The obesogenic effects of polyunsaturated fatty acids are dependent ...

The obesogenic effects of polyunsaturated fatty
acids are dependent on background diets
Tao Ma
Supervisor:
Karsten Kristiansen
Lise Madsen
Department of Biology, University of Copenhagen, Copenhagen DK-
2200, Denmark.
The Graduate School of Science, Faculty of Science, University of
Copenhagen, Denmark.
July 2011, 家

Acknowledgement
I am grateful to my supervisor Karsten Kristiansen for giving me the opportunity to continue
my research life. It is full of excitement, as all I can ask for. Thanks to his guidance and
inspiration. I would as well give my thanks to my co-supervisor Lise Madsen for conceiving,
organizing and directing my project. That is precious experiences that I can benefit from for
my future work.
We can only be a part of a team in order to achieve. I would like to thanks all the group
members in Denmark and Norway for their helps and joys they bring along, especially Qin
Hao and Rasmus Koefoed Petersen for their patience and kindness. Also, my appreciation to
Alison Keenan for her great help with the language.
And finally, there is nothing can be accomplished without the continuous support and love
from my family.
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Table of Contents
Acknowledgement ............................................................................................................................................. 2
Abstract ............................................................................................................................................................. 4
Abstract in Danish .............................................................................................................................................. 5
Introduction ....................................................................................................................................................... 6
Obesity and food intake ................................................................................................................................ 6
Polyunsaturated fatty acids in the diet ......................................................................................................... 7
Adipocyte biology .......................................................................................................................................... 7
Adipogenesis is dependent on cAMP levels. ................................................................................................. 8
Results ............................................................................................................................................................... 9
Discussion, perspectives .................................................................................................................................. 11
Low carbohydrate, high protein diet ........................................................................................................... 11
Branched-chain amino acids and mTOR ...................................................................................................... 12
Regulation of hepatic lipogenesis ................................................................................................................ 12
Fructose ....................................................................................................................................................... 13
Fish oil contaminants ................................................................................................................................... 14
References ....................................................................................................................................................... 16
Annexes ........................................................................................................................................................... 21
3

Abstract
Polyunsaturated n-3 fatty acids (n-3 PUFAs) are reported to protect against high fat diet-induced
obesity and inflammation in adipose tissue. While it has previously been reported that the
adipogenic effects of n-6 PUFAs are dependent on the macronutrient composition of the diet,
whether n-3 PUFAs metabolism is subjected to similar impact by carbohydrates and proteins in the
diet is not well explored. In the present thesis two studies are described. Isocaloric high fat diets
enriched with protein and carbohydrate with different weight ratio or with various carbohydrate
sources were fed to male C57BL/6J mice. We show that increasing the amount of sucrose at the
expense of protein in the diet correlated with increased energy efficiency and fat mass irrespective
of fat sources. We propose that this effect is a result of reduced thermogenesis in fat tissues,
combined repression of gluconeogenesis and ureagenesis in the liver. Also, high fat diets induced
glucose intolerance regardless of the adiposity of the animals. Moreover, by using carbohydrates
with differing glycemic indices we provide evidence that insulin plays a central role in promoting
adiposity and inflammation in fat tissues. This idea was strengthened further by exploring
pharmaceutical drugs to modulate insulin secretion.
In summary, the ability of background diet, namely carbohydrates and proteins, in regulating insulin
secretion significantly modulates the beneficial effects of n-3 PUFAs in development of obesity,
glucose intolerance and adipose tissue inflammation.
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Abstract in Danish
Polyumættede n-3 fedtsyrer (n-3 PUFA) er rapporteret at beskytte mod fedme og inflammation i
fedtvæv induceret af kost med højt fedtindhold. Mens det tidligere har været rapporteret, at de
adipogene effekter af n-6 PUFA er afhængige af kostens sammensætning af makronæringsstof, er
påvirkning af kulhydrater og protein i kosten på n-3 PUFA metabolisme ikke godt udforsket. I
nærværende afhandling er to undersøgelser beskrevet. C57BL/6J hanmus blev fodret med
isokaloriske dieter med højt fedtindhold beriget med protein og kulhydrat med varierende indbyrdes
vægt-forhold eller med forskellige kulhydratkilder. Vi viser, at øget sukker i kosten er korreleret
med øget energieffektivitet og fedtmasse uanset fedtkilder. Vores hypotese er, at disse observationer
er en følge af nedsat termogenese i fedtvæv, og gluconeogenese og ureagenese i leveren. Ligeledes
inducerer kost med højt fedtindhold glukose intolerance uafhængigt af dyrenes (over)vægt. Ved
brug af kulhydrater med forskellige glykæmiske indeks viser vi desuden, at insulin spiller en central
rolle for udviklingen af overvægt og inflammation i fedtvæv. Denne observation blev styrket
yderligere i forsøg med lægemidler, der direkte modulerer insulinsekretion.
Sammenfattende kan vi konkludere, at kostens indhold af kulhydrater og proteiner og evnen til at
regulere insulinsekretionen påvirker de gavnlige effekter af n-3 PUFA på udvikling af fedme,
glukose intolerance og inflammation i fedtvæv.
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Introduction
Obesity and food intake
Obesity is when excess fat is accumulated in the body, mainly in adipose tissues, to an unhealthy
extent. An adult with a body mass index (BMI) between 25 and 30 kg/m 2 is considered overweight,
while an adult with a BMI over 30 kg/m 2 is regarded as obese 1 ; nonetheless, this classification is
debatable when applied to different races 2,3 .
Utilizing the historic record provided by American obesity data 4 , we are able to track the increasing
incidence of overweight and obesity in the new world. Not trailing far behind, developing countries
are catching up with this trend, which is a direct consequence of overconsumption of energy-dense
food and engaging in a sedentary lifestyle 5 . Obesity is one of the biggest threats to public health in
the new century, as it increases the risk of developing type 2 diabetes 6 , cardiovascular disease 7 and
certain types of cancer 8 .
Consequently, considerable financial resources as well as social and scientific efforts have been
mobilized towards the war against obesity 9 . One focus point has been to promote a reduction in
dietary fat intake, as can be observed with the “light” labeled goods filling supermarket shelves.
This is mainly because fat contains more energy per unit weight than carbohydrate or protein,
9kcal/g, 4.5kcal/g and 4kcal/g respectively. By reducing the energy density of food, the average
energy intake of the general public should therefore be reduced. Besides the increase in average
energy intake over time 10 , another major alteration over the thousands of years of dietary history is
the dramatic elevation of carbohydrate content in the food at the expense of protein 11 . One recent
publication suggested that over a period of two years a low-carbohydrate diet had a similar weight
loss effect as a low-fat diet 12 . Moreover, a body of evidence pointed to the beneficial effects of high
protein diet on whole body metabolism 13 , implying that macronutrients are not simply different
with regard to their caloric value, but have other impacts on weight gain and maintenance that are
not fully elucidated.
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Polyunsaturated fatty acids in the diet
Fat comes in the form of a blend of different fatty acids, namely saturated fatty acids (SFAs),
monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs). Multiple studies
suggest that the consumption of MUFAs in place of SFAs improves insulin sensitivity 14,15 . The
increased intake of n-6 PUFAs, or higher dietary n-6 to n-3 ratio, has been implicated in promoting
many diseases including obesity and diabetes 16,17 . As shown in the work of Massiera et al., in mice,
pups from mothers fed a diet enriched with n-6 linoleic acid became obese. However, this can be
prevented by inclusion of the n-3 PUFA α-linolenic acid in the diet. The authors attributed this
phenomenon to the ability of n-6 PUFAs to promote adipogenesis 18 . In line with the animal work, a
similar outcome has been observed in a human study that revealed a higher n-6/n-3 PUFA ratio in
the umbilical cord plasma was associated with a higher incidence of obesity 19 . Furthermore, work
from our group suggests that the adipogenic nature of n-6 PUFAs is dependent on the cellular
cAMP status 20 .
The possible anti-obesity mechanisms of n-3 PUFAs include, but are not limited to: 1), competition
with n-6 PUFAs in enzymatic pathways, which may dampen the adipogenic and pro-inflammatory
effects of n-6 PUFAs 21 , as one metabolic effect from obesity is chronic low-grade inflammation.
Anti-inflammatory intervention has been shown to be helpful against metabolic dysfunction 22 . 2),
up regulation of β-oxidation and mitochondrial biogenesis. In both rodent and human settings
addition of n-3 PUFAs, particularly of marine source, increased lipid oxidation 23,24 . 3), activation of
G protein-coupled receptor 120, which exerts its anti-inflammatory effects by inhibiting both Tolllike
receptor (TLR) and tissue necrosis factor- α (TNF-α) signaling pathways in the residing
macrophages in adipose tissue 25 .
Adipocyte biology
Historically, adipose tissue was seen as a static storage unit when energy is in excess and to release
energy during food deprivation in the form of fatty acids and glycerol. When energy consumption
persistently exceeds energy expenditure, expansion of adipose tissue will occur, contributed to by
both hyperplasia and hypertrophy of adipocytes and finally culminate in obesity. Recently adipose
tissue has become appreciated as an important mediator of systemic metabolism for its role of
secreting regulatory proteins 26 . The first adipokine being identified was adipsin (Factor D) from
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1987 27 . Over the years, more and more adipokines were discovered including leptin, TNF-α,
adiponectin and so on, most of which bridge the links between obesity, inflammation and insulin
resistance 28 . Furthermore, with the rediscovery of brown adipose tissue in adult humans, people
have begun exploring the possible therapeutic role of this special fat depot 29 .
Insulin is an important driver for adipocyte differentiation and the primary anabolic hormone
promoting energy storage 30,31 . After ingesting a meal of high glycemic carbohydrate, the high
glycemia will induce pancreatic insulin secretion to promote glucose uptake in adipose tissue. As
shown in animal models, mice with adipose tissue-specific insulin receptor knock out (FIRKO mice)
are protected against obesity and remain glucose tolerant on a high-fat diet 32 . In contrast, mice
lacking the insulin receptor in muscle (MIRKO mice) maintain normal plasma glucose levels by
increasing glucose utilization in white adipose tissue (WAT) and fat mass in order to compensate
the loss of muscular glucose transport activity 33 . Furthermore, mice overexpressing facilitated
glucose transporter, member 4 (GLUT4) 34 or insulin receptor substrate 1 (Irs1) 35 in adipose tissue
are obese.
Adipogenesis is dependent on cAMP levels.
Our earlier works has shown the interplay of cAMP-elevating agents and arachidonic acid in
mediating adipogenesis 20,36 . The inclusion of n-6 fatty acid arachidonic acid (AA) in the induction
cocktail inhibited adipogenesis of 3T3-L1 preadipocytes. This inhibitory effect requires the cAMP –
elevating agent 3-isobutyl-1-methylxanthine (IBMX) in the mixture and depends on protein kinase
A (PKA) and cycloxygenase (COX) activity. During the initial phase of adipogenic induction, the
combination of IBMX and AA dramatically increases the expression levels of both COX-1 and 2,
which in turn metabolize AA towards the inhibitory prostaglandin E 2 (PGE 2 ) and F 2α (PGF 2α ) 37 .
Furthermore, when AA is present, even without an increase of cellular cAMP levels, overexpression
of COX-1 and 2 can still execute their anti-adipogenic power. On the other hand, in the absence of
IBMX, AA induces adipogenesis which was also reported previously by others, probably through
the production of adipogenic prostaglandins 18 .
In order to create a similar scenario in vivo, on top of high corn oil, different proportions of
carbohydrate and protein were included in the diets trying to manipulate the cAMP levels by
changing the plasma insulin/glucagon ratio. Indeed as we reported 38 , the insulin to glucagon ratio
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was more than 3 times higher in the mice fed with high corn oil diet enriched with sucrose than with
protein. With the different diets, significant changes in body weight gains were observed both ad
libitum and pair-fed., and were due to the different degrees of adiposity. As expected, in multiple
adipose tissues, the expression of Crem (cAMP-responsive element modulator) and Pde4b (cAMPspecific
phosphodiesterase 4b) and the phosphorylation status of CREB (cAMP-responsive element
-binding protein) were differently regulated because of the diets, which revealed an altered cAMP
level. Accordingly, raised circulating PGE 2 and PGF 2 levels were detected in mice fed with a
protein supplemented diet that correlated with increased expressions of both Cox-1 and Cox-2 in
most of fat tissues.
Taken together, it was a clear demonstration of the cAMP-PKA-COX-prostaglandin axis in
regulating adipogenesis both in vitro and in vivo. The obesogenic effect of n-6 fatty acid is
dependent on the macronutrient content of the diets.
Results
As shown previously, cAMP-dependent signaling controls the obesogenic effect of n-6 PUFAs in
mice. We want to further investigate whether this phenomenon is restricted to n-6 PUFAs, or if the
beneficial effects of n-3 PUFAs can also be affected by background diets (Annex 1). To achieve
this, we prepared isocaloric diets enriched with either fish oil or corn oil (Table 1). After 9 weeks of
feeding, to our surprise, mice fed with sucrose-supplemented diets became obese regardless of the
fat sources (Fig.1 E-F). This was linked to the differential insulin to glucagon level in plasma that
we had previously identified (Fig.1 C-D), suggesting one of the determining effects background diet
has on obesity. Furthermore, the inflammation levels in the adipose tissues were correlated with
adiposity instead of dietary fat types assessed by gene expression of macrophage and inflammatory
markers (Fig.2A).
When compared to mice fed a standard chow diet, all individuals on high fat diets had impaired
glucose tolerance regardless they were fat or lean. But the separation of the HOMA index readout
implied different mechanisms underlying the observed glucose intolerance (Fig.2B). We observed
one of the recognized beneficial effects of taking fish oil in our settings. Hepatic lipid accumulation
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was considerably less in the fish oil-fed mice than in the corn oil-fed mice (Fig.3A), but it was not
directly related to the lipogenic gene expression in their livers (Fig.3B).
Since the mice on high fat diets consumed equivalent amounts of food, the lean ones had markedly
reduced energy efficiency (Fig.3D, 4B). The following points could partly explain the differences,
1). protein-fed animals could have more brownish phenotype in inguinal WAT (iWAT) judged by
the up-regulation of Ucp1 expression, which dispenses energy as heat (Fig.3E, 4E); 2). elevated
gluconeogenesis in mice fed protein-enriched diets even in the fed state, indicated by the enhanced
glucose production during pyruvate tolerance test and hepatic gene expressions (Fig.6A and B); 3).
nitrogen metabolism was another potential contributor to the energy-wasting effect as protein
cannot be stored but must be processed immediately (Fig.6B).
In order to evaluate the role of insulin in modulating the obesogenic effects of fish oil, isocaloric
high fish oil diets with different sucrose to protein weight ratios were fed to the mice (Annex 2).
Supporting our previous findings, the fat mass development (Fig.1D), energy efficiency (Fig.1B),
inflammation levels in adipose tissues (Fig.1E) were all does-dependently correlated to sucrose
content of the diets. mRNA levels of the indicators pointing to the three energy consumption
processes, Ucp1 in the iWAT--- thermogenesis (Fig.1E); Pck1 in the live--- gluconeogenesis and
Agxt in the liver--- ureagenesis (Fig.1F), were inversely associated as earlier suggested.
By exchanging sucrose with glucose or fructose in a fish oil-supplemented diet, we further
pinpointed that the insulin stimulating agent glucose moiety in sucrose facilitated the obesitypromoting
effect of fish oil, as mice fed a fructose diet gained less WAT weights (Fig.2D) and had
lower plasma triglycerides levels (Fig.2E). However, in the same set of mice, hepatic genes related
to β-oxidation were down-regulated and the expression of lipogenic genes were increased (Fig.2F).
Our theory was further supported by including starches promote different insulin responses
(different glycemic index-GI). Although these animals did not differ in weight gain, the WAT
masses were significantly higher in high GI diet-fed mice (Fig.4A and C), and this was
accompanied by the increased expression of lipogenic genes Fasn and Scd1 (Fig.4G).
Furthermore, pharmaceutical regulators of insulin secretion were introduced to the feeding regime.
Inclusion of insulin secretagogue glybenclamide in the protein-enriched high fish oil diet did not
result in a separation of the body weight gains (Fig.5A). A tendency of fat mass differences was
observed (Fig.5B), and could be linked to observed changes in gene expression related to
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gluconeogenesis and ureagenesis (Fig.5E). On the other hand, inhibition of insulin secretion by
nifedipine did achieve the expected fat mass-reducing effect (Fig.6C)
.
Discussion, perspectives
Low carbohydrate, high protein diet
It has been suggested that a modest increase in protein ingestion combined with a low glycemic
index diet could be protective against obesity in children 39 . Further, as reported in a recent large
European cohort study, after an initial weight loss, a diet with lower glycemic index and higher
protein content is most ideal for maintaining body weight and preventing weight regain 40 . When
consuming a diet very low in carbohydrates, such as in the case of our protein-enriched mouse feed
with only 8% of calories from carbohydrates, the animals need to mobilize liver glycogen storage
and increase gluconeogenesis in order to sustain blood glucose levels. After feeding for a long
period of time, glycogen will be exhausted, and the body will have to turn to other energy sources.
Even in the fed state, these protein-fed mice still kept high rates of β-oxidation, indicated by the
high circulating levels of 2-hydroxybutyrate (Annex 1Fig.5B (1.5B)), suggesting the need to
mobilize fat and/or protein for basal metabolism. This situation, to a certain degree, mimics what
has been observed in long term fasting. During starvation, following the decrease of circulating
glucose, insulin levels drop dramatically and glucagon secretion is elevated. Although
gluconeogenesis contributes partially to the blood glucose supply, it will be prioritized for central
nervous system and red blood cells functions.
Despite the protein-fed mice kept lean as their low fat diet-fed littermates, they showed the same
degree of glucose intolerance as the obese ones on sucrose-enriched diet. If our analogy to fasting is
accurate, the glucose intolerance we have observed in lean protein-fed mice can be delineated. As
fasting reduced insulin secretion in rats, this repression can only be rescued by refeeding with diets
high in carbohydrates 41 . Although no difference was observed in the insulin tolerance test (Fig
1.s1B), there are reports both in rats and humans showing that consuming low carbohydrate, high
protein diet can attenuate the suppression of hepatic glucose output by insulin 42,43 . In order to
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esolve the interplay between insulin secretion and action, detailed studies are required to clarify the
mechanisms behind impaired glucose tolerance in these mice.
Branched-chain amino acids and mTOR
Dietary proteins are important modulators of metabolism and insulin sensitivity 44 . It has been
suggested that a modest increase in protein intake could potentially combat obesity 45 , owing at least
in part to an increased thermic effect. We have shown these processes include but are not limited to
thermogenesis in the iWAT (See annex 3), gluconeogenesis and ureagenesis in the liver. Casein has
been widely used as the main protein source in rodent feed with the supplement of L-Cystine 46 .
Given its relative high percentage of branched-chain amino acids (BCAAs) and low content of
taurine and glycine compared to fish or soy protein, special consideration needs to be taken into
account when using high amount of casein. In a separate study, we have shown by replacing casein
with salmon protein hydrolysates in the diet, rats were protected from diet-induced obesity due to
raised plasma bile acid concentrations (Annex 4). Furthermore, there are links established between
excess intake of dietary protein, especially BCAAs and insulin resistance 47,48 .
Activation of mTOR (mammalian target of rapamycin) by BCAAs, particularly leucine, could
regulate glucose uptake in skeletal muscle through the inhibitory phosphorylation of S6 kinase on
insulin receptor substrate 1 (IRS-1) 49 . Furthermore Newgard et al, had shown that by 50% increase
of BCAAs in a high fat diet, rats developed insulin resistance regardless of a low rate of weight gain,
which was connected to chronic phosphorylation of mTOR, JNK, and IRS1 Ser307 and the
accumulation of multiple acylcarnitines in muscle 50 . This observation relates to our reported results
that insulin resistance can be disconnected from adiposity. In a recent multicenter cohort
metabolomic study, elevated plasma BCAAs levels had been shown to correlate strongly with and
could predict future diabetes 51 . If BCAAs did play a role contributing to the overall effect we have
presented, protein composition in the diet needs to be thought over in the future experiment design.
Regulation of hepatic lipogenesis
Hepatic glucose and lipid metabolism are cross-regulated by multiple transcriptional regulators with
sterol regulatory element binding protein-1c (SREBP-1c) taking the central stage 52 . In the context of
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high dietary PUFAs, the modulation is less clear. PUFAs have been shown to suppress hepatic
Srebp-1c transcription 53 , mRNA stability 54 and its proteolytic activation 55 through liver X receptor
(LXR) 56 - and/or SREBP-1c 55 - mediated pathways. In our study, the suppression on Srebp-1c
transcription was only sustained when diets were supplemented with protein but abolished when
combined with sucrose (Fig1.3B, 2.7A). The most straight forward explanation would be that
increased insulin levels played a role, as multiple lines of evidence suggested the importance of
insulin in controlling expression and activity of Srebp-1c 57 . But further in our study it was
elucidated otherwise, as modulating insulin secretion by pharmaceutical drugs did not have any
effect on either Srebp-1c mRNA abundance or its activity, judged by the transcription of its target
genes Scd1, Fasn and Acaca (Fig2.5E 2.7A).
The role of glucose in the context of this story needs also to be discussed. Besides insulin,
carbohydrate ingestion can very efficiently lead to the induction of lipogenic genes 58 . However,the
main sensor of glucose carbohydrate-responsive element–binding protein (ChREBP) 59 , like
SREBP-1c, was also suppressed in the presence of PUFAs 60 . Mitro et al. has shown that LXRs
could also work as glucose sensors to regulate cholesterol metabolism genes and in some degree
lipogenic genes as well 61 . In the LXRα/β knockout model, high carbohydrate diet-induced hepatic
gene expression of Scd1 and Fasn were attenuated 62 . As a whole, they suggest that in response to
glucose stimuli, LXR-dependent pathway is indispensable for the full activation of Scd1 and Fasn
expression, while the regulation of Acaca is mostly under the control of ChREBP 60 . So in our high
fish oil feeding supplemented with different starches, the observed elevation of Scd1 and Fasn
expression with high GI diet (Fig2.4G) could be explained as under the impact of fish oil, only LXR
was still able to sense plasma glucose changes though mechanism that is still under debate 63,64 . Of
course all speculations need supporting evidence from follow up studies. And with mTORC1 65,66
and AMP-activated protein kinase (AMPK) 67,68 also in the picture now, the already intricate control
of SREBP-1c and lipogenesis is only going to be more complicated.
Fructose
The increased world consumption of fructose or high-fructose corn syrup has been associated with
the obesity epidemic 69,70 . Fructose is taken up by the liver rapidly because of the low Km of
fructokinase towards fructose 71 . Without the negative feedback on phosphofructokinase as in
glucose metabolism, the reactions continue to provide substrate for glucose and lipid production 71 .
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Although fructose has long been used to induce glucose intolerance in rats, in C57BL/6 mice
neither hyperinsulimia nor hyperglycemia is developed. It had been shown C57BL/6 mice are lessresponsive
towards fructose-induced Srebf1 activation among different strains of mice due to a
single nucleotide polymorphism in the promoter region 72 . However we failed to observe this
unresponsiveness. As others have shown in rats 73 , in our fructose-fed mice despite the presence of
fish oil in the diet, lipogenic genes were induced and β-oxidation-related targets were repressed
(Fig.2.2F). But when compared to sucrose- and glucose-fed mice, these animals gained least body
weight, had the lowest amounts of fat tissues and lowest circulating triglycerides (Fig2.2), so the
extra fat has to be either stored ectopically or burnt. The increased brown adipocyte phenotype in
the iWAT could, in part, assist this process. Although others have shown the development of
hepatic lipid accumulation in fructose-fed mice 74 , it was not the case in our hands despite elevated
liver to body weight ratios (Fig2.3A). Interestingly, the authors also reported that a nonabsorbable
antibiotics treatment could attenuate lipid accumulation in liver, suggesting potential involvement
of gut microbes in fructose metabolism 74 .
The metabolic effects of fructose do not limit to the listed above, as there have been reports
showing centrally-administrated fructose led to increased food intake in rodents 75,76 by reducing
hypothalamic malonyl–CoA levels 76 . Furthermore, the ability of fructose to cross the blood-brain
barrier indirectly supports this observation, as the orexigenic effect relies on CNS function 77 . One
interesting study, by using hyperinsulinemic-euglycemic clamps, demonstrated that sucrose feeding
was not the same as with the combination of its hydrolysis products glucose and fructose in
promoting hepatic insulin resistance 78 . This may suggest the involvement of gastric emptying or the
release of gut-secreted hormones.
Fish oil contaminants
Long chain n-3 fatty acids in fish oil are not synthesized de novo by fish themselves, but by algae
they consume in the wild or added to the feed in farmed fish colonies 79 . Along with the beneficial
nutrients, through food chains, persistent organic pollutants (POPs) are also accumulated. Despite
the effort to limit their release, POPs still persist in the environment 80 . POPs such as organochlorine
pesticides, polychlorinated biphenyls (PCBs) and dioxins are hydrophobic compounds, and
consequently they accumulate in fatty tissues, and therefore are inevitably retained in the process of
fish oil production.
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In a pair of population studies, an association was reported between serum concentrations of POPs
and insulin resistance in spite of the subjects’ diabetic status 81,82 . Furthermore, different
mechanisms were proposed in explaining this deteriorating effect, which include causing
mitochondrial dysfunction 83 and impairment of insulin actions both in vivo and in vitro (ref 84 ,
Annex 5). In order not to weigh out the beneficial effect by pollutants, purity would be a major
concern when consuming very long chain marine n-3 fatty acids. This also applies to the use of fish
oil in animal feeds 85 .
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20

Annexes
1. Ma T, Liaset B, Hao Q, Petersen RK, Fjære E, et al. (2011) Sucrose Counteracts the Antinflammatory
Effect of Fish Oil in Adipose Tissue and Increases Obesity Development in Mice.
PLoS ONE 6(6): e21647. doi:10.1371/journal.pone.0021647
2. Hao Q, Lillefosse HH, Fjære E, … Ma T, et al. (2011). Carbohydrate Source and Insulin Secretion
Modulate the Obesity Promoting Effect of Fish Oil. Manuscript in revision.
3. Madsen L, Pedersen LM, Lillefosse HH, Fjære E, … Ma T, et al. (2010) UCP1 Induction during
Recruitment of Brown Adipocytes in White Adipose Tissue Is Dependent on Cyclooxygenase
Activity. PLoS ONE 5(6): e11391. doi:10.1371/journal.pone.0011391.
4. Liaset B, Hao Q, Jørgensen H, Hallenborg P, Du ZY, Ma T, et al. (2011). Nutritional regulation of
bile acid metabolism improves pathological characteristics of the metabolic syndrome. J Biol
Chem., in press.
5. Ruzzin J, Petersen RK, Meugnier E, Madsen L, … Ma T, et al. (2010). Persistent organic
pollutant exposure leads to insulin resistance syndrome. Environ Health Perspect. 118(4):465-
71.
21

