Clinical Nutrition Experimental 12 (2017) 37e49

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Clinical Nutrition Experimental

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Deleterious effect of n-3 polyunsaturated fatty acids in

non-alcoholic steatohepatitis in the fat-1 mouse model
Diana Shefer-Weinberg a, Shlomo Sasson b, Betty Schwartz a,
Nurit Argov-Argaman c, Oren Tirosh a, *
a
School of Nutritional Sciences, Institute of Biochemistry, Food Science and Nutrition, The RH Smith Faculty of Agriculture, Food and
Environment, The Hebrew University of Jerusalem, 76100, Israel
b
The Institute for Drug Research, Department of Pharmacology, The Hebrew University, Faculty of Medicine, P.O. Box 12272,
Jerusalem 91120, Israel
c
Animal Science Department, The RH Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem,
76100, Israel

a r t i c l e i n f o s u m m a r y

Article history: Non-alcoholic fatty liver disease (NAFLD) represents a spectrum

Received 16 October 2016 of pathologies, ranging from hepatocellular steatosis to non-
Accepted 25 December 2016 alcoholic steatohepatitis (NASH), fibrosis and cirrhosis. It has
Available online 1 January 2017
been suggested that fish oil containing n-3 polyunsaturated fatty
acids (n-3 PUFA) induce beneficial effects in NAFLD. However, n-3
Keywords:
PUFA are sensitive to peroxidation that generate free radicals and
Lipids
reactive aldehydes. We aimed at determining whether changing
Fatty liver
Oxidative stress the tissue ratio of n-3 to n-6 PUFA may be beneficial or alterna-
Fat-1 tively harmful to the etiology of NAFLD. The transgenic Fat-1
n-3 PUFA mouse model was used to determine whether n-3 PUFA posi-
tively or negatively affect the development of NAFLD. fat-1mice
express the fat-1 gene of Caenorhabditis elegans, which encodes
an n-3 fatty-acid desaturase that converts n-6 to n-3 fatty acids.
Wild-type C57BL/6 mice served as the control group. Both groups
of mice were fed methionine and choline deficient (MCD) diet,
which induces NASH within 4 weeks. The study shows that NASH
developed faster and was more severe in mice from the fat-1
group when compared to control C57BL/6 mice. This was due
to enhanced lipid peroxidation of PUFA in the liver of the fat-1
mice as compared to the control group. Results of our mice study

Abbreviations: PUFA, polyunsaturated fatty acids; NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis;
MCS, methionine and choline sufficient; MCD, methionine and choline deficient; LA, linoleic acid; ALA, linolenic acid.
* Corresponding author. Institute of Biochemistry, Food Science and Nutrition, The Robert H Smith Faculty of Agriculture,
Food and Environment, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel. Fax: þ972 8 9363208.
E-mail address: oren.tirosh@mail.huji.ac.il (O. Tirosh).

http://dx.doi.org/10.1016/j.yclnex.2016.12.003
2352-9393/© 2016 Published by Elsevier Ltd on behalf of European Society for Clinical Nutrition and Metabolism. This is an
open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
38 D. Shefer-Weinberg et al. / Clinical Nutrition Experimental 12 (2017) 37e49

suggest that supplementing the diet of individuals who develop

or have fatty livers with n-3 PUFA should be carefully considered
and if recommended adequate antioxidants should be added to
the diet in order to reduce such risk.
© 2016 Published by Elsevier Ltd on behalf of European Society for
Clinical Nutrition and Metabolism. This is an open access article
under the CC BY-NC-ND license (http://creativecommons.org/
licenses/by-nc-nd/4.0/).

