Suppressive effects of natural reduced waters on alloxan-induced apoptosis and type 1 diabetes mellitus

Abstract

Insulin-producing cells express limited activities of anti-oxidative enzymes. Therefore, reactive oxygen species (ROS) produced in these cells play a crucial role in cytotoxic effects. Furthermore, diabetes mellitus (DM) development is closely linked to higher ROS levels in insulin-producing cells. Hita Tenryosui Water(®) (Hita T. W., Hita, Japan) and Nordenau water (Nord. W., Nordenau, Germany), referred to as natural reduced waters (NRWs), scavenge ROS in cultured cells, and therefore, might be a possibility as an alternative to conventional pharmacological agents against DM. Therefore, this study aimed to investigate the role of NRWs in alloxan (ALX)-induced β-cell apoptosis as well as in ALX-induced diabetic mice. NRWs equally suppressed DNA fragmentation levels. Hita T. W. and Nord. W. ameliorated ALX-induced sub-G(1) phase production from approximately 40% of control levels to 8.5 and 11.8%, respectively. NRWs restored serum insulin levels (p < 0.01) and reduced blood glucose levels (p < 0.01) in ALX-induced mice. Hita T. W. restored tissue superoxide dismutase (SOD) (p < 0.05) activity but not tissue catalase activity. Hita T. W. did not elevate SOD or catalase activity in HIT-T15 cells. Nord. W. restored SOD (p < 0.05) and catalase (p < 0.05) activity in both cultured cells and pancreatic tissue to normal levels. Even though variable efficacies were observed between Hita T. W. and Nord. W., both waters suppressed ALX-induced DM development in CD-1 male mice by administering NRWs for 8 weeks. Our results suggest that Hita T. W. and Nord. W. protect against ALX-induced β-cell apoptosis, and prevent the development of ALX-induced DM in experimental animals by regulating ALX-derived ROS generation and elevating anti-oxidative enzymes. Therefore, the two NRWs tested here are promising candidates for the prevention of DM development.

Fig. 1

Anti-apoptotic effects of various waters on alloxan-treated HIT-T15 cells. Anti-apoptotic effects were assessed by DNA fragmentation analysis (a), TUNEL assay (b), and sub-G₁ analysis (c). HIT-T15 cells were pre-incubated with various waters for 24 h, and then treated with 1 mM alloxan for 4 h. a The amount of fragmented DNA was measured. DNA was isolated and electrophoresed as described in the “Materials and methods”. The gels were stained with ethidium bromide and photographed under ultraviolet light. Similar results were obtained in three independent experiments, and a representative experiment is presented. “Marker lane” indicates a molecular weight marker (100 b.p. DNA ladder). Group designations are as follows: UPW(−) indicates ultrapure water (UPW) without alloxan; UPW(+)ALX indicates UPW plus 1 mM alloxan; CNMW(+)ALX indicates a brand of commercialized natural mineral water plus 1 mM alloxan; Nord. W.(+)ALX indicates Nordenau water plus 1 mM alloxan; Hita T. W.(+)ALX indicates Hita Tenryosui water plus 1 mM alloxan. b Anti-apoptotic effects of various waters on alloxan-treated HIT-T15 cells. Cells were pre-incubated with various waters for 24 h followed by 1 mM alloxan treatment for 4 h. Apoptotic cells were detected by the TUNEL procedure and analyzed by flow cytometry. Values represent the means ± standard error of the mean of three independent experiments. ⁺⁺⁺p < 0.001, UPW(−) compared with various waters with 1 mM alloxan; ***p < 0.001, UPW(+) compared with Hita T. W.(+)ALX or Nord. W.(+)ALX. Group designations are the same as those in a. c Effect of various waters on changes of sub-G₁ content in alloxan-treated HIT-T15 cells. HIT-T15 cells seeded on 60 mm dishes were cultured in HBSS buffer with or without 1 mM alloxan for 4 h. Cells were stained with propidium iodide and subjected to flow cytometric analysis for the measurement of the sub-G₁ phase. Cells undergoing apoptosis contain a reduced amount of DNA compared with that of cells in the G₁ phase and they exhibit an additional peak at the left side of the G₁/G₀ peak position as indicated as apoptosis (sub-G₁) in the panel. Sub-diploid apoptotic cells (sub-G₁) are expressed as a percentage on the left side of each profile

