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Effect of vitamin E on alloxan-induced mouse diabetes
Clinical Biochemistry 46 (2013) 795–798
Contents lists available at SciVerse ScienceDirect
Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
Effect of vitamin E on alloxan-induced mouse diabetes Wakana Kamimura a, Wakana Doi a, Kazunori Takemoto b, Kohji Ishihara a, Da-Hong Wang c, Hitoshi Sugiyama d, Sen-ichi Oda e, Noriyoshi Masuoka a,⁎ a
Department of Life Science, Okayama University of Science, 1–1 Ridai-cho, Kita-ku, Okayama 700-0005, Japan Kake Medical Science Education Center, Okayama 700-0005, Japan Department of Public Health, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Science, Okayama 700-8558, Japan d Center of CKD and Peritoneal Dialysis, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Science, Okayama 700-8558, Japan e Department of Zoology, Okayama University of Science, Okayama 700-0005, Japan b c
a r t i c l e
i n f o
Article history: Received 26 January 2013 Received in revised form 23 February 2013 Accepted 25 February 2013 Available online 6 March 2013 Keywords: Vitamin E Alloxan Oxidative stress Catalase Insulin Diabetes Acatalasemia
a b s t r a c t Objectives: Alloxan generates hydrogen peroxide in the body, and a small amount of alloxan administered to acatalasemic mice results in diabetes. D-α-Tocopherol (vitamin E) is an antioxidant which helps prevent excess oxidation in the body. In this study, we examined the effect of vitamin E on diabetes caused by alloxan administration in mice. Methods: Mice were maintained on a vitamin E-deprived diet and supplemented diet, respectively, for 14 weeks. Alloxan was then intraperitoneally administered, and blood glucose, glucose tolerance and the insulin level in mouse blood were examined. Results: Hyperglycemia was observed in the mice maintained on the vitamin E-deprived diet. The incidence of hyperglycemia in the mice maintained on the vitamin E-deprived diet was significantly higher than that in the mice maintained on the supplemented diet. The abnormal glucose metabolism caused by alloxan administration was ameliorated by the vitamin E-supplemented diet. Conclusions: It is deduced that vitamin E can prevent a decrease of insulin concentration in the blood in this mouse model. © 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction The reactive oxygen species generated with alloxan and reducing agents in the body selectively injure β-cells in the pancreas so as to cause diabetes [1,2]. From our previous findings [3], alloxan generated hydrogen peroxide with reduced glutathione. Acatalasemic mice, having a quite low catalase activity in blood, became diabetic with a smaller dose than those having normal catalase activity when mice were maintained on a laboratory diet. The injury caused by oxidative stress is ameliorated by the intake of antioxidants [4,5] and we chose vitamin E, D-α-tocopherol, to serve as the antioxidant in this study. The effect of D-α-tocopherol on diabetes caused by alloxan was examined.
were maintained on a laboratory diet (CE-2 diet, Clea Japan, Tokyo) and water ad libitum until the start of the experiments. D-αTocopherol was obtained from the Eisai Food & Chemical Co. Ltd. (Tokyo, Japan). The vitamin E-supplemented and deprived diets were prepared based on the composition recommended by the American Institute of Nutrition [7]. For preparation of the diets, vitamin-free casein was used. For the vitamin E-deprived diet, vitamin E was omitted from the vitamin mixture (AIN-93), and for the vitamin E-supplemented diet, D-α-tocopherol was added at 50 mg per 100 g of diet. These diets were prepared by Funabashi Farm Co. Ltd. (Chiba, Japan). Dietary samples were stored at −20 °C until use. Animal experiments
Materials and methods Materials Male mice of the C3H/AnL CS aCS a (normal) and C3H/AnL CS bCS b (acatalasemia) strains established by Feinstein, Braun, & Howard [6] ⁎ Corresponding author at: Department of Life Science, Faculty of Science, Okayama University of Science, Ridai-cho, Kita-ku, Okayama 700-0005, Japan. Fax: +81 86 256 9593. E-mail address: [email protected] (N. Masuoka).
The acatalasemic mice and normal mice (15 weeks old) were divided into two groups. One group was maintained on the vitamin E-supplemented diet and the other group the E-deprived diet. The respective dietary food and water were available ad lib for 14 weeks. Then, alloxan (200 mg/kg of body weight) was intraperitoneally administrated to each mouse using 0.106 M alloxan in saline, and mice were maintained on the same diet for one more week [3]. After fasting for 20 hs, a glucose tolerance test (GTT) was carried out, and then mice were maintained on the same diet for two additional days. After fasting, blood was collected from the heart, and heparin was used as the
0009-9120/$ – see front matter © 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clinbiochem.2013.02.016
796
W. Kamimura et al. / Clinical Biochemistry 46 (2013) 795–798