1. Introduction

n-3 and n-6 polyunsaturated fatty acids (PUFA) are essential to the normal function of every tissue
in the mammalian body. Deficiencies in these essential lipids are deleterious in disorders such as
cardiovascular disease, dyslipidaemia, the metabolic syndrome, liver and kidney abnormalities, blood
cell degeneration, reduced growth and regeneration, weak immune system, rheumatological condi-
tions, or neurological/neuropsychiatric diseases [1,2]. Therefore, it is generally recognized that
adequate supply of n-3 PUFA in the diet is essential for health. It is now commonly believed that a
balanced intake of n-3 and n-6 PUFA is beneficial due to the pro inflammatory effects of n-6 PUFA and
the anti-inflammatory effects of n-3 PUFA [1]. Indeed, different studies claim that sufficient n-3 PUFA
intake reduces incidence of heart disease and stroke, and relieved symptoms associated with ulcerative
colitis, and pain. Dietary n-3 PUFA are usually obtained from cold water fish, leafy green vegetables,
nuts and seeds such: sesame, hummus and oils such: linseed/flaxseed, soya bean, and more (Linus
Pauling Institute at Oregon State University; Essential Fatty Acids; 2010). Recommendations for n-3
PUFA supplementation to the diet have led to a big market and steady increasing consumption of these
fatty acids. Yet, some reports have suggested that the very high oxidizability of n-3 PUFA in the diet
and/or upon consumption may promote the generation of deleterious by-products that could
compromise health [3]. Turner at al. have summarized studies from cellular, animal and human trials
that have examined the effects of oxidized lipids and their potential to affect health outcomes, and also
proposed that variable levels oxidized products in fish oils may attenuate their beneficial effects and
lead to inconsistent and even conflicting outcomes in different clinical trials [4]. Thus, the advocacy for
n-3 PUFA supplementation in our diets may be challenged if these findings are relevant.
Overnutrition and sedentary life style are considered major contributing factors to the current
epidemic proportion of the metabolic syndrome: millions of overweight or obese people worldwide
are at risk to develop various symptoms of the metabolic syndrome, such as, hypertension, cardio-
vascular complications, stroke and type-2 diabetes. Metabolic parameters of the metabolic syndrome
include insulin resistance, elevated plasma triglycerides, abdominal obesity, hypercholesterolemia and
decreased levels of plasma high-density lipoprotein [5e8]. Fatty liver disease is considered the hepatic
manifestation of the metabolic syndrome can be used to demonstrate functional properties and the
nutritional value of different levels of tissue n-3 PUFA. Non-alcoholic fatty liver disease (NAFLD) and
non-alcoholic steatohepatitis (NASH) are becoming increasingly common medical problems in the
developed world with an over 30% prevalence in the general population [9]. It is predicted that by the
year 2025 over 25 million people in the USA alone may have NASH-related liver disease [10], of which
about 2e5% will develop cirrhosis. Additional metabolic insults will be at an increased risk of devel-
oping features of hepatic failure or hepatocellular carcinoma in this population.
The effect of n-3 PUFA on the development of fatty liver disease and the disease progression has
been evaluated in several studies. Casini and colleagues demonstrated that supplementation with n-3
PUFA protect the fatty liver of both rats and humans [11,12]. However, in these studies n-3 PUFA
improved the metabolic status of livers with no overt signs of liver dysfunction. On the other hand and
contrasting the beneficial reports of n-3 fatty acids role in NAFLD, Farrell and colleagues reported that
combination of n-3 PUFA with choline and methionine deficient diet dramatically increased hepatic
lipid peroxidation in mice, and n-3 PUFA did not protected against steatohepatitis but did not exac-
erbate it [13]. Additionally, supplementation of a rat diet with n-3 fatty acids obtained from 30% fish oil
 D. Shefer-Weinberg et al. / Clinical Nutrition Experimental 12 (2017) 37e49 39

