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Anti-diabetic effects of electrolyzed reduced water in streptozotocin-induced and genetic diabetic mice
Life Sciences 79 (2006) 2288 – 2292 www.elsevier.com/locate/lifescie
Anti-diabetic effects of electrolyzed reduced water in streptozotocin-induced and genetic diabetic mice Mi-Ja Kim a , Hye Kyung Kim b,⁎ a
Department of Obesity management, Graduate School of Obesity Science, Dongduk Women's University, 23-1 Wolkgukdong, Seoul, 136-714, South Korea b Department of Food and Biotechnology, Hanseo University, Sesan, 356-706, South Korea Received 28 February 2006; accepted 27 July 2006
Abstract Oxidative stress is produced under diabetic conditions and is likely involved in progression of pancreatic β-cell dysfunction found in diabetes. Both an increase in reactive oxygen free radical species (ROS) and a decrease in the antioxidant defense mechanism lead to the increase in oxidative stress in diabetes. Electrolyzed reduced water (ERW) with ROS scavenging ability may have a potential effect on diabetic animals, a model for high oxidative stress. Therefore, the present study examined the possible anti-diabetic effect of ERW in two different diabetic animal models. The genetically diabetic mouse strain C57BL/6J-db/db (db/db) and streptozotocin (STZ)-induced diabetic mouse were used as insulin deficient type 1 and insulin resistant type 2 animal model, respectively. ERW, provided as a drinking water, significantly reduced the blood glucose concentration and improved glucose tolerance in both animal models. However, ERW fail to affect blood insulin levels in STZ-diabetic mice whereas blood insulin level was markedly increased in genetically diabetic db/db mice. This improved blood glucose control could result from enhanced insulin sensitivity, as well as increased insulin release. The present data suggest that ERW may function as an orally effective antidiabetic agent and merit further studies on its precise mechanism. © 2006 Elsevier Inc. All rights reserved. Keywords: Electrolyzed reduced water; Diabetic mice; Blood glucose; Insulin; Glucose tolerance
Introduction Oxidative stress is a condition of imbalance due to excess formation of free radicals and decreased activity of antioxidant defense systems. All oxidative reactions are a continuous source of potentially cytotoxic reactive oxygen species (ROS), which play an important role in living systems both through their beneficial and detrimental effects (Halliwell and Gutteridge, 1999). Under physiological conditions, ROS are fully inactivated by an elaborated cellular and extracellular antioxidant defense system (Yu, 1994). However, under certain conditions increased generation of ROS and/or reduction of the antioxidant capacity leads to enhanced ROS activity and oxidative stress. Diabetes mellitus is characterized by increased production of ROS, sharp reduction in antioxidant defense and altered cellular ⁎ Corresponding author. Tel. +82 41 660 1454; fax: +82 41 660 1119. E-mail addresses: [email protected] (M.-J. Kim), [email protected] (H.K. Kim). 0024-3205/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2006.07.027
redox status (West, 2000). The chronic presence of high glucose levels enhances the production of ROS from protein glycation and glucose autoxidation (Feillet-Coudray et al., 1999). Therefore, supplementation with some antioxidants such as quercetin, ascorbate and β-carotene resulted in an improvement of the antioxidant status in streptozotocin (STZ)-induced diabetic rats (Mahesh and Menon, 2004; Maritim et al., 2002; Young et al., 1992). Diabetes mellitus is classified into two types, insulindependent (IDDM, type 1) and non-insulin-dependent diabetes mellitus (NIDDM, type 2). The two types of diabetes have distinct pathogenesis but hyperglycemia and various lifethreatening complications resulting from long-term hyperglycemia are the most common features. Hence, effective control of the blood glucose levels is a key step in preventing or reversing diabetic complications in both types 1 and 2 diabetic patients. However, due to limited efficacy and adverse side effects of currently available therapies, it is difficult to maintain good glycemic control in most diabetic patients. Therefore, there is a strong incentive to develop new hypoglycemic agents.
M.-J. Kim, H.K. Kim / Life Sciences 79 (2006) 2288–2292 Table 1 Fasting blood glucose, insulin and body weight changes in streptozotocin diabetic mice NC Blood glucose (mg/dl) Blood insulin (ng/ml) Initial body weight (g) Final body weight (g)
112 ± 10 0.61 ± 0.04 32.6 ± 0.2 40.4 ± 0.5
NE
DC
DE
109 ± 11 0.62 ± 0.05 32.3 ± 0.3 41.0 ± 0.7
295 ± 23⁎ 0.41 ± 0.03⁎ 31.8 ± 0.3 34.6 ± 0.5⁎
180 ± 19⁎⁎ 0.40 ± 0.02⁎ 32.4 ± 0.4 34.8 ± 0.6⁎
Data are expressed as means ± S.E.M. ⁎p b 0.01, compared with normal control (NC), ⁎⁎p b 0.01, compared with diabetic control (DC). NC; normal ICR mice control fed tap water, NE; normal mice fed ERW, DC; STZ-diabetic mice fed water, DE; STZ-diabetic mice fed ERW.