anticoagulant. Oxidative stress markers, as well as the insulin and C-peptide levels in plasma, were examined. Determination of D-α-tocopherol in the blood by HPLC The dietary food and water was available ad lib for 14 weeks. Blood was collected from the heart and the D-α-tocopherol level was measured. To 0.2 mL of plasma were added 0.8 mL water, 2.0 mL of ethanol and 5.0 mL of n-hexane. The mixture was shaken well for 5 min and the n-hexane layer was separated. The organic layer was evaporated and the residue dissolved with 0.2 mL of ethanol [8]. The fraction (20 μL) was analyzed with a reverse phase C18 column (TSK-Gel ODS-100 V, 3 μm, 4.6 × 150 mm) at 292 nm. The column was eluted at 1.0 mL/min with a linear gradient of 90% CH3CN in water to 100% CH3CN for 10 min, and then eluted with 100% CH3CN for 6 min further. Determination of glucose in blood Mice were fasted. The glucose content in the blood obtained from the tail was determined. As the blood volume for the determination of blood glucose was quite small (approximately 2 μL), the glucose contents in blood were measured with a “Glucose-Test-Ace R” apparatus (Sanwa Kagaku Kenkyusho Co., Nagoya, Japan) applying a glucose oxidase method. Glucose tolerance test After fasting, a forty percent aqueous glucose solution (5 mL/kg of body weight) was intraperitoneally administered to each mouse [3,9]. At 0 and 30 min before and 15, 30, 60, 90 and 120 min after the administration, the glucose content in the blood was measured. Determination of the insulin and C-peptide levels in blood The insulin and C-peptide plasma levels were determined using Mouse Insulin and C-peptide ELISA KITs (U-type) (Shibayagi, Gunma, Japan). Each determination was carried out according to the manufacturer's instructions. Biotin-conjugated anti-insulin antibody (45 μL) was added to each well in an antibody-coated 96-well plate. To the well, 5 μL of the sample or standard solution was added and reacted for 2 hs. Then 50 μL of peroxidase-conjugated avidin solution was added and reacted for 30 min. Chromogenic substrate solution (50 μL) was added and reacted for 30 min. The reaction was stopped and the absorbance at 450 nm (sub-wave length, 620 nm) was recorded. Microscopic studies of pancreatic tissues in the acatalasemic mice treated with alloxan
reaction was added and reacted for 15 min. The absorbance at 450 nm was recorded. Lipid peroxidation in plasma was determined using a Bioxytech LPO-586 KIT (OXIS Health Products Inc, CA, USA). Malondialdehyde and 4-hydroxyalkenals as products of lipid peroxidation were reacted with N-methyl-2-phenylindole at 45 °C. The absorbance at 586 nm was recorded. Statistical analysis Student's t-test was used to evaluate the statistical significance of difference. The difference was considered significant when p b 0.05. Results Catalase activity in the mouse erythrocytes was calculated as the difference between the hydrogen peroxide removal rate by hemolysate and the rate (0.73 μmol/s/g of hemoglobin) by hemoglobin [3,10]. The catalase activity in blood at 25 °C was 0.15 ± 0.07 μmol/s/g of Hb in the acatalasemic mice and 6.77 ± 0.62 in the normal mice. Blood concentrations of D-α-tocopherol D-α-Tocopherol was eluted for 13.15 ± 0.04 min from the HPLC column with a detection limit of 0.07 nmol/mL. After mice were maintained on the CE-2 diet, they were given a vitamin E-deprived or supplemented diet for 14 weeks. The D-α-tocopherol in mouse blood was determined by HPLC (Table 1). The concentration in the mice maintained on the vitamin E-deprived diet fell to one third of that in the mice maintained on the CE-2 diet, while the concentration in the mice maintained on the supplemented diet was twice that of the mice maintained on the CE-2 diet. These concentrations similar to those reported for mice maintained on vitamin E-supplemented or deprived diets [8]. It suggested that mice fed a vitamin E-deprived diet had a deficiency in vitamin E, while those fed the supplemented diet were saturated with vitamin E. Blood glucose in the mice maintained on the vitamin E-supplemented and deprived diets before and after alloxan treatment Before alloxan administration, the average fasting blood glucose in the normal and acatalasemic mice fed a vitamin E-supplemented or deprived diet was scarcely different. After the administration, the average fasting blood glucose of acatalasemic mice was higher than that in normal mice as previously reported [3]. Furthermore, the average fasting blood glucose of the mice fed the vitamin E-deprived diet was significantly higher than the average in the mice fed the supplemented diet (Table 2). It was also indicated the incidence of hyperglycemia in each group. The effect of vitamin E-supplemented and deprived diets on GTT
Pancreatic tissues were isolated, fixed in Bouin's fluid and embedded in paraffin. Serial sections (6 μm) were cut from each paraffinembedded tissue block, and several sections were stained with hematoxylin-eosin and mouse anti-insulin antibody (Santa Cruz Biotechnology) using the Vectastain Elite ABC Rabbit IgG Kit for visualization by light microscopy. The islets and other cells were recorded with a FX380 CCD Camera and a microscope (Olympus, Tokyo, Japan). Measurement of the oxidative stress markers The measurement of 8-oxo-2′-deoxyguanosine (8-OHdG) in plasma was carried out using an ELISA KIT (JaICA, Shizuoka, Japan). Sample or standard solution was added to each well in a 96-well plate coated with 8-OHdG, and a monoclonal antibody for 8-OHdG was added. The mixture was reacted for12 hs. Then, an enzyme-conjugated antibody was added and reacted for 1 h. The reagent solution for the color
After one week from alloxan treatment, the GTT in acatalasemic mice was examined. Differences in the blood glucose of the alloxan-
Table 1 Concentrations (nmol/mL) of D-α-tocopherol in the blood of mice maintained on the CE-2 diet, vitamin E-supplemented diet and E-deprived diet for 14 weeks. Mouse
D-α-Tocopherol in blood (nmol/mL) CE-2
VE (+)
VE (−)
Normal 3.92 ± 1.07 (n = 6) 7.30 ± 4.37 (n = 14) 1.21 ± 0.60 (n = 13) Acatalasemia 3.78 ± 1.59 (n = 6) 8.68 ± 3.34 (n = 14) 1.00 ± 0.32 (n = 7) CE-2 indicates a laboratory diet (used as a control diet). VE (+) indicates the vitamin E-supplemented diet and VE (−) the vitamin E-deprived diet.
W. Kamimura et al. / Clinical Biochemistry 46 (2013) 795–798 Table 2 Blood glucose (mg/dL) in the fasting blood from mice fed the vitamin E-supplemented [VE (+)] diet or the vitamin E-deprived [VE (−)] diet and the incidence of hyperglycemia.
797
Table 3 The concentration of insulin and C-peptide in the fasting blood of acatalasemic mice maintained on the vitamin E-supplemented or deprived diet after alloxan administration.