Sucrose Counteracts the Anti-Inflammatory Effect of Fish
Oil in Adipose Tissue and Increases Obesity
Development in Mice
Tao Ma 1 , Bjørn Liaset 2 , Qin Hao 1 , Rasmus Koefoed Petersen 1 , Even Fjære 1,2 , Ha Thi Ngo 2¤ , Haldis Haukås
Lillefosse 1,2 , Stine Ringholm 1 , Si Brask Sonne 1 , Jonas Thue Treebak 3 , Henriette Pilegaard 1 , Livar
Frøyland 2 , Karsten Kristiansen 1 *, Lise Madsen 1,2 *
1 Department of Biology, University of Copenhagen, Copenhagen, Denmark, 2 National Institute of Nutrition and Seafood Research (NIFES), Bergen, Norway,
3 Department of Exercise and Sport Sciences, University of Copenhagen, Copenhagen, Denmark
Abstract
Background: Polyunsaturated n-3 fatty acids (n-3 PUFAs) are reported to protect against high fat diet-induced obesity and
inflammation in adipose tissue. Here we aimed to investigate if the amount of sucrose in the background diet influences the
ability of n-3 PUFAs to protect against diet-induced obesity, adipose tissue inflammation and glucose intolerance.
Methodology/Principal Findings: We fed C57BL/6J mice a protein- (casein) or sucrose-based high fat diet supplemented
with fish oil or corn oil for 9 weeks. Irrespective of the fatty acid source, mice fed diets rich in sucrose became obese
whereas mice fed high protein diets remained lean. Inclusion of sucrose in the diet also counteracted the well-known antiinflammatory
effect of fish oil in adipose tissue, but did not impair the ability of fish oil to prevent accumulation of fat in the
liver. Calculation of HOMA-IR indicated that mice fed high levels of proteins remained insulin sensitive, whereas insulin
sensitivity was reduced in the obese mice fed sucrose irrespectively of the fat source. We show that a high fat diet decreased
glucose tolerance in the mice independently of both obesity and dietary levels of n-3 PUFAs and sucrose. Of note,
increasing the protein:sucrose ratio in high fat diets decreased energy efficiency irrespective of fat source. This was
accompanied by increased expression of Ppargc1a (peroxisome proliferator-activated receptor, gamma, coactivator 1 alpha)
and increased gluconeogenesis in the fed state.
Conclusions/Significance: The background diet influence the ability of n-3 PUFAs to protect against development of
obesity, glucose intolerance and adipose tissue inflammation. High levels of dietary sucrose counteract the antiinflammatory
effect of fish oil in adipose tissue and increases obesity development in mice.
Citation: Ma T, Liaset B, Hao Q, Petersen RK, Fjære E, et al. (2011) Sucrose Counteracts the Anti-Inflammatory Effect of Fish Oil in Adipose Tissue and Increases
Obesity Development in Mice. PLoS ONE 6(6): e21647. doi:10.1371/journal.pone.0021647
Editor: Aimin Xu, University of Hong Kong, China
Received March 28, 2011; Accepted June 4, 2011; Published June 28, 2011
Copyright: ß 2011 Ma et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Danish Natural Science Research Council, the Novo Nordisk Foundation, the Carlsberg Foundation, and the National
Institute of Nutrition and Seafood Research, Norway. Part of the work was carried out as a part of the research program of the Danish Obesity Research Centre
(DanORC). DanORC is supported by the Danish Council for Strategic Research (Grant NO 2101 06 0005). The funders had no role in study design, data collection
and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: kk@bio.ku.dk (KK); lise.madsen@nifes.no (LM)
¤ Current address: Department of Food Safety and Nutrition, Division of Environmental Medicine, Norwegian Institute of Public Health, Oslo, Norway
Introduction
Today it is recognized that the potentially harmful effects of
high fat diets relates to not only the amount, but also the type of
dietary fatty acids. Whereas a high intake of saturated and trans
fatty acids has been shown to be associated with increased risk of
cardiovascular diseases in several studies, intake of polyunsaturated
fatty acids (PUFAs) has been associated with lower cardiovascular
risk [1,2]. Thus, increasing the relative amount of PUFAs,
both vegetable n-6 PUFAs and marine n-3 PUFA, at the expense
of saturated fat is recommended. It is important to note, however,
that more than 85% of the total dietary PUFA intake in Western
diets today is vegetable n-6 PUFAs, mainly linoleic acid [3]. This is
largely due to the high amount of linoleic acid in corn-, sunflower-,
and soybean-oil used in both home-cooking and in industrially
prepared food [4]. Moreover, animal feeds are enriched with n-6
PUFAs, and although meat production methods are diverse, meat
fatty acid profiles will always reflect that of the animal feed [4].
Thus, the dietary n-3:n-6 PUFA ratio has decreased [3,4]. Although
the exchange of saturated fat with vegetable n-6 PUFAs
may have some beneficial effects on human health, a low n-3:n-6
PUFAs ratio is associated with a high risk of several lipid-related
disorders [2,3]. A high intake of n-6 PUFAs has also been associated
with childhood obesity, [4,5]. Animal studies have shown
that feeding mice a diet containing the n-6 PUFA, linoleic acid,
during the pregnancy-lactation period leads to obesity in the
offspring [6]. This effect, however, is prevented by inclusion of the
n-3 PUFA a-linolenic acid in the diet [6]. These findings are in
line with several studies demonstrating that dietary n-3 PUFAs are
able to limit the development of diet-induced obesity [7–13].
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Fish Oil, Sucrose and Obesity
Obesity may be considered as a state of chronic low-grade
inflammation [14,15]. Accumulated evidence strongly suggests
that low grade chronic inflammation plays a crucial role in development
of obesity related insulin resistance [16]. Furthermore,
it should be noted that continuous subcutaneous infusion of lipopolysaccharide
(LPS) is sufficient to induce adipose tissue inflammation,
insulin resistance and obesity in mice [17]. It is also well
documented that n-3 PUFAs are able to limit high fat diet-induced
inflammation in adipose tissue in rodents [18–20]. Both n-3
PUFAs and n-6 PUFAs are substrats for cyclo- and lipoxygenases
and n-3 PUFAs are traditionally assumed to act anti-innflammatory
by competitive inhibition of the biosynthesis of arachidonic
acid-derived pro-inflammatory prostaglandins of the 2-series and
furthermore, n-3 PUFA-derived prostaglandins of the 3-series
are believed to be less inflammatory [21,22]. Recent research
furthermore demonstrate that n-3 PUFAs may be converted to
anti-inflammatory cyclooxygenase-2 derived electrophilic oxoderivatives
and resolvins [21,22]. Moreover, by activation of the fatty
acid receptor, GPR120, n-3 PUFAs repress LPS- and TNFamediated
inflammatory signalling responses, and thereby increase
insulin sensitivity by repressing macrophage-induced adipose tissue
inflammation [23]. Thus, consumption of n-6 PUFAs at the
expense of n-3 PUFAs may aggravate the metabolic consequences
of obesity. Increasing the dietary intake of n-3 PUFAs is therefore
currently recommended by several health authorities.
In order to curb the increasing obesity problem, nutritionists
and authorities have largely focused on reducing fat intake, as
dietary fat contains more energy per gram than proteins and carbohydrates.
As an alternative to low energy diets, low carbohydrate
diets are becoming increasingly popular although still controversial.
The mechanisms by which such diets induce weigh loss
are still not fully elucidated, but it has been documented that high
protein diets increase energy expenditure in part due to a thermic
effect [24]. We have previously shown that the protein:sucrose
ratio in the background diet determines the adipogenic potential
of dietary n-6 PUFAs in mice [25]. Mice fed n-6 PUFAs in
combination with sucrose became obese, and had a markedly
higher feed efficiency than mice pair-fed n-6 PUFAs in combination
with proteins [25]. In fact, the high-protein fed mice needed
almost 7 times more energy to achieve a weight gain of 1 g than
mice on the high-sucrose diet [25]. The high protein diet led to
an increased glucagon/insulin ratio, concomitant with elevated
cAMP-dependent signaling, induction of COX-mediated prostaglandin
synthesis and increased expression of uncoupling protein-1
(UCP1) in inguinal subcutaneous white fat [25]. In the present
paper we aimed to investigate whether this phenomenon is
restricted to n-6 PUFAs or if the effects of dietary fats, such as fish
oils, which are considered beneficial to human health, also depend
on the background diets. Furthermore, we aimed to examine
whether the background diet exerts an influence on the ability of
n-3 PUFAs to protect against glucose intolerance and adipose
tissue inflammation.
Results
Sucrose counteracts the obesity-reducing effect of fish
oil in ad libitum fed mice
It is a general notion that intake of fish oil rich in n-3 PUFAs
limits high fat diet-induced obesity in rodents, whereas diets rich in
n-6 PUFAs have been associated with an increased propensity to
develop obesity [6,26]. As we have demonstrated that the
obesogenic effect of n-6 PUFAs is determined by the content of
carbohydrates and protein in the feed [25], we speculated whether
the effect of dietary fats considered health-beneficial, such as fish
oil, might be modulated by different background diets. To answer
this question we fed C57BL/6J male mice isocaloric high fat diets
(Table 1 and 2) containing corn oil or fish oil supplemented with
either protein or sucrose or a conventional low fat diet ad libitum for
9 weeks. Contrasting the general notion that fish oil attenuates
high fat diet-induced obesity, the mice fed the fish oil in combination
with sucrose gained as much body weight as the mice fed
corn oil and sucrose (Fig. 1A and B). When combined with
sucrose, fish oil did not reduce the weights of neither epididymal
(eWAT) nor inguinal white adipose tissue (iWAT) mass compared
with corn oil (Fig. 1E and F). Moreover, morphological analyses
demonstrated that the adipocyte size was similar in the two sucrose
fed groups (Fig. 1G). Of note, weight gain in mice fed corn oil or
fish oil plus protein were indistinguishable from that of mice fed
the low fat diet (Fig. 1A). Compared with low fat fed mice, the
weights and adipocytes sizes of eWAT and iWAT in mice fed both
high fat diets in combination with protein tended to be smaller, but
the differences did not reach statistical significance (Fig. 1E, F
and G). Thus, when combined with a high intake of sucrose fish oil
did not prevent obesity. However, high dietary protein content
prevented weight gain and obesity when combined with either
corn or fish oil.
Sucrose, but not protein or fat, strongly stimulates pancreatic
insulin secretion, and accordingly, plasma levels of insulin were
consistently higher in mice fed the sucrose-based diets than in mice
fed the protein-based diets (Fig. 1C). Conversely, the levels of
plasma glucagon were lower and hence, the insulin:glucagon ratio
was about three times higher in mice fed high sucrose than in mice
fed high protein irrespective of whether the diets contained corn
oil or fish oil (Fig. 1D). Collectively, these results indicate that
intake of sucrose and hence increased insulin secretion, abrogates
the protective effects of fish oil in relation to adipocyte hyperplasia
and hypertrophy and thereby the development obesity.
Sucrose counteracts the anti-inflammatory effect of fish
oil in adipose tissue
The ability of n-3 PUFAs to limit high fat diet-induced
inflammation in adipose tissue is well documented [18–20]. As
chronic low grade inflammation in adipose tissue is a characteristic
trait of obesity [14,15] and sucrose abrogates the anti-adipogenic
effect of fish oil, we asked whether the background diet also
attenuated the ability of n-3 PUFAs to protect against adipose
tissue inflammation. Gene expression analyses of eWAT and
iWAT revealed a striking correlation between macrophage- and
inflammatory markers and the intake of sucrose-based diets irrespectively
of the fat source (Fig. 2A). Expressions of macrophage
marker genes Emr1 (EGF-like module containing, mucin-like,
hormone receptor-like sequence 1 or F4/80) and Cd68, as well as
markers of inflammation Serpine1 (Plasminogen activator inhibitor-
1) and Ccl2 (chemokine (C-C motif) ligand 2), were significantly
higher in adipose tissue from mice fed sucrose than in mice fed
high protein or a low fat diets (Fig. 2A). Moreover, we noticed
a significant increase in the expression of Pparg (peroxisome
proliferator-activated receptor c) in eWAT in the mice fed protein
supplemented with corn oil compared with mice fed protein
supplemented with fish oil (Fig. 2A).
A high fat diet impairs glucose tolerance independent of
macronutrient composition and obesity
As adipose tissue inflammation is causally linked to development
of insulin resistance and glucose intolerance, we subjected mice fed
the different diets for 9 weeks to an intraperitoneal glucose
tolerance test (GTT). Surprisingly, the GTT demonstrated that
PLoS ONE | www.plosone.org 2 June 2011 | Volume 6 | Issue 6 | e21647

Fish Oil, Sucrose and Obesity
Table 1. Macronutrient composition in the diets.
High fish oil
High corn oil
Low energy Sucrose Protein Sucrose Protein
Protein (g/kg) 200 200 540 200 540
Casein 200 200 540 200 540
L-Cysteine 3 3 3 3 3
Carbohydrate (g/kg) 619.5 439.5 99.5 439.5 99.5
Corn starch 529.5 9.5 9.5 9.5 9.5
Sucrose 90 430 90 430 90
Fat (g/kg) 70 250 250 250 250
Soybean oil 70 70 70 70 70
Corn oil - - - 180 180
Fish oil - 180 180 - -
doi:10.1371/journal.pone.0021647.t001
the glucose tolerance was impaired both in the mice fed the
protein-based diets and in the mice fed the sucrose-based diets
(Fig. 2B). Evidently, impaired glucose tolerance was dissociated
from the state of obesity, suggesting that intake of relatively high
amounts of fat reduces glucose tolerance even if weight gain and
expression of inflammatory markers were maintained at low levels.
However, fasting glucose and insulin levels were lower in mice fed
high protein than high sucrose. Thus, calculation of HOMA-IR
indicated that the mice fed proteins remained insulin sensitive,
whereas insulin sensitivity tended to be reduced in the obese mice
fed sucrose even though the difference between sucrose and
protein fed mice did not reach statistical significance (Fig. 2B).
Sucrose does not reduce the ability of fish oil to prevent
diet-induced accumulation of fat in the liver
As the anti-inflammatory effect of n-3 PUFAs in adipose tissue is
well documented, we investigated if the high level of dietary
sucrose reduced uptake of n-3 PUFAs. Thus, GC-MS analyses
were performed to determine the fatty acid composition in red
blood cells, liver and adipose tissues. These analyses demonstrated
the expected enrichment of n-3 PUFA in lipids in red blood cells
and liver (Table 3 and 4). In adipose tissue, the enrichment of n-3
was actually higher in mice fed the sucrose-based fish oil diet than
in the protein-based fish oil diet group (Table 4). However,
inclusion of sucrose in the diet did not reduce the ability of fish oil
Table 2. Fatty acid composition in the diets.
High fish oil
High corn oil
Low energy Sucrose Protein Sucrose Protein
SFA (mg/g) 9.6 53.3 50.3 31.4 31.4
MUFA (mg/g) 17.0 54.3 54.0 65.9 65.9
PUFA (mg/g) 36.2 100.6 99.5 120.8 121.2
n-6 (mg/g) 32.7 40.6 38.9 117.3 117.8
n-3 (mg/g) 3.5 60.0 60.6 3.5 3.4
n-3/n-6 0.11 1.48 1.56 0.03 0.03
Abbreviations: SFA, saturated fatty acids; MUFA, monounsaturated fatty acids;
PUFA, polyunsaturated fatty acids.
doi:10.1371/journal.pone.0021647.t002
to prevent accumulation of fat in the liver (Fig. 3A). When sucrose
was included in the diet, lipid accumulation in livers from fish oil
fed mice was significantly lower than in livers from mice fed corn
oil (Fig. 3A). Moreover, expressions of lipogenic genes seem to be
determined by the sucrose: protein ratio independent of fat source
(Fig. 3B). Thus, the ability of fish oil, but not corn oil, to protect
against diet-induced lipid accumulation in the liver did not seem to
be directly related to the suppression of lipogenic gene expression.
Other hallmarks of n-3 PUFA actions are their ability to
increase fatty acid oxidation and to reduce plasma triacylglycerol
levels [27,28]. Plasma triacylglycerol levels were significantly
reduced in mice fed fish oil in combination with proteins, but
inclusion of sucrose abrogated this effect (Fig. 3C). The higher
plasma levels of b-hydroxybutyrate in mice fed fish oil in
combination with proteins indicated that hepatic fatty acid
oxidation was increased in these mice, and inclusion of sucrose
attenuated this effect (Fig. 3C). Together these results demonstrate
that the protein:sucrose ratio also affects the ability of fish oil to
reduce plasma levels of triacylglycerol and increase fatty acid
oxidation.
Increasing the protein:sucrose ratio in a high fat diet
decreases energy efficiency irrespective of corn or fish oil
supplementation
To verify that the obesity in mice fed fish oil in combination
with sucrose was simply not due to increased energy-intake, feed
intake was recorded and energy efficiency calculated. Obviously,
energy intake was significantly higher in mice fed high fat diets
than that of mice receiving the low fat diet (Fig. 3D). Energy intake
tended to be higher in mice receiving the sucrose diets than in
mice fed the protein-based diets, but this was not statistically
significant (Fig. 3D). Thus, energy efficiency was dramatically
increased in mice receiving sucrose compared to protein, indicating
difference in energy expenditure. A simple way to detect
differences in catabolic rate is to subject mice to fasting and
measure the resulting weight loss. Figure 3D shows that mice on
the protein-based diets lost significantly more weight during 18 h
of fasting. This supports the notion that energy expenditure is
higher in mice on a protein-based diet irrespective of whether the
diet is supplemented with corn oil or fish oil.
Expression and activation of UCP1 in brown and white adipose
tissue lead to dissipation of energy in the form of heat, and may
thus protect against diet induced obesity [29]. Gene expression
analyses of adipose tissues demonstrated that expression of Ucp1
(uncoupling protein-1), in mice fed high protein was higher in
iWAT, but not in eWAT or (iBAT) (Fig. 3E). Increased expression
of Ucp1 in iWAT in mice fed the protein-based diets was
accompanied by increased expression of Cpt1b (carnitine palmitoyltransferase-1b),
Ppargc1a (peroxisome proliferator-activated
receptor gamma coactivator 1 alpha) and Dio2 (deiodinase, iodothyronine,
type II), suggesting that iWAT adopted a more brown-like
phenotype (Fig. 3E). Thus, the lean phenotype in mice fed the high
protein diets, appears, at least in part, to result from increased
uncoupled respiration in iWAT.
The obesogenic effect of fish oil is determined by the
macronutrient composition in pair-fed mice
Since energy intake was slightly higher in mice receiving fish oil
in combination with sucrose compared with protein, we decided to
demonstrate directly that this difference was insufficient to account
for the increased adipose tissue mass. Accordingly, mice were pairfed
the isocaloric diets containing fish oil in combination with
sucrose or protein. To achieve identical energy intake we recorded
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Fish Oil, Sucrose and Obesity
Figure 1. Sucrose counteracts the obesity-reducing effect of fish oil in ad libitum fed mice. Male C57BL/6 mice (n = 8) were fed isocaloric
high fish oil or high corn oil diets with different carbohydrate and protein contents ad libitum for 9 weeks. A: Body weight development of ad libitium
fed mice. (B) Prior to termination the mice were photographed. C–D: Insulin and glucagon levels were measured in plasma in the fed state. E–G: The
weights of epididymal and inguinal white adipose tissues were recorded and sections were stained with hematoxylin and eosin. Data are presented
as means 6 SEM. Different small letters denote significant differences between the groups (P,0.05).
doi:10.1371/journal.pone.0021647.g001
the ad libitum feed intake of mice receiving the protein-based diet,
and restricted the amount of feed to mice receiving the sucrosebased
feed accordingly. Figure 4A demonstrates that even under
conditions of pair-feeding, the mice receiving high sucrose gained
dramatically more weight than those receiving a high protein diet.
Similarly, as observed in ad libitum fed mice, energy efficiency and
adipose tissue mass were significantly higher when mice were fed
sucrose (Fig. 4B and C). Moreover, energy content in the feces was
similar in both groups and the apparent digestibility was not
increased by increased sucrose amount in the diet (Fig. 4B). Plasma
levels of insulin were higher and glucagon lower in mice fed the
sucrose than protein (Fig. 4D). In iWAT, but not eWAT, we
observed a significant induction of Ppargc1a and Ucp1 expression
indicative of the transformation of iWAT into a more brown-like
depot in protein fed mice (Fig. 4E). In iBAT, expressions of Ucp1
and cyt COXII, (cytochrome c oxidase, subunit II) a marker of
mitochondrial content, were not significantly different in mice fed
protein or sucrose (Fig. 4F).
Lower expression levels of inflammatory markers in eWAT and
iWAT in protein fed mice were also confirmed (Fig. 4E). In
addition, as observed in ad libitum fed mice, glucose tolerance was
similarly affected in protein and sucrose fed mice (Fig. 5A). Fasting
levels of insulin were higher in sucrose+fish oil fed mice than the
two other groups, but an ITT test showed no significant difference
between the groups (Supp Fig. 1). Plasma levels of triacylglycerol
were lower and b-hydroxybutyrate were higher in plasma from
mice fed fish oil in combination with protein than in mice fed fish
oil in combination with sucrose (Fig. 5B). However, expression of
genes involved in fatty acid oxidation was not increased in liver
(Fig. 6B) or in muscle (not shown). Actually, expression of the
classical PPARa target Acox1 (acyl-CoA oxidase 1) was higher in
liver of sucrose fed mice (Fig. 6B). Thus, a possible increase in fatty
acid oxidation in protein fed mice as indicated by the elevated
levels of b-hydroxybutyrate did not appear to be due to increased
expression of genes involved in fatty acid oxidation. Of note,
however, higher expressions of Srebf1 (sterol regulatory element
binding transcription factor 1) as well as Acaca (acetyl-Coenzyme A
carboxylase alpha) and Fasn (fatty acid synthase), indicate that
sucrose overrides the suppressive effect of fish oil on lipogenic gene
expression.
Energy expenditure is reduced in mice fed fish oil in
combination with sucrose
Mice fed fish oil in combination with sucrose exhibited
increased weight gain and an increased feed efficiency indicative
of decreased energy expenditure. Moreover, mice fed fish oil in
combination with sucrose lost significantly less weight during 18 h
of fasting (Fig. 3C). Therefore, we examined whether energy
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Fish Oil, Sucrose and Obesity
Figure 2. A high fat diet impairs glucose tolerance independent of macronutrient composition and obesity. A: Expressions of
adipogenic and inflammatory marker genes (Pparg (peroxisome proliferator activated receptor c), Adipoq (adiponectin), Serpine1 (Plasminogen
activator inhibitor-1), Ccl2 (chemokine (C-C motif) ligand 2), Emr1 (EGF-like module containing, mucin-like, hormone receptor-like sequence 1 or F4/
80) and Cd68 (CD68 antigen)) were measured in epididymal and inguinal white adipose tissue using RT-qPCR (n = 8). B: Intraperitoneal glucose
tolerance test was performed in a separate set of mice (n = 10). Fasting glucose and insulin levels were measured to calculate HOMA-IR. Data are
presented as means 6 SEM. Different small letters denote significant differences between the groups, in 2A within the same tissue (P,0.05).
doi:10.1371/journal.pone.0021647.g002
expenditure was reduced when fish oil was combined with sucrose.
Accordingly, O 2 consumption and CO 2 production were measured
by indirect calorimetry. Figure 5C shows that O 2 consumption
both in the light and the dark periods tended to be lower
in mice fed fish oil in combination with sucrose than with protein.
As expected, mice fed fish oil in combination with sucrose had a
higher CO 2 production resulting in a statistically significant higher
RER of about 0.9 indicating a lower rate of fatty acid oxidation
(Fig. 5C).
A diet enriched with fish oil and proteins increases
gluconeogenesis
High circulating levels of insulin combined with a low level of
glucagon translate into reduced cAMP signalling in the liver.
Thus, the observed reduced expressions of Crem (cAMP responsive
element modulator), Pde4c (phosphodiesterase 4C, cAMP specific),
Ppargc1a and Pck1 (phosphoenolpyruvate carboxykinase 1, cytosolic)
as well as reduced expressions of enzymes involved in amino
acid degradation in the liver of sucrose fed mice were anticipated
(Fig. 4D). In the liver PGC1a is induced in response to elevated
levels of cAMP and plays a central role in the control of hepatic
gluconeogenesis [30–32]. In keeping with the increased expressions
of Ppargc1a and Pck1 in liver from mice fed fish oil in
combination with protein compared to sucrose, we anticipated
that gluconeogenesis was induced in the fed state in the protein fed
mice. To measure gluconeogenesis in vivo mice fed fish oil in
combination with either protein or sucrose were intraperitoneally
injected with pyruvate both after overnight fasting and in the fed
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Fish Oil, Sucrose and Obesity
Table 3. Fatty acid composition in red blood cells.
High fish oil
High corn oil
Low energy Sucrose Protein Sucrose Protein
Sum total (mg/g) 3.41±0.04 3.04±0.07 2.87±0.05 3.52±0.11 3.34±0.10
SFA (mg/g) 1.28±0.02 1.24±0.03 1.17±0.02 1.31±0.05 1.21±0.04
MUFA (mg/g) 0.49±0.01 0.34±0.01 0.35±0.01 0.41±0.01 0.40±0.01
PUFA (mg/g) 1.39±0.02 1.28±0.03 1.19±0.02 1.53±0.05 1.44±0.04
n-3 (mg/g) 0.2560.00 0.7960.02 0.7360.01 0.2160.01 0.1160.00
n-6 (mg/g) 1.1460.02 0.4960.01 0.4760.01 1.3260.05 1.3460.04
n-3/n-6 0.2260.00 1.6260.01 1.5560.28 0.1660.00 0.0860.00
Data are presented as mean 6 SEM (n = 8).
doi:10.1371/journal.pone.0021647.t003
Table 4. Fatty acid composition in organs.
High fish oil
High corn oil
Low energy Sucrose Protein Sucrose Protein
Liver
Sum total 45±4 41±1 35±2 71±10 42±3
(mg/g)
% 100 100 100 100 100
SFA (mg/g) 15±1 15±0 12±1 21±3 13±1
% 32.560.9 36.560.4 35.260.3 29.660.3 31.360.8
MUFA (mg/g) 13±2 6±1 4±0 19±4 7±1
% 29.161.6 14.962.3 11.960.7 25.661.9 17.261.3
PUFA (mg/g) 16±1 19±1 18±1 29±3 20±1
% 36.761.6 47.262.0 51.260.4 42.761.9 47.860.9
n-3 (mg/g) 3.2460.27 12.9460.51 10.8360.72 3.2860.15 1.7260.11
% 7.2560.45 31.4161.25 31.3460.35 4.9760.53 4.1060.28
n-6 (mg/g) 1361 660 760 2663 1861
% 29.461.2 15.860.8 19.960.5 37.761.4 43.760.7
n-3/n-6 0.2560.01 2.0060.03 1.5860.05 0.1360.01 0.0960.01
eWAT
Sum total 871±14 841±17 761±78 840±27 747±43
(mg/g)
% 100 100 100 100 100
SFA (mg/g) 198±6 250±8 208±23 155±8 120±7
% 22.760.3 29.760.5 27.360.6 18.460.6 16.160.2
MUFA (mg/g) 372±9 269±6 268±26 305±7 279±12
% 42.760.4 32.160.7 35.461.1 36.460.6 37.560.8
PUFA (mg/g) 295±2 308±8 271±29 375±14 341±24
% 33.960.6 36.660.5 35.560.7 44.560.4 45.460.7
n-3 (mg/g) 18.1660.47 114.8966.64 87.07613.22 8.6860.32 6.2560.54
% 2.0860.04 13.6360.59 11.2060.89 1.0360.02 0.8360.04
n-6 (mg/g) 27762 19063 181618 366614 322622
% 31.860.6 22.660.5 23.960.6 43.560.4 44.660.7
n-3/n-6 0.0760.00 0.6060.03 0.4760.05 0.0260.00 0.0260.00
Data are presented as mean 6 SEM (n = 8).
doi:10.1371/journal.pone.0021647.t004
state, and blood glucose was measured in the following 60 minutes.
In the fasted state, mice fed sucrose or protein exhibited similar
excursions, indicating similar rates of gluconeogenesis (Fig. 6A). In
fed mice, however, the rise of blood glucose following the injection
of pyruvate was dramatically faster and reached much higher levels
after 15 and 30 min in the protein fed mice than in chow fed mice
(Fig. 6A). Compared with chow fed mice the rise in blood glucose
was also increased in mice fed fish oil in combination with sucrose,
but this was not statistically significant (Fig. 6A). The decline in
blood glucose in chow fed mice remains to be explained, but this
was observed consistently. Taken together these results strongly
support the assumption that gluconeogenesis is markedly induced in
mice fed the protein-based diet.
Discussion
It is well documented that inclusion of n-3 PUFAs in high fat
diets leads to reduced development of diet-induced obesity in
rodents [7–9,11–13,33]. Unfortunately, not all studies where the
anti-obesogenic effects of fish oils are studied provide a detailed
description of the macronutrient composition. However, in
standard commercial available high fat- and very high fat diets,
starch is the most abounded carbohydrate source and the amount
of sucrose is low or absent. Here we show that a high amount of
sucrose in the diet counteracts the obesity-reducing effect of fish oil
as well as the well described anti-inflammatory effect in adipose
tissue [19,23,34,35]. Irrespective of the fatty acid source, mice fed
high protein diets remained lean whereas mice fed diets enriched
in sucrose became obese and had higher expressions of inflammatory
markers in adipose tissue. Collectively, our results demonstrate
that a high intake of sucrose abrogates the protective effects
of fish oil in development of obesity.
As dietary sucrose, but not protein or fat, stimulates secretion of
insulin from pancreatic b-cells, an increased dietary sucrose:protein
ratio will translate into an increased insulin:glucagon ratio in
the fed state. In this respect the observed higher insulin:glucagon
ratio in mice fed the sucrose-based diets than in mice fed the
protein-based diets was expected. Increased levels of insulin in fed
mice were observed irrespectively of the type of fat in the diet.
Insulin is a powerful anabolic hormone that stimulates adipocyte
differentiation and adipose tissue expansion [36]. Activation of
insulin signaling is crucial for the development of obesity [37] and
insulin receptor substrate-1 (IRS-1) transgenic mice are obese [38].
Increased insulin signaling and glucose uptake in adipose tissue
in the fed state in sucrose fed mice may thus override the
protective effect of fish oil when it comes to protection against
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Fish Oil, Sucrose and Obesity
Figure 3. Fish oil prevents diet-induced accumulation of fat in the liver. A: Total lipids were extracted from liver and separated using HPTLC.
B: Expressions of lipogenic genes (Srebf1 (sterol regulatory element binding transcription factor 1) and Acaca (acetyl-Coenzyme A carboxylase alpha)
were measured by RT-qPCR. C: Plasma triacylglycerol and b-hydroxybutyrate were measured in the fed state. D: Energy efficiency was calculated
based on energy intake and body weight gain. E: Expression levels of brown adipose tissue marker genes (Ucp1 (Uncoupling protein-1), Ppargc1a
(peroxisome proliferator-activated receptor gamma, coactivator 1 alpha), Cpt1b (carnitine palmitoyltransferase-1b) and Dio2 (deiodinase,
iodothyronine, type II) were measured in white adipose tissues using RT-qPCR. Data are presented as means 6 SEM (n = 8). Different small letters
denote significant differences between the groups, in 3E within the same tissue (P,0.05).
doi:10.1371/journal.pone.0021647.g003
obesity-development. It should also be mentioned that although
several studies have demonstrated a protective effect of fish oil in
obesity-development, it has been reported that inclusion of fish oil
increased the amount of adipose tissue mass in hyperinsulinemic
ob/ob mice [19].
Differences in the insulin:glucagon ratio and hence differences
in cAMP-dependent signaling may at least in part orchestrate the
observed differences in energy homeostasis between the sucroseand
protein-based diets regardless of whether these diet are
supplemented with corn oil or fish oil. In the liver, Ppargc1a is
induced in response to elevated levels of cAMP and plays a central
role in the control of hepatic gluconeogenesis [30–32]. High
circulating levels of insulin combined with a low level of glucagon
translate into reduced cAMP signalling in the liver. Thus, the
observed increased gluconeogenesis in the fed state in protein fed
mice may result from cAMP-mediated stimulation of Ppargc1a and
Pck-1 expression. Increased gluconeogenesis in the fed state may
contribute to the observed lower energy efficiency in protein fed
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Fish Oil, Sucrose and Obesity
Figure 4. Sucrose counteracts the obesity-reducing effect of fish oil in pair-fed mice. Male C57BL/6 mice (n = 8) were pair-fed isocaloric high
fish oil diets with different carbohydrate and protein contents for 8 weeks. A: Body weight development was followed throughout the feeding
regime. B: Energy efficiency was calculated based on energy intake, weight gain and apparent digestibility. C: The weights of different adipose tissue
depots were recorded. D: Insulin and glucagon levels were measured in plasma in the fed state. E: Inflammation and adipocyte marker genes (Pparg
(peroxisome proliferator-activated receptor c), Adipoq (adiponectin), Serpine1 (Plasminogen activator inhibitor-1), Ccl2 (chemokine (C-C motif) ligand
2), Emr1 (EGF-like module containing, mucin-like, hormone receptor-like sequence 1 or F4/80) and Cd68 (CD68 antigen) and F: thermogenesis-related
genes (Ucp1 (Uncoupling protein-1) and cyt COXII, (cytochrome c oxidase, subunit II) were measured by RT-qPCR in adipose tissues. Data are
presented as means 6 SEM. Different small letters denote significant differences between the groups, in 4E within the same tissue (P,0.05).
doi:10.1371/journal.pone.0021647.g004
mice, as 6 ATP molecules are consumed per molecule of glucose
synthesized from pyruvate, rendering gluconeogenesis an energyconsuming
process. Moreover, concomitant increased expressions
of Gpt, Got1, Agxt and Cps1 suggest that energy consuming
processes such as amino acid degradation and ureagenesis are
higher in protein than sucrose fed mice. As mammals have no
direct storage capacity for protein it needs to be metabolically
processed immediately. The high cost of urea production and
gluconeogenesis is actually often cited reasons for the higher
thermic effect of protein than other macronutrients [39,40] and
this may partly explain why diets higher in protein exert a larger
effect on energy expenditure than diets lower in protein [24].
A second mechanism by which a low sucrose:protein ratio in the
diet leads to reduced energy efficiency may be related to the observed
expression of Ucp1 in iWAT. Increased cAMP-signaling is known to
induce adaptive thermogenesis by induction of Ppargc1a and Ucp1
expression and it is well known that the UCP1 protein allows
dissipation of energy in the form of heat [41]. Of note, acute or
chronic upregulation of fatty acid oxidation alone, that is increased
fatty acid oxidation without a concomitant uncoupling of mitochondria,
has no net effect on whole-body energy expenditure or adiposity
[42]. Although Ucp1 expression was unchanged in iBAT, whole body
energy homeostasis may be influenced by increased expression in
iWAT. In fact, increased occurrence of brown-like adipocytes within
WAT depots is a feature of mouse strains resistant to dietary obesity,
such as the A/J strain [43] and reduced adiposity associated with aP2-
transgenic expression of Ucp1 is linked to increased energy dissipation
in white, but not interscapular brown, adipose tissue [29].
Conversely, inhibition of diet-induced expression of Ucp1 in iWAT
in Sv129 mice by administration of a general cyclooxygenase
inhibitor accentuates obesity-development [44].
Our finding that inclusion of sucrose abolishes the anti-obesity
effect of fish oil seems to contradict a recent study from Sato et al.
[45], as these authors demonstrated that inclusion of 5% the n-3
PUFA EPA (eicosapentaenoic acid) into a high fat-high sucrose
diet reduced body weight gain in mice. The reason for this
discrepancy is not clear, but different dietary compositions as well
as doses and type of n-3 PUFAs may account for the different
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Fish Oil, Sucrose and Obesity
Figure 5. Metabolic parameters in mice fed fish oil in combination with sucrose or protein. A. Intraperitoneal glucose tolerance test was
performed in mice pair fed fish oil enriched diets (n = 10). B. b-hydroxybutyrate, triacylglycerol, glycerol and free fatty acids were measured in pair-fed
mice in both fasted and fed state (n = 10). C. Oxygen consumption, carbon dioxide and respiratory exchange ratio were measured during a 24-h
period with indirect calorimetry (n = 8). Data are presented as means 6 SEM. Different small letters denote significant differences between the
groups, in 5B between fasted or fed state (P,0.05).
doi:10.1371/journal.pone.0021647.g005
results obtained. The amount of n-3 PUFAs used in this study is
slightly higher (6% n-3 fatty acids) than the 5% EPA used by Sato
et al.. However, whereas Sato et al. used EPA, the n-3 PUFAs used
in our study comprise a mixture (thereof 3263 g/kg and 1863 g/
kg EPA and DHA (docosahexaenoic acid), respectively). Moreover,
in fish oil as used in our study, the n-3 PUFAs are present in
the form of triacylglycerols, whereas Sato et al. used purified EPA
ethyl ester. It should also be mentioned that the main fat source in
the diets used in our study is corn-oil rich in n-6 fatty acids,
whereas Sato et al. used anhydrate milk fat containing more than
60% saturated fat. Last, the amount of sucrose used in our study is
higher than the dose used by Sato et al. It is worth noting, however,
that both the study by Sato et al. and our study demonstrated
that sucrose did not reduce the ability of fish oil and/or EPA to
prevent diet induced accumulation of fat in the liver.
A strong association between obesity and adipose tissue inflammation
exists and obesity is characterized by chronic low-grade
inflammation in adipose tissues [14,15]. In light of this it may not
be surprising that expression of macrophage and inflammatory
marker genes was elevated in obese mice compared to lean mice.
Still, as the anti-inflammatory effect of fish oil in adipose tissue is
well described [19,23,34,35], it was unexpected that the expression
of inflammatory markers was similar in adipose tissue from obese
corn oil and the fish oil fed groups. In our study the state of obesity
rather than the n-3:n-6 PUFA ratio in both feed and adipose
tissues appeared to determine the expression levels of inflammatory
markers in adipose tissue.
Chronic low grade inflammation plays an important role in
development of insulin resistance [46,47]. Pioneering work by
Storlien et al. [48], later confirmed by several others [13,23,35,
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Fish Oil, Sucrose and Obesity
Figure 6. Gluconeogenesis is increased in fed state when animals are fed fish oil supplemented with protein. A. Pyruvate tolerance
tests were performed on mice in 16 h fasted (n = 7) and fed states (n = 10). B. Hepatic gene expression (Crem (cAMP responsive element modulator),
Pde4c (phosphodiesterase 4C, cAMP specific), Ppargc1a (peroxisome proliferator-activated receptor gamma, coactivator 1 alpha), Pck1
(phosphoenolpyruvate carboxykinase 1, cytosolic), Gpt (glutamic pyruvic transaminase), Got1 (glutamate oxaloacetate transaminase 1), Agxt
(alanine-glyoxylate aminotransferase), Cps1 (carbamoyl-phosphate synthetase 1), Acox1 (acyl-CoA oxidase 1), Cpt1a (carnitine palmitoyltransferase
1a), Cpt2 (carnitine palmitoyltransferase 2), Hmgcs2 (3-hydroxy-3-methylglutaryl-Coenzyme A synthase 2), Srebf1 (sterol regulatory element binding
transcription factor 1), scd1 (stearoyl-Coenzyme A desaturase 1), Acaca (acetyl-Coenzyme A carboxylase alpha), Fasn (fatty acid synthase), was
measured using RT-qPCR (n = 8). Data are presented as means 6 SEM. Different small letters denote significant differences between the different
groups (P,0.05).
doi:10.1371/journal.pone.0021647.g006
49,50] has demonstrated that n-3 PUFAs can prevent development
of diet-induced insulin resistance in rodents. The insulin
sensitizing effect of n-3 PUFAs is generally accepted to be related
to the anti-inflammatory effect, recently demonstrated to be mediated
by the GPR120 receptor [23]. Calculation of HOMA-IR
indicated that mice fed high levels of proteins were more
insulin sensitive than mice fed sucrose, but no significant differences
was observed in an ITT. Similar to expression levels of
inflammatory markers in adipose tissue, this was irrespective of
whether the diets were supplemented with corn oil or fish oil. It
was therefore unexpected that the GTT demonstrated that mice
fed corn oil or fish oil in combination with sucrose or protein
exhibited impaired glucose tolerance irrespective of whether or not
the mice remained lean. It is possible that different mechanisms
underlay the impaired glucose tolerance observed in the sucrose
and the protein fed mice. It is likely that impaired glucose
tolerance in sucrose fed mice is related to the obese state. Of note,
in fish oil and protein fed mice, glucose tolerance was impaired
even if weight gain and inflammation were maintained at low
levels. Further studies are required to elucidate the mechanisms
underlying the impaired glucose tolerance in these mice, but the
possibility that adaption to a low carbohydrate intake with concomitant
high hepatic gluconeogenesis and glucose output should
be considered.
Seen as a whole, our study indicate that the sucrose:protein
ratio, rather than the n-6:n-3 PUFA ratio in the diet determines
development of obesity, adipose tissue inflammation and glucose
intolerance. Activation of the NF-kB system appears to represent a
link between obesity, inflammation of adipose tissue and insulin
resistance [51–53]. Insulin is able to activate NF-kB by phosporylation
of IkBa in different cell systems [54], thus, high levels of
circulating insulin may activate the NF-kB system also in adipose
tissue. Whether increased insulin levels in sucrose fed mice
translated into activation of the NF-kB system in adipose tissue in
these mice will require further investigation.
Together our results demonstrate that the background diet
exerts a crucial influence on the ability of n-3 PUFAs to protect
against development of obesity, glucose intolerance and adipose
tissue inflammation. High levels of dietary sucrose counteract the
anti-inflammatory effect of fish oil in adipose tissue and promote
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Fish Oil, Sucrose and Obesity
obesity development in mice. As the intake of sucrose in Western
societies is high and increasing dietary intake of n-3 PUFAs is
recommended by several health authorities it would be of importance
to investigate whether the background diet influences the
effect of fish oil also in humans.
Materials and Methods
Ethics Statement
All animal experiments were approved by National Animal
Health Authorities (Norwegain approval identification: 1840 and
1841). Care and handling were in accordance with local institutional
recommendations and rules. Adverse events were not
observed.
Animals and diets
Male C57BL/6JBomTac mice approximately 8 weeks of age
were obtained from Taconic Europe (Ejby, Denmark) and were
divided into groups (n = 6–10). The mice were kept at a 12 h light/
dark cycle at 28uC. After acclimatization the animals were fed ad
libitum or pair-fed experimental diets obtained from Ssniff
Spezialdiäten GmbH (Soest, Germany) described in Table 1 and
2 for 8–10 weeks. All diets were supplemented with 3 g/kg L-
cysteine, 10 g/kg choline bitartrate, 10 g/kg Vitamin mix AIN 76
A, 45 g/kg Mineral mix AIN 93 and 0.014 g/kg t-butylhydroquinone.
Mice were euthanized by cardiac puncture under
anesthesia with Isoflurane (Isoba-vet, Schering-Plough, Denmark)
using the Univentor 400 Anaesthesia Unit (Univentor Limited,
Sweeden) in the fed state and plasma prepared from blood. Tissues
were dissected out, freeze-clamped and frozen at 280uC.
Indirect calorimetry
The metabolic rate of mice was measured by indirect calorimetry
in open circuit chambers of Labmaster system (TSE Systems
GmbH, Germany). The animals were acclimated in the chambers
for 24 hours and measured continuously for another 24 hours.
Glucose, insulin and pyruvate tolerance testing (GTT, ITT
and PTT)
GTT: mice were fasted 6 hours before intraperitoneal injection
of 2 g/kg glucose in saline. ITT: mice were fasted 4 hours before
i.p. injection of 0.5 Unit/kg human recombinant insulin in saline.
PTT: mice in the fed state or mice that were fasted overnight were
injected i.p. with of 2 g/kg pyruvate in saline. Blood was collected
from the lateral tail vein at indicated time points and measured
with Bayer Contour glucometer (Bayer A/S, Denmark).
Plasma analyses
Insulin, glucagon and glucose [25] and lipid metabolites [55]
were measured in plasma as earlier described.
Lipid analyses
Total lipids were extracted from diets, red blood cells, liver and
adipose tissue samples with chloroform: methanol, 2:1 (v/v). Lipid
classes were analyzed using an automated High-Performance Thin
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3. Simopoulos AP (2002) The importance of the ratio of omega-6/omega-3
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Layer Chromatography (HPTLC) system (Camaq, Switzerland)
and separated on HPTLC plates coated with silica gel 60 F [55]
whereas fatty acid composition of total lipids was analyzed on a
capillary gas chromatograph with flame ionization detector (Perkin
Elmer, USA) [56].
Histology
Sections of adipose tissue were fixed, dehydrated, embedded in
paraffin blocks, cut into 3 mm thick section and stained with eosin
and hematoxylin as previously described [57]. Sections were visually
examined using an Olympus BX 51 binocular microscope (System
microscope, Japan), fitted with an Olympus DP50 3.0 camera.
RT-qPCR
Total RNA was purified from mouse tissue using Trizol
(Invitrogen). Reverse transcription (RT) was performed and cDNA
was analyzed in duplicates by qPCR using the ABI PRISM 7700
Sequence Detection System (Applied Biosystems) as earlier
described [58]. Primers for RT-qPCR were designed using Primer
Express 2.0 (Applied Biosystems) and are available on request.
Energy in faeces and diets
Energy content was determined in a bomb calorimeter following
the manufacturer’s instruction (Parr Instruments, Moline,
IL, USA).
Statistics
Data represent mean 6 SEM. ANOVA, post hoc pairwise
comparison: Student t-test (RT-qPCR analysis) or Tukey HSD test
(GTT, ITT and organ weights). Newman-Keuls test (nonparametric
due to non-homogenous variances) rest of data. Data were
considered statistical significant when P,0.05).
Supporting Information
Figure S1 A high fish oil diet does not impair insulin tolerance.
Male C57BL/6 mice (n = 7) were fed a low fat or isocaloric high
fish oil diets with different carbohydrate and protein contents ad
libitum for 7 weeks. A: Insulin levels were measured in the fasted
state. B: Intraperitoneal insulin tolerance test was performed. Data
are presented as means 6 SEM. Different small letters denote
significant differences between the groups.
(TIF)
Acknowledgments
The authors thank Åse Heltveit and Jan Idar Hjelle at NIFES for excellent
assistance with animal care and lipid analyses.
Author Contributions
Conceived and designed the experiments: BL KK LM. Performed the
experiments: TM BL QH RKP EF HTN HHL SR SBS JTT HP LF LM.
Analyzed the data: TM BL QH RKP EF HTN HHL SR SBS JTT HP LF
KK LM. Wrote the paper: KK LM.
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PLoS ONE | www.plosone.org 12 June 2011 | Volume 6 | Issue 6 | e21647

