Vitamin D reduces deposition of advanced glycationend-products in the aortic wall and systemic oxidativestress in diabetic rats

Erik Salum a,b,c,*, Jaak Kals b,c,d, Priit Kampus a,b,c, Tiit Salum c, Kersti Zilmer c,Marina Aunapuu e,f, Andres Arend e, Jaan Eha a,b, Mihkel Zilmer b,c

aDepartment of Cardiology, University of Tartu, 8 Puusepa Street, Tartu 51014, EstoniabEndothelial Centre, University of Tartu, 8 Puusepa Street, Tartu 51014, EstoniacDepartment of Biochemistry, Centre of Excellence for Translational Medicine, University of Tartu, 19 Ravila Street, Tartu 50411, EstoniadDepartment of Vascular Surgery, Tartu University Hospital, 8 Puusepa Street, Tartu 51014, EstoniaeDepartment of Anatomy, University of Tartu, 19 Ravila Street, Tartu 50411, Estoniaf Institute of Veterinary Medicine and Animal Sciences, Estonian University of Life Sciences, 62 Fr. Kreutzwaldi Street, Tartu 51014, Estonia

d i a b e t e s r e s e a r c h a n d c l i n i c a l p r a c t i c e 1 0 0 ( 2 0 1 3 ) 2 4 3 – 2 4 9

a r t i c l e i n f o

Article history:

Received 5 September 2012

Received in revised form

17 January 2013

Accepted 1 March 2013

Published on line 21 March 2013

Keywords:

Streptozotocin

Total antioxidant capacity

Oxidative stress index

Ne-(carboxymethyl)lysine

Cholecalciferol

a b s t r a c t

Aims: Vitamin D may have an important role in reducing the risk of cardiovascular disease.

Advanced glycation end-products (AGEs) such as Ne-(carboxymethyl)lysine (CML), have

been implicated in diabetic vascular complications via oxidative stress-mediated pathways.

We investigated the potential protective effect of vitamin D on CML accumulation in the

diabetic aortic wall. To test the effects of vitamin D on systemic oxidative stress we also

assessed liver oxidative stress index (OSI) and serum total antioxidant capacity (TAC).

Methods: Male Wistar rats were assigned to three groups: control, untreated diabetes, and

diabetes + cholecalciferol. Diabetes was induced by streptozotocin, followed by oral admin-

istration of cholecalciferol (500 IU/kg) for 10 weeks in the treatment group. Aortic CML

accumulation was determined by ELISA and immunohistochemical assays. OSI was

assessed by measuring TAC and the level of total peroxides in the liver and serum using

colorimetric assays.

Results: Untreated diabetes was associated with significantly elevated CML levels in the

aortic wall (19.5 � 3.3 vs 10.2 � 4.7 ng/mL), increased liver OSI (6.8 � 1.9 vs 3.1 � 0.7), and

reduced serum TAC (0.4 � 0.1 vs 0.8 � 0.3 mmol Trolox/L), in comparison with the control

group. Cholecalciferol significantly blocked the accumulation of CML in the aortic wall

(10.4 � 8.4 vs 19.5 � 3.3 ng/mL), decreased liver OSI (4.2 � 1.4 vs 6.8 � 1.9), and improved

serum TAC (1.0 � 0.2 vs 0.4 � 0.1 mmol Trolox/L), compared with the untreated diabetic

group.

Conclusions: Streptozotocin-diabetes resulted in increased deposition of AGEs and increased

oxidative stress in the serum and liver. Vitamin D supplementation may provide significant

protection against oxidative stress-mediated vascular complications in diabetes.

# 2013 Elsevier Ireland Ltd. All rights reserved.

Contents available at Sciverse ScienceDirect

Diabetes Researchand Clinical Practice

journal homepage: www.elsevier.com/locate/diabres

* Corresponding author at: Department of Cardiology, University of Tartu, 8 Puusepa Street, Tartu 51014, Estonia. Tel.: +3727318317.E-mail address: erik.salum@gmail.com (E. Salum).