Electrolysis of aqueous NaCl or KCl solutions by diaphragmtype electrolyzing devices produces oxidized and reduced water at the anodic and the cathodic side, respectively. Electrolyzed reduced water (ERW) exhibits high pH (pH 10–12), low dissolved oxygen (1.3–3.5 mg/l), high dissolved hydrogen (0.3 mg/l–0.6 mg/l), and significant negative redox potential (−400 to −900 mV). The ideal scavenger for ROS is “reactive hydrogen”. Since ERW has high reactive hydrogen, the usefulness of electrolyzed water in the medicinal fields is being examined. The free radical-scavenging antioxidants play an important role in the in vivo defense system against oxidative damage initiated and promoted by ROS (Mahesh and Menon, 2004; Vessal et al., 2003; Maritim et al., 2002). ERW scavenges ROS such as superoxide anion radical (O2−) and hydrogen peroxide. (Shiharata et al., 1997). Huang et al. (2003) also reported that ERW with a high reducing ability and/or direct ROS scavenging activity could be used in hemodialysis patients by strong antioxidant activity. Since oxidative damage has been implicated in the etiology of diabetic complication, ERW, a potent ROS scavenger, may have a therapeutic role in diabetic mellitus. Therefore, the present study examined the possible anti-diabetic effect of ERW in diabetic animal models. The diabetic mouse strain C57BL/ 6J-db/db (db/db) which exhibits many of the metabolic disturbances of human type 2 diabetes including hyperglycemia, obesity, and insulin resistance (Surwit et al., 1991), and STZ-induced diabetic mouse which is widely used as an animal model of type 1 diabetes were used.
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littermates (db/−) at 3 weeks of age were housed under controlled temperature (20–26 °C) and light (12 h light/dark cycle) controlled conditions. All animals had free access to standard rodent pellet food (NIH #31M, Samtako, Korea), except when fasted before experiments. Mice were divided into 4 groups (n = 10): normal control (NC), normal-ERW (NE), diabetic control (DC), and diabetic-ERW (DE). Normal and diabetic control mice were fed tap water, and normal and diabetic-ERW mice were fed ERW. Insulin deficient type 1 diabetes was induced by multiple low-dose injection of STZ (60 mg/kg/day, i.p.) dissolved in citrate buffer (0.01 M, pH 4.5) for 5 consecutive days. This dose of STZ results in significant impaired ability to produce insulin and moderate to severe hyperglycemia. Control mice received the same volume of vehicle. Tail bleeds were performed 24 h post-injection and animals with a fasting blood glucose concentration above 200 mg/dl were considered to be diabetic, and assigned as DC or DE group. The initial blood glucose and insulin concentrations of DC and DE groups before ERW administration were very similar (300 ± 5 and 298 ± 7 mg/dl, and 0.40 ± 0.02 and 0.40 ± 0.03 ng/ml, respectively). Blood glucose level and intraperitoneal glucose tolerance test (IPGT) Blood samples were collected after fasted for 4 h from the retro-orbital sinus every 7 days. Approximately 50 μl of fresh blood sample was placed on a test strip and the blood glucose content was determined in a validated One Touch Basic glucose measurement system (Lifescan Inc, USA). At the end of experimental period, mice were fasted overnight and injected with glucose (1 g/kg, i.p.). Blood samples were collected from the tail vein just prior to and 15, 30, 60, 90, and 120 min after glucose loading, and blood glucose levels were measured. Mice were killed by decapitation immediately after 120 min blood sample was taken and blood samples were taken from the cervical wound. Serum glucose and insulin concentrations were determined with commercially available kits (Sigma Co., USA).
Materials and methods Electrolyzed water The ERW was produced by AK-3000 (Nexus, Korea). This water was produced by the electrolysis of water from a municipal water system. The ERW used in this study has the following physical properties: pH 10.24 ± 0.03 and an oxidative-reduction potential of − 400.2 ± 17.3 mV. Animals and diets 8 weeks old male ICR mice (Orient, Seoul, Korea), and genetically diabetic db/db (C57BL/6J db/db, Jackson Laboratory, Bar Harbor, ME, USA) and their non diabetic heterozygous lean
Fig. 1. Effect of electrolyzed reduced water (ERW) on glucose tolerance in streptozotocin (STZ)-induced diabetic mice. Normal ICR mice fed with tap water (NC); normal mice fed with ERW (NE); STZ-diabetic mice fed with water (DC); STZ-diabetic mice fed with ERW (DE). Data are expressed as means ± S.E.M. ⁎p b 0.01, compared with diabetic control (DC).