Figure S1.
A high fish oil diet does not impair insulin tolerance. Male C57BL/6 mice (n = 7) were fed
a low fat or isocaloric high fish oil diets with different carbohydrate and protein
contents ad libitum for 7 weeks. A: Insulin levels were measured in the fasted state. B:
Intraperitoneal insulin tolerance test was performed. Data are presented as means ± SEM.
Different small letters denote significant differences between the groups.

Carbohydrate source and insulin secretion modulate the obesity promoting
effect of fish oil
Qin Hao 1, $ , Haldis Haukås Lillefosse 1, 2, $ , Even Fjære 1, 2, $ , Lene Secher Myrmel 1, 2 , Lisa Kolden
Midtbø 1, 2 , Ragnhild Jarlsby 2 , Tao Ma 1 , Bingbing Jia 1 , Rasmus Koefoed Petersen 1 , Si Brask
Sonne 1 , André Chwalibog 3 , Livar Frøyland 2 , Bjørn Liaset 2 , Karsten Kristiansen 1, * and Lise
Madsen 1, 2, * .
1 Department of Biology, University of Copenhagen, Denmark. 2 National Institute of Nutrition and
Seafood Research, Bergen, Norway. 3 Department of Basic Animal and Veterinary Sciences,
University of Copenhagen, Denmark. $ Contributed equally.
*Correspondence to: Karsten Kristiansen, Department of Biology, University of Copenhagen, Ole
Maaløes Vej 5, DK 2200 Copenhagen, Denmark. Fax +45 3522 2128; Phone: +45 3532 4443; E
mail: kk@bio.ku.dk or Lise Madsen, National Institute of Nutrition and Seafood Research, P.O.
Box 2029 Nordnes, N 5817 Bergen, Norway. Fax +47 5590 5299; Phone: +47 4147 6177; E mail:
lise.madsen@nifes.no
Number of figures: 7
Number of tables: 2
Running title: Interaction between carbohydrates and fish oil
FOOTNOTES: This work was supported by the Danish Natural Science Research Council, the
Novo Nordisk Foundation, the Carlsberg Foundation and NIFES. Part of the work was carried out
as a part of the research program of the Danish Obesity Research Centre (DanORC). DanORC is
supported by the Danish Council for Strategic Research (Grant NO 2101 06 0005). The authors
have no conflicting interests.
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ABSTRACT
Polyunsaturated n-3 fatty acids (n-3 PUFAs) are known to attenuate diet-induced obesity and
adipose tissue inflammation in rodents. In this study we aimed to investigate whether inclusion of
different carbohydrate sources modulated the effects of n-3 PUFAs. By feeding C57BL/6J mice
isocaloric high fat diets enriched with fish oil for 6 weeks, we show that increasing amounts of
sucrose in the diets dose-dependently increased energy efficiency and white adipose tissue (WAT)
mass. Mice receiving fructose had about 50% less WAT mass than mice fed a high fish oil diet
supplemented with either glucose or sucrose, indicating that the glucose moiety of sucrose was
responsible for the obesity promoting effect of sucrose. To investigate if the obesogenic effect of
sucrose and glucose was related to stimulation of insulin secretion, we combined fish oil with high
and low glycemic index (GI) starch. Mice receiving the high fish oil diet containing the low GI
starch had significantly less WAT than mice fed high GI starch. Moreover, inhibition of insulin
secretion by administration of nifedipine significantly reduced WAT mass in mice fed a high fish
oil diet in combination with sucrose. Our data show that the macronutrient composition of the diet
modulates the effects of fish oil. Fish oil combined with sucrose, glucose or high-glycemic index
starch promotes obesity and the reported anti-inflammatory actions of fish oil are abrogated.
Collectively, our data indicate that glycemic control of insulin secretion regulates the obesity
promoting effect of fish oil in combination with carbohydrates.
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Introduction
A considerable number of studies has demonstrated the beneficial effects of n-3 polyunsaturated
fatty acids (PUFAs) on lipid metabolism and lipid-related disorders in humans (1-3) and increasing
the relative amount of n-3 PUFAs is presently recommended. Moreover, diets enriched with n-3
PUFAs have been demonstrated to reduce the development of insulin resistance (4-9) and to reduce
the development of diet-induced obesity in rodents (7, 10-15). We have shown that the
macronutrient composition of the diet determines the adipogenic potential of dietary corn oil in
mice (16). Moreover, we recently demonstrated that sucrose counteracts the anti-inflammatory
effect of fish oil in adipose tissue and increases obesity development in mice (17). Mice fed a fish
oil enriched diet in combination with sucrose had markedly higher feed efficiency and required less
than 50% of the calories to achieve the same weight gain as mice pair-fed a fish oil enriched diet in
combination with protein (17).
A major difference between proteins and sucrose is the ability to cause a rise in blood
glucose and stimulate insulin secretion. Insulin is a powerful anabolic hormone that stimulates
adipocyte differentiation and adipose tissue expansion (18), and activation of insulin signaling is
crucial for the development of obesity (19). Moreover, increased insulin signaling by transgenic
expression of insulin receptor substrate-1 (IRS-1) is sufficient to induce obesity (20). Thus, it is
possible that increased insulin signaling and glucose uptake in adipose tissue in the fed state in
sucrose fed mice may override the anti-inflammatory and anti-obesity effects of fish oil.
The glucose moiety of sucrose is responsible for the rise in blood insulin upon intake
of sucrose, as fructose unlike glucose, is unable to stimulate insulin secretion (21). This in part
relates to the very low levels of Slc2a5 (solute carrier family 2 (facilitated glucose transporter),
member 5, GLUT5) in pancreatic beta-cells (22). Furthermore, fructose does not stimulate the
release of gastric inhibitory peptide which stimulates insulin secretion indirectly (23, 24). Thus, the
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ability of sucrose to counteract the beneficial effects of fish oil seems to relate to a glucosedependent
stimulation of insulin secretion. The fructose moiety of sucrose may further modulate the
effect of fish oil on the development of obesity. Thus, the increased consumption of fructose over
the past decades has been linked to development of metabolic disorders (25), and fructose is
routinely used to induce glucose intolerance in rats (26).
As different types of starch differ in their ability to increase postprandial blood
glucose and insulin secretion, different types of starch may also modulate the effect of fish oil. The
glycemic index (GI) is a measure of the ability of different types of carbohydrate based foods to
raise blood glucose levels within 2 hours (27). The interest in low GI diets as a tool in weight
management is increasing. Although reviews and meta-analyzes conclude that such diets may be
effective, their effectiveness in terms of lasting weight reduction is still a matter of debate (28-32).
Different types of starches with different GI are known to induce different responses in plasma
glucose and insulin in rodents (33). However, it is unknown whether different types of starch
modulate the effects of fish oil enriched diets. Here we have performed systematic analyses to
investigate the influence of different carbohydrate sources on the reported anti-obesity and antiinflammatory
effect of n-3 PUFAs in mice. By using isocaloric high fat diets enriched with n-3
PUFAs, we show that the amount of sucrose dose-dependently increased energy efficiency and
adiposity. Moreover, we show that nutritional and pharmacological control of insulin secretion play
a pivotal role determining the obesogenic effect of high fish oil-high carbohydrate diets.
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EXPERIMENTAL PROCEDURES
Animals and diets. Male C57BL/6JBomTac mice approximately 8 weeks of age were obtained from
Taconic Europe (Ejby, Denmark) and were divided into groups (n=6-10). The mice were kept at a
12 h light/dark cycle at thermoneutrality. After acclimation the animals were fed ad libitum or pairfed
experimental diets obtained from Ssniff (Ssniff Spezialdiäten GmbH, Soest, Germany)
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described in Table 1 and 2. The fish oil enriched diets contained 62 ± 3 g/kg n-3 fatty acids, thereof
27 ± 3 g/kg and 18 ± 3 g/kg EPA and DHA, respectively. All diets were supplemented with 3g/kg
L-cysteine, 10g/kg choline bitartrate, 10g/kg vitamin mix AIN 76 A, 45 g/kg mineral mix AIN 93
and 0.014 g/kg t-butylhydroquinone. As indicated Nifedipine (N7634, Sigma) was included in the
diet at a dose (1g/kg) earlier demonstrated to reduce plasma insulin levels in agouti mice (34). The
sulfonylurea Glybenclamide (G0639, Sigma) was used as an insulin secretagogue and was
administrated daily by intraperitoneal (i.p.) injection at a dose of 2 μg/g body weight..Control mice
received placebo. by daily i.p. injection. Mice in the fed state were euthanized by cardiac puncture
under anesthesia with isoflurane (Isoba-vet, Schering-Plough, Denmark) using the Univentor 400
Anaesthesia Unit (Univentor Limited, Sweden) and plasma was prepared from blood. Tissues were
dissected out, freeze-clamped and frozen at -80 o C. All animal experiments were approved by
National Animal Health Authorities (Denmark and Norway). Adverse events were not observed.
Energy measurements and digestibility. Male C57BL/6JBomTac mice (n=5 for each group) were
fed experimental diets ad libitum for 4 days. Faeces, urine and feed residue were collected during a
24h period before respiration measurements. The nitrogen content in feed, faeces, and urine was
determined using the Tecator-Kjeltec system 1026 (Tecator AB, Höganäs, Sweden). The gross
energy content of feed and faeces was determined using an adiabatic bomb calorimeter (System
C700, IKA Analysentechnic GmbH, Heitersheim, Germany).
Glucose and insulin tolerance testing (GTT and ITT). GTT: mice were fasted 6 hours before i.p
injection of 2 g/kg glucose in saline. ITT: mice were fasted 4 hours before i.p. injection of 0.5
Unit/kg human recombinant insulin in saline. Blood was collected from the lateral tail vein at
indicated time points and blood glucose was measured with a Bayer Contour glucometer (Bayer
A/S, Denmark).
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Plasma analysis. Insulin, glucose (16) and lipid metabolites (35) were measured in plasma as earlier
described.
Tissue lipid analysis. Total lipids were extracted from diets and liver with chloroform: methanol,
2:1 (v/v). Lipid classes were analyzed in liver samples using an automated High-Performance Thin
Layer Chromatography (HPTLC) system (Camaq, Switzerland) and separated on HPTLC plates
coated with silica gel 60 F (35). Fatty acid composition of total lipids from diets was analyzed on a
capillary gas chromatograph with flame ionization detector (Perkin Elmer, USA) (36).
Histology. Sections of adipose tissue were fixed, dehydrated, embedded in paraffin blocks, cut into
3 μm thick sections and stained with eosin and hematoxylin as described (37). Sections were
visually examined using an Olympus BX 51 binocular microscope (Olympus Corporation, Japan),
fitted with an Olympus DP50 3.0 camera.
RT-qPCR. Total RNA was purified from mouse tissue using Trizol (Invitrogen). Reverse
transcription (RT) was performed and cDNA was analyzed in duplicates by qPCR using the ABI
PRISM 7700 Sequence Detection System (Applied Biosystems) as earlier described (38). Primers
for RT-qPCR were designed using Primer Express 2.0 (Applied Biosystems) and are available on
request.
Statistics. Data represent mean ± SEM. ANOVA, post hoc pairwise comparison: Student t-test (RTqPCR
analysis) or Tukey HSD test (GTT, ITT and organ weights). Newman-Keuls test
(nonparametric due to non-homogenous variances, remaining data sets. A value of P

