Protective effect of vitamin E against alloxan-induced mouse hyperglycemia

    Protective effect of vitamin E against alloxan-induced mouse hyperglycemia Kazunori Takemoto, Wakana Doi, Noriyoshi Masuoka PII: DOI: Reference:

S0925-4439(15)00376-2 doi: 10.1016/j.bbadis.2015.12.022 BBADIS 64395

To appear in:

BBA - Molecular Basis of Disease

Received date: Revised date: Accepted date:

11 September 2015 23 November 2015 22 December 2015

Please cite this article as: Kazunori Takemoto, Wakana Doi, Noriyoshi Masuoka, Protective effect of vitamin E against alloxan-induced mouse hyperglycemia, BBA - Molecular Basis of Disease (2015), doi: 10.1016/j.bbadis.2015.12.022

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Protective effect of vitamin E against alloxan-induced mouse hyperglycemia

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of Life science, Okayama University of Science, Okayama 700-0005, Japan

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2Department

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Medical Science Education Center, Okayama 700-0005, Japan

authors contributed equally to this work.

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Kazunori Takemoto1,†, Wakana Doi2,† and Noriyoshi Masuoka2

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Abstract

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[Background] Alloxan induces oxidative stress and hyperglycemia in animal models. Acatalasemic (catalase deficiency) mice are susceptible to alloxan-induced

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hyperglycemia. As the incidence of hyperglycemia induced by alloxan was reportedly improved when mice were fed a vitamin E supplemented diet, this protective effect was examined. [Methods] Acatalasemic and normal mice fed a vitamin E supplemented diet were treated with alloxan. The pancreas was examined with microscopy. We also isolated pancreatic islets of normal mice treated with alloxan. The glucose stimulated insulin secretion was examined.

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[Results] Vitamin E powerfully ameliorated the increase in apoptosis. Vitamin E

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regulation of the glucose stimulated insulin secretion.

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increases insulin amounts secreted from pancreatic cells, but does not ameliorate the

[Conclusions] It is suggested that the difference in the mice fed vitamin E supplemented

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diet is due to an increase of insulin secretion and that vitamin E supplementation may

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have a role in helping to slow the stages of diabetes mellitus.

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Keywords; α-tocopherol, alloxan, apoptosis, insulin, beta-cell, hyperglycemia

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1. Introduction

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Alloxan is reduced to dialuric acid with intracellar glutathione (GSH), and the

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redox cycling between them generates the reactive oxygen species of the superoxide anion, hydrogen peroxide and then hydroxyl radical, and hydroxyl radical is finally

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responsible for the necrotic death of pancreatic cells [1]. As the result, alloxan induces a

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low level of insulin in the blood and diabetes in animal models. Goth and Eaton reported that acatalasemic (hereditary catalase deficiency) person increased risk of diabetes [2]. We

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examined effect of alloxan administration on normal and acatalasemic mice to examine

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the risk [3-6]. Catalase activity in acatalasemic pancreatic inlets was lower than normal

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ones, and acatalasemic mice were susceptible to alloxan-induced diabetes [4]. In microscopic studies of the pancreatic cells, alloxan induced atrophy of Langerhans islets.

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An increase of apoptosis cells by alloxan administration was observed in pancreatic islets of acatalasemic mice but was not in normal mice [4, 5]. For protection against the diabetes induced by alloxan, intakes of zinc, dimethylthiourea, 1α,25-dihydroxyvitamin D3, vitamin E and so on were reported [7-12]. Protection of normal and acatalasemic mice against the diabetes with the antioxidant vitamin E was examined [12]. However, the mechanism is still poorly understood. In this study, we examined the effect of feeding vitamin E supplemented diet on oxidative damages in the pancreas of acatalasemic

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mice induced by alloxan and also the insulin secretion ability of Langerhans islets

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isolated from alloxan-treated normal mice in the presence of vitamin E.

