Accepted Manuscript

Identification, expression analysis, and functional characterization of peptide

YY in chickens (Gallus gallus domesticus)

Koji Aoki, Makoto Kondo, Mika Okuda, Takaoki Saneyasu, Kazuhisa Honda,
Hiroshi Kamisoyama

PII: S0016-6480(16)30096-X
DOI: http://dx.doi.org/10.1016/j.ygcen.2016.04.021
Reference: YGCEN 12378

To appear in: General and Comparative Endocrinology

Received Date: 27 January 2016

Revised Date: 5 April 2016
Accepted Date: 22 April 2016

Please cite this article as: Aoki, K., Kondo, M., Okuda, M., Saneyasu, T., Honda, K., Kamisoyama, H., Identification,
expression analysis, and functional characterization of peptide YY in chickens (Gallus gallus domesticus), General
and Comparative Endocrinology (2016), doi: http://dx.doi.org/10.1016/j.ygcen.2016.04.021

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 1 Identification, expression analysis, and functional characterization of peptide YY in chickens (Gallus gallus

2 domesticus)

4 Koji Aoki, Makoto Kondo, Mika Okuda, Takaoki Saneyasu, Kazuhisa Honda, and Hiroshi Kamisoyama

6 Department of Bioresource Science, Graduate School of Agricultural Science, Kobe University, Kobe 657-8501,

7 Japan

9 Corresponding author:

10 Kazuhisa Honda, Ph.D.

11 Department of Bioresource Science,

12 Graduate School of Agricultural Science,

13 Kobe University

14 Kobe 657-8501

15 Japan

16 Tel./fax: +81 78 803 5809; E-mail address: honda@tiger.kobe-u.ac.jp

17

18

19 Acknowledgements:

20 We thank Mr. Daichi Hiramoto and Mr. Takehiro Shin (Faculty of Agriculture, Kobe University) for helpful

21 assistance.

22

23

24
1
 1 Abstract

3 Peptide YY (PYY) functions as a postprandial satiety signal in mammals. However, the genomic

4 information and physiological roles of chicken PYY have not yet been clarified, although PYY peptide was

5 isolated from chicken intestines in 1992. In this study, we identified a full-length complementary DNA (cDNA)

6 sequence encoding the chicken PYY precursor. The deduced amino acid sequence of chicken PYY was

7 completely consistent with the previously identified peptide sequence. PYY mRNA was abundantly expressed in

8 the small intestine compared with the large intestine. PYY mRNA levels in the jejunum were significantly higher

9 during ad libitum feeding compared with fasting, suggesting that intestinal PYY expression is altered in response

10 to nutritional status in chicks. Intravenous administration of PYY significantly suppressed food intake in chicks.

11 Furthermore, neuropeptide Y receptor Y2, a possible target of PYY, was expressed in various brain regions

12 including the appetite-regulating centers in chicks. This is the first evidence that the intestinal hormone PYY may

13 function as an anorexigenic hormone in chicks.

14

15 Keywords: Appetite; Chicken; Food intake; Peptide YY

2
 1 1. Introduction

3 Appetite is regulated by various circulating hormones and neuropeptides. Peptide YY (PYY), one of the

4 postprandial satiety hormones, is expressed in the gastrointestinal tract, especially in the distal intestine, in

5 mammals (Ekblad and Sundler, 2002; Ueno et al., 2008). The plasma concentration of PYY is elevated after

6 feeding (Zwirska-Korczala et al., 2007; Stadlbauer et al., 2013) and after the intraintestinal administration of

7 nutrients (Fu-Cheng et al., 1995). The peripheral injection of PYY3-36, one of the major circulating forms of PYY,

8 reduces food intake in humans and rodents (Batterham et al., 2002; Martin et al., 2004; Degen et al., 2005; Scott et

9 al., 2005; Chelikani et al., 2007). In addition, PYY knockout mice exhibit overfeeding (Batterham et al., 2006).

10 Therefore, PYY is regarded as a therapeutic target in obesity (De Silva and Bloom, 2012).

11 Two different pathways underlying the anorexigenic effect of PYY have been proposed in mammals.

12 Peripheral injection of PYY3-36 activates the neurons in the arcuate nucleus (ARC) (Blevins et al., 2008). The

13 ARC is known to be the hypothalamic appetite-regulating center receiving the humoral input from circulating

14 hormones (Joly-Amado et al., 2014). Furthermore, direct injection of PYY3-36 into the ARC decreases food intake

15 in rats (Batterham et al., 2002). PYY3-36 has been shown to cross the blood-brain barrier (Nonaka et al., 2003).

16 These findings suggest that circulating PYY is received as a humoral satiety signal in the brain. However,

17 peripheral injection of PYY3-36 activates the gastric vagal afferent nerves (Koda et al., 2005) and the neurons in

18 the nucleus of the solitary tract (NTS) (Blevins et al., 2008). NTS is known to be the site that receives neural input

19 from the gastric vagal afferent nerves (Schwartz, 2006). In addition, pretreatment by vagotomy abolishes

20 PYY3-36-induced hypophagia in rats (Koda et al., 2005). These findings suggest that circulating PYY also

21 transmits a neural satiety signal through the vagal afferent nerves into the brain.