Mice (number of mice)
Diet
Alloxan (mg/kg)
Blood glucose (mg/dL)
Incidence of hyperglycemia (%)
Mouse
Diet
Insulin (ng/L) (number of mice)
C-peptide (ng/L) (number of mice)
Normal (15) Normal (28) Normal (15) Normal (23) Acatalasemia Acatalasemia Acatalasemia Acatalasemia
VE VE VE VE VE VE VE VE
0 0 200 200 0 0 200 200
84 80 83 108 87 86 96 127
0 0 0 13 0 0 4 28
Normal Normal Acatalasemia Acatalasemia
VE(+) VE(−) VE(+) VE(−)
671 ± 282 (8) 496 ± 318 (14) 663 ± 294 (12) 399 ± 220 (14)
1594 ± 370 (8) 1543 ± 498 (9) 1126 ± 322 (7) 1053 ± 554 (10)
(30) (34) (28) (29)
(+) (−) (+) (−) (+) (−) (+) (−)
± ± ± ± ± ± ± ±
23 16 25 23* 15 17 18 77*
*
* *
VE(+): Vitamin E-supplemented diet. VE(−): Vitamin E-deprived diet. ⁎p b 0.05.
The asterisk (*) indicates p b 0.05.
treated acatalasemic mice fed the vitamin E-supplemented or deprived diet were observed (Fig. 1). The blood glucose in the mice fed the vitamin E-deprived diet after 60, 90 and 120 min from glucose administration was significantly higher than mice fed the vitamin E-supplemented diet. Blood glucose in the acatalasemic mice fed the vitamin E-deprived diet after 120 min from glucose administration was also significantly higher than that before glucose administration. The insulin and C-peptide concentrations and the index of insulin resistance (Homeostasis Model Assessment; HOMA) [11,12] The insulin and C-peptide concentrations in fasting mouse blood after alloxan administration are shown in Table 3, and the index of insulin resistance is shown in Fig. 2. The C-peptide level in the normal mouse blood was significantly higher than that in the acatalasemic blood. However, the C-peptide concentration was not affected by the diet. The insulin concentration in the vitamin E-deprived diet fed mice was lower than the mice fed the supplemented diet. The concentration in the vitamin E-deprived diet fed acatalasemic mice was only significantly lower. The index of insulin resistance was not affected by either the diet or catalase activity. Microscopic examination of pancreatic tissues in the acatalasemic mice treated with alloxan
β-cells in the islets of Langerhans were calculated and it was found that there was no difference in the number of β-cells between the acatalasemic mice on the vitamin E-supplemented and deprived diet (data not shown). Biomarkers of oxidative stress in serum The concentrations of 8-OHdG in the plasma of the normal and acatalasemic mice are indicated in Table 4. A significant increase of 8-OHdG in the blood of the mice was observed in the case of both low catalase activity and alloxan administration. However, 8-OHdG blood concentration was not affected by either the depletion or supplementation of vitamin E. On the other hand, increases in lipid peroxidation in mouse plasma were not observed in low catalase activity but in the case of alloxan administration and the depletion of vitamin E. Discussion Vitamin E consists of α-tocopherol, β-tocopherol and γ-tocopherol. Among these, D-α-tocopherol is abundantly present in food. In this study, D-α-tocopherol was added into the preparation of a vitamin E-supplemented diet. Compared to the vitamin E intake recommended by the American Institute of Nutrition, this diet contained an excess of approximately seven times the amount. After feeding for 14 weeks, there was no evident health problem with the mice maintained on the vitamin E-deprived or supplemented diet. Table 1 indicates the
Pancreatic tissues after alloxan administration subjected to immunohistochemical staining are shown in Fig. 3a and b. The numbers of
Fig. 1. Glucose tolerance test in the acatalasemic mice after alloxan administration. Mice were fed the vitamin E-supplemented or deprived diet for 14 weeks, and then alloxan was administrated. Mice were further maintained on each diet for 1 week. After fasting, GTTs were carried out. Glucose (200 g/kg of body weight) was administrated to each mouse at 0 min. ●: Blood glucose of the mice (n = 12) fed the vitamin E-supplemented diet and ○: blood glucose of the mice (n = 13) fed the E-deprived diet. *p b 0.05.
Fig. 2. The index of insulin resistance: Homeostasis Model Assessment (HOMA). Normal and acatalasemic mice were fed the vitamin E supplemented [VE(+)] or the E-deprived [VE(−)] diet for 14 weeks, and then alloxan was administrated. Mice were maintained on each diet for one week. Concentrations of insulin and glucose in fasting blood of normal mice (n = 8) fed VE(+) diet, normal mice (n = 14) fed VE(−) diet, acatalasemic mice (n = 12) fed VE(+) diet and acatalasemic mice (n = 14) fed VE(−) diet were determined. The index of insulin resistance [concentration of insulin (nM) × concentration of glucose (mM)] was calculated.
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Fig. 3. Microscopic studies of the pancreas stained with mouse anti-insulin antibody. Acatalasemic mice fed vitamin E supplemented diet or the deprived diet for 14 weeks. After alloxan administration, pancreatic tissues were stained with mouse anti-insulin antibody for visualization. A. From mice fed vitamin E supplemented diet. B. From mice fed vitamin E deprived diet.