to female SpragueeDawley rats (180e200 g), has been proposed as a model for induction of NAFLD
[14,15].
Nonetheless, different dietary supplementation with n-3 rich oils containing mixed contents of fatty
acids cannot differentiate between the effects of n-3 compared to n-6 PUFA in regards to NAFLD dis-
ease. This is not the case for the transgenic mice fat-1 model [16,17]. Fat-1 mice with the genetic
background of C57BL/J6 mice [1,2,16e19] converts n-6 fatty acids to n-3 fatty acids within the animal
tissues treated by any diet. This is an appropriate animal model that can eliminate confounding factors
of diet and would be very helpful for evaluation of the health effects of n-3 fatty acids in disease
conditions. fat-1 transgenic mouse are expressing the Caenorhabditis elegans fat-1 gene encoding an n-
3 fatty acid desaturase that converts n-6 to n-3 fatty acids (which is absent in mammals). When the
same diet is used for fat-1 mice as for the control wild type animals the model can be used to elucidate
the role of n3-PUFA lipids in NAFLD and in other diseases.
The current study was aimed to elucidate the impact of conversion of intra hepatic n-6 to n-3 fatty
acids and the changing the balance between the two PUFA types toward n-3 on the development of
NAFLD in mice (Scheme 1).

2. Experimental procedure

2.1. Chemicals and reagents

ECL Western Blot Detection Reagent (Advansta, USA), Agarose (Amresco, USA), SYBR-green PCR
master mix, Multi Scribe Reverse Transcriptase, dNTP mix, RNase inhibitor, random primers (Applied
Biosystems, USA), Difco Skim milk (BD, USA), Methanol, Formaldehyde 4% (Bio-Lab, Israel), Precision
Plus Protein All Blue Standards (Bio-Rad, Israel), TAE, Nuclease Free Water, Acrylamide solution
(Biological Industries, Israel), Loading dye (Fermentas, Canada), Fuji medical X-ray film, 100 nF (Fuji
Film, Japan), KOH, NaCl, KCl (Frutarom, Israel), DMEM medium, FCS, Penicillin, Streptomycin,
Trypsin-EDTA (Gibco, USA), MCD diet (Harlan, Israel), NaOH, Ethanol. (J.T. Baker, USA), Red load Taq
master (Larova, Germany) Sample buffer, Bovine serum albumin,SDS, Bradford reagent, Triton x-100,
Ethidium bromide, Tri-reagent, Isopropanol, Chloroform, Direct load DNA step ladder, Proteinase K,
Linolenic acid, Linoleic acid, N-acetyl-L-cysteine, Ponceau, APS, Bromophenol blue, Glycin, 2-
Mercaptoethanol, Tris, TEMED, Heparin, KH2PO4, K2HPO4, Sulfosalicylic acid, 2-Vinylpyridine, Met-
aphosphoric acid, DTNB, NaVO3, PMSF, Palmitic acid, Oleic acid, TBA, EDTA, L-Glutamine solution,

Scheme 1. Hypothesis scheme showing that oxidized omega 3 fatty acids are deleterious under non-alcoholic steatohepatitis
conditions.
40 D. Shefer-Weinberg et al. / Clinical Nutrition Experimental 12 (2017) 37e49

Hematoxylin, Eosin, (Sigma, Israel). For lipid extraction, analytical reagent grade methanol and
chloroform were purchased from (Bio-Lab Israel). For fatty acid extraction and methylation, meth-
anol analytical grade was (Bio-Lab, Israel). Petroleum ether (analytical reagent grade) was from
Gadot Lab Supplies (Netanya, Israel), and sulfuric acid (Bet Dekel Ra'anana, Israel). Retention times
were determined by injection of commercial mixtures of two fatty acid methyl ester (FAME) stan-
dards: from C14:0 to C22:6n3 (PUFA-2, Animal Source) and FAME mix of C8:0-C24:0 (Supelco, Sigma
aldrich, Israel).

