Chronic dietary n-3 PUFA intervention improves dyslipidaemia and subsequent cardiovascular complications in the JCR:LA- cp rat model of the metabolic syndrome.
ABSTRACT There is increasing interest in the potential chronic beneficial effects of dietary n-3 PUFA on the metabolic syndrome (MetS) and associated cardiovascular complications. We have recently established that increased dietary n-3 PUFA has a profound acute benefit on fasting lipids and the postprandial pro-inflammatory response in the JCR:LA-cp rat, a model of the MetS. However, it is unclear to what extent chronic dietary n-3 PUFA intervention can modulate the progression of end-stage metabolic and vascular complications. The present study aimed to determine the chronic effects of dietary n-3 PUFA supplementation on fasting and non-fasting dyslipidaemia, insulin resistance and vascular complications in the JCR:LA-cp rodent model. JCR:LA-cp rats were fed an isoenergetic lipid-balanced diet supplemented with 5 % n-3 PUFA (w/w) of the total fat (fish oil-derived EPA/DHA) for 16 weeks. Fasting and non-fasting (postprandial) plasma lipid profile was assessed. Hepatic and adipose tissue was probed for the expression of lipogenic proteins (acyl-CoA carboxylase (ACC), fatty acid synthase (FAS) and sterol regulatory element-binding protein-1 (SREBP-1)), while the activity of Jun N-terminal kinase (JNK) was assessed via Western blot to target phosphorylated JNK protein in primary enterocytes. The frequency of myocardial lesions was assessed by haematoxylin and eosin staining. Increased dietary n-3 PUFA improved both the fasting and postprandial lipid profiles (TAG, cholesterol and apoB48) in the JCR:LA-cp rat, potentially via the down-regulation of the hepatic or adipose tissue expression of lipogenic enzymes (ACC, FAS and SREBP-1). Rats fed the 5 % n-3 PUFA diet had lower (58·2 %; P < 0·01) enterocytic phosphorylated JNK protein and secreted less cholesterol (30 %; P < 0·05) into mesenteric lymph compared with the control. The chronic metabolic benefits of dietary n-3 PUFA may underlie the potential to reduce vascular complications during the MetS, including the observed reduction in the frequency (approximately 80 %) of late-stage 3 myocardial lesions.
- [show abstract] [hide abstract]
ABSTRACT: Postprandial hyperlipidemia is considered to be a substantial risk factor for atherosclerosis. Interestingly, this concept has never been supported by randomized clinical trials. The difficulty lies in the fact that most interventions aimed to reduce postprandial lipemia, will also affect LDL-C levels. The atherogenic mechanisms of postprandial lipids and lipoproteins can be divided into direct lipoprotein-mediated and indirect effects; the latter, in part, by inducing an inflammatory state. Elevations in postprandial triglycerides (TG) have been related to the increased expression of postprandial leukocyte activation markers, up-regulation of pro-inflammatory genes in endothelial cells and involvement of the complement system. This set of events is part of the postprandial inflammatory response, which is one of the recently identified potential pro-atherogenic mechanisms of postprandial lipemia. Especially, complement component 3 levels show a close correlation with postprandial lipemia and are also important determinants of the metabolic syndrome. In clinical practice, fasting TG are frequently used as reflections of postprandial lipemia due to the close correlation between the two. The use of serial capillary measurements in an out-of-hospital situation is an alternative for oral fat loading tests. Daylong TG profiles reflect postprandial lipemia and are increased in conditions like the metabolic syndrome, type 2 diabetes and atherosclerosis. Studies are needed to elucidate the role of postprandial inflammation in atherogenesis and to find new methods in order to reduce selectively the postprandial inflammatory response. Future studies are needed to find new methods in order to reduce selectively the postprandial inflammatory response.Atherosclerosis Supplements 07/2008; 9(2):39-44. · 4.33 Impact Factor
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ABSTRACT: Insulin resistance, which is a prevalent condition, has been associated with a cluster of metabolic disturbances that increase the risk of non-insulin-dependent diabetes mellitus and cardiovascular diseases. In this review article, the complexity of the etiology of insulin resistance is emphasized as it results from the interaction of genetic and environmental factors. Potential cellular defects underlying insulin resistance are discussed as well as the relation of impaired insulin action to dyslipidaemia and coronary heart disease.Current Opinion in Lipidology 09/1994; 5(4):274-89. · 5.84 Impact Factor
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ABSTRACT: Atherosclerosis-prone, insulin-resistant JCR:LA-cp male rats were treated from 6 weeks to 39 weeks of age with beta,beta'-tetramethylhexadecanedioic acid (MEDICA 16). Body weights were reduced (13%, P < .001) at 36 weeks without any accompanying decrease in food consumption. The treatment did not cause any significant change in plasma glucose or fasting insulin concentrations. There was a significant decrease in the extreme hyperplasia of the islets of Langerhans (38%, P < .05). The marked VLDL hypertriglyceridemia was decreased by 70% (P < .001), with an accompanying significant reduction in cholesterol concentrations. The severity of raised atherosclerotic lesions on the aortic arch was very markedly reduced (P < .01) in treated rats. This was accompanied by a reduction (P < .01) in the incidence of ischemic myocardial lesions. We conclude that long-term (33 weeks) MEDICA 16 treatment of an animal model for the obesity/insulin-resistant/hyperlipidemic syndrome not only markedly improved lipid metabolism, but also inhibited the development of advanced cardiovascular disease.Arteriosclerosis Thrombosis and Vascular Biology 08/1995; 15(7):918-23. · 6.34 Impact Factor