22 An in vitro binding assay revealed that neuropeptide Y receptor Y2 (Y2R), one of the G-protein-coupled

23 receptors, has the highest affinity with PYY3-36 of the neuropeptide Y receptor family in mammals (Keire et al.,

24 2002). Intravenous (Scott et al., 2005; Reidelberger et al., 2013) and intraarcuate (Abbott et al., 2005)
3
 1 administration of BIIE0246, a Y2R-specific antagonist, abolishes PYY3-36-induced hypophagia in rats. Y2R is

2 expressed in the ARC (Parker and Herzog, 1999) and vagal neurons (Koda et al., 2005). It is therefore likely that

3 both central and peripheral Y2R are involved in the PYY3-36-induced anorexigenic pathways.

4 PYY peptide was isolated from chicken intestines in 1992, and amino acid sequence analysis revealed

5 the presence of an additional N-terminal alanine residue (Conlon and OHarte, 1992) (Fig. 1). The extract of the

6 chicken intestines did not contain PYY3-36 (Conlon and OHarte, 1992). However, an in vitro binding assay

7 demonstrated that chicken PYY preferentially binds to Y2R (Salaneck et al., 2000). In addition, Y2R mRNA is

8 expressed in the brain and the peripheral tissues in chickens (Brome et al., 2006). These findings have led to the

9 hypothesis that intact-PYY influences the regulation of food intake in chickens.

10 In the present study, we identified the full-length cDNA sequence encoding the chicken PYY precursor

11 and investigated the physiological role of chicken PYY. Our findings suggest that PYY may function as an

12 anorexigenic hormone in the peripheral circulation of chickens.

13

14 2. Materials and methods

15

16 2.1. Animals and peptide

17

18 The animal experiments in this study were approved by the Institutional Animal Care and Use

19 Committee and carried out according to the Kobe University Animal Experimentation Regulation. Day-old male

20 broiler chicks (Ross 308) were purchased from a local hatchery (Ishii Co., Ltd., Tokushima, Japan). They were

21 given free access to water and a commercial chick starter diet (Feed One Holdings Co., Ltd., Kanagawa, Japan)

22 under a 23-h/1-h light:dark cycle. The 23-h/1-h light:dark cycle is commonly used in poultry industry. Chicken

23 PYY was synthesized by Bio-Synthesis, Inc. (Lewisville, TX, USA).

24
4
 1 2.2. Identification of chicken PYY nucleotide sequence

3 First-strand cDNA was synthesized from 0.5 g of total RNA from the chicken jejunum using a

4 ReverTra Ace qPCR RT Master Mix with gDNA remover (Toyobo Co., Ltd., Osaka, Japan). The coding region

5 of chicken PYY was amplified by PCR with the F1 and R1 primers (Table 1 and Fig. 2) designed according to the

6 cDNA sequence of Cuculus canorus PYY (GenBank accession number: XM_009571156.1). The PCR reaction

7 system comprised: 2 L of chicken jejunum cDNA, 5 L of SYBR Premix Ex Taq II, 0.2 L of ROX Reference

8 Dye (Takara Bio Inc., Shiga, Japan), and 0.4 L each of the 5 M F1 and R1 primers, making a final reaction

9 volume of 10 L. The PCR cycling parameters were; denaturation at 95C for 30 s; 40 cycles of 95C for 5 s,

10 60.1C for 32 s, and 72C for 30 s; and a final extension at 72C for 4 min. The resultant products were diluted

11 100-fold and used as a template in second-round PCR under the same conditions as for the first-round PCR. The

12 nucleotide sequences of the PCR products were determined by Fasmac Co., Ltd. (Kanagawa, Japan).

13 The 5- and 3-noncoding regions of chicken PYY were amplified by RACE-PCR using a GeneRacer

14 Kit (Invitrogen Corp., Carlsbad, CA, USA). Gene-specific primers (F2, F3, R2, and R3) (Table 1 and Fig. 2) were

15 designed based on the sequence information of the PCR product of the coding region of chicken PYY. Total RNA

16 isolated from chicken jejunum was used to produce RACE-ready cDNA according to the manufacturer's protocol.

17 The RACE-PCR reaction system comprised: 1 L of RACE-ready cDNA, 5 L of 10 High Fidelity PCR buffer,

18 0.5 L of Platinum Taq DNA Polymerase High Fidelity, 1 L of 10 mM dNTP Solution, 2 L of 50 mM MgSO4,

19 3 L of 10 M GeneRacer 5- or 3-primer, and 1 L of 10 M gene-specific primer (R2 or F2), making a final

20 reaction volume of 50 L. The PCR cycling parameters were denaturation at 94C for 2 min; five cycles of 94C

21 for 30 s and 72C for 1 min; 5 cycles of 94C for 30 s and 70C for 1 min; 25 cycles of 94C for 30 s, 68C for 30

22 s, and 72C for 1 min; and a final extension at 72C for 10 min. For the second-round nested PCR, 1 L of the

23 100-fold diluted solution of the first-round PCR was used as a template with 1 L of 10 M GeneRacer 5- or

24 3-nested primer and 1 L of 10 M gene-specific primer (R3 or F3). The cycling parameters were denaturation at
5
 1 94C for 2 min; 25 cycles of 94C for 30 s, 65C for 30 s, and 68C for 2 min; and a final extension at 68C for

2 10 min. The nucleotide sequences of the PCR products were determined by Fasmac Co., Ltd. (Kanagawa, Japan).

