Anti-diabetic effect of balanced deep-sea water and its mode of action in high-fat diet induced diabetic mice

Abstract

In this study, we investigated the effects of balanced deep-sea water (BDSW) on hyperglycemia and glucose intolerance in high-fat diet (HFD)-induced diabetic C57BL/6J mice. BDSW was prepared by mixing deep-sea water (DSW) mineral extracts and desalinated water to give a final hardness of 500-2000. Mice given an HFD with BDSW showed lowered fasting plasma glucose levels compared to HFD-fed mice. Oral and intraperitoneal glucose tolerance tests showed that BDSW improves impaired glucose tolerance in HFD-fed mice. Histopathological evaluation of the pancreas showed that BDSW recovers the size of the pancreatic islets of Langerhans, and increases the secretion of insulin and glucagon in HFD-fed mice. Quantitative reverse transcription polymerase chain reaction results revealed that the expression of hepatic genes involved in glucogenesis, glycogenolysis and glucose oxidation were suppressed, while those in glucose uptake, β-oxidation, and glucose oxidation in muscle were increased in mice fed HFD with BDSW. BDSW increased AMP-dependent kinase (AMPK) phosphorylation in 3T3-L1 pre- and mature adipocytes and improved impaired AMPK phosphorylation in the muscles and livers of HFD-induced diabetic mice. BDSW stimulated phosphoinositol-3-kinase and AMPK pathway-mediated glucose uptake in 3T3-L1 adipocytes. Taken together, these results suggest that BDSW has potential as an anti-diabetic agent, given its ability to suppress hyperglycemia and improve glucose intolerance by increasing glucose uptake.

Figure 1

Effect of BDSW on fasting blood glucose levels (A) and glucose tolerance (B) in mice fed the ND, HFD, and HFD with BDSW for 20 weeks. Each value represents the mean ± SEM (n = 8 per group). * P < 0.05, ** P < 0.01: Significant difference vs. HFD-fed group. ND, normal diet; HFD, high-fat diet.

Figure 2

Effects of BDSW on adipokines and cytokines levels, in plasma (A) and morphological changes in pancreas (B) of mice fed the ND, HFD, and HFD with BDSW for 20 weeks. (C) Surface area of the islets of Langerhans and intensities of staining signals for insulin and glucagon. Representative hematoxylin & eosin, glucagon, and insulin staining of the islets of Langerhans are shown at 400× magnification. Scale bar, 50 μM. Each value represents the mean ± SEM (n = 8 per group). * P < 0.05, ** P < 0.01: Significant difference vs. HFD-fed group. ND, normal diet; HFD, high-fat diet.

Figure 3

Effects of BDSW on the expression of genes involved in gluconeogenesis (A), glycogen metabolism (B), and glucose oxidation (C) in the livers of mice fed the ND, HFD, and HFD with BDSW for 20 weeks. Each value represents the mean ± SEM (n = 8 per group) in three independent experiments. * P < 0.05, ** P < 0.01: Significant difference vs. HFD-fed group. ND, normal diet; HFD, high-fat diet.

Figure 4

Effects of BDSW on the expression of genes involved in glucose uptake (A), glucose oxidation (B), and β-oxidation (C) in the muscles of mice fed the ND, HFD, and HFD with BDSW for 20 weeks. Each value represents the mean ± SEM (n = 8 per group) in three independent experiments. * P < 0.05, ** P < 0.01: significant difference vs. HFD-fed group. ND, normal diet; HFD, high-fat diet.

Figure 5

Effects of BDSW on 2-NBDG uptake in mature 3T3-L1 adipocytes. For the glucose uptake assay at different levels of BDSW hardness (A), 3T3-L1 adipocytes were preincubated in DMEM without glucose for 1 h. They were then incubated in DMEM containing BDSW at different levels of hardness, or 10 μM rosiglitazone (Rosi), and 100 nM insulin (Ins) with 20 μM 2-NBDG for 1 h. For the glucose uptake assay using several inhibitors (B), after preincubation in DMEM without glucose in the presence or absence of 10 μM LY294002, 10 μM compound C (Comp C), 10 μM rapamycin (Rapa), 2 mM nicotinamide (NA), and 10 μM rosiglitazone (Rosi) for 1 h, the cells were incubated in DMEM containing BDSW of hardness 2000 ppm only, with or without inhibitors with 20 μM 2-NBDG for 1 h. After incubation was completed, cells were lysed, and then the glucose uptake was measured using a fluorometer. Each value represents the mean ± SEM for six wells in three independent experiments. * P < 0.05, ** P < 0.01: Significant difference vs. HFD-fed group. ND, normal diet; HFD, high-fat diet; Comp C, compound C; Rapa, rapamycin; NA, nicotinamide; Rosi, rosiglitazone.

Figure 6

Effects of BDSW on the phosphorylation of AMPK for 3T3-L1 adipocyte differentiation (A), or in mature adipocytes (B). 3T3-L1 pre- and mature adipocytes were treated in media containing BDSW at different levels of hardness for eight days or 1 h, respectively, lysates (20 μg) were subjected to SDS-PAGE and western blotting analyses using anti-phospho-AMPK, anti-phospho-ACC1, and anti-phospho-IRS-1, anti-GLUT4 antibodies. β-actin was used as a loading control.

Figure 7

Effects of BDSW on the phosphorylation of AMPK in muscles (A) and livers (B) of mice fed the ND, HFD, and HFD with BDSW for 20 weeks. Each value represents the mean ± SEM (n = 8 per group). * P < 0.05: Significant difference vs. CON of HFD fed group. ND, normal diet; HFD, high fat diet.