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M.-J. Kim, H.K. Kim / Life Sciences 79 (2006) 2288–2292
tea produces an anti-hyperglycemic effect without affecting insulin secretion in STZ-diabetic mice. Cherng and Shih (2005) reported that potential hypoglycemic effects of chlorella increased exogenous insulin sensitivity without affecting endogenous insulin level in STZ-induced diabetic mice. Induction of diabetes by STZ caused significant weight loss compared with normal ICR mice (NC) because of poor utilization of nutrients. ERW administration did not significantly affect body weight in both normal (NE) and STZ-diabetic mice (DE). ROS scavengers such as green tea and quercetin were reported to reduce blood glucose concentrations in STZ-diabetic rats and mice (Mahesh and Menon, 2004; Sabu et al., 2002; Vessal et al., 2003). Sabu et al. (2002) observed the antihyperglycemic effect of green tea extract independent of changes in body weight in STZ-diabetic rats. Fig. 2. Effect of electrolyzed reduced water (ERW) on blood glucose concentrations in genetically diabetic db/db mice. Normal db/− mice fed with tap water (NC); normal db/− mice fed with ERW (NE); db/db mice fed with tap water (DC); db/db mice fed with ERW (DE). Data are expressed as means ± S.E.M. ⁎p b 0.01, compared with diabetic control (DC).
Effects of ERW on glucose tolerance in type 1 diabetic mice
Effects of ERW on fasting blood glucose and insulin level, and body weight in type 1 diabetic mice
Glucose tolerance was evaluated by IPTG after ERW consumption as a drinking water. As shown in Fig. 1, administration of 1 g glucose/kg body weight to normal mice (NC) increased blood glucose levels from 99.4 ± 8.5 to 134.2 ± 14.2 at 15 min but returned to baseline values within 60–120 min. ERW exerted no effect on the glucose tolerance curve of the normal ICR mice (NE). In contrast, STZ-induced diabetic mice (DC) demonstrated basal hyperglycemia (280 mg/dl) which remained until 120 min after glucose loading, indicating delayed glucose homeostasis in diabetic mice. ERW consumption (DE) showed definite improvement in glucose tolerance. Peak blood glucose concentration was observed after 15 min of glucose loading, and returned to below fasting level after 120 min. For the ERW treated diabetic mice (DE), the area under the curve of the blood glucose decreased compared to tap water treated diabetic mice. The present study indicates that STZ-induced diabetes and subsequent elevation of blood glucose were reversed by simultaneous administration of ERW. The elevation of glucose in STZ treated mice was due to a single strand break in pancreatic islets DNA (Omamto and Uchigata, 1981), and Shiharata et al.
STZ is widely used to induce diabetes in experimental animals, causing selective destruction of pancreatic β-islet cells, probably by a free radical-mediated mechanism which is responsible for high blood glucose level seen in STZ-induced diabetic animals (Halliwell and Gutteridge, 1999). Accordingly, STZ-induced diabetic mice (DC) developed hyperglycemia and insulin deficiency as evidenced by elevated blood glucose and reduced insulin levels (Table 1). Diabetic control mice (DC) demonstrated 3 fold increase in blood glucose concentrations over control (NC) values throughout the whole experimental period. However, following 6 weeks of ERW treatment, elevated blood glucose levels in diabetic mice were significantly reduced (p b 0.01) without any significant effect on blood insulin concentrations. There is no, or very little, insulin secretion in STZ-induced diabetic animal model. Therefore, ERW may act to increase insulin sensitivity rather than to increase insulin release. Tsuneki et al. (2004) reported that green
Fig. 3. Effect of electrolyzed reduced water (ERW) on blood insulin concentrations in genetically diabetic db/db mice. Normal db/− mice fed with tap water (NC); normal db/− mice fed with ERW (NE); db/db mice fed with tap water (DC); db/db mice fed with ERW (DE). Data are expressed as means±S.E.M. ⁎pb 0.01, compared with normal control (NC), ⁎⁎pb 0.01, compared with diabetic control (DC).
Statistical analysis All data were presented as mean ± S.E.M. Data from experiments were analyzed by ANOVA, using the statistical software SPSS (Window version 10.5). Specific comparisons were made with Tukey HSD's post-hoc test. Results and discussion The present study was designed to explore the potential role of ERW, a potent ROS scavenger, in improving hyperglycemia in two animal models of diabetes, namely the insulin deficient STZ-induced diabetic mice (type 1) and insulin resistant genetically diabetic db/db (type 2) mice.
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Effects of ERW on glucose tolerance in type 2 diabetic mice
Fig. 4. Effect of electrolyzed reduced water (ERW) on glucose tolerance in genetically diabetic db/db mice. Normal db/− mice fed with tap water (NC); normal db/− mice fed with ERW (NE); db/db mice fed with tap water (DC); db/ db mice fed with ERW (DE). Data are expressed as means ± S.E.M.