3 The full-length chicken PYY cDNA (GenBank accession number: LC102814) was amplified by PCR

4 with F4 and R4 primers (Table 1 and Fig. 2) designed based on the sequence information of the 5- and

5 3-RACE-PCR amplification products. The PCR reaction system comprised: 4 L of chicken jejunum cDNA, 5

6 L of 10 DNA Polymerase Buffer, 0.5 L of DNA Polymerase (Kaneka Corp., Osaka, Japan), 5 L of 2 mM

7 dNTPs, and 1 L each of 10 M F4 and R4 primers, making in a final reaction volume of 50 L. The PCR cycling

8 parameters were: denaturation at 94C for 2 min; 30 cycles of 95C for 20 s, 68.2C for 10 s, and 74C for 4 s.

9 The nucleotide sequences of the PCR products were determined by Fasmac Co., Ltd. (Kanagawa, Japan).

10 All PCR amplification products were electrophoresed on a 1.2% agarose gel containing GelGreen

11 Nucleic Acid Gel Stain, 10,000X in DMSO (Biotium Inc., Hayward, CA, USA) and extracted from the agarose

12 gel using a FastGene Gel/PCR Extraction Kit (NIPPON Genetics Co., Ltd., Tokyo, Japan) followed by sequence

13 analysis.

14

15 2.3. Experiment 1: Real-time PCR analysis of PYY mRNA levels in the chicken intestine

16

17 Twelve 8-day-old chicks were weighed and allocated to two groups based on body weight (six birds in

18 each group). Food was removed from six birds for 12 h, while the remaining six chicks were allowed to feed

19 freely. All chicks had free access to water. After 12 h, all chicks were sacrificed by decapitation. To investigate the

20 intestinal distribution of chicken PYY, the duodenum, jejunum, ileum, cecum, and rectum were dissected from

21 three chicks in the feeding group. In addition, to investigate the effect of fasting on PYY mRNA expression, the

22 jejunum was dissected from the remaining three chicks in the feeding group and the six chicks in the fasting group.

23 Tissues were immediately frozen in liquid nitrogen and stored at -80C until required for RNA extraction. The

24 total RNA was extracted from each section of the intestine using Sepasol-RNA I (Nacalai Tesque, Inc., Kyoto,
6
 1 Japan). First-strand cDNA was synthesized using a ReverTra Ace qPCR RT Master Mix with gDNA remover

2 (Toyobo Co., Ltd., Osaka, Japan). Complementary DNAs of chicken PYY (GenBank accession no.: LC102814)

3 and ribosomal protein S17 (RPS17) (GenBank accession no.: NM_204217) were amplified with the specific

4 primers (Table 1). The chicken PYY mRNA level was quantified in duplicate using an Applied Biosystems 7300

5 Real-Time PCR system and SYBR Premix Ex Taq II (Tli RNaseH Plus) (Takara Bio Inc., Shiga, Japan)

6 according to the supplier's recommendations.

8 2.4. Experiment 2: Effect of peripheral administration of chicken PYY on food intake in chicks

10 Peripheral administration of 1 - 10 ng/g BW (0.23 - 2.3 nmol/kg BW) PYY significantly decreased

11 food intake in goldfish (Gonzalez and Unniappan, 2010). Peripheral administration of 0.3-10 g/100g BW

12 (0.75 24.7 nmol/kg BW) PYY significantly decreased food intake in mice (Batterham et al., 2002).

13 However, preliminary experiment showed that intravascular administration of 1.5 nmol/kg BW chicken PYY

14 did not affect food intake in chicks (unpublished data). Therefore, the doses from 3 to 6 nmol/kg BW were

15 used in this experiment. Forty two 9-day-old chicks were weighed and allocated to three groups based on body

16 weight (14 birds in each group). Chicken PYY was dissolved in a 0.85% (w/v) saline solution. The peptide (0, 3,

17 or 6nmol/2 mL/kg body weight) was administered via a wing vein with ad libitum feeding. Food intake was

18 measured at 30 and 60 min after administration.

19

20 2.5. Experiment 3: Real-time PCR analysis of PYY and Y2R mRNA in the chicken brain

21

22 Four 7-day-old chicks were sacrificed by decapitation. The whole brains were collected and preserved

23 in RNAlater tissue storage reagent (Sigma-Aldrich Co., St. Louis, MO, USA) and divided into six regions

24 (telencephalon, optic lobe, cerebellum, rostral part of the brainstem, middle part of the brainstem, and caudal part
7
 1 of the brainstem) (Fig. 3). Total RNA extraction and cDNA synthesis were performed as described in Experiment

2 1. The cDNA of the chicken PYY (GenBank accession number: LC102814), RPS17 (GenBank accession number:

3 NM_204217), and Y2R (GenBank accession number: AF309091) were amplified with the specific primers (Table

4 1). Chicken PYY and Y2R mRNA levels were quantified in duplicate using an Applied Biosystems 7300

5 Real-Time PCR system and SYBR Premix Ex Taq II (Tli RNaseH Plus) (Takara Bio Inc., Shiga, Japan)

6 according to the supplier's recommendations.