2.2. Animal model

Experiments were performed on 8 week old C57BL/6J male mice (Harlan Laboratories, Israel)
and fat-1 mice (provided by Dr. Kang, Harvard, Boston, USA). All animals were maintained in
specific pathogen free room in standard cages, with controlled temperature conditions and
lighting conditions of 12 h light and 12 h of darkness. All animals had an ad libitum access to a
standard chow diet (methionine & choline sufficient diet) and water. All animals were cared ac-
cording to the guidelines of the Animal Care and Use Committee of the Hebrew University of
Jerusalem.
Mice were divided into four experimental groups: two control groups consisted of C57BL/6J and fat-
1 male mice, which were fed ad libitum the standard chow diet for 4 weeks [methionine and choline
sufficient diet (MCS)] and two groups of mice, fed a methionine and choline deficient (MCD) diet for the
same period of time n(C57BL/6J) ¼ 7, n(C57BL/6J þ MCD) ¼ 7, n(Fat-1) ¼ 5, n(Fat-1þMCD) ¼ 7. Mice
were weighed and sacrificed at the end of the experiment. Blood was collected from abdominal aorta
into vials containing 20 ml Heparin (104 mg per ml), centrifuged at 2500 rpm for 5 min and sera were
collected and liver enzyme examination was performed by an accredited chemical analyzer in Amer-
ican Lab, Herzelia Israel. Livers were snap frozen in liquid nitrogen, and kept at 80  C for further
processing.

2.3. Identification of the fat-1 gene in mice

DNA extraction from tail

Two mm sections of fat-1 mice tails were digested in 500ul digestion buffer (5 mM EDTA, pH 8.0,
200 mM NaCl, 100 mM Tris, 0.2% sodium dodecyl sulfate (SDS) and 100 mg/ml proteinase K) in
1.5 ml eppendorf tubes at 37  C overnight. 500 ml isopropanol was added to each sample, mixed
well and centrifuged at 5000 rpm for 5 min at room temperature. The pellet was mixed with 1 ml of
70% ethanol and the samples centrifuged at 12,000 rpm for 20 min at room temperature. The
supernatant was discarded and the pellet was quickly dried and dissolved with 30e50 ml nuclease
free water. Nano drop analysis was used to determine DNA concentrations and the volume needed
for 1 mg DNA.
DNA extraction from liver
Frozen liver tissues were homogenized with the abovementioned digestion buffer and kept at 37  C
overnight. Proteins and cellular debris were precipitated with 300 ml of 6 M NaCl and kept at 4  C for
15 min and then centrifuged at 25,000 g for 20 min. Aliquots of 500 ml of supernatant were transferred
to a new Eppendorf tubes containing 800 ml of isopropanol and further centrifuged at 8000 g for 5 min.
The resulting pellets were washed with 1 ml of 70% ethanol and the samples were centrifuged at
12,000 rpm for 20 min at room temperature. The supernatant was discarded and the pellet was dis-
solved with 50 jml of nuclease free water.
DNA was amplified by PCR using fat-1 gene specific primers (Fat-1 Forward50 -ATA TTC TAG ACA AGT
TTG AGG TAT GGT CGC-30 ; Fat-1 Reverse50 -ATA TAC TAG TAA GAG TTA TGG CTT TAT GCA-30 ) and the
fragments were separated by gel electrophoresis.

2.4. Tissue fat extraction and GC analysis

Frozen liver tissues were weighed, homogenized with 800 ml of a methanol:chloroform mixture
(1:2 ratio) and vortexed for several seconds. After dispersion, the mixture was agitated for two hours at
 D. Shefer-Weinberg et al. / Clinical Nutrition Experimental 12 (2017) 37e49 41

room temperature and centrifuged at 3500 rpm for 5 min. The supernatant fraction was transferred
into new, weighed vials, containing 200 ml H2O, vortexed for several seconds and placed for 30 min
allowing for two phases to separate. After removing the upper (aqueous) phase containing proteins,
the lower chloroform phase containing the extracted fat was evaporated using speed vac, for an hour at
30  C and fat weight was measured.

2.4.1. Fatty acid analysis using gas chromatography (GC)

Fatty acid composition was determined by chromatographic analysis as previously described (20).
The samples were analyzed using GC as previously described using a set of methyl-ester derivatives of
the various fatty acids [20,21]. Briefly, analysis was performed with a 6890 N gas chromatograph
(Agilent Technologies, Wilmington, DE) equipped with a fused-silica (60 m  0.25 mm ID, 0.25 mm
film) capillary column (DB-23, Agilent Technologies) under the following conditions: the oven tem-
perature was programmed from 130  C to 170  C at a rate of 27  C/min, from 170  C to 215  C at a rate of
2  C/min, held at 215  C for 8 min, from 215  C to 250  C at a rate of 40  C/min, and held at 250  C for
5 min. Run time was 37.9 min. Helium was used as the carrier gas at a flow rate of 2.21 ml/min. Flame-
ionization detector temperature was 280  C, and injector temperature was 270  C. Peak identification
was based on relative retention times of two external standards. The area of each fatty acid peak was
recorded using ChemStation software (Agilent Technologies) and fatty acid weights were calculated
using the internal standard peak area [20,21].

