ORIGINAL CONTRIBUTION

Effect of fish and krill oil supplementation on glucose tolerancein rabbits with experimentally induced obesity

Zhenya Ivanova • Bodil Bjørndal • Natalia Grigorova • Anton Roussenov • Ekaterina Vachkova •

Kjetil Berge • Lena Burri • Rolf Berge • Spaska Stanilova • Anelia Milanova • Georgi Penchev •

Rita Vik • Vladimir Petrov • Teodora Mircheva Georgieva • Boycho Bivolraski • Ivan Penchev Georgiev

Received: 14 May 2014 / Accepted: 7 October 2014

� Springer-Verlag Berlin Heidelberg 2014

Abstract

Purpose This study was conducted to investigate the

effect of fish oil (FO) and krill oil (KO) supplementation

on glucose tolerance in obese New Zealand white

rabbits.

Methods The experiments were carried out with 24 male

rabbits randomly divided into four groups: KO—castrated,

treated with KO; FO—castrated, treated with FO; C—

castrated, non-treated; NC—non-castrated, non-treated. At

the end of treatment period (2 months), an intravenous

glucose tolerance test (IVGTT) was performed in all

rabbits.

Results Fasting blood glucose concentrations in FO and

KO animals were significantly lower than in group C. The

blood glucose concentrations in FO- and KO-treated ani-

mals returned to initial values after 30 and 60 min of IV-

GTT, respectively. In liver, carnitine palmitoyltransferase 2

(Cpt2) and 3-hydroxy-3-methyl-glutaryl-CoA synthase 2

(Hmgcs2) genes were significantly increased in FO-fed

rabbits compared with the C group. Acetyl-CoA carbox-

ylase alpha (Acaca) expression was significantly reduced in

both KO- and FO-fed rabbits. In skeletal muscle, Hmgcs2

and Cd36 were significantly higher in KO-fed rabbits

compared with the C group. Acaca expression was signif-

icantly lower in KO- and FO-fed rabbits compared with the

C group.

Conclusion The present results indicate that FO and KO

supplementation decreases fasting blood glucose and

improves glucose tolerance in obese New Zealand white

rabbits. This could be ascribed to the ameliorated insulin

sensitivity and insulin secretion and modified gene

expressions of some key enzymes involved in b-oxidation

and lipogenesis in liver and skeletal muscle.

Keywords Fish oil � Krill oil � Glucose tolerance �Obesity � Gene expression � Lipogenesis

Z. Ivanova � N. Grigorova � E. Vachkova � A. Milanova �T. M. Georgieva � B. Bivolraski � I. P. Georgiev (&)

Department of Pharmacology, Animal Physiology and

Physiological Chemistry, Faculty of Veterinary Medicine,

Trakia University, 6000 Stara Zagora, Bulgaria

e-mail: iv_p63@yahoo.com

B. Bjørndal � R. Berge � R. Vik

Department of Clinical Science, University of Bergen,

5020 Bergen, Norway

A. Roussenov

Department of Internal Diseases, Faculty of Veterinary

Medicine, Trakia University, 6000 Stara Zagora, Bulgaria

K. Berge � L. Burri

Aker BioMarine Antarctic AS, Fjordalleen 16,

0115 Oslo, Norway

S. Stanilova

Molecular Biology, Immunology and Medical Genetics, Medical

Faculty, Trakia University, 6000 Stara Zagora, Bulgaria

G. Penchev

Department of Veterinary Anatomy, Histology and Embryology,

Faculty of Veterinary Medicine, Trakia University,

6000 Stara Zagora, Bulgaria

V. Petrov

Department of Veterinary Microbiology, Infection and Parasitic

Diseases, Faculty of Veterinary Medicine, Trakia University,

6000 Stara Zagora, Bulgaria

123

Eur J Nutr

DOI 10.1007/s00394-014-0782-0

Introduction

Obesity is often associated with insulin resistance (IR) and

deterioration of lipid and glucose metabolism, which are

the hallmarks of metabolic syndrome [1–6]. Briefly, IR can

be defined as an inadequate response by insulin target tis-

sues, such as skeletal muscles, adipose tissue and liver, to

insulin exposure [7–10]. The mechanisms of decreased

insulin sensitivity include reduced insulin-stimulated glu-

cose uptake and metabolism in skeletal muscle and adipose

tissue, impaired inhibition of hepatic glucose production,

i.e., gluconeogenesis and glycogenolysis, and a reduced

ability of insulin to inhibit lipolysis in adipose tissue [2, 10,

11]. In humans, IR can be manifested as one of the fol-

lowing three symptoms: diabetes mellitus type 2 (plasma

fasting glucose concentration [7 mmol/l); impaired glu-

cose tolerance (plasma glucose concentration 2 h after

glucose oral tolerance test between 7.8 and 11 mmol/l);

and impaired fasting glucose (plasma glucose concentra-

tion between 6.1 and 6.9 mmol/l).

There is a growing body of evidence showing that the

consumption of products containing marine n-3 long-chain

polyunsaturated fatty acids (n-3 LC-PUFAs) can positively

affect IR [4, 11–16].

Two possible sources of marine n-3 LC-PUFAs are fish

oil (FO) and krill oil (KO). KO is proposed as a nutritional

supplement during the last years, and an increasing number

of studies demonstrate health benefits in rodents (mice and

rats) as well as humans [14, 16–18]. KO is extracted from

Antarctic krill (Euphausia superba) and has a unique

chemical composition. Both FO and KO are rich in n-3 LC-

PUFAs, but KO contains the fatty acids predominantly in

the form of phospholipids (PL) rather than triglycerides

(TG) as in FO [16, 17]. In addition to its high content in

PL, KO contains the antioxidant astaxanthin that gives KO

its dark red color and might help to protect the unsaturated

bonds from oxidative damage [17]. The preclinical studies

have shown that exogenous KO supplementation improves

lipid profile, decreases body weight, reduces endocannab-

inoid biosynthesis, decreases liver fat infiltration and oxi-

dative stress and increases hepatic b-oxidation [14, 18–22].

The clinical studies demonstrate that KO improves blood

lipid profile, changes endocannabinoid concentration,

reduces oxidative damage and increases blood level of n-3

PUFAs [15, 23–27]. However, despite these studies indi-

cating that dietary KO supplementation has beneficial

metabolic and anti-inflammatory effects in rodents and

humans, there is little basic research investigating its effect

on glucose tolerance and insulin resistance.

