## 4 Discussion  
In this study, we clarified global protein levels under different glucose concentration media using proteome analysis. Low or Medium glucose media increased proteins involving glycolytic system, oxidative phosphorylation, and fatty acid metabolism. Consistent with the proteome observation, ATP contents were increased in myotubes under Low or Medium glucose media. Furthermore, culture in Low or Medium glucose media resulted in an increase in fast and slow _Myth_ genes at the mRNA expression and protein levels. We identified HK2 and FABP3 in this study. HK2 is an enzyme that catalyzes the first reaction in the glycolysis, producing glucose-6-phosphate from glucose. Four isoforms, HK1-4, exist in mammals, and HK2 is localized at the outer mitochondrial membrane [16]. it has been reported that HK2 can attenuate cellular senescence by maintaining carbohydrate hypermetabolism [16]. Cellular senescence promotes the age-related decline of the skeletal muscular system [17]. Conversely, it has been reported that HK2 activity is upregulated and oxidative phosphorylation is enhanced by 12-week resistance training in young men [18]. In addition, overexpression of FABP3 induces translocation of GLUT4 and enhances glucose uptake in C2C12 myocytes [19]. Based on these reports, the enhancement of glycolysis could be achieved by HK2, PFKP, and FABP3 increased in Low/Medium glucose concentrations in the culture media. Furthermore, NDUFA11, whose expression was found to be upregulated by incubation at Low/Medium glucose concentrations in the present study, is one of the subunits of mitochondrial respiratory chain complex I [14]. Mitochondrial respiratory chain complexes are composed of complex I to V, and complex I is the first enzyme in the respiratory chain reaction [20]. Specifically, it oxidizes NADH produced in the Krebs cycle in the mitochondrial matrix and uses two electrons to reduce ubiquinone to ubiquinol, thereby aiding in the process of ATP production [20]. Defects in human respiratory chain complex I cause impaired energy generation [21]. Furthermore, restoring respiratory chain complex I in mice with mitochondrial myopathy improves their symptoms [22]. These reports supported that upregulated proteins in the proteome analysis are beneficial for muscle health and provide new insights into the relationship between energy metabolism and glucose conditions. Figure 3: _Myth_ expression in C2C12 myotubes under Low, Medium, or High glucose conditions (**a**) Picture of C2C12 myotubes at 3–5 days post-differentiation under Low, Medium, or High glucose conditions. Scale bar = 50 \(\mu\)m. (**b**) mRNA expression levels of _Myd01_, _Myf5_, _Myg0_ were determined in C2C12 myotubes at 3 days post-differentiation under Low, Medium, or High glucose conditions (\(n\) = 4, each group). (**c**) mRNA expression levels of _Myh1_, _Myh2_, _Myh4_ and _Myh7_ were determined in C2C12 myotubes at 5 days post-differentiation under Low, Medium, or High glucose conditions (\(n\) = 4, each group). * \(p\) < 0.05. Mitochondria generate energy for cellular activities through ATP production via oxidative phosphorylation [23]. PGC-1\(\alpha\) is known to be a factor that enhances the biosynthesis and activity of mitochondria [24] and predicted as an up-stream regulator by IPA analysis (Figure 1d). Therefore, one of the reasons that ATP production was increased in the Low and Medium groups was that the activity of mitochondria was enhanced by the increased expression of PGC-1\(\alpha\). In addition, PGC-1\(\alpha\) is also known as a master regulator of slow type fiber genesis, and its decreased expression in the unloading condition contributes to slow muscle fiber-dominant degradation [25]. In mice overexpressing PGC-1\(\alpha\), phosphorylation of Akt1 was observed, accompanied by increased phosphorylation of FOXO03a [26]. FOXO03a is a ubiquitin ligase that is important in muscle atrophy.