Previous studies have shown that exercise increases muscle mass and strength (Armstrong et al., 1983; Wirth et al., 2003). Skeletal muscle hypertrophy is due to increase in both the cross-sectional area (hypertrophy) and the number of muscle fibers (hyperplasia). Hyperplasia and hypertrophy are closely related to both the proliferation/differentiation of satellite cells and the enhancement of protein synthesis (Appell et al., 1988; Giddings and Gonyea, 1992; Perry and Rudnick, 2000; Vierck et al., 2000).

Protein synthesis in skeletal muscle is promoted by insulin-like growth factor (IGF-1) (Barton-Davis et al., 1999). The activation of Akt/mTOR/p70S6K signal transduction pathway is partly induced by the autocrine/ paracrine response mediated by IGF-1, thereby, leads to muscular hypertrophy (Bodine et al., 2001; Glass, 2003; Rommel et al., 2001). Mechanical stimuli, such as exercise, increase IGF-1 expression and activate the Akt/mTOR/p70S6K signal transduction pathway extensively (Burry et al., 2007; Sakamoto et al., 2002, Parkington et al., 2003; Spangenburg and McBride, 2006). Sakamoto et al. reported that P-Akt increased at 1 h after 1 bout of treadmill exercise; IGF-1 increased immediately after exercise in an electrical stimulation model [Sakamoto et al., 2002]. In addition, the phosphorylation of mTOR and p70S6K, downstream of Akt, occurs after muscular contractions during a single bout of exercise (Burry et al., 2007; Parkington et al., 2003; Spangenburg and McBride, 2006). Above all, a single bout of exercise induces protein synthesis in exercised muscle.

The effects of chronic functional overload and exercise on changes in IGF-1 and Akt/mTOR/p70S6K signal transduction pathway have also been examined (Adams and Haddad, 1996; Bodine et al., 2001; DeVol et al., 1990; Thomson et al., 2006). With regard to functional overload, IGF-1 concentration increased up to 7days, but then decreased thereafter (Adams and Haddad, 1996; DeVol et al., 1990). On the other hand, activation of the Akt/mTOR/p70S6K pathway gradually increased after more than 1 week, and the activation of these proteins, especially p70S6K, correlated with the amount of muscle hypertrophy (Bodine et al., 2001; Thomson et al., 2006). Taken together, elevated IGF-1 levels and activation of Akt/mTOR/p70S6K seemed to correlate within 1 week after tenonectomy. But after one week, activation of Akt/mTOR/p70S6K was independent from IGF-1 in functional overload model.

In chronic exercise, Hameed et al. showed that during chronic exercise the concentrations of IGF-1 and its variants were elevated after 5 and 12 weeks of resistance training, respectively, in aged humans (Hameed et al., 2004). Leger et al., 2006 reported that phosphorylated Akt and mTOR levels were elevated after 8 weeks of anabolic muscular exercise in human subjects. In an animal exercise model, 20 days of exercise induced significant hypertrophy independent of contraction modes such as eccentric, isometric, and concentric (Adams et al., 2004). On the contrary, isometric and concentric, but not eccentric, modes of exercise increased muscle IGF-1 and its variant, mechano growth factor (MGF) (Adams et al., 2004). Although these results suggest that chronic exercise induces significant hypertrophy, IGF-1 elevation, and protein synthesis activation, direct comparison of IGF-1, Akt, mTOR, and p70S6K has not been examined in the same chronic exercise model.

We hypothesized that the increase in muscle mass is associated with elevation of IGF-1 mRNA content and activation of the Akt/mTOR/p70S6K pathway. We previously reported significant increases in skeletal muscle mass and muscle strength using an eccentric exercise model in the rat gastrocnemius muscle (Ochi et al., 2007). Therefore, in this study, we used this model to induce 10- and 20-day eccentric exercise in rat medial gastrocnemius. After chronic exercise, we measured the muscle wet mass, muscle fiber cross- sectional area (CSA), IGF-1 mRNA expression, and activation of the Akt/mTOR/p70S6K signal transduction pathway.