mice reached a stable state of D-α-tocopherol in the blood by feeding ad lib on the respective diets for 14 weeks. Alloxan was then intraperitoneally administrated to each mouse (Table 2). After the administration, the incidence (28%) of hyperglycemia in the acatalasemic mice fed the vitamin E-deprived diet was higher than that (13%) in the normal mice fed the vitamin E-deprived diet. However, there was scarcely hyperglycemia in the mice fed the vitamin E-supplemented diet observed. The GTT in the acatalasemic mice maintained on the vitamin E-deprived diet (Fig. 1) indicated that the mice had a state like diabetes, but in the mice fed the supplemented diet they did not. These results suggest that vitamin E prevents or ameliorates glucose metabolism abnormalities caused by alloxan administration in mice. As states like diabetes were due to contributions of pancreatic β-cell dysfunction and insulin resistance [13], we measured the insulin and C-peptide concentrations in the fasting blood after alloxan administration (Table 3). The C-peptide level in the normal mouse blood was significantly higher than that in the acatalasemic blood. It indicates that the level is affected by the catalase activity in the mice, and the result is consistent with the reduction (20%) of β-cell numbers in the Langerhans islets by alloxan administration as previously reported [13]. The C-peptide level in the mouse blood was hardly affected with vitamin E. Pancreatic β-cells of acatalasemic mice were stained with an anti-insulin antibody (Fig. 3). There was no difference β-cell number between the acatalasemic mice fed the deprived and supplemented diets. These results indicate the vitamin E in diet did not affect the destruction of the β-cells or the amount of insulin they released. HOMA (blood glucose X the insulin concentration) [11,12] indicated that none of the mice fed the supplemented or deprived diet exhibited insulin resistance (Fig. 2). Compared to the insulin concentration in the mice fed the vitamin E-supplemented diet, the concentration of acatalasemic
Table 4 Effects of alloxan administration and the depletion of vitamin E on 8-OHdG and lipid peroxidation in the blood of normal and acatalasemic mice. Mouse (number of mice)
Diet
Alloxan (mg/kg)
8-OHdG (ng/mL)
Normal (5) Normal (6) Normal (7) Acatalasemia (9) Acatalasemia (8) Acatalasemia (8)
CE-2 VE(+) VE(-) CE-2 VE(+) VE(-)
0 200 200 0 200 200
0.010 0.118 0.127 0.099 0.228 0.307
± ± ± ± ± ±
Lipid peroxidation (μM) 0.001 0.017 0.046 0.070 0.083 0.072
0.11 1.28 2.74 0.33 1.28 2.32
± ± ± ± ± ±
0.03 0.23 1.15 0.24 0.48 0.79
*
*
The asterisk (*) indicates p b 0.05. Lipid peroxidation was significantly increased by alloxan administration and vitamin E depletion, and 8-OHdG in the blood were significantly increased by alloxan administration and low catalase activity (p b 0.05).
mice fed the deprived diet was significantly lower (p = 0.022). From these results it is deduced the insulin concentration in the mice fed the vitamin E-deprived diet decreased and this resulted in hyperglycemia. This suggests that vitamin E might have potential to keep the insulin concentration in the blood. To examine the oxidative stress caused by the depletion of vitamin E, 8-OHdG and lipid peroxidation in the blood were examined (Table 4). 8-OHdG was not significantly affected by the depletion of vitamin E, but it was significantly affected by a low catalase activity and alloxan administration. On the other hand, lipid peroxidation was increased by alloxan administration and the depletion of vitamin E. As insulin molecules reach the target receptors in the body through the medium of the blood, low insulin concentration in blood may be associated with the increase of peroxidative stress. Further investigation about the decrease of insulin concentration in blood under high peroxidation conditions is currently underway. References [1] Szkudelski T. The mechanism of alloxan and streptozotocin action in B cells of the rat pancreas. Physiol Res 2001;50:537–46. [2] Lenzen S. The mechanisms of alloxan- and streptozotocin-induced diabetes. Diabetologia 2008;51:216–26. [3] Takemoto K, Tanaka M, Iwata H, Nishihara R, Ishihara K, Wang DH, et al. Low catalase activity in blood is associated with the diabetes caused by alloxan. Clin Chim Acta 2009;407:43–6. [4] Choi EJ, Bae SC, Yu R, Youn J, Sung MK. Dietary vitamin E and quercetin modulate inflammatory responses of collagen-induced arthritis in mice. J Med Food 2009;12: 770–5. [5] Yamaoka S, Kim HS, Ogihara T, Oue S, Takitani K, Yoshida Y, et al. Severe vitamin E deficiency exacerbates acute hyperoxic lung injury associated with increased oxidative stress and inflammation. Free Radic Res 2008;42:602–12. [6] Feinstein RN, Braun JT, Howard JB. Acatalasemic and hypocatalasemic mouse mutants II. Mutational variations in blood and solid tissue catalases. Arch Biochem Biophys 1967;120:165–9. [7] Reeves PG, Nielsen Jr FH, Fahey GC. AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition and hoc writing committee on the reformulation of the AIN-76A rodent diet. J Nutr 1993;123:1939–51. [8] Chi C, Hayashi D, Nemoto M, Nyui M, Urano S, Anzai K. Vitamin E-deficiency did not exacerbate partial skin reactions in mice locally irradiated with X-rays. J Radiat Res 2011;52:32–8. [9] Gao D, Li Q, Liu Z, Li Y, Liu Z, Fan Y, et al. Hypoglycemic effects and mechanisms of action of Cortex Lycii Radicis on alloxan-induced diabetic mice. Yakugaku Zasshi 2007;127:1715–21. [10] Masuoka N, Wakimoto M, Ubuka T, Nakano T. Spectrophotometric determination of hydrogen peroxide: catalase activity and rates of hydrogen peroxide removal by erythrocytes. Clin Chim Acta 1996;254:101–12. [11] Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC. Homeostasis model assessment: insulin resistance and β-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 1985;28: 412–9. [12] Zamami Y, Takatori S, Goda M, Koyama T, Iwatani Y, Jin X, et al. Royal Jelly ameliorates insulin resistance in fructose-drinking rats. Biol Pharm Bull 2008;31: 2103–7. [13] Abdul-Ghani MA, Tripathy D, DeFronzo RA. Contributions of beta-cell dysfunction and insulin resistance to the pathogenesis of impaired glucose tolerance and impaired fasting glucose. Diabetes Care 2006;29:1130–9.