Rabbits have several advantages over mice as an animal

model to study various obesity-associated metabolic

abnormalities such as dyslipidemia, atherosclerosis, MS

and IR, since their lipid profile and metabolism is similar to

that of humans (so-called LDL mammals) and differ from

mice and rats (so-called HDL mammals). In addition, they

have high levels of ApoB-containing lipoproteins and

cholesterol ester transfer proteins and are very susceptible

to the development of atherosclerosis, with lesions

resembling those in humans [28–31].

Studies in rodents, and to a lesser extend in humans,

have demonstrated that FO has positive effects on some

parameters of glucose homeostasis [4, 11, 13, 32–35].

However, no data on the effects of FO and KO on insulin

resistance and b-cell function are available in rabbits.

Therefore, this study was conducted to investigate the

effect of FO and KO supplementation on glucose tolerance

in rabbits with experimentally induced obesity by

castration.

Materials and methods

Animal experiments were conducted according to the

Guide for the Care and Use of Laboratory Animals, and the

Guidelines of the Animal Welfare Act, and were approved

by the Commission of Ethics at the Faculty of Veterinary

Medicine of Trakia University, Stara Zagora.

The experiments were carried out with 24 male New

Zealand white rabbits. At the beginning of the experiment,

they were between 3 and 3.5 months old. The animals were

housed in individual cages (80 9 60 9 40 cm). The light/

dark regime corresponded to the circadian cycle. The

rabbits were given free access to water. They were fed a

commercially available standard chow diet for adult rabbits

given as dry pellets. During the experimental period, all

rabbits received the same amount of energy by the diet

&450 kcal/daily. Before the randomization to groups,

initial body weight was determined and blood samples

were taken from each rabbit for the determination of the

pre-castrations values of blood glucose and plasma insulin.

The rabbits were randomly divided into four groups:

KO—castrated, treated with KO; FO—castrated, treated

with FO; C—castrated, full diet fed; and NC—non-

castrated.

The castration was used to induce obesity in rabbits

[31]. The castration of the rabbits was performed under

general anesthesia using Anaket 10 %. For surgery, the

rabbits were laid on their backs, the fur in the scrotal area

was depilated, and the skin was disinfected. The scrotal

wounds after castration remained open [31].

The KO and FO were kindly provided by Aker Bio-

Marine Antarctic AS Oslo, Norway. They were given as

gelatinous capsules at a dose of 600 mg omega-3 PUFA

daily for a period of 60 days.

On the day before intravenous glucose tolerance test

(IVGTT), body weight, body weight gain and body mass

Eur J Nutr

123

index (BMI) were determined as markers of obesity. BMI

was calculated using the equation = body weight (kg)/

height2 (m). The height was measured as distance between

the shoulder joint and the end of the paw at the lateral

position of the rabbit.

At the end of the treatment period of 2 months after

castration, an intravenous glucose tolerance test was per-

formed in all rabbits as previously described [31, 36, 37].

Briefly, food was removed for 12 h overnight, and a bolus

of 40 % glucose (0.6 g/kg) was injected through the ear

vein. Blood samples for the determination of glucose and

insulin concentration were obtained at 0, 10, 30, 60 and

120 min after glucose administration. The blood samples

were centrifuged immediately after the collection at

8009g for 15 min. Plasma for the determination of insulin

was stored in plastic tubes at -20 �C until assayed. Glu-

cose concentration was measured in whole blood.

Blood glucose and insulin analysis

The blood glucose concentration was measured immedi-

ately after collection of the samples with a blood glucose

monitoring system (BIONIME Gmbh, Heerbrugg, Swit-

zerland) based on the glucose oxidase method using one

drop of whole blood. Plasma insulin concentration was

measured by radioimmunoassay with a commercially

available kit adapted for rabbits (Immunotech, Prague,

Czech Republic).

Indexes of insulin sensitivity

The indexes of insulin sensitivity were calculated as

described in cats [38]. Fasting insulin (I0) and fasting

insulin to glucose (G0) ratio (I0/G0) were determined before

glucose injection. Insulin concentration (I60 min and

I120 min) and insulin to glucose ratio (I60 min/G60 min and

I120 min/G120 min) were calculated also after 60 and 120 min

after glucose infusion.

The following kinetic parameters, indicative for the

‘‘fate’’ of glucose, were estimated: half-life of plasma

glucose (t� glucose) and area under the curve (AUC) of

glucose and insulin concentrations (AUCglucose 0–120 min

and AUCinsulin 0–120 min). Kinetic parameters were calcu-

lated with Phoenix 6.01 (Pharsight Corporation, Mountain

View, USA). AUCglucose 0–120 min and AUCinsulin 0–120 min

were calculated by the trapezoidal rule. t�glucose was cal-

culated by linear regression analysis of the semilogarithmic

plot of glucose concentration versus time.

Some simplified estimates of insulin sensitivity were

calculated from insulin and glucose values at baseline:

HOMAins. res., QUICKI and the Bennett index [38–41].

HOMAins. res. was calculated using the following equations:

(1) HOMAins. res. = (I0 9 G0)/22.56 [39, 40, 42]; (2)

QUICKI = 1/logI0 ? logG0 [38, 40, 42]; and (3) Bennett

index = 1/logI0 9 logG0 [38, 41], where I0 is the amount

of fasting insulin (lU/ml) and G0 is the fasting glucose

value (mmol/l). Higher HOMAins. res. and lower QUICKI

and Bennett indexes are indicators of increased insulin

resistance.

Indexes of b-cell function

HOMAb-cell was calculated using the equation:

HOMAb-cell = (20 9 I0)/(G0 - 3.5) [39, 42].

The indexes characterizing the first or early phase of

insulin secretion and insulin secretion during the first and

second hours after glucose loading were calculated as

shown in dogs [43, 44]. The highest values of insulin and

glucose were considered peak values, and the increments of

insulin and glucose concentration above their respective

fasting levels were considered as DI and DG [43]. Early-

phase insulin secretion in response to glucose infusion was

calculated as the insulinogenic index (DI/DG), AUC for

insulin determined from 0 to 10 min (AUCinsulin 0–10 min)

and insulin to glucose ratio after 10 min (I10 min/G10 min).

Insulin secretion during the first and second hour after

IVGTT was calculated as AUCinsulin 0–60 min and

AUCinsulin 0–120 min, respectively [43, 44].

Histological examination

Materials for histological examination were taken from the

liver after the animals were killed. Briefly, after fixation in

Bouin’s fixative and in 10 % neutral-buffered formalin, the

tissue specimens were embedded in paraffin, cut into 5–6-

lm-thick section and stained with hematoxylin and eosin.

