Hydrogen improves glycemic control in type1 diabetic animal model by promoting glucose uptake into skeletal muscle

Abstract

Hydrogen (H(2)) acts as a therapeutic antioxidant. However, there are few reports on H(2) function in other capacities in diabetes mellitus (DM). Therefore, in this study, we investigated the role of H(2) in glucose transport by studying cultured mouse C2C12 cells and human hepatoma Hep-G2 cells in vitro, in addition to three types of diabetic mice [Streptozotocin (STZ)-induced type 1 diabetic mice, high-fat diet-induced type 2 diabetic mice, and genetically diabetic db/db mice] in vivo. The results show that H(2) promoted 2-[(14)C]-deoxy-d-glucose (2-DG) uptake into C2C12 cells via the translocation of glucose transporter Glut4 through activation of phosphatidylinositol-3-OH kinase (PI3K), protein kinase C (PKC), and AMP-activated protein kinase (AMPK), although it did not stimulate the translocation of Glut2 in Hep G2 cells. H(2) significantly increased skeletal muscle membrane Glut4 expression and markedly improved glycemic control in STZ-induced type 1 diabetic mice after chronic intraperitoneal (i.p.) and oral (p.o.) administration. However, long-term p.o. administration of H(2) had least effect on the obese and non-insulin-dependent type 2 diabetes mouse models. Our study demonstrates that H(2) exerts metabolic effects similar to those of insulin and may be a novel therapeutic alternative to insulin in type 1 diabetes mellitus that can be administered orally.

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.

Figure 2. The H₂ attenuation rate in pure water and several solutions.

Natural hydrogen water (NHW) and 0.17 mM (0.001%) and 0.34 mM (0.002%) saline water with 0.8 mM H₂ retained a higher hydrogen concentration than low-content (LHW) and high-content hydrogen water (HHW) at 12 and 24 hours (n = 7 for each group). Multiple comparisons were performed by Dunnett’s multipule comparison test. **P<0.01 vs LHW, ## P<0.01 vs HHW.

Figure 3. The effect of i.p. administration of H₂ on hyperglycemia in STZ-treated mice.

(A) Blood glucose in the group injected with high-content hydrogen saline (HHS) after STZ administration was significantly lower than the control group at every 7-days-interval measurement (n = 16 for each group). (B, C) Body weight and food intake every 7 day are shown. Although there was no significant difference in body weights or food intake between the control and HHS groups (n = 16 for each group), the food intake in the HHS group showed a tendency to decrease. (D, E) Blood glucose in the HHS group in the day-30 IPGTT was significantly lower than the control group at 5, 30, 60 and 120 min, and the area under the curve (AUC) of the HHS group was significantly lower than control (n = 16 for each group). (F) Membrane Glut4 in the HHS group was significantly increased compared to the control group, as determined by western blot analysis (n = 6 for each group). (G) Although there was no significant difference in cytosolic Glut4 between groups, the cytosolic Glut4 in the HHS group showed a tendency to decrease (n = 4 for each group). The bar graph shows the ratio of each protein to actin protein bands quantified by densitometric analysis. Comparisons with controls were performed by unpaired Student’s t test between two groups. *P<0.05, **P<0.01, ***P<0.001.

Figure 4. The effect of p.o. administration of H₂ on diabetes in STZ-induced type 1 diabetic mice.

(A) Blood glucose in the high content hydrogen water (HHW) and natural hydrogen water (NHW) groups at every weekly measurement showed a tendency to decrease compared to control, but, these decreases were not statistically significant (n = 6 for each group). (B, C) The AUC values of NHW group in the day-90 and day-120 IPGTTs were significantly decreased. Although the AUC values of the LHW, HHW groups in the day-90 and day-120 IPGTTs showed a tendency to decrease, these decrease were not statistically significant (n = 6 for each group). (D) As described in the Results, several mice were lost due to dehydration, so we combined the data of two groups [HHW (n = 2) and LHW (n = 2)] together. Glycated albumin in the combined LHW and HHW group (n = 4) and NHW group (n = 6) was significantly lower than the control group (n = 6). Comparisons with controls were performed by Dunnett’s multipule comparison test. *P<0.05, **P<0.01.

Figure 5. The effect of p.o. administration of H₂ on food intake in STZ-induced type 1 diabetic mice.

(A, B) Body weights and food intake were measured every 7 days. Although there was no significant difference in body weight or food intake among the 4 groups (n = 6 for each group), the food intake in the LHW, HHW and NHW groups showed a tendency to decrease in the latter part of this experiment. (C) The body weight gain in the HHW group (n = 6) and NHW group (n = 6) was significantly lower than the control group (n = 6) at 25, 26, 27, and 28 weeks of age. (D) The food intake gain in the HHW group (n = 6) was significantly lower than the control group (n = 6) at 27 weeks of age. Additionally, the food intake gain in the NHW group (n = 6) was significantly lower than the control group (n = 6) at 26, 27, and 28 weeks of age. (E) Orexigenic melanin-concentrating hormone (MCH) and orexin mRNA and anorexigenic pro-opiomelanocortin (POMC) mRNA expressions in the hypothalamus were significantly increased in the combined LHW, HHW, and NHW group (n = 10) compared with the control group (n = 6). (F) There was no significant difference in ghrelin mRNA in the stomach (the control group, n = 6; the combined LHW, HHW, and NHW group, n = 10). Comparisons with controls were performed by Dunnett’s multipule comparison test. *P<0.05.

Figure 6. The hypothetical model of H₂ action in glucose excursion.

H₂ promotes glucose uptake into skeletal muscle by stimulating Glut4 translocation by activating phosphatidylinositol-3-OH kinase (PI3K), atypical protein kinase C (aPKC), and AMP-activated protein kinase (AMPK) under conditions of severe insulin deficiency. H₂ has little effect on glucose excursion under conditions of hyperinsulinemia and insulin resistance.