Expression studies of six human obesity-related genes in seventissues from divergent pig breeds

S. Cirera*, M. S. Jensen*, V. S. Elbrønd*, S. G. Moesgaard†, B. Ø. Christoffersen†,

H. N. Kadarmideen*, K. Skovgaard‡, C. V. Bruun*, P. Karlskov-Mortensen*, C. B. Jørgensen* and

M. Fredholm**Department of Veterinary Clinical and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen,

Denmark. †Novo Nordisk A/S. Novo Nordisk Park, 2760 M�aløv, Denmark. ‡Innate Immunology Group, National Veterinary Institute,

Technical University of Denmark, Copenhagen, Denmark.

Summary Obesity has reached epidemic proportions globally and has become the cause of several

major health risks worldwide. Presently, more than 100 loci have been related to obesity

and metabolic traits in humans by genome-wide association studies. The complex genetic

architecture behind obesity has triggered a need for the development of better animal

models than rodents. The pig has emerged as a very promising biomedical model to study

human obesity traits. In this study, we have characterized the expression patterns of six

obesity-related genes, leptin (LEP), leptin receptor (LEPR), melanocortin 4 receptor (MC4R), fat

mass and obesity associated (FTO), neuronal growth regulator 1 (NEGR)1 and adiponectin

(ADIPOQ), in seven obesity-relevant tissues (liver; muscle; pancreas; hypothalamus; and

retroperitoneal, subcutaneous and mesenteric adipose tissues) in two pig breeds (production

pigs and G€ottingen minipigs) that deviate phenotypically and genetically from each other

with respect to obesity traits. We observe significant differential expression for LEP, LEPR

and ADIPOQ in muscle and in all three adipose tissues. Interestingly, in pancreas, LEP

expression is only detected in the fat minipigs. FTO shows significant differential expression

in all tissues analyzed, and NEGR1 shows significant differential expression in muscle,

pancreas, hypothalamus and subcutaneous adipose tissue. The MC4R transcript can be

detected only in hypothalamus. In general, the expression profiles of the investigated genes

are in accordance with those observed in human studies. Our study shows that both the

differences between the investigated breeds and the phenotypic state with respect to obesity/

leanness play a large role for differential expression of the obesity-related genes.

Keywords ADIPOQ, FTO, G€ottingen minipig, LEP, LEPR, MC4R, NEGR1, obesity,

Production pig

Introduction

Obesity has reached epidemic proportions globally and is

considered one of the greatest health challenges of this

century. The high morbidity and mortality rate among

obese individuals is caused by obesity-related diseases such

as metabolic syndrome, coronary artery disease and type 2

diabetes.

The clinical definition of obesity is a body mass index

(BMI) of 30 or higher, which is usually caused by an

imbalance between energy intake and energy expenditure.

The brain plays a very important role in controlling energy

balance by linking the nervous system to the endocrine

system by receiving hormonal and neuronal input from

different peripheral tissues (including adipose tissues, liver,

muscle and pancreas).

Excessive energy intake and reduced physical activity

contribute to the increasing prevalence of obesity. However,

family and twin studies have shown that genetic factors

account for 40–70% of the population variation in BMI

(Maes et al. 1997). Presently, around 100 loci have been

associated with metabolic and obesity-related traits by

genome-wide association studies (GWAS) (Herrera & Lind-

gren 2010; Speliotes et al. 2010).

The rodent model has been used extensively for obesity

studies; however, there is an increasing need for new

animal models comparatively closer to humans than

Address for correspondence

S. Cirera and M. Fredholm, Department of Veterinary Clinical and

Animal Sciences, Faculty of Health and Medical Sciences, University of

Copenhagen, Copenhagen, Denmark.

E-mail: scs@sund.ku.dk (S. Cirera) and mf@sund.ku.dk (M. Fredholm)

Accepted for publication 19 July 2013

doi: 10.1111/age.12082

1© 2013 The Authors, Animal Genetics © 2013 Stichting International Foundation for Animal Genetics

rodents. Because pigs are similar to humans in organ sizes,

metabolic features, cardiovascular systems, dietary habits

and fat deposition, resulting in many of the same dietary

health problems as in humans, they provide a valuable

model. The pig is also one of the few animals that can

voluntarily become obese (Spurlock & Gabler 2008). Char-

acterization of the genes and pathways involved in obesity

in pigs will open up new research avenues in the manage-

ment of human obesity.