8 2.6. Data analysis

10 One-way analysis of variance and Fisher's protected least significant difference test were used to

11 analyze mRNA distribution in the chicken intestine and brain and the amount of food intake. The Students t-test

12 was used to compare PYY mRNA levels in the jejunum between feeding and fasting conditions. All statistical

13 analyses were performed using a commercial software package (StatView version 5, SAS Institute, Cary, NC,

14 USA, 1998).

15

16 3. Results and Discussion

17

18 Identification of chicken PYY cDNA was performed by RT-PCR and RACE-PCR followed by the

19 nucleotide sequencing of the amplified PCR products (Fig. 2). As indicated in Fig. 4, we identified the cDNA

20 sequence encoding the chicken PYY precursor (GenBank accession number: LC102814). The chicken PYY

21 cDNA was 496 bp long, including a 39-bp 5-untranslated region, a 184-bp 3-untranslated region, and a 273-bp

22 open reading frame. The deduced protein comprised 21 amino acids of the signal peptide, 37 amino acids of the

23 chicken PYY, and 32 amino acids of the C-terminal peptide of PYY. We found that the deduced amino acid

24 sequence of PYY was completely consistent with that of the chicken PYY protein reported by Conlon and
8
 1 OHarte (1992). The deduced amino acid sequence of the chicken PYY precursor exhibited 39, 39, and 43%

2 identity with human, pig, and rat, respectively.

3 Conlon and OHarte (1992) isolated chicken PYY from the intestine, and the amino acid sequence

4 analysis revealed that the chicken PYY comprised 37 amino acids with an additional alanine residue at the

5 N-terminus compared with PYY in other classes of vertebrate (Fig. 1). The (-3, -1) rule of von Heijne (1986) is

6 available for predicting the sites of cleavage in prepropeptides. According to this rule, Conlon and OHarte (1992)

7 speculated that an amino acid substitution at the -3 position in the chicken PYY precursor leads to alteration in

8 cleavage site of signal peptide. As shown in Fig. 5, an amino acid substitution (Val to Ala) at -3 position from

9 N-terminus of mammalian PYY was found in chicken PYY precursor. Therefore, this substitution possibly leads

10 to an alternative site of signal peptide cleavage. Furthermore, chicken PYY was followed by Gly59-Lys60-Arg61 ,

11 the common site for dibasic cleavage and amidation in PYY (Keire, 2002), supporting the presence of an amidated

12 tyrosine residue at the C-terminus of chicken PYY (Conlon and OHarte, 1992).

13 We next examined the distribution of the PYY mRNA in the chicken intestine, and found that the PYY

14 mRNA level was higher in the small intestine than in the large intestine (Fig. 6). El-Salhy et al. (1982) reported

15 that PYY-immunoreactive cells were observed in the duodenum and jejunum in chickens. In mammals, PYY is

16 abundantly expressed in the large intestine compared with the small intestine (Ekblad and Sundler, 2002; Zhou et

17 al., 2006; Ueno et al., 2008). In fish, PYY is expressed particularly in the anterior sections of the digestive system

18 in Siberian sturgeon (Chen et al., 2015) and Atlantic salmon (Murashita et al., 2009), while it was detected

19 primarily in the central and posterior sections of the intestine in grass carp (Chen et al., 2013). In amphibians, high

20 expression of PYY mRNA was observed in both the small and large intestine in frogs (Sundstrm et al., 2012).

21 These findings, together with our results, demonstrate that the sites for PYY production are different among

22 vertebrates.

23 In mammals, plasma PYY concentration changes in response to feeding (Zwirska-Korczala et al., 2007;

24 Stadlbauer et al., 2013). Therefore, we next compared PYY mRNA levels in the jejunum between feeding and
9
 1 fasting conditions. PYY mRNA levels were significantly higher in chicks during ad libitum feeding than during 12

2 h-fasting (Fig. 7), suggesting that intestinal PYY expression is altered in response to nutritional status in chicks.

3 Plasma PYY concentration was significantly elevated after 2 h-refeeding in rodents (Le Roux et al. 2006; Xu

4 et al. 2011). Therefore, we further investigated the postprandial change in the mRNA levels of PYY in the

5 jejunum in chicks and found that the mRNA levels of PYY were increased after 2 h-refeeding although the

6 difference was not significant (unpublished data). Therefore, further studies will be needed to clarify the

7 postprandial change of plasma PYY concentration in chicks.

8 There is evidence that peripheral PYY functions as a satiety hormone in mammals (Murphy and Bloom,

9 2006; Coll et al., 2007; Sam et al., 2012) and fish (Gonzalez and Unniappan, 2010; Chen et al., 2013; Chen et al.,

10 2015). Therefore, we next examined the effect of intravenous administration of chicken PYY on food intake, and

11 found that PYY significantly decreased food intake in chicks in a dose-dependent manner (Fig. 8). It is therefore

12 likely that peripheral PYY may function as an anorexigenic peptide in chicks as well as in mammals. In mammals,

13 peripheral PYY3-36 transmits satiety signals to the brain via Y2R in the ARC and/or in the gastric vagal afferent

14 nerves (McGowan and Bloom, 2004; Ueno et al., 2008). In chickens, Salaneck et al. (2000) demonstrated that

15 PYY preferentially bound to Y2R. Therefore, we analyzed chicken Y2R mRNA levels in the different parts of the

16 brain, and found that Y2R mRNA was expressed in the rostral part of the brainstem including the infundibular

17 nucleus, equivalent of the mammalian ARC (Fig. 9 b). It is therefore possible that peripheral PYY suppresses food

18 intake via Y2R in the rostral part of the brainstem in chicks. Further studies are needed to elucidate whether

19 circulating PYY transmits satiety signals directly to the brain.

