Fish oil emulsion supplementation might improve quality of life of diabetic
patients due to its antioxidant and antiinflammatory properties

Lucia Laubertová, Katarı́na Koňariková, Helena Gbelcová, Zdeňka

Ďuračková, Jana Muchová, Iveta Garaiova, Ingrid Žitňanová

PII: S0271-5317(16)30819-3
DOI: doi: 10.1016/j.nutres.2017.07.012
Reference: NTR 7788

To appear in: Nutrition Research

Received date: 21 December 2016

Revised date: 26 July 2017
Accepted date: 31 July 2017

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

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 ACCEPTED MANUSCRIPT

Fish oil emulsion supplementation might improve quality of life of diabetic patients due

to its antioxidant and antiinflammatory properties

Lucia Laubertováa, Katarína Koňarikováa, Helena Gbelcováb, Zdeňka Ďuračkováa, Jana

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Muchováa, Iveta Garaiovac, Ingrid Žitňanováa*

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a
Institute of Medical Chemistry, Biochemistry and Clinical Biochemistry, Faculty of

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Medicine, Comenius University, Sasinkova 2, 813 72 Bratislava, Slovakia
b
Institute of Medical Biology, Genetics and Clinical Genetics, Faculty of Medicine,

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Comenius University, Sasinkova 4, 813 72 Bratislava, Slovakia
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c
Research and Development Department, Cultech Ltd, Port Talbot, SA12 7BZ United

Kingdom
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Email adresses:
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Lucia Laubertová: lucia.laubertova@fmed.uniba.sk

Katarína Koňariková: katarina.konarikova@fmed.uniba.sk

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Helena Gbelcová: helena.gbelcova@fmed.uniba.sk

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Zdeňka Ďuračková: zdenka.durackova@fmed.uniba.sk

Jana Muchová: jana.muchova@fmed.uniba.sk

Iveta Garaiova: ivetag@cultech.co.uk

*
Corresponding author:

Ingrid Žitňanová

Institute of Medical Chemistry, Biochemistry and Clinical Biochemistry, Faculty of

Medicine, Comenius University, Sasinkova 2, 813 72 Bratislava, Slovakia

Tel: + 421 902 840 242, + 421 2 59357 559, Fax: + 421 2 59357 557

e-mail: ingrid.zitnanova@fmed.uniba.sk

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List of abbreviations

8-oxo-G; 8-oxo-guanin

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AGEs; advanced glycation end products

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DHA; docosahexaenoic acid

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DM; diabetes mellitus

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EPA; eicosapentaenoic acid

FOE; fish oil emulsion

HG; hyperglycemic conditions

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IL-6; interleukin-6

IL-8; interleukin-8
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LPS; lipopolysaccharide

MCP-1; monocytic chemotactic protein-1

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MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

NF- κB; nuclear factor-kappaB

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NG; normoglycemic conditions

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PBS; phosphate-buffered saline

PMA; 12-myristate 13-acetate

PPARγ; peroxisome proliferator-activated receptor-γ

PUFA; polyunsaturated fatty acids

ROS; reactive oxygen species

SD; standard deviation

SOD; superoxide dismutase

TEAC; trolox equivalent antioxidant capacity

TNF; tumor necrosis factor

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Abstract

Diabetes-related complications, including cardiovascular disease, retinopathy, nephropathy

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and neuropathy, are a significant cause of increased morbidity and mortality among people

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with diabetes. Previous studies have confirmed that hyperglycemia has pro-oxidative and

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proinflammatory properties which cause diabetic complications. We hypothesized that

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supplementation of fish oil emulsion (FOE), rich in omega-3 polyunsaturated fatty acids

(PUFA), to diabetic patients might reduce hyperglycemia-induced pathological changes due

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to specific properties of fish oil emulsion. Omega-3 PUFA have a wide range of biological
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effects. In this project we have examined the potential protective effect of the fish oil

emulsion on hyperglycemia-induced oxidative stress and cytokine generation in

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monocytes/macrophages U937 system in vitro. The monocytes/macrophages U937 were

cultivated under normal- or hyperglycemic (35 mmol/L glucose) conditions, with/without

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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
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inflammation (tumor necrosis factor (TNF), interleukin-6 (IL-6), interleukin 8 (IL-8),

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monocytic chemotactic protein-1 (MCP-1)). Hyperglycemia caused reduction of antioxidant

capacity, induction of DNA damage and pro-inflammatory cytokine secretion. FOE

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

pathological mechanisms by its antioxidant and anti-inflammatory properties.

