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b-Glucan administration to diabetic rats alleviates oxidativestress by lowering hyperglycaemia, decreasingnon-enzymatic glycation and protein O-GlcNAcylation

Mirjana Mihailovica, Jelena Arambasica, Aleksandra Uskokovica, Svetlana Dinica,Nevena Grdovica, Jelena Markovica, Jelena Bauderb, Goran Poznanovica,Melita Vidakovica,*

aInstitute for Biological Research ‘‘Sinisa Stankovic’’, University of Belgrade, Bulevar despota Stefana 142, 10060 Belgrade, SerbiabCenter for Scientific Research, Teaching and Educational Activities and Human Resources, Clinical Center of Serbia, University of Belgrade,

Dr. Subotica 8, 11000 Belgrade, Serbia

A R T I C L E I N F O A B S T R A C T

Article history:

Received 21 December 2012

Received in revised form

10 April 2013

Accepted 12 April 2013

Available online 16 May 2013

Keywords:

Advanced glycation end products

Antioxidant

b-Glucan-enriched extract

Diabetes mellitus

Hyperglycaemia

O-GlcNAcylation

1756-4646/$ - see front matter � 2013 Elsevihttp://dx.doi.org/10.1016/j.jff.2013.04.005

* Corresponding author. Tel.: +381 11 2078326E-mail address: melita@ibiss.bg.ac.rs (M.Abbreviations: AGE, advanced glycation en

diabetic animal group; ND, non-diabetic animN-acetyl glucosamine; ROS, reactive oxygenSTZ, streptozotocin; UDP-GlcNAc, uridine di

This effect of commercially available b-glucan-enriched extract (BGEE) in streptozotocin-

induced diabetic rats on protein glycation and enzymatic post-translational glycosylation

with O-linked N-acetylglucosamine (O-GlcNAc) groups was examined. The BGEE-promoted

improvement of diabetic hyperglycaemia was accompanied by significantly lower serum

protein glycation. While this revealed an indirect effect of BGEE on protein glycation through

its ability to improve hyperglycaemia, the observed BGEE capability to decrease the forma-

tion of advanced glycation end-products (AGE) in an in vitro glycation process pointed to

its direct glycation-suppressive function in vivo. Compared to untreated diabetic rats,

BGEE-treated diabetic rats displayed lower levels of O-GlcNAc-modified liver and kidney

antioxidant enzymes, Mn- and CuZn superoxide dismutases and catalase, their improved

specific enzymatic activities and increased transcription of genes encoding for them. These

results show that BGEE exerts a normalizing effect on diabetes-linked protein modifications,

adding to the list of beneficial effects it could provide in diabetes management.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

In diabetes, chronic hyperglycaemia induces metabolic abnor-

malities in different cells and tissues (Giacco & Brownlee,

2010). This is characterized by mitochondrial overproduction

of reactive oxygen species (ROS) and pathological levels of

er Ltd. All rights reserved

; fax: +381 11 2761433.Vidakovic).d product; BGEE, b-glucaal group; HBP, hexosamispecies; SDS–PAGE, SDS

phosphate N-acetylgluco

oxidative stress that in due course causes tissue damage

and development of a variety of diabetic complications. Dia-

betic hyperglycaemia promotes changes in the enzymatic

post-translational glycosylation of proteins with O-linked

N-acetylglucosamine (O-GlcNAc) groups (Karunakaran &

Jeoung, 2010). Under normal circumstances, 2–5% of the

.

n-enriched extract; BSA, bovine serum albumin; CAT, catalase; D,ne biosynthetic pathway; i.p., intraperitoneal; O-GlcNAc, O-linked–polyacrylamide gel electrophoresis; SOD, superoxide dismutase;samine.

