## 4 Discussion  
Skeletal muscles are involved in movement, respiration, and the maintenance of homeostasis in the body. Thus, the loss of skeletal muscle and function can be fatal. However, muscle atrophy and sarcopenia cannot be cured using current technology, and regular exercise is the only effective strategy for maintaining muscle mass and suppressing sarcopenia. Therefore, continuous prevention and more effective pharmaceutical agents are needed. In addition, it would be more useful to consume active ingredients that prevent sarcopenia daily. The RE used in the present study induced myotube differentiation, inhibited AICAR-induced muscle cell atrophy, and further promoted hypertrophy, thereby exhibiting its potential for use as a functional substance for preventing sarcopenia. Skeletal muscles are involved in the regeneration of muscle fibers that contain mechanical, chemical, or degenerative lesions through activation, differentiation, and proliferation [32; 33]. The differentiation of myoblasts into myotubes is essential for the formation of muscle fibers involved in the development and regeneration of skeletal muscles [34]. These monocyte myoblasts proliferate, differentiate, and then fuse with existing muscle fibers to form multinucleated myotubes and myofib. Similar proliferation and differentiation of muscle cells occur during development and postnatal birth [35]. Therefore, the promotion of myoblast proliferation and differentiation and the induction of myotube hypertrophy should benefit muscle regeneration and the maintenance of muscle mass. Recent studies have reported that muscle atrophy occurs as a result of increased proteolysis, which is, in turn, caused by activation of the ubiquitin-proteasome pathway [36; 37; 38]. Indeed, the muscle-specific ubiquitin ligases MArbx/atrogin-1 and MuRF-1 are expressed early in the muscle atrophy process and are directly involved in muscle protein degradation [37; 38]. In addition, when muscle atrophy is induced, muscle-specific transcription factors that are involved in myogenic differentiation (e.g., MyoD and myogenin) are downregulated in response to muscle-specific gene expression [39; 40]. In addition, it has been reported that carnosol, a rosmary leaf extract, can also induce skeletal muscle hypertrophy by inhibiting the ubiquitin-proteasome system-dependent protein degradation pathway via suppression of E3 ubiquitin ligase MuRF-1 [10]. The injection of AICAR into mice increased FoxO1 and FoxO3 expression, and the AICAR-induced upregulation of FoxO1 was not affected by the suppression of AMPK\(\alpha\)2 [41]. The treatment of C2C12 myotubes with AICAR has been reported to induce both protein breakdown and the upregulation of FOXOO, MArbx/atrogin-1, MuRF-1, and two other FoxO target genes, namely _microtubule-associated protein IA/IB-light chain 3 (LC3)_ and _Bnip3_[42]. Meanwhile, AMPK phosphorylates FoxO3a at a site known to activate the transcription factors, thereby inducing generalized protein degradation; however, this may not necessarily affect the localization of FoxO3a to the nucleus. Treatment with AICAR also increases the binding of FoxO3 to MuRF-1 and atrogin-1 promoters [43]. The regulation of FoxO3a activity is possible by very complex mechanisms such as phosphorylation, acetylation, and methylation [44]. In particular, phosphorylation modification regulates FoxO3a activity via a cytoplasmic-nuclear shuttle mechanism. FOXOO3a is targeted for phosphorylation by numerous protein kinases, and phosphorylation leads to two opposing results. The first is a signaling pathway that causes a negative effect on FoxO3a activity. Phosphorylation through AKT/GSK/ERK/IKK after stimulation of in sulin and growth factors increases the cytoplasmic retention of FoxO3a and further induces the degradation of FoxO3a. The second is a signaling system that causes a positive effect on FoxO3a activity. In the presence of oxidant/nutrient stress, phosphorylation occurs through JNK/MST1/AMPK, promotes nuclear translocation, and increases the activity of FoxO3a.