Enzyme activity

Frozen liver samples were homogenized and the post-nuclear

fraction isolated as described earlier [45]. The assay for car-

nitine palmitoyltransferase (CPT)-2 was performed according

to Bremer [46], but with some modifications: The reaction

mix contained 17.5 mM HEPES pH 7.5, 52.5 mM KCl,

100 mM palmitoyl-CoA and 0.01 % Triton X-100. The

reaction was initiated with 100 lM [methyl-14C]-L-carnitine

(1,100 cpm/nmol), and 35 lg total protein was used [47]. The

activity of fatty acyl-CoA oxidase (ACOX)-1 and fatty acid

synthase (FAS) were measured in post-nuclear fractions using

20 lg protein and 60 lg, respectively, as described by

Madsen et al. [48] and Skorve et al. [47].

Gene expression analysis

Total RNA was purified and isolated from homogenized

pieces of liver and muscle of approximately 20 and 30 mg,

Eur J Nutr

123

respectively, using the RNAeasy Mini Kit from Qiagen

(Hilden, Germany). For muscle samples, an extra step of

proteinase K treatment was added (Qiagen). The quantity

of the RNA was measured spectrophotometrically using

NanoDrop ND-1000 (NanoDrop Products, Wilmington,

DE, USA). The purity of the RNA was assessed using

Agilent RNA 6000 Nano Kit on a Agilent 2100 Bioana-

lyzer (Agilent Technologies Inc., Santa Clara, USA). The

quality limit for further use of RNA was set to a RNA

integrity number (RIN) C7 (out of 10). cDNA was obtained

using the High Capacity Reverse Transcriptase Kit with

RNase inhibitor (Applied Biosystem, Foster City, USA).

Real-time PCR was performed on an ABI prism 7900 H

sequence detection system (Applied Biosystems) using

384-well multiply PCR plates (Sarstedt Inc., Newton,

USA), SYBR Select Master Mix from Applied Biosystems

and gene-specific primers from Sigma–Aldrich (Table 1).

Hypoxanthine phosphoribosyltransferase (Hprt1) and 18s

were used as reference genes. For 18s, primers from the

18s Genomic Control Kit (#RT-CKFT-18s) were used

(Eurogentec, Seraing, Belgium). Dilutions of pooled cDNA

were used for the standard curves. NormFinder (http://

moma.dk/normfinder-software) was used to assess the

reference genes, and hepatic mRNA levels were normal-

ized to Hprt1, while muscle tissue was normalized to 18s.

Gene names and sequences of specific primers, including

references genes, are presented in Table 1.

Statistical analysis

The statistical analyses were performed using Statistica

version 7.1 for Windows (StatSoft Inc., USA,

1984–2002). The descriptive statistical tests, including

the mean and standard error of the mean, were calculated

according to the standard methods. The ANOVA test was

used to evaluate the difference between means of glu-

cose, insulin and pharmacokinetic parameters between

groups. When the effect of groups was significant, the

differences between groups were determined by means of

the LSD test of the post hoc procedure. For the difference

between means of mRNA levels and enzyme activity,

Dunnet’s multiple comparison test, comparing all groups

to control, was used post hoc. The paired t test was

applied to assess the difference of quantitative variables

between dependent groups (concentrations of glucose and

insulin at different times of sampling). All data are pre-

sented as mean ± standard error of the mean (mean ±

SEM). The significance of differences was preset at

P \ 0.05.

Results

Body weight

There were no group differences in pre-castration body

weights (Table 2). After 60 days of treatment, the mean

body weights, body weight gain and BMI of the FO and

KO groups were lower compared with group C (Table 2).

Blood glucose and plasma insulin concentration

Pre-castration values of glucose and insulin concentration

were not different among groups (Figs. 1, 2). Fasting blood

glucose concentrations at 0 min of IVGTT in FO and KO

were significantly lower than in C animals (Fig. 1).

Exogenous glucose injection caused a sharp increase in

blood glucose concentration after 10 min, the highest val-

ues being found in group C (Fig. 1). In all groups, a gradual

decrease in glucose level was found over time. After

30 min, the glucose concentration had already returned to

the initial values in the FO group and was not statistically

different from that in NC animals. After 60 min, glucose

concentration in the KO group returned to baseline

(P [ 0.05) and was lower compared with C animals.

Fasting plasma insulin in the C group was significantly

higher when compared to the other three groups—KO, FO

and NC (Fig. 2). Glucose injection caused a marked

increase in insulin concentration after 10 min, the peak

values being found in the C and FO groups. Thereafter, an

apparent decrease in plasma insulin concentration was

noted at the 30th min. The reduction was most prominent

Table 1 Gene names and sequences of specific primers

Gene Full name Forward primer Reverse primer

Hprt Hypoxanthine phosphoribosyltransferase ccccagcgttgtgattagtg gcctcccatctccttcatca

Irs1 Insulin receptor substrate 1 actactcactgccaaggtcc atagagaaggcgaccagagc

Pklr Pyruvate kinase liver and RBC tacattgacgacgggctcat tccgcacaaaagaggcaaaa

Hmgcs2 3-Hydroxy-3-methyl-glutaryl-CoA synthase 2 (mitochondrial) cacacacaacgggaacatgt ggacacgcggaatgagaaaa

Cpt2 Carnitine palmitoyltransferase 2 agcgacaccaacaccttcaa aacgcgtggagttgaaagc

Cd36 CD36 molecule/fatty acid translocase tgctagacatcggcaagtgt agccgctttgcaaactgtaa

Acaca Acetyl-coenzyme A carboxylase alpha gggtcagtgctctcaactct actccccagcaatcattcca

Eur J Nutr

123

(more than ten times) in FO-treated animals. At the same

time point, plasma insulin levels in C animals were sig-

nificantly higher than in FO, KO and NC groups. After 60

and 120 min, there were no differences between groups.

Indexes of insulin resistance

Similar to fasting insulin, the insulin to glucose ratio at

baseline in C animals tended to be higher than in KO, FO

Table 2 Mean ± SEM of initial body weight (pre-castration), final body weight, body weight gain and body mass index (BMI) in rabbits

Parameters KO FO C NC P values

KO vs

C

KO vs

NC

FO vs

C

FO vs

NC

C vs

NC

Initial body weight (kg) 3.108 ± 0.04 3.158 ± 0.07 3.210 ± 0.03 3.085 ± 0.04 NS NS NS NS 0.08

Final body weight (kg) 3.625 ± 0.08 3.667 ± 0.08 3.994 ± 0.05 3.493 ± 0.05 0.001 NS 0.004 0.08 0.00007

Body weight gain (kg) 0.517 ± 0.09 0.508 ± 0.02 0.784 ± 0.03 0.408 ± 0.02 0.001 NS 0.001 NS 0.00005

BMI 79.887 ± 2.80 74.505 ± 2.94 88.749 ± 4.72 75.845 ± 2.53 0.07 NS 0.007 NS 0.01

All values are mean ± SEM: KO—castrated and fed krill oil; FO—castrated and fed fish oil; C—castrated non-treated; NC—non-castrated non-

treated; NS—not significant

Fig. 1 Mean ± SEM of

glucose concentrations prior to

castration (PC) and during

IVGTT in four groups of

rabbits: KO—castrated, obese

and treated with krill oil; FO—

castrated, obese and treated with

fish oil; C—castrated, obese and

non-treated; NC—non-

castrated, non-obese; a, b,

c letters indicate significant

difference between groups at a

given time point at P \ 0.05.

