Enteroendocrine profile of α-transducin and α-gustducin immunoreactive cells in the chicken (Gallus domesticus) gastrointestinal tract

Enteroendocrine profile of α-transducin and α-gustducin immunoreactive cells in the chicken (Gallus domesticus) gastrointestinal tract M. Mazzoni,∗,1 T.B. Karunaratne,† F. Sirri,‡ M. Petracci,‡ R. De Giorgio,§ C. Sternini,# and P. Clavenzani∗ ∗

Department of Veterinary Medical Sciences, University of Bologna, Ozzano Emilia, 40064 Bologna, Italy; Department of Medical and Surgical Sciences, University of Bologna, 40138 Italy; ‡ Department of Agricultural and Food Sciences, University of Bologna, Ozzano Emilia, 40064 Bologna, Italy; § Department of Medical Sciences, University of Ferrara, Nuovo Arcispedale S.Anna, in Cona, 44121 Ferrara, Italy; and # CURE/DDRC, Division of Digestive Diseases, Departments Medicine and Neurobiology, David Geffen School of Medicine, UCLA, Los Angeles, CA 90095, USA †

small intestine, a considerable subset of Gα tran or Gα gust IR cells co-expressed 5-HT in the villi of the duodenum and ileum, PYY in the villi of the jejunum, CCK or GLP-1 in the villi of the ileum, and GHR in the duodenum crypts. In the large intestine, many Gα tran or Gα gust -IR cells contained 5-HT or GLP-1 in the villi of the rectum, whereas some Gα tran /Gα gust -IR cells coexpressed PYY- or CCK-, and few Gα tran /Gα gust -IR cells were positive for GHR-IR. In the cecum, several Gα tran or Gα gust -IR cells were IR for 5-HT. Finally, many Gα tran /Gα gust cells containing 5-HT were observed in the villi and crypts of the cloaca, whereas there were few Gα tran or Gα gust /CCK-IR cells. The demonstration that Gα-subunits are expressed in the chicken GI enteroendocrine system supports the involvement of taste signaling machinery in the chicken chemosensing processes.

ABSTRACT The enteroendocrine profile and distribution patterns of the taste signaling molecules, αgustducin (Gα gust ) and α-transducin (Gα tran ) protein subunits, were studied in the gastrointestinal (GI) tract of the chicken (Gallus domesticus) using double labeling immunohistochemistry. Gα tran or Gα gust immunoreactivity was observed in enteroendocrine cells (EEC) expressing different peptides throughout the entire GI tract with different density. In the proventriculus tubular gland, Gα tran or Gα gust /gastrin (GAS) immunoreactive (-IR) cells were more abundant than Gα tran/ or Gα gust containing glucagon-like peptide-1 (GLP-1) or peptide YY (PYY), whereas only few Gα tran or Gα gust cells co-stored ghrelin (GHR) or 5-hydroxytryptamine (5-HT). In the pyloric mucosa, many Gα tran or Gα gust IR cells co-expressed GAS or GHR, with less Gα tran or Gα gust cells containing GLP-1, PYY, or 5-HT. In the

Key words: α-transducin, α-gustducin, chicken, gastrointestinal tract, enteroendocrine cells 2018 Poultry Science 0:1–10 http://dx.doi.org/10.3382/ps/pey279

INTRODUCTION

sion, is important for acquiring and choosing feeds in this species (Roura et al., 2013). Furthermore, taste is important to improve and develop new feedstuffs to obtain the best chicken products (Dey et al., 2017). α-gustducin (Gαgust ) and α-transducin (Gαtran ) are heterotrimeric G-protein signaling molecules in the intracellular cascade of bitter taste and, to a lesser extent umami and sweet, signal transduction (Behrens and Meyerhof, 2011). Gα gust and Gα tran have been described outside the oral cavity in several mammalian species (Rozengurt et al., 2006; Sutherland et al., 2007; Janssen et al., 2011; Daly et al., 2012; Widmayer et al., 2012; Mazzoni et al., 2013; 2016), and farm animals such as chicken, where 2 families of taste receptors, T1R and T2R, in addition to Gα gust and Gα tran , have been demonstrated throughout the gut (Cheled-Shoval et al., 2014, 2015; Yoshida et al., 2015; Mazzoni et al., 2016). The presence of the taste machinery in non-taste