The aim of the present study was to discriminate between

differential expression caused by state (obesity/leanness)

and by genetics (breed differences) and to establish novel

functional information using a broad range of tissues. Three

groups of pigs were used, that is, production pigs (PP), and

diet-induced lean (LMP) and obese (FMP) G€ottingen mini-

pigs. PP and MP are genetically highly divergent given that

PP have undergone strong selection for lean growth during

the last 60 years, whereas MP have not been subjected to

selection for leanness and are prone to obesity if not fed

restrictively (Bollen et al. 2005). The obesity-relevant genes

investigated were three genes implicated in rare forms of

monogenic obesity, that is, leptin (LEP), leptin receptor

(LEPR) and melanocortin 4 receptor (MC4R), as well as three

genes identified as obesity-related candidate genes in

GWAS, that is, fat mass and obesity associated (FTO), neuronal

growth regulator 1 (NEGR1) and adiponectin (ADIPOQ).

Materials and methods

Biological materials

Production pigs (PP)

Three adult Yorkshire sows and three adult Duroc sows were

housed at the animal research facilities at the University of

Copenhagen and fed to maintain a weight of around 220 kg

with standard pig feed (classic, dlg.dk) (1 kg morning and

evening). The sows were euthanized at the age of approxi-

mately4 yearswithpentobarbital and thenbled. Tissues (liver,

muscle frommusculus psoas major, pancreas, hypothalamus,

abdominal retroperitoneal, dorso-lumbar subcutaneous and

mesenteric adipose tissues) were collected immediately after

euthanasia and snap-frozen in liquid nitrogen.

G€ottingen minipigs

Seven diet-induced FMP and seven LMP female ovariecto-

mized G€ottingen minipigs were housed as described above.

The obese pigs had previously been used in pharmacological

studies with therapeutic peptides but were subjected to a

2.5-month wash-out period eliminating the effect of the

therapeutic peptides prior to the start of this study. The

minipigs were fed standard minipig chow. The LMP group

was fed restrictively 2 9 150 g a day, and the FMP group

was fed ad libitum.

Body weight was recorded before euthanasia. Body

composition was determined by the use of dual-energy

X-ray absorptiometry scanning (DEXA scanning) (Hologic

Explorer, Santax Medico) within 4 weeks before euthanasia.

The pigs were euthanized, and tissues were collected as

described above. Animal care and maintenance have been

conducted according to the Danish ‘Animal Maintenance

Act’ (Act 432 dated 09/06/2004).

RNA isolation

Homogenization of the tissues was performed using a

mortar and pestle or using a gentleMACSTM Octo Dissociator

machine (Miltenyi Biotec).

For liver, muscle and adipose tissues, the TRI Reagent®

protocol for RNA isolation was used (Molecular Research

Center, Inc.). Pancreas RNA was isolated using RNeasy mini

kit (Qiagen), andhypothalamus RNAwas isolated using Lipid

mini kit (Qiagen) following the manufacturer’s protocols.

Quantitative and qualitative measurements on RNAsamples

The amount of RNA was estimated using a NanoDrop 1000

(Thermoscientific). RNA quality was assessed by gel electro-

phoresis and by analysis on an ExperionTM system (Bio-Rad)

using Eukaryote Total RNA StdSens kit. Only RQI values >6were considered acceptable (the average values for each

tissue were as follows: liver, 9.2 � 0.37; muscle, 9 � 0.47;

pancreas, 6.8 � 0.60; hypothalamus, 8.7 � 0.52; abdomi-

nal retroperitoneal adipose tissue, 8 � 0.74; subcutaneous

adipose tissue, 8.3 � 0.79; and mesenteric adipose tissue,

8.1 � 0.51).

cDNA synthesis

Two cDNA replicates were made for each RNA sample using

Improm-IITM reverse transcriptase (Promega) and a 3:1

mixture of random hexamers/OligodT, according to the

manufacturer’s recommendations. The cDNA was diluted

1:8 prior to use in qPCR.

