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ARTICLE
Beneficial effects of long-chain n-3 fatty acids included
in an energy-restricted diet on insulin resistance
in overweight and obese European young adults
A. Ramel & A. Martinéz & M. Kiely & G. Morais &
N. M. Bandarra & I. Thorsdottir
Received: 30 October 2007 / Accepted: 2 April 2008 / Published online: 20 May 2008
#
Springer-Verlag 2008
Abstract
Aims/hypothesis Epidemiological research indicates that
long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFA)
improve insulin resistance. The aim of this study was to
investigate the effects of seafood consumption on insulin
resistance in overweight participants during energy restriction.
Methods In this 8 week dietary intervention, 324 participants
(20–40 years, BMI 27.5–32.5 kg/m
2
, from Iceland, Spain and
Ireland) were randomised by computer to one of four energy-
restricted diets (−30E%) of identical macronutrient compo-
sition but different LC n-3 PUFA content: control (n=80; no
seafood; single-blinded); lean fish (n=80;150gcod,three
times/week); fatty fish (n=84; 150 g salmon, three times/
week); (4) fish oil (n=80; daily docosahexaenoic/eicosapen-
taenoic acid capsules, no other seafood; single-blinded).
Fasting glucose, insulin, adiponectin, plasma triacylglycerol
and fatty acids in erythrocyte membrane were measured at
baseline and endpoint. Insulin resistance was calculated
using the homeostasis model assessment of insulin resistance
(HOMA-IR). Linear models with fixed effects and covariates
were used to investigate the effects of seafood consumption
on fasting insulin and HOMA-IR at endpoint in comparison
with the control group.
Results Of the partici pants, 278 (86%) completed the
intervention. Fish oil intake was a significant predictor of
fasting insulin and insulin resistance after 8 weeks, and this
finding remained significant even after including weight
loss, triacylglycerol reduction, increased LC n-3 PUFA in
membranes or adiponectin changes as covariates in the
statistical analysis. Weight loss was also a significant pre-
dictor of improvements.
Conclusions/interpretation LC n-3 PUFA consumption
during energy reduction exerts positive effects on insulin
resistance in young overweight individuals, independently
from changes in body weight, triacylglycerol, erythrocyte
membrane or adiponectin.
Trial registration: ClinicalTrials.gov NCT00315770.
Funding: The YOUNG study is part of the SEAFOODplus
Integrated Project, which is funded by the EC through the 6th
Framework Programme Contract No. FOOD-CT-2004-506359.
Keywords Glucose
.
Insulin
.
Insulin resistance
.
LC n-3 fatty acids
.
Weight loss
Diabetologia (2008) 51:1261–1268
DOI 10.1007/s00125-008-1035-7
Electronic supplementary material The online version of this article
(doi:10.1007/s00125-008-1035-7) contains supplementary material,
which is available to authorised users.
A. Ramel
:
I. Thorsdottir (*)
Unit for Nutrition Research, Landspitali-University Hospital &
Faculty of Food Science and Nutrition, Eiriksgata 29,
101 Reykjavik, Iceland
e-mail: ingathor@landspitali.is
A. Martinéz
Department of Physiology and Nutrition, University of Navarra,
Navarra, Spain
M. Kiely
Department of Food and Nutritional Sciences,
University College Cork,
Cork, Ireland
G. Morais
Department of Biochemistry, Faculty of Medical Sciences,
New University of Lisbon,
Lisbon, Portugal
N. M. Bandarra
The National Research Institute on Agriculture
and Fisheries Research,
Lisbon, Portugal
Abbreviations
DHA docosahexaenoic acid
EPA eicosapentaenoic acid
FFQ food frequency questionnaire
HOMA-IR homeostasis model assessment of
insulin resistance
LC n-3 PUFA long-chain n-3 polyunsaturated fatty
acids
Introduction
Obesity and type 2 diabetes mellitus are characterised by
impaired insulin -stimulated glucose disposal in skeleta l
muscle [1]. Fish oil (and its important long-chain n-3
polyunsaturated fatty acids (LC n-3 PUFA) eicosapentaenoic
acid (EPA) and docosahexaenoic acid (DHA) protect against
insulin resistance and obesity in rodents fed high-fat diets,
and might reduce insulin response to glucose in healthy
humans [2–4]. According to epidemiological studies, fish
consumption is inversely related to 2 h glucose levels during
follow-up, and is associated with a reduced risk of
developing impaired glucose tolerance in the elderly [5, 6].