In recent years, there has been an increasing interest in the characterization of avian energy homeostasis aimed to optimize poultry production and welfare and to better understand endocrine regulation of vertebrate energy balance. A healthy gastrointestinal (GI) tract is key to maintain good production and obtain highquality meat and eggs in poultry industry. The chicken GI tract represents about 8% of the body weight (Gracie et al., 2016) but requires 8% of the metabolized energy (Spratt et al., 1990). Taste, like smell and vi-

 C 2018 Poultry Science Association Inc. Received April 16, 2018. Accepted June 12, 2018. 1 Corresponding author: E-mail: [email protected]

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tissues might represent a chemosensory system that monitors the nutritional status (Ren et al., 2009). Indeed, nutrient-evoked GI reflexes triggered by sensory cells, either enteroendocrine cells (EEC) or brush cells, are distributed in the mucosal lining of the GI tract (Sternini, 2007). The appetite regulatory system has been a major topic of research for more than half a century (Woods, 2013). The brain integrates information generated by the gut via hormones/bioactive messengers released before or after meals and thereby regulates food intake (Woods, 2009; Sam et al., 2012; Williams and Elmquist, 2012). Many studies on the regulatory mechanisms of food intake revealed that gut hormones, such as ghrelin (GHR), cholecystokinin (CCK), glucagon-like peptide-1 (GLP-1), serotonin (or 5-hydroxytryptamine, 5-HT), and peptide YY (PYY), play critical roles in the regulation of food intake in mammals (Strader and Woods, 2005) and birds (Honda et al., 2017). GHR functions as an anorexigenic peptide in chicken since peripheral administration was able to suppress food intake (Saito et al., 2002; Oclo´ n and Pietras, 2011). Peripheral administration of CCK suppressed food intake in chick (Furuse et al., 2000). Also the intravascular injection of PYY dose-dependently significantly decreased the food intake of chicks (Aoki et al., 2017), as well as intracerebroventricular injection of 5-HT decreased food intake in the same species (Zendehdel et al., 2017). The physiological importance of GLP-1 in birds as a satiety hormone remains unsettled. Intraperitoneal administration of GLP-1 did not influence food intake in layer chicks (Tachibana et al., 2003), whereas intraperitoneal administration of this peptide suppressed food intake in the Japanese quail (Shousha et al., 2007). Gα gust and/or Gα tran immunoreactivity (-IR) has been reported mainly in EEC of the GI epithelial layer (Rozengurt et al., 2006; Sutherland et al., 2007; Janssen et al., 2011; Daly et al., 2012; Widmayer et al., 2012; Janssen and Depoortere, 2013; Mazzoni et al., 2013, 2016). This finding suggests that taste signaling molecules affect GI functions and feeding behavior through the release of substances, such as GHR, CCK, GLP-1, 5-HT, PYY, exerting orexigenic, or anorexigenic actions (Mazzoni et al., 2013, 2016). Based on their morphological appearance, the EECs may present an “open type” aspect with cytoplasmic prolongation which tends to reach the luminal surface, and “closed type” which are confined to the basal lamina and did not reach the lumen (Dockray, 2003; Sternini et al., 2008). In the present study, we used double labeling immunohistochemistry to characterize the phenotype of Gα gust and Gα tran -IR cells throughout the chicken GI tract (including cloaca) with emphasis on peptides and biogenic amines involved in satiation and body weight regulation.