Quantitative PCR primers

Primer sets for LEP, LEPR, MC4R, NEGR1 and ADIPOQ

were designed using PRIMER 3 software (http://frodo.wi.mit.

edu/cgi-bin/primer3/primer3_www.cgi). Primers were

designed over introns if possible. Primers for the FTO gene

were taken from Madsen et al. (2010). Primers for the TBP

and RPL4 (reference genes) were taken from Nygard et al.

(2007) (for details, see Table 1).

Quantitative PCR

QuantiFastTM SYBR Green PCR Kit (Qiagen) was used for the

quantitative PCR amplification following manufacturer’s

© 2013 The Authors, Animal Genetics © 2013 Stichting International Foundation for Animal Genetics, doi: 10.1111/age.12082

Cirera et al.2

recommendations. The qPCRs were performed on a

Mx3000TM (Stratagene). The PCR thermal profile was 95 °Cfor 5 min, 40 two-step cycles of 95 °C for 10 s and 60 °C for

30 s, and a melting curve analysis (55 °C–95 °C) was

performed in the last cycle to evaluate specificity of the

amplification. For each transcript, a standard curve was

created using the purified PCR product generated for each

specific primer pair in order to obtain the PCR efficiency. The

baseline adjustment method of the Mx3000/MXPRO (Strata-

gene) softwarewas used to determine the Cq in each reaction.

Expression data were analyzed using the GENEX PRO software

(Multid Analyses AB), and fold changes were calculated.

Statistical analysis

Fold change values were log2-transformed to approach

normal distribution. Subsequently, one-way analysis of

variance and a Tukey post-test were performed for each

gene in each tissue to compare within the three pig groups.

A P-value <0.05 was considered significant; P < 0.01 was

considered very significant, and P < 0.0001 was considered

extremely significant.

Histology

Paraformaldehyde-fixed retroperitoneal and subcutaneous

adipose tissues from all the animals included in this study

were prepared for histological examination following stan-

dard laboratory protocols. Subsequently, samples were

serially sectioned, collected on glass slides and stained with

hematoxylin/eosin. The sections were examined under a

light microscope (Leica DMR) and captured in LEICA APPLICA-

TION SUITE 3.6.

Results

Phenotypic characterization of the animals

Production pigs, FMP and LMP animals weighed

220 � 6.79 kg, 92.57 � 13.71 and 50.29 � 4.31 kg

respectively at slaughter. The body weights at the time of

the DEXA scanning were 49 � 4 kg and 87 � 13 kg for the

LMP and the FMP respectively (P < 0.001). The corre-

sponding body fat percentages were 26 � 4% and 42 � 4%

(P < 0.001) respectively. Production pigs animals were not

DEXA-scanned.

Expression studies

LEP was primarily expressed in the three adipose tissues

(retroperitoneal, subcutaneous and mesenteric). In these

tissues, there was highly significant differential expression

between PP and LMP as well as between PP and FMP with

lowest expression in PP. We also saw differential expression

in muscle and pancreas (Fig. 1a).Tab

le1

Primer

sequen

ces,

accessionnumber

ofthesequen

ceusedto

designprimers,

ampliconsize

andPCRefficien

cies

forallthegen

esassayed.

Gen

enam

ePrimer

sequen

ces

Accessionnumber

Ampliconsize

PCRefficien

cy(%

)

TBP1

fw:5′-AACAGTTCAGTAGTTATGAGCCAGA-3′rev:

5′-AGATGTTCTCAAACGCTTCG-3′(N

ygardetal.2007)

DQ845178

153bp

80.3

RPL41

fw:5′-CAAGAGTAACTACAACCTTC-3′rev:

5′-GAACTCTACGATGAATCTTC-3′(N

ygardetal.2007)

DQ845176

122bp

93.3

FTO

fw:5′-CAGCAGTGGCAGCTGAAATA-3′rev:

5′-TGACCAGGTCCCGAAATAAG-3′(M

adsenetal.2010)

AM905422

133bp

101.6

NEGR1

fw:5′-CGGCGGTGCTTAGGTGTTAT-3′rev:

5′-CTCGAGGATCCACTGACCAC-3′

EU189936

108bp

96.4

ADIPOQ

fw:5′-CGAGAAGGGTGAGAAAGGAG-3′rev:

5′-TAGGCGCTTTCTCCAGGTTC-3′

AY135647

123bp

86.8

LEP

fw:5′-TGACACCAAAACCCTCATCA-3′rev:

5′-ATGAAGTCCAAACCGGTGAC-5′

NM_2

13840

102bp

102.4

LEPR

fw:5′-GTATCATAGGAGTAGCCTCT-3′rev:

5′-ATGCATTCATAAAAACCATC-3′

AF0

92422

102bp

96.6

MC4R

fw:5′-GGGTGTCATAAGCCTGTTGG-3′rev:

5′-CACAGCCAGGCTACAGATGA-3′

JQ828977

103bp

99.1

1Gen

esusedas

areference

fornorm

alization.

© 2013 The Authors, Animal Genetics © 2013 Stichting International Foundation for Animal Genetics, doi: 10.1111/age.12082

Expression studies of obesity-related genes in divergent pig breeds 3

LEPR was expressed in all tissues. The highest expression

level was seen in liver where the level was comparable in

the three groups of pigs. In muscle, there was highly

significant differential expression between PP and LMP as

well as between PP and FMP and also significant differential

expression between LMP and FMP reflecting both breed-

and state-specific differential expression. Furthermore, in

two of the adipose tissues, there was breed-specific differen-

tial expression (Fig. 1b).

Among the studied genes, FTO was the one with the

lowest fold changes. However, the expression was signifi-

cantly higher in hypothalamus in all three groups of pigs

compared with the expression in the remaining six tissues.

We detected significant and/or highly significant differential

expression in all tissues. In liver, muscle, hypothalamus and

retroperitoneal adipose tissue, the differential expression

was breed specific, whereas it was state specific in pancreas.

In subcutaneous and mesenteric adipose tissues, both breed-

specific differential expression and state-specific differential

expression were observed (Fig. 1c).

The expression level of the NEGR1 gene also was

significantly higher in hypothalamus compared with the

expression level in the remaining tissues. Highly significant

breed-specific expression was detected in muscle, pancreas

and hypothalamus, and both breed-specific expression and

state-specific expression were detected in subcutaneous

adipose tissue (Fig. 1d).

ADIPOQ was the gene with the highest fold change

detected. It was significantly higher expressed in the three

adipose tissues in all three groups of pigs. Expression was

state specific in liver and hypothalamus because in these

two tissues, expression was only observed in FMP. In muscle

and mesenteric adipose tissue, highly significant expression

and significant breed-specific expression respectively were

observed, whereas in subcutaneous adipose tissue both

breed- and state-specific differential expression were

observed (Fig. 1e).

MC4R was expressed at very low level in all tissues except

hypothalamus. In most samples, the expression levels were

below the limit of detection (Cq >33). Furthermore, the

expression levels varied between samples resulting in high

standard deviations. Consequently, this gene was excluded

from Fig. 1.

Histology

Examination of histological preparations from retroperito-

neal and subcutaneous adipose tissues from the three

groups of pigs showed that in general retroperitoneal

adipose tissue was composed of a majority of fat cells with

only a minor amount of intercellular connective tissue,

whereas subcutaneous adipose tissue was richer in connec-

tive tissue. Most of the cells in both adipose tissues in LMP

and FMP were hexagonal, whereas in PP, in particular in

the retroperitoneal adipose tissue, many cells were round.

In Fig. 2, representative histological sections are shown

providing an impression of the variation between the three

animal groups. In retroperitoneal adipose tissue, cell size

differed between the three groups, that is, cell sizes were in

the range of 75–125 lm, 100–200 lm and 40–100 lm in

LMP, FMP and PP respectively.

Discussion

The great boom of the obesity epidemic starting around the

1980s underlines the impact of the modern lifestyle factors

like high-calorie diets in the Western world and the

decreased amount of physical exercise. However, there is

substantial evidence that obesity is highly heritable in

human cohorts (Maes et al. 1997). Identifying genes and

variants related to obesity will open new avenues in the

management of obesity in humans for which no efficient

treatment, apart from major surgery, currently exists.