Chronic dietary n-3 PUFA intervention improves dyslipidaemia and
subsequent cardiovascular complications in the JCR:LA-cp rat model
of the metabolic syndrome
Jing Lu1, Faye Borthwick1, Zahra Hassanali1, Ye Wang1, Rabban Mangat1, Megan Ruth1, Danni Shi1,
Anja Jaeschke2, James C. Russell3, Catherine J. Field3, Spencer D. Proctor1* and Donna F. Vine1*
1Metabolic and Cardiovascular Diseases Laboratory, Alberta Institute for Human Nutrition, 4–10 Agriculture Forestry
Centre, University of Alberta, Edmonton, AB, Canada T6G 2P5
2Department of Physiology, University of Cincinnati, Cincinnati, OH, USA
3Alberta Institute for Human Nutrition, 4–10 Agriculture Forestry Centre, University of Alberta, Edmonton, AB, Canada
(Received 6 April 2010 – Revised 23 November 2010 – Accepted 25 November 2010)
There is increasing interest in the potential chronic beneficial effects of dietary n-3 PUFA on the metabolic syndrome (MetS) and associated
cardiovascular complications. We have recently established that increased dietary n-3 PUFA has a profound acute benefit on fasting lipids
and the postprandial pro-inflammatory response in the JCR:LA-cp rat, a model of the MetS. However, it is unclear to what extent chronic
dietary n-3 PUFA intervention can modulate the progression of end-stage metabolic and vascular complications. The present study aimed
to determine the chronic effects of dietary n-3 PUFA supplementation on fasting and non-fasting dyslipidaemia, insulin resistance and
vascular complications in the JCR:LA-cp rodent model. JCR:LA-cp rats were fed an isoenergetic lipid-balanced diet supplemented with
5% n-3 PUFA (w/w) of the total fat (fish oil-derived EPA/DHA) for 16 weeks. Fasting and non-fasting (postprandial) plasma lipid profile
was assessed. Hepatic and adipose tissue was probed for the expression of lipogenic proteins (acyl-CoA carboxylase (ACC), fatty acid
synthase (FAS) and sterol regulatory element-binding protein-1 (SREBP-1)), while the activity of Jun N-terminal kinase (JNK) was assessed
via Western blot to target phosphorylated JNK protein in primary enterocytes. The frequency of myocardial lesions was assessed by haema-
toxylin and eosin staining. Increased dietary n-3 PUFA improved both the fasting and postprandial lipid profiles (TAG, cholesterol and
apoB48) in the JCR:LA-cp rat, potentially via the down-regulation of the hepatic or adipose tissue expression of lipogenic enzymes
(ACC, FAS and SREBP-1). Rats fed the 5% n-3 PUFA diet had lower (58·2%; P,0·01) enterocytic phosphorylated JNK protein and secreted
less cholesterol (30%; P,0·05) into mesenteric lymph compared with the control. The chronic metabolic benefits of dietary n-3 PUFA may
underlie the potential to reduce vascular complications during the MetS, including the observed reduction in the frequency (approximately
80%) of late-stage 3 myocardial lesions.
Key words: CVD: Dietary interventions: Dyslipidaemia: Metabolic syndrome
The metabolic syndrome (MetS) is a pre-diabetic state,
characterised by obesity, insulin resistance (IR) and dys-
lipidaemia(1). Lipid abnormalities of the MetS include
low plasma HDL-cholesterol, increased LDL-cholesterol,
abnormal apoB level and raised blood TAG (hypertri-
acylglycerolaemia)(1,2). Emerging evidence also suggests
that postprandial TAG and chylomicron (CM) concen-
trations are strongly correlated with cardiovascular risk(3).
The long-term complications of the MetS include
lesion development(1,4), and are often characterised in
stages of severity (i.e. stage 1 to stage 4(5–7)) including
early areas of cell lysis through to inflammatory cell
infiltration and scarring.
n-3 PUFA, such as EPA (20:5) and DHA (22:6), have
been reported to have beneficial effects in states of the
MetS and cardiovascular complications(8,9). Studies have
reported that dietary fish oil (rich in n-3 PUFA) can
reduce cardiovascular risk factors, including repression
of plasma insulin, glucose and cholesterol levels(10,11).
*Corresponding authors: Dr S. D. Proctor, fax þ1 780 492 6358, email email@example.com; Dr D. F. Vine, fax þ1 780 492 9270,
Abbreviations: CM, chylomicron; IR, insulin resistance; JNK, Jun N-terminal kinase; JNK-P, phosphorylated Jun N-terminal kinase; LBD, lipid-balanced diet;
MetS, metabolic syndrome; SREBP, sterol regulatory element-binding protein.
British Journal of Nutrition (2011), page 1 of 11
q The Authors 2011
British Journal of Nutrition
Evidence from human clinical trials suggests that n-3 PUFA
reduce acute atherosclerotic infarction, and hence lower
the risk of CHD(12,13). Furthermore, an investigation of
the protective effect of n-3 PUFA on isoproterenol-induced
myocardial infarction in rats has reported a marked
reduction in the level of lipid components (cholesterol,
TAG and NEFA) in the plasma and heart tissue(14).
In addition, a human trial conducted by Metcalf et al.(15)
has reported dietary n-3 PUFA to be readily incorporated
into cardiomyocyte phospholipids, potentially exerting
beneficial outcomes by consequent effects on myocardial
membrane function. Despite this, there is no compre-
hensive histological evidence regarding the improvement
of dietary n-3 PUFA on myocardial lesions and the
long-term beneficial vascular effects.
Recently, our group has reported that acute (3-week)
dietary n-3 PUFA supplementation can reduce weight
gain, and improve postprandial lipid metabolism and
JCR:LA-cp rat, a model of the MetS(16). The JCR:LA-cp rat,
homozygous for the corpulent trait (cp/cp), exhibits
symptoms of the MetS, enhanced hepatic VLDL and
intestinal CM overproduction, as well as end-stage com-
plications such as ischaemic myocardial lesions(5,6,17–22).
Consequently, the aim of the present study was to (1)
assess the long-term (chronic) effects of dietary n-3 PUFA
supplementation on dyslipidaemia, IR and myocardial
lesion development in the JCR:LA-cp rat and (2) investigate
the putative mechanistic effects of dietary n-3 PUFA, specifi-
cally on intestinal enterocytes, in the obese JCR:LA-cp rat.