(1997) suggested that ERW scavenges ROS and protects DNA from oxidative damage. Therefore, we speculate that the observed effects of ERW on STZ-diabetic mice are primarily due to the promotion of insulin sensitivity in peripheral tissues, such as skeletal muscles and adipocytes rather than increasing insulin release in pancreas as evidenced by unchanged insulin levels in ERW administered mice. Effects of ERW on body weight, fasting blood glucose, and insulin level in type 2 diabetic mice Since genetically diabetic db/db mice exhibit obesity, the body weights of db/db mice (DC) were significantly higher than control mice (NC). Administration of ERW did not affect body weight in both diabetic db/db (DE) and control mice (NE) (data not shown). Blood glucose levels after 4 h fasting were measured on days 1, 7, 14, and 29 after daily administration of ERW or tap water. The diabetic db/db mice exhibit its diabetic characteristics after 4 weeks of age. Therefore, blood glucose concentrations on 1st day of experiment (3 weeks of age) were similar in all groups. However, blood glucose levels of diabetic mice (DC) were markedly increased at day 7 compare to control mice (NC), and ERW consumption (DE) significantly lowered the blood glucose levels (Fig. 2). On day 29, blood glucose levels in ERW supplied db/db mice (DE) were 41% lower than those of water supplied db/db group (DC). The values of blood glucose levels in NC, NE, DC and DE group were 128 ± 29, 125.4 ± 3.9, 490.1 ± 32.4 and 287.9 ± 34.5, respectively. Blood insulin levels of diabetic mice (DC) were more than 2-fold higher than control mice (NC) indicating insulin resistance in type 2 diabetes (Fig. 3). Interestingly, ERW administration also increased insulin level in diabetic mice (DE) without any effect in control mice (NE), suggesting elevated insulin release in diabetic mice. The hypoglycemic effects of ERW in type 2 diabetic mice model may be associated with the improvement of pancreatic β-cell function.
Glucose tolerance in diabetic db/db mice (DC) exhibited significantly higher level during all time points determined (Fig. 4). After glucose loading, the increase in serum glucose concentrations in diabetic mice was very slow, while normal mice exhibited sharp increase in glucose level with peak concentration at 15 min, indicating delayed glucose homeostasis in db/db mice. ERW administration fails to affect glucose tolerance in both diabetic db/db mice and control mice. STZ-induced and genetically diabetic db/db mice are the most commonly used animal models in experimental diabetes research. Injected STZ has been reported to accumulate in the pancreatic islets (Tjalve et al., 1976). Excessive production of nitric oxide and the subsequent increase in local oxidative stress are some suggested mechanisms in the destruction of pancreatic β-cells and the development of insulin dependent diabetes mellitus (Tanaka et al., 1995; Montilla et al., 1998). The protective mechanism of ERW results from active atomic hydrogen with high reducing ability, which can contribute to ROS scavenging activity, and may participate in the redox regulation of cellular function (Shiharata et al., 1997). Therefore, ERW administration improved islet β-cell function resulting in increased release of circulating insulin (type 2 diabetic db/db mice) and improved insulin sensitivity (type 1 diabetic STZ-treated mice and type 2 diabetic mice), and thus, ameliorate hyperglycemia and delay the development of diabetes in these diabetic mice model. To our knowledge, this is the first report showing different anti-hyperglycemic effect in various diabetic animal models mediated by ERW in vivo. Conclusion The present study shows that administration of ERW, a potent free radical scavenger, has an anti-hyperglycemic effect on an insulin deficient (STZ-diabetic mice) and insulin resistant animal model (db/db mice). This improved blood glucose control could result from enhanced insulin sensitivity, as well as increased insulin release. The molecular mechanism of ERW anti-diabetic effect is worthwhile to explore further. Acknowledgements The support of this study by Hanseo University is gratefully acknowledged. References Cherng, J.Y., Shih, M.F., 2005. Potential hypoglycemic effects of chlorella in streptozotocin-induced diabetic mice. Life Sciences 77, 980–990. Feillet-Coudray, C., Rock, E., Coudray, C., Grzelkowska, K., Azais-Braesco, V., Dardevet, D., Mazur, A., 1999. Lipid peroxidation and antioxidant status in experimental diabetes. Clinica Chimica Acta 284, 31–34. Halliwell, B., Gutteridge, J.M.C., 1999. Free Radicals in Biology and Medicine. Oxford University Press, pp. 20–37. Huang, K., Yan, C., Lee, K., Chien, C., 2003. Reduced hemodyalisis-induced oxidative stress in end-stage renal disease patients by electrolyzed reduced water. Kidney International 64, 704–714.
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Mahesh, T., Menon, V.P., 2004. Quercetin allievates oxidative stress in streptozotocin-induced diabetic rats. Phytotherapy Research 18, 123–127. Maritim, A., Dene, B.A., Sanders, R.A., Watkins III, J.B., 2002. Effects of β-carotene on oxidative stress in normal and diabetic rats. Journal of Biochemical and Molecular Toxicology 16, 203–208. Montilla, P., Vagas, J.F., Tunez, I.F., Munez, M.C., Cabrera, E.S., 1998. Oxidative stress in diabetic rats induced by streptozotocin. Journal of Pineal Research 25, 94–100. Omamto, H., Uchigata, Y., 1981. STZ induced DNA strand breaks and poly (ADP ribose) synthesis in pancreatic islets. Nature 294, 284–286. Sabu, M.C., Smitha, K., Kuttan, R., 2002. Anti-diabetic activity of green tea polyphenols and their role in reducing oxidative stress in experimental diabetes. Journal of Ethnopharmacology 83, 109–116. Shiharata, S., Kabayama, S., Nakano, M., Miura, T., Kusumoto, K., Morisawa, S., Katakura, Y., 1997. Electrolyzed-reduced water scavenges active oxygen species and protects DNA from oxidative damage. Biochemical and Biophysical Research Communications 234, 269–274. Surwit, R.S., Seldin, M.F., Kuhn, C.M., Cochrane, C., Feinglos, M.N., 1991. Control of expression of insulin resistance and hyperglycemia by different genetic factors in diabetic C57BL/6J mice. Diabetes 40, 82–87.