20 We have analyzed the mRNA levels of PYY and Y2R in the hypothalamus and the caudal part of the

21 brainstem in both feeding and fasting chicks and found that 24 h-fasting did not affect the mRNA levels of them

22 (unpublished data). Therefore, brain PYY does not play important roles in the appetite regulation in chicks. In

23 mammals, several roles of brain PYY have been proposed in mammals, such as regulating olfactory neurogenesis

24 (Doyle et al., 2012), attenuating depression-like behavior (Painsipp et al., 2011), and inducing locomotor and
10
 1 exploratory behaviors (Edelsbrunner et al., 2009). As shown in Fig. 9, we found that PYY and Y2R mRNA were

2 widely distributed in the brains of the chicks. The mRNA level of PYY in the cerebellum was lower than in other

3 parts of the brain. The level of Y2R mRNA was significantly high in the optic lobe but significantly low in the

4 cerebellum. In probably most avian species the majority of retinal ganglion cells project to the optic tectum,

5 and the tectum projects bilaterally to the thalami nucleus rotundus, which itself sends fibers to the ipsilateral

6 ectostriatum (Gntrkn, 2000). It is therefore possible that visual information is transmitted via Y2R in the

7 optic lobes in chicks. Further studies are needed to clarify this point.

9 4. Conclusion

10

11 In the present study, we identified the cDNA sequence encoding the chicken PYY precursor. PYY mRNA was

12 highly expressed in the small intestine, and its expression changed significantly in response to the nutritional

13 status in chicks. Peripheral administration of PYY significantly decreased food intake in chicks. Our findings

14 suggest that peripheral PYY may function as an anorexigenic hormone in chicks.

15

16 References

17

18 Abbott C.R., Small C.J., Kennedy A.R., Neary N.M., Sajedi A., Ghatei M.A., Bloom S.R., 2005. Blockade of the

19 neuropeptide Y Y2 receptor with the specific antagonist BIIE0246 attenuates the effect of endogenous

20 and exogenous peptide YY(3-36) on food intake. Brain Res. 1043, 139-144.

21 Batterham R.L., Cowley M.A., Small C.J., Herzog H., Cohen M.A., Dakin C.L., Wren A.M., Brynes A.E., Low

22 M.J., Ghatei M.A., Cone R.D., Bloom S.R., 2002. Gut hormone PYY(3-36) physiologically inhibits

23 food intake. Nature 418, 650-654.

24 Batterham R.L., Heffron H., Kapoor S., Chivers J.E., Chandarana K., Herzog H., Le Roux C.W., Thomas E.L.,
11
 1 Bell J.D., Withers D.J., 2006. Critical role for peptide YY in protein-mediated satiation and body-weight

2 regulation. Cell Metab. 4, 223-233.

3 Blevins J.E., Chelikani P.K., Haver A.C., Reidelberger R.D., 2008. PYY(3-36) induces Fos in the arcuate nucleus

4 and in both catecholaminergic and non-catecholaminergic neurons in the nucleus tractus solitarius of

5 rats. Peptides 29, 112-119.

6 Brome T., Sjdin P., Fredriksson R., Boswell T., Larsson T.A., Salaneck E., Zoorob R., Mohell N., Larhammar

7 D., 2006. Neuropeptide Y-family receptors Y6 and Y7 in chicken. Cloning, pharmacological

8 characterization, tissue distribution and conserved synteny with human chromosome region. FEBS J.

9 273, 2048-2063.

10 Chelikani P.K., Haver A.C., Reidelberger R.D., 2007. Effects of intermittent intraperitoneal infusion of salmon

11 calcitonin on food intake and adiposity in obese rats. Am. J. Physiol. Regul. Integr. Comp. Physiol. 293,

12 1798-1808.

13 Chen H., Zhang X., Hao J., Chen D., Liu J., Gao Y., Zhu J., Wu H., Lin F., Pu Y., Yuan D., Wei R., Zhou C., Wang

14 T., Li Z., 2015. Molecular cloning, expression analysis, and appetite regulatory effect of peptide YY in

15 Siberian sturgeon (Acipenser baerii). Gene 563, 172-179.

16 Chen Y., Shen Y., Pandit N.P., Fu J., Li D., Li J., 2013. Molecular cloning, expression analysis, and potential food

17 intake attenuation effect of peptide YY in grass carp (Ctenopharyngodon idellus). Gen. Comp.

18 Endocrinol. 187, 66-73.

19 Coll A.P., Farooqi I.S., O'Rahilly S., 2007. The hormonal control of food intake. Cell 129, 251-262.

20 Conlon J.M., O'Harte F., 1992. The primary structure of a PYY-related peptide from chicken intestine suggests an

21 anomalous site of cleavage of the signal peptide in preproPYY. FEBS Lett. 313, 225-228.