Materials and methods
Animal model and experimental procedures
Male obese (cp/cp) JCR:LA-cp rats were raised in our
established breeding colony at the University of Alberta
(Edmonton, AB, Canada), as described previously(5). Rats
were weaned at 6 weeks, and allowed to age until
8 weeks of age. Obese rats (cp/cp) were randomly
isoenergetic lipid-balanced diet (LBD; n 8) (1% (w/w)
cholesterol; 15% (w/w) total fat; polyunsaturated:saturated
fat ratio 0·4) or a LBD supplemented with 5% n-3 PUFA
(of the total fat n 8; 1% (w/w) cholesterol, 15% (w/w)
total fat; polyunsaturated:saturated fat ratio 0·4 and 5%
(w/w) fish oil-derived EPA/DHA) (Table 1), for 3 or
16 weeks, as indicated throughout. Note that the equiva-
lent amount of n-3 PUFA from 5% fish oil treatment in
rats used in the present study equates to approximately
5g fish oil/d for a 10 460kJ/d (2500kcal/d) human diet.
Food consumption and body weight were recorded
throughout the study. At 22 weeks of age, an oral fat
challenge test was performed on all rats, as described
previously(22). At 24 weeks of age, rats were fasted over-
night and killed under isoflurane anaesthesia. Plasma and
serum were collected via cardiac puncture. Liver, heart
and inguinal fat pads were weighed and snap-frozen in
liquid N2at 2808C for subsequent analysis. Animal care
and experimental procedure were conducted in accord-
ance with the Canadian Council on Animal Care (Ottawa,
ON, Canada) and approved by the University of Alberta
Animal Care and Use Committee (ACUC-Livestock).
Assessment of postprandial lipidaemia
At 22 weeks of age and following a 16h overnight fast, rats
were subjected to an oral fat challenge test in order to
assess non-fasting lipid metabolism(22). In brief, all rats con-
sumed a 5g pellet prepared from 5001 laboratory chow,
consisting of 49% carbohydrate, 24% crude protein, 10%
moisture, 6·5% minerals, 6% fibre and 4·5% fat, and further
supplemented with 25% (w/w) dairy fat from double
cream (raising the total fat content of the 5g meal to
approximately 30% (w/w) of the total meal)(16,22). Blood
samples were collected (from the tail) into tubes containing
Na2EDTA at time intervals (0, 2, 4 and 6h) following pellet
consumption. Plasma and serum were separated by cen-
trifugation (3901g, 48C, 10min). Aliquots of plasma were
immediately stored at 2808C for biochemical analyses.
Mesenteric lymph duct cannulation and nascent lymph
To determine the effect of n-3 PUFA on the secretion of
CM in mesenteric lymph, the superior mesenteric lymph
duct of obese JCR:LA-cp rats was cannulated following
consumption of a control LBD (n 5) or a 5% n-3 PUFA
Table 1. Nutrient and lipid summaries for both dietary groups*
Control diet (LBD) 5% n-3 PUFA diet
Nutrient summary (per kg)
Non-nutritive cellulose (g)
Vitamin mixture (g)
Mineral mix (g)
Linseed oil (g)
Sunflower oil (g)
Fish oil (g)
Lipid summary (% of fat)
Total polyunsaturated fat
Total saturated fat
Total EPA þ DHA
LBD, lipid-balanced control diet; P:S, polyunsaturated:saturated fat.
* Fatty acid composition of the LBD and the diet supplemented with 5% fish oil
containing EPA and DHA; 5% n-3 PUFA diet, as determined by GC as described
J. Lu et al.2
British Journal of Nutrition
diet (n 5) for 3 weeks(23). Mesenteric lymph was collected
into EDTA-coated vacutainers for 5h following infusion
of intralipid (Kabi Pharmacia, Uppsala, Sweden). The
concentrations of TAG and total cholesterol were measured
as described below for the plasma.
Isolation of primary jejunal enterocytes and quantification
of apoB48 and Jun N-terminal kinase protein
Primary jejunal enterocytes were isolated from the intestine
of obese rats fed either a control LBD or a 5% n-3 PUFA
diet for 3 weeks, as per the Weiser method(24)of isolation
and fractionation, as described previously(23). The protein
concentration of the isolated enterocyte fractions (no.
1–10) was determined, and immuno-Western blot analysis
was used to probe the expression of apoB48 protein
(as below) along the intestinal villus, from enterocyte frac-
tion no. 1 (tip of the villus) to enterocyte fraction no. 10
(crypt of the villus). Pooled enterocyte fractions (no. 1–10)
were assessed for Jun N-terminal kinase (JNK) activity
by Western blotting, to target phosphorylated (active)
JNK (JNK-P) protein. Briefly, cell extracts were prepared
in Triton lysis buffer and examined (50mg protein) by
immunoblot analysis, to target JNK-P (catalogue no. 9252;
Cell Signalling, Beverly, MA, USA) and JNK (catalogue
no. 558268; Pharmingen, San Diego, CA, USA).
Plasma biochemical determination
The biochemical lipid profile of obese rats fed either a
control LBD or a 5% n-3 PUFA diet was assessed using
commercially enzymatic kits, as described previously(16);
including plasma TAG (catalogue no. 998-40391/994-40491;
Wako Pure Chemicals, USA, Inc., Richmond, VA, USA), total
(catalogue no. 993-00404/999-00504; Wako) and HDL-
cholesterol (catalogue no. 258-20; Diagnostic Chemicals
Limited, Charlottetown, PE, Canada). Plasma glucose levels
were assessed as per the glucose oxidase method (catalogue
no. 220-32; Diagnostic Chemicals Limited). Insulin was
analysed by a solid-phase, two-site enzyme immunoassay
(catalogue no. 10-1137-01; Mercodia AB, Uppsala, Sweden).
Plasma adiponectin (catalogue no. 44-ADPR-0434; Alpco
Diagnostics, Salem, NH, USA) and leptin (catalogue no.
22-LEP-E06; ALPCO) concentrations were determined using
commercially available rodent-specific immunoassays.
