Professional Documents
Culture Documents
10.1016/j.nutres.2017.07.012: Nutrition Research
10.1016/j.nutres.2017.07.012: Nutrition Research
10.1016/j.nutres.2017.07.012: Nutrition Research
Fish oil emulsion supplementation might improve quality of life of diabetic
patients due to its antioxidant and antiinflammatory properties
PII: S0271-5317(16)30819-3
DOI: doi: 10.1016/j.nutres.2017.07.012
Reference: NTR 7788
Please cite this article as: Laubertová Lucia, Koňariková Katarı́na, Gbelcová He-
lena, Ďuračková Zdeňka, Muchová Jana, Garaiova Iveta, Žitňanová Ingrid, Fish
oil emulsion supplementation might improve quality of life of diabetic patients due
to its antioxidant and antiinflammatory properties, Nutrition Research (2017), doi:
10.1016/j.nutres.2017.07.012
This is a PDF file of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof
before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that
apply to the journal pertain.
ACCEPTED MANUSCRIPT
Fish oil emulsion supplementation might improve quality of life of diabetic patients due
T
Muchováa, Iveta Garaiovac, Ingrid Žitňanováa*
P
RI
a
Institute of Medical Chemistry, Biochemistry and Clinical Biochemistry, Faculty of
SC
Medicine, Comenius University, Sasinkova 2, 813 72 Bratislava, Slovakia
b
Institute of Medical Biology, Genetics and Clinical Genetics, Faculty of Medicine,
NU
Comenius University, Sasinkova 4, 813 72 Bratislava, Slovakia
MA
c
Research and Development Department, Cultech Ltd, Port Talbot, SA12 7BZ United
Kingdom
ED
Email adresses:
PT
Ingrid Žitňanová
Tel: + 421 902 840 242, + 421 2 59357 559, Fax: + 421 2 59357 557
e-mail: ingrid.zitnanova@fmed.uniba.sk
1
ACCEPTED MANUSCRIPT
List of abbreviations
8-oxo-G; 8-oxo-guanin
T
AGEs; advanced glycation end products
P
DHA; docosahexaenoic acid
RI
DM; diabetes mellitus
SC
EPA; eicosapentaenoic acid
IL-8; interleukin-8
ED
LPS; lipopolysaccharide
2
ACCEPTED MANUSCRIPT
Abstract
T
and neuropathy, are a significant cause of increased morbidity and mortality among people
P
with diabetes. Previous studies have confirmed that hyperglycemia has pro-oxidative and
RI
proinflammatory properties which cause diabetic complications. We hypothesized that
SC
supplementation of fish oil emulsion (FOE), rich in omega-3 polyunsaturated fatty acids
NU
to specific properties of fish oil emulsion. Omega-3 PUFA have a wide range of biological
MA
effects. In this project we have examined the potential protective effect of the fish oil
FOE for 72 h. We have focused on specific markers of oxidative stress (antioxidant capacity;
superoxide dismutase (SOD) activity; oxidative damage to DNA, proteins and lipids) and
CE
significantly increased antioxidant capacity of cells as well as SOD activity and significantly
reduced TNF, IL-6, IL-8 and MCP-1 release. No effect was observed on oxidative damage to
DNA, proteins and lipids. Our results indicate that FOE can reduce hyperglycemia-induced
Keywords
3
ACCEPTED MANUSCRIPT
1. Introduction
Diabetes mellitus (DM) is one of the major debilitating human metabolic diseases at present.
T
It is a group of human metabolic diseases with characteristic symptoms of hyperglycemia,
P
chronic inflammation and insulin resistance [1]. The chronic hyperglycemia in diabetes is
RI
associated with different mechanisms such as activation of protein kinase C, the polyol and
SC
hexosamine pathways and advanced glycation end products (AGEs) formation. All of these
NU
and glycation stress, promote reactive oxygen species (ROS) accumulation [2].
MA
It is suggested that oxidative stress represents a major pathophysiological link between
ED
progression of diabetes mellitus and the onset of diabetic complications. These complications
affect many tissues and organs, causing retinopathy, nephropathy, neuropathy, cardiovascular
PT
diseases, peripheral vascular diseases, stroke and periodontal pathologies [3]. Factors that
may contribute to increased oxidative stress in diabetes mellitus include increased formation
CE
of ROS such as superoxide and hydrogen peroxide, increased protein carbonylation including
AC
protein glycation (gluco-oxidation) and lipid peroxidation, increased oxidative DNA damage
and antioxidant deficiencies [4]. In addition, it has been reported that not only oxidative stress
but also inflammation plays a central role in diabetic tissue damage [1]. Elevated levels of
pro-inflamatory cytokines have been reported in various diabetic and insulin resistant states
[5]. The generation of free radicals, induced by hyperglycemia, stimulated the production of
interleukin 8 (IL-8) and monocytic chemotactic protein-1 (MCP-1) as well as the expression
4
ACCEPTED MANUSCRIPT
Alternative approaches based on diet therapy have been of interest in many laboratories.