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To investigate whether sucrose dose-dependently counteracts the obesity protective effect of fish
oil, we fed for 6 weeks C57BL/6J mice high fat diets containing 25 w% fat, thereof 62 ± 3 g/kg n-3
fatty acids, in combination with four diets of different carbohydrate:protein ratios (Table 1). All
diets contained 25.12 kJ/g. Body weight gain and white adipose tissue (WAT) mass increased in
parallel with the increase in the sucrose:protein ratio in the feed (Fig 1A and D). The weight of
interscapular brown adipose tissue (iBAT), liver and tibialis anterior muscle were not changed (not
shown). Total energy intake was not statistically significantly different in the four groups, and thus,
energy efficiency increased dose-dependently in response to the increased amount of dietary
sucrose (Fig 1B). Moreover, expressions of inflammatory markers, such as Serpine1 (serine (or
cysteine) peptidase inhibitor, clade E, member 1 or plasminogen activator inhibitor-1, PAI-1), Ccl2
(Ccl2 chemokine (C-C motif) ligand 2, MCP1), Cd68 (CD68 antigen) and Emr1 (EGF-like module
containing, mucin-like, hormone receptor-like sequence 1, F4/80) were increased in both eWAT
and iWAT in the obese mice, suggesting that sucrose attenuated the anti-inflammatory effect of fish
oil (Fig 1E). The expressions of Pparg (Peroxisome proliferator-activated receptor gamma), Srebf1
(sterol regulatory element binding transcription factor 1) and Fasn (Fatty acid synthase) did not
change significantly (Fig 1E). Mice receiving the highest amount of sucrose also lost significantly
less body weight during a 24h fast (Fig 1C) than mice receiving the lowest amount of sucrose,
indicative of a lower metabolic rate in high-sucrose fed mice.
Energy may be lost as heat by the uncoupling activity of UCP1 (uncoupling protein 1
(mitochondrial, proton carrier)) expressed in brown adipocytes, and increased expression of Ucp1
can prevent diet-induced obesity. Therefore, we measured Ucp1 expression in both white and
brown adipose tissue depots. In iBAT Ucp1 expression was similar in all groups, but the expression
of Ucp1 was significantly reduced in iWAT in mice fed the high amount of sucrose (Fig 1C and E).
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This indicates that adipocytes in iWAT from mice receiving a low amount of sucrose and a high
amount of protein had a more brownish phenotype.
We have previously demonstrated that a high fat diet in combination with a high
protein:sucrose ratio translated into a high glucagon:insulin ratio, increased cAMP-signaling and
expression of Ppargc1a (peroxisome proliferative activated receptor, gamma, coactivator 1 alpha)
leading to increased gluconeogenesis and amino acid degradation in liver (16, 17). Here we show
that sucrose dose-dependently reduced expression of Ppargc1a, Pck1 (phosphoenolpyruvate
carboxykinase 1, cytosolic) and Agxt (alanine-glyoxylate aminotransferase) in the liver (Fig 1F).
Expression of the lipogenic gene, Fasn, was increased whereas expressions of enzymes involved in
fatty acid oxidation were unchanged when the sucrose:protein ratio was increased (Fig 1F).
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The glucose moiety of sucrose is responsible for the obesity promoting effect of sucrose.
To investigate whether the obesity promoting effect of sucrose fed in combination with fish oil was
depended on the glucose or fructose moiety of sucrose, we prepared diets where fish oil was
combined with sucrose, glucose or fructose (Table 2). C57BL/6J mice were fed these diets adlibitum.
After 7 weeks mice receiving fish oil in combination with fructose had gained less weight
than mice receiving fish oil in combination with sucrose or glucose (Fig 2A). Energy intake was
slightly, but not statistically significantly lower in mice fed the fructose supplemented diets and
hence, energy efficiency was significantly reduced (Fig 2B). Calculation of digestibility
demonstrated a minor, but not significantly reduced digestibility of protein and fat in fructose fed
mice (Fig 2C). Of note, the mice receiving fructose had about 50% less white adipose tissue mass,
iWAT, pWAT and eWAT, than mice receiving sucrose or glucose, combined with a tendency
towards a slight decrease in the weight of the tibialis anterior muscle (Fig 2D).
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We have previously provided evidence that the different obesogenic effect of corn oil
fed in combination with either protein or sucrose is related to the effect of the macronutrient
composition on hormonal status (16). As sucrose and glucose, unlike fructose, stimulate insulin
secretion, we measured plasma levels of glucose and insulin in the fed state. As expected, plasma
glucose and insulin were lower in fructose fed mice (Fig 2E). As in mice fed a high fat diet in
combination with proteins, expressions of Ppargc1a and Pck1 in the liver were increased (Fig 2F).
However, unlike mice fed a high protein diet, expression of genes involved in amino acid
degradation was not increased in fructose fed mice (Fig 2F). Plasma levels of 2-hydroxybutyrate, a
marker for fatty acid β-oxidation, were similar in all groups, but the expression of genes involved in
hepatic fatty acid oxidation was reduced in livers from fructose fed mice (Fig 2F). Hepatic
expression of lipogenic genes was higher in fructose fed mice (Fig 2F). Importantly, however,
excess lipid accumulation was not seen in liver or tibialis anterior muscle (Fig 3). The finding that
Ucp1 expression was strongly induced in iWAT, but not iBAT in fructose fed mice, indicates that
energy, as observed in protein fed mice, may be dissipated in the form of heat. Indeed, a higher
expression of markers for brown adipocytes such as Ppargc1a and Cox2 (cytochrome c oxidase
subunit II) suggested a higher number of brown-like adipocytes in iWAT (Fig 3C).
Fructose feeding is frequently used to induce glucose intolerance in rats (26, 39) and
pro-inflammatory cytokines such as CCL2 produced by both adipocytes and infiltrating
macrophages are causally linked to the development of glucose intolerance (40, 41). As expression
of inflammatory markers was significantly higher in mice fed fish oil in combination with glucose
or sucrose than fructose (Fig 3C), a glucose tolerance test was performed in mice fed the sucrose,
glucose and fructose-based diets. Of note, blood glucose levels reached higher concentrations in
glucose compared to fructose fed mice and the area under the curve (AUC) was significantly higher
in glucose than both sucrose and fructose fed mice (Fig 3D).
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High glycemic index starch increases the adipogenic potential of fish oil.
To further investigate whether other types of carbohydrates, such as starch with different capacity to
stimulate insulin secretion are able to modulate the adipogenic potential of fish oil, we prepared
isocaloric diets (Table 2) where fish oil was combined with low or high GI starch (100%
amylopectin vs 60% amylose/40% amylopectin) demonstrated to induce the expected differences in
postprandial blood glucose levels in a meal tolerance test when combined with a high fat diet (33,
42). After 7 weeks of feeding, body weights were similar in mice receiving fish oil in combination
with either high- or low GI starch (Fig 4A). Energy intake was also similar, and no statistical
significant difference in energy efficiency was found (Fig 4A). However, mice fed fish oil in
combination with the low GI starch had significantly lower levels of insulin and less white adipose
tissue (Fig 4 B and C). Furthermore, expressions of inflammatory markers tended to be higher in
adipose tissues from mice fed high GI starch (Fig 4E). The reduced adiposity observed in mice
receiving fish oil in combination with the low GI starch was not due to decreased digestibility, as
digestibility of the diet containing the low GI starch was slightly higher than digestibility of the diet
containing the high GI starch (Fig 4D). Expressions of genes involved in fatty acid oxidation,
gluconeogenesis and amino acid degradation in the liver were similar in the two groups of mice, but
expressions of the lipogenic genes Scd1 (stearoyl-Coenzyme A desaturase 1) and Fasn were
reduced in mice fed low GI starch (Fig 4F). Of note, expression of Ucp1 was similar in iBAT, but
significantly higher in iWAT in mice fed the low GI starch (Fig 4E and F). Altogether, results from
the experiments combining fish oil with glucose or fructose as well as high or low GI starch
suggested that stimulation of insulin secretion increases the obesogenic effect of a diet enriched
with dietary fish oil.
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The obesity promoting effect of sucrose in combination with fish oil is associated to increased
insulin secretion.
To investigate if increased insulin secretion is able to increase the obesogenic effect of fish oil when
the carbohydrate load is low, we fed mice a diet enriched with fish oil in combination with proteins.
To increase insulin secretion, half of the mice were injected daily with the sulfonylurea class drug
glybenclamide (43, 44), whereas the second half were injected with placebo. Energy intake, body
weight gain (Fig 5A) and energy efficiency (not shown) were not affected by glybenclamide, but
the amount of white adipose tissue mass tended to increase although not statistically significantly
(Fig 5B). However, expression of Pparg was significantly increased in both eWAT and iWAT (Fig
5C). As insulin levels in the fed state only tended to increase, a glucose tolerance test was
performed to validate whether the dose of glybenclamide used was sufficient to increase insulin
levels. As expected, glucose tolerance was improved by daily injections of glybenclamide (Fig 5D).
This was not accompanied by reduced expressions of markers for adipose tissue inflammation (Fig
5C). In accordance with unchanged energy efficiency, Ucp1 expression was not significantly
changed. However, glybenclamide treatment reduced expression of Pck1 and genes involved in
amino acid degradation and ureagenesis (Fig 5E). Together, these results demonstrate that increased
insulin secretion is unable to significantly increase the obesogenic effect of fish oil when the
carbohydrate load is low. It is thus likely that a certain threshold level of carbohydrates is required
in order for insulin to ellicit the obesogenic potential of fish oil.
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If insulin promotes the obesogenic potential of fish oil in presence of carbohydrates,
inhibition of insulin secretion should attenuate the adipogenic effect of sucrose in combination with
fish oil. Since insulin secretion can be reduced by treatment with nifedipine (34) we supplemented a
diet enriched with fish oil and sucrose with nifedipine. Inclusion of nifedipine did not influence
body weight gain (Fig 6A), or feed intake (not shown). Consequently, energy efficiency was not
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different (not shown). As expected, inclusion of nifedipine in the sucrose enriched high-fish oil diet
resulted in lower levels of plasma insulin in the fed state (Fig 6B). Importantly, the mice receiving
the nifedipine-supplemented diet had lower adipose tissue mass and adipocytes had a normal
appearance (Fig 6C). When nifedipine was added to a standard low fat diet, no effect on adipose
tissue mass was observed (not shown), and expression of inflammatory markers in white adipose
tissue was, with the exception of Serpine1 in iWAT, not reduced (Fig 6D). In line with unchanged
energy efficiency, we did not detect any increased expression of Ucp1 in iWAT in nifedipine
treated mice (Fig 6D). Moreover, nifedipine did not increase hepatic expression of Pck1 or genes
involved in lipogenesis or amino acid degradation (Fig 7A). Thus, inhibition of insulin secretion
was able to partly attenuate the obesogenic effect of sucrose in combination with fish oil, but did
not reduce energy efficiency. Furthemore, nifedipine did not improve the reduced glucose tolerance
observed after feeding fish oil in combination with sucrose, but a insulin tolerance test indicated
that insulin sensitivity was improved (Fig 7B). Together, our data indicate that an inhibition of
insulin secretion attenuates the adipogenic effect of sucrose in combination with fish oil. However,
inhibition of insulin secretion is insufficient to reduce energy efficiency.
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DISCUSSION
Due to the well described beneficial effects of n-3 PUFAs on lipid metabolism and lipid-related
disorders in humans, increasing the relative amount of n-3 PUFAs in diet or as supplement is
presently recommended by Health Authorities. The potential use of n-3 PUFAs in weight control in
humans is not fully explored, but the ability of these fatty acids to reduce the development of diet-
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induced obesity rodents is well described (7, 10-15).
In this study we demonstrate that the
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composition of the diet determines the adipogenic potential of fish oil. In particular, we demonstrate
that a high dose of fish oil promoted obesity when combined with sucrose, glucose or high GI
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starch. Thus, many if not all reported beneficial effects of fish oil intake might be diminishes or
completely abrogated by a simultaneous intake of high GI carbohydrates.
Insulin is the primary anabolic hormone promoting energy storage in the fed state and
is an important driver for adipocyte differentiation (18). Of note, mice lacking insulin receptors in
adipose tissue (FIRKO mice), are protected against obesity and remain glucose tolerant on a highfat
diet (19). Further, white adipose tissue mass is dramatically reduced in Irs1 -/- Irs2 -/- doubleknockout
mice (45). In contrast, mice lacking the insulin receptor in muscle (MIRKO mice) have
increased glucose utilization in WAT (46), and exhibit a more than 50% increase in fat mass and
adipocyte number (47). Similarly, mice overexpressing Slc2a4 (solute carrier family 2 (facilitated
glucose transporter), member 4, GLUT4) (48) or Irs1 (20) in adipose tissue are obese. Finally,
treating normal rats with insulin via osmotic minipumps increases Slc2a4 expression, glucose
utilization and de novo fatty acid synthesis in adipose tissue, which are accompanied by weight gain
and increased adipose tissue mass (49-51). The importance of insulin secretion and insulin levels in
circulation is further supported by the finding that daily injections of insulin increases weight gain
in transgenic mice expressing agouti in adipose tissue (52). Of note, unlike the traditional agouti
mice, mice with transgenic expression of the agouti gene driven by the aP2 promoter do not get
obese unless triggered by insulin (52). Transgenic mice treated with insulin gained significantly
more weigh than their wild type littermates, and the authors suggested that the daily insulin
treatment mimicked the hyperinsulinemia normally observed in mice expressing the agouti gene
ubiquitously (52). Moreover, weight gain is a well recognized side effect of type 2 diabetic drugs
that increase insulin sensitivity (53, 54). Together these observations suggest that hyperinsulinemia
is a contributing factor to development of obesity and reducing hyperinsulinemia would thus
possible counteract obesity development. In keeping with this view, inhibition of insulin secretion
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by administration of nifedipine attenuated the adipogenic effect of fish oil in combination with
sucrose.
Increasing insulin secretion alone, however, is not sufficient to promote obesity
development, as mice receiving glybenclamide in combination with proteins and fish oil did not
become obese. Furthermore, increased insulin secretion by glybenclamide in the presence of low
levels of carbohydrates in the feed was insufficient to increase the adipogenic potential of fish oil. It
has been demonstrated that a high-fat diet is unable to increase adipose tissue mass in the absence
of carbohydrates (55, 56).
In our study nifedipine did not reduce adipose tissue mass in low fat fed mice (not
shown). However, as emphasized above, inclusion of nifedipine attenuated hyperinsulinemia and
obesity induced by fish oil in combination with sucrose. These findings are in line with the study by
Kim et al (34) demonstrating that nifedipine attenuated agouti-induced hyperinsulinemia and
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obesity.
Hyperinsulinemia is an early event also in obese Zucker rats, and attenuation of
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hyperinsulinemia using diazoxide reduces obesity development in obese- but not lean Zucker rats
independently of feed intake (57). Together our results suggest that the increased insulin secretion
contribute to the obesity promoting effect of fish oil when combined with sucrose.
The obesity promoting effect of increased insulin secretion is further supported by our
finding that glucose, but not fructose is the obesity promoting moiety of sucrose. Unlike sucrose
and glucose, fructose does not stimulate insulin secretion from pancreatic beta-cells (26) and mice
receiving fish oil in combination with fructose had less adipose tissue mass than mice receiving fish
oil in combination with glucose or sucrose. Mice receiving fructose in combination with fish oil
were also less glucose intolerant than mice fed fish oil in combination with sucrose or glucose.
Fructose is commonly used to induce glucose intolerance in rats and some mice strains also develop
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metabolic syndrome in response to high fructose feeding (58). However, in C57BL/6 mice neither
hyperinsulinemia nor hyperglycemia developed in response to high fructose feeding (58).
The obesity promoting effect of fish oil can also be affected by different type of starch
as mice receiving fish oil in combination with a high GI starch had higher adipose tissue mass than
mice receiving fish oil in combination with low GI starch. These results may also relate to the
effect of the different type of starch on postprandial glucose levels and insulin secretion (33, 42,
59). Low GI diets are becoming popular in weight management also in humans although their
effectiveness in terms of lasting weight reduction is not commonly accepted (28-32). The lack of
acceptance may, however, partly be due to insufficient power of early studies as a meta-analysis has
indicated that diets in which there was a reduction in the glycemic index produced moderately more
weight loss than control low fat diets in humans (32). However, a recent large European study
demonstrated that intake of low glycemic index carbohydrates combined with a modest increase in
protein content improved maintenance of weight loss (60).
Obviously, increased adipose tissue mass is related to energy-intake. However, as
demonstrated here, the macronutrient composition can influence energy efficiency, and mice
consuming the same amount of calories end up with quite different amounts of adipose tissue. Of
note, increasing the amount of sucrose from 13 to 43% led to an approximately 5-fold higher energy
efficiency. Circulating insulin levels may also indirectly influence energy efficiency. Hepatic PGC-
1α is a central target of the insulin/glucagon axis regulating activation of the entire gluconeogenesis
cascade in liver (61-63) and a dose-dependent decrease in Ppargc1a and Pck1 expression was
observed in response to increasing intake of dietary sucrose. This might be of importance regarding
energy efficiency as gluconeogenesis requires ATP and activation of gluconeogenesis reduces feed
efficiency as protein is converted to glucose at a cost of 16-20 kJ/g protein (64). Moreover,
increased catabolism of amino acids requires ATP for disposal of nitrogen as urea at an energy-cost
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of ca 5.4 kJ/g urea. Consistent with this notion, we have observed that fish oil combined with a high
level of protein and low levels of sucrose induces hepatic expression of Ppargc1a that was
accompanied by an induction of mRNAs encoding enzymes involved in catabolism of glucogenic
amino. Thus, increased energy cost by increased gluconeogenesis and ureagenesis probably
contribute to the reduced feed-efficiency observed by a high protein diet.
In mice fed increasing amounts of protein at the expense of sucrose, we observed a
dose-dependent increase in Ucp1 expression in iWAT. Others and we have recently demonstrated
that cyclooxygenase-dependent induction of UCP1 expression in WAT counteracts diet-induced
obesity (65, 66). The importance of UCP1 expressing brown-like adipocyte in WAT for regulation
of energy expenditure is further underscored by aP2-UCP1 transgenic mice in which endogenous
Ucp1 expression and respiration were reduced in iBAT, whereas UCP1 expression, respiration and
total oxidative capacity were induced in WAT, and this was sufficient to account for the observed
changes of total energy balance (67). Thus, UCP1-dependent uncoupled respiration in iWAT in
combination with increased energy cost from gluconeogenesis may account for the reduced energyefficiency
observed when sucrose levels in the diet were low.
If the background diet determines the adipogenic potential of fish oil also in humans,
this is of great concern, as the intake of refined sugars from sources such as soft-drinks has
increased dramatically during the last several decades (68). Moreover, n-3 supplements are often
taken in combination with morning meals containing high-glycemic index carbohydrates such as
cereal, bread and orange juice. Thus, comprehensive studies of the interaction between dietary
macronutrients and fish oil in humans seem warranted.
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FIGURE LEGENDS
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FIGURE 1. Macronutrient composition determines the obesogenic effect of fish oil. Male
C57BL/6 mice (n=8) were pair-fed isocaloric high fat diets with different carbohydrate/protein
ratios for 6 weeks. (A) Body weight development in pair-fed mice is shown as relative increase. (B)
Energy efficiency was calculated as body weight gain divided by feed energy intake. (C) Weight
reduction during 24h fasting was measured and Ucp1 (Uncoupling protein-1) and Dio2 (deiodinase,
iodothyronine, type II) expressions were measured by RT-qPCR. (D-F) Adipose tissue and liver
were dissected out and the weights were recorded. Expressions of Pparg (peroxisome proliferator
activated receptor gamma), Srebf1 (sterol regulatory element binding transcription factor 1), Fasn
(fatty acid synthase), Ucp1 (Uncoupling protein-1), Serpine1 (Plasminogen activator inhibitor-1),
Ccl2 (chemokine (C-C motif) ligand 2), Cd68 (CD68 antigen) and Emr1 (EGF-like module
containing, mucin-like, hormone receptor-like sequence 1 or F4/80) were measured in epididymal
and inguinal white adipose tissue and expressions of Acox1 (acyl-CoA oxidase 1), Cpt1a (carnitine
palmitoyltransferase 1a), Fasn (fatty acid synthase), Ppargc1a (peroxisome proliferator-activated
receptor gamma, coactivator 1 alpha), Pck1 (phosphoenolpyruvate carboxykinase 1, cytosolic) and
Agxt (alanine-glyoxylate aminotransferase) were measured in liver using RT-qPCR and normalized
to Tbp (TATA-box binding protein). Data are presented as mean ± SEM. Different small letters
denote significant differences (P

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with different carbohydrate sources ad-libitum for 8 weeks. (A) Body weight development is shown
as relative increase. (B) Energy efficiency was calculated as body weight gain divided by feed
intake. (C) Digestibility of energy, nitrogen, organic material and fat were calculated in a separate
set of mice (n=5). (D-F) Blood was collected, organs were dissected out and the weights were
recorded. Hormones and lipid parameters were measured in plasma and expressions of Acox1 (acyl-
CoA oxidase 1), Cpt1a (carnitine palmitoyltransferase 1a), Cpt2 (carnitine palmitoyltransferase 2),
Hmgcs2 (3-hydroxy-3-methylglutaryl-Coenzyme A synthase 2), Srebf1 (sterol regulatory element
binding transcription factor 1), Scd1 (stearoyl-Coenzyme A desaturase 1), Acaca (acetyl-Coenzyme
A carboxylase alpha), Fasn (fatty acid synthase), Crem (cAMP responsive element modulator),
Pde4c (phosphodiesterase 4C, cAMP specific), Ppargc1a (peroxisome proliferator-activated
receptor gamma, coactivator 1 alpha), Pck1 (phosphoenolpyruvate carboxykinase 1, cytosolic),
Gpt1a (glutamic pyruvic transaminase 1a), Got1 (glutamate oxaloacetate transaminase 1, soluble),
Agxt (alanine-glyoxylate aminotransferase) and Cps1 (carbamoyl-phosphate synthetase 1) were
measured in liver using RT-qPCR and normalized to Tbp (TATA-box binding protein). Data are
presented as mean ± SEM. Different small letters denote significant differences (P

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subunit II), Serpine1 (Plasminogen activator inhibitor-1), Ccl2 (chemokine (C-C motif) ligand 2),
Cd68 (CD68 antigen) and Emr1 (EGF-like module containing, mucin-like, hormone receptor-like
sequence 1 or F4/80) were measured in epididymal and inguinal white adipose tissue by RT-qPCR
and normalized to Tbp. (D) In a separate set of mice (n=9), an intraperitoneal glucose tolerance and
insulin tolerance test were performed. Data are presented as mean ± SEM. Different small letters
denote significant differences (P

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carboxykinase 1, cytosolic), Got1 (glutamate oxaloacetate transaminase 1, soluble), Agxt (alanineglyoxylate
aminotransferase) and Cps1 (carbamoyl-phosphate synthetase 1) were measured in liver
using RT-qPCR and normalized to Tbp (TATA-box binding protein). Data are presented as mean ±
SEM. Different small letters denote significant differences (P

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gamma, coactivator 1 alpha), Pck1 (phosphoenolpyruvate carboxykinase 1, cytosolic), Gpt1a
(glutamic pyruvic transaminase 1a), Got1 (glutamate oxaloacetate transaminase 1, soluble), Agxt
(alanine-glyoxylate aminotransferase) and Cps1 (carbamoyl-phosphate synthetase 1) were measured
in liver using RT-qPCR and normalized to Tbp (TATA-box binding protein). Data are presented as
mean ± SEM. Different small letters denote significant differences (P

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nifedipine supplementation (1 g/kg) diet for 4 weeks. (A) Expressions of Acox1 (acyl-CoA oxidase
1), Cpt1a (carnitine palmitoyltransferase 1a), Cpt2 (carnitine palmitoyltransferase 2), Hmgcs2 (3-
hydroxy-3-methylglutaryl-Coenzyme A synthase 2), Srebf1 (sterol regulatory element binding
transcription factor 1), Scd1 (stearoyl-Coenzyme A desaturase 1), Acaca (acetyl-Coenzyme A
carboxylase alpha), Fasn (fatty acid synthase), Crem (cAMP responsive element modulator), Pde4c
(phosphodiesterase 4C, cAMP specific), Ppargc1a (peroxisome proliferator-activated receptor
gamma, coactivator 1 alpha), Pck1 (phosphoenolpyruvate carboxykinase 1, cytosolic), Gpt1a
(glutamic pyruvic transaminase 1a), Got1 (glutamate oxaloacetate transaminase 1, soluble), Agxt
(alanine-glyoxylate aminotransferase) and Cps1 (carbamoyl-phosphate synthetase 1) were measured
in liver using RT-qPCR and normalized to Tbp (TATA-box binding protein). (B) Intraperitoneal
glucose tolerance test and insulin glucose tests were performed. Data are presented as mean ± SEM.
Different small letters denote significant differences (P

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TABLE 1.
Macronutrient composition in the diets
Data represent weight % (g /100g)
High fish oil
Sucrose
13% 23% 33% 43%
Protein 500 400 300 200
Casein 500 400 300 200
L-Cysteine 3 3 3 3
Carbohydrate 189,5 289,5 389,5 489,5
Corn starch 9,5 9,5 9,5 9,5
Cellulose 50 50 50 50
Sucrose 130 230 330 430
Fat 250 250 250 250
Soybean oil 70 70 70 70
Fish oil 180 180 180 180
Energy (kJ/g) 25.12 25.12 25.12 25.13
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TABLE 2.
Macronutrient composition in the diets
Data represent weight % (g /100g)
493
High fish oil
Low
energy Sucrose Fructose Glucose Low GI High GI
Protein 200 200 200 200 200 200
Casein 200 200 200 200 200 200
L-Cysteine 3 3 3 3 3 3
Carbohydrate 669,5 489,5 489,5 489,5 489,5 489,5
Corn starch 529,5 9,5 9,5 9,5 9,5 9,5
Cellulose 50 50 50 50 50 50
Amylose 204
Amylopectin 136 340
Sucrose 90 430 90 90 90 90
Fructose 340
Glucose 340
Fat 70 250 250 250 250 250
Soybean oil 70 70 70 70 70 70
Fish oil 180 180 180 180 180
24

Energy (kJ/g) 17.16 25.12 25.12 25.12 25.12 25.12
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Acknowledgments:
We thank Alison Keenan for the helpful comments and suggestions during preparation of the
manuscript. Moreover, we thank Åse Heltveit and Jan Idar Hjelle at NIFES for their excellent
assistance with animal care and lipid analyses.
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Authors´ contribution:
B. L., K. K. and L. M. designed research. Q. H., H. H. L., E. F., L. S. M., L. K. M., R. J., T. M., B.
J., R. K. P., S. B. S., A. C., L. F., B. L. and L. M. conducted research and analyzed data. K. K. and
L. M. had primary responsibility for the final content. All authors read and approved the final
manuscript.
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31

UCP1 Induction during Recruitment of Brown Adipocytes
in White Adipose Tissue Is Dependent on
Cyclooxygenase Activity
Lise Madsen 1,2 *, Lone M. Pedersen 3 , Haldis Haukaas Lillefosse 1,2 , Even Fjære 1,2 , Ingeborg Bronstad 4 , Qin
Hao 1 , Rasmus K. Petersen 1 , Philip Hallenborg 1 , Tao Ma 1 , Rita De Matteis 5 , Pedro Araujo 2 , Josep
Mercader 6 , M. Luisa Bonet 6 , Jacob B. Hansen 7 , Barbara Cannon 8 , Jan Nedergaard 8 , Jun Wang 1,9 , Saverio
Cinti 10 , Peter Voshol 11 , Stein Ove Døskeland 4 , Karsten Kristiansen 1,9 *
1 Department of Biology, University of Copenhagen, Copenhagen, Denmark, 2 National Institute of Nutrition and Seafood Research, Bergen, Norway, 3 Department of
Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark, 4 Department of Biomedicine, University of Bergen, Bergen, Norway,
5 Department of Biomolecular Sciences, University of Urbino, Urbino, Italy, 6 Laboratory of Molecular Biology, Nutrition and Biotechnology, Universitat de les Illes Balears,
and CIBER de Fisiopatología de la Obesidad y Nutrición (CIBERobn), Palma de Mallorca, Spain, 7 Department of Biomedical Sciences, University of Copenhagen,
Copenhagen, Denmark, 8 The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden, 9 BGI-Shenzhen, Shenzhen, China, 10 Department of Molecular
Pathology and Innovative Therapies, University of Ancona, Ancona, Italy, 11 Metabolic Research Laboratories, University of Cambridge, Cambridge, United Kingdom
Abstract
Background: The uncoupling protein 1 (UCP1) is a hallmark of brown adipocytes and pivotal for cold- and diet-induced
thermogenesis.
Methodology/Principal Findings: Here we report that cyclooxygenase (COX) activity and prostaglandin E 2 (PGE 2 ) are
crucially involved in induction of UCP1 expression in inguinal white adipocytes, but not in classic interscapular brown
adipocytes. Cold-induced expression of UCP1 in inguinal white adipocytes was repressed in COX2 knockout (KO) mice and
by administration of the COX inhibitor indomethacin in wild-type mice. Indomethacin repressed b-adrenergic induction of
UCP1 expression in primary inguinal adipocytes. The use of PGE 2 receptor antagonists implicated EP 4 as a main PGE 2
receptor, and injection of the stable PGE 2 analog (EP 3/4 agonist) 16,16 dm PGE 2 induced UCP1 expression in inguinal white
adipose tissue. Inhibition of COX activity attenuated diet-induced UCP1 expression and increased energy efficiency and
adipose tissue mass in obesity-resistant mice kept at thermoneutrality.
Conclusions/Significance: Our findings provide evidence that induction of UCP1 expression in white adipose tissue, but not
in classic interscapular brown adipose tissue is dependent on cyclooxygenase activity. Our results indicate that
cyclooxygenase-dependent induction of UCP1 expression in white adipose tissues is important for diet-induced
thermogenesis providing support for a surprising role of COX activity in the control of energy balance and obesity
development.
Citation: Madsen L, Pedersen LM, Lillefosse HH, Fjære E, Bronstad I, et al. (2010) UCP1 Induction during Recruitment of Brown Adipocytes in White Adipose Tissue
Is Dependent on Cyclooxygenase Activity. PLoS ONE 5(6): e11391. doi:10.1371/journal.pone.0011391
Editor: Aimin Xu, University of Hong Kong, China
Received May 19, 2010; Accepted May 30, 2010; Published June 30, 2010
Copyright: ß 2010 Madsen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Danish Natural Science Research Council, the Novo Nordisk Foundation and the Carlsberg Foundation. Part of the
work was carried out as a part of the research program of the Danish Obesity Research Centre (DanORC). DanORC is supported by The Danish Council for Strategic
Research (Grant No 2101 06 0005). CIBER de Fisiopatologia de la Obesidad y Nutrición is an initiative of the ISCIII. The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: kk@bio.ku.dk (KK); lise.madsen@nifes.no (LM)
Introduction
The two types of adipose tissues, white (WAT) and brown (BAT),
have opposite functions in whole body energy homeostasis. Whereas
white adipocytes store excess energy as fat, brown adipocytes
contain a large number of mitochondria dedicated to convert fat
into heat through uncoupled respiration. The uncoupling of
respiration and the resulting heat dissipation depend on the
expression of the uncoupling protein 1 (UCP1). UCP1 is an integral
membrane protein unique to brown adipocyte mitochondria, where
it acts as a proton channel to uncouple oxidative phosphorylation by
dissipating the proton gradient across the inner mitochondrial
membrane [1]. In mice, an increased content of UCP1 in adipose
tissue mitochondria is strongly linked to protection against dietinduced
obesity. This is true whether increased UCP1 expression is
induced by transgenic expression of UCP1 itself [2;3], of forkhead
box 2 (FOXC2) [4], of PR domain containing 16 (PRDM16) [5] or
by disruption of the RIIb subunit of protein kinase A [6;7],
eukaryotic translation initiation factor E4-binding protein 1 (4E-
BP1) [8], cell death inducing DFFA like effector A and C (Cidea and
Cidec/Fsp27) [9], the p160 coregulator TIF2 [10] or retinoblastoma
Rb [11–13].
PLoS ONE | www.plosone.org 1 June 2010 | Volume 5 | Issue 6 | e11391