Keywords

fish oil emulsion; monocytes; macrophages; hyperglycemia; oxidative stress; cytokines

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1. Introduction

Diabetes mellitus (DM) is one of the major debilitating human metabolic diseases at present.

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It is a group of human metabolic diseases with characteristic symptoms of hyperglycemia,

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chronic inflammation and insulin resistance [1]. The chronic hyperglycemia in diabetes is

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associated with different mechanisms such as activation of protein kinase C, the polyol and

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hexosamine pathways and advanced glycation end products (AGEs) formation. All of these

pathways, in association with hyperglycemia-induced mitochondrial dysfunction, oxidative

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and glycation stress, promote reactive oxygen species (ROS) accumulation [2].
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It is suggested that oxidative stress represents a major pathophysiological link between
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progression of diabetes mellitus and the onset of diabetic complications. These complications

affect many tissues and organs, causing retinopathy, nephropathy, neuropathy, cardiovascular
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diseases, peripheral vascular diseases, stroke and periodontal pathologies [3]. Factors that

may contribute to increased oxidative stress in diabetes mellitus include increased formation
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of ROS such as superoxide and hydrogen peroxide, increased protein carbonylation including
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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

pro-inflammatory cytokines such as tumor necrosis factor (TNF), interleukin-6 (IL-6),

interleukin 8 (IL-8) and monocytic chemotactic protein-1 (MCP-1) as well as the expression

of nuclear factor-kappaB (NF- κB) [6].

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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

eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). An increasing number of

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studies have demonstrated that omega-3 polyunsaturated fatty acids (PUFA), specifically

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EPA and DHA can suppress inflammation and have a beneficial role in a variety of

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inflammatory human diseases, including diabetes, atherosclerosis, asthma, and arthritis.

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Omega-3 PUFA have a wide range of biological effects, including benefits on lipoprotein

metabolism, platelet function, endothelial function and vascular reactivity, inflammatory

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markers, cytokine production, coagulation, and fibrinolysis [4]. Omega-3 PUFA have also
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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,
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particularly EPA and DHA, compared with the non-emulsified form of the fish oil [7].
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In the treatment of DM, it is necessary to examine the relationship between pathological

mechanisms and diabetic complications. The pathogenesis of diabetic complications has not
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been sufficiently clarified yet. Successful therapy depends on detailed knowledge of the role
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of hyperglycemia, oxidative stress and inflammation in the pathogenesis of diabetic

complications, and the effects of therapeutics on pathogenic mechanisms.

Our in vitro experiments were based on previous studies reporting an important role of

hyperglycemia-induced oxidative stress and inflammation in the development of diabetic

complications. Reduction of these pathological changes should by an important step in

therapy of diabetic complications. In diabetic patients decreased level of antioxidants were

found, the damage to macromolecules and increased level of cytokines. We have

hypothesized that the fish oil emulsion, rich in omega-3 PUFA, has a potential protective

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effect against hyperglycemia-induced oxidative stress and inflammation due to various

biological activities of omega-3 PUFA and increased absorption of fatty acids.

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2. Methods and materials

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2.1. Cell culture

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The monocyte cells U937 (American Type Culture Collection, Manassas, USA) were

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cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum,
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100 U/mL penicillin and 100 µg/mL streptomycin (Sigma-Aldrich, Switzerland) at 37 °C in a

humidified atmosphere containing 5% CO2 and 95% air.

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2.2. Hyperglycemic conditions and fish oil emulsion

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To mimic hyperglycemic conditions in diabetes [8], the U937 cells (monocytes/macrophages)

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were incubated in a high glucose medium. The cells (5 x 105 cells/mL) were cultured under
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normoglycemic (NG) or hyperglycemic (HG, 35 mmol/L glucose) conditions, in the absence

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,

Sigma-Aldrich, Switzerland), and a similar volume of PBS was added to controls.

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2.3. Proliferation of cells

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MTT assay was used to evaluate cell proliferation (MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-

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diphenyltetrazolium bromide) [9]. The procedure by Price and McMillan was used to convert

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results of MTT assay to cell number [10]. After incubation of cells with/without FOE and

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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.

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Lysates were cleared by centrifugation, and the supernatant was aliquoted and stored at
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−80°C. Micro BCA Protein Assay Kit (Thermo Scientific, USA) was used to determine the

protein concentration of cell lysates.