Accordingly, we have focused on the possible benefit of a fish oil emulsion (FOE) rich in
T
studies have demonstrated that omega-3 polyunsaturated fatty acids (PUFA), specifically
P
EPA and DHA can suppress inflammation and have a beneficial role in a variety of
RI
inflammatory human diseases, including diabetes, atherosclerosis, asthma, and arthritis.
SC
Omega-3 PUFA have a wide range of biological effects, including benefits on lipoprotein
NU
markers, cytokine production, coagulation, and fibrinolysis [4]. Omega-3 PUFA have also
MA
demonstrated beneficial effects in reducing oxidative stress and improving the antioxidant
system. Pre-emulsification of the fish oil leads to increased absorption of fatty acids,
ED
particularly EPA and DHA, compared with the non-emulsified form of the fish oil [7].
PT
mechanisms and diabetic complications. The pathogenesis of diabetic complications has not
CE
been sufficiently clarified yet. Successful therapy depends on detailed knowledge of the role
AC
Our in vitro experiments were based on previous studies reporting an important role of
hypothesized that the fish oil emulsion, rich in omega-3 PUFA, has a potential protective
5
ACCEPTED MANUSCRIPT
T
2. Methods and materials
P
RI
2.1. Cell culture
SC
The monocyte cells U937 (American Type Culture Collection, Manassas, USA) were
NU
cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum,
MA
100 U/mL penicillin and 100 µg/mL streptomycin (Sigma-Aldrich, Switzerland) at 37 °C in a
were incubated in a high glucose medium. The cells (5 x 105 cells/mL) were cultured under
AC
or presence of the fish oil emulsion (10 – 250 μg/mL) and in PBS for 24, 48 and 72 h.
Mannitol (35 mmol/L, Sigma-Aldrich, Switzerland) was used as an osmolarity control. Fish
oil emulsion (FOE) was obtained from Obsidian Research Limited (currently Cultech
Limited), Port Talbot, UK. The composition of fish oil emulsion was: 45% fish oil (8.1%
EPA (20:5) 5.9% DHA (22:6)), 28% sugar, 25% water, 0.6% emulsifiers, 0.5% natural lemon
oil, 0.5% antioxidant (50% natural mixed tocopherols), 0.2% preservative (potassium
sorbate), 0.2% citric acid. Fish oil emulsion was diluted with phosphate-buffered saline (PBS,
6
ACCEPTED MANUSCRIPT
T
MTT assay was used to evaluate cell proliferation (MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-
P
diphenyltetrazolium bromide) [9]. The procedure by Price and McMillan was used to convert
RI
results of MTT assay to cell number [10]. After incubation of cells with/without FOE and
SC
PBS (24, 48, and 72 h) the medium was removed and the cells were rinsed with PBS. To
prepare cell lysates, sonification was used with 3 cycles (5s on / 5s off) at 50% power at 4 °C.
NU
Lysates were cleared by centrifugation, and the supernatant was aliquoted and stored at
MA
−80°C. Micro BCA Protein Assay Kit (Thermo Scientific, USA) was used to determine the
Antioxidant capacity of cells was determined in cell lysates by the TEAC method (Trolox
CE
Equivalent Antioxidant Capacity method) [11]. The blue-green radical cation was generated
AC
antioxidants into colorless form. The antioxidant capacity of the cells was measured at 735
Concentration of protein carbonyls in cell lysates was measured by ELISA [12]. The cell
7
ACCEPTED MANUSCRIPT
Switzerland) with spectrophotometric detection at 492 nm. Results are expressed in nmol
T
carbonyls/mg proteins.
P
RI
2.6. Isoprostanes
SC
Isoprostane levels were determined in cell lysates by the 8-Isoprostane ELISA Kit (Cayman
NU
Chemical, USA) according to the manufacturer´s protocol. Results are expressed in pg/mg
MA
proteins.
ED
SOD activity was determined in cell lysates by the superoxide dismutase (SOD) activity
activity was expressed in U/mg proteins, where 1U SOD is defined as 50% inhibition of
AC
Cells (3 x 105 cells/mL) were cultured in normo- or hyperglycemic conditions and treated
with/without FOE (10 – 250 μg/mL) and PBS for 24, 48 and 72 h. Oxidative damage to DNA
8
ACCEPTED MANUSCRIPT
Sigma-Aldrich, Switzerland) which can cleave oxidatively modified purines [13]. Results are
T
2.9. Cell differentiation and cytokine secretion
P
RI
To differentiate cells into macrophages, monocytes U937 (1 x 106 cells/mL) were cultured
SC
in normo- or hyperglycemic conditions and differentiated with phorbol 12-myristate 13-
acetate (PMA, 20 nmol/L, Sigma-Aldrich, Switzerland) for 24 h [14] and washed three times
NU
in PBS, followed by administration of FOE (10 – 250 μg/mL) to differentiated cells. After 2,
MA
16 and 18 h incubation with/without FOE, cells were stimulated with lipopolysaccharide
from E. coli (LPS, 200 ng/mL, Sigma-Aldrich, Switzerland) for 24 h. During stimulation with
ED
lipopolysaccharide from E. coli, the culture medium contained PBS not FOE therefore we
have described only time of preincubation with FOE. Cytokine release was monitored in
PT
The concentration of selected cytokines (TNF, IL-6, IL-8, MCP-1) in culture medium was
AC
measured by ELISA. All cytokines were determined using matched antibody pairs (Sigma-
peroxidase conjugate secondary antibody were used for spectrophotometric detection at 490
9
ACCEPTED MANUSCRIPT
Data obtained from each study were analyzed by a two way ANOVA with Tukey's multiple
comparison tests. The correlation analysis was performed by using StatsDirect® 2.3.7
(StatsDirect. Sales, Sale, Cheshire M33 3UY, UK). Results are presented as the means ±
T
standard deviations (SD) and represent at least 3 independent experiments. Each experiment
P
was performed with three replicates. The level of significance was defined as a/bP < 0.05,
RI
aa/bb
P ≤ 0.01, aaa/bbbP < 0.001.