0168-8227/$ – see front matter # 2013 Elsevier Ireland Ltd. All rights reserved.http://dx.doi.org/10.1016/j.diabres.2013.03.008

d i a b e t e s r e s e a r c h a n d c l i n i c a l p r a c t i c e 1 0 0 ( 2 0 1 3 ) 2 4 3 – 2 4 9244

1. Introduction

Advanced glycation end-products (AGEs) are irreversibly

formed during glycation and oxidation of proteins [1]. Because

of its abundance in uncontrolled diabetes mellitus (DM), glucose

is assumed to be a major source of glycation and AGE formation.

AGEs can form covalent cross-links between the extracellular

matrix proteins in the vascular wall and alter their function,

particularly of collagen and elastin [2,3]. AGE cross-linking of

collagen has been associated with increased stiffness of the

arterial wall and impaired arterial haemodynamics [4].

Increases in the levels of Ne-(carboxymethyl)lysine (CML), a

major antigenic structure of AGEs [5], have been correlated with

the severity of diabetic complications [6,7], suggesting that CML

may be an important biomarker in the assessment of vascular

integrity in DM. Considering the emerging evidence about the

adverse effects of AGEs on the vascular properties, therapies

aimed at inhibiting AGEs may provide significant improvement

of diabetic vascular complications.

Oxidative stress is regarded as an important factor in the

pathogenesis and progression of DM and its associated

cardiovascular complications [8]. In diabetes, persistent

hyperglycemia is associated with increased production of

reactive oxygen species (ROS) [9] with concomitant depletion

of intrinsic antioxidant defense mechanisms [10], rendering

the organism more susceptible to oxidative damage. The

imbalance between oxidants and antioxidants could be largely

responsible for the functional and morphological damages to

the blood vessels associated with uncontrolled hyperglycemia

[11]. Oxidative reactions also enhance the formation of AGEs

and their accumulation in cardiovascular tissue [12], raising a

potential link between diabetic vascular complications and

deposition of AGEs.

Agents that enhance the antioxidant defense and reduce

the damage caused by oxidative stress to the blood vessels

may be beneficial in the treatment of diabetic vascular

complications. Several studies have demonstrated that vita-

min D may reduce lipid peroxidation [13,14] and maintain a

steady level of glutathione (GSH), a potent intracellular

antioxidant [15]. Calcitriol, the active form of vitamin D, has

been shown to improve vascular endothelial function by

modulating the expression of radical generating and scaveng-

ing enzymes, thus preventing the overproduction of ROS [16].

In our previous study, we demonstrated that chronic

supplementation of vitamin D improves aortic wall remodel-

ling in streptozotocin-induced diabetes [17]. To test the

possible ways how vitamin D may have protective effects

concerning diabetes-induced vascular abnormalities we in-

vestigated in the current study the effect of vitamin D on CML

deposition in the aortic wall, and its potential antioxidative

influence by assessing systemic oxidative stress-related

parameters in the serum and liver in a rat model of type 1 DM.

2. Methods

2.1. Animals

Male Wistar rats (n = 24, age 4 months), purchased from Harlan

Laboratories (Harlan Laboratories, Inc., The Netherlands),

were used in the experiments. The animals were housed in

a room maintained at 21 � 2 8C under a standard 12-h light/12-

h dark cycle and were given normal rat chow and tap water

ad libitum. All experimental procedures were approved by the

Estonian National Board of Animal Experiments and were

conducted in accordance with the European Communities

Directive (86/609/EEC).

2.2. Experimental protocol

Rats were randomly assigned to one of three groups: control,

diabetic, and cholecalciferol-treated diabetic group. Diabetes

was induced by an intraperitoneal administration of strepto-

zotocin (STZ) 50 mg/kg (Sigma–Aldrich, St. Louis, MO, USA),

freshly dissolved in 0.9% saline solution. Blood samples from

the tail vein were obtained 48 h later and glucose levels were

measured with a glucometer (Glucocard X-meter, Arkray Inc.,

Japan). All rats in the diabetic groups had blood glucose level

>15 mmol/L. Supplementation of cholecalciferol (Sigma–

Aldrich, St. Louis, MO, USA) 12.5 mg (500 IU) kg�1 body weight,

dissolved in 0.3 ml olive oil, was started immediately after

confirmation of diabetes. Cholecalciferol was administered

orally every other day for 10 weeks.

