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: [email protected]
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
ble
3In
suli
nre
sist
ance
ind
exes
inra
bb
its
Ind
exes
of
insu
lin
sen
siti
vit
yA
bb
rev
iati
on
sK
OF
OC
NC
Pv
alu
es
KO
vs
CF
Ov
sC
KO
vs
NC
FO
vs
NC
Cv
sN
C
Insu
lin
/glu
cose
rati
oat
bas
elin
eI 0
/G0
0.4
3±
0.1
00
.50
±0
.09
4.5
4±
3.1
10
.53
±0
.04
0.0
70
.08
NS
NS
0.0
7
Insu
lin
/glu
cose
rati
oat
60
and
12
0m
in
I 60/G
60
0.2
7±
0.0
40
.31
±0
.07
0.8
9±
0.2
90
.76
±0
.05
0.0
10
.02
0.0
30
.04
NS
I 120/G
120
0.2
7±
0.0
50
.42
±0
.09
0.7
7±
0.1
80
.63
±0
.06
0.0
10
.04
0.0
3N
SN
S
HO
MA
insu
lin
-res
ista
nce
ind
ex0
.76
±0
.23
0.9
5±
0.1
91
7.1
5±
12
.39
1.3
6±
0.0
80
.07
0.0
8N
SN
S0
.07
QU
ICK
Iin
dex
5.5
6±
2.4
73
.04
±0
.40
1.7
7±
0.1
22
.56
±0
.08
0.0
4N
S0
.09
NS
NS
Ben
etin
dex
4.7
7±
2.4
72
.22
±0
.40
0.8
3±
0.1
41
.68
±0
.08
0.0
4N
S0
.08
NS
NS
t 1/2
glu
cose
39
.23
±1
.48
35
.64
±1
.41
58
.43
±7
.12
35
.54
±2
.77
0.0
07
0.0
01
NS
NS
0.0
01
AU
Cin
suli
n0?
120m
in5
96
.07
±7
71
,78
9.9
±4
90
2,6
59
.9±
68
8.0
72
6.1
±4
4.3
0.0
07
0.0
01
NS
NS
0.0
01
AU
Cglu
cose
0?
120m
in1
,09
2.6
9±
57
.93
98
0.2
3±
34
.81
,26
2.9
±2
6.2
71
,00
8.9
±2
6.3
0.0
10
.00
02
NS
NS
0.0
00
6
All
val
ues
are
mea
n±
SE
M:
KO
—ca
stra
ted
and
fed
kri
llo
il;
FO
—ca
stra
ted
and
fed
fish
oil
;C
—ca
stra
ted
no
n-t
reat
ed;
NC
—n
on
-cas
trat
edn
on
-tre
ated
;N
S—
no
tsi
gn
ifica
nt
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
4b
-cel
lfu
nct
ion
ind
exes
inra
bb
its
Ind
exes
ofb-
cell
fun
ctio
nA
bb
rev
iati
on
sK
OF
OC
NC
Pv
alu
es
KO
vs
CF
Ov
sC
KO
vs
NC
FO
vs
NC
Cv
sN
C
Insu
lin
og
enic
ind
exD
I/D
G1
.53
±0
.57
.65
±2
.82
.45
±1
.50
.77
±0
.03
NS
0.0
3N
S0
.01
NS
Insu
lin
/glu
cose
rati
oat
10
min
I 10m
in/G
10m
in1
.15
±0
.35
.10
±1
.83
.60
±1
.00
.66
±0
.03
NS
NS
NS
0.0
10
.05
HO
MA
b-c
el
lindex
19
.4±
4.0
21
.5±
3.5
14
6.6
±9
8.4
19
.7±
1.7
0.0
80
.08
NS
NS
0.0
7
AU
Cin
suli
n0?
10m
inA
UC
ins
0?
10
min
lU/m
lm
in1
17
.8±
23
.94
76
.4±
14
5.9
56
6.8
±2
35
.47
8.3
±2
.70
.03
NS
NS
0.0
40
.02
AU
Cin
suli
n0?
60m
inA
UC
ins
0?
60
min
lU/m
lm
in4
69
.4±
71
.01
,64
1.7
±4
93
2,2
24
.5±
56
54
38
.1±
19
.40
.00
5N
SN
S0
.03
0.0
03
AU
Cin
suli
n60?
120m
inA
UC
ins
60?
120
min
lU/m
lm
in1
89
.3±
37
.42
08
.13
±4
0.0
67
2.6
8±
20
54
27
.8±
32
.50
.00
60
.00
8N
SN
SN
S
All
val
ues
are
mea
n±
SE
M:
KO
—ca
stra
ted
and
fed
kri
llo
il;
FO
—ca
stra
ted
and
fed
fish
oil
;C
—ca
stra
ted
no
n-t
reat
ed;
NC
—n
on
-cas
trat
edn
on
-tre
ated
;N
S—
no
tsi
gn
ifica
nt
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.
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