Tanaka, Y., Schimazu, H., Sato, N., Mori, M., Scimomura, Y., 1995. Involvement of spontaneous nitric oxide production in the diabetogenic action of streptozotocin. Pharmacology 50, 69–73. Tjalve, H., Wilander, E., Johansson, E., 1976. Distribution of labeled streptozotocin in mice. The Journal of Endocrinology 69, 455–456. Tsuneki, H., Ishizuka, M., Terasawa, M., Wu, J., Sasaoka, T., Kimura, I., 2004. Effects green tea on blood glucose levels and serum proteomic patterns in diabetic (db/db) mice and glucose metabolism in healthy humans. BMC Pharmacology 4, 18–27. Vessal, M., Hemmati, M., Vasei, M., 2003. Antidiabetic effects of quercetin in streptozotocin-induced diabetic rats. Comparative Biochemistry and Physiology. C 135, 357–364. West, I.C., 2000. Radicals and oxidative stress in diabetes. Diabetic Medicine 17, 171–180. Yu, B.P., 1994. Cellular defenses against damage from reactive oxygen species. Physiological Reviews 74, 139–162. Young, I.S., Torney, J.J., Trimble, E.R., 1992. The effect of ascorbate supplementation on oxidative stress in the streptozotocin diabetic rat. Free Radical Biology & Medicine 12, 41–46.
Anti-diabetic effects of electrolyzed reduced water in streptozotocin-induced and genetic diabetic mice Mi-Ja Kim a , Hye Kyung Kim b,⁎ a
Department of Obesity management, Graduate School of Obesity Science, Dongduk Women's University, 23-1 Wolkgukdong, Seoul, 136-714, South Korea b Department of Food and Biotechnology, Hanseo University, Sesan, 356-706, South Korea Received 28 February 2006; accepted 27 July 2006
Abstract Oxidative stress is produced under diabetic conditions and is likely involved in progression of pancreatic β-cell dysfunction found in diabetes. Both an increase in reactive oxygen free radical species (ROS) and a decrease in the antioxidant defense mechanism lead to the increase in oxidative stress in diabetes. Electrolyzed reduced water (ERW) with ROS scavenging ability may have a potential effect on diabetic animals, a model for high oxidative stress. Therefore, the present study examined the possible anti-diabetic effect of ERW in two different diabetic animal models. The genetically diabetic mouse strain C57BL/6J-db/db (db/db) and streptozotocin (STZ)-induced diabetic mouse were used as insulin deficient type 1 and insulin resistant type 2 animal model, respectively. ERW, provided as a drinking water, significantly reduced the blood glucose concentration and improved glucose tolerance in both animal models. However, ERW fail to affect blood insulin levels in STZ-diabetic mice whereas blood insulin level was markedly increased in genetically diabetic db/db mice. This improved blood glucose control could result from enhanced insulin sensitivity, as well as increased insulin release. The present data suggest that ERW may function as an orally effective antidiabetic agent and merit further studies on its precise mechanism. © 2006 Elsevier Inc. All rights reserved. Keywords: Electrolyzed reduced water; Diabetic mice; Blood glucose; Insulin; Glucose tolerance
Introduction Oxidative stress is a condition of imbalance due to excess formation of free radicals and decreased activity of antioxidant defense systems. All oxidative reactions are a continuous source of potentially cytotoxic reactive oxygen species (ROS), which play an important role in living systems both through their beneficial and detrimental effects (Halliwell and Gutteridge, 1999). Under physiological conditions, ROS are fully inactivated by an elaborated cellular and extracellular antioxidant defense system (Yu, 1994). However, under certain conditions increased generation of ROS and/or reduction of the antioxidant capacity leads to enhanced ROS activity and oxidative stress. Diabetes mellitus is characterized by increased production of ROS, sharp reduction in antioxidant defense and altered cellular ⁎ Corresponding author. Tel. +82 41 660 1454; fax: +82 41 660 1119. E-mail addresses: [email protected] (M.-J. Kim), [email protected] (H.K. Kim). 0024-3205/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2006.07.027
redox status (West, 2000). The chronic presence of high glucose levels enhances the production of ROS from protein glycation and glucose autoxidation (Feillet-Coudray et al., 1999). Therefore, supplementation with some antioxidants such as quercetin, ascorbate and β-carotene resulted in an improvement of the antioxidant status in streptozotocin (STZ)-induced diabetic rats (Mahesh and Menon, 2004; Maritim et al., 2002; Young et al., 1992). Diabetes mellitus is classified into two types, insulindependent (IDDM, type 1) and non-insulin-dependent diabetes mellitus (NIDDM, type 2). The two types of diabetes have distinct pathogenesis but hyperglycemia and various lifethreatening complications resulting from long-term hyperglycemia are the most common features. Hence, effective control of the blood glucose levels is a key step in preventing or reversing diabetic complications in both types 1 and 2 diabetic patients. However, due to limited efficacy and adverse side effects of currently available therapies, it is difficult to maintain good glycemic control in most diabetic patients. Therefore, there is a strong incentive to develop new hypoglycemic agents.