Fig. 2

Effect of NRWs on cellular SOD and catalase production in alloxan-treated HIT-T15 cells. HIT-T15 cells were pretreated with various waters for 24 h prior to 4 h treatment with 1 mM alloxan. Cells were harvested and processed for SOD activity (a) and catalase activity (b) as described in the “Materials and methods” section. Values represent the means ± standard error of the mean of three independent experiments. ⁺p < 0.05, ⁺⁺⁺p < 0.001, UPW(−) compared with various waters with alloxan treatment; *p < 0.05, ***p < 0.001, UPW(+)ALX compared with Hita T. W.(+)ALX or Nord. W.(+)ALX. Group designations are the same as those in Fig. 1a

Fig. 3

Schematic representation of the in vivo study protocol with alloxan and various waters. Six-week-old CD-1 mice were treated with various waters for up to 9 weeks. Alloxan was administered intraperitoneally (I.P.) at the indicated time points (↓). Blood samples were taken at the indicated time points and used for blood glucose assay (indicated by ↑). Adapted from Li et al. (2011)

Fig. 4

The effect of natural mineral waters on serum insulin levels of alloxan-administered CD-1 mice. Blood samples were collected via orbital sinus puncture technique at the 9th week. Values are means ± standard error of the mean (n = 8). **p < 0.01, mice administered ultrapure water compared with those given natural mineral water followed by alloxan administration. Group designations are the same as those in Fig. 1a

Fig. 5

The effect of various waters on blood glucose levels of normal and diabetic CD-1 mice induced by alloxan. CD-1 mice were treated as described in the study protocol (Fig. 3). Each value denotes the mean ± standard error of the mean (n = 8). ⁺⁺p < 0.01,⁺⁺⁺p < 0.001, control (ultrapure water without alloxan) compared with various waters with alloxan. **p < 0.01, ultrapure water with alloxan compared with various waters with alloxan. Group designations are the same as those in Fig. 1a

Fig. 6

Effect of natural mineral waters on superoxide dismutase and catalase production in tissue isolated from alloxan-treated mice. a, b Mice were pretreated with various waters followed by alloxan injection. Nine weeks later, the pancreas was excised from mice for enzyme activity measurements. Values represent the means ± standard error of the mean of three independent experiments. ⁺p < 0.05, UPW(−)ALX compared with various waters with alloxan treatment; *p < 0.05, UPW(+)ALX compared with Hita T. W. (+)ALX or Nord. W.(+)ALX. Group designations are the same as those in Fig. 1a

Fig. 7

The effect of various waters with or without alloxan on body weight (a), and food (b) and water (c) consumption. a Body weight was measured at the time of experimental initiation (designated as 0 weeks), and at the 4th and 8th weeks. Body weight was compared between alloxan-treated and non-treated mice. Values are means ± standard error of the mean calculated from the data of mice that survived. *p < 0.05, UPW(+)ALX compared with Hita T. W.(+)ALX or Nord. W.(+)ALX. For food and water consumption measurements, one group was placed in three cages in which three mice were placed in each cage. Consumed food and water were measured as the total weight per cage. The time course of food (b) consumption in normal and alloxan-injected mice was measured at the indicated time points (0–9 weeks). Food consumption is expressed as means ± standard error of the mean (n = 3). ⁺⁺p < 0.01, ⁺⁺⁺p < 0.001, UPW(+)ALX compared with various waters with alloxan injection. ^#p < 0.05, Nord. W.(+)ALX compared with CNMW(+)ALX. ^$p < 0.05, Nord. W.(+)ALX compared with UPW(+)ALX. Group designations are as follows: open circle ultrapure water (UPW) without alloxan; closed circle UPW plus 1 mM alloxan; open square a brand of commercialized natural mineral water plus 1 mM alloxan; open diamond Nordenau water plus 1 mM alloxan; closed square Hita Tenryosui water plus 1 mM alloxan. The time course of water (c) consumption in normal and alloxan-injected mice was measured at the indicated time points. Water consumption is expressed as means ± standard error of the mean (n = 3). ⁺⁺p < 0.01, ⁺⁺⁺p < 0.001, UPW(ALX) compared with various waters with alloxan injection. ^#p < 0.05, ^##p < 0.01, CNMW(+)ALX compared with Nord. W.(+)ALX or Hita T. W.(+)ALX. Group designations are the same as those in b