*P \ 0.05, **P \ 0.01 and

***P \ 0.001 indicate

significance of difference within

the groups compared to start

Fig. 2 Mean ± SEM of insulin

concentrations prior to

castration (PC) and during

IVGTT in four groups of

rabbits: KO—castrated, obese

and treated with krill oil; FO—

castrated, obese and treated with

fish oil; C—castrated, obese and

non-treated; NC—non-

castrated, non-obese; a, b,

c letters indicate significant

difference between groups at a

given time point at P \ 0.05.

*P \ 0.05 indicate significance

of difference within the groups

compared to start

Eur J Nutr

123

and NC groups (Table 3). AUCglucose 0–120 min in group C

was significantly higher than in KO, FO and NC animals.

The half-life of plasma glucose (t� glucose) in KO and FO

groups was significantly lower than in group C and similar

to that in NC group. AUCinsulin 0–120 min in C was signifi-

cantly higher than in KO and NC groups.

The HOMAins. res. index in the C group tended to be

higher than in KO, FO and NC groups, while no differences

between KO, FO and NC rabbits were found.

Indexes of b-cell function

The insulinogenic index (DI/DG) in FO animals was sig-

nificantly higher than in C, KO and NC groups (Table 4).

HOMA-b-cell index in group C was higher when compared

to KO, FO and NC groups. The first phase of insulin

secretion measured as AUCinsulin 0–10 min in the C group was

higher than in KO and NC groups. Insulin secretion during

the first hour after glucose injection (AUCinsulin 0–60 min) in C

and FO was significantly higher as compared to NC. Insulin

secretion during the second hour in the C group, measured

as AUCinsulin 60–120 min, was significantly higher than after

KO and FO treatment.

Gene expression in liver and skeletal muscle

The hepatic expression of genes involved in lipid and

glucose metabolism was determined. The PPARa-regu-

lated gene, carnitine palmitoyltransferase 2 (Cpt2), was

significantly increased in FO-fed rabbits as well as NC

rabbits, compared with the C group (Fig. 3a). Another

PPARa-regulated gene, 3-hydroxy-3-methyl-glutaryl-

HMG CoA synthase 2 (Hmgcs2), was also significantly

increased in FO-fed rabbits and NC rabbits compared

with C (Fig. 3b). The fatty acid transporter Cd36 was,

however, not affected by any of the treatments (Fig. 3c).

The rate-limiting gene in lipogenesis, acetyl-CoA car-

boxylase alpha (Acaca), was significantly reduced at the

mRNA level in both KO- and FO-fed rabbits (Fig. 3d).

The protein encoded by the gene pyruvate kinase (Pklr1)

is the rate-limiting step in glycolysis, and its mRNA level

was significantly reduced compared with C in FO-fed

rabbits (Fig. 3e). The gene expression of insulin receptor

substrate 1 (Irs1) was not significantly affected by KO or

FO treatment (Fig. 3f).

In skeletal muscle, the Cpt2 mRNA level was signifi-

cantly higher in NC rabbits, while Hmgcs2 and Cd36 were

significantly higher in KO-fed and NC rabbits compared

with the C group (Fig. 4a–c). Acaca expression was sig-

nificantly lower in KO- and FO-fed rabbits and signifi-

cantly higher in NC rabbits compared with the C group

(Fig. 4d). The gene expression of Irs1 was not significantly

affected (Fig. 4e). Ta

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Eur J Nutr

123

Hepatic enzyme activity

Liver CPT2 activity and ACOX1 activity were not affected

by KO or FO diets compared with the C diet (Fig. 5a, b).

FO demonstrated a significantly lower level of FAS

activity compared with C (Fig. 5c).

Histological examination

Light microscopy of liver samples in rabbits from group C

showed bright vacuoles in the hepatocytes, indicating

marked fat infiltration. No fat infiltration was found in liver

samples from FO- and KO-treated animals, the histological

pictures being similar to that in non-castrated non-treated

rabbits (Fig. 6).

Discussion

In the current study, we used a model of obesity based on

the castrated male New Zealand white rabbits developed in

our laboratory. The generation of this model has been

previously described [31]. This is the first study demon-

strating the effect of KO and FO on glucose homeostasis

parameters in this model of obesity in rabbits. The major

finding in this study was that FO and KO supplementation

decreased fasting blood glucose and improved glucose

tolerance in obese New Zealand rabbits. In addition, FO

and KO affected the expression levels and/or activity of

some key enzymes involved in b-oxidation and lipogenesis

in liver and skeletal muscle.

The results from IVGTT indicated marked hyperinsuli-

nemia in castrated non-treated rabbits, which is in accor-

dance with our previous results [31] and is an important

feature of early stage of decreased insulin sensitivity in

obesity [7, 9, 49, 50]. This was further confirmed by the

calculated indexes of b-cell function (HOMAb-cell,

AUCinsulin 0–10 min, AUCinsulin 0–60 min, AUCinsulin 60–120 min),

showing a compensatory increase in insulin secretion both at

fasting and during the first 2 h after glucose loading after

IVGTT. The significant reduction in plasma insulin con-

centration at 0 min and after 30 min during the IVGTT and

the observed changes in some of the simplified indexes of

insulin resistance after treatment (Io/Go ratio; HOMAins. res.;

AUCglucose 0–120 min; AUCinsulin 0–120 min and t1/2 glucose)

suggest improvement of glucose tolerance in FO- and

KO-treated animals.

Previously obtained results in our laboratory showed

that castration in male New Zealand white rabbits caused a

marked increase in body weight [31]. In addition, they

developed obesity resembling human visceral type of

obesity which is one of the main predisposing factor for

insulin resistance in humans [3–7, 51, 52], as well as inTa

ble

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Eur J Nutr

123

rabbits [30, 31, 37]. In our current study, the castrated

rabbits (C) received the same amount of energy by the diet

as non-castrated rabbits (NC) but gained significantly more

weight proving that castration is an obesogenic factor.