SC
3. Results
NU
MA
3.1. Cell proliferation
ED
At the beginning of evaluating the effect of fish oil emulsion on oxidative stress and
inflammation, we performed the monocytes U937 proliferation study by the MTT assay. The
PT
high glucose concentration as well as different concentrations of the FOE (10 – 250 µg/mL)
had no effect on cell proliferation during 72 h incubation (Fig. 1). The number of cells only
CE
cells (Fig. 2). In high concentration of glucose, antioxidant capacity did not change with time.
Antioxidant capacity of cells increased with the FOE concentrations and decreased with time
of incubation and this time-dependent trend was the same as in normoglycemic conditions.
10
ACCEPTED MANUSCRIPT
T
effect on SOD activity in monocytes U937 (Fig. 3). In normoglycemic, as well as in
P
hyperglycemic conditions without the FOE, there was a time-dependent decrease in SOD
RI
activity. The fish oil emulsion showed no significant effect on SOD activity on the first and
SC
second day of incubation. After 72 h of incubation, the two highest concentrations of fish oil
emulsion (100 and 250 µg/mL) caused a significant increase in SOD activity. Time-
NU
dependent changes of SOD activity affected by the two highest tested concentrations of FOE
MA
showed S-curved typical for biological systems.
ED
Oxidative damage to DNA in monocytes U937 was evaluated by measuring levels of 8-oxo-
all incubation times (Fig. 4). Oxidative damage to DNA was time-dependent under both
AC
was measured in monocytes affected with FOE. This damage was not prevented by any
Antioxidant potential of the FOE against protein oxidation in monocytes U937 was studied in
terms of protein carbonyl formation. Hyperglycemic conditions and FOE had no effect on
11
ACCEPTED MANUSCRIPT
oxidative damage to proteins when compared to normoglycemic conditions (Fig. 5). Time of
T
3.6. Oxidative damage to lipids
P
RI
Oxidative damage to lipids in monocytes U937 was evaluated by determination of 8-
SC
isoprostane levels after 24h, 48h and 72h incubation in normo- or hyperglycemic conditions
and treated with/without FOE. High concentration of glucose and FOE had no effect on 8-
NU
isoprostane levels in U937 cells when compared to normoglycemic conditions (Fig. 6). Time
MA
of incubation had no significant effect on protein carbonyl formation.
ED
there was a time-dependent increase in TNF secretion in response to LPS treatment (Fig. 7).
AC
After 16 h and 18 h incubation, the high concentration of glucose significantly increased TNF
release compared with normoglycemic conditions. After 2 h – 18h incubation of cells with
the emulsion, the two lowest concentrations of emulsion significantly decreased TNF
secretion. The time-dependent increasing trend of TNF secretion was the same in each tested
group.
12
ACCEPTED MANUSCRIPT
hyperglycemic conditions (Fig. 8). The highest concentration of fish oil emulsion (250
µg/mL) significantly reduced IL-6 secretion during all incubation times. The time-dependent
T
decreasing trend of IL-6 secretion was the same in each tested group.
P
RI
3.9. IL-8 release
SC
Release of IL-8 from monocytes-derived macrophages was significantly stimulated by LPS in
NU
hyperglycemic conditions at all incubation times. The fish oil emulsion had no significant
MA
effect on LPS-stimulated IL-8 secretion when compared to controls in hyperglycemic
conditions (Fig. 9). However, IL-8 release was increased nonsignificantly with respect to
ED
hyperglycemic controls at two highest FOE concentrations (100 and 250 µg/mL). There was
the same time-dependent increasing trend of IL-8 secretion in each tested group.
PT
monocytes-derived macrophages (Fig. 10). The three highest emulsion concentrations (50,
100 and 250 µg/mL) after 2h incubation and the four highest concentrations after 16 h and 18
normoglycemic conditions were not significantly changed during the incubation periods.
4. Discussion
13
ACCEPTED MANUSCRIPT
In this project we have examined a potential protecting effect of the FOE against oxidative
T
Our results indicate the reduced antioxidant capacity of monocytes U937 under
P
hyperglycemic conditions, as well as significant oxidative damage to DNA and the secretion
RI
of pro-inflammatory cytokines from monocyte-derived macrophages. In contrast, the FOE
SC
can increase antioxidant capacity of monocytes U937, as well as SOD activity and reduce the
NU
concentration. Nevertheless, the DNA may not be protected against oxidation by the FOE.
