Ai

KD

a

ARRAA

KAACFP

1

bhlbtic

bromtimccrt

0h

Peptides 37 (2012) 13–17

Contents lists available at SciVerse ScienceDirect

Peptides

j ourna l ho me pa ge: www.elsev ier .com/ locate /pept ides

comparative study of the central effects of melanocortin peptides on foodntake in broiler and layer chicks

azuhisa Honda ∗, Takaoki Saneyasu, Shin Hasegawa, Hiroshi Kamisoyamaepartment of Bioresource Science, Graduate School of Agricultural Science, Kobe University, Kobe 657-8501, Japan

r t i c l e i n f o

rticle history:eceived 30 May 2012eceived in revised form 25 June 2012ccepted 25 June 2012vailable online 1 July 2012

a b s t r a c t

Broiler chicks eat more food than layer chicks. However, the causes of the difference in food intake inthe neonatal period between these strains are not clear. In this study, we examined the involvementof proopiomelanocortin (POMC)-derived melanocortin peptides �-, �- and �-melanocyte-stimulatinghormones (MSHs) in the difference in food intake between broiler and layer chicks. First, we comparedthe hypothalamic mRNA levels of POMC between these strains and found that there was no significant

eywords:ppetitevianhickenseeding behaviorOMC

difference in these levels between broiler and layer chicks. Next, we examined the effects of centraladministration of MSHs on food intake in these strains. Central administration of �-MSH significantlysuppressed food intake in both strains. Central administration of �-MSH significantly suppressed foodintake in layer chicks, but not in broiler chicks, while central administration of �-MSH did not influencefood intake in either strain. It is therefore likely that the absence of the anorexigenic effect of �-MSHmight be related to the increased food intake in broiler chicks.

© 2012 Elsevier Inc. All rights reserved.

. Introduction

Broiler chickens have been genetically selected for their largeody weight and high growth rate, and this genetic selectionas led to wide variations in food intake between broiler and

ayer chickens. There is evidence that differences in food intakeetween broiler and layer chickens are observable in the neona-al period [11,15]. However, the causes of the difference in foodntake in the neonatal period between these strains are notlear.

It is generally accepted that appetite is regulated by the balanceetween two orexigenic peptides (neuropeptide Y and agouti-elated protein) and proopiomelanocortin (POMC, a precursorf neuropeptides)-derived anorexigenic melanocortin peptides inammals [14]. However, there is evidence that central adminis-

ration of agouti-related protein does not influence food intaken broiler chicks [28]. We previously showed that hypothalamic

RNA levels of neuropeptide Y and its receptors are lower in broilerhicks than layer chicks, and that increase in food intake in broilerhicks cannot be explained by the difference in neuropeptide Y

esponsiveness [20]. These findings suggest that the differences inhe orexigenic effects of neuropeptide Y and agouti-related protein

∗ Corresponding author. Tel.: +81 78 803 5809; fax: +81 78 803 5809.E-mail address: honda@tiger.kobe-u.ac.jp (K. Honda).

196-9781/$ – see front matter © 2012 Elsevier Inc. All rights reserved.ttp://dx.doi.org/10.1016/j.peptides.2012.06.015

between broiler and layer chicks are not related to the increase infood intake in broiler chicks.

POMC neurons in the hypothalamus play an important rolein transducing afferent inputs, i.e., hormonal and metabolic sig-nals, into a neuronal anorexigenic response in mammals and birds[14,18,23]. The hypothalamic POMC mRNA level is increased bythe peripheral anorexigenic hormone [3,7,13,24] and reduced byfasting [6,14] in mammals and chickens. Central administrationof the POMC-derived neuropeptides �-, �- and �-melanocyte-stimulating hormones (MSHs) significantly reduced food intake inPOMC-null mice [29]. We recently showed that central admin-istration of �-MSH significantly reduced food intake in broilerchicks, while �- and �-MSH did not influence food intake [19].There is evidence that central administration of �-MSH also sig-nificantly reduces food intake in layer chicks [7,27]. However,the effects of �- and �-MSH on food intake in layer chicks havenot yet been examined. It is therefore possible that the absenceof the anorexigenic effects of �- and �-MSH in broiler chicks isinvolved in the difference in food intake between broiler and layerchickens.

