## 4 Discussion  
In this study, we factored the effect of exercise into motoneuron activity and endocines and established two in vitro exercise models for the research of transcription using clenbuterol treatment and EPS experiments. We analyzed global transcriptional regulation by RNA-seq in both models. The IPA analyses summarized the RNA-seq results and are shown in Figure 5a,b. RNA-seq analyses revealed that clenbuterol regulated hypertrophic effect and mitogenesis pathway and EPS-regulated sarcomeric gene transcription to achieve fiber type shift (Figure 5c). _Papgcla_ and _Irs2_ are important genes to achieve the effect of exercise, mitogenesis and insulin/IGF-1 signaling. The induction of _Papgcla_ and _Irs2_ by clenbuterol was observed only in low glucose media (Figure 1). The precise mechanism causing the difference  
Figure 5: Models of clenbuterol treatment and EPS. (**a**) Pathway diagram for CREB1 and mitogenesis of clenbuterol treatment analyzed by IPA; (**b**) pathway diagram for cardiac hypertrophy of EPS analyzed by IPA; (**c**) a model of exercise regulation of acute and slow transcriptional changes. between high glucose media and low glucose media is not clear. High glucose concentration is reported to induce phosphorylation of HDAC by CaMKII and nuclear export of HDAC5 in C2C12 myotubes [33]. HDAC5 is a transcriptional suppressor, therefore nuclear export of HDAC5 might be directly or indirectly related with induction of _Ppargca_ and _Irs2_ by clenbuterol. To examine the physiological meaning of the impaired induction of _Ppargca_ and _Irs2_ by adrenaline, diabetic patients or animal models would be useful to test induction of _Ppargca_ and _Irs2_ by exercise in future research. Exercise affects the whole body through myokines, bioactive molecule secreted from skeletal muscles, which include Irisin, IGF-I, II6 and so on. EPS was reported to increase the mRNA expression of _I16_, _Cxc11_, and _Cxc15_ in C2C12 myotubes and human primary myoblasts [31; 34]. Consistent with previous reports, our RNA-seq analysis of 24 h EPS also identified up-regulation of _I16_, _Cxc11_ and _Cxc15_. In addition, clenbuterol treatment surprisingly induced _I16_, _Cxc11_ and _Cxc15_ gene expression (Figures 1 and 2). Although the meaning of the existence of common target genes is not clear, those genes might act as myokines to inform the other tissues expressing their corresponding receptors about the occurrence of transcriptional changes in skeletal muscles. Clenbuterol treatment induced gene expression of _Nr4a3_, _Irs2_, and _Ppargca_ within 1 h, whereas EPS needed longer to induce _Myh7_ and _Myh2_ gene expression. The gene induction speed was different between clenbuterol treatment and EPS. Transcription is closely related with epigenome, chemical modification without DNA sequence change such as methylation of DNA, acetylation and methylation of histone proteins and turnover of histone protein variants. Depending of epigenetic modification, the speed of transcriptional suppression and activation is different and acetylation of histone responds faster than H3K9 methylation or DNA methylation [35]. The difference of gene induction speed might be explained from the point of epigenetic regulation. As for epigenetic analysis of exercise, exercise led to enhanced hypoacetylation of histone 3 lysine 27 [36]. Recently, it was also reported that chronic exercise training induces histone turnover to histone variant H3.3 and enhanced gene responses to exercise [37]. However, the responsible biological signal for epigenetic modification is not clear. As another possibility, different sets and number of transcription factors might explain the difference in transcriptomic responses. It was reported that CREB/ATF/API-, MYC/MAX- and E2F-related transcription factors are associated with the acute response genes in vivo exercise and IRF- and STAT-related transcription factors were associated with chronic response genes [38; 39]. For the advanced molecular mechanistic analyses, our in vitro exercise models would be useful.