MA
This study demonstrates that hyperglycemia leads to decreased antioxidant capacity of cells
ED
during 24, 48 and 72 h of incubation. Our findings are in a good agreement with other studies
reporting that acute hyperglycemic incidents such as an oral glucose tolerance test or a food
PT
can reduce the antioxidant capacity of plasma in both normal and diabetic individuals and can
The fish oil emulsion caused a concentration – dependent increase of cells´ antioxidant
capacity. The current results agree with the findings of several studies which indicate that the
serum antioxidant status was improved following omega-3 PUFA administration [19-21]. A
potential mechanism of action of long chain polyunsaturated fatty acids assumes that they act
as a “sink” to trap free radicals, hence becoming oxidized themselves [22]. Other possible
mechanisms could operate enhancing the antioxidant effects of fish oil. Anderson et al.
(2014) described that omega-3 PUFA increased antioxidant capacity in the myocardium via
that omega-3 PUFA are able to displace arachidonic acid in cell membranes. They can
14
ACCEPTED MANUSCRIPT
different structure from those derived from arachidonic acid [24]. Fish oil emulsion
T
containing omega-3 PUFA might therefore influence the potency of arachidonic acid-derived
P
mediators which are often much less biologically active than those produced from
RI
arachidonic acid.
SC
SOD plays an important role in cellular protection against oxidative stress by catalysing the
NU
conversion of the superoxide anion (O2.-) to hydrogen peroxide [25]. Imbalance between the
MA
formation and elimination of superoxide anions by enzymes in diabetes leads to
for 72 h, the same result we had obtained in our previous study [16]. However, after 72 h fish
PT
oil emulsion significantly increased SOD activity. In accordance with the present study,
several researchers described that supplementation with fish oil rich in omega-3 PUFA
CE
increased the SOD activity [19-20, 26-28]. Marugan and Pari (2007) found that fish oil
AC
contains a free radical scavenging activity, which could exert a beneficial effect against
pathological alterations caused by the presence of O2.- and hydroxyl radical (OH·). The
[29]. Basquets-Cortés et al. (2016) described no changes on the antioxidant enzyme Mn-SOD
protein levels after DHA diet supplementation on peripheral blood mononuclear cells
(PBMCs) [30]. We assume that the EPA and DHA changed the SOD activity of pre-existing
15
ACCEPTED MANUSCRIPT
The comet analysis showed hyperglycemia-induced oxidative damage to the DNA in the
monocytic cell line U937. Several studies have found a significant correlation between DNA
damage and hyperglycemia [31-34]. Kuppan et al. (2010) found that hyperglycemia
T
significantly elevated levels of DNA damage in the monocytic cell line THP-1 [35]. When
P
the damage to DNA is higher than the cellular capacity to repair it, the accumulation of errors
RI
can overcome the cell resulting in cell death or fixation of genome mutations that can be
SC
transmitted to future cell generations [36]. The emulsion of fish oil had no protective effect
against oxidative damage to DNA induced by hyperglycemia. Müllner et al. (2013) showed a
NU
similar result that the healthy diet including PUFAs did not improve genome damage in
MA
peripheral blood lymphocytes from patients with diabetes mellitus type 2 [37].
ED
protein carbonyls formation in liver and kidney cytosolic fractions from diabetic rats [39].
lymphocytes of diabetic patient [40-41]. On the other hand, Du et al. (2003) reported no
increase in protein carbonyls in rMC-1 cells incubated in 25 mmol/L glucose for 5 days [42].
because the modified proteins can be degraded by proteasome systems. Prolonged exposure
16
ACCEPTED MANUSCRIPT
accumulation of protein carbonyls. Recent studies have demonstrated that high glucose and
diabetes are modulators of proteasome activity [43-44]. In eukaryotic cells, the majority of
T
intracellular proteins are degraded by the ubiquitin-proteasome system. Aghdam et al. (2013)
P
determined that high glucose treatment mediated impairment of glucose increased ubiquitin-
RI
proteasome activity in retinal endothelial cells and it was responsible for an increased level of
SC
total ubiquitin-conjugate [45]. Physiological carbonyls are also potent glycating agents that
are formed during lipid peroxidation, they are glycolytic intermediates, and can react with
membrane integrity, fluidity, and permeability. Isoprostanes are the second most widely
formation of ROS. They are bound to membrane phospholipids and they are released by
CE
phospholipase into blood circulation [47]. One of the isoprostanes, 8-isoprostane, is used as a
AC
cells during 24, 48 and 72 h of incubation. Vincent et al. (2005) described significantly
increased amount of 8-isoprostnes in dorsal root ganglion of rat neurons after 1 h incubation
in hyperglycemic conditions and this amount persisted during next 5h [48]. Although several
studies showed the protective effect of omega-3 PUFA, especially EPA and DHA, against
lipids peroxidation in diabetic animals and humans [49-50], our results did not confirm these
17
ACCEPTED MANUSCRIPT
Several studies indicate that hyperglycemia-induced inflammation is one of the key factors
T
orchestrating these effects. High glucose leads to the activation of the master switch of
P
inflammation. The adverse effects of hyperglycemia on innate immunity manifest through the
RI
regulation of macrophages cytokine secretion [7, 51]. Similar to other studies, we found that
SC
hyperglycemia induces secretion of the pro-inflammatory cytokines TNF, IL-6, IL-8 and
NU
MA
In the present study we found that fish oil emulsion decreased the release of TNF, IL-6 and
emulsion concentration and time of incubation with cells. Fish oil emulsion had no effect on
IL-8 secretion. Long chain fatty acids influence inflammation through a variety of
PT
these compositions can modify cell signaling, membrane fluidity and the formation of lipid
CE
mediators. Cells involved in the inflammatory response are typically rich in the arachidonic
AC
acid, but the contents of arachidonic acid, EPA and DHA can be altered through oral
patients with Type 1 and Type 2 diabetes [57]. Pro-inflammatory cytokines are widely
recognised markers of vascular inflammation. Elevated levels of TNF, IL-6 and other
patients [58]. IL-8 mediates monocyte recruitment and firm adhesion to the endothelium of
arteries [59]. MCP-1 exerts its effects through binding to G-protein-coupled receptors on the
18
ACCEPTED MANUSCRIPT
surface of leukocytes targeted for activation and migration that mediates atherosclerosis and
other cardiovascular diseases [60]. Adkins et al. (2010) suggested that the decreased release
of TNF, IL-6 and MCP-1 is the underlying cardioprotective mechanism of omega-3 PUFA
T
[61].