In the present study, we examined the effects of centraladministration of �-, �- and �-MSH on food intake in layer andbroiler chicks in order to clarify the involvement of POMC-derived

melanocortin peptides in the difference in food intake betweenthese strains. The results demonstrate that �-MSH significantlysuppresses food intake only in layer chicks and that �-MSH doesnot influence food intake in either strain.

1 ptides 37 (2012) 13–17

2

2

(Gw(pfm�tOp

2l

dtffztKDHc(f3snsGTfis

2c

gcbeiFtbbv

2c

gcaad

Arb

itra

ry u

nit

0

0.5

1.0

Layer Broiler

4 K. Honda et al. / Pe

. Materials and methods

.1. Animals and peptides

Day-old male layer chicks (White Leghorn) and broiler chickschunky) were purchased from a local hatchery (Ghen Corporation,ifu, Japan and Ishii Co., Ltd., Tokushima, Japan, respectively). Theyere given free access to water and a commercial chick starter diet

Nippon Formula Feed Mfg. Co., Ltd., Kanagawa, Japan). Room tem-erature was maintained at 32 ± 2 ◦C. All experimental proceduresollowed the guidelines for the care and use of experimental ani-

als at the Rokkodai Campus of Kobe University in Japan. Human-MSH (H-SYSMEHFRWGKPV-OH) that has identical sequence to

he chicken �-MSH, chicken �-MSH (H-DGGSYRMRHFRWHAPLKD-H) [26], and chicken �-MSH (H-YVMSHFRWNKFG-OH) [26] wereurchased from Peptide Institute, Inc. (Osaka, Japan).

.2. Experiment 1: hypothalamic POMC mRNA level in broiler andayer chicks

Broiler and layer chicks were sacrificed by decapitation at 8ays of age. Their brains were removed within 1 min of decapita-ion, weighed, frozen on powdered dry ice and stored at −80 ◦Cor further analysis. The hypothalamus was dissected from therozen brain (by referring to a stereotaxic atlas drawn by Kuen-el and Masson [8]) and weighed. Total RNA was extracted fromhe hypothalamus using the Sepazol-RNA I (Nacalai Tesque, Inc.,yoto, Japan). First-strand cDNA was synthesized from 5 �g ofNase I (Ambion Inc., Austin, TX, USA)-treated total RNA using theigh Capacity RNA-to-cDNA Kit (Applied Biosystems Inc., Fosterity, CA, USA) with random primers. Complementary DNA of POMCGenBank accession no. NM 001031098) was amplified with theollowing primers: POMC sense, 5′-AGA TGG AGA AGG GTT GGA A-′; POMC antisense, 5′-CGT TGG GGT ACA CCT TGA-3′. As an internaltandard, chicken ribosomal protein S17 mRNA (GenBank accessiono. NM 204217) was also amplified, using the following primers:ense, 5′-GCG GGT GAT CAT CGA GAA GT-3′; antisense, 5′-GCG CTTTT GGT GTG GAA GT-3′. SYBR® Premix Ex Taq was purchased fromakara Bio Inc. (Shiga, Japan), and mRNA expression was quanti-ed in triplicate using the Applied Biosystems 7300 Real-Time PCRystem according to the supplier’s recommendations.

.3. Experiment 2: effect of central administration of ˛-MSH onumulative food intake in broiler and layer chicks

Eight-day-old chicks from each strain were divided into threeroups. �-MSH was dissolved in a 0.85% (w/v) saline solutionontaining 0.1% (w/v) Evans Blue. The peptides were intracere-roventricularly administered according to the method of Davist al. [5] at a volume of 10 �l after 3 h of fasting. Chicks were admin-stered either the �-MSH (40 or 400 pmol) or saline (as a control).ood intake was measured at 30, 60 and 120 min after adminis-ration. At the end of the experiment, the chicks were sacrificedy decapitation. Verification of peptide administration was madey observation of the presence of Evans Blue dye in the lateralentricle.