2.3. Laboratory parameters

At 10 weeks, the animals were anaesthetised with a mixture of

fentanyl (0.07 mg/kg, Gedeon-Richter Plc., Hungary), midazo-

lam (5 mg/kg, Roche Pharma AG, Germany), and ketamine

(75 mg/kg, Vetoquinol Biowet Sp. z.o.o., Poland) administered

subcutaneously. Blood samples were taken from the tail vein

for assessment of glucose levels. Anaesthetised animals were

subjected to cardiac puncture for blood withdrawal, followed

by cervical dislocation. One part of blood sample was used for

assessment of glycated haemoglobin (HbA1c) and glucose

levels, and the remaining portion was centrifuged at 3000 rpm

for 15 min to obtain serum. Samples of liver and aortic tissue

were collected, soaked in ice-cold 0.9% NaCl, snap-frozen in

liquid nitrogen, and stored at �60 8C until use.

Calcium (Ca) concentration in the serum was determined by

a colorimetric test (Calcium liquicolor, HUMAN Gesellschaft fur

Biochemica und Diagnostica mbH, Germany). Serum albumin

levels were measured using a colorimetric test (Albumin

liquicolor, HUMAN Gesellschaft fur Biochemica und Diagnos-

tica mbH, Germany). Serum Ca levels were corrected for

albumin concentrations using the following formula: corrected

Ca = serum Ca + 0.02 � (40 � serum albumin) [18].

Serum 25-hydroxyvitamin D [25(OH)D] levels were mea-

sured using a radioimmune assay (25-Hydroxyvitamin D, 125I

Ria Kit, Diasorin Corporation, USA).

2.4. Oxidative stress markers

Liver samples were homogenised in ice-cold 0.9% NaCl

solution and centrifuged at 15,000 � g for 10 min at 4 8C.

The insoluble pellets were discarded and the supernatants

were used for analysis. Total peroxide concentrations (TPX)

were measured with an OxyStat colorimetric assay kit

(Biomedica Gruppe, Austria), which assesses the colour

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change produced by the reaction of total peroxides in the

sample with peroxidase. The data were expressed as nmol/

mg protein, with protein concentration measured spectro-

photometrically using the Lowry method [19].

Total antioxidant capacity (TAC) was measured in the

serum and liver samples using a colorimetric assay (Randox

Laboratories Ltd., UK). This assay is based on the decolorisa-

tion of 2,20-azinobis(3-ethylbenzothiazoline 6-sulfonate)

(ABTS) radical action. The TAC value was expressed as an

equivalent of the millimolar concentration of Trolox (a soluble

vitamin E analogue) solution. Oxidative stress index (OSI), an

indicator of the redox balance between oxidation and

antioxidation, was expressed as the percent ratio of TPX to

TAC [20].

Aortic strips (10 mm) were homogenised in ice-cold 0.9%

NaCl solution, using a blade type homogeniser (Tekmar

Tissumizer, Cincinnati, OH, USA) and centrifuged at

10,000 � g for 10 min at 4 8C. The insoluble pellets were

discarded and the supernatants were used for analysis. The

concentration of CML was assessed using a commercial

enzyme-linked immunosorbent (ELISA) test OxiSelectTM N-

epsilon-(Carboxymethyl) Lysine ELISA Kit (Cell Biolabs, Inc.,

San Diego, CA, USA). Ten mg/ml of total protein extracts were

adsorbed onto a 96-well plate at 4 8C overnight. The absor-

bance of each well was read using a microplate reader (Tecan

Sunrise, Tecan GmbH, Austria) using 450 nm as the primary

wavelength. The concentration of CML was calculated by

comparison to a standard curve consisting of known con-

centrations of CML-BSA. Results are presented as nanogram of

CML per millilitre of solution.