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2.4. Antioxidant capacity

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Antioxidant capacity of cells was determined in cell lysates by the TEAC method (Trolox
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Equivalent Antioxidant Capacity method) [11]. The blue-green radical cation was generated
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by oxidation of 2,2´ -azino-bis(-3-ethylbenzothiazoline-6-sulfonic acid) with potassium

persulfate (Sigma-Aldrich, Switzerland) and reduced in the presence of hydrogen-donating

antioxidants into colorless form. The antioxidant capacity of the cells was measured at 735

nm and expressed in mmol trolox/L/mg proteins.

2.5. Protein carbonyls

Concentration of protein carbonyls in cell lysates was measured by ELISA [12]. The cell

lysates were derivatized with 2,4-dinitrophenylhydrazine (Sigma-Aldrich, Switzerland).

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Polyclonal antibody anti-dinitrophenyl-rabbit-IgG (Sigma-Aldrich, Switzerland) was used

followed by a polyclonal goat antirabbit-IgG antibody peroxidase conjugate (Sigma-Aldrich,

Switzerland) with spectrophotometric detection at 492 nm. Results are expressed in nmol

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carbonyls/mg proteins.

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2.6. Isoprostanes

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Isoprostane levels were determined in cell lysates by the 8-Isoprostane ELISA Kit (Cayman

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Chemical, USA) according to the manufacturer´s protocol. Results are expressed in pg/mg
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proteins.
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2.7. Superoxide dismutase (SOD) activity

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SOD activity was determined in cell lysates by the superoxide dismutase (SOD) activity

assay kit (Sigma-Aldrich, Switzerland) according to the manufacturer´s protocol. SOD

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activity was expressed in U/mg proteins, where 1U SOD is defined as 50% inhibition of
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WST-1 (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium,

monosodium salt) reduction to water-soluble formazan by a superoxide anion.

2.8. Oxidative damage to DNA

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

was evaluated by modified comet assay using formamidopyrimidine-DNA glycosylase (Fpg,

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Sigma-Aldrich, Switzerland) which can cleave oxidatively modified purines [13]. Results are

expressed in 8-oxo-guanine/106 guanins.

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2.9. Cell differentiation and cytokine secretion

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To differentiate cells into macrophages, monocytes U937 (1 x 106 cells/mL) were cultured

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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

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in PBS, followed by administration of FOE (10 – 250 μg/mL) to differentiated cells. After 2,
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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
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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
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culture medium [8, 15].

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The concentration of selected cytokines (TNF, IL-6, IL-8, MCP-1) in culture medium was
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measured by ELISA. All cytokines were determined using matched antibody pairs (Sigma-

Aldrich, Switzerland). Microwell plates were coated with samples of cultured

medium/standards containing the target cytokine. Specific polyclonal antibodies and a

peroxidase conjugate secondary antibody were used for spectrophotometric detection at 490

nm [16]. Results are expressed in ng/ml/106 cells.

2.10. Statistical analyses

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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 ±

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standard deviations (SD) and represent at least 3 independent experiments. Each experiment

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was performed with three replicates. The level of significance was defined as a/bP < 0.05,

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aa/bb
P ≤ 0.01, aaa/bbbP < 0.001.

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3. Results

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3.1. Cell proliferation
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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
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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
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increased with the incubation time in each tested group.

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3.2. Antioxidant capacity of cells

The antioxidant capacity of monocytes U937 grown in normoglycemic conditions was

significantly higher compared to the cells incubated in high glucose medium. In

normoglycemic conditions there was a time-dependent decrease in antioxidant capacity of

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.

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3.3. Superoxide dismutase (SOD) activity

After 24 h, 48 h and 72 h incubation in hyperglycemic conditions, hyperglycemia had no

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effect on SOD activity in monocytes U937 (Fig. 3). In normoglycemic, as well as in

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hyperglycemic conditions without the FOE, there was a time-dependent decrease in SOD

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activity. The fish oil emulsion showed no significant effect on SOD activity on the first and

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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-

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dependent changes of SOD activity affected by the two highest tested concentrations of FOE
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showed S-curved typical for biological systems.
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3.4. Oxidative damage to DNA

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Oxidative damage to DNA in monocytes U937 was evaluated by measuring levels of 8-oxo-

guanin (8-oxo-G). High glucose medium significantly increased formation of 8-oxo-guanin at

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all incubation times (Fig. 4). Oxidative damage to DNA was time-dependent under both
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normo- and hyperglycemic conditions. The same time-dependent increase of 8-oxoguanine

was measured in monocytes affected with FOE. This damage was not prevented by any

concentrations of the FOE used.