An ecological study indicates that the prevalence of type 2
diabetes in men in the Nordic countries is significantly and
inversely associated with both the n-3 PUFA and EPA
content of milk, and positively associated with the ratio of
n-6/n-3 fatty acids in milk [7]. According to a meta-analysis
of published clinical trials, significant dose–response effects
of EPA on HbA
1c
and triacylglycerol, and of DHA on
fasting blood glucose levels, HbA
1c
and triacylglycerol, have
been demonstrated in individuals with type 2 diabetes [8].
However, several intervention studies concerning LC n-3
PUFA supplementation and insulin resistance have reported
negative outcomes, which require further clarification [9].
The mechanism responsible for the possible fish oil-
induced prevention of insulin resistance is unclear, but
different studies have demonstrated a strong association
between elevated triacylglycerol concentration (plasma and/
or tissues) and insulin resistance [10]. In animal models,
insulin resistance is significantly correlated with hepatic or
plasma triacylglycerol content [11, 12]. Fish oil intake has
been shown to decrease plasma and liver triacylglycerol
levels and VLDL-triacylglycerol secretion, and to suppress
postprandial hypertriacylglycerolaemia [13].
Increasing evidence also suggests that the fatty acid
composition of membrane phospholipids of insulin target
tissues, such as liver, fat pad and skeletal muscle, is a critical
factor that influences both insulin secretion and its biological
actions (e.g. changes in membrane fluidity, diacylglycerol
second messenger function) [14, 15]. The fatty acid profile of
muscle membrane phospholipids has been associated with
insulin sensitivity in rodents [16, 17] and humans [18–21].
These studies demonstrated a positive correlation between a
high content of LC n-3 PUFA and insulin sensitivity.
Adiponectin is an adipocyte-derived hormone that
stimulates glucose utilisation and fatty acid oxidation in
muscle and decreases hepatic gluconeogenesis, and a low
plasma level of this protein is an independent risk factor for
the future development of type 2 diabetes [22]. Moreover,
an association between circulating adiponectin and plasma
n-3 PUFA was recently found in healthy humans [23].
Based o n this evidence, it has been hypothesised that
possible protective effects of EPA and DHA may involve
induction of adiponectin. In animal research, the intake of
diets rich in EPA and DHA (5.3% of total energy intake)
leads to elevated systemic concentrations of adiponectin,
largely independent of food intake or adiposity [24].
To investigate the effects of the consumption of seafood
containing different amounts of LC n-3 PUFA on insulin
resistance, we conducted a randomise d controlled dietary
intervention trial in overweight and obese young adults
from three European countries. The goals of the present
study were to investigate whether seafoo d intake during
energy restriction impro ves fasting insulin and insulin
resistance (homeostasis model assessment of insulin resis-
tance [HOMA-IR]). We also paid particular attention to
plasma triacylglycerol, fatty acid composition of membrane
phospholipids, and plasma adiponectin levels, to estimate
their combined or independent contribution to changes in
insulin sensitivity during this 8 week trial.