MATERIAL AND METHODS A total of 5 1-day-old Ross 308 male chicks were reared on a pen floor using wood shavings as litter material and fed a commercial diet. At 40 d of age, 4 chickens with a homogeneous live body weight (2.8 ± 0.1 kg) were sacrificed by cervical dislocation according to the legislation on the protection of animals used for scientific purposes (Directive 2010/63/EU). The GI tract from the proventriculus to the cloaca was carefully excised. Specimens of the GI tract, including the proventriculus gastritis (in the middle part of the regio glandularis), gizzard (pars pylorica), duodenum (ansa duodeni), jejunum (proximal to the diverticulum vitellinum or Meckel’s diverticulum), ileum (between ileocecal ligament), cecum (in the corpus ceci), rectum (between the ileum and cloaca), and cloaca (approximately urodeum compartment), were collected (the anatomical nomenclature is from Nomina Anatomica Avium) (Baumel et al., 1993). After a brief washing with 0.01 M phosphate buffer saline (PBS), tissues were fixed in 4% paraformaldehyde in phosphate buffer (0.1 M, pH 7.2) for 48 h at 4◦ C. Fixed tissues were dehydrated in a graded series of ethanol and embedded in paraffin. Transverse paraffin sections were cut at 5 μm thickness with a microtome. To avoid counting the same cells in serial sections, each seventh section was mounted onto poly-L-lysine-coated slides for immunohistochemistry.

Immunohistochemistry Paraffin sections were processed for double immunofluorescence using antibodies directed to Gα tran or Gα gust and specific EEC markers such as GHR, gastrin (GAS)/CCK, 5-HT, GLP-1, and PYY shown in Table 1. Briefly, sections were deparaffinized with xylene, rehydrated with graded ethanol, and heat-treated in a microwave (2 cycles at 750 W, 5 min each) in sodium citrate buffer (pH 6.0) to retrieve the antigenicity. Sections were incubated in 10% appropriate normal serum in 0.01 M PBS (1 h at room temperature) to prevent non-specific bindings, and subsequently incubated overnight with primary antibodies diluted in PBS and 5% of normal serum. After primary antibody incubation, a mixture of fluorescein isothiocyanate-conjugated and tetramethyl rhodamine isothiocyanate-conjugated (Alexa Fluor 594- and 488-conjugated, respectively) secondary antibodies diluted in PBS (Table 1) was added for 1 h at room temperature. Finally, slides were washed in PBS and cover slipped with buffered glycerol, pH 8.6.

Antibody Specificity Specificity of Gα tran and Gα gust has been previously demonstrated by preabsorption test (Mazzoni et al., 2016). GHR, 5-HT, PYY, GLP-1, and GAS/CCK

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TASTE SIGNALING MOLECULES IN THE CHICKEN GUT Table 1. List and dilutions of primary and secondary antibodies. Primary antibodies

Code

Species

Dilution

Supplier

α -Transducin α -Gustducin Cholecystokinin/Gastrin Ghrelin 5-hydroxitryptamine Peptide YY Glucagon-like pepetide-1

sc-390 sc-395 CCK/GAS # 9303 AM26736PU-N ab16007 PAB17185 GLP-1(1–36) # 9153

rabbit rabbit mouse mouse mouse guinea pig mouse

1:200 1:200 1:2000 1.200 1:200 1:500 1:600

Santa Cruz Santa Cruz CURE/DDRC Acris Abcam Abnova CURE/DDRC

Dilution 1:600 1:1000 1:500 1:100

Supplier Mol. Probes Mol. Probes Calbiochem Chemicon/Millipore

Secondary antibodies Alexa 594-conjugated goat anti-mouse IgG Alexa 488-conjugated donkey anti-rabbit IgG FITC-conjugated goat anti-rabbit IgG TRITC-conjugated goat anti-guinea pig IgG

CURE/DDRC, UCLA, Los Angeles, CA. Acris Antibodies GmbH OriGene Company, Schillerstraße 5, Herford, Germany. Abnova, Jhouzih St., Neihu District, Taipei, Taiwan. Abcam, Cambridge, UK. Santa Cruz Biotecnology, Inc., CA. Abnova, Jhouzih St. Neihu District, Taipei City, Taiwan. Calbiochem-Novabiochem Corporation, San Diego, CA. Molecular Probes, Eugene, OR. Chemicon International, Temecula, CA. FITC, fluorescein isothiocyanate; TRITC, tetramethyl rhodamine isothiocyanate.

antibody specificity was assessed by preabsorption with an excess of the homologous peptide (GHR, sc-10,368 P, Santa Cruz, CA; 5-HT, H9523, Sigma-Chemicals, St. Louis, MO; PYY, 059–06, Phoenix Pharm. Inc., Burlingame, CA, GLP-1, MBS826668, MyBiosource Inc., San Diego, CA, GAS/CCK, MBS425779, MyBiosource Inc., San Diego, CA, respectively). Since our monoclonal antibody cannot discriminate the close Cterminus homology of CCK and GAS, it was not possible to establish the actual presence of CCK-IR cells in the duodenum. In addition, further specificity tests for secondary antibodies were performed by omitting the primary antibodies. These data were not shown.