3.5. Oxidative damage to proteins

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

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oxidative damage to proteins when compared to normoglycemic conditions (Fig. 5). Time of

incubation had no effect on protein carbonyl formation.

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3.6. Oxidative damage to lipids

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Oxidative damage to lipids in monocytes U937 was evaluated by determination of 8-

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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-

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isoprostane levels in U937 cells when compared to normoglycemic conditions (Fig. 6). Time
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of incubation had no significant effect on protein carbonyl formation.
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3.7. TNF release

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We studied the anti-inflammatory potential of the fish oil emulsion on high-glucose-treated

monocytes-derived macrophages. In both normoglycemic and hyperglycemic conditions

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there was a time-dependent increase in TNF secretion in response to LPS treatment (Fig. 7).
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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.

3.8. IL-6 release

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Secretion of IL-6 from monocytes-derived macrophages was significantly induced by LPS in

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

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decreasing trend of IL-6 secretion was the same in each tested group.

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3.9. IL-8 release

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Release of IL-8 from monocytes-derived macrophages was significantly stimulated by LPS in

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hyperglycemic conditions at all incubation times. The fish oil emulsion had no significant
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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
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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.
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3.10. MCP-1 release

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Hyperglycemic conditions significantly stimulated LPS-induced secretion of MCP-1 from

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

h of incubation prevented the release of this cytokine. The levels of MCP-1 in

normoglycemic conditions were not significantly changed during the incubation periods.

Time-depending trend of MCP-1 released in cells affected by hyperglycemic conditions and

FOE showed S-curved typical for biological systems.

4. Discussion

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In this project we have examined a potential protecting effect of the FOE against oxidative

stress and inflammation induced by hyperglycemic conditions in monocytes/macrophages.

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Our results indicate the reduced antioxidant capacity of monocytes U937 under

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hyperglycemic conditions, as well as significant oxidative damage to DNA and the secretion

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of pro-inflammatory cytokines from monocyte-derived macrophages. In contrast, the FOE

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can increase antioxidant capacity of monocytes U937, as well as SOD activity and reduce the

secretion of pro-inflammatory cytokines from differentiated macrophages depending on its

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concentration. Nevertheless, the DNA may not be protected against oxidation by the FOE.
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This study demonstrates that hyperglycemia leads to decreased antioxidant capacity of cells
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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
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can reduce the antioxidant capacity of plasma in both normal and diabetic individuals and can

increase oxidative stress in diabetic patients [17-18].

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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

peroxisome proliferator-activated receptor-γ (PPARγ) activation [23]. It was also reported

that omega-3 PUFA are able to displace arachidonic acid in cell membranes. They can

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compete for enzymes (cyclooxygenase and lipoxygenase) generating eicosanoids such as

thromboxanes, prostaglandins and leukotrienes resulting in formation of products with

different structure from those derived from arachidonic acid [24]. Fish oil emulsion

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containing omega-3 PUFA might therefore influence the potency of arachidonic acid-derived

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mediators which are often much less biologically active than those produced from

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arachidonic acid.

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SOD plays an important role in cellular protection against oxidative stress by catalysing the

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conversion of the superoxide anion (O2.-) to hydrogen peroxide [25]. Imbalance between the
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formation and elimination of superoxide anions by enzymes in diabetes leads to

atherosclerosis and microvascular complications [19]. We found no statistically significant

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difference in SOD activity in cells cultured in normoglycemic and hyperglycemic conditions

for 72 h, the same result we had obtained in our previous study [16]. However, after 72 h fish
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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
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increased the SOD activity [19-20, 26-28]. Marugan and Pari (2007) found that fish oil
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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

increased activity of SOD accelerates dismutation of superoxide (O2.-) to hydrogen peroxide

[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

SOD protein rather than the SOD expression.

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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

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significantly elevated levels of DNA damage in the monocytic cell line THP-1 [35]. When

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the damage to DNA is higher than the cellular capacity to repair it, the accumulation of errors

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can overcome the cell resulting in cell death or fixation of genome mutations that can be

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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

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similar result that the healthy diet including PUFAs did not improve genome damage in
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peripheral blood lymphocytes from patients with diabetes mellitus type 2 [37].
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By measuring the levels of protein carbonyls we monitored oxidative damage to proteins in

U937 cells cultured in hyperglycemic conditions for 72 h. Protracted exposure of proteins to

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hyperglycemia can lead to spontaneous modifications, such as oxidation, glycation or

nitration. Proteins can be converted to carbonyls by a variety of oxidative mechanisms [38].