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2. Materials and Methods

2.1. Chemicals and animals

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Male mice of the C3H/AnL CSaCSa (normal) and C3H/AnL CSbCSb (acatalasemia)

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strains established by Feinstein et al. [13] were kindly provided by Professor K. Ogino, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical

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Science. Mice were propagated and maintained on a laboratory diet (CE-2 diet, Clea

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Japan, Tokyo, Japan) and water ad libitium until the experiments. The experimental

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procedure was approved by the Ethics Review Committees for Animal Experimentation of Okayama University of Science. Alloxan monohydrate and the other reagents were

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purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

2.2. Microscopic study of pancreas in mice fed a vitamin E supplemented diet for 14 weeks Vitamin E feeding experiments were previously carried out, and the procedure is presented in [12]. Male mice were maintained on vitamin E supplemented and depleted diets for 14 weeks, respectively, and then alloxan monohydrate (200 mg/kg of body

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weight) was intraperitoneally administrated [14]. Seven days after alloxan

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administration, mice were sacrificed and each animal’s pancreatic tissues were isolated,

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fixed in Bouin’s fluid and embedded in paraffin. Serial sections (4 μm) were cut from each paraffin-embedded tissue block. Anti-insulin antibody stains were carried out with H-86,

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sc-9168, Santa Cruz Biotechnology and Vectastain Elite ABC rabbit IgG, Vector Lab. for

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visualization. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining was carried out using the In Situ Cell Death Detection Kit (Roche

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Diagnostics Japan) [15]. The apoptosis incidence was calculated from the

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TUNEL-positive cells per the total cells in the pancreatic islets. Sections were also

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stained with Protein-carbonyl immunohistochemical stain [16] for visualization by light microscopy (Cosmo Bio. Co., Ltd, Tokyo, Japan). After staining, the stained cells in each

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tissue section were observed with IX 71 microscopy equipped with a DP 50 Camera (Olympus, Tokyo, Japan), and these cells were counted manually from the images. For the measurements in the photographs, the Image J program (NIH) was used.

2.3. Isolation of Pancreatic Islets in the Pancreas from alloxan-treated normal Mice As a previous study [6] had indicated that alloxan induced severe deterioration of pancreas in mice and that isolation of Langerhans islets from alloxan-treated

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acatalasemic mice was quite difficult compared to normal mice, we chose

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alloxan-treated normal mice and isolated the Langerhans islets. Alloxan monohydrate

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(200 mg/kg of body weight) was administrated to normal mice, and mice were sacrificed one week after alloxan administration. Islets were isolated from the pancreas [17].

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Five mL of collagenase solution (type IV, 160 U/ml, Worthington Biochem. Corp., NJ,

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USA) in Hank’s balanced salt solution (pH 7.4, Sigma-Aldrich Co. LLC) was injected into the common bile duct of each mouse. The pancreas was taken out and incubated

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with 1 mL of collagenase solution at 37˚C for 30 min. Digested pancreatic tissue was

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washed three-times with 5 mL of Hank’s balanced salt solution. The suspension was

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centrifuged at 700 X g for 10 min. using a discontinuous gradient of Ficoll (Nacalai Tesque, Kyoto) at concentrations of 25 %, 23 %, 20 % and 11 % (2.0, 2.0, 2.0 and 1.0 mL,

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respectively) in Hank’s solution. Islets were taken up with a micropipette from the interface between the 20 % and 11 % layers, and centrifuged. To the precipitate, 5 mL of RPIM 1640 medium (pH 7.4, Invitrogen Corp., CA, USA) were added twice and washed out. Purified islets were cultured in 5 mL of RPMI 1640 medium at 37˚C under air containing 5% CO2 for 60 min and used for the experiments.

2.4. Assay of Insulin Secretion under Glucose Stimulation [18]

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According to the manufacturer’s instructions (Pancreatic islet culture kit: Cosmo Bio.

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Co., Ltd), 0.5 mL of RPIM 1640 medium containing 3 mM glucose (pH 7.4) was added to

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ten purified islets, and then the suspension was incubated at 37˚C for 60 min under 5% CO2. After centrifugation, the supernatant was removed. To the residue (the

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precipitated islets), a mixture containing 0.495 mL of 3 mM glucose in RPIM 1640

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medium and 5 µL of 0-10 mM α-tocopherol (the final concentration of which was 0, 10 or 100 µM) in DMSO was added, and the suspension was incubated at 37˚C for 6 0 min

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under 5% CO2. The suspension was centrifuged again and the supernatant was