Contents lists available at SciVerse ScienceDirect
Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
Effect of vitamin E on alloxan-induced mouse diabetes Wakana Kamimura a, Wakana Doi a, Kazunori Takemoto b, Kohji Ishihara a, Da-Hong Wang c, Hitoshi Sugiyama d, Sen-ichi Oda e, Noriyoshi Masuoka a,⁎ a
Department of Life Science, Okayama University of Science, 1–1 Ridai-cho, Kita-ku, Okayama 700-0005, Japan Kake Medical Science Education Center, Okayama 700-0005, Japan Department of Public Health, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Science, Okayama 700-8558, Japan d Center of CKD and Peritoneal Dialysis, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Science, Okayama 700-8558, Japan e Department of Zoology, Okayama University of Science, Okayama 700-0005, Japan b c
a r t i c l e
i n f o
Article history: Received 26 January 2013 Received in revised form 23 February 2013 Accepted 25 February 2013 Available online 6 March 2013 Keywords: Vitamin E Alloxan Oxidative stress Catalase Insulin Diabetes Acatalasemia
a b s t r a c t Objectives: Alloxan generates hydrogen peroxide in the body, and a small amount of alloxan administered to acatalasemic mice results in diabetes. D-α-Tocopherol (vitamin E) is an antioxidant which helps prevent excess oxidation in the body. In this study, we examined the effect of vitamin E on diabetes caused by alloxan administration in mice. Methods: Mice were maintained on a vitamin E-deprived diet and supplemented diet, respectively, for 14 weeks. Alloxan was then intraperitoneally administered, and blood glucose, glucose tolerance and the insulin level in mouse blood were examined. Results: Hyperglycemia was observed in the mice maintained on the vitamin E-deprived diet. The incidence of hyperglycemia in the mice maintained on the vitamin E-deprived diet was significantly higher than that in the mice maintained on the supplemented diet. The abnormal glucose metabolism caused by alloxan administration was ameliorated by the vitamin E-supplemented diet. Conclusions: It is deduced that vitamin E can prevent a decrease of insulin concentration in the blood in this mouse model. © 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction The reactive oxygen species generated with alloxan and reducing agents in the body selectively injure β-cells in the pancreas so as to cause diabetes [1,2]. From our previous findings [3], alloxan generated hydrogen peroxide with reduced glutathione. Acatalasemic mice, having a quite low catalase activity in blood, became diabetic with a smaller dose than those having normal catalase activity when mice were maintained on a laboratory diet. The injury caused by oxidative stress is ameliorated by the intake of antioxidants [4,5] and we chose vitamin E, D-α-tocopherol, to serve as the antioxidant in this study. The effect of D-α-tocopherol on diabetes caused by alloxan was examined.
were maintained on a laboratory diet (CE-2 diet, Clea Japan, Tokyo) and water ad libitum until the start of the experiments. D-αTocopherol was obtained from the Eisai Food & Chemical Co. Ltd. (Tokyo, Japan). The vitamin E-supplemented and deprived diets were prepared based on the composition recommended by the American Institute of Nutrition [7]. For preparation of the diets, vitamin-free casein was used. For the vitamin E-deprived diet, vitamin E was omitted from the vitamin mixture (AIN-93), and for the vitamin E-supplemented diet, D-α-tocopherol was added at 50 mg per 100 g of diet. These diets were prepared by Funabashi Farm Co. Ltd. (Chiba, Japan). Dietary samples were stored at −20 °C until use. Animal experiments
Materials and methods Materials Male mice of the C3H/AnL CS aCS a (normal) and C3H/AnL CS bCS b (acatalasemia) strains established by Feinstein, Braun, & Howard [6] ⁎ Corresponding author at: Department of Life Science, Faculty of Science, Okayama University of Science, Ridai-cho, Kita-ku, Okayama 700-0005, Japan. Fax: +81 86 256 9593. E-mail address: [email protected] (N. Masuoka).
The acatalasemic mice and normal mice (15 weeks old) were divided into two groups. One group was maintained on the vitamin E-supplemented diet and the other group the E-deprived diet. The respective dietary food and water were available ad lib for 14 weeks. Then, alloxan (200 mg/kg of body weight) was intraperitoneally administrated to each mouse using 0.106 M alloxan in saline, and mice were maintained on the same diet for one more week [3]. After fasting for 20 hs, a glucose tolerance test (GTT) was carried out, and then mice were maintained on the same diet for two additional days. After fasting, blood was collected from the heart, and heparin was used as the
0009-9120/$ – see front matter © 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clinbiochem.2013.02.016
796
W. Kamimura et al. / Clinical Biochemistry 46 (2013) 795–798
anticoagulant. Oxidative stress markers, as well as the insulin and C-peptide levels in plasma, were examined. Determination of D-α-tocopherol in the blood by HPLC The dietary food and water was available ad lib for 14 weeks. Blood was collected from the heart and the D-α-tocopherol level was measured. To 0.2 mL of plasma were added 0.8 mL water, 2.0 mL of ethanol and 5.0 mL of n-hexane. The mixture was shaken well for 5 min and the n-hexane layer was separated. The organic layer was evaporated and the residue dissolved with 0.2 mL of ethanol [8]. The fraction (20 μL) was analyzed with a reverse phase C18 column (TSK-Gel ODS-100 V, 3 μm, 4.6 × 150 mm) at 292 nm. The column was eluted at 1.0 mL/min with a linear gradient of 90% CH3CN in water to 100% CH3CN for 10 min, and then eluted with 100% CH3CN for 6 min further. Determination of glucose in blood Mice were fasted. The glucose content in the blood obtained from the tail was