22 Degen L., Oesch S., Casanova M., Graf S., Ketterer S., Drewe J., Beglinger C., 2005. Effect of peptide YY3-36 on

23 food intake in humans. Gastroenterology 129, 1430-1436.

24 De Silva A., Bloom S.R., 2012. Gut Hormones and Appetite Control: A Focus on PYY and GLP-1 as Therapeutic
12
 1 Targets in Obesity. Gut and Liver 6, 10-20.

2 Doyle K.L., Hort Y.J., Herzog H., Shine J., 2012. Neuropeptide Y and peptide YY have distinct roles in adult

3 mouse olfactory neurogenesis. J. Neurosci. Res. 90, 1126-1135.

4 Edelsbrunner M.E., Herzog H., Holzer P., 2009. Evidence from knockout mice that peptide YY and neuropeptide

5 Y enforce murine locomotion, exploration and ingestive behaviour in a circadian cycle- and

6 gender-dependent manner. Behav. Brain Res. 203, 97-107.

7 Ekblad E., Sundler F., 2002. Distribution of pancreatic polypeptide and peptide YY. Peptides 23, 251-261.

8 El-Salhy M., Wilander E., Grimelius L., Terenius L., Lundberg J.M., Tatemoto K., 1982. The distribution of

9 polypeptide YY (PYY) - and pancreatic polypeptide (PP) - immunoreactive cells in the domestic fowl.

10 Histochemistry 75, 25-30.

11 Fu-Cheng X., Anini Y., Chariot J., Voisin T., Galmiche J.P., Roz C., 1995. Peptide YY release after intraduodenal,

12 intraileal, and intracolonic administration of nutrients in rats. Pflugers Archiv 431, 66-75.

13 Gonzalez R., Unniappan S., 2010. Molecular characterization, appetite regulatory effects and feeding related

14 changes of peptide YY in goldfish. Gen. Comp. Endocrinol. 166, 273-279.

15 Gntrkn O., 2000. Sensory Physiology: Vision, in: Whittow G.C. (Eds.), Strukie's Avian Pysiology. Academic

16 Press, New York, pp. 1-19.

17 Joly-Amado A., Cansell C., Denis R.G., Delbes A.S., Castel J., Martinez S., Luquet S., 2014. The hypothalamic

18 arcuate nucleus and the control of peripheral substrates. Best Pract. Res. Clin. Endocrinol. Metab. 28,

19 725-737.

20 Keire D.A., Bowers C.W., Solomon T.E., Reeve J.R. Jr., 2002. Structure and receptor binding of PYY analogs.

21 Peptides 23, 305-321.

22 Koda S., Date Y., Murakami N., Shimbara T., Hanada T., Toshinai K., Niijima A., Furuya M., Inomata N., Osuye

23 K., Nakazato M., 2005. The role of the vagal nerve in peripheral PYY3-36-induced feeding reduction in

24 rats. Endocrinology 146, 2369-2375.

13
 1 Le Roux C.W., Batterham R.L., Aylwin S.J., Patterson M., Borg C.M., Wynne K.J., Kent A., Vincent R.P.,

2 Gardiner J., Ghatei M.A., Bloom S.R., 2006. Attenuated peptide YY release in obese subjects is

3 associated with reduced satiety. Endocrinology 147, 3-8.

4 Martin N.M., Small C.J., Sajedi A., Patterson M., Ghatei M.A., Bloom S.R., 2004. Pre-obese and obese agouti

5 mice are sensitive to the anorectic effects of peptide YY(3-36) but resistant to ghrelin. Int. J. Obes. Relat.

6 Metab. Disord. 28, 886-893.

7 McGowan B.M., Bloom S.R., 2004. Peptide YY and appetite control. Curr. Opin. Pharmacol. 4, 583-588.

8 Murashita K., Kurokawa T., Nilsen T.O., Rnnestad I., 2009. Ghrelin, cholecystokinin, and peptide YY in Atlantic

9 salmon (Salmo salar): molecular cloning and tissue expression. Gen. Comp. Endocrinol. 160, 223-235.

10 Murphy K.G., Bloom S.R., 2006. Gut hormones and the regulation of energy homeostasis. Nature 444, 854-859.

11 Nonaka N., Shioda S., Niehoff M.L., Banks W.A., 2003. Characterization of blood-brain barrier permeability to

12 PYY3-36 in the mouse. J. Pharmacol. Exp. Ther. 306, 948-953.

13 Painsipp E., Herzog H., Sperk G., Holzer P., 2011. Sex-dependent control of murine emotional-affective

14 behaviour in health and colitis by peptide YY and neuropeptide Y. Br. J. Pharmacol. 163, 1302-1314.

15 Parker R.M., Herzog H., 1999. Regional distribution of Y-receptor subtype mRNAs in rat brain. Eur. J. Neurosci.

16 11, 1431-1448.

17 Reidelberger R., Haver A., Chelikani P.K., 2013. Role of peptide YY(3-36) in the satiety produced by gastric

18 delivery of macronutrients in rats. Am. J. Physiol. Endocrinol. Metab. 304, 944-950.