.4. Experiment 3: effect of central administration of ˇ-MSH onumulative food intake in broiler and layer chicks

Eight-day-old chicks from each strain were divided into threeroups, and either the �-MSH (40 or 400 pmol) or saline (as a

ontrol) was intracerebroventricularly administered as describedbove. Food intake was measured at 30, 60 and 120 min afterdministration and verification of peptide injection was made asescribed above.

Fig. 1. Hypothalamic POMC mRNA levels in broiler and layer chicks. Values aremeans ± S.E.M. for eight chicks in each group.

2.5. Experiment 4: effect of central administration of �-MSH oncumulative food intake in broiler and layer chicks

Eight-day-old chicks from each strain were divided into threegroups, and either the �-MSH (40 or 400 pmol) or saline (as a con-trol) was intracerebroventricularly administered as described inExperiment 1. Food intake was measured at 30, 60 and 120 minafter administration and verification of peptide injection was madeas described in Experiment 1.

2.6. Experiment 5: hypothalamic melanocortin 4 receptor mRNAlevel in broiler and layer chicks

Hypothalamic mRNA level of melanocortin 4 receptor (MC4R)was analyzed as described in Experiment 1. Complementary DNAof MC4R (GenBank accession no. NM 001031514) was amplifiedwith the following primers: sense, 5′-CCT CGG GAG GCT GCT ATGA-3′; antisense, 5′-GAT GCC CAG AGT CAC AAA CAC TT-3′.

2.7. Data analysis

Data from Experiments 1 and 5 was analyzed by Student’s t test.Data from Experiments 2–4 were analyzed by the Tukey–Kramertest at each time point. All statistical analyses were performed usingthe commercial package (StatView version 5, SAS Institute, Cary,NC, USA, 1998).

3. Results

There was no significant difference in the hypothalamic POMCmRNA levels between these strains (Fig. 1). Central administra-tion of �-MSH significantly suppressed food intake in both strains(Fig. 2). Central administration of �-MSH significantly suppressedfood intake in layer chicks, but not in broiler chicks (Fig. 3). Gamma-MSH did not affect food intake in both strains (Fig. 4). There was nosignificant difference in hypothalamic MC4R mRNA levels betweenbroiler and layer chicks (Fig. 5).

4. Discussion

We first examined the hypothalamic mRNA levels of POMC inbroiler and layer chicks. There was no significant difference in thehypothalamic POMC mRNA levels between these strains (Fig. 1),suggesting that the hyperphagia in broiler chicks is not caused bythe reduction in hypothalamic POMC mRNA levels. This view is sup-

ported by a recent publication; layer and broiler chicks exhibitedsimilar hypothalamic expression of POMC at 7 days of age [30].

We next examined the effects of central administration of MSHson food intake in these strains. Central administration of �-MSH

K. Honda et al. / Peptides 37 (2012) 13–17 15

Saline40 pmol400 pmol

0

2

4

6

8

10

Cu

mu

lati

ve

food

in

tak

e (g

)

(8)(9)(6)

Time after administration (min)

0

1

2

3

4

5

Cu

mu

lati

ve

food

in

tak

e (g

)

(6)(6)(8)

Broiler

Layer

30 60 120

Fig. 2. Effect of central administration of �-MSH on food intake in broiler and layerchicks. Values are means ± S.E.M. The number of chicks used is shown in parenthe-ses. Groups with different letters are significantly different (p < 0.05) at each timepoint.

Fig. 3. Effect of central administration of �-MSH on food intake in broiler and layerchicks. Values are means ± S.E.M. The number of chicks used is shown in parenthe-ses. Groups with different letters are significantly different (p < 0.05) at each timepoint.

Cu

mu

lati

ve

food

in

tak

e (g

)

0

2

4

6

8

10

(8)(7)(10)

0

1

2

3

4

5

Cu

mu

lati

ve

food

in

tak

e (g

)

(8)(8)(8)

Time after administration (min)

Broiler

Layer

30 60 120

Saline40 pmol400 pmol

Fig. 4. Effect of central administration of �-MSH on food intake in broiler and layerchicks. Values are means ± S.E.M. The number of chicks used is shown in parenthe-ses.

significantly suppressed food intake in both strains (Fig. 2). Theseresults are in good agreement with previous studies [7,19,27].