determined. As the blood volume for the determination of blood glucose was quite small (approximately 2 μL), the glucose contents in blood were measured with a “Glucose-Test-Ace R” apparatus (Sanwa Kagaku Kenkyusho Co., Nagoya, Japan) applying a glucose oxidase method. Glucose tolerance test After fasting, a forty percent aqueous glucose solution (5 mL/kg of body weight) was intraperitoneally administered to each mouse [3,9]. At 0 and 30 min before and 15, 30, 60, 90 and 120 min after the administration, the glucose content in the blood was measured. Determination of the insulin and C-peptide levels in blood The insulin and C-peptide plasma levels were determined using Mouse Insulin and C-peptide ELISA KITs (U-type) (Shibayagi, Gunma, Japan). Each determination was carried out according to the manufacturer's instructions. Biotin-conjugated anti-insulin antibody (45 μL) was added to each well in an antibody-coated 96-well plate. To the well, 5 μL of the sample or standard solution was added and reacted for 2 hs. Then 50 μL of peroxidase-conjugated avidin solution was added and reacted for 30 min. Chromogenic substrate solution (50 μL) was added and reacted for 30 min. The reaction was stopped and the absorbance at 450 nm (sub-wave length, 620 nm) was recorded. Microscopic studies of pancreatic tissues in the acatalasemic mice treated with alloxan
reaction was added and reacted for 15 min. The absorbance at 450 nm was recorded. Lipid peroxidation in plasma was determined using a Bioxytech LPO-586 KIT (OXIS Health Products Inc, CA, USA). Malondialdehyde and 4-hydroxyalkenals as products of lipid peroxidation were reacted with N-methyl-2-phenylindole at 45 °C. The absorbance at 586 nm was recorded. Statistical analysis Student's t-test was used to evaluate the statistical significance of difference. The difference was considered significant when p b 0.05. Results Catalase activity in the mouse erythrocytes was calculated as the difference between the hydrogen peroxide removal rate by hemolysate and the rate (0.73 μmol/s/g of hemoglobin) by hemoglobin [3,10]. The catalase activity in blood at 25 °C was 0.15 ± 0.07 μmol/s/g of Hb in the acatalasemic mice and 6.77 ± 0.62 in the normal mice. Blood concentrations of D-α-tocopherol D-α-Tocopherol was eluted for 13.15 ± 0.04 min from the HPLC column with a detection limit of 0.07 nmol/mL. After mice were maintained on the CE-2 diet, they were given a vitamin E-deprived or supplemented diet for 14 weeks. The D-α-tocopherol in mouse blood was determined by HPLC (Table 1). The concentration in the mice maintained on the vitamin E-deprived diet fell to one third of that in the mice maintained on the CE-2 diet, while the concentration in the mice maintained on the supplemented diet was twice that of the mice maintained on the CE-2 diet. These concentrations similar to those reported for mice maintained on vitamin E-supplemented or deprived diets [8]. It suggested that mice fed a vitamin E-deprived diet had a deficiency in vitamin E, while those fed the supplemented diet were saturated with vitamin E. Blood glucose in the mice maintained on the vitamin E-supplemented and deprived diets before and after alloxan treatment Before alloxan administration, the average fasting blood glucose in the normal and acatalasemic mice fed a vitamin E-supplemented or deprived diet was scarcely different. After the administration, the average fasting blood glucose of acatalasemic mice was higher than that in normal mice as previously reported [3]. Furthermore, the average fasting blood glucose of the mice fed the vitamin E-deprived diet was significantly higher than the average in the mice fed the supplemented diet (Table 2). It was also indicated the incidence of hyperglycemia in each group. The effect of vitamin E-supplemented and deprived diets on GTT
Pancreatic tissues were isolated, fixed in Bouin's fluid and embedded in paraffin. Serial sections (6 μm) were cut from each paraffinembedded tissue block, and several sections were stained with hematoxylin-eosin and mouse anti-insulin antibody (Santa Cruz Biotechnology) using the Vectastain Elite ABC Rabbit IgG Kit for visualization by light microscopy. The islets and other cells were recorded with a FX380 CCD Camera and a microscope (Olympus, Tokyo, Japan). Measurement of the oxidative stress markers The measurement of 8-oxo-2′-deoxyguanosine (8-OHdG) in plasma was carried out using an ELISA KIT (JaICA, Shizuoka, Japan). Sample or standard solution was added to each well in a 96-well plate coated with 8-OHdG, and a monoclonal antibody for 8-OHdG was added. The mixture was reacted for12 hs. Then, an enzyme-conjugated antibody was added and reacted for 1 h. The reagent solution for the color
After one week from alloxan treatment, the GTT in acatalasemic mice was examined. Differences in the blood glucose of the alloxan-
Table 1 Concentrations (nmol/mL) of D-α-tocopherol in the blood of mice maintained on the CE-2 diet, vitamin E-supplemented diet and E-deprived diet for 14 weeks. Mouse
D-α-Tocopherol in blood (nmol/mL) CE-2
VE (+)
VE (−)
Normal 3.92 ± 1.07 (n = 6) 7.30 ± 4.37 (n = 14) 1.21 ± 0.60 (n = 13) Acatalasemia 3.78 ± 1.59 (n = 6) 8.68 ± 3.34 (n = 14) 1.00 ± 0.32 (n = 7) CE-2 indicates a laboratory diet (used as a control diet). VE (+) indicates the vitamin E-supplemented diet and VE (−) the vitamin E-deprived diet.
W. Kamimura et al. / Clinical Biochemistry 46 (2013) 795–798 Table 2 Blood glucose (mg/dL) in the fasting blood from mice fed the vitamin E-supplemented [VE (+)] diet or the vitamin E-deprived [VE (−)] diet and the incidence of hyperglycemia.
797
Table 3 The concentration of insulin and C-peptide in the fasting blood of acatalasemic mice maintained on the vitamin E-supplemented or deprived diet after alloxan administration.