P
RI
Some ex vivo studies described that omega-3 PUFA reduced production of pro-inflammatory
SC
cytokines including TNF, IL-1 and IL-6 following lipopolysaccharide stimulation of
monocytes/lymphocytes [41, 62-63]. Oh et al. (2010) showed that DHA inhibited TNF
NU
production in the Raw 264.7 mouse macrophage cell line [64]. EPA inhibited NF-κB
MA
activation in the THP-1 cell line [65] as well as in human monocytes [66]. A double-blind
intervention study among 60 patients with coronary heart disease found that IL-6 was
ED
significantly reduced by fish oil compared to rapeseed oil supplementation [67]. In vitro
studies also showed that DHA but not EPA decreased the expression of pro-inflammatory
PT
cytokines, cell adhesion molecules and monocyte adhesion to endothelial cells [4].
CE
Our results as well as results from previous studies confirm our hypothesis of protective
AC
appropriate antioxidant response, the system becomes overwhelmed leading to the production
of reactive molecules that can cause cellular damage and are responsible for the late stage
complications of diabetes [35]. We have found that the fish oil emulsion used in our study
had no protective effect against oxidative damage to DNA, however it increased antioxidative
by reducing the release of some pro-inflammatory cytokines. Further studies are required to
19
ACCEPTED MANUSCRIPT
examine whether these fish oil emulsion properties are associated with an improvement of
T
A limitation of our study is that the experimental design is affected by the selection of our
P
control. In addition, our results might be attributed not only to the fish oil emulsion but also
RI
to the emulsifier and other additives present in it. Another limitation is the experimental
SC
model system used in our study. We have not confirmed that hyperglycemia caused the
NU
isoprostanes. Longer time of incubation or other markers of damage to proteins and lipids
MA
should be chosen. We have used cell cultures to examine FOE effects as a baseline for
the effects of FOE on diabetic patients. Therefore, our findings must be confirmed in animal
Acknowledgment
CE
This study was financially supported by the Mind & Health, civil association and by the
AC
References
[1] Yang RH, Lin J, Hou XH, Cao R, Liu HQ, Ji A L, Wang F. Effect of docosahexaenoic
20
ACCEPTED MANUSCRIPT
2014;274:218-28.
[2] Fiorentino TV, Prioletta A, Zuo P, Folli F. Hyperglycemia-induced oxidative stress and
T
its role in diabetes mellitus related cardiovascular diseases. Curr Pharm Des
P
2013;19(32):5695-703.
RI
[3] King GL. The role of inflammatory cytokines in diabetes and its complications. J
SC
Periodontol 2008;79(8):1527-34.
[4] Mori TA. Dietary n-3 PUFA and CVD; a review of the evidence. Proc Nutr Soc
2014;73(1):57-64.
NU
MA
[5] de Luca C, Olefsky JM. Inflammation and insulin resistance. FEBS Lett
2008;582(1):97-105.
ED
[6] Jain SK, Rains J, Croad J, Larson B, Jones K. Curcumin supplementation lowers TNF-
α, IL-6, IL-8, and MCP-1 secretion in high glucose-treated cultured monocytes and
PT
blood levels of TNF-α, IL-6, MCP-1, glucose, and glycosylated hemoglobin in diabetic
[7] Garaiova I, Guschina IA, Plummer SF, Tang J, Wang D, Plummer NT. A randomised
AC
cross-over trial in healthy adults indicating improved absorption of omega-3 fatty acids
[8] Jain SK, Kannan K. Chromium chloride inhibits oxidative stress and TNF-alpha
[9] Mosman T. Rapid colorimetric assay for cellular growth and survival; Application to
2):55-63.