19 Salaneck E., Holmberg S.K., Berglund M.M., Boswell T., Larhammar D., 2000. Chicken neuropeptide Y receptor

20 Y2: structural and pharmacological differences to mammalian Y2. FEBS Lett. 484, 229-234.

21 Sam A.H., Troke R.C., Tan T.M., Bewick G.A., 2012. The role of the gut/brain axis in modulating food intake.

22 Neuropharmacology 63, 46-56.

23 Schwartz G.J., 2006. Integrative capacity of the caudal brainstem in the control of food intake. Philos. Trans. R.

24 Soc. Lond. B Biol. Sci. 361, 1275-1280.

14
 1 Scott V., Kimura N., Stark J.A., Luckman S.M., 2005. Intravenous peptide YY3-36 and Y2 receptor antagonism in

2 the rat: effects on feeding behavior. J. Neuroendocrinol. 17, 452-457.

3 Stadlbauer U., Arnold M., Weber E., Langhans W., 2013. Possible mechanisms of circulating PYY-induced

4 satiation in male rats. Endocrinology. 154, 193-204.

5 Sundstrm G., Xu B., Larsson T.A., Heldin J., Bergqvist C.A., Fredriksson R., Conlon J.M., Lundell I., Denver

6 R.J., Larhammar D., 2012. Characterization of the neuropeptide Y system in the frog Silurana tropicalis

7 (Pipidae): three peptides and six receptor subtypes. Gen. Comp. Endocrinol. 177, 322-331.

8 Ueno H., Yamaguchi H., Mizuta M., Nakazato M., 2008. The role of PYY in feeding regulation. Regul. Pept. 145,

9 12-16.

10 Von Heijne G., 1986. A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 14,

11 4683-4690.

12 Xu J., McNearney T.A., Chen J.D., 2011. Impaired postprandial releases/syntheses of ghrelin and PYY(3-36) and

13 blunted responses to exogenous ghrelin and PYY(3-36) in a rodent model of diet-induced obesity. J.

14 Gastroenterol. Hepatol. 26, 700-705.

15 Zhou J., Hegsted M., McCutcheon K.L., Keenan M.J., Xi X., Raggio A.M., Martin R.J., 2006. Peptide YY and

16 proglucagon mRNA expression patterns and regulation in the gut. Obesity 14, 683-689.

17 Zwirska-Korczala K., Konturek S.J., Sodowski M., Wylezol M., Kuka D., Sowa P., Adamczyk-Sowa M., Kukla

18 M., Berdowska A., Rehfeld J.F., Bielanski W., Brzozowski T., 2007. Basal and postprandial plasma

19 levels of PYY, ghrelin, cholecystokinin, gastrin and insulin in women with moderate and morbid obesity

20 and metabolic syndrome. J. Physiol. Pharmacol. 58, 13-35.

21

22 Figure captions

23

24 Fig. 1. Alignment of peptide YY (PYY) of chicken and other classes of vertebrate. Highlights indicate amino
15
 1 acids identical to those of human PYY. GenBank accession numbers: chicken (P29203.1); human (NP_004151.3);

2 rat (NP_001029252.1); tortoise (XP_007061964.1); lizard (XP_003222691.1); frog (P29204.1); salmon

3 (NP_001132995.1); tilapia (XP_003446444.1).

5 Fig. 2. Polymerase chain reaction (PCR) strategy for identifying complementary DNA (cDNA) sequence of

6 chicken PYY

8 Fig. 3. Schematic drawing of the chicken brain. The brain was divided into the six regions (telencephalon, optic

9 lobe, cerebellum, rostral part of the brainstem, middle part of the brainstem, and caudal part of the brainstem).

10

11 Fig. 4. Nucleotide sequence and deduced amino acid sequence of chicken peptide YY (PYY). The amino acid

12 sequences of the predicted signal peptide (21 residues) and the mature protein (37 residues) are denoted by dotted

13 and solid lines, respectively.

14

15 Fig. 5. Alignment of peptide YY (PYY) precursors of chicken and mammals. Highlights indicate amino acids

16 identical to those of deduced chicken PYY. GenBank accession numbers: human (NP_004151.3); pig

17 (AEY83942.1); rat (NP_001029252.1).

18

19 Fig. 6. Distribution of peptide YY (PYY) mRNA in the chicken intestine. Data represent means SEM (n = 3).

20 Groups with different letters are significantly different (P < 0.05).

21

22 Fig. 7. Comparison of peptide YY (PYY) mRNA levels in the chicken jejunum between feeding and fasting

23 conditions. Data represent means SEM (n = 6 in both groups). * Significant with respect to the feeding group (P

24 < 0.05).
16
 1

2 Fig. 8. Effect of intravenous administration of chicken peptide YY (PYY) on food intake in chicks. Data

3 represent means SEM (n = 13-14 in each group). Groups with different letters are significantly different (P <

4 0.05).

6 Fig. 9. Distribution of peptide YY (PYY) (a) and neuropeptide Y receptor Y2 (Y2R) (b) mRNAs in the chicken

7 brain. Data represent means SEM (n = 4). Groups with different letters are significantly different (P < 0.05).