Mice (number of mice)
Diet
Alloxan (mg/kg)
Blood glucose (mg/dL)
Incidence of hyperglycemia (%)
Mouse
Diet
Insulin (ng/L) (number of mice)
C-peptide (ng/L) (number of mice)
Normal (15) Normal (28) Normal (15) Normal (23) Acatalasemia Acatalasemia Acatalasemia Acatalasemia
VE VE VE VE VE VE VE VE
0 0 200 200 0 0 200 200
84 80 83 108 87 86 96 127
0 0 0 13 0 0 4 28
Normal Normal Acatalasemia Acatalasemia
VE(+) VE(−) VE(+) VE(−)
671 ± 282 (8) 496 ± 318 (14) 663 ± 294 (12) 399 ± 220 (14)
1594 ± 370 (8) 1543 ± 498 (9) 1126 ± 322 (7) 1053 ± 554 (10)
(30) (34) (28) (29)
(+) (−) (+) (−) (+) (−) (+) (−)
± ± ± ± ± ± ± ±
23 16 25 23* 15 17 18 77*
*
* *
VE(+): Vitamin E-supplemented diet. VE(−): Vitamin E-deprived diet. ⁎p b 0.05.
The asterisk (*) indicates p b 0.05.
treated acatalasemic mice fed the vitamin E-supplemented or deprived diet were observed (Fig. 1). The blood glucose in the mice fed the vitamin E-deprived diet after 60, 90 and 120 min from glucose administration was significantly higher than mice fed the vitamin E-supplemented diet. Blood glucose in the acatalasemic mice fed the vitamin E-deprived diet after 120 min from glucose administration was also significantly higher than that before glucose administration. The insulin and C-peptide concentrations and the index of insulin resistance (Homeostasis Model Assessment; HOMA) [11,12] The insulin and C-peptide concentrations in fasting mouse blood after alloxan administration are shown in Table 3, and the index of insulin resistance is shown in Fig. 2. The C-peptide level in the normal mouse blood was significantly higher than that in the acatalasemic blood. However, the C-peptide concentration was not affected by the diet. The insulin concentration in the vitamin E-deprived diet fed mice was lower than the mice fed the supplemented diet. The concentration in the vitamin E-deprived diet fed acatalasemic mice was only significantly lower. The index of insulin resistance was not affected by either the diet or catalase activity. Microscopic examination of pancreatic tissues in the acatalasemic mice treated with alloxan
β-cells in the islets of Langerhans were calculated and it was found that there was no difference in the number of β-cells between the acatalasemic mice on the vitamin E-supplemented and deprived diet (data not shown). Biomarkers of oxidative stress in serum The concentrations of 8-OHdG in the plasma of the normal and acatalasemic mice are indicated in Table 4. A significant increase of 8-OHdG in the blood of the mice was observed in the case of both low catalase activity and alloxan administration. However, 8-OHdG blood concentration was not affected by either the depletion or supplementation of vitamin E. On the other hand, increases in lipid peroxidation in mouse plasma were not observed in low catalase activity but in the case of alloxan administration and the depletion of vitamin E. Discussion Vitamin E consists of α-tocopherol, β-tocopherol and γ-tocopherol. Among these, D-α-tocopherol is abundantly present in food. In this study, D-α-tocopherol was added into the preparation of a vitamin E-supplemented diet. Compared to the vitamin E intake recommended by the American Institute of Nutrition, this diet contained an excess of approximately seven times the amount. After feeding for 14 weeks, there was no evident health problem with the mice maintained on the vitamin E-deprived or supplemented diet. Table 1 indicates the
Pancreatic tissues after alloxan administration subjected to immunohistochemical staining are shown in Fig. 3a and b. The numbers of
Fig. 1. Glucose tolerance test in the acatalasemic mice after alloxan administration. Mice were fed the vitamin E-supplemented or deprived diet for 14 weeks, and then alloxan was administrated. Mice were further maintained on each diet for 1 week. After fasting, GTTs were carried out. Glucose (200 g/kg of body weight) was administrated to each mouse at 0 min. ●: Blood glucose of the mice (n = 12) fed the vitamin E-supplemented diet and ○: blood glucose of the mice (n = 13) fed the E-deprived diet. *p b 0.05.
Fig. 2. The index of insulin resistance: Homeostasis Model Assessment (HOMA). Normal and acatalasemic mice were fed the vitamin E supplemented [VE(+)] or the E-deprived [VE(−)] diet for 14 weeks, and then alloxan was administrated. Mice were maintained on each diet for one week. Concentrations of insulin and glucose in fasting blood of normal mice (n = 8) fed VE(+) diet, normal mice (n = 14) fed VE(−) diet, acatalasemic mice (n = 12) fed VE(+) diet and acatalasemic mice (n = 14) fed VE(−) diet were determined. The index of insulin resistance [concentration of insulin (nM) × concentration of glucose (mM)] was calculated.
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Fig. 3. Microscopic studies of the pancreas stained with mouse anti-insulin antibody. Acatalasemic mice fed vitamin E supplemented diet or the deprived diet for 14 weeks. After alloxan administration, pancreatic tissues were stained with mouse anti-insulin antibody for visualization. A. From mice fed vitamin E supplemented diet. B. From mice fed vitamin E deprived diet.