Methods
Participants A total of 324 overweight individuals (138
men, 18 6 women) were recruited to our study SEAFOOD-
plus YOU NG (http://www.seafoodplus.org, accessed 4
April 2008) through advertisements on hospital and univer-
sity noticeboards, 140 from Iceland, 120 from Spain and 64
from Ireland. All subjects were screened for inclusion and
exclusion criteria. The inclusion criteria were a BMI in the
range of 27.5–32.5 kg/m
2
, age 20–40 years and a waist
circumference of ≥94 cm and ≥80 cm for men and women,
respectively. Exclusion criteria were weight change (±3 kg)
in response to a weight loss diet within 3 months before the
start of the study, use of supplements containing n-3 fatty
acids, calcium or vitamin D during the last 3 months, allergy
to fish, pregnancy or lactation, drug treatment for hyperten-
sion, hyperlipidaemia and diabetes mellitus. None of the
participants was diagnosed with type 2 diabetes, but 12
participants had impaired fasting glycaemia (>5. 6 and
<6.9 mmol/l). About 86% (n=278) of the participants
completed the intervention. The study was approved by
the National Bioethical Committee in Iceland (04–031), the
1262 Diabetologia (2008) 51:1261–1268
Ethical Committee of the University of Navarra in Spain
(24/2004) and the Clinical Research Ethics Committee of
the Cork University Hospital in Ireland. The study followed
the Helsinki guidelines (as revised in 2000), and all
participants gave their written consent. Power calculations
for the study were based on weight loss, the primary
endpoint of the study. It was estimated that a participation
rate of 70–80% allowed the detection of approximately 1 kg
difference in weight loss between the four diet groups,
assuming an sSD of 3 kg, a p value for significance of <0.05
and a statistical power of >0.8.
Study design This study was a randomised controlled
dietary intervention trial. It was conducted at the Land-
spitali-University Hospital in Reykjavik, Iceland, the
University College of Cork, Ireland, and the University of
Navarra in Pamplona, Spain, and took place during the
period from April 2004 to November 2005. The interven-
tion lasted for 8 consecutive weeks, during which time the
participants were instructed to follow a diet restricted in
terms of energy to 70% of their normal intake, which was
determined by calculating energy expenditure by Harris–
Benedict equations [25], using a correction factor to take
into account the overweight status of the subjects [26] and
an adjustment for physical activity level [27].
The participants were random ly assigned to one of the
following four diets, which varied with respect to the
amount of LC n-3 PUFA:
Diet 1: no seafood (control; 6×500 mg sunflower oil
capsules/day, Loders Croklaan [Lipid Nutrition],
Wormerveer, the Netherlands; encapsulated by
Banner Pharmacaps, Tilburg, the Netherlands);
Diet 2: lean fish (150 g cod, three times/week);
Diet 3: fatty fish (150 g salmon, three times/week); or
Diet 4: fish oil capsules (6×500 mg capsules/day, Loders
Croklaan, encapsulated by Banner Pharmacaps).
A chart of participant flow through the study is presented
in Electronic supplementary material Fig. 1.
Diet group 1 and 4 were single-blinded. For compara-
bility, the diets prescribed were of identical macronutrient
composition: total fat (~30% of total energy), carbohydrate
(~50% of total energy), protein (~20% of total energy) and
dietary fibre (~20–25 g). Each diet provided different
amounts of LC n-3 PUFA: the placeb o capsules in diet
group 1 provided 0 g/day, cod in diet 2 provided 0.26 g/
day, salmon in diet group 3 provided 2.1 g/day, and fish oil
capsules p rovided 1.3 g/da y. Each subject re ceive d a
detailed meal plan to follow, as well as recipe booklets
and instructions, so as to minimise differences between
diets in terms of sources of fat (other than LC n-3 PUFA),
fruit and vegetable consumption and meal frequency. The
physical activity level of the participants remained un-
changed during the intervention. (For more information on
the intervention see Thorsdottir et al. [28].) To measure
compliance, seafood intake was assessed by a validated
food frequency questionnaire (FFQ) and dietary intake was
assessed by 2 day weighed food records before baseline
(habitual diet) and in week 6 or later of the intervent ion trial
[29]. Compliance was also assessed by analysing n-3 and n-
6 fatty acids in erythrocyte phospholipids in fasting blood
samples, as previously reported [28]. Results showed good
compliance with the intervention diets.
Anthropometric measurements All measurements were
done using standard procedu res, as outlined in a research
protocol approved and used by all centres participating in
the study. Anthropometrical measurements were performed
at baseline and endpoi nt of the study. Body weight was
measured in light underwear on a calibrated scale (model
no. 708; Seca, Hamburg, Germany). The height of the
subjects was measured with a calibrated stadiometer. Fat
mass and fat-free mass were assessed by bioelectrical
impedance analysis (BIA; Bodystat 1500; Bodystat, Doug-
las, Isle of Man, British Isles).