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collected for analysis as the sample was stimulated with 3 mM glucose. To the residue, a

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mixture containing 0.495 mL of 20 mM glucose and 5 µL of α-tocopherol in DMSO was added and the mixture was incubated at 37˚C for a further 60 min under 5% CO2. The

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suspension was centrifuged and the supernatants were collected for analysis as the sample stimulated with 20 mM glucose. Insulin release from the islets was also reacted in the mixture containing 0.495 mL of 30 mM glucose and 5 µL of α-tocopherol in DMSO for a further 60 min. These supernatants were stored at −80˚C until analysis. The insulin levels in the supernatant were determined using Mouse Insulin ELISA kits (Shibayagi), and the total DNA content in each sample was measured with a CyQUANT assay (Invitrogen). In order to estimate the function of the islets for glucose stimulation,

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the GSIS index from the islets was calculated. The index was obtained by dividing the

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insulin level stimulated with 20 mM glucose by the level stimulated with 3 mM glucose.

2.5. Statistics

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Student’s t-test was used to evaluate significant differences. The difference was considered

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significant when p < 0.05.

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3. Results

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The catalase activity in acatalasemic mouse blood at 25˚C was 0.15 ± 0.09 μmol/s/g of Hb and the

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activity in normal mouse blood was 6.89 ± 0.57 μmol/s/g of Hb [19]. After alloxan administration, numbers and size of insulin positive cells in acatalasemic

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mice decreased 87 ± 6 and 72 ± 5 % of insulin positive cells in control mice, respectively [6], and the volume of insulin positive cells was 63 % of the control. When acatalasemic mice fed vitamin E supplemented diet, numbers and size of insulin positive cells after alloxan administration were indicated in Figure 1, and the volume was improved from 61 % to 66 % of the control by feeding the vitamin E diet.

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(B)

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(A)

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Figure 1 Relative numbers and mean size of insulin positive cells in acatalasemic mice

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fed vitamin E supplemented diet or the depleted diet

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(A) Numbers of insulin positive cells. The number of the control mice (100 %) was 41.7

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± 2.4 / µm2. (B) Mean size of insulin positive cells. The size of control mice was 90.2 ± 1.7 µm2. Number in parenthesis indicated the number of mice. Bars of each symbol

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indicate the SE. * indicates p<0.05, and ** indicates p<0.01.

Apoptosis in the mouse pancreas is summarized in Table 1. When alloxan was administered, apoptosis level in pancreas of the acatalasemic mice fed the vitamin E depleted diet was significantly increased. By feeding the vitamin E supplemented diet, incidence of apoptosis was kept to the control level.

Table 1. Incidence of apoptosis in the pancreatic tissues isolated from alloxan 9

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administrated normal and acatalasemic mice

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0.9 ± 0.3

1.0 ± 0.4

+

0.1 ± 0.1

1.2 ± 0.7

Alloxan (-)

Alloxan (+)

1.3 ± 0.4

4.5 ± 0.9*

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Alloxan (+)

1.7 ± 0.3

1.3 ± 0.5

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Alloxan (-)

Acatalasemic mice (5)

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Normal mice (5)

in diet

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Apoptosis (%)

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Vitamin

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*: p < 0.05 compared to the vitamin E supplemented group.

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Protein-carbonyl immunohistochemical staining of the pancreas isolated from the

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alloxan-treated acatalasemic mice is shown in Figure 2. As the result of alloxan administration, a mild increase of oxidized protein was observed on the inside of the

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islets in the alloxan administered group. It is evident that the increase by alloxan administration decreased when the mice were fed the vitamin E supplemented diet.

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alloxan-treated acatalasemic mice

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Figure 2 Protein-carbonyl immunohistochemical staining of the pancreas isolated from

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Effect of vitamin E on insulin release from the Langerhans islets isolated from alloxan-treated normal mice stimulated with 3 and 20 mM glucose was indicated in

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Table 2. The values from the lack of any addition of vitamin E are consistent with previous data [6]. The amount of insulin secreted was increased by higher concentrations of vitamin E. The GSIS index was calculated. Compared to the index (4.6 ± 2.6) from no alloxan- treated mice, these indexes were low.