Cyclooxygenases and UCP1
Although it has been estimated that 50 g of brown adipocytes
would be sufficient to burn 20% of the daily energy intake [14],
BAT has traditionally been considered to be virtually absent and
of no physiological relevance in adult humans. This view has
recently changed dramatically with the demonstration of functional
BAT in adult humans [15–19] adding to the observation of
brown-like multilocular adipocytes expressing UCP1 interspersed
within human WAT [20–22]. Actually, UCP1 mRNA has been
detected in all adipose tissues in adult humans, and it has been
estimated that 1 in 100–200 adipocytes in human intraperitoneal
adipose tissue expresses UCP1 [23].
Classic interscapular brown adipocytes and brown-like adipocytes
found in WAT depots appear to originate from distinct
lineages. Brown pre-adipocytes derived from the interscapular
region (iBAT) demonstrate myogenic gene expression [24] and
classic brown adipocytes arise from Myf5 expressing progenitors
[25]. In contrast, brown-like adipocytes appearing in white
adipose tissue by b-adrenergic stimulation (‘‘brite adipocytes’’)
appear to originate from another lineage, much closer to white
adipocytes [26–29] and display different molecular markers [30].
Several lines of evidence suggest that the number of brown-like
adipocytes in WAT depots might influence whole body energy
balance. Increased occurrence of brown-like adipocytes within
WAT depots is a feature of mouse strains resistant to dietary
obesity, such as the A/J strain [31;32], and reduced adiposity
associated with aP2-transgenic expression of UCP1 is linked to
increased energy dissipation in white, but not interscapular brown,
adipose tissue [33]. Human obesity is associated with a reduced
expression of UCP1 and other thermogenesis related genes in
WAT depots [34;35]. Thus, identification of factors controlling
induction of UCP1 expression and an increase in the number of
brown-like adipocytes in white depots obviously deserves further
attention.
It is intriguing that the cold-induced occurrence of brown-like
adipocytes and UCP1 requires the presence of the b 3 -adrenoceptor
in previously white adipose tissue, but not in interscapular
brown adipose tissue [36]. Furthermore, the presence of the b 3 -
adrenoceptor is required for full stimulation of energy expenditure
and oxygen consumption in white adipose tissue [37].
Adipocytes from lean rats have higher isoprenalin-stimulated
prostaglandin E 2 (PGE 2 ) synthesis, than adipocytes from obese
Zucker rats [38]. We therefore hypothesized that prostaglandins
or related products synthesized by cyclooxygenases (COXs)
might be involved in the recruitment of brown adipocytes in
white depots. The COXs have previously been implicated in
adipogenesis [39–41], but no specific role has been assigned. Here,
we demonstrate that COX activity is crucially involved in the
induction of UCP1 expression in WAT providing further evidence
for a role of COXs in the control of energy balance and obesity
development. In view of the worldwide epidemic of obesity and
associated metabolic disorders it is obviously of importance to
identify pathways that can be manipulated genetically or
pharmacologically and regulate the induction of UCP1 expression
and recruitment of brown-like adipocytes in white adipose tissues.
Results
COX1 and COX2 protein expression is upregulated in
iWAT during cold treatment
When mice are kept at 28uC, close to thermoneutrality, the
majority of the adipocytes in the inguinal white adipose depot
(iWAT) – the major subcutaneous depot in the mouse – appear as
UCP1-negative, spherical unilocular adipocytes [42]. In iWAT
from warm-acclimated mice, only endothelial cells and macrophages
stained positive for COX1 and COX2, respectively
(Figure 1A and B). However, when mice were transferred to a
cold environment, the emerging multilocular adipocytes stained
positive for COX1 and COX2. In particular, cells that appeared
to be in a transition state between uni- and multilocular cells
stained strongly (Figure 1A and B). Western blotting demonstrated
increased expression of COX1 and COX2 in iWAT, and also in
iBAT, after cold exposure (Figure 1C). Real time qPCR analysis
verified that genes preferentially expressed in brown vs. white
adipose tissue, such as UCP1, peroxisome proliferator activated
receptor gamma coactivator 1a (PGC1a), type II thyroxine
deiodinase (Dio2), cytochrome C oxidase subunit 8b (Cox8b),
epithelial V like antigen 1 (Eva1) and Cidea were all highly
induced in iWAT upon cold exposure, whereas expression of 4E-
BP1 and of nuclear receptor interacting protein 140 (Nrip1/
RIP140) was reduced (Figure 1D). Immunohistochemical staining
of iBAT from cold-treated mice demonstrated that adipocytes
stained positive for COX2, whereas only endothelial cells stained
positive for COX1 (Figure S1A). The lack of COX1 and COX2
expression in adipocytes from iBAT in warm-acclimated mice was
verified by analysis of protein and RNA isolated from fractionated
adipose tissue, in which COX1 and COX2 was detected solely in
the stromal vascular fraction (Figure S1B).
Inhibition of COX activity represses induction of UCP1
expression
Differentiated mouse embryo fibroblasts (MEFs) lacking the
retinoblastioma (Rb) gene, resemble brown or brown-like
adipocytes in demonstrating b-adrenergic induction of UCP1
expression [43]. To achieve a robust induction of UCP1
expression, differentiated Rb 2/2 MEFs were treated with a
combination of isoproterenol and 9-cis retinoic acid [44]. Just as
cold exposure increased COX1 and COX2 mRNA and protein
levels in brown-like adipocytes (Figure 1), isoproterenol/9-cis
retinoic acid treatment increased COX1 and COX2 mRNA and
protein expression (Figure 2A and B) in this model system.
Upregulation of COX1 and COX2 expression in Rb 2/2
adipocytes was accompanied by increased production of PGE 2 ,
the primary prostaglandin produced by mature adipocytes
[45;46], but not of PGF 2a and 6-keto-PGF 1a (Figure 2C). This
indicates that Rb 2/2 adipocytes resemble mature adipocytes in
producing PGE 2 as the major prostaglandin species.
To investigate the importance of COX activity for induction of
UCP1 expression, we treated differentiated Rb 2/2 adipocytes
with isoproterenol/9-cis retinoic acid in the absence or presence of
the general COX inhibitor indomethacin. Indomethacin prevented
induction of UCP1 mRNA and protein expression (Figure 2D
and E), thus suggesting the intriguing possibility that COX activity
is required for induction of UCP1.
To examine if COX activity was required also in primary
adipocytes, we induced cells from the stromal vascular fraction of
iBAT and iWAT to differentiate and then treated the mature
adipocytes with the b-adrenergic agonist isoproterenol in the
absence and presence of indomethacin. Interestingly, indomethacin
inhibited isoproterenol-induced UCP1 expression in cells
derived from iWAT but not from iBAT (Figure 3A), indicating
that COX activity is required for b-adrenergic induction of UCP1
expression in adipocytes from iWAT, but not in iBAT adipocytes.
In keeping with this notion, indomethacin only marginally
attenuated induction of UCP1 expression in the WT-1 cell model
representing interscapular brown adipocytes (Text S1, Figure S2)
[47].
To investigate the role of COX activity during induction of
UCP1 expression in iWAT and iBAT in vivo, we treated warm-
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Cyclooxygenases and UCP1
Figure 1. Cold exposure induces COX1 and COX2 expression in iWAT and iBAT. Sv129 mice were warm-acclimated at 28–30uC for 6 days
and then transferred to 4–6uC. Samples for cryosections, RNA and protein extractions were prepared from iBAT and iWAT after 2, 4 and 6 days at 4–
6uC. A–B. Representative COX1 (A) and COX2 (B) immunoreactivity in iWAT from mice kept at 28–30uC for 6 days and after 6 days of cold exposure. C.
Proteins were isolated from warm-acclimated mice (lane 1) and after 2, 4 and 6 days of cold exposure. COX1 and COX2 expression were determined
by Western blotting. D. RNA was harvested from iBAT and iWAT from individual mice (n = 4 in each group) that were warm-acclimated or coldexposed
for 6 days. Expressions of genes were measured by RT-qPCR in duplicates and normalized to TBP (TATA box binding protein). The bars
represent mean 6 standard error. * indicates statistical difference (p,0.05) compared to expression in warm acclimated mice.
doi:10.1371/journal.pone.0011391.g001
acclimated mice with the COX inhibitor indomethacin and
transferred the mice to 4uC. Measurements of rectal temperature
revealed that mice treated with indomethacin had slightly, but
significantly lower body temperature (Figure 3B). As expected,
UCP1 expression was induced in both iBAT and iWAT in
vehicle-treated mice (Figure 3C and D). While indomethacin
treatment only slightly attenuated cold-induced UCP1 expression
in iBAT, it almost completely prevented the induction of UCP1
expression in iWAT (Figure 3C and D). Thus, COX activity
appeared to be necessary for cold-induced UCP1 expression in
iWAT, but not in iBAT. In addition, indomethacin treatment
attenuated cold-induced enhancement of PGC1a, Dio2,Cox8b,
Eva1 and Cidea expression in iWAT, while preventing coldinduced
repression of RIP140 and 4E-BP1 expression in iWAT
(Figure 3D).
Forced expression of COX2 induces UCP1 expression in
Rb 2/2 adipocytes
Since indomethacin attenuated b-adrenergically stimulated
UCP1 expression in Rb 2/2 adipocytes and primary inguinal
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Cyclooxygenases and UCP1
Figure 2. Indomethacin prevents isoproterenol-induced UCP1 expression in Rb-negative adipocytes. Rb-negative mouse embryo
fibroblasts were induced to differentiate as described in experimental procedures. Differentiated cells were treated with vehicle or isoproterenol
(100 nM) and 9-cis-retinoic acid (1 mM) for 24 h. Indomethacin (1 mM) was included when indicated in the figure. A and D. RNA was isolated and
expressions of genes were measured by RT-qPCR in duplicates and normalized to TBP. C. The levels of prostaglandin E 2 ,F 2a and 6-keto-prostaglandin
F 1a were determined in cell medium using ELISA kits after 24 h. B and E. Proteins were harvested and expressions of COX1, COX2 and UCP1 were
measured by Western blotting. The bars represent mean 6 standard error. The experiments were performed in triplicates and performed 3–5 times.
* indicates statistical significant difference (p,0.05).
doi:10.1371/journal.pone.0011391.g002
adipocytes, but not in WT-1 cells and primary interscapular
brown adipocytes, we again used Rb 2/2 adipocytes as a model
system for ‘‘brite’’ adipocytes. To investigate the relative
importance of COX1 and COX2 activities in mediating induction
of UCP1 expression in such cells, we treated Rb 2/2 adipocytes
with isoproterenol/9-cis retinoic acid in the absence and presence
of selective COX1 and COX2 inhibitors. As shown in Figure 4A,
selective inhibition of COX1 and COX2 with SC560 or NS398,
respectively, partially prevented UCP1 induction, whereas a
combination of these inhibitors or treatment with the non-selective
inhibitor indomethacin fully prevented UCP1 induction. Accordingly,
activities of both COX1 and COX2 seem necessary for full
UCP1 induction.
To further examine the relative importance of COX1 and
COX2 for prostaglandin synthesis and UCP1 expression, these
enzymes were retrovirally expressed both singly and in combination
in Rb 2/2 MEFs (Figure 4B). The cells were induced to
differentiate, and on day 8, the medium was replaced by fresh
medium, which was harvested 24 h later and analyzed for PGE 2
content. The level of PGE 2 was higher when the cells were
transduced with COX2 alone or in combination with COX1,
than with COX1 alone (Figure 4C). These results, together with
the fact that PGE 2 formation in adipose tissue in COX2 KO
mice is significantly lower than in COX1 KO mice [48], point to
COX2 expression as being of major importance for PGE 2
production. In accordance with this, forced expression of COX1
alone was unable to induce UCP1 expression (Figure 4D).
However, UCP1 expression was significantly induced by forced
expression of COX2 alone or in combination with COX1
(Figure 4D). Increased expression of UCP1 was accompanied
with increased expression of PGC1a, Dio2, Cox8b, Eva1 and
Cidea, as well as reduced expression of RIP140, but not 4E-BP1
(Figure 4D).
Cold-induced UCP1 expression is attenuated in iWAT in
COX2 KO mice
To confirm the importance of COX2 for UCP1 induction in
iWAT, wild-type and COX2 KO mice were challenged with a
cold environment after warm acclimation. The wild-type mice
defended their body temperature better than the COX2 KO mice
(Figure 5A). The COX2 KO mice develop severe nephropathy
and are susceptible to peritonitis in early life [49]; therefore, KO
and wild-type littermates 6 weeks of age were used in this
experiment. Unfortunately, we were unable to collect sufficient
amounts of iWAT from these young mice to detect UCP1 or COX
by Western blotting. However, as expected, cold-induced UCP1
mRNA expression was attenuated in iWAT in COX2 KO mice
(Figure 5B). Cold-induced expression of Dio2 and Cidea was also
attenuated in iWAT in the COX2 KO mice and PGC1a
expression also tended to be attenuated (Figure 5B). Moreover,
the cold-induced reduction of RIP140 expression was prevented in
the COX2 KO mice (Figure 5B). Expression of Cox8b, Eva1 and
4E BP1 was, however, not significantly different in iWAT from
wild-type and COX2 KO mice, suggesting that inhibition of both
COX1 and COX2 might be necessary to attenuate cold-induced
changes in the expression of these genes. As expected, we observed
no differences in UCP1 expression in iBAT in COX2 KO and
wild-type mice, and surprisingly, cold-treated COX2 KO mice
had significantly higher expression of PGC1a in iBAT than did
wild-type mice (Figure 5B).
PGE 2 induces UCP1 expression via activation of the EP 3 /
EP 4 receptors
PGE 2 is reported to mediate its action by interacting with four
subtypes of PGE receptors, the EP 1 ,EP 2 ,EP 3 and EP 4 receptors
[50], but may also bind to the prostaglandin F (FP) receptor with an
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Cyclooxygenases and UCP1
Figure 3. Indomethacin prevents cold-induced UCP1 expression in iWAT. A. Stromal vascular fractions (SVFs) were isolated from mouse
iWAT and iBAT, cultured and induced to differentiate as described in experimental procedures. Differentiated adipocytes were treated with vehicle or
isoproterenol (100 nM) in the absence or presence of indomethacin (1 mM) for 24 h. Expression of UCP1 was measured by RT-qPCR in duplicates and
normalized to PPARc. The bars represent mean 6 standard deviation. The experiment was performed in triplicates and repeated 3 times. B–D. C57Bl/
6 mice were warm-acclimated at 28–30uC for 10 days and then transferred to 4–6uC for 48 h. Mice were injected with vehicle or indomethacin
(2.5 mg/kg) 2 h prior transfer to 4uC and thereafter every 12 h. Rectal temperature was measured before the mice were transferred and after 24 and
48 h (B). Protein and RNA extractions were isolated after 48 h. UCP1 expression was measured by Western blotting (C) and expressions of genes were
measured by RT-qPCR and normalized to TBP (D). The bars represent mean 6 error (n = 5–6). * indicates statistical significant difference (p,0.05).
doi:10.1371/journal.pone.0011391.g003
affinity that is only 10–30 fold lower than that of PGF 2a [51]. In
order to probe the relative importance of these receptors in
mediating the possible effect of PGE 2 on induction of UCP1,
expression of the EP and FP receptors was measured in adipose
tissue and in Rb 2/2 adipocytes. All receptors were expressed in
both white and brown adipose tissue, whereas no expression of the
EP 3 receptor could be detected in Rb 2/2 adipocytes (Figure 6A),
implying a minor if any role of this receptor in mediating the PGE 2
response in those cells. Thus, Rb 2/2 adipocytes were treated with
isoproterenol and 9-cis retinoic in the absence or presence of
AL8810, AH6809, or AH23848, that are FP-, EP 1 /EP 2 and EP 4
receptor antagonists, respectively. Isoproterenol-stimulated UCP1
expression was not affected by the FP receptor antagonist, but
slightly attenuated by the EP 1 /EP 2 receptor antagonist and strongly
attenuated by the EP 4 antagonist (Figure 6B). Reduced expression
of UCP1 was accompanied by reduced expression of PGC1a and
Cidea (Figure 6B). To verify the importance of PGE 2 signaling via
the EP 4 receptor with a possible minor contribution by the EP 3
receptor, mice were injected with an EP 3 /EP 4 receptor agonist [52],
the stable PGE 2 analogue 16,16-dimethyl-PGE 2 As predicted,
qPCR analysis revealed that UCP1 expression was induced in
iWAT, but not in iBAT (Figure 6C). Together, the in vitro and in vivo
results suggest that PGE 2 -induced UCP1 expression at least in part
is mediated via the EP 3 /EP 4 receptors with EP 4 being the
predominant receptor involved.
Inhibition of COX activity increases adiposity and energy
efficiency in obesity resistant Sv129 mice
Diet-induced thermogenesis protects several mouse strains
against obesity [53–55]. Since it appears that the protection
against diet-induced obesity is related to increased occurrence of
brown-like adipocytes in white depots [56;57], we aimed to
investigate the hypothesis that indomethacin could also attenuate
diet-induced UCP1 expression and thereby increase the propensity
for diet-induced obesity in Sv129 mice. Since it was recently
demonstrated that UCP1-deficient mice become obese when
housed at thermoneutrality [58], we predicted that the most
pronounced effect of COX inhibition would be observed for mice
kept under thermoneutral conditions. Accordingly, we fed Sv129
mice a very high-fat diet with or without indomethacin
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Cyclooxygenases and UCP1
Figure 4. UCP1 expression is induced by forced expression of COX2 alone or in combination with COX1 in cultured cells. A. Rb 2/2
MEFs were induced to differentiate as described in experimental procedures. Differentiated adipocytes were treated with vehicle or isoproterenol
(100 nM) and 9-cis-retinoic acid (1 mM) in the absence and presence of the COX1 inhibitor SC560 (50 nM) or the COX2 inhibitor NS398 (5 mM), alone
or in combination, or with indomethacin (1 mM), for 24 h. Expression of UCP1 was measured by RT-qPCR. The bars represent mean 6 standard error.
The experiment was performed in triplicates and repeated 2 times. B–D. Rb 2/2 MEFs were retrovirally transduced with empty vector, vector
encoding COX1 or COX2, or both. The transduced cells were selected and induced to differentiate and analyzed for COX1 and COX2 expression by
Western blotting (B). PGE 2 levels were measured in cell media (C). RNA was isolated on day 8 and expressions of genes were measured by RT-qPCR
(D). The bars represent mean 6 standard error. Different letters indicate statistically significant difference (p,0.05). The experiments were performed
in triplicates.
doi:10.1371/journal.pone.0011391.g004
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Cyclooxygenases and UCP1
Figure 5. Cold-induced UCP1 expression is attenuated in iWAT in COX2 KO mice. A B COX2 KO mice and wild-type littermates were warmacclimated
at 28–30uC for 10 days and then transferred to 4–6uC for 48 h. Rectal temperature was measured before transfer and after 24 and 48 h (A).
RNA was extracted and expressions of genes were measured by RT-qPCR (B). The bars represent mean 6 standard error (n = 5–6). * indicates
statistical significant difference between wild-type and KO mice (p,0.05).
doi:10.1371/journal.pone.0011391.g005
supplementation for 4 weeks while keeping the mice at 28–30uC.
As demonstrated in Figure 7A, indomethacin supplementation led
to a higher weight gain. Energy intake was slightly, but not
statistically significantly lower (data not shown). However, energy
intake relative to body weight gain was significantly lower in mice
that received the indomethacin-supplemented high-fat diet
(Figure 7A). Mice fed the diet supplemented with indomethacin
also had more WAT in different depots, but not iBAT (Figure 7B).
Histological analysis revealed that the adipocytes in both iWAT
and iBAT appeared normal, but adipocytes in iWAT in mice fed
the high-fat diet supplemented with indomethacin were slightly
larger (Figure 7C and D). As expected, feeding mice a high-fat diet
lead to augmented expression of UCP1 in both iBAT and iWAT
in vehicle-treated mice (Figure 7E). Importantly, diet-induced
UCP1 expression was prevented in iWAT, but not in iBAT in the
indomethacin-treated mice (Figure 7E). Reduced expression of
UCP1 in iWAT in mice fed a high-fat diet supplemented with
indomethacin was accompanied with reduced expression of
Cox8b. Expression of PGC1a, Dio2, Eva1, Cidea, 4E-BP1 and
RIP140 was not affected by inclusion of indomethacin with the
high-fat diet (Figure 7E). Collectively, these results underscore the
notion that inhibition of COX activity attenuates the acquisition of
‘‘brite’’ adipocytes in white adipose depots with an accompanying
increase in feed efficiency leading to accumulation of more adipose
tissue. Obviously, other mechanisms may contribute to the
increase in feed efficiency, but the lack of ‘‘brite’’ adipocyte
recruitment seems a key player.
Discussion
The unique energy-dissipating ability of UCP1 makes control of
its expression and activation potential targets for the development
of novel drugs for the treatment of obesity and obesity-associated
diseases. Here we present evidence that COX activity and COXderived
PGE 2 are intimately linked to induction of UCP1
expression in iWAT, but not in iBAT. Thus, cold-induced
expression of UCP1 in iWAT was repressed in mice treated with
the general COX inhibitor indomethacin, and in COX2 KO
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Cyclooxygenases and UCP1
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Cyclooxygenases and UCP1
Figure 6. UCP1 expression is attenuated by an EP 4 receptor antagonist in Rb 2/2 adipocytes and induced by the EP4 receptor
agonist 16,16dmPGE 2 in vivo. A.Expressions of EP 1 ,EP 2 ,EP 3 ,EP 4 and FP receptors were measured by RT-qPCR in iBAT and iWAT isolated from
warm- and cold-acclimated mice, and in Rb 2/2 adipocytes treated with vehicle or isoproterenol (100 nM) and 9-cis-retinoic acid (1 mM). B. Rb 2/2
adipocytes were treated with vehicle or isoproterenol (100 nM) and 9-cis-retinoic acid (1 mM) by RT-qPCR in absence and presence of AL8810,
AH6809, or AH23848, which are FP, EP 1 /EP 2 and EP 4 receptor antagonists, respectively. Expressions of genes were measured by RT-qPCR. The bars
represent mean 6 standard error. Different letters indicate statistically significant differences (p,0.05). C. C57BL/6J mice were subcutaneously
injected with vehicle or 16,16dmPGE 2 (50 mM/kg) every 12 h for 48 h. Expressions of genes were measured by RT-qPCR. The bars represent mean 6
standard error (n = 5). * indicates statistical significant difference between vehicle and 16,16 dmPGE 2 treated mice (p,0.05).
doi:10.1371/journal.pone.0011391.g006
mice. Also, injection of a stable analog of the COX2 downstream
product PGE 2 , 16,16-dimethyl-PGE 2 , induced UCP1 expression
in iWAT. Forced expression of COX2, alone or in combination
with COX1, induced UCP1 expression in a cell model resembling
inguinal adipocytes. Finally, the inhibition of COX activity not
only attenuated diet-induced UCP1 expression in iWAT, but also
increased weight gain in Sv129 mice kept at thermoneutrality.
The association between diet-induced thermogenesis and the
recruitment of brown adipose tissue was first noted more than 30
years ago and believed to involve the classical brown adipose tissue
located in the interscapular region [59]. The anti-obesity role of
UCP1 was challenged by the finding that UCP1 KO mice were
not obese [60]. However, the recent demonstration that UCP1
ablation per se induced obesity when the mice were kept at
thermoneutrality [61] clearly indicates that UCP1 is important in
diet-induced energy dissipation at thermoneutrality. Our data
indicate that inhibition of COX activity increased weight gain and
concomitantly attenuated diet-induced UCP1 expression in
iWAT, but not iBAT in Sv129 mice kept at thermoneutrality,
pointing to a novel role of COX activity in the control of energy
balance and the development of obesity. These results are in line
with our earlier observation that enhanced cAMP signaling in
response to an increased glucagon/insulin ratio led to an increased
COX-mediated PGE 2 production. This was accompanied by
increased expression of UCP1 in iWAT, but not in iBAT, and
decreased feed efficiency [62]. Although neither COX1 KO nor
COX2 KO mice are obese, COX2 +/2 KO mice have more
adipose tissue than wild-type littermates when fed an obesogenic
diet [48]. The reason why COX2 +/2 , but not COX2 KO mice
were reported to be prone to obesity is not clear. However, these
studies were not performed at thermoneutrality [48]. A similar
phenomenon is actually seen in GLUT4 KO mice, where the
majority of GLUT4 2/+ , but not GLUT4 2/2 mice develops
diabetes [63]. Moreover, release of PGE 2 from adipose tissue in
COX2 +/2 mice was reported to be reduced compared to adipose
tissue from wild-type mice [48] and adipose tissue cultures
obtained from obese rats have lower PGE 2 release rates than
cultures from lean rats [38]. In addition, microsomal prostaglandin
α
Figure 7. Indomethacin prevents high-fat diet-induced UCP1 expression in iWAT but not iBAT in the obesity-resistant Sv129 mouse
strain. Mice were fed a very high-fat diet (VHF) with or without indomethacin supplementation (16 ppm) for 4 weeks at a temperature of 28–30uC.
One group of mice was killed before the experiment started. A. Body weight gain and energy intake relative to body weight gain. B. Different
adipose tissue depots were dissected and weighed. C and D. Representative paraffin-embedded representative sections from iWAT and iBAT were
stained with hematoxylin and eosin. The scale bars represent 50 mM. E. Expressions of genes in iWAT and iBAT were measured by RT-qPCR. The bars
represent mean 6 standard error (n = 6). * indicates statistical significant difference (p,0.05) between different groups.
doi:10.1371/journal.pone.0011391.g007
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Cyclooxygenases and UCP1
E synthase1 (mPGES1) expression is reported to be downregulated
in iWAT and eWAT in obese mice [64], suggesting a
dysregulation of prostaglandin synthesis in obesity.
PGE 2 mediates its action by interacting with four subtypes of
PGE 2 receptors, the EP 1 ,EP 2 ,EP 3 and EP 4 receptors [50]. Using
the Rb 2/2 brown-like adipocytes, we show that b-adrenergic
stimulation of UCP1 expression is attenuated by an EP 4 receptor
antagonist. This combined with our finding that injection of the
EP 3 /EP 4 receptor agonist 16,16-dimethyl-PGE 2 increased expression
of UCP1 in iWAT indicates that the action of PGE 2 is
predominantly mediated via the EP 4 receptor with a possible minor
contribution by the EP 3 receptor. Most EP 4 KO mice die shortly
after birth, and no adipose tissue phenotype has been reported for
the few surviving pups [65]. Similarly, to our knowledge, no adipose
tissue phenotype has been reported for the EP 3 KO mice.
Collectively, our findings strongly indicate that both cold- and
diet-induced expression of UCP1 in iWAT, but not in iBAT,
requires COX activity and most likely PGE 2 formation.
Furthermore, our results point to differential roles of induction
of UCP1 expression in iWAT and iBAT in the context of diet- and
cold-induced thermogenesis. We suggest that whereas UCP1 in
iWAT plays an important role in protection against obesity, UCP1
in iBAT is essential for temperature adaptation. Upon cold
challenge, body temperatures were only slightly lower in wild-type
mice treated with indomethacin and in COX2 KO mice
compared to non-treated wild-type mice. This is in line with the
earlier finding that UCP1 expression is blunted in iWAT, but not
in iBAT, in cold-adapted b 3 adrenoreceptor KO mice [66].
The importance of COX in diet-induced expression of UCP1 in
iWAT and diet-induced thermogenesis is underscored by our
demonstration that inclusion of the general COX inhibitor
indomethacin in the diet augmented high-fat diet-induced obesity
in Sv129 mice kept at thermoneutrality irrespectively of UCP1
expression in iBAT. Increased expression of UCP1 in WAT with
accompanying increased thermogenic activity coupled with unchanged
or even reduced BAT activity has been observed in several
genetically modified lean mouse models such as RIP140 [67],
Caveolin 1 [68], Fsp27 [69], hormone sensitive lipase [70] and
vitamin D receptor [71] KO mice. Further, in RIIb mice [72], pRbdeficient
mice [11;73] and in mice overexpressing FOXC2 [74],
protection against diet-induced obesity is accompanied by an
increased RIa/RIIb ratio, rendering PKA somewhat more sensitive
to cAMP, which is accompanied by an increased occurrence of
brown adipocytes in WAT. Last, it should be recalled that in aP2-
UCP1 transgenic mice, both endogenous UCP1 expression and
respiration are actually reduced in iBAT [75]. UCP1 expression,
respiration and total oxidative capacity are, however, strongly
induced in WAT and the oxidative capacity of WAT is sufficient for
the changes of total energy balance induced by the transgene [76].
In keeping with the earlier notion that i) mouse strains that have
more UCP1-expressing adipocytes in their WAT depots are
protected against diet-induced obesity [77;78] and ii) brown-like
multilocular adipocytes expressing UCP1 are detected interspersed
within white adipose tissue in humans [20;21;79], we suggest that
factors influencing UCP1 expression in white adipose tissue are of
particular importance for the regulation of energy balance and the
development of obesity also in humans.
Materials and Methods
Ethics Statement
The animal experiments were approved by the Norwegian
Animal Health Authorities, ID 819 and 888. Care and handling
were in accordance with local institutional recommendations.
Cell culture, transduction and differentiation
Mouse embryo fibroblasts (MEFs) were prepared from wild-type
and Rb 2/2 embryos [80]. The cells were grown and differentiated
in AmnioMax Medium as described earlier [81]. Retrovira
expressing pLXSN-hygro, pBabe-puro, pLXSN-COX1 or
pBabe-COX2 were harvested from Phoenix–Eco cells, plated at
30–40% confluence in DMEM supplemented with 10% fetal
bovine serum, and transductions performed as described [82].
Isolation of the stromal vascular fraction and adipocytes
from mice
The stromal vascular fraction and adipocytes were obtained
from iWAT and iBAT dissected from 8-week old C57BL/6J mice
as earlier described [83]. Contaminating erythrocytes were
eliminated from the stromal-vascular fraction by a wash with
sterile distilled water. Cells were plated and induced to
differentiate as described [83].
Cold acclimation experiments
Groups (n = 5–8) of 10-week old male mice were acclimated at a
temperature of 28–30uC for at least 1 week and transferred to 4uC
for 1, 2, 3 or 6 days. Where relevant, mice were injected with
indomethacin (2.5 mg/kg) 2 h prior transfer to 4uC. The mice
received a dose of indomethacin every 12 h. Injections were
performed subcutaneously from a 0.75 mg/ml solution. Final dose
was 5 mg/kg/day. Control mice received vehicle. Animals were
housed individually with a 12 h light/dark cycle and free access to
pellet food and water. Mice used for immunohistochemical
analyses were immediately perfused intracardially with 4%
paraformaldehyde. iBAT, iWAT, lung and skin were dissected
and frozen for immunohistochemistry on cryosections. For
morphology experiments, the mice were immediately perfused
with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 for
5 min. COX2 KO mice (B6;129P2 Ptgs2 tm1Unc) and corresponding
wild-type littermates were obtained from Taconic. C57BL/6J
used in indomethacin experiments were obtained from Møllegård
breeding laboratories.
16,16dmPGE 2 injections
Male, C57BL/6J approx 10-weeks old from Møllegård
breeding laboratories, Denmark were divided into two groups
(n = 5). The mice received a dose of 50 mg/kg 16,16dmPGE 2 every
12 h for 48 h. Injections were performed subcutaneously and the
total dose was 0.1 mg/kg/day. Control mice received vehicle.
Animals were housed individually with a 12 h light/dark cycle and
free access to pellet food and water.
High-fat feeding
Male Sv129 mice, 11 weeks old, were obtained from Taconic.
The mice were acclimated for 1 week at a temperature of 28–30uC
and divided into three groups (n = 6 in each). One group of mice
was sacrificed before dietary intervention while the remaining
mice were fed a very high-fat diet (ssniff EF R/M acc D12492)
with or without indomethacin supplementation (16 ppm) for 4
weeks at a temperature of 28–30uC. Body weight and feed intake
were recorded twice a week. Mice were anesthetized using
isoflurane, cardiac puncture was performed and mice were killed
by cervical dissociation. Tissues were immediately frozen in liquid
N 2 .
Real time qPCR
Total RNA was extracted from cultured cells or mouse tissue
using TRIzol (Invitrogen). Reverse transcription and qPCR were
PLoS ONE | www.plosone.org 10 June 2010 | Volume 5 | Issue 6 | e11391