2.5. Immunohistochemistry

Three-mm thick paraffin sections mounted on poly-L-lysine

coated SuperFrost slides (Menzel-Glaser, Germany) were

deparaffinised and rehydrated. Peroxidase activity was

blocked by 0.6% H2O2 (Merck, Germany) in methanol (Merck,

Germany). Then the sections were washed in tap water and in

PBS (pH = 7.4; Gibco, Invitrogen, USA) for 10 min, treated with

normal 1.5% goat serum (Gibco, Invitrogen Corporation, USA)

for 20 min at room temperature and incubated with the first

antibody: anti-carboxymethyl lysine (mouse monoclonal

antibody [CML26], abcam, UK) diluted 1:50 overnight at 4 8C

in the humidity chamber. On the next day the sections were

incubated with the biotinylated horse anti-mouse secondary

Table 1 – Body weights and serum laboratory values at the en

Group Body weight(g)

Blood glucose(mmol/L)

HbA1c(%)

Control 456 � 26 6.3 � 1.6 4.0 � 0.1

Diabetes 374 � 52** 28.3 � 3.9** 10.3 � 0.7**

Diabetes +

vitamin D

348 � 43** 28.5 � 5.9** 9.5 � 1.3**

Data are expressed as mean � SD (n = 8 per group).

HbA1c, glycated haemoglobin; 25(OH)D, 25-hydroxyvitamin D; Corrected

using the following formula: corrected calcium = serum calcium + 0.02 �* p < 0.05 vs control.** p < 0.01 vs control.*** p < 0.001 vs diabetes + vitamin D.

antibody for 30 min at room temperature. After a wash step

the sections were incubated with the avidin-biotin peroxidase

complex ELITE system (Vectastain Elite ABC Kit, Vector

Laboratories Inc., Burlingame, USA) for 30 min. Peroxidatic

activity was detected with 3,30-diaminobenzidine [DAB]

(Vector Laboratories Inc., USA) and the sections were counter-

stained with hemalaun, dehydrated and mounted with DPX

(Fluka, Switzerland). The labelling was expressed on a semi-

quantitative scale ranging from 0 to 4 (0 – no staining, 1 – weak

staining, 2 – moderate staining, 3 – strong staining, 4 – very

strong staining). Two independent observers in a blinded

fashion performed the evaluation. Immunohistochemistry

negative controls were performed by omitting the primary

antibody (mouse IgG was used in place of the primary

antibody).

2.6. Statistical analysis

Results are expressed as means � standard deviation (SD).

Differences between the groups were evaluated using the one-

way analysis of variance (ANOVA) followed by Tukey’s post

hoc analysis for multiple comparisons of group means. Semi-

quantitative data were compared by the Kruskal–Wallis one-

way ANOVA followed by Mann–Whitney U test. Differences

were considered to be statistically significant when p was

<0.05. All statistical comparisons were performed with the

Statistica software (version 8; StatSoft, USA).

3. Results

The basic parameters and biochemical results are presented

in Table 1. Rats in both diabetic groups presented with

significantly lower body weights and increased levels of

blood glucose and HbA1c. Serum 25(OH)D levels were

significantly decreased in untreated diabetic rats and

completely restored by vitamin D supplementation. Serum

Ca and albumin-corrected Ca levels were not different

between all groups, indicating that calcium homeostasis

was not affected by short-term changes in circulating

vitamin D levels.

Untreated diabetes produced significantly higher levels of

CML in the aortic wall homogenates and OSI in the liver (Figs. 1

and 2). The levels of serum TAC were significantly decreased in

untreated diabetic rats compared to non-diabetic rats (Fig. 3).

d of the study.

25(OH)D(nmol/L)

Calcium(mmol/L)

Albumin(g/L)

Corrected calcium(mmol/L)

140 � 21*** 2.6 � 0.2 39.3 � 2.7 2.6 � 0.3

108 � 38*,*** 2.5 � 0.2 30.8 � 3.2** 2.6 � 0.3

494 � 125 2.6 � 0.3 33.4 � 5.1** 2.7 � 0.2

calcium, calcium concentrations adjusted to albumin concentrations

(40 � serum albumin).

Fig. 1 – Diabetes was associated with significantly higher

levels of CML in the aortic wall that was prevented by

vitamin D supplementation. CML, Ne-

(carboxymethyl)lysine. Data are mean W SD (n = 8 per

group).