J O U R N A L O F F U N C T I O N A L F O O D S 5 ( 2 0 1 3 ) 1 2 2 6 – 1 2 3 4 1227

glucose imported into cells is converted through the hexosa-

mine biosynthetic pathway (HBP) to uridine diphosphate N-

acetylglucosamine (UDP-GlcNAc), which is used to catalyze

the addition of a single O-GlcNAc group to serine or threonine

residues of intracellular proteins and thereby modify cell sig-

nalling activities. Being in part affected by the concentration

of produced UDP-GlcNAc and depending on the glucose

concentrations in cells, O-GlcNAc modifications adjust

cellular activity to glucose availability (Parker, Lund, Taylor,

& McClain, 2003). Consequently, in diabetes alterations in

O-GlcNAc modifications significantly affect normal cell sig-

nalling pathways and functioning (Issad & Kuo, 2008). Incor-

porated in the adverse effects of hyperglycaemia is also the

increased formation of advanced glycation end products

(AGE) by the non-enzymatic addition of glucose to proteins

through a series of reactions between the carbonyl group on

reducing sugars and protein amino groups (Rahbar & Figarola,

2002). While the initial reaction in this process, the formation

of fructose–lysine products is reversible, subsequent rear-

rangements of the protein side chain lead to the formation

of irreversible protein AGE products and reactive intermedi-

ates, including ROS (Gutierrez, Baez, Cortez, & Cardenas,

2011). AGE are generated through normal metabolism and

ageing, however in diabetes, excess glucose promotes AGE

formation above normal levels, inducing cumulative, long-

term changes that contribute to altered functioning at the cell

and tissue levels (Rahbar, 2007).

Acute hyperglycaemia in type 1 diabetes is treated by

direct administration of insulin. In patients with type 2 diabe-

tes, blood sugar levels can be regulated by oral hypoglycaemic

therapy. The dietary intake of b-glucans, a diverse group of

polysaccharides of D-glucose monomers linked by b-glyco-

sidic bonds and found in cell walls of fungi, yeast, oat, barley

and bacteria (Chen & Seviour,2007; Kuda, Toshiki Enomoto, &

Yano, 2009), was found to provide considerable potential for

diabetes management. By delaying stomach emptying,

decreasing appetite and reducing food intake so that dietary

glucose is absorbed more gradually (Saris, 2003), the b-glucans

attenuate the postprandial rise in blood glucose levels (Lo,

Tsai, Wasser, Yang, & Huang, 2006; Peng et al., 2012).

While the dietary intake of b-glucans benefits the treat-

ment of diabetes, the mechanisms behind their effects re-

main to be elucidated. Previously, we reported that the

administration of a commercially available grain food supple-

ment enriched with b-glucans (BGEE) to streptozotocin

(STZ)-induced diabetic rats, resulted in a systemic adjustment

of redox disturbance and reduction of DNA damage through

free radical scavenging and antioxidant activities of BGEE.

We proposed that the consequent stimulation of cellular

pro-survival mechanisms, observed as increased Akt kinase

activity and decreased pro-caspase-3 degradation, could

potentially improve the organism’s resistance to the onset

of different diabetic complications (Mihailovic et al., 2013).

The aim of the present study was to examine further the ef-

fects of administration of BGEE on the molecular events that

contribute to disrupted cell and tissue functioning in STZ-

induced diabetic rats. In the present work we focused on

hyperglycaemia-induced protein modifications: total AGE

formation, the non-enzymatic glycation status of serum pro-

teins, and the enzymatic post-translational O-GlcNAcylation

of liver and kidney antioxidant enzymes. Our results reveal

additional effects of b-glucans on hyperglycaemia-induced

pathological mechanisms manifested as the described modi-

fications of proteins.

2. Materials and methods

2.1. Materials

The b-glucan-enriched extract (BGEE) from cereal grains is for

human consumption and is produced by Essentia s.r.o.,

Poprad, in the Slovak Republic, using stringent quality control

to ensure stable purity and potency of the standardized prep-

aration. The BGEE examined herein was supplied by Agrobiv

d.o.o. (Rijeka, Croatia). The BGEE is a concentrate of coloid

micronized b-glucan (P80%). Based on methylation analysis,

the material was characterized as a 1,3 b-D-glucan with a

small number of 1,6 and 1,4 linked glucose residues. The

maximal protein, fat and sodium contents in the standard-

ized dry substance are 20; 10 and 15 mg/g, respectively.

Routine microbiological testing did not reveal the presence

of any contaminants. The quality of the BGEE is covered by

the Essentia certificate. Streptozotocin (STZ) was obtained

from MP Biomedicals (Solon, OH, USA).