The concentration of intestinally derived CM particles
was determined by quantification of plasma apoB48,
using an adapted immuno-Western blotting procedure, as
described previously(25,26). Briefly, total plasma was sepa-
rated by SDS-PAGE on a 3–8% Tris-acetate polyacrylamide
NuPagewgel (InVitrogen, Camarillo, CA, USA). Separated
proteins were transferred to a polyvinylidene fluoride
membrane (0·45mM, ImmobilonPe; Millipore, Billerica,
MA, USA). Membranes were incubated with a goat
polyclonal antibody to apoB (1:100; catalogue no. sc-11795;
Santa-Cruz Biotechnology, Inc., Santa Cruz, CA, USA),
which recognises both apoB100 and apoB48 isoforms.
Detection was achieved using an anti-goat secondary anti-
body (catalogue no. sc-2304; Santa-Cruz Biotechnology,
nescence (ECL) advance; Amersham Biosciences, Little
Chalfont, Bucks, UK); intensity was quantified using
linear densitometric comparison with a known mass of
purified rodent apoB48 protein.
Measurement of lipogeneic gene targets
Total RNA was isolated from both hepatic and adipose
tissue collected from obese (cp/cp) rats of both diet groups
(TRIzol; InVitrogen) and reverse-transcribed into comp-
lementary DNA using MultiScribee Reverse transcriptase
(High-Capacity cDNA Reverse Transcription Kit; Applied
Biosystems, Foster City, CA, USA). The expression of
acyl-CoA carboxylase (ACC), fatty acid synthase (FAS),
PPARa, PPARg and sterol regulatory element-binding
protein-1 (SREBP-1) mRNA, relative to the housekeeping
gene ACTB (b-actin), was measured by quantitative
real-time PCR, using the StepOnee Plus Real-Time PCR
system (Applied Biosystems) and StepOnee Software
(version 2). PCR contained complementary DNA template,
100nM of commercially available, pre-mixed target-specific
primers and TaqmanwFAMe-labelled probe (Applied
Biosystems) for ACC (reference sequence NM _022193.1;
catalogue no. Rn01456582_m1), ACTB (NM_031144.2; cata-
logue no. Rn00667869_m1), FAS (NM_017332.1; catalogue
no. Rn01463550_m1), PPARa (NM_013196.1; catalogue no.
no. Rn01495769). Thermal cycling conditions were as
follows: 958C for 20s, followed by forty cycles of 958C for
1s and 608C for 20s. Gene quantification was assessed rela-
tive to ACTB mRNA, utilising the comparative 22DCtmethod.
Measurement of lipogenic-related proteins
Proteins from liver and adipose homogenates were sepa-
rated by SDS-PAGE electrophoresis on 3–8% Tris-acetate
polyacrylamide gels (InVitrogen), transferred to a poly-
vinylidene fluoride membrane (described previously) and
incubated with antibodies for the following target proteins:
anti-ACC goat polyclonal (1:20000; catalogue no. sc-11795;
Santa-Cruz Biotechnology, Inc.); anti-FAS goat mono-
clonal (1:20000; catalogue no. sc-55580; New England
BioLabs, Acton, MA, USA), anti-SREBP-1 mouse monoclonal
(1:20000; catalogue no. sc-13551; Santa-Cruz Biotech-
nology, Inc.); anti-actin goat polyclonal (1:20000; catalogue
no. A5441; Sigma-Aldrich, St Louis, MO, USA); antibodies.
ondary antibodies and the ECL advance detection system
(described previously; Amersham Biosciences). Results are
expressed as a ratio of target protein:b-actin protein.
Benefits of PUFA to CVD in the JCR:LA-cp rat3
British Journal of Nutrition
Heart histology and myocardial lesion analysis
Hearts were fixed in formalin, embedded in a single para-
ffin block, sectioned and then stained with haematoxylin
and eosin as described previously(6). Heart sections
were examined blindly by an experienced observer, and
the number of ischaemic lesions was identified in each of
the sections. Myocardial lesions were categorised as
stage 1 through to stage 4, as described previously(5–7,27);
stage 1, necrotic areas; stage 2, cell lysis with long-term
inflammatory infiltration; stage 3, nodules of long-term
inflammatory infiltration; stage 4, old scarred lesions.
The number of lesions determined from sections of indi-
vidual hearts was aggregated, and the mean incidence for
each group was calculated.
Statistical analysis was performed using GraphPad Prism
software, version 4.0. Data were tested for normal distri-
bution, and significant differences between the obese
(cp/cp) LBD and obese (cp/cp) 5% n-3 PUFA groups
were determined using Student’s t test or repeated-
measures ANOVA followed by Bonferroni post hoc tests,
when appropriate. All results are expressed as means
with their standard errors and the number of independent
experiments as indicated in the figure legends. The level
of significance was set at P,0·05.
Food intake, body fat and organ weight
Following a 16-week n-3 PUFA intervention, despite no
reduction in food intake, 5% n-3 PUFA-supplemented
obese rats had significantly lower body weight (12–17%;
P,0·05) compared with the obese control rats (Table 2;
Fig. 1). Those rats supplemented with 5% n-3 PUFA
(16 weeks) had a reduced ratio of inguinal fat-pad weight:
body weight compared with the obese control rats
(P,0·001) (Fig. 2). Furthermore, the weight of the liver iso-
lated from obese rats supplemented with 5% n-3 PUFA
Table 2. Physical and fasting biochemical parameters of obese (cp/cp)
male JCR:LA-cp rats fed a lipid-balanced control diet (LBD) or a 5%
n-3 PUFA diet
(Mean values with their standard errors, n 8)
cp/cp LBDcp/cp 5% n-3 PUFA
Food consumption (g)
Body weight (g)
Fasting glucose (mg/l)
Fasting insulin (pmol/l)
Fasting cholesterol (mg/l)
Fasting TAG (mg/l)
Fasting apoB48 (mg/ml)
* Mean values were significantly different from those of the obese control (cp/cp)
Body weight (g)
Fig. 1. Body weight of obese control (
, 16 weeks) obese JCR:LA-cp rats. Values are means, with standard
errors represented by vertical bars (n 8). *Mean values were significantly
different from those of the lipid-balanced diet cp/cp control group (P,0·05).