Fig. 3 – Serum total antioxidant capacity was significantly

decreased in diabetic rats and completely restored by

treatment with vitamin D. Data are mean W SD (n = 8 per

group).

d i a b e t e s r e s e a r c h a n d c l i n i c a l p r a c t i c e 1 0 0 ( 2 0 1 3 ) 2 4 3 – 2 4 9246

Vitamin D effectively prevented CML accumulation in the

aortic tissue (Fig. 1) and reduced the levels of OSI in the liver,

compared to untreated diabetic rats (Fig. 2). Furthermore,

significant improvement of serum TAC was observed in the

diabetic treated group where TAC was restored to a higher

level than that in the control group (Fig. 3).

Immunohistochemical detection of CML demonstrated

strong immunostaining in all layers of the aortic wall in the

untreated diabetes group (Fig. 4). In the diabetic treated group

strong staining was still seen in the intima and adventitia, but

the thickest layer, the media, showed moderate staining.

Similar staining pattern was found in the control group, where

immunostaining was moderate in intima and adventitia,

while the medial layer stained weakly (Table 2).

4. Discussion

In our previous study, we demonstrated that chronic

supplementation of vitamin D to STZ-diabetic rats provides

Fig. 2 – Oxidative stress index in the liver was significantly

increased in untreated diabetes and improved by vitamin

D. Oxidative stress index is expressed as the percent ratio

of TPX to TAC. TPX, total peroxide level; TAC, total

antioxidant capacity. Data are mean W SD (n = 8 per group).

NS = non-significant difference.

significant protection against diabetes-induced alterations in

the arterial structure [17]. In this study, we have assessed the

effects of vitamin D on the aortic wall deposition of AGEs and

oxidative stress status in the blood and liver in a rat model of

type 1 DM. The major findings of this study extend our

previous data regarding that the vasoprotective effects of

vitamin D could be mediated, at least in part, via inhibition of

the accumulation of AGEs in the aortic wall in the context of

improved systemic antioxidant capacity and reduced oxida-

tive stress in the liver, the key organ in the metabolism and

circulation of vitamin D.

AGE formation in DM could be responsible for development

of vascular complications by several mechanisms. In the

arterial wall, accumulation of AGEs may adversely affect

vasodilation either by increased stiffness of structural

proteins [3,4] or by disturbing the smooth muscle response

to nitric oxide [21]. The levels of AGEs in tissues reflect the

severity of these complications while therapeutic interven-

tions aimed at reducing AGEs may inhibit or delay their

progression [22,23]. Our findings of increased deposition of

CML, the major advanced glycation adduct, in the aortic wall

induced by untreated experimental DM are consistent with

Table 2 – Estimation of the immunostaining intensity ofcarboxymethyl lysine in the layers of the aortic wall.

Group Intima Media Adventitia

Control 1.40 � 0.41 0.79 � 0.37 1.96 � 0.39

Diabetes 2.79 � 0.39* 2.07 � 0.45* 2.86 � 0.24*

Diabetes + vitamin D 2.79 � 0.39* 1.30 � 0.27** 2.50 � 0.35

The staining intensity was expressed by a scale ranging from 0 to 4

(0 – no staining, 1 – weak staining, 2 – moderate staining, 3 – strong

staining, 4 – very strong staining). The scores were summed and

the values are presented as means � SD (n = 8 per group).* p < 0.01 vs control.** p < 0.01 vs untreated diabetes, Mann–Whitney U-test.

Fig. 4 – Immunohistochemical localisation of Ne-(carboxymethyl)lysine (CML) in the wall of the aorta of the

diabetes + cholecalciferol group (A), untreated diabetes group (B) and control group (C). Strong immunostaining was found

in all layers of the aorta in the diabetes group (B), while in diabetes + cholecalciferol group immunostaining in the medial

layer was moderate (A), similar to the staining intensity of the control group (C). Staining was not seen in negative controls,

where the primary antibody was omitted (D). Diaminobenzidine + hemalaun.

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reports from animal [3,24] and clinical studies [4,23] describing

increased levels of AGEs in the arterial wall associated with

structural and functional disturbances. We confirmed these

results by using a second, immunohistochemical method, and

found that CML was localised in the intima, media, and

adventitia with prominent stainings in the intimal and

adventitial layers.