Cell Counting Cell counting was performed with a ×40 objective lens using a Zeiss Axioplan microscope (Carl Zeiss, Oberkochen, Germany) with appropriate filter cubes to discriminate different wave fluorescence. Images were collected with a Polaroid DMC digital photocamera (Polaroid, Cambridge, MA) and minimal adjustment to brightness and contrast was performed with Corel Photo Paint and Corel Draw (Corel, Dublin, Ireland). Cell counting was done in a blind fashion by 2 expert investigators (MM and Claudia Vallorani). For each chicken, Gα tran or Gα gust -IR cells showing the expression of different EEC markers were counted in 30 random villi (or folds) and, when they were present, in 30 intestinal glands (crypts) in the GI tract. In the proventriculus, as well as in endoluminal epithelium, positive cells were also observed in the simple tubular glands of the lamina propria; for this reason the glandular IR cells were counted in 18 random microscope fields (0.28 mm2 each field), for a total area of 5 mm2 . Only villi/glands/crypts located perpendicularly to the mucosal surface were counted. The values were pooled for each GI collected tract (proventriculum, gizzard pars pylorica, duodenum, and all other segments

under investigation) and expressed as mean ± standard deviation.

RESULTS Distribution of Gαtran or Gαgust -IR Cells in the GI Tract Gα tran or Gα gust -IR cells were distributed throughout the whole chicken GI tract, confirming and extending our previous studies (Mazzoni et al., 2016). Gα tran or Gα gust -IR cells were observed in the endoluminal epithelium and tubular glands of the proventriculus, as well as in the villi of the pyloric mucosa and along the villus-crypt axis of the small and large intestine and cloaca. Most Gα tran or Gα gust -IR cells had the morphological appearance of “open-type” EECs with an elongated (“pear-like”), homogenous cytoplasm and 2 cytoplasmic prolongations, one reaching the lumen and the other the basal lamina. Other Gα tran or Gα gust -IR cells had the “closed-type” EEC appearance with a round shape without cytoplasmic prolongations.

Distribution of Colocalized Gαtran or Gαgust in the Proventriculus and Pyloric Mucosa Double immunostainings demonstrated colocalization of Gα tran or Gα gust -IR with various endocrine cells markers. In the proventriculus tubular gland, most Gα tran or Gα gust -IR cells contained GAS/CCK-IR, less co-expressed PYY- or GLP-1-IR, and only few costored GHR- or 5-HT-IR (Figures 1A–H and 2A). In the pyloric mucosa, many cells co-expressed Gα tran or Gα gust -IR and GAS/CCK-IR, less showed Gα tran or Gα gust - and GHR-IR (Figures 2B and 3A–D), and even less Gα tran or Gα gust and GLP-1 (Figure 3E and F), PYY, or 5-HT-IR. In the proventriculus and pyloric mucosa, most of the EECs containing either Gα tran or Gα gust -IR had an “open-type” morphology, whereas those located in

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most cases, an elongated or pear shape (Figure 4E and F), while those located in the crypts showed rounded shape. Gα tran or Gα gust /PYY-IR cells were well represented in the duodenum, jejunum, and ileum (Figure 2F). The greatest number was found in correspondence to the jejunal and ileal villi. Gα tran or Gα gust /PYY-IR cells showed histological characteristics similar to those containing GLP-1-IR. Most Gα tran or Gα gust /GAS/CCK-IR cells were observed in the jejunal and ileal villi (Figures 2G and 4G and H) and less in the crypts. Since our monoclonal antibody cannot discriminate the close C-terminus homology of CCK and GAS, it was not possible to establish the actual abundance of CCK-IR cells in the duodenum.