Central administration of �-MSH significantly suppressed foodintake in layer chicks (Fig. 3). On the other hand, central admin-istration of �-MSH did not affect food intake in broiler chicks(Fig. 3). It is therefore likely that the absence of the anorexigeniceffect of �-MSH might be related to the difference in food intakebetween broiler and layer chicks. We previously showed that cen-tral administration of human �-MSH in broiler chicks potentlysuppresses food intake [17]. We confirmed that central adminis-tration of human �-MSH in layer chicks potently suppressed foodintake as well as �-MSH-induced anorexia (data not shown). Thereis evidence that central administration of human �-MSH in ratssignificantly reduced food intake, whereas human �-MSH mutant,which has only one amino acid substitution, had no effect on foodintake [4]. It is therefore likely that only two amino acid substitu-tions in chicken �-MSH have attenuated the anorexigenic action of�-MSH in chickens.

Gamma-MSH did not show any significant anorexigenic effect inbroiler or layer chicks (Fig. 4), suggesting that �-MSH is not involvedin the central regulation of food intake in chickens. In mammals, dif-

ferent results for the anorexigenic effect of �-MSH were reported.For example, Millington et al. reported that central administrationof �-MSH strongly suppressed food intake in rats [12]. However,

Arb

itra

ry u

nit

0

2

4

Layer Broiler

1

3

Fig. 5. Hypothalamic MC4R mRNA levels in broiler and layer chicks. Values aremeans ± S.E.M. for eight chicks in each group.

1 ptides

Tt�tii

aMtd9Mt�fislpctiliiScfiotiwtl

occTwiw�a

5

etficwciai

A

fS

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

6 K. Honda et al. / Pe

ung et al. showed that equimolar amounts of �-MSH were ableo reduce food intake, but to a lesser degree than that seen with-MSH [29]. In addition, Abott et al. reported that central adminis-

ration of either �-MSH or �-MSH significantly reduced food intaken rats when given in the early light phase, whereas �-MSH did notnfluence food intake [1].

In mammals, the anorexigenic effect of �- and �-MSH is medi-ted by hypothalamic MC4R [2,9]. In chickens, MC3R, MC4R, andC5R are expressed in the brain [16,25]. Ling et al. reported

hat HS014, which is selective for the mammalian MC4R [22],isplays clear selectivity for the chicken MC4R with more than0-fold higher affinity as compared with the chicken MC3R andC5R [10]. Recently, we showed that HS014 completely reversed

he anorexigenic effect of �-MSH in chicks [19], suggesting that-MSH suppresses food intake via MC4R in chicks. Thus, wenally analyzed the mRNA levels of hypothalamic MC4R in bothtrains. However, there was no significant difference in hypotha-amic MC4R mRNA levels between broiler and layer chicks (Fig. 5,

= 0.159), although the mean value of MC4R mRNA level in layerhicks was higher than that in broiler chicks. It is therefore possiblehat MC4R is not involved in the different effects of �-MSH on foodntake between broiler and layer chicks. However, MC4R proteinevels on the target neurons of POMC neurons were not investigatedn the present study. In addition, up to now, chicken �-MSH bind-ng affinity to the melanocortin receptors has not been examined.harma et al. reported polymorphisms in MC3R and MC4R and asso-iation with production traits in a commercial broiler line [21]. Thisnding and our results raise the hypothesis that the protein levelsf melanocortin receptors and/or chicken �-MSH binding affinity tohe melanocortin receptors on the target neurons of POMC neuronsn broiler chicks was lower than that in layer chicks. Further study

ill be needed to clarify this point and the mechanisms underlyinghe different effects of �-MSH on food intake between broiler andayer chicks.

In the present study, we use 8-day-old chicks. The body weightf broiler chicks (about 150–160 g) was heavier than that of layerhicks (about 80 g). However, brain weights of broiler and layerhicks are not much different between two strains at 8 days of age.herefore, we did not decide the doses of MSHs based on their bodyeight. The results in the present study were quite similar to those

n our previous study using 4-day-old broiler chicks (average bodyeight was about 75 g) [19]. Thus, the ineffectiveness of 400 pmol-MSH would not be due to the difference in their body weight orge between two strains.