Biochemical measurements Participants were instructed to
avoid strenuous exercise and alcohol consumption the day
before the drawing of blood samples at baseline and
endpoint, which were analysed for fasting concentrations
of blood glucose (mmol/l), insulin (pmol/l), plasma triacyl-
glycerol (mmol/l) and adiponectin (
μg/ml). All the blood
analyses were performed centrally. Insulin was measured
with electrochemiluminescence immunoassay (ECLIA) on
a Modular Analytics E170 system from Roche Diagnostics
(Manheim, Germany). Plasma triacylglycerol and glucose
were analys ed using an enzymatic colorimetric assay and
an automated analyzer (Hitachi 911; Roche Diagnostics).
Adiponectin was measured with a radioimmunoassay kit
developed by Linco Research (St Charles, MO, USA).
Fatty acid composition in extracted erythrocyte membrane
phospholipids was analysed by gas chromatography under
the conditions described previously [30].
Insulin resis tance In the present study we used the update d
HOMA model (known as HOMA2, computer model) to
calculate insulin resistance (referred to as HOMA-IR), as
described previously [31], which can be downloaded as a
calculator or Excel spread sheet from the homepage of the
Diabetes Trials Unit, University of Oxford, UK (http://
www.dtu.ox.ac.uk/homa, accessed 4 April 2008).
Statistical analysis The data were entered into the SPSS
statistical package, version 11.0 (SPSS, Chicago, IL, USA).
Wilcoxon test was used to calculate whether there were
significant changes in the variables between baseline and
Diabetologia (2008) 51:1261–1268 1263
endpoint. The baseline characteristics of the groups were
compared using linear models with fixed effects (country, sex,
diet group) and covariate (age). To find out whether diet groups
predict endpoint fasting insulin and HOMA-IR after 8 weeks,
linear models with fixed effects (country, sex, diet group) and
covariates (age, baseline value of the relevant outcome
variable) were constructed. To find out whether changes in
body weight, triacylglycerol, membrane lipid fatty acids or
adiponectin can explain possible effects of diet groups on
outcomes, the blood biochemical and anthropometrical varia-
bles were entered as additional covariates in separate linear
models. Insulin and HOMA-IR values were log-transformed
for this analysis. Results from the linear models are shown as
parameter estimates where the cod, salmon and fish oil groups
were compared separately with the control group. The
numbers in the parameter estimates (B, lower confidence limit,
higher confidence limit) were back-transformed and are shown
as 1 – B,1– lower confidence limit, 1 – higher confidence
limit, respectively, thus giving percentage differences between
groups with respect to endpoint variables. A p value of less
than 0.05 was regarded as statistically significant.
Results
Baseline anthropometric variables, hormones, HOMA-IR
and fatty acid values are reported in Table 1. Despite
randomisation, the cod group had higher triacylglycerol
levels at baseline than the control group (p=0.017), and all
intervention groups had lower EPA values at baseline than
the control group ( p=0.004–0.026). Other baseline param-
eters did not significantly differ between groups. During the
intervention, the body weight of participants decreased
(−5.2±3.2 kg, p<0.001, n=278), as did fasting insulin,
glucose, triacylglycerol and adiponectin levels and HOMA-
IR, whereas LC n -3 PUFA content in membrane lipids
significantly increased (all p<0.001; Table 2).
Linear models
The following variables were entered into the linear models
to find out whether diet groups can predict endpoint fasting
insulin (Table 3) and HOMA IR (Table 4 ): sex, country, diet
groups, age and baseline value of the dependent variable.