M.-J. Kim, H.K. Kim / Life Sciences 79 (2006) 2288–2292 Table 1 Fasting blood glucose, insulin and body weight changes in streptozotocin diabetic mice NC Blood glucose (mg/dl) Blood insulin (ng/ml) Initial body weight (g) Final body weight (g)
112 ± 10 0.61 ± 0.04 32.6 ± 0.2 40.4 ± 0.5
NE
DC
DE
109 ± 11 0.62 ± 0.05 32.3 ± 0.3 41.0 ± 0.7
295 ± 23⁎ 0.41 ± 0.03⁎ 31.8 ± 0.3 34.6 ± 0.5⁎
180 ± 19⁎⁎ 0.40 ± 0.02⁎ 32.4 ± 0.4 34.8 ± 0.6⁎
Data are expressed as means ± S.E.M. ⁎p b 0.01, compared with normal control (NC), ⁎⁎p b 0.01, compared with diabetic control (DC). NC; normal ICR mice control fed tap water, NE; normal mice fed ERW, DC; STZ-diabetic mice fed water, DE; STZ-diabetic mice fed ERW.
Electrolysis of aqueous NaCl or KCl solutions by diaphragmtype electrolyzing devices produces oxidized and reduced water at the anodic and the cathodic side, respectively. Electrolyzed reduced water (ERW) exhibits high pH (pH 10–12), low dissolved oxygen (1.3–3.5 mg/l), high dissolved hydrogen (0.3 mg/l–0.6 mg/l), and significant negative redox potential (−400 to −900 mV). The ideal scavenger for ROS is “reactive hydrogen”. Since ERW has high reactive hydrogen, the usefulness of electrolyzed water in the medicinal fields is being examined. The free radical-scavenging antioxidants play an important role in the in vivo defense system against oxidative damage initiated and promoted by ROS (Mahesh and Menon, 2004; Vessal et al., 2003; Maritim et al., 2002). ERW scavenges ROS such as superoxide anion radical (O2−) and hydrogen peroxide. (Shiharata et al., 1997). Huang et al. (2003) also reported that ERW with a high reducing ability and/or direct ROS scavenging activity could be used in hemodialysis patients by strong antioxidant activity. Since oxidative damage has been implicated in the etiology of diabetic complication, ERW, a potent ROS scavenger, may have a therapeutic role in diabetic mellitus. Therefore, the present study examined the possible anti-diabetic effect of ERW in diabetic animal models. The diabetic mouse strain C57BL/ 6J-db/db (db/db) which exhibits many of the metabolic disturbances of human type 2 diabetes including hyperglycemia, obesity, and insulin resistance (Surwit et al., 1991), and STZ-induced diabetic mouse which is widely used as an animal model of type 1 diabetes were used.
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littermates (db/−) at 3 weeks of age were housed under controlled temperature (20–26 °C) and light (12 h light/dark cycle) controlled conditions. All animals had free access to standard rodent pellet food (NIH #31M, Samtako, Korea), except when fasted before experiments. Mice were divided into 4 groups (n = 10): normal control (NC), normal-ERW (NE), diabetic control (DC), and diabetic-ERW (DE). Normal and diabetic control mice were fed tap water, and normal and diabetic-ERW mice were fed ERW. Insulin deficient type 1 diabetes was induced by multiple low-dose injection of STZ (60 mg/kg/day, i.p.) dissolved in citrate buffer (0.01 M, pH 4.5) for 5 consecutive days. This dose of STZ results in significant impaired ability to produce insulin and moderate to severe hyperglycemia. Control mice received the same volume of vehicle. Tail bleeds were performed 24 h post-injection and animals with a fasting blood glucose concentration above 200 mg/dl were considered to be diabetic, and assigned as DC or DE group. The initial blood glucose and insulin concentrations of DC and DE groups before ERW administration were very similar (300 ± 5 and 298 ± 7 mg/dl, and 0.40 ± 0.02 and 0.40 ± 0.03 ng/ml, respectively). Blood glucose level and intraperitoneal glucose tolerance test (IPGT) Blood samples were collected after fasted for 4 h from the retro-orbital sinus every 7 days. Approximately 50 μl of fresh blood sample was placed on a test strip and the blood glucose content was determined in a validated One Touch Basic glucose measurement system (Lifescan Inc, USA). At the end of experimental period, mice were fasted overnight and injected with glucose (1 g/kg, i.p.). Blood samples were collected from the tail vein just prior to and 15, 30, 60, 90, and 120 min after glucose loading, and blood glucose levels were measured. Mice were killed by decapitation immediately after 120 min blood sample was taken and blood samples were taken from the cervical wound. Serum glucose and insulin concentrations were determined with commercially available kits (Sigma Co., USA).
