Figure 1. The effect of H₂ on glucose uptake into C2C12 cells.

(A) 2-DG uptake into C2C12 cells after 30 or 60 min exposure to high content hydrogen water (HHW) was significantly increased over control (n = 6 for each group). (B) Natural hydrogen water (NHW) significantly increased 2-DG uptake into C2C12 cells over control, while degassed NHW did not increase 2-DG uptake (n = 7 for each group). (C, D, E) After incubation with or without each pharmacological inhibitor for 30 min, the cells were exposed to pure water or SHW for another 30 min. The addition of LY-2940002, a phosphatidylinositol-3-OH kinase (PI3K) inhibitor, at 1.0 × 10⁻⁶ M significantly decreased the 2-DG uptake into C2C12 cells compared with HHW alone (n = 6 for each group). The addition of chelerythrine, a protein kinase C (PKC) inhibitor, at 1.0 × 10⁻⁶ M significantly decreased the 2-DG uptake into C2C12 cells compared with HHW alone (n = 6 for each group). The addition of Compound C (6-[4-(2-piperidin-1-ylethoxy)-phenyl]-3-pyridin-4-ylpyrazolo[1,5-a] pyrimidine), an AMP-activated protein kinase (AMPK) inhibitor, at 1.0 × 10⁻⁶ M significantly decreased the 2-DG uptake into C2C12 cells compared with HHW alone (n = 10 for each group). (F, G) Western blot analysis was performed as described in the Materials and Methods. HHW increased membrane Glut4 (n = 6 for each group) and phosphorylated AMPK (p-AMPK) (n = 13 for each group) in C2C12 cells. (H, I) There was no significant difference in total AMPK in C2C12 cells (n = 8 for each group) or membrane Glut2 in Hep-G2 cells between groups (n = 6 for each group). Comparisons with controls were performed by unpaired Student’s t test between two groups and Dunnett’s multipule comparison test among more than two groups. *P<0.05, **P<0.01, ***P<0.001.