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We found no significant effect of hyperglycemia or fish oil emulsion on protein carbonyls

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formation over 72 h. Similarly, Portero-Otín (1999) determined no significant increase in

protein carbonyls formation in liver and kidney cytosolic fractions from diabetic rats [39].

The literature describes inconsistent results on protein carbonyl formation in hyperglycemic

conditions. Several clinical studies demonstrate increased protein carbonyl formation in

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].

We hypothesized that 72 h or 5 days of cultivation cells in hyperglycemic conditions is too

short period to demonstrate the effect of hyperglycemia on protein carbonyl formation

because the modified proteins can be degraded by proteasome systems. Prolonged exposure

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to hyperglycemic conditions might result in the damage of proteasome leading to

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

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intracellular proteins are degraded by the ubiquitin-proteasome system. Aghdam et al. (2013)

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determined that high glucose treatment mediated impairment of glucose increased ubiquitin-

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proteasome activity in retinal endothelial cells and it was responsible for an increased level of

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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

proteins to form AGEs directly [46].

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Oxidative damage to lipids can cause disturbance of membrane organisation and alteration of
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membrane integrity, fluidity, and permeability. Isoprostanes are the second most widely

described product of lipid peroxidation. They represent a complex group of prostaglandin F

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2α -like compounds generated by a nonenzymatic peroxidation of PUFA during increased

formation of ROS. They are bound to membrane phospholipids and they are released by
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phospholipase into blood circulation [47]. One of the isoprostanes, 8-isoprostane, is used as a
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marker of oxidative damage to lipids.

We have found no effect of hyperglycemic conditions on levels of 8-isoprostanes in U937

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

findings. We found no effects of FOE on the 8-isoprostane levels.

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Several studies indicate that hyperglycemia-induced inflammation is one of the key factors

that contribute to diabetic complications and monocytes/macrophages are important in

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orchestrating these effects. High glucose leads to the activation of the master switch of

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inflammation. The adverse effects of hyperglycemia on innate immunity manifest through the

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regulation of macrophages cytokine secretion [7, 51]. Similar to other studies, we found that

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hyperglycemia induces secretion of the pro-inflammatory cytokines TNF, IL-6, IL-8 and

MCP-1 from monocytes-derived macrophages [7, 16, 52-56].

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In the present study we found that fish oil emulsion decreased the release of TNF, IL-6 and

MCP-1 from monocytes-derived macrophages under hyperglycemic condition depending on

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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
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mechanisms mostly associated with changes in composition of cell membranes. Changes in

these compositions can modify cell signaling, membrane fluidity and the formation of lipid
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mediators. Cells involved in the inflammatory response are typically rich in the arachidonic
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acid, but the contents of arachidonic acid, EPA and DHA can be altered through oral

administration of EPA and DHA [24].

Devaraj et al. (2007) described increased production of pro-inflammatory cytokines in

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

inflammatory cytokines were detected in atherosclerotic plaques of diabetic and nondiabetic

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

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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

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[61].

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Some ex vivo studies described that omega-3 PUFA reduced production of pro-inflammatory

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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

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production in the Raw 264.7 mouse macrophage cell line [64]. EPA inhibited NF-κB
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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
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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
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cytokines, cell adhesion molecules and monocyte adhesion to endothelial cells [4].
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Our results as well as results from previous studies confirm our hypothesis of protective
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effect of FOE against hyperglycemia-induced oxidative stress and inflammation in

monocytes/macrophages U937. There is a considerable evidence that hyperglycemia

ultimately leads to increased oxidative stress and inflammation. In the absence of an

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

protection of cells over 72 h incubation. Moreover, it exhibited anti-inflammatory properties

by reducing the release of some pro-inflammatory cytokines. Further studies are required to

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examine whether these fish oil emulsion properties are associated with an improvement of

clinical signs in patients with diabetes mellitus.