Animals
Male Wistar rats (age, 12 weeks; body mass, 303-365 g; n = 24) were randomly assigned into 4 groups: 5 sessions of eccentric exercise group (ECC5S, n = 6); 5 sessions of sham-operated control group (CON5S, n = 6); 10 sessions of eccentric exercise group (ECC10S, n = 6); 10 sessions of sham-operated control group (CON10S, n = 6). They were housed individually in a steel case maintained at 22-24°C and 12-h light/dark cycle. The right hindlimb of each animal was shaved, and the animals were anesthetized with isoflurance during the exercise. Water and food were available ad libitum. No significant differences in age or mass were found between the groups (as shown in Table 1). The study was approved by the Ethical Committee for Animal Experiments at the University of Tokyo.

CSA of medial gastrocnemius muscle fibers
A part of each frozen sample was subjected to hematoxylin-eosin (H&E) staining (Sakura Finetek Japan Co. Japan). Before staining, the muscle sample was sectioned at 10 μm thickness at -20°C. Sections from each group were placed on the same glass slide to eliminate variability in staining conditions. After staining, the fibers were observed under a light microscope. CSA of muscle fibers was determined using NIH image (version 1.61; National Institutes of Health, USA). We measured 200 fibers from each animal.

Eccentric exercise for medial gastrocnemius muscle
The eccentric exercise system of the rat ankle joint was similar to the system described in our previous study (Ochi et al., 2007). Rats were anesthetized and firmly fixed on the platform of an isokinetic dynamometer in a prone position, and the triceps surae muscle of the right hindlimb was electrically stimulated. Rats in the eccentric exercise group were trained every second day for 10 days (5 sessions in total; ECC5S) or 20 days (10 sessions in total; ECC10S). The stimulus voltage was adjusted to produce submaximal isometric twitch torque through an adhesive surface electrode (7.5 mm × 7.5 mm) connected to an electric stimulator and isolator (Nihon Koden, Japan). The muscle was then stimulated for 3 s to cause submaximal tetanic contraction. One second after the onset of stimulation, forced isokinetic dorsi-flexion was given to the ankle joint to cause an eccentric contraction of the triceps surae muscle. The speed and the range of forced lengthening were 30°/s and from 0° (defined as the angle at which the sole of the foot and tibial bone are orthogonally positioned) to 45°, respectively. Each exercise session consisted of 4 sets of 5 contractions, with a 5-min interval between each set. The sham-operated groups (CON5S and CON10S) were only anesthetized, not exercised. Within 12 h after the last exercise session, the muscle specimens were dissected, weighed, and immediately frozen in liquid N2 and stored at -80°C until analysis.

Analysis of mRNA by real-time reverse transcriptase-polymerase chain reaction (RT-PCR)
The expressions of IGF-1 and MGF mRNAs in medial gastrocnemius muscle was measured using real-time (RT-PCR) as described by Nakazato et al., 2007. The RNA isolation and RT-PCR methods were performed according to the manufacturer's instructions (RNeasy MiniKit, QIAGEN, USA, High-capacity cDNA Archive kit, Applied Biosystems, USA); RT-PCR was performed with a master mix in a final volume of 12.5 μl (SYBR Green PCR Master Mix, Applied Biosystems, USA). Amplification was carried out on an ABI Prism system (Applied Biosystems, USA) using the following program: 2 min at 50°C and 10 min at 95°C, followed by 15 s at 95°C and 1 min at 60°C for 40 cycles. External standard curves were used for quantitative analysis, and the mRNA levels of each target gene were normalized to those of 18S, which served as the control. The primers used were 5'-GCATTGTGGATGAGTGTTGC-3' (forward primer) and 3'-GGCTCCTCCTACATTCTGTA-5' (reverse primer) for IGF-1 (GenBank: X06043) and 5'-GCATTGT GGATGAGTGTTGC-3' (forward primer) and 3'-CTTTTCTTGTGTGTCGATAGG-5' (reverse primer) for MGF-1 (GenBank: X06108) based on Adams et al., 2004. Since the amplicon ranges in the sharing region of IGF variants, mixture of all IGF variants mRNAs were evaluated by this primer set. Data were analyzed using a comparative critical threshold (Ct) method where the amount of target gene was normalized to that of the endogenous control gene.