2.5. Lipid peroxidation

2.5.1. Thiobarbituric acid reactive species assay

Liver tissues were weighed, homogenized in 1 ml of 10% (w/v) TCA and centrifuged at 5000 rpm for
10 min at 4  C. Two ml of thiobarbituric acid (0.375%, w/v) were mixed with 500 ml of the upper phase
and incubated for 15 min in boiling water. The absorbance was measured at 532 nm. The lower phase
was dissolved with 500 ml 0.3 M KOH for protein determination.

2.5.2. Conjugated dienes assay

Liver tissue was extracted for fat content determination using Folch method. Lipids were dissolved
with 1 ml hexane and absorbance of conjugated diene was measured at 234 nm.

2.6. Western blot analysis and real time -PCR

Performed as previously reported [22].

2.7. Liver histology

Oil red staining of tissue section was performed on 5 mm frozen liver tissue sections.

2.8. Cell culture and in vitro treatment

AML-12 hepatocytes (ATCC) were treated with different concentrations of free fatty acids for 18 h as
previously reported [23,24].

2.9. Statistical analysis

All values were expressed as mean values þ SEM. Comparisons between two groups were per-
formed with student's t-test. For multiple groups, statistical analysis was performed using the statis-
tical computer program, SPSS version 17.0 (SPSS, USA) with One-way ANOVA-tests. In experiments
with multiple groups common letters were used in order to indicate significance. Means with different
letters are significantly different at p < 0.05.
42 D. Shefer-Weinberg et al. / Clinical Nutrition Experimental 12 (2017) 37e49

3. Results

3.1. Pro-oxidant effect of n-3 linolenic acid in AML12 hepatocytes

The effect on steatosis in AML-12 cells of linolenic acid (ALA), an essential n-3 fatty acids with lower
potential to be oxidized (compared to longer n-3 fatty acids) was evaluated. The cells were incubated
with different combinations of fatty acid for 18 h. Intracellular lipid levels were evaluated using Nile red
staining and flow cytometry analysis. Figure 1A and 1B shows that the total lipid accumulation was not
altered by the addition of n-3 fatty acid, also there was no significant change in intracellular ROS
production as evaluated by H2DCF oxidation.

3.1.1. In vitro lipid peroxidation levels: conjugated dienes formation

In order to examine whether ALA modulates lipid peroxidation in steatotic hepatocytes, formation
of conjugated dienes was evaluated. Figure 2 shows that in AML12 cells following exposure to ALA a
trend for (P ¼ 0.1) increased level of conjugated dienes was observed as compared to cells treated with
the essential n-6 linoleic acid (LA). These findings indicate a higher intracellular oxidizability of n-3
PUFA as compared to n-6 PUFA.

Fig. 1. The effect of alpha linolenic acid (ALA) on total lipid and ROS accumulation in AML12 hepatocytes. AML12 cells were
incubated for 18 h with different fatty acids mixtures (oleic acid (OA): palmitic acid (PA); 2:1, OA: PA: ALA 1:1:1 in complex with
BSF), total concentration 1 mM. Steatosis, evaluated by Nile red (A), and ROS, evaluated by DCFH oxidation (B). Cells were analyzed
by flow cytometry. Means with different letters in each group differ significantly at P < 0.05. n ¼ 3.
 D. Shefer-Weinberg et al. / Clinical Nutrition Experimental 12 (2017) 37e49 43

Fig. 2. The effect of alpha linolenic acid (ALA) on lipid peroxidation evaluated by conjugated dienes formation. AML12 cells were
incubated for 18 h with 1 mM of different kinds of fatty acid mixture as described in the legend to Fig. 1. At the end of incubation the
cell fat was extracted using Folch reagent (methanol: chloroform; 1:2). Conjugated dienes levels were determined at 234 nm
(ε ¼ 28*103 M1 cm1). t-test at P ¼ 0.1. n ¼ 3.