Cyclooxygenases and UCP1
performed in duplicates as described earlier [83]. Primer
sequences are available on request.
Western blotting
Preparation of extracts from mouse tissues or whole cell dishes,
electrophoresis, blotting, visualization and stripping of membranes
were performed as described [84]. Primary antibodies used were
goat anti-COX1, goat anti-COX2, rabbit anti-UCP1 and rabbit
anti-TFIIB antibodies (Santa Cruz Biotechnology). Secondary
antibodies were horseradish peroxidase-conjugated anti-mouse,
anti-goat or anti-rabbit antibodies obtained from DAKO.
Immunohistochemistry
COX1 (M-20; sc 1754) and COX2 (M-19; sc 1747) antibodies
were obtained from Santa Cruz Biotechnology, diluted 1:300 on
cryosections and 1:100 on paraffin-embedded sections (for
COX1). Lung [85] and skin [86] were used as positive control
for both COX1 and COX2 antibodies.
Histological analyses
Parts of adipose tissue were fixed in 4% formaldehyde in PB
buffer for 24 h, washed in PB, dehydrated in ethanol, embedded
in paraffin after 2610 min xylen treatment. Sections (8 mm thick)
of the embedded tissue sections were subjected to standard
hematoxylin and eosin staining.
Supporting Information
Text S1
Experimental.
Found at: doi:10.1371/journal.pone.0011391.s001 (0.04 MB
DOC)
Figure S1 COX1 and COX2 are mainly expressed in the
stromal-vascular fraction of iBAT in warm-acclimated mice. A.
Sv129 mice were warm-acclimated at 28–30uC for 6 days and
then transferred to 4–6uC. Samples for cryosections were prepared
from iBAT and iWAT after four days of cold exposure.
Representative COX2 immunoblots in iBAT B. iBAT from
warm-acclimated mice was fractionated into SVF and adipocyte
fractions, respectively. Expression levels of COX1 and COX2
were determined by RT-qPCR and Western blotting.
Found at: doi:10.1371/journal.pone.0011391.s002 (15.01 MB
EPS)
Figure S2 Inhibition of COX does not prevent isoproterenol/9-
cis-retinoic acid-induced UCP1 expression in WT-1 cells.
Differentiated WT-1 cells were treated with a combination of
isoproterenol (100 nM) and 9-cis-retinoic acid (1 mM) for 24 h.
When included, indomethacin (1 mM) were added 2 h before
isoproterenol and 9-cis retinoic acid. UCP1 and PGE1a
expressions were determined by RT-qPCR.
Found at: doi:10.1371/journal.pone.0011391.s003 (2.44 MB EPS)
Author Contributions
Conceived and designed the experiments: LM KK. Performed the
experiments: LM LMP HHL EF IB QH RKP PH TM RDM PA JM
MLB JBH BC JN JW SC PV SOD. Analyzed the data: LM LMP HHL EF
RKP PH TM PA JM MLB JBH BC JN JW SC PV SOD KK. Wrote the
paper: LM LMP BC JN SOD KK.
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JBC Papers in Press. Published on June 16, 2011 as Manuscript M111.234732
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M111.234732
Nutritional Regulation of Bile Acid Metabolism is Associated with
Improved Pathological Characteristics of the Metabolic Syndrome
Bjørn Liaset 1* , Qin Hao 2 , Henry Jørgensen 3 , Philip Hallenborg 4 , Zhen-Yu Du 1 , Tao Ma 2 , Hanns-
Ulrich Marschall 5 , Mogens Kruhøffer 6 , Ruiqiang Li 7 , Qibin Li 7 , Christian Clement Yde 3 ,
Gabriel Criales 1 , Hanne C. Bertram 8 , Gunnar Mellgren 9, 10 , Erik Snorre Øfjord 11 , Erik-Jan
Lock 1 , Marit Espe 1 , Livar Frøyland 1 , Lise Madsen 1,2 , Karsten Kristiansen 2*
1 National Institute of Nutrition and Seafood Research, Bergen, Norway, 2 Department of Biology,
University of Copenhagen, Denmark, 3 Department of Animal Health, Welfare and Nutrition, Aarhus
University , Denmark, 4 Department of Biochemistry and Molecular Biology, University of Southern
Denmark, Denmark, 5 Institute of Medicine, Sahlgrenska Academy, University of Gothenburg,
Sweden, 6 AROS Applied Biotechnology, Aarhus, Denmark, 7 Beijing Genomic Institute, Shenzhen,
Peoples Republic of China, 8 Department of Food Science, Aarhus University, Denmark, 9 Institute of
Medicine, University of Bergen, Norway, 10 Hormone Laboratory, Haukeland University Hospital,
Norway, 11 Center for Clinical Trials, Bergen, Norway.
*Correspondence to: Bjørn Liaset, National Institute of Nutrition and Seafood Research, P.O.
Box 2029 Nordnes, N-5817 Bergen, Norway. Fax: +47 55 90 52 99; Phone: +47 46 81 12 97;
E-mail: bli@nifes.no
or
Karsten Kristiansen, Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, DK-2200
Copenhagen, Denmark. Fax +45 3522 2128; Phone +45 6011 2408; E mail: kk@bio.ku.dk
Running title: Bile acid metabolism is regulated by dietary protein source
Bile acids (BAs) are powerful
regulators of metabolism, and mice treated
orally with cholic acid are protected from
diet-induced obesity, hepatic lipid
accumulation, and increased plasma triacyl
glycerol (TAG) and glucose levels. Here, we
show that plasma BA concentration in rats
was elevated by exchanging the dietary
protein source from casein to salmon
protein hydrolysate (SPH). Importantly, the
SPH-treated rats were resistant to dietinduced
obesity. SPH-treated rats had
reduced fed state plasma glucose and TAG
levels, and lower TAG in liver. The elevated
plasma BA concentration was associated
with induction of genes involved in energy
metabolism and uncoupling, Dio2, Pgc-1α
and Ucp1, in interscapular brown adipose
tissue. Interestingly, the same
transcriptional pattern was found in white
adipose tissue depots of both abdominal and
1
subcutaneous origin. Accordingly, rats fed
SPH-based diet exhibited increased wholebody
energy expenditure and heat
dissipation. In skeletal muscle, expressions
of the PPARβ/δ target genes (Cpt-1b,
Angptl4, Adrp, and Ucp3) were induced.
Pharmacological removal of BAs by
inclusion of 0.5wt% cholestyramine to the
high-fat SPH diet attenuated the reduction
in abdominal obesity, the reduction in liver
TAG and the decrease in non-fasted plasma
TAG and glucose levels. Induction of Ucp3
gene expression in muscle by SPH treatment
was completely abolished by cholestyramine
inclusion. Taken together, our data provide
evidence that bile acid metabolism can be
modulated by diet, and that such
modulation may prevent/ameliorate
characteristic features of the metabolic
syndrome.
Downloaded from www.jbc.org by guest, on July 7, 2011
Copyright 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

Bile acids (BAs) are synthesized in the
liver from cholesterol. After their synthesis,
they are conjugated to the amino acids taurine
or glycine in a species-dependent manner (1).
Conjugation of bile acids increases their
solubility and facilitates their secretion into
bile (2). In the intestine, the bile acids aid in
the absorption of lipophilic nutrients (3).
Normally, the bile acids are efficiently taken
up by the enterocytes of the small intestine,
transported back to the liver, and are thus
conserved through the enterohepatic
circulation (4). Bile acids are activators of the
nuclear receptor farnesoid X receptor α
(Fxr/Nr1h4) (5-7). The main physiological role
of Fxr is to maintain bile acid homeostasis and
to regulate genes encoding enzymes involved
in bile acid synthesis and transport.
In addition to bile acid homeostasis,
Fxr is important for normal lipid metabolism.
Fxr -/- mice display higher triacylglycerol
(TAG) concentrations in serum and liver (8,9)
and have an increased synthesis of
apolipoprotein (Apo) B-containing lipoproteins
(9). In line with these findings, bile acid
treatment has been reported, in a Fxrdependent
manner, to induce liver very-lowdensity
lipoprotein receptor (Vldlr)
transcription (10) and to prevent hepatic TAG
accumulation, VLDL secretion and elevated
serum TAG concentration in mice (11). Thus,
bile acids and Fxr play central roles in VLDL
lipoprotein metabolism and in the control of
TAG levels.
Glucose metabolism is also regulated
by bile acids and mice treated with cholic acid
are protected from diet-induced hyperglycemia
(12) and have down-regulated liver expression
of the gluconeogenic phosphoenolpyruvate
carboxykinase (Pck1) gene (13,14).
Furthermore, Fxr -/- mice are glucose intolerant
and insulin resistant, and insulin signaling is
blunted in several tissues (15-17). Activation
of Fxr with a synthetic ligand, GW4064, or
hepatic over-expression of constitutively active
Fxr lowered blood glucose in both diabetic
db/db and wild-type mice (15). It is therefore
evident that bile acid signaling and Fxr
function are essential for normal glucose
regulation.
Bile acids are also endogenous
activators of the G-protein coupled receptor
Tgr5 (also referred to as GB37/ M-BAR/
Gpbar1) (18,19). Stimulation of Tgr5 by bile
2
acids has been shown to increase energy
expenditure and to protect mice from dietinduced
obesity and glucose intolerance (20).
Consistent with the role of Tgr5 in the control
of energy expenditure, female Tgr5 -/- mice
show increased adiposity when challenged
with a high fat diet (21). Also, Tgr5 activation
by bile acids has been reported to promote
production and release of glucagon-like
peptide 1 (GLP-1) in enteroendocrine cell lines
(22) and in mice (23), adding another aspect of
bile acid treatment for the prevention of dietinduced
glucose intolerance and insulin
resistance. Thus, bile acid signaling through
Tgr5 might be important for the maintenance
of normal energy homeostasis and insulin
sensitivity.
The benefits of activating Tgr5 or Fxr
for the prevention of the metabolic syndrome
have stimulated the development of synthetic
ligands for these receptors (24,25). Another
strategy to increase Tgr5 and/or Fxr signaling
would be to alter endogenous BA metabolism.
As hepatic bile acid conjugation is important
for secretion of BAs into bile (2), and rats
conjugate BAs to both taurine and glycine with
high efficiency (26), the dietary levels of these
amino acids might be crucial for BA
conjugation and secretion (27). Feeding rats
casein-based diets supplemented with either
glycine or taurine led to reduced plasma
cholesterol and liver TAG concentrations,
whereas only taurine supplementation reduced
plasma TAG levels (28). Furthermore, taurine
administration has been reported to induce
hepatic protein expression of the canalicular
transporter proteins ATP-binding-cassette b11,
Abcb11 (also called Bsep) and Abcc2 (also
called Mrp2). Concomitantly, bile flow and
taurocholate excretion was induced in
experiments with perfused rat livers (29). In
rats fed low-fat diets, we have previously
shown that BA metabolism can be modulated
by dietary proteins with different endogenous
glycine and taurine contents (30).
Thus, evidence is accumulating that
BA metabolism can be modulated by dietary
levels of glycine and taurine in normal-energy
diets. However, development of the metabolic
syndrome is tightly associated with intake of
energy-dense diets. Therefore, the present
study was undertaken in order to test the
hypothesisis that rats treated with a taurineand
glycine-rich protein source, salmon protein
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hydrolysate (SPH), would be protected from
developing high-fat diet-induced pathological
characteristics of the metabolic syndrome. We
demonstrate that bile acid metabolism can be
modulated by the protein source in high-fat fed
rats, and that such a modulation may protect
against development of the metabolic
syndrome.
EXPERIMENTAL PROCEDURES
Indirect calorimetric measurements – A
separate set of rats (n=6/ group) was fed
experimental diets for 17 days. Heat
production was calculated from gas exchange
measurements, as previously described (32).
Gas exchange was determined twice, each time
for 22 hours. From the average gas exchange
measurements, heat production was calculated
by the respiratory quotient method, and
reported per 24 h.
Animals - Male Wistar Hannover GALAS
(HanTac:WH) were obtained from Taconic
Europe (Ejby, Denmark) and divided into
experimental groups (n=6). The rats were kept
at a 12-h light/ dark cycle in a temperaturecontrolled
room at 22 °C. After
acclimatization, the animals were fed
experimental diets with either SPH or casein as
the sole protein source (Supplemental Table 1).
The composition of the protein sources has
been described elsewhere (31). Feed intake
and body weight were recorded throughout the
experiments and faeces were collected the last
5 days. The rats were killed by cardiac
puncture under anaesthesia (0.23 mg/ kg BW
Fentanyl (Janssen) and 0.45 mg/ kg BW
Dormitor Vet (Orion Pharma)). Heparinplasma
and EDTA-plasma containing aprotinin
were prepared from blood. Tissues were
dissected out and weighted. A portion of the
liver was used for sub-cellular fractionation
and measurement of mitochondrial carnitine
palmitoyl transferase-1 capacity, and portions
of interscapular brown adipose tissue (iBAT)
and epididymal white adipose tissue (eWAT)
were homogenized and used for determination
of palmitoyl-Coenzyme A oxidation capacity.
The rest of the tissues were freeze-clamped
and frozen at -80 o C. All animal experiments
were approved by the National State Board of
Biological Experiments with Living Animals
(Norway and Denmark).
Whole body composition – After 40 days of
feeding, rats were anaesthetized (0.4 mg
Dormitor Vet (Medetomidin Hydrochlorid)/ kg
BW rat (Orion Pharma, Espoo, Finland), and
whole body composition was determined by a
dual X-ray absorptiometry scanner equipped
with a small animal option (Discovery QDR
Series, Hologic, Bedford, MA, USA).
3
Plasma metabolomics - A separate set of rats
(n=5/ group) was fed experimental diets for 25
days. After termination in the fed state, 200 µl
heparin plasma of each sample was mixed with
a solution of 400 µl 0.9% saline and 20% D 2 O.
Measurements were performed at 310 K on a
on a Bruker Avance III 600 spectrometer,
operating at a 1 H frequency of 600.13 MHz,
and equipped with a 5-mm 1 H TXI probe
(Bruker BioSpin, Rheinstetten, Germany). 1 H
NMR spectra were acquired using the Carr-
Purcell-Meiboom-Gill (CPMG) spin-echo
pulse sequence with water suppression. All
spectra were referenced to the lactate doublet
signal at 1.33 ppm. The spectra were
segmented into 0.013 ppm bins and each of the
bins was integrated. The reduced spectra
excluding the residual water signal were
normalized to the whole spectrum. Principal
component analysis (PCA) and partial least
squares regression discriminate analysis (PLS-
DA) was performed using the Unscrambler
software version 9.8 (Camo, Oslo, Norway) to
elucidate biochemical differences between predefined
classes. Martens' uncertainty test was
applied to find significant variables on the full
cross-validated data (33,34).
Bile acid measurements - For total bile acid
measurements in feces, bile acids were
extracted according to Suckling et al (35).
Amounts of total bile acids in fecal extracts
and in non-fasted EDTA-plasma were
determined enzymatically by the 3-αhydroxysteroid
dehydrogenase reaction
(Dialab, Vienna, Austria). Bile acids in liver
samples were extracted and analyzed using
gas-chromatography-mass spectrometry and
electrospray-mass spectrometry as previously
described in detail (36).
Real time RT-qPCR - Total RNA was purified
tissues using Trizol, and cDNA was
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synthesized from individual rats. Gene
expression was determined in individual
samples by real-time qPCR using ABI PRISM
7700 Sequence Detection System (Applied
Biosystems) carried out in 96-well plates and
in duplicate as earlier described (37). Primers
for real-time PCR (Supplemental Table 2)
were designed using Primer Express 2.0
(Applied Biosystems).
Liver microarray analysis- The following
procedures were all performed according to
Affymetrix standard procedures. Briefly, equal
amounts of RNA isolated from livers were
pooled (n=6) and 5 µg of total RNA was used
as starting material for the target preparation.
First and second strand cDNA synthesis were
performed using the SuperScript II System
(Invitrogen) according to the manufacturers’
instructions except using an oligo-dT primer
containing a T7 RNA polymerase promoter
site. Labeled aRNA was prepared using the
BioArray High Yield RNA Transcript
Labeling Kit (Enzo) using Biotin labeled CTP
and UTP (Enzo) in the reaction together with
unlabeled NTP´s. Unincorporated nucleotides
were removed using RNeasy columns
(Qiagen). Fifteen µg of cRNA were
fragmented, loading onto the Affymetrix Rat
Genome RAE 230 2.0 probe array cartridge
and hybridized for 16h. The arrays were
washed and stained in the Affymetrix Fluidics
Station and scanned using a confocal laserscanning
microscope (GeneChip® Scanner
3000 System with Workstation and
AutoLoader). The raw images files from the
quantitative scanning were analyzed by the
Affymetrix Gene Expression Analysis
Software (MAS 5.0) resulting in cell files
containing background corrected probe values.
Liver microarray KEGG pathway analysis –
Liver microarray data was used to identify
metabolic pathways regulated by treatments,
using the KEGG resource (38,39), release 43.
Genes with at least two-fold change in
expression level between the two treatment
groups were taken as differentially expressed
genes (DEGs). The enrichment of these DEGs
among KEGG pathways was measured by twotailed
Fisher’s exact test. The pathways with P-
value less than 0.05 were considered as
statistically significant and were further
studied.
Histological analyses – Lipids in cryosections
of frozen liver samples were stained by the
standard Oil Red-O method. Parts of the
epididymal white adipose tissue (eWAT) were
subjected to standard hematoxylin and eosin
staining as previously described (40).
Plasma measurements - Lipids, metabolites
and enzyme activities in plasma were
determined by commercially available
enzymatic kits: alanine aminotransferase
(ALAT), total cholesterol, HDL-cholesterol,
LDL-cholesterol (Dialab, Vienna, Austria),
glucose, TAG (MaxMat, Montpellier, France),
OH-butyrate (Randox, Crumlin, United
Kingdom). Hormones were measured in EDTA
plasma containing aprotonin: insulin by an
ELISA kit (DRG Diagnostics, Germany) and
glucagon by a Radioimmunoassay (RIA) Kit
(LINCO Research, ST. Charles, MO, USA).
Liver glycogen content – Glycogen in liver
samples was converted to glucose by treatment
with amyloglucosidase as described elsewhere
(41). Glucose content after the treatment was
determined using a commercial glucoses
quantification kit (Dialab, Vienna, Austria).
Liver lipid analyses - Total lipids were
extracted from liver samples with chloroform:
methanol, 2:1 (v/v). The lipid classes (TAG,
cholesterol and sterol esters) were analyzed on
an automated Camaq HPTLC system and
separated on HPTLC silica gel 60 F plates as
previously described (42).
Liver mitochondrial preparation and CPT I
assay – Liver mitochondria-enriched fractions
were prepared as previously described (30).
Freshly prepared mitochondrial enriched
fractions were used for determination of
carnitine-palmitoyl transferase capacity, as
previously described (43), with or without
addition of the CPTI inhibitor malonyl-CoA
(5µM).
Liver cytosolic preparation and total
glutathione determination – After removal of
the mitochondrial enriched fraction, the
homogenates were further centrifuged at
100 000g av for 90 minutes at 4 o C. The
supernatant collected after this centrifugation
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4

was used as the liver cytosolic fraction and
total glutathione (GSH) content was
determined by a colorimetric assay, Bioxytech
GSH-400 (OXISResarch TM , Portland, OR,
USA).
Adipose tissue fatty acid oxidation capacity –
samples from eWAT and iBAT was
homogenized in a glycine-glycine buffer as
previously described (44). The homogenates
were centrifuged at 1000 g for 5 minutes and
the supernatant was collected (post-nuclear
fraction). Fatty acid oxidation capacity was
measured in the post-nuclear fractions by the
acid-soluble product method using radiolabeled
palmitoyl-Coenzyme A as the
substrate, as previously described in detail
(30).
In vitro adipocyte differentiation and fatty acid
oxidation – The stromal vascular fraction was
isolated from eWAT dissected out from rats as
earlier described (37). Contaminating
erythrocytes were eliminated from the stromalvascular
fraction by a wash with sterile
distilled water. Cells were plated and induced
to differentiate as described (37).
Differentiated cells were treated with
increasing concentrations of taurocholic acid in
the medium for 24h. Afterwards, the cells were
scraped and homogenized in a buffer
containing 0.25 M sucrose, 2 mM EGTA and
10 mM Tris/HCl, pH 7.4. Fatty acid oxidation
capacity was evaluated by the total amount of
acid-soluble products using radio-labeled
palmitoyl-Coenzyme A as the substrate, as
previously described in detail (30).
Energy in faeces and diets - The gross energy
content was determined in a bomb calorimeter
following the manufacturer’s instruction (Parr
Instruments, Moline, IL, USA).
Statistical analysis – Data are presented as
mean+S.E. Analysis of variance was
performed by post-hoc pairwise comparison
Student’s t-test or Tukey’s HSD test. Data that
failed to show homogeneity in variance by
Levenes’ test were tested by the nonparametric
tests Mann-Whitney U test or
Kruskal-Wallis test. Data were considered
statistically different at P

the SPH+c’am treated groups (Fig 1I).
However, the lean body mass including bones
was borderline reduced in the SPH-treated
(231±4 g, P = 0.052) but was significantly
reduced in the SPH+cholestyramine-treated
(223±1 g, P = 0.005), relative to the casein-fed
rats (259±7 g).
Since bile acids are known to increase
energy expenditure and energy dissipation in
the form of heat (20), we considered it likely
that the reduced fat content in the SPH-treated
rats was accompanied by higher energy
dissipation. To demonstrate this we determined
energy expenditure in the rats by indirect
calorimetry. Indeed, SPH-treated rats had
significantly higher O 2 consumption and heat
production, as well as CO 2 elimination relative
to the casein treated rats (Fig 1K-M).
Importantly, and providing further evidence for
a role of the increased plasma BA
concentration with regard to the increased
energy expenditure, pharmacological lowering
of plasma BAs attenuated heat production, and
led to higher body fat content at an equal
energy intake (Fig 1J-M). The higher CO 2
production in the SPH-treated rats was
independent of the reduced bodyweight in
these animals, as total CO 2 production
(Liter/24h) was higher in the SPH, relative to
the casein fed rats, 8.69 ± 0.15 and 8.09 ±
0.11, respectively (P = 0.03). The
corresponding value for the SPH+c’am treated
rats was 8.31 ± 0.18, which was not
statistically different from neither the SPH nor
the casein treated rats.
Nutritional regulation of endogenous
BA metabolism modulates fat oxidation
capacity in brown and white adipose tissues.
Mice ingesting cholic acid supplemented diets
have increased fat oxidation and energy
dissipation as heat in iBAT, which is important
to prevent high-fat diet-induced adiposity (20).
To elucidate whether nutritional modulation of
plasma BAs could induce fat oxidation in
iBAT, we measured ex vivo fatty acid
oxidation capacity in iBAT. In agreement with
the increased whole-body heat production,
fatty acid oxidation capacity was significantly
higher in iBAT from SPH fed rats compared
with rats fed casein (Fig 2A). The metabolic
effect of bile acids on energy expenditure is
critically dependent on the cAMP-inducible
thyroid activating enzyme type 2
6
iodothyronine deiodinase (Dio2) and is lost in
Dio2 -/- mice (20). Expression of Dio2 has been
linked to expression of the thyroid-responsive
gene, uncoupling protein 1 (Ucp1) (45).
Exogenously added bile acids have also been
shown to increase the expression of
peroxisome proliferator-activated receptor γ
coactivator 1α (Pgc-1α) in brown adipose
tissue (20), a key regulator of mitochondrial
biogenesis and energy expenditure (46,47).
Here we show that the higher heat production
and fatty acid oxidation capacity in iBAT was
accompanied by induction of genes encoding
Ucp1 and Dio2 whereas Pgc-1α expression
was not significantly increased (Fig. 2B-D).
Even though brown adipose tissue is
considered the major adipose tissue to dissipate
chemical energy in the form of heat (48), white
adipose tissue is plastic and under certain
conditions the expression of Pgc-1α and Ucp1
can be induced (49-51). Under conditions with
increased intracellular cAMP signaling, studies
in mice suggest that induced expression of
Ucp1 in white adipose tissues is associated
with a lean and healthy phenotype (40,52-54).
As BAs are known to increase intracellular
cAMP levels, we speculated whether the
increased plasma BA concentrations could also
alter the phenotype of WAT. Indeed, the SPHtreated
rats displayed higher expressions of
Ucp1, Dio2 and Pgc-1α in white adipose
tissues of subcutaneous origin (inguinal,
iWAT) and of abdominal origin, eWAT and
MeWAT (Fig. 2E-G). These data supported
the idea that not only iBAT, but also white
adipose tissues contributed to the lean
phenotype in the SPH-treated rats.
To further corroborate the hypothesis
that nutritional regulation of plasma BA levels
was of importance for the WAT phenotype, we
investigated WAT from the rats treated with
SPH and SPH+c’am. Pharmacological removal
of BAs from circulation attenuated the
reduction in abdominal WAT mass (eWAT +
MeWAT + perirenal/retroperitoneal WAT)
(Fig. 2H). Furthermore, ex vivo fat oxidation
capacity and expression of Ucp1, Dio2 and
Pgc-1α were lower in eWAT from SPH+c’am,
relative to the SPH treated rats, even though
the difference did not reach significant levels
(Fig. 2I, J). The lower plasma BA
concentrations were also accompanied by
larger adipocyte cell sizes in eWAT (Fig. 2K).
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In order to demonstrate that bile acids could
induce fat oxidation in white adipocytes, we
isolated preadipocytes from the eWAT depot,
and treated differentiated adipocytes with
increasing concentrations of taurocholic acid
for 24hrs. Compared to untreated cells, a
physiologic relevant dose of 25 µM
taurocholic acid significantly raised fat
oxidation capacity in the adipocytes (Fig 2L).
Nutritional regulation of endogenous
BA metabolism alters TAG concentrations in
liver and plasma. Oral bile acid treatment
lowers TAG concentrations in mouse liver (11)
and blood (11,55). Moreover, BA treatment
has been reported to down-regulate fatty acid
de novo synthesis via repression of Sterol
regulatory-element binding protein 1c (Srebp-
1c) and its downstream lipogenic target genes
in mouse primary hepatocytes and liver
(11,56). We found no significant differences in
hepatic gene-expressions of Srebp-1c (24±4 vs
34±4, P=0.18), Acetyl-CoA carboxylase 1
(Acc1) (36±2 vs 43±2, P=0.10), fatty acid
synthase (Fas) (3.8±0.8 vs 5.7±1.3, P=0.10)
between the high-fat SPH and high-fat casein
treated rats, respectively. Others have reported
that in HepG2 cells, BA treatment represses
transcription of microsomal triglyceride
transfer protein (MTP) and APO B, both
important for hepatic secretion of VLDL (57).
However, the high-fat SPH-treated rats had
higher liver expression of ApoB (6.3±0.6 vs
4.1±0.2, P=0.009) and Mtp (6.6±0.5 vs
5.0±0.2, P=0.14), compared to high-fat casein
treated rats, respectively. Our results obtained
using rat therefore deviate from the reported
findings in mice and HepG2 cells.
To gain further information on the
mechanisms by which nutritional regulation of
BA metabolism modulates hepatic TAG
metabolism we used a microarray approach.
To identify functional differences between
treatment groups, we performed a Kegg
pathway analysis. Six Kegg metabolic
pathways were significantly altered, and four
tended (P

Accordingly, when we measured
mitochondrial CPT-1 capacity in the presence
of 5 µM malonyl-CoA, we observed a stronger
inhibition in SPH-treated rats than in casein
treated rats, even though the difference not was
significant (Fig. 4G). Furthermore, the fasting
plasma levels of the ketone-body β-
hydroxybutyrate, a marker of liver
mitochondrial fatty acid oxidation was
significantly elevated (Fig. 4H) and liver TAG
deposition was lower in the SPH-treated rats
(Fig. 4I, J), further supporting that the SPHtreated
rats had higher liver fat catabolism.
Exogenous bile acid supplementation (11), as
well as the activation of the PPAR β/δ receptor
(61) prevents increased plasma TAG through
inhibition of hepatic VLDL secretion. Higher
liver Vldlr expression might be one underlying
mechanism to the lower plasma VLDL
amounts, as both BA-treatment (10) and
expression and activation of the
PPARβ/δ receptor (62) might induce Vldlr
transcription. As the rats given the high-fat
SPH diet had both elevated plasma BA
concentrations and induction of Pparβ/δ gene
expression in liver, we hypothesized that
plasma TAG would be decreased in these
animals. As predicted, the plasma TAG
concentrations were reduced in the non-fasted
state (Fig. 4K), and this decrease was
accompanied by a pronounced elevation in
liver Vldlr expression (Fig. 4L). To verify that
plasma VLDL concentration actually was
reduced, we analyzed plasma samples by
nuclear magnetic resonance (NMR), which
confirmed that SPH-treated rats had decreased
VLDL amounts (Fig. 4M). The NMR analysis
also suggested that plasma LDL- or HDLcholesterol
concentrations were elevated in the
SPH-treated rats, but no significant differences
were observed in plasma cholesterol levels by
standard clinical chemistry methods (Fig. N,
O).
Our data supported the hypothesis that
nutritional regulation of endogenous BA
metabolism could modulate hepatic fatty acid
oxidation capacity, liver TAG storage and
plasma non-fasted TAG-rich VLDL
concentrations. To investigate further the
potential role of BAs in relation to the
observed effects on TAG metabolism, we
examined the rats treated with
SPH+cholestyramine. Indeed, pharmacological
removal of BAs by the cholestyramine
treatment attenuated the reduction in liver and
non-fasted plasma TAG concentrations (Fig.
4P, Q), and attenuated the elevation in liver
mitochondrial fat oxidation capacity and nonfasted
plasma β-hydroxybutyrate (Fig. 4R, S).
We conclude that nutritional regulation of
endogenous BA metabolism contributes
significantly to the observed modulation of
lipid metabolism introduced by SPH treatment.
Nutritional regulation of endogenous
BA metabolism modulates skeletal muscle
Ucp3 expression. PPARβ/δ is abundantly
expressed in skeletal muscle, a peripheral
tissue that accounts for approximately 40% of
total body mass. Activation of PPARβ/δ leads
to increased expression of genes involved in
lipid catabolism and energy uncoupling in
skeletal muscle (63-65). One of the genes
robustly induced by PPARβ/δ activation is
Ucp3, and mice over-expressing the human
UCP3 in skeletal muscle are protected from
diet-induced adiposity (66). In keeping with
the strong hepatic Pparβ/δ induction
combined with the lean phenotype of the SPH
treated rats, we hypothesized that
PPARβ/δ signaling was altered also in skeletal
muscle of the SPH treated rats. As in liver,
skeletal muscle expressions of Pparα and its
target genes Acox1 were down-regulated in the
SPH treated rats (Fig. 5A). In contrast,
expression of Pparβ/δ , and its downstream
target genes Adrp and angiopoietin-like 4
(Angptl4, also called fasting-induced adipose
factor, Fiaf) were significantly induced (Fig.
5B). Concomitantly, Cpt-1b, Ucp2 and in
particular Ucp3 expressions were elevated
(Fig. 5C). To further corroborate a regulatory
role for BAs on skeletal muscle Ucp3
expression, we measured the Ucp3 expression
in the SPH+c’am treated rats. Of note, skeletal
muscle Ucp3 induction by SPH treatment was
completely abolished by reducing plasma BA
concentrations with cholestyramine (Fig. 5D).
Our data indicate that besides the known
regulatory effects of BAs on liver and adipose
tissue metabolism, skeletal muscle fatty acid
oxidation and uncoupling may also be
regulated through altered bile acid metabolism.
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8