2.2. Animals

Experiments were performed on 2.5-month-old adult albino

Wistar rats weighing 220–250 g. All animal procedures were

approved by the Committee for Ethical Animal Care and Use

of the Institute for Biological Research, Belgrade, which acts

in accordance with the Guide for the Care and Use of Labora-

tory Animals, published by the US National Institute of Health

(NIH Publication No. 85/23 revised in 1986).

2.3. Experimental design

The experimental model of diabetes mellitus in rats was in-

duced by intraperitoneal (i.p.) injections of STZ (40 mg/kg/

day) for five consecutive days. STZ was dissolved immediately

before use in sodium citrate buffer (0.1 M, pH 4.5). The blood

glucose level was measured 24 h after the last STZ injection.

Diabetic condition was defined by a fasting blood glucose

level that exceeded 20 mmol/L. Blood samples were obtained

from the tail vein from rats that fasted overnight. Glucose was

measured with a blood glucose meter (Accu-Chech Active,

Roche Diagnostics Scientific Office, Cairo, Egypt). In our

hands, the described treatment with multiple low doses of

STZ did not cause animal death. The rats were randomly di-

vided into four groups: (i) the non-diabetic group (ND) which

received an equivalent volume of citrate buffer (i.p.) for five

consecutive days (n = 7); (ii) the BGEE-treated non-diabetic

group which received a daily dose of 80 mg BGEE suspended

in water/kg (i.p.) for 4 weeks (n = 7); (iii) the diabetic group

(D) which received STZ as described above; these rats were

left untreated throughout the four week period (n = 8); (iv)

the diabetic group, treated with BGEE (n = 8) which received

a daily dose of BGEE as described above, lasting four weeks

and starting from the last day of STZ administration.

1228 J O U R N A L O F F U N C T I O N A L F O O D S 5 ( 2 0 1 3 ) 1 2 2 6 – 1 2 3 4

2.4. Determination of general indicators of diabetes

The serum was obtained after blood clotting and centrifuga-

tion at 2000·g for 10 min. Blood glucose concentrations

were measured using serum obtained from rats at the

beginning of the experimental procedure, 24 h after the

administration of the last STZ injection, and after 4 weeks

of BGEE treatment, using a commercial kit (Gluco-quant

Glucose/HK, Boehringer Mannheim, Germany). Haemoglobin

(Hb) was determined according to Drabkin and Austin

(1935). Glycated Hb (GlyHb) was determined by the colori-

metric assay according to Parker, England, DaCosta, Hess,

and Goldstein (1981).

2.5. Detection of glycated serum proteins by thefructosamine assay

The non-enzymatic glycation of serum proteins was mea-

sured according to Johnson, Metcalf, and Baker (1983). Ali-

quots of serum (50 ll) were added to 450 ll of 100 mM

carbonate buffer (pH 10.8) containing 0.5 mM nitro blue tetra-

zolium (NBT). The samples were incubated for 1 h at 37 �C.

The reduction of NBT was measured at 595 nm.

2.6. Formation of fructosamines during in vitro glycation

Reaction mixtures of the bovine serum albumin (BSA)

(20 mg/ml)/glucose (1 M)/fructose (1 M) system (1 ml), dis-

solved in 20 mM Tris–HCl (pH 7.4), in the presence or ab-

sence of BGEE (50–200 mg/ml) were incubated at 37 �C for

7 days (Brownlee, Vlassara, Kooney, Ulrich, & Cerami,

1986). The incubated solution was mixed with 0.5 mM NBT

(0.5 ml) dissolved in 100 mM carbonate puffer pH 10.8.

The reduction of NBT was measured at 595 nm according

to the formula: % Inhibition ¼ ððAblank� AtestÞ=AblankÞ � 100.

Ablank is the absorbance of the BSA/glucose system without

the test sample; Atest is the absorbance of BSA/glucose sys-

tem in the solution with the test sample.

2.7. AGE formation in vitro

BSA (20 mg/ml) was incubated with glucose (1 M) and fructose

(1 M) in Tris–HCl pH 7.4 containing 0.02% sodium azide. All

of the reagents and the BGEE (50–200 mg/ml) were filtered

through filter paper and each of the mixtures was incu-

bated at 37 �C for 7 days. The formation of AGE was mea-

sured by the fluorescence intensity at an excitation

wavelength of 330 nm and an emission wave-length of

410 nm with a Luminescence spectrometer LS50B (Perkin–El-

mer Ltd., Buckinghamshire, England) (Lee, Yim, Chock, Yim,

& Kang, 1998).