21
ACCEPTED MANUSCRIPT
[10] Price P, McMillan J. Use of the tetrazolium assay in measuring the response of human
T
an improved ABTS radical cation decolorization assay. Free Radic Biol Medic
P
1991;26(9-10);1231-7.
RI
[12] Buss IH, Chan TP, Sluis KB, Domigan NM, Winterbourn CC. Protein carbonyl
SC
measurement by a sensitive ELISA method. Free Radic Biol Med 1997;23(3):361-6.
[13] Collins AR, Dobson VL, Dušinská M, Kennedy G, Štetina R. The comet assay; what
NU
can it really tell us? Mutat Res 1997;375(2):183-93.
MA
[14] García A, Serrano A, Abril E, Jimenez P, Real LM, Cantón C, et al. Differential effect
[15] Guha M, Mackman N. LPS induction of gene expression in human monocytes. Cell
PT
Signal 2001;13(2):85-94.
defences are reduced during the oral glucose tolerance test in normal and non-insulin-
[18] Vincent AM, Russell JW, Low P, Feldman EL. Oxidative stress in the pathogenesis of
[19] Mahmoudabadi MM, Rahbar AR. Effect of EPA and vitamin C on superoxide
22
ACCEPTED MANUSCRIPT
[20] Garsía-Alonso FJ, Jorge-Vidal V, Periago MJ. Effect of consumption of tomato juice
enriched with n-3 polyunsaturated fatty acids on the lipid profile, antioxidant biomarker
status, and cardiovascular disease risk in healthy women. Eur J Nutr 2012;51(4): 415-
T
24.
P
[21] Thorlaksdottir AY, Skuladottir GV, Petursdottir AL, Tryggvadottir L, Oqmundsdottir
RI
HM, Eygjord JE, et al. Positive association between plasma antioxidant capacity and n-
SC
3 PUFA in red blood cells from women. Lipids 2006;41(2):119-25.
NU
antioxidants. Pharm Res 2008;57:451-5.
MA
[23] Anderson EJ, Thayne KA, Harris M, Shaikh SR, Darden TM, Lark DS et.al. Do fish oil
omega-3 fatty acids enhance antioxidant capacity and mitochondrial fatty acid
oxidation in human atrial myocardium via PPARγ activation? Antioxid Redox Signal
ED
2014;21(8):1156-63.
PT
[24] Calder PC. Omega-3 fatty acids and inflammatory processes. Nutrients 2010;2(3):355-
74.
CE
[25] Zanatta AL, Miranda DT, Dias BC, Campos RM, Massaro MC, Michellotto, PV, et al.
AC
Fish oil supplementation decreases oxidative stress but does not affect platelet-
[26] Rahman I, Biswas SK, Kirkham PA. Regulation of inflammation and redox signaling
[27] Song JH, Miyazawa T. Enhanced level of n-3 fatty acid in membrane phospholipids
induced lipid peroxidation in rats fed dietary docosahexaenoic acid oil. Atherosclerosis
2001;155(1):9-18.
23
ACCEPTED MANUSCRIPT
T
[29] Murugan P, Pari L. Influence of tetrahydrocurcumin on erythrocyte membrane and
P
antioxidant status in experimental type 2 diabetic rats. J Ethnopharmacol 2007;113:479-
RI
86.
SC
[30] Busquets-Cortés C, Capó X, Martorell M, Tur JA, Sureda A, Pons A. Training
NU
antioxidant capabilities synergistically with dietary docosahexaenoic supplementation.
MA
Oxid Med Cell Longev 2016; 2016:8950384. doi: 10.1155/2016/8950384.
[31] Choi SW, Benzie IF, Lam CS, Chat SW, Lam J, Yiu C, et al. Inter-relationships
ED
between DNA damage, ascorbic acid and glycaemic control in Type 2 diabetes
in the comet assay and antioxidant capacity in diabetic patients. Mutat Res 1998;398(1-
2):151-61.
24
ACCEPTED MANUSCRIPT
[36] Moreli JB, Santos JH, Rocha CR, Damasceno DC, Morceli G, Rudge MV, et al. DNA
damage and its cellular response in mother and fetus exposed to hyperglycemic
T
[37] Müllner E, Brath H, Tofere D, Adrigan S, Bulla MT, Stieglmayer R, et al. Genome
P
damage in peripheral blood lymphocytes of diabetic and non-diabetic individuals after
RI
intervention with vegetables and plant oil. Mutagenesis 2013;28(2):205-11.
SC
[38] Martín-Gallán P, Carrascosa A, Gussinyé M, Domíngez C. Biomarkers of diabetes-
associated oxidative stress and antioxidant status in young diabetic patients with or
NU
without subclinical complications. Free Radic Biol Med 2003;34(12):1563-74.
MA
[39] Portero-Otín M, Pamplona R, Ruiy MC, Cabiscol E, Prat J, Bellmunt MJ. Diabetes
[40] Dayanand CD, Vegi PK, Kutty AV. Protein carbonyl content as a stable oxidative
al. Oxidative stress, glutathione status, sirtuin and cellular stress response in type 2
25
ACCEPTED MANUSCRIPT
[44] Powell SR, Samuel SM, Wang P, Divald A, Thirunavukkarasu M, Koneru S, et al.
