whether the expression of leucine transporters would be induced by voluntary running and reduced by inactivity following the exercise in rat skeletal muscle. Thirty male Fisher 344 × Brown Norway F1 hybrid rats were obtained at 21 days of age. Rats were separated into those with access to running wheels (n=15, wheel lock, WL) and those without access to the wheels (n=15, SED). Forty days after the beginning, wheels were locked at 06:00 and rats were killed 5, 53 or 173 h after locking of wheels. LAT1, CD98, SNAT2, and PAT1 mRNAs in soleus, gastrocnemius, and EDL muscles were measured by qRT‐PCR. Soleus muscle weight was increased by voluntary running and reduced by inactivity following voluntary running. The expression of mRNAs for LAT1 and PAT1 was also induced by voluntary running and reduced by inactivity following it. These results suggested the expression of leucine transporters would be involved in a muscle hypertrophy and atrophy induced by physical activity and inactivity, respectively.

Rapid induction of REDD1 expression by endurance exercise in rat skeletal muscle

An acute bout of exercise induces repression of protein synthesis in skeletal muscle due in part to reduced signaling through the mammalian target of rapamycin complex 1 (mTORC1). Previous studies have shown that upregulated expression of regulated in DNA damage and development (REDD) 1 and 2 is an important mechanism in the regulation of mTORC1 activity in response to a variety of stresses. This study investigated whether induction of REDD1/2 expression occurs in rat skeletal muscle in response to a burst of endurance exercise. In addition, we determined if ingestion of glucose or branched chain amino acids (BCAA) before exercise changes the expression of REDD1/2 in muscle. Rats ran on a motor-driven treadmill at a speed of 28 m min −1 for 90 min, and then the gastrocnemius muscle was removed and analyzed for phosphorylation of the eukaryotic initiation factor (eIF) 4E binding protein 1 (4E-BP1) and expression of REDD1/2. Exercise repressed the mTORC1-signaling pathway regardless of the ingestion of nutrients before the exercise, as shown by dephosphorylation of 4E-BP1. In addition, exercise induced the expression of REDD1 mRNA (∼8-fold) and protein (∼3-fold). Exercise-induced expression of REDD1 was not affected by the ingestion of glucose or BCAA. Expression of REDD2 mRNA was not altered by either exercise or nutrients. These findings indicated that enhanced expression of REDD1 may be an important mechanism that could partially explain the downregulation of mTORC1 signaling, and subsequent inhibition of protein synthesis in skeletal muscle during exercise.

Stanozolol treatment decreases the mitochondrial ROS generation and oxidative stress induced by acute exercise in rat skeletal muscle

Anabolic androgenic steroids are used in the sport context to enhance muscle mass and strength and to increase muscle fatigue resistance. Since muscle fatigue has been related to oxidative stress caused by an exercise-linked reactive oxygen species (ROS) production, we investigated the potential effects of a treatment with the anabolic androgenic steroid stanozolol against oxidative damage induced on rat skeletal muscle mitochondria by an acute bout of exhaustive exercise. Mitochondrial ROS generation with complex I- and complex II-linked substrates was increased in exercised control rats, whereas it remained unchanged in the steroid-treated animals. Stanozolol treatment markedly reduced the extent of exercise-induced oxidative damage to mitochondrial proteins, as indicated by the lower levels of the specific markers of protein oxidation, glycoxidation, and lipoxidation, and the preservation of the activity of the superoxide-sensitive enzyme aconitase. This effect was not due to an enhancement of antioxidant enzyme activities. Acute exercise provoked changes in mitochondrial membrane fatty acid composition characterized by an increased content in docosahexaenoic acid. In contrast, the postexercise mitochondrial fatty acid composition was not altered in stanozolol-treated rats. Our results suggest that stanozolol protects against acute exercise-induced oxidative stress by reducing mitochondrial ROS production, in association with a preservation of mitochondrial membrane properties.

