β-Glucan administration to diabetic rats alleviates oxidative stress by lowering hyperglycaemia,...
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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
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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: [email protected] (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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