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A limitation of our study is that the experimental design is affected by the selection of our

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control. In addition, our results might be attributed not only to the fish oil emulsion but also

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to the emulsifier and other additives present in it. Another limitation is the experimental

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model system used in our study. We have not confirmed that hyperglycemia caused the

damage to proteins and lipids by determination of formation of protein carbonyls and 8-

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isoprostanes. Longer time of incubation or other markers of damage to proteins and lipids
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should be chosen. We have used cell cultures to examine FOE effects as a baseline for

understanding pathological changes in diabetic patients. However, it is necessary to examine

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the effects of FOE on diabetic patients. Therefore, our findings must be confirmed in animal

models and the human.

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Acknowledgment
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This study was financially supported by the Mind & Health, civil association and by the
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Obsidian Research Limited (currently Cultech Limited), Port Talbot, UK.

IG is an employee of Cultech Ltd. and had no role in the data analysis.

All other authors declare that they have no conflict of interest.

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Legends to the figures

Fig.1. Influence of hyperglycemia and the FOE on the proliferation 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 – cells in

hyperglycemic conditions incubated with different concentrations of the fish oil emulsion (10

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– 250 µg/mL). All values are reported as means ± SD of triplicates, each repeated 3 times

(n=3), (two way ANOVA with Tukey's multiple comparison tests).

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Fig.2. Influence of hyperglycemia and the FOE on antioxidant capacity of monocytes U937.

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C-NG – control cells in normoglycemic conditions, C-HG - control cells in hyperglycemic

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conditions, C-PBS- control cells in hyperglycemic conditions with PBS. FOE – cells in

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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

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compared with C-NG, bP < 0.05, bbP ≤ 0.01, bbbP ≤ 0.001- compared with C-PBS (two way
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ANOVA with Tukey's multiple comparison tests). All values are reported as means ± SD of

triplicates, each repeated 3 times (n=3).

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Fig. 3. Influence of hyperglycemia and the FOE on superoxide dismutase activity in

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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
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– cells in hyperglycemic conditions incubated with different concentrations of the fish oil
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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

reported as means ± SD of triplicates, each repeated 3 times (n=3).

Fig. 4. Influence of hyperglycemia and the FOE on levels of 8-oxo-guanin formation in

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-

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C-HG compared with C-PBS (two way ANOVA with Tukey's multiple comparison tests). All

values are reported as means ± SD of triplicates, each repeated 3 times (n=3).

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Fig. 5. Influence of hyperglycemia and the FOE on levels of protein carbonyls in monocytes

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U937. C-NG – control cells in normoglycemic conditions, C-HG - control cells in

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hyperglycemic conditions, C-PBS- control cells in hyperglycemic conditions with PBS. FOE

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– 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

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repeated 3 times (n=3), (two way ANOVA with Tukey's multiple comparison tests).
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Fig. 6. Influence of hyperglycemia and the FOE on levels of isoprostenes in monocytes

U937. C-NG – control cells in normoglycemic conditions, C-HG - control cells in

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hyperglycemic conditions, C-PBS- control cells in hyperglycemic conditions with PBS. FOE
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– 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
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repeated 3 times (n=3), (two way ANOVA with Tukey's multiple comparison tests).
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Fig. 7. Influence of hyperglycemia and the FOE on TNF release from monocytes-derived

macrophages. 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 - 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

repeated 3 times (n=3).

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Fig. 8. Influence of hyperglycemia and the FOE on IL-6 release from monocytes-derived

macrophages. C-NG – control cells in normoglycemic conditions, C-HG - control cells in

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hyperglycemic conditions, C-PBS- control cells in hyperglycemic conditions with PBS. FOE

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– cells in hyperglycemic conditions incubated with different concentrations of the fish oil

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emulsion (10 – 250 µg/mL). Differences are considered significant at aP < 0.05 - C-HG

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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

repeated 3 times (n=3).

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Fig. 9. Influence of hyperglycemia and the FOE on IL-8 release from monocytes-derived

macrophages. C-NG – control cells in normoglycemic conditions, C-HG - control cells in

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hyperglycemic conditions, C-PBS - control cells in hyperglycemic conditions with PBS. FOE
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– 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
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compared with C-NG (two way ANOVA with Tukey's multiple comparison tests). All values
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are reported as means ± SD of triplicates, each repeated 3 times (n=3).

Fig. 10. Influence of hyperglycemia and the FOE on MCP-1 release from monocytes-derived

macrophages. 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 aaaP ≤ 0.001- C-HG

compared with C-NG, bP < 0.05, bbP ≤ 0.01 - compared with C-PBS (two way ANOVA with

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Tukey's multiple comparison tests). All values are reported as means ± SD of triplicates, each

repeated 3 times (n=3).

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