Response of mitochondrial fusion and fission protein gene expression to exercise in rat skeletal muscle

The purpose of this study was to investigate the changes in the gene expression of Mitofusion (Mfn) 1 and 2 and Fission 1 (Fis1) and mitochondrial energy metabolism in response to altered energy demand during prolonged exercise in rat skeletal muscle. Male Sprague–Dawley rats were subjected to an acute bout of treadmill running at various durations and killed immediately or during recovery. Mfn1/2 and Fis1 mRNA and protein contents, reactive oxygen species (ROS) generation, state 3 and state 4 respiration rates, trans-innermembrane potential and ATP synthase activity were measured in isolated muscle mitochondria. We found that (1) Mfn1/2 mRNA contents were progressively decreased during 150 min of exercise, along with decreased Mfn 1 protein levels. Fis1 mRNA and protein contents showed significant increases after 120–150 min of exercise. These changes persisted through the recovery period up to 24 h. (2) Mitochondrial ROS generation and state 4 respiration showed progressive increases up to 120 min, but dropped at 150 min of exercise. (3) State 3 respiration rate and respiratory control index were unchanged initially but decreased at 150 and 120 min of exercise, respectively, whereas ATP synthase activity was elevated at 45 min and returned to resting level thereafter. Our data suggested that the gene expression of mitochondrial fusion and fission proteins in skeletal muscle can respond rapidly to increased metabolic demand during prolonged exercise, which could significantly affect the efficiency of oxidative phosphorylation.

MyoD and Myogenin mRNA Levels after Single Session of Treadmill Exercise in Rat Skeletal Muscle

[Purpose] We examined mRNA levels of MyoD and myogenin in rat skeletal muscle after a single session of treadmill running. [Subjects] A total of 26 male Sprague-Dawley rats aged 4 weeks were used in this study. [Methods] Rats were run on a 16° decline for 30 min. Treadmill speeds were 0, 16, 20, 24 and 28 m/min. At 72 h post-exercise, soleus (SOL) and extensor digitorum longus (EDL) muscles were extracted. The expression of MyoD and myogenin mRNA were analyzed by reverse transcription polymerase chain reaction (RT-PCR) and quantitative RT-PCR. [Results] MyoD and myogenin mRNA expressions were detected in all groups. Significant differences in myogenin levels were apparent in SOL between 16 m/min and 28 m/min. MyoD levels in SOL at 16, 24, 28 m/min were lower, while myogenin levels at 20, 24 and 28 m/min were higher, when compared with the 0 m/min group, although significant differences were not seen. [Conclusion] These results suggest that a single session of exercise has little effect on proliferation of satellite cells, or myotube production.

Analysis of exercise‐induced Na+–K+ exchange in rat skeletal muscle in vivo

We aimed to quantify the Na(+)-K(+) exchange occurring during exercise in rat skeletal muscle in vivo. Intracellular Na(+) and K(+) content, Na(+) permeability ((22)Na(+) influx), Na(+)-K(+) pump activity (ouabain-sensitive (86)Rb(+) uptake) and Na(+)-K(+) pump alpha(2) subunit content ([(3)H]ouabain binding) were measured. Six-week-old rats rested (control animals) or performed intermittent running for 10-60 min and were then killed or were killed at 15 or 90 min following 60 min exercise. In the soleus muscle, intracellular Na(+) was 80% higher than in control rats after 60 min exercise, was still elevated (38%) after 15 min rest and returned to control levels after 90 min rest. Intracellular K(+) showed corresponding decreases after 15-60 min exercise, returning to control levels 90 min postexercise. Exercise induced little change in Na(+) and K(+) in the extensor digitorum longus muscle (EDL). In soleus, the exercise-induced rise in Na(+) and reduction in K(+) were augmented by pretreatment with ouabain or by reducing the content of muscular Na(+)-K(+) pumps by prior K(+) depletion of the animals. Fifteen minutes after 60 min exercise, ouabain-sensitive (86)Rb(+) uptake in the soleus was increased by 30% but was unchanged in EDL, and there was no effect of exercise on [(3)H]ouabain binding measured in vitro or in vivo in either muscle. In conclusion, in the soleus, in vivo exercise induces a rise in intracellular Na(+), which reflects the excitation-induced increase in Na(+) influx and leads to augmented Na(+)-K(+) pump activity without apparent change in Na(+)-K(+) pump capacity.

Melatonin inhibits the expression of the inducible isoform of nitric oxide synthase and nuclear factor kappa B activation in rat skeletal muscle.