T
[45] Aghdam SY, Gurel Z, Ghaffarieh A, Sorenson CM, Sheibani N. High glucose and
P
diabetes modulate cellular proteasome function; Implications in the pathogenesis of
RI
diabetes complications. Biochem Biophys Res Commun 2013;432(2):339-44.
SC
[46] Ahmed N, Babaei-Jadidi R, Howell SK, Beisswenger PJ, Thornalley PJ. Degradation
NU
diabetes. Diabetologia 2005;48(8):1590-603.
MA
[47] Milne GL, Yin H, Hardz KD, Davies SS, Roberts LJ. Isoprostane generation and
[48] Vincent AM, McLean LL, Backus C, Feldman EL. Short-term hyperglycemia produces
[49] Hünkar T, Aktan F, Ceylan A, Karasu C. Effects of cod liver oil on tissue antioxidant
CE
2002;20(4):297-302.
AC
[50] Merzouk H, Khan NA. Implication of lipids in macrosomia of diabetic pregnancy: can
n-3 polyunsaturated fatty acids exert beneficial effects? Clin Sci 2003;105(5):519-29.
[51] Dasu MR, Devaraj S, Zhao L, Hwang DH, Jialal I. High glucose induces toll-like
2008;57(11):3090-8.
[52] Guha M, Bai W, Nadler JL, Natarajan R. Molecular mechanisms of tumor necrosis
26
ACCEPTED MANUSCRIPT
2014;10(1):223-35.
T
[54] Jain SK, Rains J, Croad J, Larson B, Jones K. Curcumin supplementation lowers TNF-
P
alpha, IL-6, IL-8, and MCP-1 secretion in high glucose-treated cultured monocytes and
RI
blood levels of TNF-alpha, IL-6, MCP-1, glucose, and glycosylated hemoglobin in
SC
diabetic rats. Antioxid Redox Signal 2009;11(2):241-9.
[55] Xiu F, Stanojci M, Diao L, Jeschke MG. Stress hyperglycemia, insulin treatment, and
NU
innate immune cells. Int J Endocrinol 2014;2014:486403. doi:10.1155/2014/486403.
MA
[56] Dasu MR, Devaraj S, Jialal I. High glucose induces IL-1beta expression in human
E346.
[57] Devaraj S, Cheung AT, Jialal I, Griffen SC, Nguyen D, Glaser N, et al. Evidence of
PT
[58] Desfaits AC, Serri O, Renier G. Normalization of plasma lipid peroxides, monocyte
AC
[59] Srinivasan S, Yeh M, Danziger EC, Hatley ME, Riggan AE, Leitinger N, et al. Glucose
2003;92(4):371-7.
[60] Wang YC, Hsieh CC, Kuo HF, Tsai MK, Yang SN, Kuo CH, et al. Effect of vitamin
27
ACCEPTED MANUSCRIPT
[61] Adkins Y, Kelley DS. Mechanisms underlying the cardioprotective effects of omega-3
[62] Calder PC. Polyunsaturated fatty acids, inflammation, and immunity. Lipids 2001;
T
36(9):1007-24.
P
[63] Mori TA, Beilin LJ. Omega-3 fatty acids and inflammation. Curr Atheroscler Rep
RI
2004;6(6):461-7.
SC
[64] Oh DY, Talukdar S, Bae EJ, Imamura T, Morinaga H, Fan W, et al. GPR120 is an
dependent upon time and dose-response elements associated with LPS stimulation in
ED
[67] Seirstad SL, Seljeflot I, Johansen O, Hansen R, Haugen M, Rosenlund G, et al. Dietary
AC
Fig.1. Influence of hyperglycemia and the FOE on the proliferation of monocytes U937. C-
conditions, C-PBS- control cells in hyperglycemic conditions with PBS. FOE – cells in
hyperglycemic conditions incubated with different concentrations of the fish oil emulsion (10
28
ACCEPTED MANUSCRIPT
– 250 µg/mL). All values are reported as means ± SD of triplicates, each repeated 3 times
T
Fig.2. Influence of hyperglycemia and the FOE on antioxidant capacity of monocytes U937.