2.8. Preparation of liver and kidney homogenates

The liver and kidneys from all experimental animals were ex-

cised and homogenized in 0.25 M sucrose, 0.1 M EDTA and

0.05 M Tris–HCl, pH 7.4. After sonication, the homogenate

was centrifuged at 100,000·g in a Beckmann rotor Ti 50 for

90 min. Aliquots of the supernatants were stored at �80 �C

and used for the determination of CAT and SOD activities

and for detection of O-GlcNAc-modified proteins.

2.9. Immobilization of O-GlcNAc-modified proteins withwheat germ agglutinin

To detect O-GlcNAc-modified proteins, 100 lg of liver and kid-

ney homogenates in a total volume of 100 ll lysis buffer, were

first pre-cleared with 50 ll of 50% (v/v) of non-conjugated aga-

rose beads for 2 h at 4 �C to eliminate non-specific interac-

tions of proteins with the agarose beads (Gandy, Rountree, &

Bijur, 2006). The agarose beads were collected by centrifuga-

tion in a microcentrifuge (20,817·g for 1 min). The superna-

tant was transferred to a new reaction tube and incubated

with 50 ll of 50% (v/v) wheat germ agglutinin (WGA)-

conjugated agarose beads, (Sigma–Aldrich, St. Louis, MO,

USA), for 20 h at 4 �C. The WGA-conjugated agarose beads

were collected by centrifugation and washed three times in

lysis buffer. Immobilized proteins were eluted by boiling in

Laemmli buffer (in 30 ll for 5 min). Equal aliquots of eluted

proteins of all four experimental groups were separated by

12% SDS–polyacrylamide gel electrophoresis (SDS–PAGE).

The protein gels were subjected to silver staining or were

electroblotted to PVDF membranes (Hybond-P, Amersham

Pharmacia Biotech, Uppsala, Sweden) and analyzed by immu-

noblot analysis.

2.10. SDS–PAGE and immunoblot analysis

Twenty microgram of liver and kidney homogenates were

separated by sodium dodecyl sulphate–polyacrylamide gel

electrophoresis (SDS–PAGE) (Laemmli, 1970). Immunoblot

analysis was performed by the procedure of Towbin,

Staehelin, and Gordon (1979) using goat-CuZn superoxide dis-

mutase (CuZnSOD) and rabbit Mn superoxide dismutase

(MnSOD) (Santa Cruz Biotechnology, Santa Cruz, CA, USA)

and catalase (CAT) (Abcam, Cambridge Science Park, Cam-

bridge, UK) polyclonal antibodies. Immunoreactive bands

were identified by an enhanced chemiluminescence (ECL)

detection system (Santa Cruz Biotechnology, Santa Cruz, CA,

USA) according to the manufacturer’s instructions.

2.11. Determination of SOD and CAT activities

Liver and kidney homogenates were used for the determina-

tion of SOD and CAT. Total SOD (tot SOD) activity was mea-

sured by the epinephrine method (Misra & Fridovich, 1972),

and was expressed as U/g wet mass. MnSOD activity was per-

formed after preincubation with 8 mmol/L KCN; CuZnSOD

activity was calculated from the difference between total

SOD and MnSOD activities. CAT activity was determined

according to Beutler (1982) by the rate of H2O2 decomposition

and expressed as lmol H2O2/min/g wet mass.

2.12. Real time quantitative (q) PCR (RT-qPCR)

Total RNA was isolated from liver and kidney tissue

using RNeasy Mini Kit (Qiagen, Hilden, Dusseldorf, Germany)

according to the protocol recommended by the manufacturer.

mRNA levels were quantitatively determined on an ABI Prism

Fig. 1 – The effect of BGEE administration on fasting blood

glucose concentration and serum protein glycation. (A)

Blood glucose level; (B) level of glycated haemoglobin; (C)

serum protein glycation levels. ND – non-diabetic control

group; ND + BGEE � BGEE-treated non-diabetic group; D–

diabetic group; D + BGEE � BGEE-treated diabetic group.