Analysis of signal transduction markers by western blotting
The medial gastrocnemius muscle samples were macerated in liquid N₂ and homogenized in 50 mM Tris-Cl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 0.5% sodium dodecyl sulfate (SDS), 1% deoxycholate, 0.1% Triton X-100, 1% Nonidet P-40 (NP-40), 0.05% mercaptoethanol, 10 mg/ml PMSF, 0.5 mg/ml leupeptin, 0.2 mg/ml aprotinin, and 1mM Na₃VO₄. The homogenate was centrifuged at 15,000 × g for 25 min at 4°C. Protein concentrations were determined using a protein concentration determination kit (Protein Assay II, Bio-Rad, Richmond, CA, USA). An equivalent 25 µg of total protein extract from each sample was mixed with the sample buffer, boiled, and loaded onto SDS polyacrylamide gels. Proteins were separated by discontinuous PAGE through 12.5% acrylamide gels. The samples were electrophoretically separated at 180mA for 90min and then transferred onto PVDF membranes (ATTO, Japan). The membranes were blocked with PBS containing 5% skim milk and incubated for 1 h. All primary antibodies were incubated overnight at 4°C: a monoclonal anti-Akt (P) (Cell Signaling Technology, USA), a polyclonal anti-mTOR (P) (Cell Signaling Technology, USA), and a monoclonal anti-p70S6K (Cell Signaling Technology, USA), each diluted 1/1,000. Membranes were washed and incubated for 10 min 2 times at room temperature. The secondary HRP-conjugated goat anti-mouse or anti-rabbit IgG was used at a dilution of 1:10,000 (Santa-Cruz, Biotechnology, USA). HRP activity was detected by chemiluminescence (Supersignal West Dura; Pierce, USA) using a chemiluminescence detector (AE6961, ATTO, Japan) and quantified using CS Analyzer (ATTO). To minimize the effect of reaction conditions, samples from each gel contained each experimental group.

Statistical analyses
All values are expressed as means and standard deviations. One-way analysis of variance (ANOVA) was used to compare the CSA of muscle fibers in 10 sessions. Two-way ANOVA followed by Bonferroni test was used for the whole body mass, muscle wet mass, muscle wet mass per body mass, expression of IGF-1 mRNA, and tissue concentrations of Akt (P), mTOR (P), and p70S6K (P). The significance level was set at p < 0.05.

The wet mass and relative wet mass of muscles and CSA of muscle fibers
Both medial gastrocnemius muscle mass and the muscle mass per body mass in ECC5S did not increase significantly, whereas those in ECC10S were significantly greater than those in CON10S, with a concomitant increase in the CSA of muscle fibers (p < 0.05; Table 1 and Figure 1). In addition, those in the ECC10S samples were also significantly larger than those in the ECC5S group (p < 0.01).

Expression of IGF-1 mRNA and MGF mRNA
Expression of IGF-1 mRNA in the gastrocnemius was determined by RT-PCR, and it was found to be significantly greater in ECC10S than that in CON10S and ECC5S (p < 0.05); Figure2A). On the other hand, the MGF mRNA expressions in both ECC5S and ECC10S were significantly higher than that in each period-matched control (p < 0.01; Figure 2B).

Phosphorylation of signal transduction proteins
The tissue concentrations of Akt (P), mTOR (P), and p70S6K (P) were determined by using western blot analysis. The tissue concentration of Akt (P) in ECC10S was significantly higher than those in both CON10S and ECC5S (p < 0.01; Figure 3). The tissue concentration of mTOR (P) in ECC10S showed a significant increase when compared with their age-matched controls (p < 0.01; Figure 4). The tissue concentration of p70S6K in ECC10S was significantly higher than in CON10S and ECC5S (p < 0.001; Figure 5).