The improved glucose homeostasis observed in our

study could partly be attributed to PPARs activation by the

omega-3 PUFAs in FO and KO. Two main mechanisms of

an insulin sensitizing effect of PPARc activation in adipose

tissue have been revealed: Stimulation of adipocyte dif-

ferentiation leading to protection of non-adipose tissue

(skeletal muscle, liver) against excessive lipid accumula-

tion and stimulation of an adequate secretion of some

adipokines such as adiponectin and leptin which are

important mediators of insulin action in insulin-sensitive

Fig. 3 Hepatic mRNA

expression of genes involved in

lipid and glucose metabolism in

castrated control rabbits (C),

castrated rabbits fed krill oil

(KO) or fish oil (FO), or non-

castrated rabbits (NC).

a Carnitine palmitoyltransferase

2 (Cpt2), b HMG CoA synthase

2 (Hmgcs2), c Cd36, d acetyl-

CoA carboxylase alpha (Acaca),

e pyruvate kinase (Pklr1),

f insulin receptor substrate 1

(Irs1). Mean values ± SEM are

shown (n = 4–6). One-way

ANOVA followed by Dunnet’s

post hoc test was used to assess

statistical significance, and

values significantly different

from control are indicated by

*P \ 0.05, **P \ 0.01 and

***P \ 0.001

Fig. 4 Muscle mRNA

expression of genes involved in

lipid and glucose metabolism in

castrated control rabbits (C),

castrated rabbits fed krill oil

(KO) or fish oil (FO), or non-

castrated rabbits (NC).

a Carnitine palmitoyl

transferase 2 (Cpt2), b HMG

CoA synthase 2 (Hmgcs2),

c Cd36, d acetyl-CoA

carboxylase alpha (Acaca),

e insulin receptor substrate 1

(Irs1). Mean values ± SEM are

shown (n = 4–6). One-way

ANOVA followed by Dunnet’s

post hoc test was used to assess

statistical significance, and

values significantly different

from control are indicated by

*P \ 0.05 and **P \ 0.01

Eur J Nutr

123

tissue [32, 53, 54]. In contrast to group C, where the his-

tological analysis showed an abundant liver fat infiltration,

the liver samples of FO- and KO-supplemented rabbits

were similar to that of the NC group, which may in part

explain the improved insulin sensitivity in these rabbits

since fatty liver and simple steatosis in humans is often

accompanied by insulin resistance and deterioration in

glucose metabolism [55, 56]. Our data are in accordance

with the results in rats and mice showing that FO and KO

suppress hepatic steatosis by decreasing TG accumulation

in liver [14, 18, 58]. This effect of omega-3 PUFAs might

be due to the combined effects of inhibition of lipogenesis

and stimulation of fatty acid oxidation [57–59]. Omega-3

PUFAs down-regulate the mature form of sterol regulatory

element-binding protein 1, which is the main activator of

genes encoding for lipogenic enzymes such as fatty acid

synthase, acetyl-CoA carboxylase and stearoyl-CoA

desaturase [57, 58]. At the same time, omega-3 PUFAs up-

regulate the activity of PPARa in liver, which is the main

activator of b-oxidation [58, 60]. This was confirmed by

our data on the expression levels of some hepatic genes

involved in lipid metabolism. For example, the hepatic

gene expression of one of the rate-limiting enzymes in de

novo lipogenesis, acetyl-CoA carboxylase, was signifi-

cantly lower in rabbits fed FO and KO than in C. However,

the hepatic activity of the other key enzyme in fatty acid

synthesis, the fatty acid synthase complex (FAS), was

affected by FO only. Therefore, the explanation of these

results needs additional studies as the histological analysis

of liver samples showed that in contrast to group C, no fat

infiltration in both FO- and KO-supplemented groups was

found. We found that FO increased expression of the

PPARa-dependent gene Cpt2 in liver and to a lesser extend

in skeletal muscle and Hmgs2 in liver. In addition to its role

as a rate-limiting enzyme in ketogenesis and cholesterol

synthesis, recently it has been shown that mitochondrial

Hmgs2 is involved in stimulation of b-oxidation by its

ability to interact with PPARa and act as co-activator to

up-regulate transcription from PPRE of its own gene [61,

62]. FO and KO did not significantly affect the concen-

tration of mRNA encoding for the transmembrane fatty

acid transporting protein—CD36 in liver. At the same time,

KO increased the expression of Cd36 and Hmgs2 genes in

skeletal muscles. In FO- and KO-supplemented animals,

there were no significant changes in Irs-1 expression in

liver and skeletal muscle, suggesting no marked effect of

exogenous FO and KO on the early steps of the insulin

transduction pathway. Therefore, the observed ameliora-

tion of glucose tolerance and fasting blood glucose con-

centration by FO and KO might be at least in part due to

the enhanced intracellular transport and oxidation of non-

esterified fatty acid in skeletal muscles and/or liver, as their

acyl-CoA derivatives are one of the main factors decreas-

ing insulin sensitivity [7, 9, 63]. The results of Neschen

et al. [32] showed that omega-3 fatty acids protect from

high-fat-induced hepatic insulin resistance in a PPARaactivation manner in rats. Interestingly, in liver, CPT-2 was

increased by FO at the mRNA level only while the activity

of this enzyme did not change after KO and FO adminis-

tration. In addition, no effect of treatment on ACOX-1

Fig. 5 Hepatic enzyme activity

of carnitine palmitoyl

transferase 2 (CPT2) (a), fatty

acyl-CoA oxidase (ACOX)-1

(b), and fatty acid synthase

(FAS) (c) in castrated control

rabbits (C), castrated rabbits fed

krill oil (KO) or fish oil (FO), or

non-castrated rabbits (NC).

Mean values ± SEM are shown

(n = 5–6). One-way ANOVA

followed by Dunnet’s post hoc

test was used to assess statistical

significance, and values

significantly different from

control are indicated by

*P \ 0.05

Eur J Nutr

123

activity was found. We hypothesize that this is due to the

fact that omega-3 PUFAs are ligands of PPARs and as such

they predominantly regulate gene expressions at the tran-

scriptional level, while the activity of CPT-2 and other

enzymes involved in lipid and glucose metabolism is

probably also regulated at posttranscriptional, translational

or posttranslational levels. Therefore, in order to prove this

hypothesis, additional studies are needed to measure the

same parameters at mRNA and at protein levels. The

reduction in gene expression of the key enzyme of gly-

colysis, PKLR1, indicated decreased glucose degradation

in liver and, therefore, some glucose-sparing effect of

omega-3 PUFA treatment, especially in the form of FO. As

such glucose remains available as an energy source (for

example in neurons and red blood cells) or for glycogen

synthesis in skeletal muscle and liver.