The values are means ± S.E.M. from three separate

J O U R N A L O F F U N C T I O N A L F O O D S 5 ( 2 0 1 3 ) 1 2 2 6 – 1 2 3 4 1229

7000 Sequence Detection system (Applied Biosystems, Foster

City, CA, USA) using SYBR-green technology. The sequences

of the utilized primers (Invitrogen) were: 5 0-CAG ATC ATG

CAG CTG CAC CA-3 0 and 5 0-TCA GTC CAG GCT GAA GAG

CA-3 0 for forward and reverse primers of the rat MnSOD gene,

respectively; 5 0-GCA GAA GGC AAG CGG TGA AC-3 0 and

5 0-CGG CCA ATG ATG GAA TGC TC-50 for forward and reverse

primers of the rat CuZnSOD gene, respectively; 5 0-GCG AAT

GGA GAG GCA GTG TAC-3 0 and 5 0-GAG TGA CGT TGT CTT

CAT TAG CAC TG-3 0 for forward and reverse primers of the

rat CAT gene, respectively. To perform the real time PCR reac-

tion, 10 ll Maxima SYBR Green/ROX qPCR Master Mix (2·)

were mixed with 400 ng of cDNA template. Program of the

quantitative RT-PCR comprised an initial step at 50 �C for

2 min, followed by a step of initial denaturation at 95 �Cfor 10 min and a subsequent 2-step PCR program at 95 �C for

15 s and 58 �C for 60 s for 40 cycles. PCR reactions were carried

out in triplicates. Negative controls lacking the template were

in use in all RT-qPCR reactions. The results were presented as

2�dCt, where dCt was difference between Ct values of specific

gene and endogenous control (b-actin).

2.13. Statistical analysis

Mean and standard error values (S.E.) were determined for all

the parameters studied. Results were statistically analyzed by

analysis of variance (ANOVA). Duncan’s multiple range test

was performed to determine significant differences between

experimental groups.

experiments. Means that do not share a common letter are

significantly different between groups (p < 0.05).

Fig. 2 – The effect of BGEE on non-enzymatic glycation

in vitro. (A) Measurement of fructosamines formation in the

presence of different BGEE concentration (50–200 mg/ml). (B)

Formation of AGE in the BSA/glucose/fructose system was

measured in the presence of different concentrations of

BGEE (50–200 mg/ml). The values are means ± S.E.M. from

three separate experiments.

3. Results

3.1. The effect of BGEE administration on thenon-enzymatic glycation of proteins

The blood glucose concentration was significantly increased

in the diabetic animal group (38 mmol/L) (Fig. 1A). After the

treatment of diabetic rats with BGEE, the glucose concentra-

tion decreased to 19.9 mmol/L.

The increased concentration of glucose in the diabetic

group promoted non-enzymatic protein glycation, revealed

as 2-fold increases in glycohaemoglobin (GlyHb) (Fig. 1B)

and concentrations of glycated serum proteins (Fig. 1C) com-

pared to the intact control group. After the treatment of

diabetic rats with BGEE, the concentrations of GlyHb and gly-

cated serum proteins decreased, but remained 1.2-fold and

1.4-fold above the control values, respectively. Treating

healthy rats with BGEE did not significantly affect the concen-

trations of GlyHb and glycated serum proteins.

Examination of the in vivo outcome of BGEE administration

on protein glycation was accompanied by investigations of

the in vitro effect of BGEE on the formation of fructosamines

during glycation. We followed the reduction of nitro blue tet-

razolium (NTB) after seven days of incubation in a BSA/

glucose/fructose system. As can be seen on Fig. 2A, the reduc-

tion of NTB, which is directly related to the ability of BGEE to

suppress glycation, was BGEE dose-dependent. The ability of

BGEE to affect the formation of total AGE was assessed

in vitro by monitoring the production of fluorescent products

and the fluorescence intensity attributed to the formation of

glycophore groups, a feature of AGE (Gutierrez et al., 2011).

1230 J O U R N A L O F F U N C T I O N A L F O O D S 5 ( 2 0 1 3 ) 1 2 2 6 – 1 2 3 4

The results presented on Fig. 2B show that BGEE significantly

suppressed the fluorescence intensity in a dose-dependent

manner.