10

17
Fig. 1

Chicken A Y P P K P E S P G D A A S P E E I A Q Y F S A L R H Y I N L V T R Q R Y
Human - Y P I K P E A P G E D A S P E E L N R Y Y A S L R H Y L N L V T R Q R Y
Rat - Y P A K P E A P G E D A S P E E L S R Y Y A S L R H Y L N L V T R Q R Y
Tortoise - Y P P K P E N P G D D A S P E E M A K Y F S A L R H Y I N L V T R Q R Y
Lizard - Y P P K P E S P G E D A S P E E M A K Y F S A L R H Y I N L V T R Q R Y
Frog - Y P P K P E N P G E D A S P E E M T K Y L T A L R H Y I N L V T R Q R Y
Salmon - Y P P K P E N P G E D A P P E E L A K Y Y T A L R H Y I N L I T R Q R Y
Tilapia - Y P P K P E N P G E D A P P E E L A K Y Y T A L R H Y I N L I T R Q R Y
Fig. 2
Fig. 3
 Fig. 4

GA A A GC GT C C C C T GT GC T GT C A C C C C A C T GC C T GGC A C C A T GGC GC T GT C CC C GC GC C GC 60

M A L S P R R 7

GT T C T GC C C GC GT T GGC GC T GT GC GC GC T GC T GT GC GC C GC C GC GT A T C C A C C GA A GC C C 120

V L P A L A L C A L L C A A A Y P P K P 27

GA A A GC C C C GGA GA C GC C GC GT C C C C GGA GGA GA T C GC GC A GT A C T T C T C GGC GC T GC GC 180

E S P G D A A S P E E I A Q Y F S A L R 47

C A T T A C A T C A A C C T GGT C A C GC G GC A GC GGT A T GGGA A GC GC A GC A GC T C T GC GC C T GC A 240

H Y I N L V T R Q R Y G K R S S S A P A 67

GT GT C A GT GC A GC C C A T C GGT C C C C GC A GT GA T GC GC T GT GGT C C GA C A T CGA C GA T GA C 300

V S V Q P I G P R S D A L W S D I D D D 87

T C C A C GT GGT GA C C GC C C C C C C A C A C C C C C A C C C C C A A A A C C C C C C C T C A C C C C A C A A A T 360

S T W 90

T GT C C C C A T C T C T C C A T GT T GC T C C C A T C C GA GT GGGGGT GC A GC A C C GGGGGC T GC A T G 420

A GC A C T GC A T GGGGGGC A C C C C GA GT GC C C GGGGGT GT C C C A C GGGGGGGGC A C T C A C A C 480

A C A C A C A C A C A C A T C C 496
 Fig. 5

Chicken M A L S P R - - R V L P A L A L C A L L C - - - - A A A Y P P K P E S P G D A A S P E E I A Q Y F
Human M V F V R R P W P A L T T V L L A L L V C L G A L V D A Y P I K P E A P G E D A S P E E L N R Y Y
Pig M V T V R R P W P A M A T V L L G L L I C L G T L V D A Y P A K P E A P G E D A S P E E L S R Y Y
Rat M V A V R R P W P V M V A M L L V L L A C L G A L V D A Y P A K P E A P G E D A S P E E L S R Y Y

Mature PYY

Chicken S A L R H Y I N L V T R Q R Y G K R S S S A P A V S V Q P I G P R S D A L - - W S D I D D D S T W
Human A S L R H Y L N L V T R Q R Y G K R D G P D T L L S K T F F P D - G E D R P V R S R S E G P D L W
Pig A S L R H Y L N L V T R Q R Y G K R D S P D A L L S K L L F P E - G E D R P V K S W P E G A Y L W
Rat A S L R H Y L N L V T R Q R Y G K R E V P A A L F S K L L F T D D S E N L P F R S R P E G V D Q W
Fig. 6
Fig. 7
Fig. 8
Fig. 9
1 Table 1. Sequence of primers

Name Sequence

Identification of chicken PYY

cDNA

F1 5'-ATG GTG TGT CCC CGC-3'

R1 5'-TCA CCA CGC AGA GTC ATC-3'

F2 5'-CGT TCC ACC GAA GCC CGA AAG C-3'

R2 5'-CCG CGT GAC CAG GTT GAT GTA ATG-3'

F3 5'-CCG GAG GAG ATC GCG CAG TAC T-3'

R3 5'-GCT TTC GGG CTT CGG TGG ATA CG-3'

F4 5'-AGC GTC CCC TGT GCT GTC A-3'

R4 5'-GGG AGC AAC ATG GAG AGA TGG-3'

Real-time PCR analysis

PYY forward 5'-AGG AGA TCG CGC AGT ACT TCT C-3'

PYY reverse 5'-TGC TGC GCT TCC CAT ACC-3'

Y2R forward 5'-CTG GTT TGC GTG GTT GTT GT-3'

Y2R reverse 5'-CTA GCT GGA AGG CAT GGA ATG-3'

RPS17 forward 5'-GCG GGT GAT CAT CGA GAA GT-3'

RPS17 reverse 5'-GCG CTT GTT GGT GTG GAA GT-3'

18
1 Highlights:

2 Complementary DNA sequence encoding the chicken PYY precursor was identified.

3 PYY mRNA was abundantly expressed in the small intestine in chicks.

4 Intravenous administration of PYY significantly suppressed food intake in chicks.

5 PYY may function as an anorexigenic hormone in chicks.

19