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Table 2. Effect of vitamin E on the insulin secretion of the Langerhans islets isolated

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GSIS* (Insulin /DNA) X 103

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vitamin E

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from alloxan-treated normal mice.

GSIS Index**

3 mM glucose

20 mM glucose

30 mM glucose

0.00 (8)

18.5 ± 6.2

34.1 ± 12.3

46.5 ± 17.1

1.34 ± 0.8

0.01 (7)

58.5 ± 18.9***

88.2.± 34.6

171.4 ± 130.7

1.83 ± 0.6

0.10 (6)

121.7 ± 59.9***

175.4 ± 62.7

1.14 ± 0.6

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（mM）

121.2 ± 58.7

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The islets were isolated 7 days after normal mice were treated with alloxan. The

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number in parenthesis indicates the number of mice. * Glucose stimulated Insulin

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secretion, ** GSIS index = each insulin concentration at 20 mM glucose/the concentration at 3 mM glucose. ***: p<0.05 compared to the values at 0.00 mM vitamin

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E and 3 mM glucose.

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4. Discussion

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Acatalasemic mouse is the susceptible to alloxan-induced hyperglycemia, and the

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susceptibility is due to low catalase activity in pancreas compared to normal ones [4]. When

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acatalasemic mice were fed a vitamin E supplemented diet for 14 weeks [12], the

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hyperglycemia and the insulin level in the blood induced by alloxan administration were significantly improved. Volume of insulin positive cells in acatalasemic mouse pancreas

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was strongly decreased (to 63 %) by alloxan administration [6]. By feeding the vitamin E

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supplemented diet, volume of the positive cells is increased (from 61 to 66 %), but the

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increased volume is low compared to the decrease induced by alloxan administration

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(Figure 1). Protein-carbonyl immunohistochemical staining of the pancreas isolated from the alloxan-treated acatalasemic mice is shown in Figure 2. The amount of oxidized protein was

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increased by alloxan administration. When mice were fed the vitamin E diet, only a mild increase of oxidized protein on the inside of islets was observed. The cellular apoptosis in the acatalasemic islets induced by alloxan was ameliorated by the feeding of a vitamin E supplemented diet (Table 1), such as an Aspergillus awamori-fermented burdock root diet [5, 20]. However, as the incidence of apoptosis was small (about 4 %) [4, 5], it was deduced that apoptosis induced by alloxan did not play a main role to decrease blood insulin level. To further examine effect of vitamin E, we isolated pancreatic cells from alloxan-treated 13

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normal mice and examined the GSIS in the presence of 0 - 0.1 mM vitamin E, since the

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concentration of vitamin E in the blood of mice fed a vitamin E supplemented diet was

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approximately 0.01 mM [12]. The GSIS is shown in Table 2. The GSIS index was calculated since it is useful for characterizing the progression of the stages of diabetes

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mellitus [21]. Alloxan induced low insulin secretion from the pancreas and a low GSIS

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index [6]. Vitamin E did not affect the index, suggesting that vitamin E did not improve the regulation of GSIS. However, vitamin E dramatically enhanced insulin secretion

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from the alloxan-treated Langerhans islets. Such an increase in insulin secretion would

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provide protection against the low insulin level in the blood caused by alloxan [10-12].

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Therefore, it is suggested that vitamin E supplementation might be useful for slowing the stages of diabetes mellitus, though vitamin E did not fully prevent the oxidative

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damages induced by alloxan. Further study is necessary, how the increase in the insulin secretion is induced by vitamin E and impacts the uptake of glucose to pancreas.

5. Conclusion Alloxan induces the redox cycling and in turn generates reactive oxygen species to result necrosis in pancreas. Apoptosis in pancreas is a hallmark of severe oxidative stress, and the vitamin E intake prevented this apoptosis. Vitamin E strongly enhances

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insulin secretion from pancreas and improves incidence of hyperglycemia. It is

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suggested that vitamin E supplements may be helpful in slowing the stages of diabetes

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mellitus.

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Highlight

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1. Vitamin E prevents the apoptosis in pancreas caused by oxidative stress.

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2. Protection of hyperglycemia by vitamin E is due to an increase of insulin secretion.

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3. Vitamin E may be useful for slowing the stages of diabetes mellitus.

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