Nutritional regulation of endogenous
BA metabolism modulates high-fat dietinduced
hyperglycemia. Oral BA treatment
reduces hyperglycemia in mice (12), possibly
through reduced hepatic glucose output by
repression of the Pck1 gene expression (13,14).
In addition, PPARβ/δ activation reduces
hepatic glucose output and improves peripheral
glucose disposal (67). In the present study, the
SPH-treated rats both had elevated plasma BA
levels and induction of Pparβ/δ target gene
expression in liver and skeletal muscle.
Therefore, we decided to examine whether
glucose metabolism was improved in these
animals. The non-fasted plasma insulin and
liver Pck1 expression were lower in the SPHtreated
rats (Fig. 6A, B). Hepatic glycogen
concentrations were significantly higher (Fig. 6
C), whereas non-fasted glucose was
significantly lower in the SPH-treated rats
(Fig. 6D). No difference was observed in
fasting plasma glucose levels (Fig. 6D). The
lower fed-state plasma insulin and glucose
concentrations, as well as the reduced hepatic
Pck-1 expression in the SPH fed rats might
indicate that these animals were more insulinsensitive,
relative to those fed casein. This
notion is in agreement with a recent report in
which rats fed a high-fat SPH-diet had
increased whole-body insulin-sensitivity
determined by the hyperinsulinemiceuglycemic
clamp technique (68). In the
present study, pharmacological removal of
BAs attenuated the reduction in non-fasted
blood glucose (Fig. 6E). Therefore, our data
support the hypothesis that nutritional
modulation of endogenous BA metabolism
may regulate blood glucose concentrations.
Possible determinants of the elevated
plasma BA levels in the SPH treated rats. In
the present study, the SPH treated rats had
elevated plasma BA levels. Increased
circulating BAs might be due to cholestasis or
increased bile acid synthesis. The plasma
levels of alanine aminotransferase, although
slightly elevated, did not indicate
hepatocellular damage in the SPH fed rats (Fig.
7A). Furthermore, liver Ucp2 gene expression,
recently shown to be up-regulated in bile duct
obstructed rats (69), was borderline (P=0.053)
down-regulated in the SPH fed rats (Fig. 7B).
Hence, it is unlikely that the elevated plasma
BA level was due to cholestasis in the present
9
study.
Supplementing taurine (70,71) or
glycine (70) at doses of 50g/ kg diet to caseinbased,
atherogenic diets (containing 1wt%
cholesterol and 0.25 wt% cholic acid) have
previously been shown to increase fecal BA
excretion in rats. Furthermore, the rats treated
with the taurine-supplemented diet had higher
liver GSH content, decreased liver cholesterol
concentration and higher cholesterol-7a
hydroxylase (Cyp7a1) mRNA levels and
enzyme activity (71). In the present study, the
SPH diet provided taurine (1.9g/kg diet),
whereas the casein diet was free of taurine.
Also, the glycine level differed and was 23 and
4 g/ kg diet in the SPH and casein diets,
respectively (Supplemental Fig. 1). The SPHtreated
rats had significantly reduced liver
cholesterol and sterol ester concentrations (Fig.
7C). However, the SPH fed rats had lower liver
expression of genes involved in the de novo
bile acid synthesis, Cyp7a1, and in bile acid
conjugation, Bile acid Coenzyme A: amino
acid N-acyltransferase, (Baat) (Fig. 7D).
Therefore, our data does not support that a
higher BA synthesis was the underlying factor
for the elevated plasma BA concentrations.
Secretion of bile acids into bile is
partly dependent on bile acid availability and
transport, and partly determined by the
availability and transport of phospholipids,
cholesterol, glutathione and bicarbonate, as
reviewed in (72). In the present study, the liver
expression of genes involved in canalicular
bile flow generation was either increased (Atpbinding
cassette b4, Abcb4, also called Mdr2),
tended (P=0.09) to be increased (Abcb11/
Bsep), or was statistically unaltered
(Abcb1b/Mdr1 and Abcc2/ Mrp2) in the SPHtreated
rats (Fig. 7E). Furthermore, the liver
gene expression of Glutathione synthase (Gss),
as well as the liver concentration of glutathione
(GSH), a primary osmotic driving force in
hepatic bile formation (73), was increased by
SPH treatment (Fig. 7F, G). However, fecal
BA excretion measured over 5 days did not
differ between the treatments (Fig. 7H). Thus,
our liver data indicate that the SPH fed rats had
increased biliary BA secretion, yet the fecal
BA excretion was unaltered. If this was the
case, the SPH-fed rats must have had an
efficient intestinal BA re-uptake. In humans
with a normal hepatic function, the major
determinant of circulating bile acid
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concentration is their rate of intestinal
absorption (74). In the present study, the
hepatic amount of α-muricholic acid, a bile
acid of intestinal origin (75) was significantly
higher in the SPH-fed rats (Fig. 1B). Based on
the higher hepatic presence of intestinalderived
BA species, as well as the fact that the
SPH + cholestyramine treated rats exhibited
reduced plasma BA levels, we conclude that
the increased plasma BA level in the SPH-fed
rats was likely due to higher intestinal influx.
DISCUSSION
Bile acids strongly affect metabolism
and energy-expenditure in mice (11,12,20).
From studies in rats, it is known that
supplementing low-fat, casein-based diets with
rather high doses of glycine and/or taurine
affects bile acid metabolism (29,70,71).
Previously, we have reported that exchanging
casein with a glycine and taurine rich proteinsource
modulated BA metabolism in low-fat
fed rats (30). Here we show that choice of
dietary protein source is sufficient to increase
plasma BA levels also in high-fat fed rats.
Importantly, the elevated plasma BA level was
accompanied with attenuated diet-induced
obesity and ameliorated characteristics of the
metabolic syndrome.
Little is known about the role(s) that
endogenous BA metabolism may play in the
development of the metabolic syndrome.
However, a few recent publications support
such a role for endogenous bile acid
metabolism. Bile secretion is impaired in both
the Zucker (fa/fa) rat (76) and in the ob/ob
mice (77), two animal models extensively
studied as they both develop metabolic features
resembling the metabolic syndrome.
Furthermore, in lean and fat mouse lines
developed from the same founder population
by long-term divergent selection for low- or
high body fat %, the lean mice exhibited
higher hepatic Cyp8b1 and Abcb11 gene
expressions, and had increased blood BA
concentrations, relative to the obese mice (78).
Even though the underlying mechanisms need
to be further elucidated, our data strongly
indicate a regulatory role for endogenous bile
acid metabolism on the development of the
metabolic syndrome.
We used diets in which the casein was
fully exchanged with SPH. Obviously,
10
differences in dietary amino acids other than
taurine and glycine, such as the level of the
branched-chain amino acids might have
affected the outcome in the present study
(Supplemetal Fig. 1). Moreover, we recognize
that taurine has metabolic effects beyond bile
acid metabolism, such as anti-oxidant capacity
(79). However, the taurine tissue level in the
rats (Supplemetal Fig 1) excludes the
possibility that the observed differences were
due to increased tissue taurine concentrations.
Since addition of cholestyramine reduced
plasma BA levels and attenuated the beneficial
effects on adiposity, TAG metabolism and
hyperglycemia, we conclude that parts of the
beneficial effects found by SPH treatment was
caused by nutritional regulation of endogenous
BA metabolism.
Our results provide evidence that
nutritional regulation of bile acid metabolism
may attenuate characteristics of the metabolic
syndrome in high-fat fed rats. The relevance of
our findings for man remains to be elucidated.
However, it has been reported that obese
subjects had a lower post-prandial bile-acid
response, relative to normal weight subjects
(80). Furthermore, subjects that had previously
undergone gastric bypass, showed higher
circulating bile acid levels, relative to both
obese and severely obese objects. Also, total
bile acids were inversely correlated with 2hr
post meal-glucose and fasting TAG levels (81).
In another study with objects subjected to
bariatric surgery, circulating bile acid and
GLP-1 levels increased post surgically, relative
to pre-surgical baseline levels (82). Finally, it
was reported in a human intervention study
with cross-over design that subjects on a high
fat, high protein diet had increased fasting
plasma bile acid concentrations as compared to
subjects on a high-fat diet alone. The elevation
in plasma BA levels was accompanied by
reduced hepatic lipids and increased fasting
plasma concentrations of β-hydroxybutyrate
(83). Thus, nutritional regulation of
endogenous BA metabolism may also be
related to development of the metabolic
syndrome in man.
In conclusion, we provide compelling
evidence that plasma bile acid levels can be
modulated by the dietary protein source in
high-fat treated rats. Increased levels of plasma
BAs were associated with a significant
reduction in diet-induced obesity and resulted
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in increased whole body energy expenditure
and dissipation of energy in the form of heat.
Concomitantly, fed-state plasma glucose and
TAG concentrations were reduced. Thus,
protein source dependent increase in plasma
BA levels appears to have the potential to
attenuate pathological characteristics of the
metabolic syndrome in the high-fat fed rats.
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11

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14

FOOTNOTES
This work was carried out as a part of the ‘DOCMAR’ research program funded by Innovation
Norway and RUBIN. This work was supported by the Danish Natural Science Research Council, The
Novo Nordisk Foundation, The Carlsberg Foundation and performed as part of the research program
of the Danish Obesity Research Centre, supported by the Danish Council for Strategic Research, Grant
2101-06-0005. Financial support was also received from the University of Bergen, Program
Committee on Nutrition, from the Eckbo Foundation and from the Norwegian Research Council,
Grant 200515-I30.
ACKNOWLEDGMENTS
We thank Aase Heltveit and Lars Erik Pindard for help during the animal studies. We also thank Guro
K. Christensen and Jacop Wessels for valuable technical assistance.
FIGURE LEGENDS
FIGURE 1. Elevated plasma bile acid concentration is an underlying factor for the increased
energy expenditure and decreased adiposity elicited by salmon protein hydrolysate (SPH)
feeding.(A-F) Male Wistar rats (n=6) were fed high-fat diets (45 kcal% fat) ad libitum for 46 days
with either SPH or casein as the sole protein source. (A-B) SPH-fed rats had elevated plasma bile
acids (BAs), whereas total liver BAs were unchanged. (C-E) The SPH-fed rats showed reduced body
weight gain. Energy efficiency, calculated as bodyweight gain per energy intake, and white adipose
tissue masses were reduced by SPH treatment. (F) Energy digestibility was equal in SPH and casein
fed rats. (G-M) Three groups of rats (n=6) were pair-fed the SPH-diet, the casein diet and the SPHdiet
with 0.5wt% cholestyramine (c’am). (G-I) Inclusion of cholestyramine to the SPH-diet attenuated
the increase in plasma BA concentrations, without modulating liver total BAs or growth. (J) Inclusion
of cholestyramine attenuated the reduction in body fat mass determined by dual-X ray absorptiometry
(DEXA). (K-L) Three groups of rats (n=6) were pair-fed the SPH-diet, the casein diet and the SPHdiet
with 0.5wt% cholestyramine (c’am) and energy expenditure was calculated by indirect
calorimetry. Cholestyramine treatment attenuated the increase in O2 consumption, CO2 elimination and
heat production. Data are presented as mean + S.E. Significant differences from casein-fed rats are
denoted by * (P

FIGURE 3. Liver microarray analysis. Male Wistar rats (n=6) were fed high-fat diets (45 kcal% fat)
ad libitum for 46 days with either SPH or casein as the sole protein source. RNA from individual rats
was pooled within experimental groups, and the pooled samples were analyzed by Affymetrix arrays.
(A) Data were analyzed and significantly altered KEGG pathways (P

fed the same diets, plus the SPH-diet with 0.5wt% cholestyramine (c’am) for 46 days.
Pharmacological removal of bile acids by 0.5 wt% cholestyramine attenuated the glucose lowering
effect of SPH. Pck1 mRNA levels are normalized to General transcription factor IIB (Gtf2b). Data are
presented as mean + S.E. Significant difference from casein-fed rats are denoted by * (P

Figure 1
A
30
Plasma BA
**
B
1,5
Liver BAs
SPH
Casein
C
300
Growth chart
µmol/ L
20
10
0
SPH
Casein
µmol/ tissue
1,0
0,5
0,0
**
Gram BW
200
100
0
0 10 20 30 40 50
Days of feeding
Casein
SPH
D
Energy efficiency
E
Adipose tissue masses
F
Energy digestibility
Gram BW/ MJ intake
21
14
7
0
**
SPH
Casein
g tissue
6
4
2
0
**
**
eWAT MeWAT iBAT
SPH
Casein
% of ingested energy
96
64
32
0
SPH
Casein
G
µmol/ L
J
Gram
54
36
18
0
90
60
30
0
Plasma BA
**
SPH SPH +
c'am
Body fat
**
SPH SPH +
c'am
Casein
Casein
H
µmol/ tissue
K
Liter O2/ kg BW/ 24h
3
2
1
0
60
50
40
Liver total BAs
SPH SPH +
c'am
O 2 consumed
**
*
SPH SPH +
c'am
Casein
Casein
I
Gram BW
L
Liter CO2/ kg BW/ 24h
350
300
250
200
150
55
45
35
Growth chart
0 10 20 30 40 50
CO 2 eliminated
**
*
SPH SPH +
c'am
Days of feeding
Casein
M
kJ/ 24h/ kg BW 0.75
780
720
660
600
SPH SPH +
c'am
Casein
SPH
SPH + c'am
Heat production
**
*
Casein
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18

Figure 2
A
nmol/ min/ mg prot
2,4
1,6
0,8
iBAT Palm-CoA ox
*
B
Ucp1/ Gtf2b
360
240
120
iBAT Ucp1
**
C
Dio2/ Gtf2b
6
4
2
iBAT Dio2
*
D
Pgc-1α/ Gtf2b
3,9
2,6
1,3
iBAT Pgc-1α
0,0
SPH
Casein
0
SPH
Casein
0
SPH
Casein
0,0
SPH
Casein
E
Relative to casein
3,6
2,4
1,2
0
**
WAT Ucp1
iWAT eWAT MeWAT
F
Relative to casein
7,5
5,0
2,5
0,0
WAT Dio2
*
*
*
iWAT eWAT MeWAT
G
Relative to casein
2,4
1,6
0,8
0
WAT Pgc-1α
* *
iWAT eWAT MeWAT
SPH
Casein
SPH
Casein
SPH
Casein
H
Gram
K
30
20
10
0
Abdominal fat
**
SPH SPH +
c'am
Casein
I
Relative to casein
eWAT Palm-CoA ox
1,4
1,2
1,0
0,8
SPH SPH +
c'am
eWAT adipocyte cell size
SPH SPH + c'am Casein
Casein
J
Rel expression
9
6
3
0
L
eWAT gene-expression
Ucp1 Dio2 Pgc-1a
Relative to control
1,8
1,2
0,6
0
In vitro adipocyte Palm-CoA
oxidation
SPH
SPH + c'am
Casein
* ** *
0 25 75 200
Taurocholic acid, µmol/L
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19

Figure 3
A
KEGG Pathway
>2 fold Changed
in this pathway
Total expressed
genes in this
pathway
Fisher P-
value
Genes induced by SPH treatment
Type I diabetes mellitus 6 19 0,015 RT1-Aw2, RT1-A3, RT1-T24-1, -
RT1-CE2, RT1-CE15, RT1-CE10
Genes repressed by SPH
treatment
PPAR signaling pathway 10 49 0,024 Cd36, Fabp5, Fabp2, Cpt1a, Angptl4, Cyp7a1, Apoa2, Scd1
Fads2, Me1
Cell adhesion molecules (CAMs) 10 50 0,027 RT1-Aw2, RT1-A3, RT1-T24-1, RT1-CE2, Ptprm, Igsf4a, Jam3
RT1-CE15, RT1-CE10, Cldn1
Nicotinate and nicotinamide metabolism 4 11 0,031 - Enpp3, Enpp2, Pbef1, Aox1
Bile acid biosynthesis 5 18 0,038 Hadhb Adh7, Adh1, Srd5a1, Cyp7a1
Sulfur metabolism 3 7 0,044 - Smp2a, Sult2a1, Sult2a2_predicted
Antigen processing and presentation 7 35 0,058 RT1-Aw2,RT1-A3,RT1-T24-1,RT1-CE2, Klrd1
RT1-CE15,RT1-CE10
Terpenoid biosynthesis 2 3 0,058 Fdps, Fdft1 -
Jak-STAT signaling pathway 8 47 0,085 Akt2, Socs2, Stat3, Stat5b, Stam2, Ghr Il6ra, RGD1560373_predicted
Hematopoietic cell lineage 5 25 0,100 Itga5, Itga5_mapped, Cd36, Thpo Il6ra, Cd1d1
B
Signal log ratio < -2 -2,0 -1,5 -1,0 -0,5 0.5 1.0 1,5 2,0 > 2
Function
Gene
Cellular lipid import
Fatty acid binding
cd36 antigen
lipase, hepatic
low density lipoprotein receptor
low density lipoprotein receptor-related protein 3
low density lipoprotein receptor-related protein 4
low density lipoprotein receptor-related protein associated protein 1
very low density lipoprotein receptor
fatty acid binding protein 2, intestinal
fatty acid binding protein 4, adipocyte
fatty acid binding protein 5, epidermal
fatty acid binding protein 6, ileal (gastrotropin)
Fatty acid activation acyl-CoA synthetase long-chain family member 3
acyl-CoA synthetase long-chain family member 4
acyl-CoA synthetase long-chain family member 5
acyl-CoA synthetase long-chain family member 6
Mitochondrial fatty acid oxidation
carnitine palmitoyltransferase 1, liver
carnitine palmitoyltransferase 1b
solute carrier family 25 (carnitine/acylcarnitine translocase), member 20
carnitine palmitoyltransferase 2
acyl-Coenzyme A dehydrogenase, short/branched chain
acyl-Coenzyme A dehydrogenase, very long chain
enoyl coenzyme A hydratase 1, peroxisomal/mitochondrial
HADH (trifunctional protein), alpha subunit
HADH (trifunctional protein), beta subunit
mitochondrial acyl-CoA thioesterase 1
dodecenoyl-coenzyme A delta isomerase
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Krebs/ TCA-cycle
citrate synthase
aconitase 2, mitochondrial
isocitrate dehydrogenase 3, gamma
malate dehydrogenase, mitochondrial
pyruvate dehydrogenase kinase, isoenzyme 2
pyruvate dehydrogenase kinase, isoenzyme 4
Ketone body metabolism acetyl-coenzyme A acetyltransferase 1
3-hydroxy-3-methylglutaryl CoA lyase
20

Figure 4
A
6
Liver Pparα
B
21
Liver mRNA levels
C
2,1
Liver Ppar β/δ
**
D
33
Liver Adrp
**
Ppar α/ Gtf2b
4
2
*
Rel expression
14
7
*
SPH
Casein
Ppar β/δ/ Gtf2b
1,4
0,7
Adrp/ Gtf2b
22
11
0
SPH
Casein
0
Cpt-1a
Acox1
0,0
SPH
Casein
0
SPH
Casein
E
nmol/ mg prot/ min
Liver mit CPT-1
5,1
3,4
1,7
0,0
*
SPH
Casein
F
Acc2/ Gtf2b
0,9
0,6
0,3
0,0
Liver Acc2
**
SPH
Casein
G
% inhibition
CPT-1 inhibition by
malonyl-CoA
0
-15
-30
-45
SPH
Casein
H
mmol/ L
Plasma OH-butyrate
*
1,5
1,0
0,5
0,0
Fasted
Fed
SPH
Casein
I
mg/ tissue
M
Regression coefficient
270
180
90
0
40
30
20
10
0
-10
Liver TAG
**
SPH
(R 2 =0.92)
Casein
J
Liver Oil red-O
staining
SPH
Casein
Fed plasma metabolomic by NMR
Unassigned peaks
(not in casein)
Tyr
Lipid (unsaturated)
Choline
Creatine
Pyruvate or oxalacetate
K
mmol/ L
3
2
1
0
Plasma TAG
Fasted
*
Lipid (mainly HDL or LDL)
Fed
SPH
Casein
L
Vldlr/ Gtf2b
N
mmol/ L
O
36
24
12
2,4
1,6
0,8
0,0
0,9
0
Liver Vldlr
*
SPH
Fed plasma
cholesterol
Total
HDL
Casein
Fed plasma
cholesterol
SPH
Casein
Downloaded from www.jbc.org by guest, on July 7, 2011
-20
-30
Glycoprotein
Lipid (mainly VLDL)
mmol/ L
0,6
0,3
SPH
Casein
-40
9 8 7 6 5 4 3 2 1
Chemical shift (ppm)
0,0
LDL
HDL/total
P
mg/ tissue
240
160
80
0
Liver TAG
SPH SPH +
c'am
Casein
Q
mmol/ L
Fed plasma TAG
4,8
3,2
1,6
0,0
**
SPH SPH +
c'am
Casein
R
nmol/ mg prot/ min
Liver mit Palm-CoA
oxidation
2,1
1,9
1,7
1,5
SPH SPH +
c'am
Casein
S
mmol/ L
Fed plasma OHbutyrate
0,51
0,34
0,17
0,00
SPH SPH +
c'am
Casein
21

Figure 5
A
Ppar α related
B
Ppar β/δ related
Relative expression
1,8
1,2
0,6
0
* *
Ppar a Acox Mcad
SPH
Casein
Relative expression
2,7
1,8
0,9
0
** *
**
Ppar b/d Adrp Angptl4
SPH
Casein
C
Relative expression
5,1
3,4
1,7
0,0
Fatty acid oxidation and uncoupling
*
*
**
Cpt-1b Ucp2 Ucp3
SPH
Casein
D
Relative expression
2,4
1,6
0,8
0
Ppar β/δ and Ucp3
*
##
Ppar b/d
Ucp3
SPH
SPH+c'am
Casein
Figure 6
A
pmol/ L
D
mmol/ L
81
54
27
0
18
12
6
Fed state hormones
*
Insulin Glucagon
Plasma glucose
**
SPH
Casein
SPH
Casein
B
E
Pck1/ Gtf2b
mmol/ L
1,5
1,0
0,5
0,0
12,6
8,4
4,2
Liver Pck1
*
C
mg/ g liver
Liver glycogen
105
70
35
**
0
SPH Casein
SPH Casein
Fed plasma glucose
*
SPH
SPH + c'am
Casein
Downloaded from www.jbc.org by guest, on July 7, 2011
0
Fasted
Fed
0,0
SPH
SPH + c'am Casein
22

Figure 7
A
Plasma ALAT
B
Liver Ucp2
C
Liver cholesterol
U/ L
39
26
13
0
**
SPH
Casein
Relative expression
1,2
0,8
0,4
0
SPH
Casein
mg/ tissue
36
24
12
0
**
Cholesterol
**
Sterol esters
SPH
Casein
D
Relative expression
F
Relative expression
1,2
0,8
0,4
0
1,8
1,2
0,6
0,0
Bile acid synthesis
Cyp7a1
**
Baat
SPH
Casein
Liver expression GSH
synthezising genes
**
Gclc Gclm Gss
SPH
E
Relative expression
Casein
2,7
1,8
0,9
0
G
µmol/ mg protein
Apical hepatocyte transporters
Abcb11/
Bsep
0,18
0,12
0,06
0,00
Abcc2 /
Mrp2
Liver GSH
*
SPH
**
Abcb4/
Mdr 2
Casein
Abcb1b/
Mdr1
H
µmol/ 5 days
51
34
17
0
SPH
Casein
Fecal bile acids
SPH
Casein
Downloaded from www.jbc.org by guest, on July 7, 2011
23

Research
Persistent Organic Pollutant Exposure Leads to Insulin Resistance Syndrome
Jérôme Ruzzin, 1 Rasmus Petersen, 2,3 Emmanuelle Meugnier, 4 Lise Madsen, 1,3 Erik-Jan Lock, 1 Haldis Lillefosse, 1
Tao Ma, 2,3 Sandra Pesenti, 4 Si Brask Sonne, 3 Troels Torben Marstrand, 5 Marian Kjellevold Malde, 1 Zhen-Yu Du, 1
Carine Chavey, 6 Lluis Fajas, 6 Anne-Katrine Lundebye, 1 Christian Lehn Brand, 7 Hubert Vidal, 4 Karsten Kristiansen, 3
and Livar Frøyland 1
1 National Institute of Nutrition and Seafood Research (NIFES), Bergen, Norway; 2 Department of Biochemistry and Molecular Biology,
University of Southern Denmark, Odense, Denmark; 3 Department of Biology, University of Copenhagen, Copenhagen, Denmark;
4 INSERM U-870, INRA (Institut national de la recherche agronomique) U-1235, Lyon 1 University, Institut National des Sciences
Appliquées de Lyon and and Hospices Civils de Lyon, Oullins, France; 5 The Bioinformatics Centre, Department of Biology and Biotech
Research and Innovation Centre, University of Copenhagen, Copenhagen, Denmark; 6 Institut de Recherche en Cancérologie de
Montpellier, INSERM U896 Metabolism and Cancer Laboratory, Montpellier, France; 7 Department of Insulin Pharmacology, Histology
and Delivery, Novo Nordisk A/S, Maaloev, Denmark
Bac k g r o u n d: The incidence of the insulin resistance syndrome has increased at an alarming rate
worldwide, creating a serious challenge to public health care in the 21st century. Recently, epidemiological
studies have associated the prevalence of type 2 diabetes with elevated body burdens of
persistent organic pollutants (POPs). However, experimental evidence demonstrating a causal link
between POPs and the development of insulin resistance is lacking.
Objective: We investigated whether exposure to POPs contributes to insulin resistance and metabolic
disorders.
Met h o d s: Sprague-Dawley rats were exposed for 28 days to lipophilic POPs through the consumption
of a high-fat diet containing either refined or crude fish oil obtained from farmed Atlantic
salmon. In addition, differentiated adipocytes were exposed to several POP mixtures that mimicked
the relative abundance of organic pollutants present in crude salmon oil. We measured body weight,
whole-body insulin sensitivity, POP accumulation, lipid and glucose homeostasis, and gene expression
and we performed micro array analysis.
Res u l t s: Adult male rats exposed to crude, but not refined, salmon oil developed insulin resistance,
abdominal obesity, and hepatosteatosis. The contribution of POPs to insulin resistance was
confirmed in cultured adipocytes where POPs, especially organochlorine pesticides, led to robust
inhibition of insulin action. Moreover, POPs induced down-regulation of insulin-induced gene-1
(Insig-1) and Lpin1, two master regulators of lipid homeostasis.
Con c l u s i o n: Our findings provide evidence that exposure to POPs commonly present in food
chains leads to insulin resistance and associated metabolic disorders.
Key w o r d s : contaminants, farmed salmon, metabolic syndrome, nonalcoholic fatty liver,
obesity, pollution, public health, type 2 diabetes. Environ Health Perspect 118:465–471
(2010). doi:10.1289/ehp.0901321 [Online 19 November 2009]
factors can only partly explain the worldwide
explosive prevalence of insulin resistance–
associated metabolic diseases. We therefore
sought to elucidate whether the exposure to
POPs present in a food matrix could contribute
to insulin resistance and metabolic
disorders.
POPs accumulate in the lipid fraction of
fish, and fish consumption represents a source
of POP exposure to humans (Dougherty et al.
2000; Hites et al. 2004; Schafer and Kegley
2002). Therefore, certain European countries
have dietary recommendations to limit the
consumption of fatty fish per week (Scientific
Advisory Committee on Nutrition 2004).
On the other hand, n-3 polyunsaturated fatty
acids present in fish oil have a wide range of
beneficial effects (Jump 2002), including protection
against high-fat (HF) diet– induced
insulin resistance (Storlien et al. 1987).
Accordingly, we fed rats an HF diet containing
either crude (HFC) or refined (HFR) fish
oil obtained from farmed Atlantic salmon and
Despite international agreements intended to
limit the release of persistent organic pollutants
(POPs) such as organochlorine pesticides,
polychlorinated biphenyls (PCBs),
polychlorinated dibenzo-p-dioxins (PCDDs),
and polychlorinated dibenzofurans (PCDFs),
POPs still persist in the environment and food
chains (Atlas and Giam 1981; Dougherty et al.
2000; Fisher 1999; Jorgenson 2001; Schafer
and Kegley 2002; Van den Berg 2009). Most
human populations are exposed to POPs
through consumption of fat-containing food
such as fish, dairy products, and meat (Fisher
1999). Humans bioaccumulate these lipophilic
and hydrophobic pollutants in fatty tissues
for many years because POPs are highly
resistant to metabolic degradation (Fisher
1999; Kiviranta et al. 2005). The physiological
impact associated with chronic exposure
to low doses of different mixtures of POPs is
poorly understood, but epidemiological studies
have reported that Americans, Europeans, and
Asian patients with type 2 diabetes accumulated
greater body burdens of POPs, including
2,3,7,8‐tetrachlorodibenzo-p-dioxin (TCDD),
2,2´,4,4´,5,5´-hexachlorobiphenyl (PCB153),
coplanar PCBs (PCB congeners 77, 81,
126, and 169), p,p´-diphenyldichloroethene
(DDE), oxychlordane, and trans-nonachlor
(Fierens et al. 2003; Henriksen et al. 1997;
Lee et al. 2006; Rignell-Hydbom et al. 2007;
Turyk et al. 2009; Wang et al. 2008).
The incidences of type 2 diabetes and the
insulin resistance syndrome have increased at
a globally alarming rate, and > 25% of adults
in the United States have been estimated to
be affected by metabolic abnormalities associated
with insulin resistance (Ford et al. 2004).
Impaired insulin action is a central dysfunction
of the insulin resistance syndrome characterized
by abdominal obesity and defects in
both lipid and glucose homeostasis, increasing
the risk for developing type 2 diabetes,
cardio vascular diseases, non alcoholic fatty
liver disease, polycystic ovarian disease, and
certain types of cancer (Biddinger and Kahn
2006; Reaven 2005). Although a sedentary
lifestyle and consumption of high-fat food
are considered major contributors to insulin
resistance and obesity, these conventional risk
Address correspondence to J. Ruzzin, National
Institute of Nutrition and Seafood Research, N-5817
Bergen, Norway. Telephone: (0047) 41450448. Fax:
(0047) 559052 99. E-mail: jerome.ruzzin@nifes.no
Supplemental Material is available online
(doi:10.1289/ehp.0901321 via http://dx.doi.org/).
We thank J. Burén for technical assistance on primary
adipocyte investigations, Å. Heltveit for animal
care, J.I. Hjelle for analysis of hepatic lipids,
J. Wessel for assistance on tissue harvest, K. Heggstad
for his expertise on contaminant analysis, J. Rieusset
for advice on histology investigation, and C. Debard
for real-time PCR analysis. We thank T. Fenchel,
P. Grandjean, and E. Lund for discussions and comments
on the manuscript.
This work was supported by grants from the
European Research Council, the Research Council
of Norway, the Danish Natural Science Research
Council, The Diabetes Association, and the Novo
Nordisk Foundation.
C.L.B., employed at Novo Nordisk (a leading
manu facturer of insulin analogs and diabetes management
care products), holds shares in the company,
and contributed independently with the clamp studies.
The Novo Nordisk Foundation supports basic science
independently of the company interests of Novo
Nordisk A/S. The remaining authors declare they have
no competing financial interest.
Received 11 August 2009; accepted 19 November
2009.
Environmental Health Perspectives • v o l u m e 118 | number 4 | April 2010 465