On the other hand, it has been found that in high-fat-fed

rats FO improved glucose tolerance by enhancing insulin

secretion from pancreatic b-cells, which was also con-

firmed in an in vitro study on pancreatic islets [12]. Our

results are in line with this data as FO-treated rabbits

exhibited enhanced first phase of insulin secretion

(AUCinsulin 0–10 min), greater insulinogenic index (DI/DG),

describing the increment of insulin after glucose infusion,

and enhanced insulin secretion during the first and sec-

ond hour after glucose injection during the IVGTT

(AUCinsulin 0–60 min and AUCinsulin 60–120 min).

In a recent study, Tandy et al. [14] found that dietary

KO supplementation reduced hepatic steatosis and blood

glucose concentration in high-fat-fed mice in a dose-

dependent manner. It has been suggested that omega-3

PUFA ingested in the form of phospholipids, as in KO, is

characterized by a higher bioavailability and tissue uptake

probably resulting in different molecular effects and tissue

specificities [16–18, 20]. It has been reported that the

effects of KO on lipid and glucose metabolism were

stronger than those observed with FO, which may be

attributed to an increased efficacy of omega-3 PUFA in PL

form (KO) compared with the TG form (FO) and to their

different tissue distribution [16]. However, in our study, the

A: C B: NC

C: KO D: FO

Fig. 6 Morphological features of liver. a Castrated non-treated

rabbits (group C); b non-castrated (group NC); c castrated and fed

krill oil (group KO); d castrated fed fish oil (group FO); vc vena

centralis; arrow normal hepatocytes; arrowhead hepatocytes with fat

infiltration; H/E (bar a, b, c, d = 25 lm, 940)

Eur J Nutr

123

effects of KO and FO on fasting glucose and glucose tol-

erance in obese rabbits were similar. In humans, it has been

found that the metabolic effects of KO were essentially

similar to those of FO, but at a lower dose of EPA and

DHA [15]. In rabbits, however, FO and KO have similar

effects on glucose homeostasis parameters with the same

dose of EPA and DHA.

One limitation of this investigation is that the differen-

tial effects of KO and FO compared with castrated non-

treated rabbits (group C) could be mediated by the differ-

ences in body weight. In addition, we determined the

concentration of key enzymes and factors involved in lipid

and glucose metabolism predominantly at mRNA level.

Therefore, more studies are needed to measure the same

parameters at protein level in order to better understand the

molecular mechanism of improved glucose homeostasis

after FO and KO treatment.

In conclusion, the present results indicated that both FO

and KO supplementation decreased fasting blood glucose,

improved glucose tolerance and reduced hepatic steatosis

in obese New Zealand white rabbits. These effects could be

ascribed to the ameliorated insulin sensitivity and insulin

secretion by omega-3 PUFAs as shown by the changes in

calculated indexes of insulin resistance and b-cell function

and the modified gene expression levels of key enzymes

and factors involved in b-oxidation and lipogenesis in liver

and skeletal muscles. We found that the effects of FO and

KO on investigated glucose homeostasis parameters were

similar, suggesting the eventual use of KO as an alternative

source of omega-3 PUFA in individuals with metabolic

syndrome and obesity to improve insulin resistance and b-

cell function.

Acknowledgments The technical assistance of our students from

Faculty of Veterinary Medicine in Stara Zagora—Vladimir Gospo-

dinov, Anton Stoynev and Ivan Simeonov, in maintaining our

experimental animals and during performance of intravenous glucose

tolerance test was very much appreciated.

Conflict of interest The authors declare no conflict of interest.

References

1. Grundy SM, Brewer HB, Cleeman JI, Smith SC, Lenfant C

(2004) Definition of metabolic syndrome. Report of the National

Heart, Lung and Blood Institute/American Heart Association

conference on scientific issues related to definition. Circulation

109:433–438

2. Carpentier YA, Portois L, Malaisse WJ (2006) N-3 fatty acids

and the metabolic syndrome. Am J Clin Nutr 83:1499–1504

3. Despres J, Lemieux I (2006) Abdominal obesity and metabolic

syndrome. Nature 444:881–887

4. Lombardo YB, Hein G, Chicco A (2007) Metabolic syndrome:

effects of n-3 PUFAs on a model of dyslipidemia, insulin resis-

tance and adiposity. Lipids 42:427–437

5. Stumvoll M (2007) Metabolic syndrome in humans. In: Furl M

(ed) Proceedings of the 13-th international conference production

diseases in farm animals, Leipzig, pp 207–213

6. Hajer GR, van Haeften WT, Visseren FLJ (2008) Adipose tissue

dysfunction in obesity, diabetes, and vascular diseases. Eur Heart

J 29:2959–2971

7. Weiss R (2007) Fat distribution and storage: how much, where,

and how. Eur J Endocrinol 157:39–45

8. Dimitrova S, Penchev Georgiev I (2007) Relative contribution of

decreased insulin sensitivity to deterioration of glucose homeo-

stasis. Bulg J Vet Med 10:205–221

9. Corcoran MP, Fava SL, Fielding RA (2007) Skeletal muscle lipid

deposition and insulin resistance: effect of dietary fatty acids and

exercise. Am J Clin Nutr 85:662–677

10. Schenk S, Saberi M, Olefsky JM (2008) Insulin sensitivity:

modulation by nutrients and inflammation. J Clin Invest

118:2992–3002

11. Fedor D, Kelley DS (2009) Prevention of insulin resistance by

n-3 polyunsaturated fatty acids. Curr Opin Clin Nutr Metab Care

12:138–146

12. Ajiro K, Sawamura M, Ikeda K, Nara Y, Nishimura M, Ishida H,

Seino Y, Yamori Y (2000) Beneficial effects of fish oil on glu-

cose metabolism in spontaneously hypertensive rats. Clin Exp

Pharmacol Physiol 27:412–415

13. Lombardo YB, Chicco A (2006) Effects of dietary polyunsatu-

rated n-3 fatty acids on dyslipidemia and insulin resistance in

rodents and humans. A review. J Nutr Biochem 17:1–13

14. Tandy S, Chung RWS, Wat E, Kamili A, Berge R, Griinari M,

Cohn JS (2009) Dietary krill oil supplementation reduces hepatic

steatosis, glycemia, and hypercholesterolemia in high-fat-fed

mice. J Agric Food Chem 57:9339–9345

15. Ulven S, Kirkhus B, Lamglait A, Basu S, Elind E, Haider T,

Berge K, Vik H, Pedersen J (2011) Metabolic effects of krill oil

are essentially similar to those of fish oil but at lower dose of EPA

and DHA, in healthy volunteers. Lipids 46:37–46

16. Burri L, Hoem N, Banni S, Berge K (2012) Marine omega-3

phospholipids: metabolism and biological activities. Int J Mol Sci

13:15401–15419

17. Burri L, Berge K, Wibrand K, Berge RK, Barger JL (2011)

Differential effects of krill oil and fish oil on the hepatic tran-

scriptome in mice. Front Genet 2:1–8

18. Ferramosca A, Conte A, Burri L, Berge K, De Nuccio F, Giudetti

AM, Zara V (2012) A krill oil supplemented diet suppresses

hepatic steatosis in high-fat fed rats. PLoS One 7:38797

19. Piscitelli F, Carta G, Bisogno T, Murru E, Cordeddu L, Berge K,

Tandy S, Cohn JS, Griinari M, Banni S, Di Marzo V (2011)