Fasting insulin and HOMA-IR were significantly lower in the
fish oil group than in the control group at endpoint (16.4%, p=
0.025 and 17.2%, p=0.022, respectively). To find out
whether the effects of diet groups on endpoint fasting insulin
and HOMA-IR can be explained by changes in weight loss,
triacylglycerol reduction, increased EPA/DHA content in
membrane lipids or adiponectin, these anthropometric and
blood chemical variables were entered as additional cova-
Table 1 Baseline data of the participants
Parameter Control Cod Salmon Fish oil
Body weight (kg)
min. 70.1 70.5 67.3 66.7
25 79.9 83.5 82.7 79.2
50 86.7 88.6 88.3 84.8
75 95.4 95.9 98.0 91.6
max. 120.2 117.8 122.3 109.7
Fasting glucose (mmol/l)
min. 3.94 3.67 3.83 4.00
25 4.68 4.61 4.72 4.61
50 4.97 4.83 5.17 4.89
75 5.26 5.28 5.44 5.22
max. 6.06 6.72 5.89 6.94
Fasting insulin (pmol/l)
min. 27.1 20.8 26.4 18.1
25 47.9 47.2 50.7 48.6
50 66.0 61.1 68.8 62.5
75 86.8 87.5 93.8 84.0
max. 279.2 154.2 254.2 184.7
HOMA-IR
min. 0.50 0.37 0.49 0.33
25 0.89 0.88 0.95 0.91
50 1.18 1.15 1.30 1.17
75 1.56 1.64 1.74 1.58
max. 5.10 2.82 4.63 3.47
Adiponectin (μg/ml)
a
min. 3.2 3.4 2.9 4.6
25 8.2 7.8 8.0 8.7
50 12.4 10.7 10.3 10.8
75 17.1 15.7 15.6 15.4
max. 34.8 26.8 38.4 30.4
EPA (%)
b,c
min. 0.29 0.22 0.37 0.22
25 0.78 0.75 0.74 0.79
50 1.23 0.95 0.93 1.08
75 1.83 1.34 1.24 1.33
max. 5.90 2.80 3.17 4.86
DHA (%)
b
min. 0.99 2.32 2.65 2.37
25 4.98 4.67 4.88 5.22
50 5.92 5.61 5.88 5.90
75 6.48 6.53 6.49 6.68
max. 11.3 7.8 9.1 11.4
Triacylglycerol (mmol/l)
a,d
min. 0.32 0.50 0.44 0.33
25 0.71 0.84 0.86 0.67
50 0.91 1.11 1.07 0.97
75 1.27 1.61 1.54 1.41
max. 4.40 4.34 3.25 4.51
Values are shown as minimum (min.), maximum (max.) and the
quartile cut-off values
a
Fasting values
b
In erythrocyte membrane phospholipids
c
All intervention groups had lower EPA values at baseline than the control
group (p= 0.004–0.026) when corrected for country , sex and age)
d
Significant difference between cod and control group, p=0.017,
when corrected for country, sex and age)
1264 Diabetologia (2008) 51:1261–1268
riates. The differences between the fish oil group and control
remained significant (values 15.8% and 16.1% lower than
control, respectively), and weight loss was revealed to be an
additional significant predictor of both outcomes each kg
weight loss predicted a 3.4% and 3.5% reduction, respec-
tively). Triacylglycerol reduction, EPA changes and adipo-
nectin changes did not contribute significantly in the linear
models. The results of the linear models show estimated
changes in endpoint insulin (Table 3) and HOMA-IR
(Table 4)inresponsetoanincreaseinEPA;theuseof
DHA instead did not change the results.
Discussion
In this randomised dietary intervention trial we investigated
the effects of seafood intake and weight loss on fasting insulin
and HOMA-IR in young overweight and obese European
adults from three different countries following an energy-
restricted diet. Participants consumed cod (150 g, three times/
week), salmon (150 g, three times/week), fish oil (6×500 mg
capsules/day) or placebo capsules (6×500 mg capsules/day),
but otherwise consumed diets of identical macronutrient
composition and percentage energy restriction [28]. During
these 8 weeks, average fasting insulin decrea sed and
HOMA-IR improved in the participants.