. Conclusion

In summary, we first compared the hypothalamic mRNA lev-ls of POMC between broiler and layer chicks, and then examinedhe effects of central administration of melanocortin peptides onood intake in these strains. There were no significant differencesn hypothalamic POMC mRNA levels between broiler and layerhicks. �-MSH significantly suppressed food intake in both strains,hereas the anorexigenic effect of �-MSH was found only in layer

hicks. Central administration of �-MSH did not influence foodntake in either strain. It is therefore likely that the absence of thenorexigenic effect of �-MSH might be related to the increased foodntake in broiler chicks.

cknowledgements

This work was supported by a Grant-in-Aid (Number 50091947)or Scientific Research (A) from the Ministry of Education, Culture,ports, Science, and Technology of Japan.

[

[

37 (2012) 13–17

References

[1] Abbott CR, Rossi M, Kim M, AlAhmed SH, Taylor GM, Ghatei MA, et al. Investiga-tion of the melanocyte stimulating hormones on food intake. Lack of evidenceto support a role for the melanocortin-3-receptor. Brain Res 2000;869:203–10.

[2] Adan RA, Tiesjema B, Hillebrand JJ, La Fleur SE, Kas MJ, De Krom M. The MC4receptor and control of appetite. Br J Pharmacol 2006;149:815–27.

[3] Benoit SC, Air EL, Coolen LM, Strauss R, Jackman A, Clegg DJ, et al. Thecatabolic action of insulin in the brain is mediated by melanocortins. J Neurosci2002;22:9048–52.

[4] Biebermann H, Castaneda TR, Van Landeghem F, Von Deimling A, Escher F,Brabant G, et al. A role for beta-melanocyte-stimulating hormone in humanbody-weight regulation. Cell Metab 2006;3:141–6.

[5] Davis JL, Masuoka DT, Gerbrandt LK, Cherkin A. Autoradiographic distribu-tion of l-proline in chicks after intracerebral injection. Physiol Behav 1979;22:693–5.

[6] Hen G, Yosefi S, Simchaev V, Shinder D, Hruby VJ, Friedman-Einat M.The melanocortin circuit in obese and lean strains of chicks. J Endocrinol2006;190:527–35.

[7] Honda K, Kamisoyama H, Saneyasu T, Sugahara K, Hasegawa S. Centraladministration of insulin suppresses food intake in chicks. Neurosci Lett2007;423:153–7.

[8] Kuenzel WJ, Masson MA. Stereotaxic atlas of the brain of the chick (Gal-lus domesticus). Baltimore/London: The Johns Hopkins University Press;1988.

[9] Lee YS, Challis BG, Thompson DA, Yeo GS, Keogh JM, Madonna ME, et al. APOMC variant implicates beta-melanocyte-stimulating hormone in the controlof human energy balance. Cell Metab 2006;3:135–40.

10] Ling MK, Hotta E, Kilianova Z, Haitina T, Ringholm A, Johansson L, et al. Themelanocortin receptor subtypes in chicken have high preference to ACTH-derived peptides. Br J Pharmacol 2004;143:626–37.

11] Mahagna M, Nir I. Comparative development of digestive organs, intestinaldisaccharidases and some blood metabolites in broiler and layer-type chicksafter hatching. Br Poult Sci 1996;37:359–71.

12] Millington GWM, Tung YCL, Hewson AK, O’rahilly S, Dickson SL. Differentialeffects of alpha-, beta- and gamma(2)-melanocyte-stimulating hormones onhypothalamic neuronal activation and feeding in the fasted rat. Neuroscience2001;108:437–45.

13] Mizuno TM, Kleopoulos SP, Bergen HT, Roberts JL, Priest CA, Mobbs CV.Hypothalamic pro-opiomelanocortin mRNA is reduced by fasting and in ob/oband db/db mice, but is stimulated by leptin. Diabetes 1998;47:294–7.