Effect of dietary krill oil supplementation on the endocannabi-

noidome of metabolically relevant tissues from high fat-fed mice.

Nutr Metab (Lond) 8:51

20. Ferramosca A, Conte L, Zara V (2011) A krill oil supplemented

diet reduces the activities of the mitochondrial tricarboxylate

carrier and of the cytosolic lipogenic enzymes in rats. J Anim

Physiol Anim Nutr 96:295–306

21. Vigerust NF, Bjorndal B, Bohov P, Brattelid T, Svardal A, Berge

RK (2012) Krill oil versus fish oil in modulation of inflammation

and lipid metabolism in mice transgenic for TNF-alpha. Eur J

Nutr 52:1315–1325

22. Grimstad T, Bjorndal B, Cacabelos D, Aasprong OG, Jans-

sen E, Omdal R, Svardal A, Hausken T, Bohov P, Portero-Otin

M, Pamplona R, Berge RK (2012) Dietary supplementation of

krill oil attenuates inflammation and oxidative stress in

experimental ulcerative colitis in rats. Scand J Gastroenterol

47:49–58

23. Bunea R, El Farrah K, Deutsch L (2004) Evaluation of the effects

of Neptune krill oil on the clinical course of hyperlipidemia.

Altern Med Rev 9:420–428

Eur J Nutr

123

24. Maki KC, Reeves MS, Farmer M, Griinari M, Berge K, Vik H,

Hubacher R, Rains TM (2009) Krill oil supplementation increa-

ses plasma concentrations of eicosapentaenoic and docosahexa-

enoic acids in overweight and obese men and women. Nutr Res

29:609–615

25. Skarpanska-Stejnborn A, Pilaczynska-Szczesniak L, Basta P,

Foriasz J, Arlet J (2010) Effects of supplementation with Neptune

krill oil (Euphasia superba) on selected redox parameters and

pro-inflammatory markers in athletes during exhaustive exercise.

