## 4 Discussion  
BCAA supplementation has beneficial effects on exercise endurance and muscle recovery after injurious exercise [26; 27]. Various trials utilizing porcine blood waste by enzymatic hydrolysis have shown significant increases in free amino acids and bioactive peptides, including BCAAs [2; 28; 29; 30]. However, the effects of PWBPH on exercise function and skeletal muscle protein differentiation remain unknown. In the present study, we demonstrated that chronic administration of PWBPH enhanced exercise function in mice. Interestingly, PWBPH showed greater effects on exercise function and muscle mass in the group administered the lower dose of 250 mg/kg and the Leu administration group (positive control group). Previous studies showed that treatment of BCAAs had weaker effects on biomarkers of muscle gain compared with mixtures of BCAAs and other amino acids in mice and humans [31; 32; 33]. These observations suggest that PWBPH enhances exercise function at the optimal dose, and its effect is not only dependent on the amount of BCAAs. Skeletal myogenesis is highly regulated by MRFs, including MyoD and myogenin, which  
Figure 4: Effects of PWBPH treatment on the Akt/mTOR signaling pathway in vivo and in vitro. (**A**) Phosphorylation levels of Akt, mTOR, and p70S6K in the Gas muscle of the control, PWBPH 250 mg/kg, and Leu 500 mg/kg groups were determined by Western blotting. (**B**) The band intensities were measured using ImageJ software. Data are shown as the means \(\pm\) SD (\(n=3\)). * \(p\) \(<\) 0.05 vs. control group. C2C12 cells were treated with DM alone, or DM supplemented with PWBPH (50–200 mg/mL), or Leu (50 uM) for 4 days. (**C**) Phosphorylation levels of Akt, mTOR, and p70S6K were determined by Western blotting. (**D**) The band intensities were measured using ImageJ software. Data are shown as the means \(\pm\) SD from three independent experiments. * \(p\) \(<\) 0.05 vs. control group. precisely regulate myogenic cell differentiation in a highly complex process involving cell cycle arrest and multinucleated myotube formation [34]. MyoD-null mice in limb skeletal muscles had a regeneration deficient phenotype in vivo as well as a defective differentiation phenotype in vitro [35; 36]. Myofiber deficiencies are observed in myogenin-null mice and removing myogenin before embryonic muscle development, suggesting myogenin has a key role in muscle growth [37]. In the present study, PWBPH treatment increased the expression of MRFs, including MyoD and myogenin, both in vivo and in vitro. PWBPH treatment also increased the myofiber CSA of Gas muscle and hypertrophy of C2C12 myotube. A previous study showed that regular resistance exercise or Leu intake had effects on satellite cell activity and skeletal muscle hypertrophy, with induction of MyoD and myogenin expression in rats [38]. Leu was also reported to have a potential role in improving muscle growth and expression of muscle growth-related genes, including MyoD and myogenin in fish [39]. The Akt/mTOR signaling cascade is involved in muscle protein synthesis induced by nutritional interventions and physical activity, such as BCAAs and resistance exercise [8; 24]. Muscle protein synthesis is essential for increasing muscle strength and mass [40]. Therefore, exercise performance can be enhanced by the activation of the Akt/mTOR signaling pathway and increased expression of proteins related to power stroke. Denervation or immobilization of mice and rats induced atrophy via increased expression of both atrogin and MuRF in skeletal muscles. Deficiency in either atrogin or MuRF suppressed denervation-induced muscle fiber diameter and skeletal muscle weight loss in mice. In the present study, PWBPH treatment significantly induced the phosphorylation of Akt, mTOR, and p70S6K both in vivo and in vitro. PWBPH did not affect atrogin or MuRF protein expression in mice but increased muscle mass, indicating that the balance between protein synthesis and degradation regulates the maintenance of muscle mass.