14] Morton GJ, Cummings DE, Baskin DG, Barsh GS, Schwartz MW. Central ner-vous system control of food intake and body weight. Nature 2006;443:289–95.

15] Nir I, Nitsan Z, Mahagna M. Comparative growth and development of the diges-tive organs and of some enzymes in broiler and egg type chicks after hatching.Br Poult Sci 1993;34:523–32.

16] Ka S, Lindberg J, Strömstedt L, Fitzsimmons C, Lindqvist N, Lundeberg J, et al.Extremely different behaviours in high and low body weight lines of chicken areassociated with differential expression of genes involved in neuronal plasticity.J Neuroendocrinol 2009;21:208–16.

17] Kamisoyama H, Honda K, Saneyasu T, Sugahara K, Hasegawa S. Corticotropin-releasing factor is a downstream mediator of the beta-melanocyte stim-ulating hormone-induced anorexigenic pathway in chicks. Neurosci Lett2009;458:102–5.

18] Richards MP, Proszkowiec-Weglarz M. Mechanisms regulating feed intake,energy expenditure, and body weight in poultry. Poult Sci 2007;86:1478–90.

19] Saneyasu T, Honda K, Kamisoyama H, Nakayama Y, Ikegami K, Hasegawa S.Alpha-melanocyte stimulating hormone plays an important role in the regu-lation of food intake by the central melanocortin system in chicks. Peptides2011;32:996–1000.

20] Saneyasu T, Honda K, Kamisoyama H, Ikura A, Nakayama Y, Hasegawa S. Neu-ropeptide Y effect on food intake in broiler and layer chicks. Comp BiochemPhysiol A 2011;159:422–6.

21] Sharma P, Bottje W, Okimoto R. Polymorphisms in uncoupling protein,melanocortin 3 receptor, melanocortin 4 receptor, and pro-opiomelanocortingenes and association with production traits in a commercial broiler line. PoultSci 2008;87:2073–86.

22] Schiöth HB, Mutulis F, Muceniece R, Prusis P, Wikberg JE. Discovery ofnovel melanocortin4 receptor selective MSH analogues. Br J Pharmacol1998;124:75–82.

23] Schwartz MW, Woods SC, Porte Jr D, Seeley RJ, Baskin DG. Central nervoussystem control of food intake. Nature 2000;404:661–71.

24] Shiraishi J, Yanagita K, Fujita M, Bungo T. Central insulin suppresses feedingbehavior via melanocortins in chicks. Domest Anim Endocrinol 2008;34:223–8.

25] Takeuchi S, Takahashi S. Melanocortin receptor genes in the chicken—tissuedistributions. Gen Comp Endocrinol 1998;112:220–31.

26] Takeuchi S, Teshigawara K, Takahashi S. Molecular cloning and characteriza-tion of the chicken pro-opiomelanocortin (POMC) gene. Biochim Biophys Acta

1999;1450:452–9.

27] Tachibana T, Oikawa D, Takahashi H, Boswell T, Furuse M. The anorexic effectof alpha-melanocyte-stimulating hormone is mediated by corticotrophin-releasing factor in chicks. Comp Biochem Physiol A: Mol Integr Physiol2007;147:173–8.

ptides

[

[

K. Honda et al. / Pe

28] Tachibana T, Sugahara K, Ohgushi A, Ando R, Kawakami S, Yoshimatsu T,

et al. Intracerebroventricular injection of agouti-related protein attenuatesthe anorexigenic effect of alpha-melanocyte stimulating hormone in neonatalchicks. Neurosci Lett 2001;305:131–4.

29] Tung YC, Piper SJ, Yeung D, O’Rahilly S, Coll AP. A comparative study of thecentral effects of specific pro-opiomelanocortin (POMC)-derived melanocortin

[

37 (2012) 13–17 17

peptides on food intake and body weight in POMC null mice. Endocrinology

2006;147:5940–7.

30] Yuan L, Ni Y, Barth S, Wang Y, Grossmann R, Zhao R. Layer and broiler chicksexhibit similar hypothalamic expression of orexigenic neuropeptides but dis-tinct expression of genes related to energy homeostasis and obesity. Brain Res2009;1273:18–28.