3.2. Pro-steatotic effect of n-3 PUFA in NASH: MCD model

3.2.1. Fat accumulation in the livers of treated animals

n-3 PUFA were suggested to potentiate lipid beta oxidation and metabolism [13], we therefore
asked whether n-3 PUFA could protect against the deleterious effects of the MCD diet and decrease
liver fat accumulation. Surprisingly, the highest percentage of steatotic hepatocytes was found in fat-1
mice supplemented with the MCD diet. In contrast, fat-1 mice supplemented with the regular diet had
the lowest levels of fat (Fig. 3A).
Using GC analysis the absolute amounts of different fatty acids in the liver was evaluated. The fat-1
mice had a more balanced ratio between n-6 and n-3 (Fig. 3B, Table 1). As could be expected mice with
MCD diet had increased accumulation of fatty acids in the liver (Table 1).

3.2.2. Liver histology

Histological examination of the liver sections demonstrated significant accumulation of lipids in
fat-1 mice fed the MCD diet for 4 weeks. Oil red staining (Fig. 4) showed a marked difference between
the MCS diet control groups and MCD diet treated groups. The histological results support the total
lipid content detected by the quantitative lipid extraction and liver weight measurement. Increase in
macrovesicular steatosis in fat-1 mice treated with MCD diet compared to C57BL/J6 mice was
observed.

3.3. Pro-oxidant effect of n-3 PUFA in NASH: MCD model

3.3.1. Levels of liver enzymes in the serum

Figure 5 shows that there were no significant differences in serum hepatic enzyme activities in
C57BL/J6 as compared to fat-1 mice fed MCS diet (control diet). The activity levels of hepatic enzymes
in the blood remained within the normal physiological range.
However, both C57BL/J6-and fat-1 MCD diet-fed groups, exhibited significantly elevated serum liver
enzyme activities, indicating liver damage. Furthermore, the MCD diet-fed fat-1 mice had significantly
higher hepatic enzymes levels in the serum, indicating more intensive liver damage compared to wild
type C57BL/J6 mice (Fig. 5).
44 D. Shefer-Weinberg et al. / Clinical Nutrition Experimental 12 (2017) 37e49

Fig. 3. Increased fat accumulation in fat-1 mice: Evaluation of total fat in mice livers (fat percentage). Livers of fat-1 and C57BL/J6
mice treated or not with MCD diet were evaluated. Frozen liver tissues were weighed and extracted using Folch reagent dried and
analyzed by weight (A), and by Gas chromatography and ratio of quantitative measurements of specific fatty acids was calculated (B).
Means with different letters in each group statistically differ at p < 0.05.

Table 1
Accumulation of fatty acids in mice livers following treatment for 4 weeks with MCD diet and MCS diet.

C18:2n6 cis9,12 (LA) C20:4n6 (AA) C18:3n3 cis12,15 C20:5n3 (EPa) C22:6n3 (DHA)
[mg fat/gr liver] [mg fat/gr liver] (ALA) [mg fat/gr liver] [mg fat/gr liver] [mg fat/gr liver]

C57BL/6 1395.33±86.54C 704.12±116.15B 42.13±3.4B 17.52±3.61C 325.00±73.30C

C57BL/6þMCD 2896.54±447.14B 1329.67±154.52A 21.96±3.0C 8.76±0.71D 640.99±64.28B
Fat-1 1235.33±250.75C 586.65±110.34B 43.29±4.96B 46.28±4.41B 553.81±64.62C,B
Fat-1þMCD 5257±794.6A 1239.82±100.12A 71.52±9.41A 104.50±11.05A 1358.34±110.5A

Means that are inculcated with different letters are significantly different, p < 0.05.