Distribution and Colocalization of Gαtran or Gαgust Cells in the Large Intestine In the large intestine we have observed many Gα tran or Gα gust cells that contained 5-HT or GLP-1-IR in the villi of the rectum, less that co-expressed PYY or GAS/CCK-IR and only few that contained GHR-IR. In the cecum, several Gα tran or Gα gust -IR cells contained 5HT-IR (Figures 2C–G and 5A–D).

Figure 1. Colocalization of Gα tran or Gα gust -IR (A–E, arrows), with gastrin/cholecystokinin (GAS/CCK) (B, arrows), ghrelin (GHR) (D, arrows), and peptide YY (F, arrows) in the chicken proventriculus. G and H show some roundish Gα tran -IR cells (typical closed-type morphology, arrowheads) that do not contain peptide YY (PYY): these PYY-IR cells occur alternately “open-type” (arrow) and “closed-type” (asterisks) enteroendocrine cells. A–H: scale bars = 50 μ m.

the inner part of the mucosa displayed a “closed-type” shape (Figure 3A and B).

Distribution of Colocalized Gαtran or Gαgust in the Small Intestine In the small intestine, we observed Gα tran or Gα gust /GHR-IR cells in all segments especially in the crypts of the duodenum, while fewer cells were observed in the villi (Figure 2C). All these cells showed an elongated or round shaped morphology in the same villus (Figure 4A and B). Compared to other intestinal segments, a substantial subset of Gα tran and Gα gust -IR cells co-expressed 5-HTIR especially in the villi of duodenum (Figures 2D and 4C and D) and ileum. Generally, Gα tran and Gα gust /5HT-IR cells were also visualized in the crypts. The Gα tran and Gα gust /5-HT-IR cells located in the villi showed an “open-type” appearance, whereas those located in the crypts exhibit a “closed-type” aspect. Gα tran or Gα gust /GLP-1-IR cells were found mainly in the villi of the ileum (Figure 2E): these cells showed, in

Distribution of Colocalized Gαtran or Gαgust in the Cloaca A significant number of Gα tran or Gα gust cells containing 5-HT-IR were observed in the villi and crypts of the cloaca, whereas only few cells were Gα tran or Gα gust and GAS/CCK-IR (Figures 2C–G and 5E–H).

DISCUSSION Our data show that Gα gust - and Gα tran -IR cells are distributed throughout the chicken GI tract and comprise EECs containing multiple peptides/signaling molecules including GHR, GAS/CCK, 5-HT, GLP-1, and PYY confirming and expanding previous studies in mammals, chicken, and fish (Sutherland et al., 2007; Janssen et al., 2011; Steinert et al., 2011; Latorre et al., 2013; Mazzoni et al., 2013, 2016; Cheled-Shoval et al., 2014, 2015). These findings suggest that endoluminal stimuli (i.e., chemicals, nutrients, and even noxae) evoke the release of different hormones/bioactive messengers by EECs through the activation of taste receptors, which might represent one of the initial molecular mechanisms underlying chemosensitivity in the chicken gut. (Sternini, 2007). The array of chemical messengers contained in EECs expressing Gα gust and Gα tran provides indirect evidence for the involvement of taste signaling molecules in multiple functions, including motility, secretion, and feeding. In mammals, GHR is stored in EECs of the gastric mucosa (Kojima et al., 1999; Lee et al., 2002; Kojima

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Figure 2. Graphs in A–G show the mean number of Gα tran or Gα gust -IR cells containing amines/peptides in the different segments of the chicken gastrointestinal tract. In the A histograms (proventriculus), the values refer to a total area of 10 mm2 . In the B graph, the values represent the mean number of Gα tran or Gα gust -IR cells colocalized with amine/peptide evaluated in 30 villi. The other histograms represent the mean number of Gα tran or Gα gust -IR cells evaluated in 30 villi (or folds) and in 30 intestinal glands (crypts) co-express ghrelin (GHR) (C), 5-hydroxytryptamine (5-HT) (D), glucagon-like peptide-1 (GLP-1) (E), peptide YY (PYY) (F), and gastrin/cholecystokinin (GAS/CCK) (G), respectively. Values are expressed as means + SD.

and Kangawa, 2005; Kaiya et al., 2007) and exerts a variety of biological functions, ranging from regulation of growth hormone release, food intake, and GI motility to reproduction and cardiovascular functions (Date et al., 2000; Masuda et al., 2000; Wren et al., 2000; Nakazato et al., 2001; Yamazaki et al., 2002; Nagaya and Kangawa, 2003; Fern´ andez-Fern´ andez et al., 2005).