J Hum Kinet 25:49–57

26. Banni S, Carta G, Murru E, Cordeddu L, Giordano E, Sirigu AR,

Berge K, Vik H, Maki KC, Di Marzo V, Griinari M (2011) Krill

oil significantly decreases 2-arachidonoylglycerol plasma levels

in obese subjects. Nutr Metab (Lond) 8:7

27. Schuchardt JP, Schneider I, Meyer H, Neubronner J, von Schacky

C, Hahn A (2011) Incorporation of EPA and DHA into plasma

phospholipids in response to different omega-3 fatty acid for-

mulations—a comparative bioavailability study of fish oil vs. krill

oil. Lipids Health Dis 10:145

28. Fan J, Unoki H, Kojima N, Sun H, Shimoyamada H, Deng H,

Okazaki M, Shikama H, Yamada N, Watanabe T (2001) Over-

expression of lipoprotein lipase in transgenic rabbits inhibits diet-

induced hypercholesterolemia and atherosclerosis. J Biol Chem

276:40071–40079

29. Kitajima S, Morimoto M, Liu E, Koike T, Higaki Y, Taura Y,

Mamba K, Itamoto K, Watanabe T, Tsutsumi K, Yamada N, Fan

J (2004) Overexpression of lipoprotein lipase improves insulin

resistance induced by a high-fat diet in transgenic rabbits. Dia-

betologia 47:1202–1209

30. Kawai T, Ito T, Ohwada K, Mera Y, Matsushita M, Tomoike H

(2006) Hereditary postprandial hypertriglyceridemic rabbit exhibits

insulin resistance and central obesity. A novel model of metabolic

syndrome. Arterioscler Thromb Vasc Biol 26:2752–2757

31. Georgiev IP, Georgieva TM, Ivanov V, Dimitrova S, Kanelov I,

Vlaykova T, Tanev S, Zaprianova D, Dichlianova E, Penchev G,

Lazarov L, Vachkova E, Roussenov A (2011) Effects of castra-

tion-induced visceral obesity and antioxidant treatment on lipid

profile and insulin sensitivity in New Zealand white rabbits. Res

Vet Sci 90:196–204

32. Neschen S, Morino K, Dong J, Wang-Fischer Y, Cline G, Ro-

manelli AJ, Rossbacher JC, Moore I, Regittnig W, Munoz DS,

Kim JH, Shulman GI (2007) N-3 fatty acids preserve insulin

sensitivity in vivo in a peroxisome proliferator-activated recep-

tor-a-dependent manner. Diabetes 56:1034–1041

33. Kopecky J, Rossmeisl M, Flachs P, Kuda O, Brauner P, Jilkova

Z, Stankova B, Tvrzicka E, Bryhn M (2009) N-3 PUFA: bio-

availability and modulation of adipose tissue function. Proc Nutr

Soc 68:361–369

34. Buckley JD, Howe PR (2009) Anti-obesity effects of long-chain

omega3 polyunsaturated fatty acids. Obes Rev 10:648–659

35. Yamazaki RK, Brito GA, Coelho I, Pequitto DC, Yamaguchi AA,

Borghetti G, Schiessel DL, Kryczyk M, Machado J, Rocha RE,

Aikawa J, Iagher F, Naliwaiko K, Tanhoffer RA, Nunes EA,

Fernandes LC (2011) Low fish oil intake improves insulin sen-

sitivity, lipid profile and muscle metabolism on insulin resistant

MSG-obese rats. Lipids Health Dis 10:66

36. Liu E, Kitajima S, Higaki Y, Morimoto M, Sun H, Watanabe T,

Yamada N, Fan J (2005) High lipoprotein lipase activity

increases insulin sensitivity in transgenic rabbits. Metabolism

54:132–138

37. Zhao S, Chu Y, Zhang C, Lin Y, Xu K, Yang P, Fan J, Liu E

(2007) Diet-induced central obesity and insulin resistance in

rabbits. J Anim Physiol Anim Nutr 92:105–111

38. Appleton DJ, Rand JS, Sunvold GD (2005) Basal plasma insulin

and homeostasis model assessment (HOMA) are indicators of

insulin sensitivity in cats. J Feline Med Surg 7:183–193

39. Mattheeuws D, Rottiers R, Kaneko JJ, Vermeulen A (1984)

Diabetes mellitus in dogs: relationship of obesity to glucose

tolerance and insulin response. Am J Vet Res 45:98–103

40. Chen H, Sullivan G, Quon MJ (2005) Assessing the predictive

accuracy of QUICKI as a surrogate index for insulin sensitivity

using a calibration model. Diabetes 54:1914–1925

41. Ciampelli M, Leoni F, Cucinelli F, Mancuso S, Panunzi S, De

Gaetano A, Lanzone A (2005) Assessment of insulin sensitivity

from measurements in the fasting state and during an oral glucose

tolerance test in polycystic ovary syndrome and menopausal

patients. J Clin Endocrinol Metab 90:1398–1406

42. Wallace TM, Levy JC, Matthews DR (2004) Use and abuse of

HOMA modeling. Diabetes Care 27:1487–1495

43. Larson BT, Lawler DF, Spitznagel EL, Kealy RD (2003)

Improved glucose tolerance with lifetime diet restriction favor-

ably affects disease and survival in dogs. J Nutr 133:2887–2892

44. Slavov E, Penchev Georgiev I, Dzhelebov P, Kanelov I, Ando-

nova M, Georgieva TM, Dimitrova S (2010) High-fat feeding and

Staphylococcus intermedius infection impair beta cell function

and insulin sensitivity in mongrel dogs. Vet Res Commun

34:205–215

45. Berge RK, Flatmark T, Osmundsen H (1984) Enhancement of

long-chain acyl-CoA hydrolase activity in peroxisomes and

mitochondria of rat liver by peroxisomal proliferators. Eur J

Biochem 141:637–644

46. Bremer J (1981) The effect of fasting on the activity of liver

carnitine palmitoyltransferase and its inhibition by malonyl-CoA.

Biochim Biophys Acta 665:628–631

47. Skorve J, al-Shurbaji A, Asiedu D, Bjorkhem I, Berglund L,

Berge RK (1993) On the mechanism of the hypolipidemic effect

of sulfur-substituted hexadecanedioic acid (3-thiadicarboxylic

acid) in normolipidemic rats. J Lipid Res 34:1177–1185

48. Madsen L, Rustan AC, Vaagenes H, Berge K, Dyroy E, Berge

RK (1999) Eicosapentaenoic and docosahexaenoic acid affect

mitochondrial and peroxisomal fatty acid oxidation in relation to

substrate preference. Lipids 34:951–963

49. Nelson RW, Himsel CA, Feldman EC, Bottoms GD (1990)

Glucose tolerance and insulin response in normal-weight and

obese cats. Am J Vet Res 51:1357–1362

50. Thiess S, Becskei C, Tomsa K, Lutz TA, Wanner M (2004)

Effects of high carbohydrate and high fat diet on plasma

metabolite levels and on i.v. glucose tolerance test in intact and

neutered male cats. J Feline Med Surg 6:207–218

51. Appleton DJ, Rand JS, Sunvold GD (2001) Insulin sensitivity

decreases with obesity, and lean cats with low insulin sensitivity

are at greatest risk of glucose intolerance with weight gain.

J Feline Med Surg 3:211–228

52. Hays NP, Galassetti PR, Coker RH (2008) Prevention and

treatment of type 2 diabetes: current role of lifestyle, natural

product, and pharmacological intervention. Pharmacol Ther

118:181–191

53. Sharma AM, Staels B (2007) Peroxisome proliferator-activated

receptor gamma and adipose tissue-understanding obesity-related

changes in regulation of lipid and glucose metabolism. J Clin

Endocrinol Metab 92:386–395

54. Rosenson RS (2008) Effect of fenofibrate on adiponectin and

inflammatory biomarkers in metabolic syndrome patients. Obes-

ity 17:504–509

55. Banerji MA, Buckley MC, Chaiken RL, Gordon D, Lebovitz HE,

Kral JG (1995) Liver fat, serum triglycerides and visceral adipose

tissue in insulin-sensitive and insulin-resistant black men with

NIDDM. Int J Obes Relat Metab Disord 19:846–850

56. Hwang JH, Stein DT, Barzilai N, Cui MH, Tonelli J, Kishore P,

Hawkins M (2007) Increased intra-hepatic triglycerides is associated

with peripheral insulin resistance: in vivo MR imaging and spec-

troscopy studies. Am J Physiol Endocrinol Metab 293:1663–1669

Eur J Nutr

123

57. Kim HJ, Takahashi M, Ezaki O (1999) Fish oil feeding decreases

mature sterol regulatory element-binding protein 1 (SREBP-1) by

down-regulation of SREBP1c mRNA in mouse liver. J Biol

Chem 274:25892–25898

58. Jump DB (2008) N-3 polyunsaturated fatty acid regulation of

hepatic gene transcription. Curr Opin Lipidol 19:242–247

59. Buckley JD, Howe PR (2010) Long-chain omega-3 polyunsatu-

rated fatty acids may be beneficial for reducing obesity—a

review. Nutrients 2:1212–1230

60. Burri L, Thoresen GH, Berge RK (2010) The role of PPARa acti-

vation in liver and muscle. PPAR Res. doi:10.1155/2010/542359

61. Kostiuk MA, Keller BO, Berthiaume LG (2010) Palmitoylation

of ketogenic enzyme HMGCS2 enhances its interaction with

PPARa and transcription at the Hmgcs2 PPRE. FASEB J

24:1914–1924

62. Vila-Brau A, De Sousa-Coelho AL, Mayordomo C, Haro D,

Marrero PF (2011) Human HMGCS2 regulates mitochondrial

fatty acid oxidation and FGF21 expression in HepG2 cell line.

J Biol Chem 286:20423–20430

63. Yu C, Chen Y, Cline GW, Zhang D, Zong H, Wang Y, Bergeron

R, Kim JK, Cushman SW, Cooney GJ, Atcheson B, White MF,

Kraegen EW, Shulman GI (2002) Mechanism by which fatty

acids inhibit insulin activation of insulin receptor substrate-1

(IRS-1)-associated phosphatidylinositol 3-kinase activity in

muscle. J Biol Chem 277:50230–50236

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