3.2. The effect of BGEE on O-GlcNac protein modificationsin the liver and kidneys of diabetic rats

O-GlcNAc-modified proteins eluted from WGA-conjugated

agarose beads from liver and kidneys of control and diabetic

rats are shown on Fig. 3. Visual inspection of O-GlcNAc-

modified protein profiles obtained after SDS–PAGE from

the liver and kidneys of diabetic rats reveals qualitative

differences when compared to matching samples from

the non-diabetic group (Fig. 3A). An overall relative increase

in O-GlcNAcylated protein levels is observed in diabetic rats,

notably in the liver. The treatment of diabetic rats with BGEE

promoted an overall relative decrease of O-GlcNAc-modified

protein levels in the liver and kidneys (Fig. 3A). As the O-Glc-

NAc-modified protein profiles from non-diabetic and BGEE-

treated non-diabetic rats were apparently identical (Fig. 3A),

it can be concluded that the administration of BGEE to control

rats does not significantly influence protein O-GlcNAc

modification.

Immunoblot analysis of the O-GlcNAc-protein fractions

(Fig. 3B) revealed that diabetes promoted the post-

Fig. 3 – The effect of the BGEE treatment on the level of

O-GlcNAc-modified proteins in liver and kidney. (A)

Electrophoretic profiles (silver staining) of O-GlcNAc-

modified proteins from liver and kidney of control and

diabetic rat homogenates eluted from WGA-conjugated

agarose beads. (B) Immunoblot analysis with anti-CAT,

anti-CuZnSOD and anti-MnSOD antibodies of O-GlcNAc-

modified proteins from liver and kidneys of control and

diabetic rats eluted from WGA-conjugated agarose beads.

ND – non-diabetic control group; ND + BGEE � BGEE-treated

non-diabetic group; D – diabetic group; D + BGEE � BGEE-

treated diabetic group.

translational O-GlcNAc-modification of antioxidant enzymes.

Both non-diabetic and BGEE-treated non-diabetic groups

(Fig. 3B) were completely devoid of O-GlcNAc-modified CAT

and CuZnSOD and MnSOD species. Treatment of diabetic rats

with BGEE caused a significant decrease in O-GlcNAc-MnSOD

in the liver and complete absence of O-GlcNAc-modified

MnSOD in diabetic kidney (Fig. 3B). In comparison, the

BGEE treatment exerted a less pronounced effect on O-

GlcNAc-CuZnSOD levels in the liver and kidneys. Treatment

of diabetic rats with BGEE brought about a slight reduction

of O-GlcNAc-CAT in the liver and a more pronounced reduc-

tion in the kidneys. Treating healthy rats with BGEE did not

promote changes in O-GlcNacylation (Fig. 3B).

3.3. The effect of BGEE administration on antioxidantenzyme activities in the liver and kidneys under diabeticconditions

As can be seen on Fig. 4A, the activities of total SOD, CuZn-

SOD and MnSOD were 1.4-fold lower in the livers of diabetic

rats. The treatment of diabetic rats with BGEE promoted in-

creased activities of these enzymes to nearly control levels

(Fig. 4A). The highest activities of total SOD, CuZnSOD and

MnSOD were observed in BGEE-treated non-diabetic rats

(1.2-fold increases for total SOD and CuZnSOD, and a 1.5-

fold increase for MnSOD compared to their respective con-

trol levels). In diabetic rats, liver CAT activity was 1.9-fold

lower compared to the appropriate control. In BGEE-treated

diabetic rats, CAT activity was slightly improved but re-

mained 1.5-fold lower than in the control (Fig. 4A). There

was no statistically significant difference between CAT

activities in the BGEE-treated non-diabetic rats and intact

controls.

In the kidneys of diabetic rats, the activities of total SOD

and CuZnSOD were 1.2-fold, and of MnSOD 1.4-fold lower

than in control rats (Fig. 4B). Treating diabetic rats with

BGEE recovered SOD activities to their respective control

levels. The activity of CAT in the kidney was about 2-fold

lower in diabetic rats. Although the treatment of diabetic

rats with BGEE increased CAT activity, it remained 1.5-fold

lower than in the control (Fig. 4B). Treating healthy rats

with BGEE did not promote changes in kidney antioxidant

enzyme activities.