, cp/cp) and 5% n-3 PUFA-fed
Inguinal fat pads:body weight
cp/cp5% n-3 PUFA
Fig. 2. Ratio of the weight of inguinal fat pads:body weight of either obese
(cp/cp) control JCR:LA-cp rats or JCR:LA-cp rats fed a 5% n-3 PUFA diet
(16 weeks). Values are means, with standard errors represented by vertical
bars (n 8). ***Mean value was significantly different from that of the lipid-
balanced diet cp/cp control group (P,0·001).
Fasting adiponectin (mg/l)
cp/cp 5% n-3 PUFA
Fig. 3. Fasting plasma adiponectin concentration of either obese (cp/cp) or
5% n-3 PUFA (16 weeks)-fed JCR:LA-cp rats. Values are means with standard
errors represented by vertical bars (n 8). *Mean value was significantly different
from that of the lipid-balanced diet cp/cp control group (P,0·005).
J. Lu et al.4
British Journal of Nutrition
(16 weeks) was significantly decreased (P,0·05) com-
pared with the obese control rats (data not shown). The
weight of the heart isolated from rats of both groups was
unaltered by dietary n-3 PUFA (data not shown).
Fasting biochemical profile
Treatment with 5% n-3 PUFA for 16 weeks significantly
lowered fasting plasma leptin and insulin concentrations
(P,0·05; Table 2). In addition, treatment with 5% n-3
PUFA increased (improved) fasting plasma adiponectin
concentration (P,0·05; Fig. 3), but did not significantly
reduce fasting glucose concentration (Table 2). Further-
more, fasting plasma cholesterol and TAG concen-
trations were reduced in rats fed the 5% n-3 PUFA diet
(16 weeks), compared with the obese control rats (P,0·05)
Non-fasting (postprandial) lipid response
Consistent with previous results from acute dietary n-3
PUFA supplementation in JCR:LA-cp rats, the postprandial
lipid response (measured as area under the curve) for
TAG was significantly lower in obese rats fed a 5% n-3
PUFA diet chronically for 16 weeks compared with the
obese control rats (54%; P,0·05; Fig. 4(a)), apoB48 (69%;
P,0·01; Fig. 4(b)); and total cholesterol (38%; P,0·001;
Expression of enterocytic apoB48 protein and secretion of
chylomicron lymphatic cholesterol and TAG
The abundance of enterocyte-specific apoB48 (number
of CM particles) was significantly lower (43·8%; P,0·05)
in obese JCR:LA-cp rats fed a 5% n-3 PUFA diet for
3 weeks, compared with the obese control rats (Fig. 5).
Obese JCR:LA-cp rats fed a 5% n-3 PUFA diet (3 weeks)
(P,0·05) into mesenteric lymph compared with rats fed
the control diet (Fig. 6(a)). Interestingly, by comparison,
the secretion of CM-TAG, into mesenteric lymph, was
increased (1·7-fold; P,0·05) in those rats fed a 5% n-3
PUFA diet (Fig. 6(b)).
Plasma TAG (µg/ml)
Plasma cholesterol (µg/ml)
Plasma apoB48 (µg/ml)
Fig. 4. Postprandial response of plasma TAG (a), apoB48 (b) and total
cholesterol (c) (area under the curve) of control (
), (16 weeks) JCR:LA-cp rats, following an oral fat challenge.
Values are means with standard errors represented by vertical bars (n 8).
Mean values were significantly different from those of the lipid-balanced
diet cp/cp control group: *P,0·05, **P,0·01, ***P,0·001.
, cp/cp) and 5% n-3
apoB48 mass/5µg of protein
1023456789 10 11
Fig. 5. Enterocyte-specific apoB48 protein expression in obese (
JCR:LA-cp rats fed either a control lipid-balanced diet or a 5% n-3 PUFA
( )-enriched diet for 3 weeks. Primary jejunal enterocytes were isolated
as per the Weiser method of isolation and fractionation, as described pre-
viously(23). Immuno-Western blot analysis probed the expression of apoB48
protein (as per Methods) along the intestinal villus from enterocyte fraction 1
(tip of the villus) to enterocyte fraction 10 (crypt of the villus). Values are
means, with standard errors represented by vertical bars (n 5). *Mean values
were significantly different between total enterocyte and apoB48 mass of
5% n-3 PUFA cp/cp v. lipid-balanced diet (LBD) cp/cp control (P,0·05).
†Mean values were significantly different in enterocyte fraction 5-specific
apoB48 protein of 5% n-3 PUFA cp/cp v. LBD cp/cp control (P,0·05).
Benefits of PUFA to CVD in the JCR:LA-cp rat5
British Journal of Nutrition
Expression of genes involved in lipogenesis and fatty acid
The expression of both hepatic and adipose ACC, FAS,
SREBP-1, PPARa and PPARg mRNA is reported in Fig. 7(a)
(hepatic) and Fig. 8(a) (adipose). Hepatic specific ACC
(25%; P,0·05) and SREBP-1 (61·3%; P,0·01) mRNA levels
were significantly reduced in obese rats fed a 5% n-3 PUFA
diet (16 weeks) compared with the control rats (Fig. 7(a)).
Similarly, adipose-specific expression of SREBP-1 mRNA
was also significantly lower (37%; P,0·05) in those obese
rats fed a 5% n-3 PUFA diet (16 weeks) (Fig. 8(a)). The
gene expression of PPARa/g and ACC was unaltered in
the adipose tissue of obese rats fed a 5% n-3 PUFA diet,
while FAS mRNA was significantly up-regulated (1·9-fold;
P,0·05) compared with the obese control rats (Fig. 8(a)).
Protein expression of lipogenic enzymes
The abundance of hepatic ACC, FAS and SREBP-1 proteins
is reported in Fig. 7(b) and (c). The abundance of hepatic
FAS protein was significantly lower in rats fed a 5% n-3
PUFA diet for 16 weeks (P,0·01; Fig. 7(b) and (c)), relative
to the obese control rats. However, ACC protein was
not significantly altered in livers from 5% n-3 PUFA-fed
rats, relative to the obese control rats (Fig. 7(b) and (c)).