GHR has also been identified in many non-mammalian species, such as bullfrogs, chickens, turtles, and rainbow trout (Kaiya et al., 2011). Recent studies showed that GHR has an anti-lipogenic action (Buyse et al., 2009), and elicits motor function of the GI tract particularly in the middle intestine of birds (Kitazawa et al., 2007, 2009). Notably, in contrast to mammals, a number

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Figure 3. Gα tran or Gα gust -IR (A, C, and E, arrows) cells containing gastrin/cholecystokinin (GAS/CCK) (B, arrows), ghrelin (GHR) (D, arrows), or glucagon-like peptide-1 (GLP-1) (F, arrows) in the chicken pyloric mucosa. The Gα tran or Gα gust /GAS/CCK-labeled cells (A and B) observed in the villi surface epithelium show typical opentype (arrows) whereas those located at the base of the villi display a closed-type (asterisks) enteroendocrine cells morphology. C and D show enteroendocrine cells of the pyloric mucosa co-expressing Gα gust and GHR (arrows), and E and F show Gα gust containing GLP-1-IR (arrows). Arrowheads in D and F point to GHR-(D) and GLP-1-IR (F) cells that do not contain Gα gust -IR. A–F: scale bars = 50 μ m.

of studies suggest that GHR functions as an anorexigenic peptide in birds. For example, peripheral administration of GHR suppressed food intake in broiler chicks (Geelissen et al., 2006; Buyse et al., 2009; Oclo´ n and Pietras, 2011). One can speculate that different stimuli including foodstuff might induce opposite effects, orexigenic or antiorexigenic, in different species, through the activation of distinct taste receptors. Further studies are awaited to shed light on this intriguing functional diversity of GHR in the chicken and other species. PCR data have revealed that GHR mRNA is distributed primarily in the proventriculus, followed by the brain, lungs, pancreas, spleen, and intestines, in 8day-old layer chickens and in 3-wk-old broiler chickens (Kaiya et al., 2002; Richards and McMurtry, 2008). In the chicken, Wada et al. (2003) detected GHR mRNA expression in the proventriculus, pylorus, and duodenum. GHR-IR cells are found in the mucosal layer of the proventriculus (Wada et al., 2003; Neglia et al., 2005; Yamato et al., 2005) and intestine (Neglia et al., 2005; Scanes and Pierzchala-Koziec, 2014). In the present study, we observed that the majority of Gα gust - and Gα tran /GHR-IR cells were closed- and very few opentype cells. As indicated by Solcia et al. (2000), it is possible that the opened-cell type is influenced by the food content and pH in the lumen, whereas the closed-cell

Figure 4. Gα tran or Gα gust -IR colocalized with ghrelin (GHR) (A and B, arrows) or 5-hydroxytryptamine (5-HT)-IR (C and D, arrows) in the chicken duodenum. In A and B, the asterisks indicate a Gα tran IR cell not containing GHR, whereas the arrowheads show a GHR-IR cell negative for Gα tran -IR. The arrows in the photomicrographs (E– H) show Gα tran (E) and Gα gust -IR (G) cells co-expressing glucagonlike peptide-1 (GLP-1) (F) or CCK (H) in the chicken ileum. In E and F, arrowheads point to a Gα tran -IR cell that does not contain GLP-1. Frequently, the Gα tran /Gα gust -IR cells localized in the surface epithelium of the villi exhibit a typical open-type morphology (G and H, arrows). A, B, E, F, G, and H: scale bars = 50 μ m. C and D scale bars = 100 μ m.