3.3.2. Increased damage and lipid peroxidation in livers of fat-1 mice treated with MCD diet
Figure 6A shows significantly higher levels of conjugated dienes formation in fat-1mice fed the MCD
diet for 4 weeks (Fig. 6A). These results suggest that livers containing high levels of n-3 PUFA are more
susceptible to liver lipid peroxidation compared to liver that are richer in n-6 PUFA. Figure 6B shows
that fat-1 mice treated with MCD diet had the highest and the most significant production of advanced
lipid peroxidation end products such as Malondialdehyde (MDA) as was evaluated by the TBARS assay.
 D. Shefer-Weinberg et al. / Clinical Nutrition Experimental 12 (2017) 37e49 45

Fig. 4. Increased fat accumulation in Fat-1 mice: liver histology and oil red staining. Images of liver sections stained with oil red.
C57BL/J6 and fat-1 mice treated or not with MCD diet for 4 weeks were evaluated. Liver morphology and fat accumulation were
evaluated by oil-red-O staining (red) for lipids at 40 magnification bars indicate 50 mm.

4. Discussion

Non-alcoholic fatty liver disease (NAFLD) represents a spectrum of diseases ranging from hepato-
cellular steatosis through steatohepatitis to fibrosis and irreversible cirrhosis. The prevalence of NAFLD
has risen rapidly in parallel with the dramatic rise in obesity and diabetes and is rapidly becoming the
most common cause of liver disease in Western countries [18,20]. In this study we aim at elucidating
the effect of n-3 PUFA on NAFLD progression. To this end we used the fat-1 mouse model that expresses
the fat-1 gene of C. elegans, which encodes an n-3 fatty-acid desaturase that converts n-6 to n-3 fatty
acids allowing to test the direct effect of n-3 fatty acids in NAFLD and NASH without confounding
effects of n-3PUFA rich diet.
Hitherto there is no approved pharmacological treatment to the disease and the main recom-
mendation is a better and healthier life style. In obese patients, who make up the majority of patients
with NASH, current therapies are centered on weight loss and exercise programs [25]. n-3 PUFA
nutrition has been proposed as a potential supportive treatment [2].
Kang's et al. [17] showed that a balanced ratio of n-6 to n-3 PUFA, and particularly balanced AA/
(EPA þ DPA þ DHA) ratio, in all of the organs of fat-1 mice. n-3 PUFA in organs of the fat-1 mice is
enriched compared to the WT mice without additional dietary n-3 PUFA [26]. Accordingly, we also
found similar enrichment in n-3 PUFA (see Table 1). Notably, the comparison between C57BL/J6 mice
and fat -1 mice shows that the former had higher amounts of LA and AA compared to lower amount of
ALA, EPA and DHA in the liver. Furthermore a more balanced ratio AA/(EPA þ DHA) is observed. MCD
diet increase the absolute amount of FA in the liver most probably due to the formation of fatty liver
and prevention of lipoproteins excretion by the liver [27,28].
46 D. Shefer-Weinberg et al. / Clinical Nutrition Experimental 12 (2017) 37e49

Fig. 5. Increased liver damage in fat-1 mice: serum hepatic enzymes activities. Blood was collected from the abdominal aorta of fat-
1 and C57BL/J6 mice treated or not with MCD diet. Serum was separated and liver enzyme activity of alanine aminotransferase (ALT)
(A), and aspartate aminotransferase (AST) (B) was determined. Means with different letters are statistically different at p < 0.05.