The most important finding of our study is that the fish oil
diet reduces fasting insulin and improves HOMA-IR to a
significantly greater extent than the control diet (between
15.8% and 17.2%, depending on the statistical model) and to
an extent similar to that observed with a weight loss of 4.7 kg
(calculated by dividing the B coefficients of the fish oil group
and weight loss from the linear models). Animal research,
ecological and epidemiological studies have shown associ-
ations between LC n-3 PUFA intake and insulin sensitivity
[2, 5, 6, 7], but, according to reviews [9, 32], there is
relatively little evidence from interventions that n-3 PUFA
supplementation has positive effects on insulin sensitivity in
humans. An early experimental study reported positive
effects of 3 g/day (in total) of EPA and DHA on insulin
sensitivity (measured by in vivo insulin-stimulated glucose
uptake by simultaneous infusions of glucose and insulin) in
six individuals with type 2 diabetes [33]. Another study [34],
which investigated the combined effects of fish consumption
and weight loss on cardio vascul ar risk factors in 69
overweight patients, found that the greatest decreases in
fasting insulin and glucose occurred in the fish+weight-loss
group. However, there were no independent effects of fish
on glucose or insulin. A similar intervention study involving
116 overweight insulin-resistant women showed independent
(from weight loss) effects of LC n-3 PUFA supplementation
on triacylglycerol and adiponectin levels, but not insulin
sensitivity [35]. The Kuopio, Aarhus, Naples, Wollongong
Table 2 Changes in weight and hormone and fatty acid levels during
the 8 week intervention
Parameter Control Cod Salmon Fish oil
Body weight (kg)
min. 2.2 −1.4 −0.7 −0.7
25 −2.6 −3.5 −3.1 −2.9
50 −4.5 −5.4 −5.6 −5.2
75 −5.9 −7.2 −7.4 −7.8
max. −11.6 −12 −15.3 −12.7
Fasting glucose (mmol/l)
min. 0.94 1.50 1.06 0.67
25 0.11 0.17 0.11 0.17
50 −0.00 −0.11 −0.11 −0.17
75 −0.28 −0.28 −0.50 −0.29
max. −0.89 −1.67 −1.06 −2.06
Fasting insulin (pmol/l)
min. 51.4 99.3 51.4 63.2
25 9.7 3.5 −4.2 −5.6
50 −9.7 −9.7 −17.4 −16.0
75 −31.3 −22.9 −32.6 −27.8
max.
−112.5 −70.1 −123.6 −94.5
HOMA-IR
min. 0.99 1.91 0.95 1.17
25 0.15 0.08 −0.04 −0.08
50 −0.15 −0.18 −0.32 −0.30
75 −0.56 −0.46 −0.61 −0.50
max. −2.07 −1.29 −2.29 −1.80
Adiponectin (μg/ml)
a
min. 6.7 6.4 8.0 5.2
25 0.9 1.2 0.7 1.0
50 −0.5 −0.2 −0.9 −0.5
75 −2.2 −1.9 −2.1 −2.1
max. −7.3 −7.8 −7.7 −7.2
EPA (%)
b
min. −5.1 −1.8 −1.4 −3.4
25 −0.6 −0.3 0.2 0.3
50 −0.2 −0.1 0.7 0.6
75 0.0 0.1 1.2 1.0
max. 4.5 1.9 2.7 4.8
DHA (%)
b
min. −6.7 −5.3 −3.0 −4.9
25 −1.0 0.2 0.4 0.0
50 −0.2 0.7 1.5 0.7
75 0.4 1.4 2.3 1.4
max. 5.9 3.4 6.2 4.5
Triacylglycerol (mmol/l)
a
min. 0.92 0.76 0.65 1.05
25 0.22 0.03 0.05 0.13
50 −0.02 −0.14 −0.21 −0.10
75 −0.21 −0.43 −0.52 −0.34
max. −2.21 −1.97 −1.80 −2.98
Values are shown as minimum (min.), maximum (max.) and the
quartile cut-off values
a
Fasting values
b
In erythrocyte membrane phospholipids
Diabetologia (2008) 51:1261–1268 1265
and Uppsala (KANWU) study, a controlled multi-centre
isoenergetic dietary intervention study focusing on dietary fat
composition but not weight loss in 162 individuals, showed
that decrea sin g die tar y s atura ted fatty acid intak e an d
increasing monounsaturated fatty acid intake improves insulin
sensitivity, but no effect of fish oil supplementation on insulin
sensitivity was found, despite reduced plasma triacylglycerol
concentrations [36]. Similarly, a recent study investigating 29
India n Asians, a group particularly suscep tible to the
metabolic syndrome and type 2 diabetes, did not find an
effect of LC n-3 PUFA supplementation on insulin resistance,
even though supplementation was associated with improved
fasting and postprandial plasma triacylglycerol metabolism
[37]. In comparison with the above-mentioned studies, the
present study investigated a greater number of subjects. The
higher statistical power can explain our significant findings.