type is affected by hormones, local factors, neuronal stimulation, and mechanical stress. An abundant population of Gα gust - or Gα tran EEC cells contains 5-HT, a biogenic amine that has been shown to exert a key role in the control of eating behavior and body weight. 5-HT is known to decrease food intake in virtually all mammalian species, and experimental conditions that increase 5-HT release decrease feeding behavior (Simansky, 1996; De Vry and Schreiber, 2000; Da Silva et al., 2004). In chickens, 5-HT is also involved in the regulation of various physiological functions, including inhibition of food intake (Denbow et al., 1982, 1983; Blundell, 1984; Saadoun and Cabrera, 2002). Intracerebroventricular injection of 5HT induces a potent decrease in food intake in chickens and turkeys (Denbow 1989; Zendehdel et al., 2017). Studies with agonists demonstrated that 5-HT acts on different receptor subtypes (De Vry and Schreiber, 2000; Saadoun and Cabrera, 2002; Da Silva et al.,

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Figure 5. Gα tran -IR (A, C, E, G, arrows) colocalized with 5-hydroxytryptamine (5-HT) (B, arrows), glucagon-like peptide-1 (GLP-1) (D, arrows), 5-HT (F, arrows), or gastrin/cholecystokinin (GAS/CCK) (H, arrows) in the large intestine and cloaca. Generally, in the cecum and rectum epithelium surface, the Gα tran -IR cells exhibit an open type enteroendocrine cells morphology (A–D, arrows), whereas, in some cases, the positive cells located in the crypts show a closed type morphology (C and D, arrowheads). Most Gα gust -IR cells located in the cloaca folds and crypts (E and H, arrows) co-express 5-HT or GAS/CCK. A, B, C, D, G, and H: scale bars = 50 μ m. E and F scale bars = 100 μ m.

2004). The results obtained in this study confirmed that Gα gust - and Gα tran /5-HT cells show a similar trend to what previously reported by several authors on the distribution of 5-HT-IR cells in the proventriculus, pylorus, and GI tract (Salvi et al., 1995; Rawdon and Adrew, 1999; Aksoy and Cinar, 2009; Gomi et al., 2015) and cloaca (Salvi et al., 1996). Gα gust - or Gα tran EEC cells containing GLP-1 are abundant in the proventriculus and large intestine but are also present in the small intestine, matching at least in part previous studies using immunohistochemical and morphometrical techniques and reporting GLP-1 containing EECs in the chicken proventriculus (Scanes and Pierzchala-Koziec, 2014) and small intestine (Hiramatsu et al., 2003, 2005; Watanabe et al., 2014). GLP-1 is a 30-amino-acid peptide derived from the precursor, proglucagon, and secreted after food ingestion from EECs distributed throughout the intestinal epithelium (Drucker, 2006; Balkan, 2008). GLP-1 is

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referred to as an “incretin hormone” with potent insulin-releasing activity and inhibition of GI motility and secretion. This peptide is also a physiological regulator of appetite and food intake (Furuse et al., 1997; Holst, 2007). The role of chicken GLP-1 remains largely unknown. Although some effects of GLP-1 in mammals and birds are established, e.g., the inhibition of food intake (Furuse et al., 1997; Tachibana et al., 2003, 2005; Huang et al., 2013), whether GLP-1 has the same insulinotropic activity in chickens is unknown (Huang et al., 2013). The chicken, unlike mammals, lacks the T1R2 subunit that, together with the T1R3 subunit, detects sugar and sweeteners. This feature suggests that carbohydrates are either detected by different pathways or that they can be sensed by the functional dimer T1R3 alone (Cheled-Shoval et al., 2015). Furthermore, GLP-1 is also released in response to T2R stimulation (Jeon et al., 2008) and T2R activation has been shown to be involved in glucose homeostasis (Dotson et al., 2008). PYY, a postprandial satiety hormone that is expressed in the mammalian GI tract, especially in the distal intestine (Ekblad and Sundler, 2002; Ueno et al., 2008), is also expressed in many Gα gust - or Gα tran EEC cells especially in the small intestine and proventriculum, but also in the large intestine. PYY peptide was isolated from chicken intestines in 1992, and amino acid sequence analysis revealed the presence of an additional N-terminal alanine residue (Conlon and O’Harte, 1992). Aoki et al., (2017) identified a full-length complementary DNA sequence encoding the chicken PYY precursor and showed that PYY mRNA was abundantly expressed in the small intestine compared to the large intestine, which is in line with our observation of more abundant Gα gust - or Gα tran /PYY cells in the small intestine than in the large intestine The same authors report that the intestinal PYY expression is altered in response to the nutritional status in chicks and intravenous administration of PYY significantly suppressed food intake in chicks. The diverse actions of neuropeptide Y, PYY, and pancreatic polypeptide are suggested to be mediated through multiple neuropeptide Y receptors (Michel et al., 1998), all of which belong to G protein-coupled receptors and share high structural similarity. It is likely that PYY released by Gα gust - or Gα tran /PYY EEC cells upon activation by endoluminal contents affects feeding behavior in the chicken through the activation of these distinct types. Our results showing high number of Gα tran or Gα gust /GAS/CCK-IR cells in the pyloric mucosa are consistent with previous studies reporting GAS/CCKIR cells in the proventriculus (Aksoy and Cinar, 2009), gizzard and antrum (gizzard–duodenum junction) (Salvi et al., 1995), and small intestine (Salvi et al., 1996). GAS is secreted by endocrine cells in birds and stimulates both hydrochloric acid and pepsin secretion (Dimaline et al., 1986; Dimaline and Lee, 1990; Campbell et al., 1994; Rawdon and Andrew, 1999). Recently, Reid and Dunn (2017) by means of PCR and in