This study investigated whether the induction of inducible nitric oxide synthase (iNOS) produced by acute exercise in rat skeletal muscle could be prevented by melatonin and whether iNOS down-regulation was related to inhibition of nuclear factor kappaB (NF-kappaB) activation. Male Wistar rats received melatonin i.p. at a dose of 1.0 mg/kg body weight 30 min before being exercised for 60 min on a treadmill at a speed of 25 m/min and a 10% slope. Exercise caused a significant induction of iNOS protein levels and a marked activation of NF-kappaB that were significantly prevented in rats treated with melatonin. Exercise also resulted in increased IkappaB kinasealpha (IKKalpha) and phosphorylated IkappaBalpha protein levels, whereas IkappaBalpha content decreased. These effects were blocked by melatonin administration. The increase in the muscle concentration of thiobarbituric acid reactive substances and in the oxidized/reduced glutathione ratio induced by exercise was partially prevented by melatonin. Our data indicate that melatonin has potent protective effects against damage caused by acute exercise in rat muscle, preventing oxidative stress, NF-kappaB activation and iNOS over-expression. These findings support the view that melatonin treatment, by abolishing the IKK/NF-kappaB signal transduction pathway, might block the production of noxious mediators involved in the inflammatory process.

Mitochondrial H2O2 Production Is Reduced with Acute and Chronic Eccentric Exercise in Rat Skeletal Muscle

Oxidative stress with acute/chronic exercise has been so far examined using exercise involving a combination of concentric and eccentric contractions, but skeletal muscles are likely to be injured to a greater extent by pliometric contractions. In the present study, the effects of acute and chronic bouts of downhill running exercise on mitochondrial hydrogen peroxide (H2O2) generation (fluorimetric detection of a dimer with homovanillic acid in presence of horseradish peroxidase) and oxygen consumption in conjunction with antioxidant enzymes activity were examined. The results show that acute eccentric exercise was accompanied by a significantly reduced mitochondrial H2O2 production that is likely due to a decrease in complex I of the electron transport chain (ETC). On the other hand, eccentric training leads to positive adaptations, reflected by a higher citrate synthase activity and decreased mitochondrial H2O2 production. The decrease in mitochondrial H2O2 cannot be attributed to alterations in antioxidant capacities but rather to changes in mitochondrial membrane composition characterized by an increased polyunsaturated to saturated fatty acids ratio, and decreased contents in arachidonic acid and plasmalogens. These results suggest that changes in mitochondrial membrane properties with eccentric training can affect H2O2 production by muscle mitochondria. It is hypothesized that these changes resulted in a mild uncoupling sufficient to reduce electron back flow through complex I of the ETC, the major generator of reactive oxygen species by skeletal muscle mitochondria.

Immediate response of mammalian target of rapamycin (mTOR)-mediated signalling following acute resistance exercise in rat skeletal muscle.

The purpose of the present investigation was to determine whether mammalian target of rapamycin (mTOR)-mediated signalling and some key regulatory proteins of translation initiation are altered in skeletal muscle during the immediate phase of recovery following acute resistance exercise. Rats were operantly conditioned to reach an illuminated bar located high on a Plexiglass cage, such that the animals completed concentric and eccentric contractions involving the hindlimb musculature. Gastrocnemius muscle was extracted immediately after acute exercise and 5, 10, 15, 30 and 60 min of recovery. Phosphorylation of protein kinase B (PKB) on Ser-473 peaked at 10 min of recovery (282% of control, P < 0.05) with no significant changes noted for mTOR phosphorylation on Ser-2448. Eukaryotic initiation factor (eIF) 4E-binding protein-1 (4E-BP1) and S6 kinase-1 (S6K1), both downstream effectors of mTOR, were altered during recovery as well. 4E-BP1 phosphorylation was significantly elevated at 10 min (292%, P < 0.01) of recovery. S6K1 phosphorylation on Thr-389 demonstrated a trend for peak activation at 10 min following exercise (336%, P = 0.06) with ribosomal protein S6 phosphorylation being maximally activated at 15 min of recovery (647%, P < 0.05). Components of the eIF4F complex were enhanced during recovery as eIF4E association with eIF4G peaked at 10 min (292%, P < 0.05). Events regulating the binding of initiator methionyl-tRNA to the 40S ribosomal subunit were assessed through eIF2B activity and eIF2 alpha phosphorylation on Ser-51. No differences were noted with either eIF2B or eIF2 alpha. Collectively, these results provide strong evidence that mTOR-mediating signalling is transiently upregulated during the immediate period following resistance exercise and this response may constitute the most proximal growth response of the cell.