Significant weight loss was achieved in our study, and it can
be speculated that LC n-3 PUFA exert their positive health
effects on insulin sensitivity best during weight loss;
however, similar or greater amounts of weight loss were
achieved in similar studies [34, 35] that did not report
significant effects of LC n-3 PUFA on insulin sensitivity.
We cannot explain why endpoint fasting insulin and
HOMA-IR values for the salmon group were not significantly
different from those for the control group, despite the fact that
salmon provided the highest dose of LC n-3 PUFA in our
Table 3 Estimated changes
a
in endpoint insulin
b
relative to the control group
Model B
e
95% CI
e
p value
Unadjusted model
Cod group
c
−0.014 1.007 −1.208 0.978
Salmon group
c
0.535 1.493 −0.583 0.327
Fish oil group
c
1.139 1.986 0.160 0.025
Adjusted model
Cod group
c
−0.069 0.951 −1.271 0.901
Salmon group
c
0.632 1.625 −0.549 0.276
Fish oil group
c
1.097 1.972 0.076 0.037
Weight loss (kg) 0.236 0.340 0.125 <0.001
Triacylglycerol change (mmol/l) −0.021 0.569 −0.667 0.948
Increase in EPA
d
−0.056 0.243 −0.375 0.715
Adiponectin decrease (μg/ml) 0.063 0.181 −0.049 0.276
a
All models are corrected for baseline values of insulin, age, sex and country
b
Log-transformed
c
Compared with control group
d
In erythrocyte membrane phospholipids
e
B Coefficients are presented as 1 – back-transformed B, e.g. endpoint insulin and is 16.4% lower in the fish oil group than in the control group,
and each kg weight loss results in a 3.4% lower endpoint insulin (for all groups)
Table 4 Estimated changes
a
in endpoint HOMA-IR
b
to the control group
Model B
e
95% CI
e
p value
Unadjusted model
Cod group
c
0.004 0.152 −0.172 0.966
Salmon group
c
0.075 0.217 −0.093 0.359
Fish oil group
c
0.172 0.295 0.027 0.022
Adjusted model
Cod group
c
−0.012 0.137 −0.187 0.886
Salmon group
c
0.089 0.234 −0.083 0.290
Fish oil group
c
0.161 0.289 0.011 0.036
Weight loss (kg) 0.035 0.050 0.019 <0.001
Triacylglycerol change (mmol/l) −0.001 0.086 −0.095 0.990
Increase in EPA
d
−0.010 0.034 −0.056 0.668
Adiponectin decrease (μg/ml) 0.009 0.026 −0.008 0.296
a
All models are corrected for baseline values of HOMA-IR, age, sex and country
b
Log-transformed
c
Compared with control group
d
In erythrocyte membrane phospholipids
e
B coefficients are presented as 1 – back-transformed B, e.g. endpoint HOMA-IR 17.2% lower in the fish oil group than in the control group, and
each kg weight loss results in a 3.5% lower HOMA-IR (for all groups)
1266 Diabetologia (2008) 51:1261–1268
study. The estimated improvements for the salmon group
were lower than those for the fish oil group (7.5–10.5%), but
were not significantly different (p=0.191–0.334, analyses
not shown). The LC n-3 PUFA from salmon were
bioavailable, as indicated by increases in DHA and EPA in
erythrocyte membrane. It can be speculated that other con-
stituents in salmon could counteract the effects of LC n-3
PUFA. However, it should be mentioned that predicted
changes in the salmon group were the same direction as
those in the fish oil group, but they did not reach statistical
significance. This is in contrast to the cod group, which did
not show any effects compared with the control group.