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situ hybridization showed high expression of the GAS gene in the pylorus, less in other intestinal segments of the chicken. On the other hand, the proventriculus best resembles the mammalian monogastric stomach in form and function, and so is sometimes referred to as the “true stomach” (Zaher et al., 2012). The strict delineation of avian GAS within the “antrum” region observed here resembles primary mammalian GAS production at the pyloric antrum which suggests homology of these GI structures in birds and mammals (Reid and Dunn, 2017). Gα gust - and Gα tran EEC cells containing CCK, a GI hormone/peptide that is also abundantly expressed in the brain of mammals (Miyasaka and Funakoshi, 2003; Strader and Woods, 2005), are abundant in different regions including proventriculus, pyloric region, and small intestine but less abundant in the large intestine. CCK has multiple effects on the GI system including gallbladder contraction, gut motility, gastric emptying, and the secretion of gastric acid and pancreatic enzymes (Miyasaka and Funakoshi, 2003; Strader and Woods, 2005). Additionally, numerous studies have documented a role of CCK in the induction of satiety. For example, peripheral injection of CCK suppressed feeding behavior in mammals such as rats (Gibbs and Smith, 1977) and humans (Kissileff et al., 1981), and so does central injection in sheep (Della-Fera and Baile, 1980), rats (Fekete et al., 1981), and goldfish (Kang et al., 2010). Furuse et al., (2000) reported that central administration of GAS/CCK family decreased chick food intake, while Tachibana et al. (2012) showed that Nterminal amino acids and the sulfation of tyrosine are important for the anorexigenic effect of CCK8S after intraperitoneal and intracerebroventricular administration in chicks. CCK has also been purified from the chicken (Fan et al., 1987), and CCK-like immunoreactivity is found in the chicken small intestine (Alison, 1989; Salvi et al., 1996). Finally, Reid and Dunn (2017) observed GI CCK transcripts dispersed throughout the chicken intestine, in particularly, in the proximal ileum. In summary, our study demonstrates that G-proteins involved in chemosensory transmission are expressed in the chicken GI tract enteroendocrine system, suggesting their participation in chemosensing processes. Nutrients may elicit the release of different bioactive messengers (mainly peptides), which directly, or via neural reflexes, contribute to the control of GI functions and nutrient intake in the chicken (Song et al., 2013). A better understanding of the mechanisms involved in luminal chemosensitivity in the chicken may provide a new basis for feeding formulations to be applied in poultry.

ACKNOWLEDGMENTS The present work was supported by NIH grants P30 DK41301 Imaging and Stem Cell Biology Core and R01 DK 098447 (CS). We are grateful to Dr. Claudia Vallorani, Department of Medical Veterinary Sciences,

University of Bologna, for her valuable technical assistance with immunohistochemistry and cell counting.

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