Ruzzin et al.
investigated the metabolic impacts of POPs
and their ability to interfere with n-3 polyunsaturated
fatty acids.
Materials and Methods
Tissue RNA from liver of rats fed HFC and
HFR was extracted using Trizol, and microarray
analysis was performed using the Operon
Rat Oparray. Levels of specific mRNA were
quantified using real-time polymerase chain
reaction (PCR) as described previously (Rome
et al. 2008). 3T3-L1 cells were exposed to
different POP mixtures, and we measured
insulin-stimulated glucose uptake and mRNA
expression of target genes. Details of the
methods are available in the Supplemental
Material (doi:10.1289/ehp.0901321).
Animals. All experimental protocols
described below were approved by the
Norwegian State Board of Biological
Experiments with Living Animals, and the
animals were treated humanely and with
regard for alleviation of suffering. Male
Sprague-Dawley rats (Taconic, Ry, Denmark)
weighing 200–250 g were housed with a
12-hr light/dark cycle and with free access
to food and tap water. Animals were fed a
standard diet (chow; 17% fat-derived calories,
3.4 kcal/g) or an HF diet (65% fatderived
calories, 5.5 kcal/g) for 28 days
(Lavigne et al. 2001). Two additional HF
diets were made by substituting corn oil (20%
wt/wt) with either crude or refined salmon
oil. Crude salmon oil was obtained by heating
the rest raw material of farmed Atlantic
salmon to 92°C and separating oil from water
and solid material. Refined salmon oil was
obtained by bleaching, carbon filtering, and
deodorizing the crude oil. HF, HFC, and
HFR diets were supplemented with cellulose
(50 g/kg), choline bitartrate (2 g/kg),
American Institute of Nutrition (AIN) vitamin
mixture 76 (14 g/kg), and AIN mineral
mixture 76 (67 g/kg) (MP Biochemicals,
Inc, Illrich, France) to meet the daily nutrient
requirement levels of adult rats (Reeves et al.
1993). Fatty acid composition of HF, HFC,
and HFR diets was analyzed as previously
described (Jordal et al. 2007).
Hepatic lipids. We determined levels of
triacylglycerol, diacyl glycerol, and total cholesterol
in frozen liver samples of overnight-fasted
rats using high-performance thin-layer chromatography
as described previously (Berntssen
et al. 2005). Frozen (O.C.T. compound;
Sakura Finetek Europe, Zoeterwoude, the
Netherlands) and fixed (paraffin-embedded)
liver sections were stained with Oil red O and
hematoxylin and eosin (H&E), respectively.
Determination of POP levels. We measured
levels of POPs as described previously
(Berntssen et al. 2005; Julshamn et al. 2004).
Determination of insulin action in peripheral
tissues. We used soleus muscles and
epididymal fat of overnight-fasted animals
to assess insulin-stimulated glucose uptake
as described previously (Buren et al. 2002;
Ruzzin et al. 2005).
Hyperinsulinemic–euglycemic clamps.
Animals were catheterized, and hyperinsulinemic–euglycemic
clamps were performed
7 days later (Brand et al. 2003; Ruzzin
et al. 2007). After a 6-hr fasting period,
conscious unrestrained catheterized animals
were infused with a prime (6 µCi) continuous
(0.1 µCi/min for basal; 0.17 µCi/min for
clamp) infusion of [3- 3 H]glucose from –90 to
120 min for assessment of whole-body glucose
disappearance (R d ) and appearance (R a ) using
Steele’s non–steady-state equations (Steele
et al. 1956). The hyperinsulinemic–euglycemic
clamp was performed (0–120 min) by a
continuous infusion of human insulin (3 mU/
kg/min) (Actrapid, Novo Nordisk, Bagsvaerd,
Denmark), and euglycemia (~ 115 mg/dL)
was maintained by variable infusion rates of
a 20% non labeled glucose solution [glucose
infusion rate (GIR)]. At the end of the clamp,
rats were given a lethal dose of pento barbital
sodium; liver, epididymal fat, and gastrocnemius
muscles were removed, frozen in liquid
nitrogen, and stored at –80°C for determination
of POP levels. Plasma glucose and insulin
levels were analyzed by the glucose oxidase
method (YSI 2300 STAT Plus glucose analyzer;
YSI Incorporated, Yellow Spring, OH,
USA) and an enzyme-linked immunosorbent
assay kit (DRG Instruments, Marburg,
Germany), respectively. To determine plasma
[3- 3 H]glucose, plasma was deproteinized,
dried to remove tritiated water, resuspended
in water, and counted in biodegradable scintillation
fluid (Nerliens Meszansky, Oslo,
Norway) on a beta scintillation counter (Tri-
Carb 1900TR; Packard, Meriden, CT, USA).
All samples were run in duplicate. Hepatic
glucose production (HGP) was calculated as
tracer-determined R a minus GIR.
Insulin resistance was further assessed by
the homeostasis model assessment of insulin
resistance (HOMA-IR) index as described by
Lee et al. (2008).
Cultured adipocyte studies. We used cultured
and differentiated 3T3-L1 cells (Petersen
et al. 2008) to assess insulin-stimulated glucose
uptake and mRNA expression of target genes.
On day 8 of the differentiation program, cells
were exposed to vehicle (dimethyl sulfoxide)
or POP mixtures for 48 hr, and glucose uptake
was assessed.
Cytotoxicity. Membrane integrity of
POP-treated adipocytes was determined
by the release of lactate dehydrogenase into
cell medium by a Tox7 kit (Sigma-Aldrich,
Leirdal, Norway).
Statistical analysis. We examined differences
between groups for statistical significance
using analysis of variance (ANOVA)
with the least-square difference post hoc
test. We used one-class statistical analysis of
microarray to identify differentially expressed
genes (Tusher et al. 2001) between HFC- and
HFR-fed rats. We determined statistical significance
of the real-time PCR results using
the Student’s t-test, and the threshold for significance
was set at p ≤ 0.05.
Results
Characteristics of animals exposed to POPs.
As we expected, concentrations of POPs were
consistently much higher in the HFC diet
than in the HFR diet [Supplemental Material,
Table 1 (doi:10.1289/ehp.0901321)], whereas
the contents of n-3 polyunsaturated fatty acids
and other fatty acids were similar in the two
diets because both the crude and the refined
fish oils were obtained from the same batch
of farmed salmon (Supplemental Material,
Table 2 (doi:10.1289/ehp.0901321).
After 28 days, rats fed the HFC diet
appeared normal, although they tended to
gain more weight than rats fed the HFR
diet despite similar daily energy intake
(Figure 1A,B). Intake of the HFC diet, but not
HFR diet, enhanced the accumulation of visceral
adipose tissue induced by HF consumption
(Figure 1C,D). Profound dys regulation
in lipid homeo stasis was further observed in
livers of HFC-fed rats, which exhibited elevated
levels of triacyl glycerol, diacyl glycerol,
and total cholesterol compared with HF-fed
rats; livers of HFR-fed rats tended to exhibit
a reduced lipid accumulation (Figure 1E–G).
Histological examinations highlighted the
development of severe hepatosteatosis in rats
fed HFC (Figure 1H) and confirmed that the
presence of POPs in salmon oil provokes significant
impairment of lipid metabolism.
To gain further insight into the phenotypical
changes of animals exposed to POPs,
we performed a comparison of gene expression
profiles in the liver of rats fed the HFC
and HFR diets, using oligonucleo tide microarrays.
The expression of genes involved in
drug metabo lism was affected, indicating
dietary POP exposure [Supplemental Material,
Table 3 (doi:10.1289/ehp.0901321)]. We
also observed major differences for genes
involved in lipid metabo lism and for several
genes linked to lipid deposition (Supplemental
Material, Table 3). Of interest, POPs induced
robust down-regulation of insulin-induced
gene-1 (Insig-1) and Lpin1, two master regulators
of lipo genesis and synthesis of triglyceride
and cholesterol (Croce et al. 2007;
Engelking et al. 2004; Finck et al. 2006; Lee
and Ye 2004). Real-time PCR analysis confirmed
the strong repression of Lpin 1 and
Insig-1 genes in the liver of rats consuming the
HFC diet (Table 1). Similarly, in adipose tissue
of HFC-fed rats, expression of Lpin1 and
Insig-1 genes was repressed compared with
466 v o l u m e 118 | number 4 | April 2010 • Environmental Health Perspectives

POP exposure provokes insulin resistance
HFR-fed animals [mean ± SE, 78 ± 8 vs. 55 ±
5 (n = 9, p = 0.02) for Insig-1 and 98 ± 11 vs.
64 ± 8 (n = 9, p = 0.03) for Lpin1 for HFRand
HFC-fed rats, respectively]. Furthermore,
POPs induced a significant increase in the
expression level of SREBP1C (sterol regulatory
element-binding protein 1C), the master
regulator of the lipogenic pathway, and
FAS (fatty acid synthase), a well-known target
gene of SREBP1C (Table 1). Interestingly, the
hepatic expression of LXRα (liver X receptor
alpha) was not affected, suggesting that the
oxy sterol pathway was not modified by POP
exposure (Table 1). Altogether, these results
demonstrate that POP exposure signifi cantly
affects the expression of critical genes involved
in the regulation of lipid homeostasis. Gene
set enrichment analysis further revealed significant
effects on several biological pathways
[Supplemental Material, Table 4 (doi:10.1289/
ehp.0901321)]. This analysis demonstrated
a highly significant up-regulation of pathways
designated “pathogenic Escherichia coli
infection” (EPEC/EHEC). The core genes
up-regulated in the pathways include TLR5,
ROCK2, CD14, and YWHAZ, a gene encoding
a member of the 14-3-3 family of proteins
reported to interact with insulin receptor substrate-1
and thereby regulating insulin signaling.
Similarly, the roles of toll-like receptors,
Body weight gain (g)
200
160
120
80
40
0
*
Chow
HF
HFR
HFC
Energy intake
(kcal/day)
200
160
120
80
40
0
*
* *
Chow
HF
Visceral fat (g)
30
25
20
15
10
5
0
*
*
**
Triacylglycerol
(mg/g)
120
100
80
60
40
20
0
*
*
**
Diacylglycerol (mg/g)
1.2
0.8
0.4
0
*
*
Total cholesterol
(mg/g)
**
20
15
**
10
5
0
* *
HFR
HFC
Chow
HF HFR HFC
Oil red O
100 µM
100 µM 100 µM 100 µM
H&E
200 µM
200 µM 200 µM 200 µM
50 µM 50 µM 50 µM 50 µM
Figure 1. Characteristics of rats fed salmon oil containing POPs. Body weight gain (A) and daily energy intake (B) in rats fed chow or the HF, HFR, or HFC diets
over a 4-week period. (C) Exposed ventral view of a representative rat from each diet group showing increased visceral adipose tissue after consumption of
the HFC diet. (D) Quantification of visceral fat (epididymal and perirenal fat pads). (E–G) Levels of hepatic triacylglycerol (E), diacyl glycerol (F), and total cholesterol
(G). (H) Representative histological sections of liver stained with Oil red O (top) or H&E at low (middle) and high (bottom) magnifications; the three sections
for each treatment group are from the same liver sample. All data are shown as mean ± SE; n = 8–9.
*p < 0.02 compared with control. **p < 0.04 compared with HF.
Environmental Health Perspectives • v o l u m e 118 | number 4 | April 2010 467

Ruzzin et al.
CD14, and rho kinases in regulating insulin
signaling and establishment of insulin resistance
in response to chronic low-grade inflammation
are well documented (Begum et al.
2002; Cani et al. 2007; Furukawa et al. 2005;
Petersen et al. 2008; Tzivion et al. 2001).
Effects of POPs on insulin action in vivo.
Next, we assessed the impacts of POPs on
whole-body insulin action. In the basal state,
intake of the HFC diet exacerbated the hyperinsulinemia
induced by HF consumption,
whereas animals fed HFR and control diets
had similar plasma insulin levels (Figure 2A).
Basal plasma glucose levels were similar in
all groups (Figure 2B), but the HOMA-IR
index was significantly increased in rats fed
Table 1. Real-time PCR determination of mRNA expression of a set of relevant genes in the liver of rats
fed HFR or HFC diets (n = 9 per group).
HFR HFC p-Value
Genes related to mitochondrial function
PGC1α 0.73 ± 0.3 0.05 ± 0.02 0.043
PPARα (peroxisome proliferator-activated receptor α) 76 ± 7 75 ± 18 0.988
CS (citrate synthase) 316 ± 19 214 ± 10 0.002
SDHA (succinate dehydrogenase) 74 ± 2 63 ± 4 0.038
MCAD (medium chain acyl CoA dehydrogenase) 332 ± 30 170 ± 18 0.003
Genes related to lipogenesis
SREBP1C 3.0 ± 0.3 4.6 ± 0.6 0.021
LXRα 50 ± 3 51 ± 7 0.932
FAS 1.1 ± 0.1 1.9 ± 0.2 0.01
Lpin 1 96 ± 17 22 ± 10 0.0017
Insig-1 123 ± 23 43 ± 12 0.0071
Plasma insulin
(ng/mL)
GIR
(mg/kg/min)
Clamp HGP
(mg/kg/min)
Glucose uptake
(mmol/kg ww/30 min)
3
2
1
0
15
10
5
0
15
10
5
0
5
4
3
2
1
0
*
#
#
**
##
0 2,000
#
Insulin (µU/mL)
#
Chow
HF
HFR
HFC
#
Plasma glucose
(mg/dL)
Basal HGP
(mg/kg/min)
Clamp R d
(mg/kg/min)
Glucose uptake
(fmoL/1,000 cells/min)
Figure 2. Effects of salmon oil and POPs on insulin action and glucose metabolism evaluated by hyperinsulinemic–euglycemic
clamps performed in rats fed chow or HF, HFR, or HFC diets over a 4-week period.
(A) Basal insulinemia. (B) Basal glycemia. (C) GIR. (D) Basal HGP. (E) HGP during the clamps. (F) Glucose
disposal rate (R d ). (G) Insulin-stimulated glucose uptake in soleus muscles. (H) Insulin-stimulated glucose
uptake in primary adipocytes. All data are shown as mean ± SE; n = 6–9.
*p < 0.04 compared with chow control. **p < 0.04 compared with HF. # p < 0.05 compared with HFR. ## p < 0.03 compared
with HF.
160
120
80
40
0
15
10
5
0
15
10
5
0
8
6
4
2
0
0
#
Insulin (µU/mL)
2,000
##
the HFC diet (7.1 for control rats, 11.2 for
rats fed HF, 8.4 for rats fed HFR, and 15.5
for rats fed HFC; p < 0.04).
The performance of hyperinsulinemic–​
euglycemic clamp, the gold standard for
investigating and quantifying insulin resistance
(Kraegen et al. 1983), revealed that the
consumption of the HFC diet aggravated
HF-induced reduced GIR, whereas HFR-fed
rats showed no impairment of insulin action
compared with control rats (Figure 2C).
Reduced GIR reflects decreased insulinmediated
suppression of HGP, reduced insulinstimulated
peripheral glucose disposal rates,
or both. Analysis of these parameters revealed
that basal HGP was similar in all groups
(Figure 2D), whereas suppression of HGP
by insulin was impaired in animals consuming
both HFC and HF diets (Figure 2E).
Moreover, intake of HFC led to impaired
insulin-mediated glucose disposal in peripheral
tissues, which mainly include skeletal muscles
and adipose tissue (Figure 2F). To investigate
this further, we determined the rates of glucose
uptake in isolated soleus muscles and primary
adipocytes. We found that insulin-stimulated
glucose uptake was reduced to a similar extent
in skeletal muscle of animals fed HFC and
HF diets (Figure 2G). In contrast, rats fed
the HFR diet were protected against muscle
insulin resistance (Figure 2G). In adipose tissue,
the ability of insulin to stimulate glucose
uptake was impaired in both the HFR and HF
groups, and this metabolic defect was worsened
by the consumption of the HFC diet
(Figure 2H). Thus, exposure to POPs present
in HFC exacerbated the deleterious metabolic
effects of HF and attenuated the protective
effects of n-3 polyunsaturated fatty acids,
which indicates that the presence of environmental
organic contaminants may influence
the outcomes of food and dietary products.
There is growing evidence that mitochondrial
dysfunction contributes to insulin resistance
(Lowell and Shulman 2005). To assess
the impact of POPs on hepatic mitochondrial
content, we measured mitochondrial
DNA levels by quantitative polymerase chain
reaction (qPCR), using primers specific for
the COXII gene, and determined the ratio
between mitochondrial DNA and nuclear
DNA as previously validated (Bonnard et al.
2008). We found no apparent modification
of the amount of mitochondrial DNA in the
liver of the animals fed HFC (ratio COXII/
PPIA, 1.1 ± 0.2 (mean ± SE) for rats fed HFR
and 0.9 ± 0.1 for rats fed HFC, p = 0.189).
However, despite this apparent lack of change
in mitochondrial content, we observed significant
reduction in the expression of several
genes related to mitochondrial function,
such as PGC1α (peroxisome proliferatoractivated
receptor gamma-coactivator-1
alpha), citrate synthase, medium-chain acyl
468 v o l u m e 118 | number 4 | April 2010 • Environmental Health Perspectives

POP exposure provokes insulin resistance
CoA dehydrogenase, and SDHA (succinate
dehydrogenase) (Table 1), indicating the presence
of alterations in mitochondrial function
and oxidative capacities in the liver of the rats
exposed to POPs.
Analysis of POPs distribution in these
animals revealed that whereas both liver and
adipose tissue stored organochlorine pesticides,
indicator PCBs, mono-ortho-substituted
PCBs, and non–ortho-substituted PCBs,
the liver preferentially retained PCDDs or
PCDFs [Supplemental Material, Table 5
(doi:10.1289/ehp.0901321)].
Effects of POPs on insulin action in vitro.
To further demonstrate the contribution of
lipophilic POPs to the development of insulin
resistance–associated metabolic disturbances,
we exposed differentiated adipocytes
to a POP mixture that mimicked the relative
abundance of organic contaminants found
in crude salmon oil. Incubation of adipocytes
with this POP mixture impaired the
ability of insulin to stimulate glucose uptake
(Figure 3A), which is in agreement with the
reduced insulin–stimulated glucose uptake
observed in adipose tissue of rats fed the
HFC diet (Figure 2H). We then determined
whether POP exposure, as observed in rats
fed the HFC diet, could affect the expression
of Lpin1 and Insig-1 mRNA in cultured
adipo cytes. After 48-hr treatment with the
POP mixture, Lpin1 and Insig-1 mRNA levels
were dose-dependently reduced in adipocytes
[Supplemental Material, Figure 1
(doi:10.1289/ehp.0901321)], which confirms
the ability of POPs to interfere with key
regulators of lipid metabolism. Importantly,
the metabolic defects observed in adipocytes
exposed to POPs were independent of cytotoxicity,
as demonstrated by the absence of
an increased release of lactate dehydrogenase
into the cell culture media (Supplemental
Material, Figure 2). Altogether, these findings
clearly establish the capacity of POPs to
impair insulin action and associated metabolic
abnormalities in a cell-autonomous manner.
Humans and other organisms are chronically
exposed to a variety of organic pollutants.
To investigate which POPs contributed significantly
to the impairment of insulin action,
we incubated adipocytes with different POP
mixtures. Although adipocytes exposed to a
PCDD or PCDF mixture showed normal
insulin action (Figure 3B,C), those exposed
to non-ortho- substituted and mono-orthosubstituted
PCB mixtures had reduced insulin
action (Figure 3D,E). Impaired insulin action
was independent of the total toxic equivalent
(TEQ) concentration (Van den Berg et al.
2006) of the mixtures; up to 6.027ng WHO
2005 TEQ/mL for the PCDF mixture compared
with 0.0016ng WHO 2005 TEQ/mL
for the mono-ortho-PCB mixture. These findings
demon strate that risk assessment based
on TEQ assigned to dioxins and dioxin-like
PCBs (Van den Berg et al. 2006) is unlikely
to reflect the risk of insulin resistance. Further
investigations showed that insulin-stimulated
glucose uptake was dramatically reduced in
adipocytes treated with both the mixture of
organochlorine pesticides (Figure 3F) and
dichloro diphenyl trichloroethanes (DDTs)
(Figure 3G), whereas the mixture of indicator
PCBs had less inhibitory effects on insulin
action (Figure 3H).
Relative glucose uptake
(fold increase)
Relative glucose uptake
(fold increase)
Relative glucose uptake
(fold increase)
Relative glucose uptake
(fold increase)
7
6
5
4
3
2
1
0
7
6
5
4
3
2
1
0
7
6
5
4
3
2
1
0
7
6
5
4
3
2
1
0
POPs
PCDFs
Vehicle
1 nM
10 nM
100 nM
* *
TEQ (ng WHO
2005 TEQ/mL)
0.060
0.603
6.027
Mono-ortho-PCBs
TEQ (ng WHO
2005 TEQ/mL)
0.0002
0.0016
1.0158
* *
* * *
* * *
*
* *
* *
*
*
Discussion
In this study, we demonstrate for the first time
a causal relationship between POPs and insulin
resistance in rats. In vivo, chronic exposure to
low doses of POPs commonly found in food
chains induced severe impairment of wholebody
insulin action and contributed to the
development of abdominal obesity and hepatosteatosis.
Treatment in vitro of differentiated
adipocytes with nano molar concentrations of
POP mixtures mimicking those found in crude
7
6
5
PCDDs
TEQ (ng WHO
2005 TEQ/mL)
0.004
0.043
* * *
4 0.425
3
2
1
0
7 Non-ortho-PCBs
TEQ (ng WHO
6
*
2005 TEQ/mL)
5 0.010
0.103
4 1.025
3
* * *
2
*
*
1
* *
* *
0
Relative glucose uptake
(fold increase)
Relative glucose uptake
(fold increase)
Relative glucose uptake
(fold increase)
DDTs
7
PCBs
Vehicle
Vehicle
1 nM
6
10 nM
10 nM
5
100 nM
100 nM
1 µM
4
3
*
*
*
*
*
2
*
*
*
* *
* 1
*
* * ** * *
0
0 1 3 10 30
0 1 3 10 30
Insulin (nM)
*
* * *
*
*
*
Relative glucose uptake
(fold increase)
Figure 3. Effects of POPs on insulin action in adipocytes shown as the ability of differentiated 3T3-L1 adipocytes to
take up radioactive-labeled glucose in response to insulin measured after 48 hr exposure to several POP mixtures
found in crude oil from farmed Atlantic salmon. (A) POP mixture, (B) PCDD mixture, (C) PCDF mixture, (D) non–
ortho-substituted PCB mixture, (E) mono–ortho-substituted PCB mixture, (F) Pesticide mixture, (G) DDT mixture,
or (H) PCB mixture. Concentrations of POP mixtures are shown according to the highest contaminant compound
present in the mixture, as well as the World Health Organization (WHO) 2005 TEQ for dioxins and dioxin-like PCBs
(Van den Berg et al. 2006). Glucose uptake was determined in eight parallel wells for each mixture and for each
concentration. Data are expressed as relative glucose uptake and presented as mean ± SE.
*p < 0.05 compared with vehicle (dimethyl sulfoxide)-treated cells.
7
6
5
4
3
2
1
0
Organochlorine pesticides
Vehicle
1 nM
10 nM
100 nM
* * * *
* *
Insulin (nM)
* * *
*
* *
*
* *
*
Environmental Health Perspectives • v o l u m e 118 | number 4 | April 2010 469

Ruzzin et al.
salmon oil induced a significant inhibition of
insulin-dependent glucose uptake. These data
provide compelling evidence that exposure to
POPs increases the risk of developing insulin
resistance and metabolic disorders.
Despite intense investigations and establishment
of both preventive and therapeutic
strategies, insulin resistance–associated meta bolic
diseases such as type 2 diabetes, obesity, and
non alcoholic fatty liver disease have reached
alarming proportions worldwide (Angulo
2002; Ford et al. 2004; Zimmet et al. 2001).
By 2015, the World Health Organization
(WHO) estimates that > 1.5 billion people
will be overweight and that 338 million people
will die from chronic diseases such as diabetes
and heart disease (WHO 2005). Although
physical inactivity and regular intake of highenergy
diets are recognized contributors (Hill
and Peters 1998; Roberts and Barnard 2005),
these lifestyle factors can only partially explain
the explosive and uncontrolled global increase
in metabolic diseases. Recently, the development
of insulin resistance and inflammation
was found to be exacerbated in humans and
animals exposed to air pollution (Kelishadi
et al. 2009; Sun et al. 2009). Furthermore,
the widespread environmental contaminant
bisphenol A was reported to impair pancreatic
beta cells and trigger insulin resistance (Alonso-
Magdalena et al. 2006). Our data, together
with the finding that type 2 diabetics accumulate
significant body burdens of POPs (Lee
et al. 2006), provide additional evidence that
global environmental pollution contributes to
the epidemic of insulin resistance–associated
metabolic diseases.
Although rats chronically fed the HFC
diet for 28 days were exposed to a relatively
high intake of organic pollutants, the
POPs
Nuclear
receptors
(AhR, CAR, PXR, )
TLR5, ROCK2,
CD14, and YWHAZ
PGC1α
Insig-1
Lpin1
SREBP1c
FAS
concentrations of PCDDs/PCDFs and indicator
PCBs in adipose tissue of these animals
did not exceed those observed in Northern
Europeans 40–50 years of age (Kiviranta et al.
2005), thereby indicating that doses of POP
exposure sufficient to induce detrimental
health effects were not excessive. Whether the
exposure to lower levels of POPs would induce
similar detrimental effects as those observed in
the present study remains to be investigated.
Dietary interventions are current strategies
to prevent or treat metabolic diseases,
and nutritional guidelines are usually based
on energy density and glycemic index of the
diet; however, the levels of POPs present in
food has received less attention. Given that
POPs are ubiquitous in food chains (Fisher
1999), such under estimation may interfere
with the expected beneficial effects of some
dietary recommendations and lead to poor
outcomes. For instance, the presence of POPs
in food products may, to some extent, explain
the conflicting results regarding the protective
effects of n-3 polyunsaturated fatty acids
against the incidence of myocardial infarction
(Guallar et al. 1999; Rissanen et al. 2000).
Overall, better understanding of the interactions
between POPs and nutrients will help
improve nutritional education of patients with
insulin resistance syndrome.
To protect consumer health, the presence
of contaminants in food is internationally
regulated. In the European Union legislation,
certain POPs including dioxins and dioxin-like
PCBs are regulated in foodstuffs (European
Union 2006). Risk assessment of these organic
pollutants is based on the ability of individual
compounds to produce hetero geneous toxic
and biological effects through the binding of
the aryl hydrocarbon receptor. Interestingly,
Chronic low-grade
inflammation
Mitochondrial function
Fatty acid oxidation
Lipogenesis
Insulin resistance
syndrome
Figure 4. Schematic representation of the possible mechanisms behind the development of the insulin
resistance syndrome induced by POP exposure. POPs may activate nuclear receptors including aryl
hydrocarbon receptor (AhR), pregnane X receptor (PXR), constitutive androstane receptor (CAR), or yet
unknown receptors. POP exposure may induce the regulation of genes involved in the inflammatory pathway,
mitochondrial function, lipid oxidation, and lipogenesis, thereby contributing to the development of
the insulin resistance syndrome.
we found that cultured adipo cytes exposed to a
PCDF or PCDD mixture have normal insulin
action, even though the TEQ of these mixtures
could be up to 3,500 times higher than the
TEQ of the non-ortho-substituted and monoortho-substituted
PCB mixtures that impaired
insulin action. These findings demonstrate that
risk assessment based on WHO TEQs assigned
to dioxins and dioxin-like PCBs is unlikely to
reflect the risk of insulin resistance and the possible
development of metabolic disorders.
Although the production of organochlorine
pesticides has been restricted since
the 1970s, the global production and use of
pesticides are poorly controlled (Jorgenson
2001; Nweke and Sanders 2009), and the
presence of these environmental chemicals in
seafood still remains unregulated in European
countries (European Union 2008). Of the
POP mixtures tested in vitro, organochlorine
pesticides were the most potent disruptors of
insulin action. This powerful inhibitory effect
of pesticides on insulin action likely explains
the common finding emerging from several
independent cross-sectional studies reporting
an association between type 2 diabetes and
the body burdens of p,p´-DDE, oxychlordane,
or trans-nonachlor (Lee et al. 2006; Rignell-
Hydbom et al. 2007; Turyk et al. 2009).
Therefore, widespread pesticide exposure to
humans appears to be of particular global concern
in relation to public health.
We draw two main conclusions from
these observations. First, exposure to POPs
present in the environment and food chains
are capable of causing insulin resistance and
impair both lipid and glucose metabolism,
thus supporting the notion that these chemicals
are potential contributors to the rise in
prevalence of insulin resistance and associated
disorders (Figure 4). Second, although beneficial,
the presence of n-3 polyunsaturated fatty
acids in crude salmon oil (in the HFC diet)
could not counteract the deleterious metabolic
effects induced by POP exposure. Altogether,
our data provide novel insights regarding the
ability of POPs to mediate insulin resistance–
associated metabolic abnormalities and provide
solid evidence reinforcing the importance
of international agreements to limit the release
of POPs to minimize public health risks.
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