It is an open question whether the accumulation of AGEs in

vascular tissue can be attributed mainly to the chronically

increased blood glucose levels and to what extent is elevated

oxidative stress involved in this process. It has been proposed

that persistent hyperglycaemia promotes a pro-oxidant

environment via auto-oxidation of glucose [25] which

exhausts the potential of the endogenous antioxidant system

[9,10]. AGEs also have a causative role in oxidative damage

through binding to the receptor for AGEs (RAGE). The binding

of AGEs to RAGE initiates many of the downstream effects,

including superoxide radical generation and apoptosis [26,27].

In agreement with previous studies [9,28,29], we show that the

susceptibility to oxidation is significantly increased in the

diabetic liver, as evidenced by increased levels of OSI.

Moreover, significantly lower levels of serum TAC in the

untreated diabetic rats reflect a severely compromised

systemic antioxidant response to the increased oxidative

stress. TAC assay is used widely to assess the synergistic

effects of different antioxidants to neutralise free radicals [30].

Our results are in accordance with those reported by other

investigators, demonstrating decreased plasma radical-trap-

ping potential in experimental [28] and clinical diabetes

[10,31].

Certain AGEs (e.g. pentosidine) are directly derived from

the non-enzymatic reactions between proteins and carbohy-

drates, while a combination of glycation and oxidation

reactions is required for the formation of CML [32]. In our

study, vitamin D inhibited the CML deposition in the medial

layer of the aortic wall in the presence of ongoing hypergly-

caemia. This implies that vitamin D is involved in other

important mechanisms of CML formation. To our best of

knowledge, this is the first study demonstrating such an

inhibitory effect of vitamin D on CML accumulation. The

mechanism of this effect remains to be established, but it is

possible that vitamin D interferes with the formation of

reactive oxygen species by upregulation of antioxidant

enzymes [13,33]. Furthermore, in vitro studies have shown

that vitamin D reduces endoplasmic reticulum stress [34] and

downregulates the expression of RAGE [35].

d i a b e t e s r e s e a r c h a n d c l i n i c a l p r a c t i c e 1 0 0 ( 2 0 1 3 ) 2 4 3 – 2 4 9248

The liver is central to the vitamin D metabolism and

circulation in that liver converts absorbed vitamin D into

25(OH)D, the circulating form vitamin D, which is the best

indicator of vitamin D status in the organism [36]. Thus, we

aimed to assess the impact of vitamin D pooled in the liver on

the increased oxidative stress, as seen in the diabetic rats. We

found that supplementation of vitamin D was able to reduce

OSI to the level that of normal rats, clearly demonstrating the

antioxidative potential of vitamin D. These results are in line

with those by Hamden et al. [13] who showed that oxidative

stress in the diabetic liver may be improved by calcitriol, the

active form of vitamin D. Moreover, we found that treatment

of diabetic rats with vitamin D restored the serum TAC to a

level that was significantly higher than that found in control

rats. Detailed explanation to the antioxidative effects of

vitamin D cannot be provided, but these may include

stabilisation of the plasma membrane against lipid peroxida-

tion [12] or upregulation of antioxidant systems, including

GSH, GSH peroxidase, and superoxide dismutase, via its

nuclear receptors [13,33].

Our findings underscore the importance of oxidative and

antioxidative status in the development of diabetic vascular

complications. Indeed, among diabetic populations, a consid-

erable variance in the rates of AGE accumulation, despite

similar blood glucose and HbA1c levels, has been demonstrat-

ed which may be attributed to individual variations in

oxidative stress status [6]. Furthermore, antioxidants are

known to decrease CML formation [37] which, again, empha-

sises the essential role of oxidative stress in the advanced

glycation processes.

In conclusion, the findings of the present study indicate

that vitamin D may have a role in reducing different

diabetes-related complications. Particularly, we demon-

strate that treatment with vitamin D reduces the accumula-

tion of AGEs in the medial layer of the aortic wall and

oxidative stress at a systemic and end organ level. Thus, our

results support the understanding that vitamin D supple-

mentation provides an important protection from the

oxidative damage associated with the development of

diabetic vascular complications.

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgement

This study was supported by the Estonian Science Foundation

grants Nos. 9094 and 8273 and by Target Financing (Nos.

0180001s07 and 0180012s11) by the European Union through

the European Regional Development Fund.

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