3.4. The effect of BGEE administration to diabetic rats onantioxidant enzyme transcription in the liver and kidneys

Under diabetic conditions, the levels of expression of genes

encoding for MnSOD, CuZnSOD and CAT in the liver de-

creased 1.4-, 1.5- and 2.2-fold, respectively, compared to the

control (Fig. 5A). The BGEE treatment restored MnSOD and

CuSOD expression to control levels, while CAT mRNA re-

mained 1.5-fold lower than in the control group. There was

no statistically significant difference between the mRNA lev-

els in the BGEE-treated non-diabetic rats and intact controls.

Compared to the control group, in the diabetic group kidney

MnSOD, CuZnSOD and CAT mRNA levels were 3.4-, 1.8- and

2.3-fold lower, respectively (Fig. 5B). While the treatment of

diabetic rats with BGEE promoted increased expression of

Fig. 4 – The effect of BGEE administration on liver (A) and kidney (B) antioxidant enzyme activities. ND – non-diabetic control

group; ND + BGEE � BGEE-treated non-diabetic group; D – diabetic group; D + BGEE � BGEE-treated diabetic group. Values are

means ± S.E.M. for the indicated number of animals for each group. Means that do not share a common letter are significantly

different between groups (p < 0.05).

J O U R N A L O F F U N C T I O N A L F O O D S 5 ( 2 0 1 3 ) 1 2 2 6 – 1 2 3 4 1231

all three genes, they remained lower than in control rats (1.6-,

1.3- and 1.4-fold lower, respectively; Fig. 5B). Administration

of BGEE to control rats did not significantly influence the

examined mRNA levels.

4. Discussion

The direct and indirect consequences of hyperglycaemia

manifest as a sequence of events in which oxidative stress as-

sumes an important role in causing significant disruptions of

normal cellular processes. In diabetes, glucose oxidation is

the primary source of free radicals, whereas increased AGE

generation under hyperglycaemic conditions in the course

of which toxic ROS intermediates are released represents a

secondary source of pro-oxidant signals (Gutierrez et al.,

2011; Rahbar, 2007; Rahbar & Figarola, 2002). In diabetes,

increased concentrations of free radicals, together with ele-

vated AGE levels, trigger via the RAGE axis cycles of oxidative

injury/upregulation of inflammatory signalling pathways and

stimulation of pro-apoptotic signals (Yan, Ramasamy, &

Schmidt, 2010). This complex process lies at the root of an

ongoing pathogenic cell dysfunction that leads to diverse dia-

betic complications, such as retinopathy, neuropathy,

nephropathy and osteoporosis (Bhatti et al., 2005; Mendez,

2003; Resmi, Pekcetin, & Guner, 2001). Consequently, it has

been argued that therapeutic interventions aimed at prevent-

ing the formation of AGE have a vital role in delaying the

development of diabetic-complications (Cohen, Ziyadeh, &

Chen, 2006). In our study, BGEE administration to diabetic rats

substantially lowered hyperglycaemia and the levels of gly-

cated serum proteins and haemoglobin. This in vivo effect of

the administered BGEE is consonant with the finding that

BGEE decreases the formation of fructosamines and total

AGE in an in vitro glycation process. We therefore conclude

that BGEE can bring about a decrease of AGE levels indirectly,

through its ability to lower hyperglycaemia, and by a direct ef-

fect on the process of AGE formation. These findings indicate

that BGEE can play an important supportive role in therapeu-

tic interventions targeting the AGE–RAGE axis.

Other indicators that point to the alleviation of aberrant

cell functioning in BGEE-treated diabetic rats were: upregu-

lated antioxidant enzyme gene transcription, lower levels of

enzymatically modified O-GlcNAc-antioxidant enzymes and

their improved enzymatic activities in the liver and kidneys.