NASH is associated with increased activities of in liver enzymes in the serum and the accumulation
of fat in the hepatocytes. Serum taken from fat-1 mice fed the MCD diet had increased levels of alanine
aminotransferase and aspartate aminotransferase activity in comparison with the C57BL/J6 control
group mice fed a similar diet. In mice supplemented with MCS diet, there were no difference between
the two strains of mice and the level of liver enzyme activities in the serum was low. These findings
agree with Tipoe et al. who demonstrated that unsaturated fatty acid in the diet with 30% of calories
from fish oil lead to an increase in serum alanine aminotransferase in rats [14,15].
In addition to liver damage, the highest percentage of accumulation of fat in the livers was observed
in the fat-1 group fed the MCD diet, while when fed the MCS diet fat-1 mice had the lowest percentage
of fat of all groups.
It has been suggested that n-3 PUFA function in the healthy livers as a fat lowering agent takes place
most probably due to the upregulation of PPARa, which induces fatty acid oxidation and reduction of
SREBP-1 [29,30]. Our results suggest that this is unlikely to occur under the necro-inflammation stress
conditions induced by the MCD diet. The MCD diet-induced NASH can lead to oxidative stress and
decrease antioxidant protection [27,28]. Therefore, n-3 PUFA will be subjected to lipid peroxidation
under such conditions and will facilitate increased liver damage and increased steatosis.
Liver histology and Oil red staining demonstrated higher liver fat content in MCD diet-fed to the fat-
1 group compared to WT, it is known that unsaturated fatty acids activate triglyceride synthesis and by
 D. Shefer-Weinberg et al. / Clinical Nutrition Experimental 12 (2017) 37e49 47

Fig. 6. Increased lipid peroxidation in fat-1 mice. Livers of fat-1 and C57BL/J6 mice treated or not with MCD diet were evaluated for
lipid peroxidation. Conjugated dienes formation levels. Fat was extracted from frozen liver tissue and the protein concentration was
determined using Bradford assay. Conjugated dienes levels were determined at 234 nm (ε ¼ 28*103 M1 cm1). Means with different
letters in each group differ at p < 0.05. (A), Determination of thiobarbituric acid reacting substances (TBARS) levels. Frozen liver
tissues homogenized in 1 ml 10% TCA, centrifuged at 5000 RPM, for 10 min at 4  C. 2 ml TBA (0.375 g %) were mixed with 500 ml of
the upper phase and incubated for 15 min in boiling water. The absorbance was measured at 532 nm (ε ¼ 1.56*105 M1 cm1) (B).
Protein concentration was determined using Bradford assay. Means with different letters in each group differ at p < 0.05.

that may lead to increased steatosis as was demonstrated for oleic acid [24,31]. These finding strongly
suggest that the common view on the beneficial effects of n-3 PUFA for lowering triglycerids is highly
dependent on the dosage of n-3 PUFA, and that over-consumption of these fatty acids may be detri-
mental and enhance the induction and progression of NAFLD when the disease is active.
Numerous studies have shown that free fatty acids can generate toxic by-products under increasing
oxidative stress [20]. Moreover, the onset of oxidative stress have also been linked to lipid peroxidation
accumulation in the fatty livers [32]. Tipoe et al. showed an increase in nitrotyrosine proteins, a marker
of oxidative stress [14]. Larter et al. group fed mice with n-3 PUFA-enriched fish oil diet in the MCD
model (compared with mice fed the corresponding diet supplemented with olive oil). Though in their
study n-3 PUFA feeding activated PPARa and suppressed hepatic de novo lipogenesis, while it failed to
prevent the development of steatohepatitis. Moreover, they observed an extensive accumulation of
oxidized lipids (TBARs) in fish oil combined with MCD diet-fed animals [13]. Grundt et al. followed
subjects who suffered from an acute myocardial infarctions and received high doses of n-3 PUFA in
their diets. One of his findings in this long-term, post infarct study was an increase in lipid peroxi-
dation, measured as TBAeMDA by HPLC compared with the a control group of patients who consumed
48 D. Shefer-Weinberg et al. / Clinical Nutrition Experimental 12 (2017) 37e49

n-6 FA rich corn oil [33]. The study may point to a potential damaging side of high dose of n-3 PUFA in
the diet.
In conclusion, we demonstrate that high concentrations n-3 PUFA are strong inducers of lipid
peroxidation and liver damage under inflammatory conditions. Caution should be considered when
recommending it to NASH patients.

Conflict of interest

None.

Acknowledgment

This study was supported by a Grant from the Center of Nutrigenomics and Functional Foods of the
Faculty of Agriculture, Food and Environment to O.T. and S.S.

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