Mechanisms underlying increases in SR Ca2+-ATPase activity after exercise in rat skeletal muscle.

Prolonged exercise followed by a brief period of reduced activity has been shown to result in an overshoot in maximal sarcoplasmic reticulum (SR) Ca(2+)-ATPase activity [maximal velocity (V(max))] in rat locomoter muscles (Ferrington DA, Reijneveld JC, Bär PR, and Bigelow DJ. Biochim Biophys Acta 1279: 203-213, 1996). To investigate the functional significance and underlying mechanisms for the increase in V(max), we analyzed Ca(2+)-ATPase activity and Ca(2+) uptake in SR vesicles from the fast rat gastrocnemius muscles after prolonged running (RUN) and after prolonged running plus 45 min of low-intensity activity (RUN+) or no activity (REC45) and compared them with controls (Con). Although no differences were observed between RUN and Con, both V(max) and Ca(2+) uptake were higher (P < 0.05) by 43 and 63%, respectively, in RUN+ and by 35 and 34%, respectively, in REC45. The increase in V(max) was accompanied by increases (P < 0.05) in the phosphorylated enzyme intermediate measured by [gamma-(32)P]ATP. No differences between groups for each condition were found for the fluorescent probes FITC and (N-cyclohexyl-N(1)-dimethylamino-alpha-naphthyl)carbodiimide, competitive inhibitors of the nucleotide-binding and Ca(2+)-binding sites on the enzyme, respectively. Similarly, no differences for the Ca(2+)-ATPase were observed between groups in nitrotyrosine and phosphoserine residues, a measure of nitrosylation and phosphorylation states, respectively. Western blots indicated no changes in relative isoform content of sarcoendoplasmic reticulum (SERCA)1 and SERCA2a. It is concluded that the increase in V(max) of the Ca(2+)-ATPase observed in recovery is not the result of changes in enzyme nitroslyation or phosphorylation, changes in ATP and Ca(2+)-binding affinity, or changes in protein content of the Ca(2+)-ATPase.

Endurance training improves responsiveness to insulin and modulates insulin signal transduction through the phosphatidylinositol 3-kinase/Akt-1 pathway.

Endurance training increases insulin-stimulated muscle glucose transport and leads to improved metabolic control in diabetic patients. To analyze the effects of endurance training on the early steps of insulin action in muscle of rats. Male rats submitted to daily swimming for 6 weeks were compared with sedentary controls. At the end of the training period, anesthetized animals received an intravenous (i.v.) injection of insulin and had a fragment of their gastrocnemius muscle excised for the experiments. Associations between insulin receptor, insulin receptor substrates (IRS)-1 and -2 and phosphatidylinositol 3-kinase (PI3-kinase) were analyzed by immunoprecipitation and immunoblotting. Akt-1 serine phosphorylation and specific protein quantification were detected by immunoblotting of total extracts, and IRS-1/IRS-2-associated PI3-kinase activity were determined by thin-layer chromatography. Insulin-induced phosphorylation of IRS-1 and IRS-2 increased respectively by 1.8-fold (P<0.05) and 1.5-fold (P<0.05), whereas their association with PI3-kinase increased by 2.3-fold (P<0.05) and 1.9-fold (P<0.05) in trained rats as compared with sedentary controls, respectively. The activity of PI3-kinase associated with IRS-1 and IRS-2 increased by 1.8-fold (P<0.05) and 1.7-fold (P<0.05) respectively, in trained rats as compared with their untrained counterparts. Serine phosphorylation of Akt-1/PKB increased 1.7-fold (P<0.05) in trained rats in response to insulin. These findings were accompanied by increased responsiveness to insulin as demonstrated by a reduced area under the curve for insulin during an i.v. glucose tolerance test, by increased glucose disappearance rate during an insulin tolerance test, and by increased expression of glucose transporter-4. The increased responsiveness to insulin induced by chronic exercise in rat skeletal muscle may result, at least in part, from the modulation of the insulin signaling pathway at different molecular levels.