In the uncontrolled diabetic state, lower activities of liver

and kidney antioxidant enzymes MnSOD, CuZnSOD and

CAT, were in part the result of Mn-, CuZnSOD and CAT gene

downregulation. While low levels of oxidative stress elicit

an adaptive response which includes the induction of antiox-

idant genes, a chronic shift in the intracellular redox status to

more oxidant conditions is associated with decreased

DNA-binding activities of a number of transcription factors

that participate in the regulation of antioxidant genes (Morel

& Barouki, 1999). O-GlcNAcylation is a dynamic and reversible

post-translational modification analogous to phosphorylation

which affects normal protein functions at several levels,

including DNA binding (Gao, Miyazaki, & Hart, 2003), pro-

tein–protein interactions (Yang et al., 2001) and enzymatic

activities (Parker et al., 2003). We propose that the administra-

tion of BGEE to diabetic rats lifted a repressive oxidative

impairment of transcription factor functions, as well as the

repressive effect of post-translational O-GlcNAcylation of pro-

teins that mediate antioxidant gene regulation.

The ability of O-GlcNAcylation to inhibit the enzymatic

activity of CuZnSOD was documented earlier (Oda et al.,

1994), suggesting that the O-GlcNAcylation of antioxidant en-

zymes was responsible for the observed lowering of their

enzymatic activities in diabetic rats. Recently, it was sug-

gested that the hyperglycaemia-induced activation of the

hexosamine pathway, the resulting increased O-GlcNAc levels

and inhibition of cytosolic enzymatic activity are caused by a

hyperglycaemia-induced overproduction of superoxide in the

mitochondria (Zeidan & Hart, 2010). In light of the obtained

prominent increases in Mn- and CuZnSOD activities and

Fig. 5 – The effect of BGEE administration on the relative transcription level of antioxidant enzymes. Liver (A) and kidney

(B) antioxidant enzyme’s mRNA determined by RT-qPCR. ND – non-diabetic control group; ND + BGEE � BGEE-treated

non-diabetic group; D – diabetic group; D + BGEE � BGEE-treated diabetic group. Values are means ± S.E.M. for the indicated

number of animals for each group. Means that do not share a common letter are significantly different between groups

(p < 0.05).

1232 J O U R N A L O F F U N C T I O N A L F O O D S 5 ( 2 0 1 3 ) 1 2 2 6 – 1 2 3 4

associated decline in O-GlcNAcylation (notably of MnSOD in

the liver) after BGEE administration to diabetic rats, it can

be assumed that the suppression of SOD O-GlcNAcylation

and resulting increased enzymatic activity, and improved

superoxide clearance were mutually supportive processes.

This is mirrored in our previous report where we described

a link between decreased protein O-GlcNAcylation and an im-

proved redox status in diabetic rats treated with a-lipoic acid

(Mihailovic et al., 2012). Being a mechanism through which

the nutritional environment of the cell regulates cellular

activities, the process of protein O-GlcNAcylation depends

on the availability of glucose (Wells, Vosseller, & Hart, 2001).

Protracted elevation of O-GlcNAcylation has been implicated

as a pathogenic contributor to glucose toxicity and insulin

resistance in diabetes-related complications (Karunakaran &

Jeoung, 2010). In BGEE-treated diabetic rats, the ability of BGEE

to lower hyperglycemia effectively lifted the negative feed-

back mediated by elevated glucose, by lowering O-GlcNAcyla-

tion-linked signalling.

5. Conclusion

BGEE administration to diabetic rats improves hyperglyca-

emia which in turns promotes a significant reduction in

systemic levels of non-enzymatically glycated protein spe-

cies (AGE precursors), and lowers the ratios of O-GlcNAc-

associated antioxidant protein species. As a result of

the partial suppression of O-GlcNAc-modifications of the

J O U R N A L O F F U N C T I O N A L F O O D S 5 ( 2 0 1 3 ) 1 2 2 6 – 1 2 3 4 1233

antioxidant enzymes SOD and CAT, as well as upregulation of

their genes, antioxidant enzyme activities were significantly

increased in diabetic rats after BGEE application. The result-

ing improvement of antioxidant enzyme activities is expected

to play a vital role in alleviating the oxidative stress that

accompanies diabetes. This should impact positively on vari-

ous detrimental cellular functions, delaying the progression

of the pathophysiological complications in diabetes. These re-

sults point to the considerable potential of b-glucans in diabe-

tes management.

Acknowledgements

This work was supported by the Ministry of Education,

Science and Technological Development of the Republic of

Serbia, Grant No. 173020.

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