Materials and methods Electrolyzed water The ERW was produced by AK-3000 (Nexus, Korea). This water was produced by the electrolysis of water from a municipal water system. The ERW used in this study has the following physical properties: pH 10.24 ± 0.03 and an oxidative-reduction potential of − 400.2 ± 17.3 mV. Animals and diets 8 weeks old male ICR mice (Orient, Seoul, Korea), and genetically diabetic db/db (C57BL/6J db/db, Jackson Laboratory, Bar Harbor, ME, USA) and their non diabetic heterozygous lean
Fig. 1. Effect of electrolyzed reduced water (ERW) on glucose tolerance in streptozotocin (STZ)-induced diabetic mice. Normal ICR mice fed with tap water (NC); normal mice fed with ERW (NE); STZ-diabetic mice fed with water (DC); STZ-diabetic mice fed with ERW (DE). Data are expressed as means ± S.E.M. ⁎p b 0.01, compared with diabetic control (DC).
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tea produces an anti-hyperglycemic effect without affecting insulin secretion in STZ-diabetic mice. Cherng and Shih (2005) reported that potential hypoglycemic effects of chlorella increased exogenous insulin sensitivity without affecting endogenous insulin level in STZ-induced diabetic mice. Induction of diabetes by STZ caused significant weight loss compared with normal ICR mice (NC) because of poor utilization of nutrients. ERW administration did not significantly affect body weight in both normal (NE) and STZ-diabetic mice (DE). ROS scavengers such as green tea and quercetin were reported to reduce blood glucose concentrations in STZ-diabetic rats and mice (Mahesh and Menon, 2004; Sabu et al., 2002; Vessal et al., 2003). Sabu et al. (2002) observed the antihyperglycemic effect of green tea extract independent of changes in body weight in STZ-diabetic rats. Fig. 2. Effect of electrolyzed reduced water (ERW) on blood glucose concentrations in genetically diabetic db/db mice. Normal db/− mice fed with tap water (NC); normal db/− mice fed with ERW (NE); db/db mice fed with tap water (DC); db/db mice fed with ERW (DE). Data are expressed as means ± S.E.M. ⁎p b 0.01, compared with diabetic control (DC).
Effects of ERW on glucose tolerance in type 1 diabetic mice
Effects of ERW on fasting blood glucose and insulin level, and body weight in type 1 diabetic mice
Glucose tolerance was evaluated by IPTG after ERW consumption as a drinking water. As shown in Fig. 1, administration of 1 g glucose/kg body weight to normal mice (NC) increased blood glucose levels from 99.4 ± 8.5 to 134.2 ± 14.2 at 15 min but returned to baseline values within 60–120 min. ERW exerted no effect on the glucose tolerance curve of the normal ICR mice (NE). In contrast, STZ-induced diabetic mice (DC) demonstrated basal hyperglycemia (280 mg/dl) which remained until 120 min after glucose loading, indicating delayed glucose homeostasis in diabetic mice. ERW consumption (DE) showed definite improvement in glucose tolerance. Peak blood glucose concentration was observed after 15 min of glucose loading, and returned to below fasting level after 120 min. For the ERW treated diabetic mice (DE), the area under the curve of the blood glucose decreased compared to tap water treated diabetic mice. The present study indicates that STZ-induced diabetes and subsequent elevation of blood glucose were reversed by simultaneous administration of ERW. The elevation of glucose in STZ treated mice was due to a single strand break in pancreatic islets DNA (Omamto and Uchigata, 1981), and Shiharata et al.
STZ is widely used to induce diabetes in experimental animals, causing selective destruction of pancreatic β-islet cells, probably by a free radical-mediated mechanism which is responsible for high blood glucose level seen in STZ-induced diabetic animals (Halliwell and Gutteridge, 1999). Accordingly, STZ-induced diabetic mice (DC) developed hyperglycemia and insulin deficiency as evidenced by elevated blood glucose and reduced insulin levels (Table 1). Diabetic control mice (DC) demonstrated 3 fold increase in blood glucose concentrations over control (NC) values throughout the whole experimental period. However, following 6 weeks of ERW treatment, elevated blood glucose levels in diabetic mice were significantly reduced (p b 0.01) without any significant effect on blood insulin concentrations. There is no, or very little, insulin secretion in STZ-induced diabetic animal model. Therefore, ERW may act to increase insulin sensitivity rather than to increase insulin release. Tsuneki et al. (2004) reported that green
Fig. 3. Effect of electrolyzed reduced water (ERW) on blood insulin concentrations in genetically diabetic db/db mice. Normal db/− mice fed with tap water (NC); normal db/− mice fed with ERW (NE); db/db mice fed with tap water (DC); db/db mice fed with ERW (DE). Data are expressed as means±S.E.M. ⁎pb 0.01, compared with normal control (NC), ⁎⁎pb 0.01, compared with diabetic control (DC).
Statistical analysis All data were presented as mean ± S.E.M. Data from experiments were analyzed by ANOVA, using the statistical software SPSS (Window version 10.5). Specific comparisons were made with Tukey HSD's post-hoc test. Results and discussion The present study was designed to explore the potential role of ERW, a potent ROS scavenger, in improving hyperglycemia in two animal models of diabetes, namely the insulin deficient STZ-induced diabetic mice (type 1) and insulin resistant genetically diabetic db/db (type 2) mice.