Both precursor (125kDa) and mature (68kDa) hepatic
SREBP-1 proteins were reduced in the 5% n-3 PUFA diet
group (16 weeks) compared with the obese control
group (P,0·01) (Fig. 7(b) and (c)). Adipose tissue-specific
expression of ACC and SREBP-1 protein (125 kDa) was not
significantly altered in rats fed a 5% n-3 PUFA diet (Fig. 8(b)
and (c)). Furthermore, treatment with 5% n-3 PUFA signi-
ficantly reduced FAS protein expression in the adipose
(P,0·05) compared with the obese control rats (Fig. 8(b)
cp/cp5% n-3 PUFA
cp/cp 5% n-3 PUFA
Fig. 6. Secretion of chylomicron-associated (a) cholesterol and (b) TAG into
mesenteric lymph. Mesenteric lymph cannulation procedures were carried
out as described previously(23). Cholesterol and TAG secretion (mg/ml) into
mesenteric lymph was compared in obese (cp/cp) JCR:LA-cp rats fed a 5%
n-3 PUFA diet v. those fed a control lipid-balanced diet (LBD) for 3 weeks.
Values are means, with standard errors represented by vertical bars (n 5).
*Mean value was significantly different from that of the LBD cp/cp control
group in all cases (P,0·05).
mRNA:β-actin mRNA (×103)
5 % n-3 PUFA
Fig. 7. Hepatic gene and protein expression of lipogenic enzymes in
JCR:LA-cp rats in response to long-term feeding (16 weeks) of 5% n-3
PUFA ( ). (a) The expression of ACC, FAS, SREBP-1, PPARa and PPARg
mRNA, relative to the housekeeping gene b-actin, in the livers of both obese
( , cp/cp) and 5% n-3 PUFA-fed groups. ((b) and (c)) Protein abundance of
ACC, FAS, precursor SREBP-1 (approximately 125kDa) and mature
SREBP-1 (approximately 68kDa) protein,
expression, in the livers of the obese (cp/cp) control and 5% n-3 PUFA diet
groups. Values are means, with standard errors represented by vertical
bars (n 8). Mean values were significantly different from those of the lipid-
balanced diet cp/cp control group: *P,0·05, **P,0·01.
J. Lu et al.6
British Journal of Nutrition
Enterocytic phosphorylated Jun N-terminal kinase protein
The protein expression of phosphorylated JNK (active)
in the enterocytes of obese JCR:LA-cp rats fed a 5%
n-3 PUFA diet for 3 weeks compared with the obese
control group (Fig. 9).
Frequency of myocardial lesions
Representative images of the stages (stages 1–4) of
myocardial lesions assessed are shown in Fig. 10. The
frequency of stages of myocardial lesion development
(stages 1–4) for all four treatment groups is reported in
Fig. 11. Supplementation with n-3 PUFA for 16 weeks in
the obese JCR:LA-cp rat had no effect on the frequency
of early stage 1 lesions (Fig. 11). Most notably, the 5%
n-3 PUFA diet significantly reduced the number of late
stage 3 (areas of chronic inflammatory infiltration) lesions
(83·3%; P,0·05) in obese rats compared with the control
group (Fig. 11). Additionally, in the hearts of rats supple-
mented with a 5% n-3 PUFA diet, no stage 4 lesions
were detected (Fig. 11).
The main objective of the present study was to investigate
the impact of chronic (16 weeks) dietary intervention
with n-3 PUFA on pre-existing hyperinsulinaemia, dys-
lipidaemia and ischaemic lesion development, in the
JCR:LA-cp rat model. Our findings show that chronic
feeding of a diet with increased n-3 PUFA can improve
both metabolic parameters and vascular complications
associated with the MetS.
The chronic effect of n-3 PUFA on body weight and fat
While obesity is known to increase the risk of developing
type 2 diabetes and cardiovascular complications, the
mRNA:β-actin mRNA (×103)
5 % n-3 PUFA
approximately 125 kDa
Fig. 8. Adipose-specific gene and protein expression of lipogenic enzymes in
JCR:LA-cp rats in response to long-term feeding (16 weeks) of 5% n-3
PUFA ( ). (a) Expression of ACC, FAS, SREBP-1, PPARa and PPARg
mRNA, relative to the housekeeping gene b-actin, in the adipose tissue of
obese ( , cp/cp) and 5% n-3 PUFA-fed JCR:LA-cp rats. ((b) and (c)) Protein
abundance of ACC, FAS and precursor SREBP-1 (approximately 125kDa)
protein, relative to b-actin protein expression, in the adipose tissue of the
obese (cp/cp) control and 5% n-3 PUFA diet groups. Values are means, with
standard errors represented by vertical bars (n 8). Mean values were signifi-
cantly different from those of the lipid-balanced diet cp/cp control group:
(arbitrary density units)
5% n-3 PUFA
5% n-3 PUFA
Fig. 9. Activity of Jun N-terminal kinase (JNK) was assessed via Western
blot to target phosphorylated JNK (JNK-P) protein. Enterocyte extracts
(50mg protein) were examined by immunoblot analysis, utilising antibodies
from Cell Signalling (JNK-P) and Pharmingen (JNK). JNK-P protein was
measured in obese (cp/cp) JCR:LA-cp rats fed either a 5% n-3 PUFA diet or
a control lipid-balanced diet (LBD) for 3 weeks; a representative blot and
graph, presented as a measure of arbitrary density units, are shown. Values
are means, with standard errors represented by vertical bars (n 5). **Mean
value was significantly different from that of the LBD cp/cp control group in all
Benefits of PUFA to CVD in the JCR:LA-cp rat7
British Journal of Nutrition
long-term effect of dietary fat consumption in the process
of development of diabetes, IR and dyslipidaemia remains
controversial. Thorsdottir et al.(28)demonstrated in a study
of overweight men that the inclusion of fish oil (n-3
PUFA) in the diet induced a greater weight loss (1kg)
over 4 weeks, than those on a diet without fish oil(28).
An additional study in overweight hypertensive subjects,
which showed a weight-loss programme incorporating
fish meals rich in n-3 PUFA, was more effective in
reducing weight loss, serum lipids and glucose–insulin
metabolism, than either treatment alone(29). Interestingly,
and consistent with our previous finding in this rat
model(16), we observed that increased dietary n-3 PUFA
reduced body-weight gain in the absence of any alteration
in food intake.