mice reached a stable state of D-α-tocopherol in the blood by feeding ad lib on the respective diets for 14 weeks. Alloxan was then intraperitoneally administrated to each mouse (Table 2). After the administration, the incidence (28%) of hyperglycemia in the acatalasemic mice fed the vitamin E-deprived diet was higher than that (13%) in the normal mice fed the vitamin E-deprived diet. However, there was scarcely hyperglycemia in the mice fed the vitamin E-supplemented diet observed. The GTT in the acatalasemic mice maintained on the vitamin E-deprived diet (Fig. 1) indicated that the mice had a state like diabetes, but in the mice fed the supplemented diet they did not. These results suggest that vitamin E prevents or ameliorates glucose metabolism abnormalities caused by alloxan administration in mice. As states like diabetes were due to contributions of pancreatic β-cell dysfunction and insulin resistance [13], we measured the insulin and C-peptide concentrations in the fasting blood after alloxan administration (Table 3). The C-peptide level in the normal mouse blood was significantly higher than that in the acatalasemic blood. It indicates that the level is affected by the catalase activity in the mice, and the result is consistent with the reduction (20%) of β-cell numbers in the Langerhans islets by alloxan administration as previously reported [13]. The C-peptide level in the mouse blood was hardly affected with vitamin E. Pancreatic β-cells of acatalasemic mice were stained with an anti-insulin antibody (Fig. 3). There was no difference β-cell number between the acatalasemic mice fed the deprived and supplemented diets. These results indicate the vitamin E in diet did not affect the destruction of the β-cells or the amount of insulin they released. HOMA (blood glucose X the insulin concentration) [11,12] indicated that none of the mice fed the supplemented or deprived diet exhibited insulin resistance (Fig. 2). Compared to the insulin concentration in the mice fed the vitamin E-supplemented diet, the concentration of acatalasemic
Table 4 Effects of alloxan administration and the depletion of vitamin E on 8-OHdG and lipid peroxidation in the blood of normal and acatalasemic mice. Mouse (number of mice)
Diet
Alloxan (mg/kg)
8-OHdG (ng/mL)
Normal (5) Normal (6) Normal (7) Acatalasemia (9) Acatalasemia (8) Acatalasemia (8)
CE-2 VE(+) VE(-) CE-2 VE(+) VE(-)
0 200 200 0 200 200
0.010 0.118 0.127 0.099 0.228 0.307
± ± ± ± ± ±
Lipid peroxidation (μM) 0.001 0.017 0.046 0.070 0.083 0.072
0.11 1.28 2.74 0.33 1.28 2.32
± ± ± ± ± ±
0.03 0.23 1.15 0.24 0.48 0.79
*
*
The asterisk (*) indicates p b 0.05. Lipid peroxidation was significantly increased by alloxan administration and vitamin E depletion, and 8-OHdG in the blood were significantly increased by alloxan administration and low catalase activity (p b 0.05).
mice fed the deprived diet was significantly lower (p = 0.022). From these results it is deduced the insulin concentration in the mice fed the vitamin E-deprived diet decreased and this resulted in hyperglycemia. This suggests that vitamin E might have potential to keep the insulin concentration in the blood. To examine the oxidative stress caused by the depletion of vitamin E, 8-OHdG and lipid peroxidation in the blood were examined (Table 4). 8-OHdG was not significantly affected by the depletion of vitamin E, but it was significantly affected by a low catalase activity and alloxan administration. On the other hand, lipid peroxidation was increased by alloxan administration and the depletion of vitamin E. As insulin molecules reach the target receptors in the body through the medium of the blood, low insulin concentration in blood may be associated with the increase of peroxidative stress. Further investigation about the decrease of insulin concentration in blood under high peroxidation conditions is currently underway. References [1] Szkudelski T. The mechanism of alloxan and streptozotocin action in B cells of the rat pancreas. Physiol Res 2001;50:537–46. [2] Lenzen S. The mechanisms of alloxan- and streptozotocin-induced diabetes. Diabetologia 2008;51:216–26. [3] Takemoto K, Tanaka M, Iwata H, Nishihara R, Ishihara K, Wang DH, et al. Low catalase activity in blood is associated with the diabetes caused by alloxan. Clin Chim Acta 2009;407:43–6. [4] Choi EJ, Bae SC, Yu R, Youn J, Sung MK. Dietary vitamin E and quercetin modulate inflammatory responses of collagen-induced arthritis in mice. J Med Food 2009;12: 770–5. [5] Yamaoka S, Kim HS, Ogihara T, Oue S, Takitani K, Yoshida Y, et al. Severe vitamin E deficiency exacerbates acute hyperoxic lung injury associated with increased oxidative stress and inflammation. Free Radic Res 2008;42:602–12. [6] Feinstein RN, Braun JT, Howard JB. Acatalasemic and hypocatalasemic mouse mutants II. Mutational variations in blood and solid tissue catalases. Arch Biochem Biophys 1967;120:165–9. [7] Reeves PG, Nielsen Jr FH, Fahey GC. AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition and hoc writing committee on the reformulation of the AIN-76A rodent diet. J Nutr 1993;123:1939–51. [8] Chi C, Hayashi D, Nemoto M, Nyui M, Urano S, Anzai K. Vitamin E-deficiency did not exacerbate partial skin reactions in mice locally irradiated with X-rays. J Radiat Res 2011;52:32–8. [9] Gao D, Li Q, Liu Z, Li Y, Liu Z, Fan Y, et al. Hypoglycemic effects and mechanisms of action of Cortex Lycii Radicis on alloxan-induced diabetic mice. Yakugaku Zasshi 2007;127:1715–21. [10] Masuoka N, Wakimoto M, Ubuka T, Nakano T. Spectrophotometric determination of hydrogen peroxide: catalase activity and rates of hydrogen peroxide removal by erythrocytes. Clin Chim Acta 1996;254:101–12. [11] Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC. Homeostasis model assessment: insulin resistance and β-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 1985;28: 412–9. [12] Zamami Y, Takatori S, Goda M, Koyama T, Iwatani Y, Jin X, et al. Royal Jelly ameliorates insulin resistance in fructose-drinking rats. Biol Pharm Bull 2008;31: 2103–7. [13] Abdul-Ghani MA, Tripathy D, DeFronzo RA. Contributions of beta-cell dysfunction and insulin resistance to the pathogenesis of impaired glucose tolerance and impaired fasting glucose. Diabetes Care 2006;29:1130–9.