By using our exercise model, we showed that 20 days, but not 10 days, of eccentric exercise causes an increase in muscle mass (Table 1). Since no changes were observed in the medial gastrocnemius muscle mass or the muscle per body mass ratio after 5 exercise sessions, we conclude that 5 exercise sessions are inadequate to cause muscular hypertrophy in this model system. On the other hand, significant hypertrophy was observed after 10 exercise sessions, and it was accompanied by a significant increase in the expression of IGF-1 mRNA and activation of the Akt/mTOR/p70S6K pathway. Taken together, these results suggest that the IGF-1/Akt/mTOR signaling pathway is associated with significant muscle hypertrophy in chronic exercise. Future work using this model should be performed with more attention on the timing of dissection. Dissections in this study were performed within 12 h after the last exercise session. The present results suggest that the observed increases in IGF-1 mRNA, mTOR (P), Akt (P), and p70S6k (P) after 10 exercise sessions may be maintained for up to 12 h.

In this study, the expression of IGF-1 mRNA was quantified. Although no changes were observed after 5 exercise sessions, a significant increase was observed after 10 exercise sessions (p < 0.05; Figure 2A). These results are consistent with IGF-1 responses after anabolic exercise in humans (Bamman et al., 2001; Hameed et al., 2004). Repeated bouts of eccentric contractions n a rat gastrocnemius model also induced higher expression of IGF-1 mRNA after 10 exercise sessions than in controls, but the observed difference was not statistically significant (Adams et al., 2004). In the present study, significant expression of IGF-1 mRNA was observed after 10 sessions of eccentric exercise. Since the range of forced lengthening in our model and that in Adams et al., 2004 were different (45° and 20°, respectively), the expression of MGF after both 5 and 10 sessions showed significant increases (Figure 2B). That was not in the case of Adams et al., 2004. Since MGF is defined as the loading sensitive IGF-1 isoform, the MGF increase might be the cause of greater mechanical loading in comparison to Adams et al., 2004.

The time course change of IGF-1 mRNA is another point to be considered. In a functional overload model, IGF-1 was only observed within 1 week (Adams and Haddad, 1996; Ishido et al., 2008). Since the timing of increase in IGF-1 mRNA was associated with significant muscular hypertrophy, we hypothesize that the activation of IGF-1 plays a role in the onset of muscle hypertrophy during chronic strength exercise.

Previous studies have shown that a single session of muscular exercise causes a transient increase in activation of the Akt/mTOR/p70S6K signal pathway within working muscles (Burry et al., 2007; Parkington et al., 2003; Sakamoto et al., 2002; Spangenburg and McBride, 2006). However, the relevance between IGF-1 and Akt/mTOR/p70S6K to muscle hypertrophy is controversial. Previous studies have examined the relationship between IGF-1 and Akt/mTOR/p70S6K by using functional loading and exercise models (Adams, 2002; Hornberger et al., 2006; Ishido et al., 2008; O'Neil et al., 2009; Spangenburg et al., 2008). Spangenburg et al. reported that activation of the IGF-1 receptor was not necessary for muscular hypertrophy and had no influence on the activation of Akt and p70S6k (Spangenburg et al., 2008). In addition, it has been shown that phospholipase D (PLD) and phosphatidic acid (PA) activate mTOR and p70S6K, independent of IGF-1 (Hornberger et al., 2006). Conversely, it showed that IGF-1 activates the Akt/mTOR/p70S6K pathway in chronic models (Adams, 2002; Ishido et al., 2008). Although we believe that IGF-1 and the Akt/mTOR/p70S6K pathway play a role in muscle hypertrophy, we also hypothesize the presence of redundant pathways such as PLD and PA pathways.

• We confirmed that the rat muscular exercise model using originally-developed equipment increased the wet mass of the medial gastrocnemius muscle and cross-sectional areas of muscle fibres in 10 sessions (20 days) but not in 5 sessions (10days).
• We clarified that the increases of muscle mass and CSA of muscle fibers were accompanied by IGF-1 mRNA expression, the phosphorylated Akt, mTOR, and p70S6K.
• These results suggest that muscular hypertrophy in our model was achieved after 10 sessions of exercise and associated with the activation of IGF-1/Akt/mTOR/p70S6K signal pathway.