Clinical data also suggest that type 2 diabetic patients
subjected to a diet rich in n-3 PUFA have reduced
abdominal subcutaneous fat, in addition to improved
IR(30). Consistent with this, inguinal fat (intra-abdominal)
depots were significantly lower in n-3 PUFA dietary
groups in the present study. Additional animal studies
have also shown that dietary supplementation with fish
oil (n-3 PUFA) can make an impact upon fatty acid pro-
portions and distribution in subcutaneous and visceral
fat(31,32). Indeed, it has been suggested previously that
n-3 PUFA may not be as readily deposited, but more
freely oxidised within the adipose tissue, implying that
n-3 PUFA may at least have a partial protective effect
against weight gain per se(33–35).
The chronic effect of n-3 PUFA on glucose, insulin
resistance and adipokines
Studies have indicated that dietary fish oil (n-3 PUFA) may
act to normalise and/or improve the storage of lipids and
glucose oxidation within skeletal muscle. Evidence sup-
ports the notion that hypolipidaemic effects of n-3 PUFA
act to reduce lipid utilisation within skeletal muscle, restore
glucose oxidation and normalise insulin sensitivity(36).
In the present study, JCR:LA-cp rats from the n-3 PUFA
diet groups had significantly improved fasting plasma
insulin (but only a trend towards improved glucose levels),
implying improved IR, consistent with previous data(37).
Furthermore, n-3 PUFA supplementation significantly
decreased plasma leptin levels. Existing evidence suggests
leptin to be a contributor to the hypolipidaemic benefits of
n-3 PUFA, reducing TAG biosynthesis and enhancing
b-oxidation(38). While the effects of n-3 PUFA on plasma
and tissue leptin levels remain controversial(38), studies
have shown that n-3 PUFA supplementation may reduce
leptin mRNA expression(38). Plasma leptin levels have
also been shown to be reduced in rodents supplemented
with dietary n-3 PUFA (fish oil), with corresponding
decreases in visceral adipose tissue(39). The ability of n-3
PUFA to reduce plasma leptin levels in our IR rodent
model may occur directly via suppression of leptin
mRNA expression (not measured in the present study).
Alternatively, it may be possible that plasma leptin could
be modulated indirectly via the parallel reductions in
plasma insulin, inguinal (intra-abdominal) fat weight and
It is well established that there is a negative corre-
lation between the concentration of plasma adiponectin
(an adipokine with anti-diabetic properties) and BMI(40).
Consistent with this, fasting adiponectin concentration
was significantly enhanced in rats supplemented with 5%
n-3 PUFA, while body weight was profoundly reduced.
It may be that n-3 PUFA influence the expression of
Fig. 10. Representative micrographs of ischaemic lesions in the hearts
of obese JCR:LA-cp rats (age 24 weeks). (a) Stage 1: area of necrosis
with no long-term inflammatory cell infiltration in the left ventricle. (b)
Stage 2: area of long-term inflammatory cell infiltration, without visible cell
lysis, in the trabecular muscle. (c) Stage 3: area of active inflammatory
cell activity and cell lysis in the lower trabecular muscle. (d) Stage 4:
early scarred lesion with a small number of inflammatory cells or fibro-
blasts in the upper penvalvular region of the heart. All images were cap-
tured at a magnification of 2£ after haematoxylin and eosin staining of
Heart lesion frequency
Stage 1Stage 2 Stage 3 Stage 4
Fig. 11. Frequency of myocardial lesions in the hearts of JCR:LA-cp rats
from obese control ( , cp/cp) and 5% n-3 PUFA ( ) diet groups (16 weeks).
(a) Stage 1 lesions; (b) stage 2 lesions; (c) stage 3 lesions; (d) stage 4
lesions. Values are means, with standard errors represented by vertical bars
(n 8). *Mean values were significantly different from those of the lipid-
balanced diet cp/cp control group in all cases (P,0·05).
J. Lu et al.8
British Journal of Nutrition
adipokines (such as leptin and adiponectin) via direct
interaction with transcription factors, or indirectly via
mechanisms that control fatty acid oxidation, synthesis
and/or storage(41); but this remains to be defined.
Potential mechanisms of n-3 PUFA via regulation of
SREBP are sterol-responsive transcription factors. SREBP
are synthesised in their precursor form (approximately
125kDa) in the endoplasmic reticulum, before cleavage
to their active (nuclear) form (approximately 68kDa),
in response to low cellular sterol levels, regulating the
expression of lipid-related genes, including lipogenic
enzymes (FAS and ACC)(42). In particular, the SREBP-1 iso-
form is selective for genes involved in fatty acid synthesis:
ACC, FAS; stearoyl-CoA desaturase-1(43). In the present
study, chronic dietary n-3 PUFA reduced both gene
and protein expression of hepatic precursor and mature
SREBP-1, also down-regulating lipogenic enzyme expre-
ssion of hepatic ACC mRNA and FAS protein, consistent
with the expression of SREBP-1(44). Studies have shown
that PUFA can act as a competitive antagonist for the
liver X receptor, a nuclear receptor responsive to endo-
genous oxysterols, in vitro(45). We also know that inhi-
bition of the binding of the liver X receptor/retinoid X
receptor heterodimer to the liver X receptor response
element, in the promoter region of SREBP-1c, can suppress
the expression of SREBP-1c(45). More recently, the same
group has reported that the primary mechanism underlying
PUFA-induced SREBP-1c suppression, in fact, occurs at
the proteolytic processing level in vivo(46). The ability of
PUFA to suppress SREBP-1 may also be dependent on
the level of the incorporation of PUFA into cellular lipids,
as recently suggested by Di Nunzio et al.(47).
The hypolipidaemic effect of n-3 PUFA may arise either
via the reduced expression of SREBP-1, reducing lipogen-
esis and cholesterol biosynthesis, as discussed above, or
via activation of the common PUFA-activated transcription
factor, PPARa(42,48,49), promoting fatty acid oxidation.