P
C-NG – control cells in normoglycemic conditions, C-HG - control cells in hyperglycemic
RI
conditions, C-PBS- control cells in hyperglycemic conditions with PBS. FOE – cells in
SC
hyperglycemic conditions incubated with different concentrations of the fish oil emulsion (10
aa
– 250 µg/mL). Differences are considered significant at P ≤ 0.01, aaaP ≤ 0.001- C-HG
NU
compared with C-NG, bP < 0.05, bbP ≤ 0.01, bbbP ≤ 0.001- compared with C-PBS (two way
MA
ANOVA with Tukey's multiple comparison tests). All values are reported as means ± SD of
monocytes U937. C-NG – control cells in normoglycemic conditions, C-HG - control cells in
hyperglycemic conditions, C-PBS- control cells in hyperglycemic conditions with PBS. FOE
CE
– cells in hyperglycemic conditions incubated with different concentrations of the fish oil
AC
emulsion (10 – 250 µg/mL). Differences are considered significant at bP < 0.05 - compared
with C-PBS (two way ANOVA with Tukey's multiple comparison tests). All values are
monocytes U937. C-NG – control cells in normoglycemic conditions, C-HG - control cells in
hyperglycemic conditions, C-PBS - control cells in hyperglycemic conditions with PBS. FOE
– cells in hyperglycemic conditions incubated with different concentrations of the fish oil
emulsion (10 – 250 µg/mL). Differences are considered significant at aP < 0.05, aaP ≤ 0.01-
29
ACCEPTED MANUSCRIPT
C-HG compared with C-PBS (two way ANOVA with Tukey's multiple comparison tests). All
T
Fig. 5. Influence of hyperglycemia and the FOE on levels of protein carbonyls in monocytes
P
U937. C-NG – control cells in normoglycemic conditions, C-HG - control cells in
RI
hyperglycemic conditions, C-PBS- control cells in hyperglycemic conditions with PBS. FOE
SC
– cells in hyperglycemic conditions incubated with different concentrations of the fish oil
emulsion (10 – 250 µg/mL). All values are reported as means ± SD of triplicates, each
NU
repeated 3 times (n=3), (two way ANOVA with Tukey's multiple comparison tests).
MA
Fig. 6. Influence of hyperglycemia and the FOE on levels of isoprostenes in monocytes
hyperglycemic conditions, C-PBS- control cells in hyperglycemic conditions with PBS. FOE
PT
– cells in hyperglycemic conditions incubated with different concentrations of the fish oil
emulsion (10 – 250 µg/mL). All values are reported as means ± SD of triplicates, each
CE
repeated 3 times (n=3), (two way ANOVA with Tukey's multiple comparison tests).
AC
Fig. 7. Influence of hyperglycemia and the FOE on TNF release from monocytes-derived
hyperglycemic conditions, C-PBS- control cells in hyperglycemic conditions with PBS. FOE
– cells in hyperglycemic conditions incubated with different concentrations of the fish oil
emulsion (10 – 250 µg/mL). Differences are considered significant at aP < 0.05 - C-HG
compared with C-NG, bP < 0.05, bbP ≤ 0.01 - compared with C-PBS (two way ANOVA with
Tukey's multiple comparison tests). All values are reported as means ± SD of triplicates, each
30
ACCEPTED MANUSCRIPT
Fig. 8. Influence of hyperglycemia and the FOE on IL-6 release from monocytes-derived
T
hyperglycemic conditions, C-PBS- control cells in hyperglycemic conditions with PBS. FOE
P
– cells in hyperglycemic conditions incubated with different concentrations of the fish oil
RI
emulsion (10 – 250 µg/mL). Differences are considered significant at aP < 0.05 - C-HG
SC
compared with C-NG, bP < 0.05 - compared with C-PBS (two way ANOVA with Tukey's
multiple comparison tests). All values are reported as means ± SD of triplicates, each
hyperglycemic conditions, C-PBS - control cells in hyperglycemic conditions with PBS. FOE
PT
– cells in hyperglycemic conditions incubated with different concentrations of the fish oil
emulsion (10 – 250 µg/mL). Differences are considered significant at aP < 0.05 - C-HG
CE
compared with C-NG (two way ANOVA with Tukey's multiple comparison tests). All values
AC
Fig. 10. Influence of hyperglycemia and the FOE on MCP-1 release from monocytes-derived
hyperglycemic conditions, C-PBS - control cells in hyperglycemic conditions with PBS. FOE
– cells in hyperglycemic conditions incubated with different concentrations of the fish oil
emulsion (10 – 250 µg/mL). Differences are considered significant at aaaP ≤ 0.001- C-HG
compared with C-NG, bP < 0.05, bbP ≤ 0.01 - compared with C-PBS (two way ANOVA with
31
ACCEPTED MANUSCRIPT
Tukey's multiple comparison tests). All values are reported as means ± SD of triplicates, each
P T
RI
SC
NU
MA
ED
PT
CE
AC
32
ACCEPTED MANUSCRIPT
T
RIP
SC
NU
MA
ED
PT
Figure 1
CE
AC
33
ACCEPTED MANUSCRIPT
T
RIP
SC
NU
MA
ED
PT
Figure 2
CE
AC
34
ACCEPTED MANUSCRIPT
T
RIP
SC
NU
MA
ED
PT
Figure 3
CE
AC
35
ACCEPTED MANUSCRIPT
T
RIP
SC
NU
MA
ED
PT
Figure 4
CE
AC
36
ACCEPTED MANUSCRIPT
T
RIP
SC
NU
MA
ED
PT
Figure 5
CE
AC
37
ACCEPTED MANUSCRIPT
TP
RI
SC
NU
MA
ED
PT
Figure 6
CE
AC
38
ACCEPTED MANUSCRIPT
T
RIP
SC
NU
MA
ED
PT
Figure 7
CE
AC
39
ACCEPTED MANUSCRIPT
T
RIP
SC
NU
MA
ED
PT
Figure 8
CE
AC
40
ACCEPTED MANUSCRIPT
T
RIP
SC
NU
MA
ED
PT
Figure 9
CE
AC
41
ACCEPTED MANUSCRIPT
T
RIP
SC
NU
MA
ED
PT
Figure 10
CE
AC
42