Unexpectedly, the positive effects of LC n-3 PUFA on the
outcome variables during the 8 week intervention cannot be
explained by reductions in weight loss or triacylglycerol, or
changes in membrane fatty acids or adiponectin. It has long
been recognised that fish oil can decrease plasma and liver
triacylglycerol levels and VLDL-triacylglycerol secretion,
and suppress postprandial hypertriacylglycerolaemia [13].
However, the inclusion of plasma triacylglycerol in our
linear models did not alter the strength of fish oil as a
significant predictor of insulin sensitivity.
In several cross-sectional studies, measures of insulin
sensitivity are negatively correlated with the percentage of
individual long-chain polyunsaturated fatty acids in the
phospholipid fraction of muscle in patients or healthy subjects
[18, 19]. An animal study investigated the effects of fish oil
intake on membrane composition and insulin sensitivity in
rats and showed that incorporation of n-3 fatty acids into
adipocyte membrane phospholipids was higher in rats fed fish
oil than in the control group, and insulin action was positively
correlated with the fatty acid unsaturation index in membrane
phospholipids [38]. Furthermore, a recent human intervention
study showed that increased incorporation of LC n-3 PUFA
and reduced saturated fatty acids in skeletal muscle mem-
brane phospholipids is associated with better insulin sensitiv-
ity [21]. According to our linear models, changes in membrane
fatty acid composition do not play an independent role in the
improvements in insulin sensitivity in our participants. It is
possible that phospholipid composition in erythrocytes does
not reflect phospholipid composition in muscle [39]and/orthe
study duration of 8 weeks was too short to allow a full steady
state to be achieved with respect to phospholipid fatty acid
composition of skeletal muscle, liver and depot fat.
Adiponectin is a hormone secreted by the adipose tissue,
and its administration enhances insulin action in animals [40].
Cross-sectional studies have shown adiponectin levels to be
associated with insulin sensitivity [41, 42]. When we use our
baseline data to calculate correlations between insulin
sensitivity and adiponectin (results not shown), we see the
same associations as in the aforementioned cross-sectional
studies. However, according to the fitted statistical models,
changes in adiponectin did not predict changes in insulin
sensitivity. It could be that during the intervention, other
factors, namely fish oil intake and weight loss, had a much
stronger effect on insulin sensitivity then the small changes
in adiponectin, such that effects of adiponectin were not
visible in our analysis.
Limitations A common limitation of a dietary intervention
trial is blinding. In our study the fish oil and control groups
were single-blinded, whereas the cod and salmon groups were
not (fish was received as a fillet). Another limitation is the
uncertainty of whether the dietary intakes during the study
period were as prescribed. This risk was minimised by the
intense support provided by the staff and frequent contact with
the participants via phone and personal visits. Compliance
was tested during the intervention trial using an FFQ,
validated for assessing frequency of seafood consumption
[29], at the endpoint of the study, and measuring erythrocyte
fatty acid composition at the end of the intervention.
Changes in n-3 fatty acids in erythrocyte membrane were
in accordance with fatty acid composition of the diets, and
results from the FFQ confirmed good compliance.
Conclusion Fish oil consumption during an 8 week energy-
restricted diet exerts positive effects on fasting insulin and on
a measure of insulin resistance in young overweight or obese
individuals. These effects are independent from weight loss,
changes in plasma triacylglycerol, erythrocyte membrane
EPA/DHA or adiponectin concentrations. Weight loss is an
additional independent predictor of improvements in individ-
uals who consume fish oils. The present randomised dietary
intervention trial supports previous epidemiological evidence,
and demonstrates the importance of LC n-3 PUFA consump-
tion for improvement of insulin resistance and, possibly, for
the prevention of type 2 diabetes in addition to weight-
lowering strategies. Future intervention studies are required
to confirm the results of this study.
Acknowledgements This work is included in the SEAFOODplus
YOUNG, co-ordinated by Prof. Inga Thorsdottir, being part of the
SEAFOODplus Integrated Project, which is funded by the European
Commission through the 6th Framework Programme Contract with Ref.
FOOD-CT-2004–506359. Thanks are given to the EU Commission for
financial support, and to the volunteers who participated in the study.
Duality of interest The authors declare that there is no duality of
interest associated with this manuscript.
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