However, in the present study, we found that hepatic-
and adipose-specific expression of PPAR (a/g) mRNA
was not significantly regulated in response to chronic
dietary n-3 PUFA.
Furthermore, while dietary n-3 PUFA induced a marked
reduction in hepatic SREBP-1 gene and protein abundance,
only a modest suppression of adipose-specific SREBP-1
mRNA was observed. The differential PUFA-mediated regu-
lation of SREBP-1 between tissue types in the JCR:LA-cp
model is consistent with that observed in other rodent
models(50,51). In the present study, we observed a greater
abundance of hepatic SREBP-1 mRNA compared with
adipose-specific SREBP-1. By contrast, we report greater
PPARg mRNA in adipose compared with hepatic tissue.
As reviewed by Kersten(52), data suggest that SREBP-1
regulates lipogenic genes in the liver, while PPARg is
essential for the regulation of lipogenesis in the adipose.
Thus, PUFA-induced regulation of lipogenesis may exhibit
tissue specificity, via hepatic SREBP-1 and PPARg in the
The acute effect of n-3 PUFA on intestinal enterocytes
We have shown that both acute(16)and long-term n-3 PUFA
intervention clearly exert beneficial effects specifically in
our model to improve postprandial lipids, therefore
suggesting that the ability of n-3 PUFA to improve post-
prandial metabolism, during conditions of IR, may occur
via the direct action of n-3 PUFA on intestinal CM secretion
(Figs. 5 and 6). For this reason, we initiated a subsequent
acute (3-week) n-3 PUFA intervention to assess potential
mechanistic effects of n-3 PUFA, directly on intestinal
enterocyte CM production, associated lymphatic lipid
observed that 3 weeks (short-term) of dietary intervention
with n-3 PUFA was sufficient to reduce the intestinal
production of apoB48 (CM) (Fig. 5), but also suppressed
the subsequent lymphatic cholesterol secretion (Fig. 6).
This is complementary to the observed improvement
(reduction) to both fasting and postprandial plasma
apoB48 (both acute and chronic) and cholesterol (chronic)
reported in our previous acute study(16)and the present
long-term (chronic) study. Consistent with clinical obser-
vations, neither long-term nor acute(16)intervention with
n-3 PUFA lowered fasting plasma LDL concentrations, sug-
gestive of preferential n-3 PUFA-induced improvement to
intestinally derived lipoprotein fractions. Interestingly,
while the n-3 PUFA diet lowered fasting and postprandial
plasma TAG, we observed an increase in lymphatic TAG
secretion from the intestine following acute n-3 PUFA
supplementation (Fig. 6). We hypothesise that there may
be an increase in CM particle size and/or enhanced
clearance of TAG, although this remains to be elucidated.
Most recently, n-3 PUFA have been reported to exert
potent anti-inflammatory effects that improve IR and
other symptoms of the MetS in mice, via binding to G
protein-coupled receptor 120(53). This study reported n-3
PUFA (DHA specifically) to block both NF-kB and JNK
pathways, reversing IR induced by a high-fat diet(53).
Supportive of this notion, in the present study, n-3 PUFA
appear to improve associated insulin signalling pathways
in the intestine by reducing the activity of enterocyte-
specific JNK (Fig. 9). Collectively, these data support the
hypothesis that n-3 PUFA may act directly on the intestine
to improve insulin signalling and lower non-fasting lipid
contribution following a lipid excursion.
The chronic effect of n-3 PUFA on myocardial lesion
Development of ischaemic lesions, secondary to vascular
damage or dysfunction, is a major endpoint of CVD(54).
Benefits of PUFA to CVD in the JCR:LA-cp rat9
British Journal of Nutrition
JCR:LA-cp rats develop lesions spontaneously, as a result of
the hyperinsulinaemic and hyperlipidaemic status of these
rodents(6,55). Rats from the present study were killed at a
relatively early age, and may explain the low frequency
of stage 4 (advanced scarred lesions) heart lesions com-
pared with previous studies(5,27). However, rats from the
present study presented with a large number of early
stage 2 lesions, probably due to the high lipid and choles-
terol load, in the control diet. Dietary n-3 PUFA (5%) in
the JCR:LA-cp rats resulted in fewer stage 3 myocardial
lesions, consistent with the complete absence of stage 4
lesions in these animals, as well as an amelioration of myo-
cardial histology; evidence that n-3 PUFA may inhibit CVD
progression. It may be reasonable to suggest that the vascu-
lar improvement observed, in response to dietary n-3 PUFA,
is due to chronic improvement to metabolic status, inclu-
ding non-fasting lipids, visceral fat, insulin sensitivity, or
potentially via a direct effect of n-3 PUFA on the heart.
In conclusion, the present study provides evidence that
chronic increased dietary n-3 PUFA has beneficial effects
on both hepatic and intestinal lipid metabolism, IR, body
weight and myocardial ischaemic lesion frequency in the
obese JCR:LA-cp rat. Dietary n-3 PUFA may confer
additional therapeutic potential to lower the risk of athero-
sclerotic progression in patients with metabolic disorders
over the long term. Future directions should focus on
additional animal and clinical studies to help verify a bene-
ficial target dose for human therapy and to further define
the mechanistic pathways behind the action of n-3 PUFA.
the Alberta Diabetes Institute (S. D. P.), a NSERC Discovery
grant (S. D. P. and D. F. V.) and a HSFC Grant-in-aid
(S. D. P.). S. D. P. is a HSFC New Investigator. The authors
wish to declare no conflict of interest. We wish to thank
technical assistance throughout the present study. We
also wish to acknowledge Anja Jaeschke (University of
Cincinnati, Cincinnati, OH, USA) for her contribution to the
and designed the experiments. J. L., Z. H., Y. W., R. M.,M. R.,
D. S., F. B. and J. C. R. performed the experiments. J. L.,
S. D. P., J. C. R., F. B. and D. F. V. analysed the data. F. B.,
S. D. P. and J. L. prepared the manuscript.
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Benefits of PUFA to CVD in the JCR:LA-cp rat11
British Journal of Nutrition