In the present study, we used three different pig groups:

production pigs and lean and fat G€ottingen minipigs. The

two production pig breeds, Yorkshire and Duroc, have been

selected for leanness for many years (Cleveland et al. 1982),

whereas the G€ottingen minipig is predisposed to obesity

when fed ad libitum (Bollen et al. 2005). However, the males

do not get as obese as the females (Christoffersen et al.

2007) comparable to what is seen in humans (Kanter &

Caballero 2012). Because of the extreme differences in

phenotype between G€ottingen minipigs and production pigs,

it can be hypothesized that different variants of genes

underlying obesity have been fixed in the respective breeds,

potentially resulting in differences in transcription levels of

genes involved in fat deposition.

The expression of six obesity-related genes, LEP, LEPR,

MC4R, FTO, NEGR1 and ADIPOQ, was investigated.

Mutations in LEP, LEPR and MC4R giving rise to morbid

obesity have been detected in the human population

(Dougkas et al. 2013). These three genes have been

implicated as obesity candidate genes in QTL and associ-

ation studies in pigs (e.g. Switonski et al. 2010). Leptin is

an adipocyte-specific hormone that regulates adipose tissue

mass through hypothalamic effects on satiety and energy

expenditure (Campfield et al. 1995). We found highly

significant differential expression of LEP in all three

adipose tissues showing a prominent breed-specific pattern

with the highest expression in MP. We also saw differential

expression in muscle and pancreas; however, in these

tissues, the expression seemed to be influenced by state,

that is, the expression was highest or only detectable in

FMP.

Expression of LEPR was detected in all tissues comparable

to what is seen in humans, with significant differential

expression between breeds and state in muscle and between

breeds in subcutaneous and mesenteric adipose tissues. The

high expression of LEPR in liver in all three groups of pigs

supports the notion that leptin can affect lipid metabolism

© 2013 The Authors, Animal Genetics © 2013 Stichting International Foundation for Animal Genetics, doi: 10.1111/age.12082

Cirera et al.4

(a) (c)

(b) (d)

(e)

-

- - -

- - -

-

------

Figure 1 qPCR results in the different tissues and pig groups for the 6 genes analyzed. Error bars are standard deviations (SD). Under each graph, the

P-values from Tukey post-test for each comparison are specified. PP, production pigs; LMP, lean Gottingen minipigs; FMP, obese Gottingen minipigs;

-, under limit of detection; ns, not significant; a, only expressed in FMP; b, only expressed in PP.

© 2013 The Authors, Animal Genetics © 2013 Stichting International Foundation for Animal Genetics, doi: 10.1111/age.12082

Expression studies of obesity-related genes in divergent pig breeds 5

independent of its effect on food intake and energy

expenditure as shown in ob/ob mice (Singh et al. 2009).

FTO is a nuclear protein of the AlkB-related non-heme

iron and 2-oxoglutarate-dependent oxygenase superfamily

(Gerken et al. 2007). We found differential expression in all

tissues included in the study with the highest expression in

hypothalamus. The differential expression was breed spe-

cific in liver, muscle, hypothalamus and retroperitoneal

adipose tissue, whereas it was both breed and state specific

in subcutaneous adipose tissue. A study of FTO expression

in subcutaneous adipose tissue from morbidly obese

women has shown that expression of this gene is reduced

in obese women compared with control subjects (Terra

et al. 2010). Although it was not statistical significant,

when comparing the expression in LMP and FMP in

subcutaneous and mesenteric adipose tissue, the same

trend was observed (Fig. 1c). Remarkably, the expression

was more than a log2-fold change higher in pancreas in

FMP compared with PP and LMP, indicating that expres-

sion in this tissue is influence by obesity status and

suggesting that the pancreas is also an intermediate in FTO

communication.

Studies of FTO in humans and rodents have mainly

focused on the expression in adipose tissues and hypothal-

amus. Thus, our study provides a broader view on FTO

expression in both lean and obese individuals.

NEGR1 has a role in neuronal outgrowth (Sch€afer et al.

2005) and is highly expressed in cortex and hypothalamus

(a) (b)

(c) (d)

(e) (f)

Figure 2 Representative histological sections

of adipose tissues from the three groups of

pigs (a and b, LMP; c and d, FMP; and d and e,

PP). Retroperitoneal adipose tissue is present

in the left and subcutaneous adipose tissue in

the right column. Notice that the subcutane-

ous adipose tissue is richer in connective tissue

when compared to the retroperitoneal and the

variation of the size of the fat cells between

the three groups. All figures are at the same

magnification (Bar = 100 lm).