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Effects of ERW on glucose tolerance in type 2 diabetic mice
Fig. 4. Effect of electrolyzed reduced water (ERW) on glucose tolerance in genetically diabetic db/db mice. Normal db/− mice fed with tap water (NC); normal db/− mice fed with ERW (NE); db/db mice fed with tap water (DC); db/ db mice fed with ERW (DE). Data are expressed as means ± S.E.M.
(1997) suggested that ERW scavenges ROS and protects DNA from oxidative damage. Therefore, we speculate that the observed effects of ERW on STZ-diabetic mice are primarily due to the promotion of insulin sensitivity in peripheral tissues, such as skeletal muscles and adipocytes rather than increasing insulin release in pancreas as evidenced by unchanged insulin levels in ERW administered mice. Effects of ERW on body weight, fasting blood glucose, and insulin level in type 2 diabetic mice Since genetically diabetic db/db mice exhibit obesity, the body weights of db/db mice (DC) were significantly higher than control mice (NC). Administration of ERW did not affect body weight in both diabetic db/db (DE) and control mice (NE) (data not shown). Blood glucose levels after 4 h fasting were measured on days 1, 7, 14, and 29 after daily administration of ERW or tap water. The diabetic db/db mice exhibit its diabetic characteristics after 4 weeks of age. Therefore, blood glucose concentrations on 1st day of experiment (3 weeks of age) were similar in all groups. However, blood glucose levels of diabetic mice (DC) were markedly increased at day 7 compare to control mice (NC), and ERW consumption (DE) significantly lowered the blood glucose levels (Fig. 2). On day 29, blood glucose levels in ERW supplied db/db mice (DE) were 41% lower than those of water supplied db/db group (DC). The values of blood glucose levels in NC, NE, DC and DE group were 128 ± 29, 125.4 ± 3.9, 490.1 ± 32.4 and 287.9 ± 34.5, respectively. Blood insulin levels of diabetic mice (DC) were more than 2-fold higher than control mice (NC) indicating insulin resistance in type 2 diabetes (Fig. 3). Interestingly, ERW administration also increased insulin level in diabetic mice (DE) without any effect in control mice (NE), suggesting elevated insulin release in diabetic mice. The hypoglycemic effects of ERW in type 2 diabetic mice model may be associated with the improvement of pancreatic β-cell function.
Glucose tolerance in diabetic db/db mice (DC) exhibited significantly higher level during all time points determined (Fig. 4). After glucose loading, the increase in serum glucose concentrations in diabetic mice was very slow, while normal mice exhibited sharp increase in glucose level with peak concentration at 15 min, indicating delayed glucose homeostasis in db/db mice. ERW administration fails to affect glucose tolerance in both diabetic db/db mice and control mice. STZ-induced and genetically diabetic db/db mice are the most commonly used animal models in experimental diabetes research. Injected STZ has been reported to accumulate in the pancreatic islets (Tjalve et al., 1976). Excessive production of nitric oxide and the subsequent increase in local oxidative stress are some suggested mechanisms in the destruction of pancreatic β-cells and the development of insulin dependent diabetes mellitus (Tanaka et al., 1995; Montilla et al., 1998). The protective mechanism of ERW results from active atomic hydrogen with high reducing ability, which can contribute to ROS scavenging activity, and may participate in the redox regulation of cellular function (Shiharata et al., 1997). Therefore, ERW administration improved islet β-cell function resulting in increased release of circulating insulin (type 2 diabetic db/db mice) and improved insulin sensitivity (type 1 diabetic STZ-treated mice and type 2 diabetic mice), and thus, ameliorate hyperglycemia and delay the development of diabetes in these diabetic mice model. To our knowledge, this is the first report showing different anti-hyperglycemic effect in various diabetic animal models mediated by ERW in vivo. Conclusion The present study shows that administration of ERW, a potent free radical scavenger, has an anti-hyperglycemic effect on an insulin deficient (STZ-diabetic mice) and insulin resistant animal model (db/db mice). This improved blood glucose control could result from enhanced insulin sensitivity, as well as increased insulin release. The molecular mechanism of ERW anti-diabetic effect is worthwhile to explore further. Acknowledgements The support of this study by Hanseo University is gratefully acknowledged. References Cherng, J.Y., Shih, M.F., 2005. Potential hypoglycemic effects of chlorella in streptozotocin-induced diabetic mice. Life Sciences 77, 980–990. Feillet-Coudray, C., Rock, E., Coudray, C., Grzelkowska, K., Azais-Braesco, V., Dardevet, D., Mazur, A., 1999. Lipid peroxidation and antioxidant status in experimental diabetes. Clinica Chimica Acta 284, 31–34. Halliwell, B., Gutteridge, J.M.C., 1999. Free Radicals in Biology and Medicine. Oxford University Press, pp. 20–37. Huang, K., Yan, C., Lee, K., Chien, C., 2003. Reduced hemodyalisis-induced oxidative stress in end-stage renal disease patients by electrolyzed reduced water. Kidney International 64, 704–714.
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