## 3 Results  
### Proteome Analysis in C2C12 Myotubes under Low, Medium or High Glucose Conditions  
C2C12 myotubes were cultured in Low (1.0 g/L), Medium (2.0 g/L), and High (4.5 g/L) glucose media. At 5 days post-differentiation, proteome analysis was performed to examine the intracellular dynamics of the C2C12 myotubes at different glucose concentrations in the culture media (Table S1). Principal component analysis (PCA) revealed three clusters  
\begin{table}
\begin{tabular}{c c c} \hline
**Gene Name** & **Forward (5’-3’)** & **Reverse (5’-3’)** \\ \hline _Myod1_ & AGGACACGCGCACACTTCCT & TCTCGAAGCCCTCAT \\ _Myf5_ & AGACGCCTGAAAGAGTCAA & GTTCTCACCCTGTTCCCTC \\ _Myog_ & CTACAGGCGTCTCACCCTC & ACGATGACCTAAGAGAGAGGTG \\ _Myh1_ & AATCAAAGGTCAAGGCCTACAA & GAATTGTGACGAGTTGACAT \\ _Myh2_ & CAGCACAGGCTGAGGAGG & CTGCGTCTGGTATCC \\ _Myh4_ & GCTGAGGAGGCTGAGAGAC & GTGTGAAACCTCTCGGCTCTT \\ _Myh7_ & AGTCCAGGGTCAACAAGCTG & TTCACCCTAAAAGGGCTGTTG \\ _18S rRNA_ & CATTCGAAACGTCTGCCCTA & CCTGCCTGCCTTCCTTGGA \\ \hline \end{tabular}
\end{table}
Table 1: Sequence of the oligonucleotides for qRT-PCR.  
of High, Medium, and Low groups (Figure 1a). The contribution ratio of PC1 and PC2 are 0.27 and 0.14, respectively. The \(p\)-value was corrected by empirical Bayes method. In total, 113 proteins were identified as differentially expressed proteins with statistical significance (q < 0.05) and were classified by their expression patterns into two major groups (Figure 1b). Compared with High, Low and Medium showed similar protein expression patterns. Comparison between Low and Medium did not show any statistically significant difference. The differentially expressed proteins were analyzed by KEGG pathway and IPA analysis (Figure 1c,d). The KEGG and IPA results showed that glycolysis, oxidative phosphorylation, and fatty acid metabolism were increased in the Medium and Low groups compared to the High group. It is noted that PPARC1A was suggested as an upstream regulator in Medium condition (Figure 1d). Specifically, in the Medium and Low groups, the glycolytic pathways included hexokinase 2 (HK2), which catalyzes the reaction of glucose-to-glucose hexaphosphate, and phosphofructokinase (PFKP), which catalyzes the reaction of fructose hexaphosphate to fructose 1,6-bisphosphate was increased (Figure 1e). Moreover, the expression of NDUFA11, a subunit of mitochondrial complex 1 of the electron transport system [14], and the expression of FABP3, which is involved in the intracellular transport of fatty acids [15], were also increased (Figure 1e). Proteome analyses suggested that Low or Medium glucose media induces the metabolic change.  
Figure 1: Proteome analysis with C2C12 myotubes under Low, Medium, or High glucose conditions. (**a**) Principal components analysis with proteome analysis of C2C12 myotubes under Low, Medium, or High glucose conditions (\(n=3\), each group). (**b**) Hierarchical clustering of 113 Differentially expressed proteins in proteome analysis of C2C12 myotubes under Low, Medium, or High glucose conditions (\(n=3\), each group). The scale value is logarithm of fold change to the base 2. (**c**) KEGG pathway analysis for upregulated proteins in Low or Medium glucose condition. (**d**) Left: Pathway diagram for Low glucose compared with High glucose condition analyzed by IPA. Right: Pathway diagram for Medium glucose compared with High glucose condition analyzed by IPA. Upregulation and downregulated pathways are indicated in orange and blue, respectively. Directly regulated and indirectly regulated are indicated in solid lines and dotted lines, respectively. (**e**) Protein levels of HK2, Nduta11, Fabp3, Pfkm were obtained from proteome analysis of C2C12 myotubes under Low, Medium, or High glucose conditions (\(n=3\), each group). * \(p<0.05\), ** \(p<0.01\).