© 2013 The Authors, Animal Genetics © 2013 Stichting International Foundation for Animal Genetics, doi: 10.1111/age.12082

Cirera et al.6

(Willer et al. 2009). A meta-analysis of 15 GWAS for BMI

has identified a deletion upstream of NEGR1 as a candidate

causal variant associated with BMI (Willer et al. 2009).

Furthermore, NEGR1 has been shown to be expressed in

subcutaneous adipose tissue where it is suggested to act as a

central ‘hub’ in an obesity-related network (Walley et al.

2012). The expression profile we saw in pigs is concordant

with the results seen in humans, that is, NEGR1 is highly

expressed in hypothalamus and also expressed in other

tissues. Both in hypothalamus, pancreas and muscle, there

is breed-specific significant differential expression. We also

saw differential expression in subcutaneous adipose tissue

that seems to be affected both by breed and obesity status,

validating the role of NEGR1 in pathways involved in the

regulation of obesity.

Adiponectin, encoded by ADIPOQ, is a hormone

secreted by adipocytes regulating energy homeostasis

and glucose and lipid metabolism. We found the highest

expression of ADIPOQ in the three adipose tissues. In

agreement with the studies conducted by Matsuzawa et al.

(2011) and Jacobi et al. (2004), we found significant

differential expression in subcutaneous and mesenteric

adipose tissues. We also saw differential expression in

muscle. Remarkably, ADIPOQ expression was not detect-

able in liver and hypothalamus in PP and LMP, but it was

high in the FMP.

MC4R is the gene with the most frequent prevalence of

multiple polymorphisms related to obesity (Lubrano-

Berthelier et al. 2003). MC4R is expressed primarily in the

brain, regulating food intake and possibly energy expendi-

ture. A mis-sense mutation has been identified in the

porcine MC4R gene associated with back fat thickness,

growth rate and food intake (Kim et al. 2004). In our study,

expression levels in the investigated tissues, except for

hypothalamus, were below the range at which differential

expression can confidently be detected. In hypothalamus,

the MC4R transcript tends to be down-regulated in both

FMP and LMP compared with PP, in agreement with several

studies carried out in humans and mice where down-

regulation has been seen in obese individuals.

The expression of all the genes investigated in this study

was significantly altered in one or more of the adipose

tissues, confirming the functional link between these genes

and obesity. Because the expression profile for some of the

genes (LEPR, FTO, NEGR1, ADIPOQ) differed in the three

adipose tissues, our results indicate that metabolism differs

between the different adipose compartments of the body.

This also was supported by the histological findings, which

show structural differences both between tissues and pig

groups. Functional studies demonstrating that uptake of

triglycerides are higher in omental than in retroperitoneal

adipose tissue lend further support to these findings (M�arin

et al. 1992). Human studies also have shown that, when

adipocytes enlarge in relation to a gain of body weight, the

activity of low density lipoprotein increases (reviewed by

Wajchenberg 2000). This is in accordance with both our

histological findings and with the results obtained on

differential expression of LEP, that is, the largest adipose

cells are found in FMP where we also saw the highest LEP

expression.

Our study clearly shows that both breed- and lean/obese-

state differences have a high impact on differential expres-

sion of obesity-related genes. This is in agreement with

findings in different strains of rats (Lange et al. 2012). The

breed differences detected in our study reflect the obvious

phenotypic differences between the obesity-prone and

obesity-resistant (lean) phenotypes.

In summary, the expression profiles of the investigated

genes in pig in general are in accordance with those

observed in human studies. However, because we have

included a broader range of tissues, we have generated

novel information, indicating that some of the genes are

involved in metabolic pathways exceeding those that have

been described previously.

Competing interests

The authors declare that they have no competing interests.

Acknowledgements

We would like to thank Minna Jakobsen for excellent

technical assistance in the qPCR experiment and Helle

Anita V. Ruby in the histology work respectively. The LMP

were funded by the SHARE Foundation and Danielsen’s

Foundation. The FMP were donated by Novo Nordisk A/S,

Denmark.

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