Localization of ghrelin-like peptide in the gastrointestinal tract of the golden apple snail (Pomacea canaliculata) and changing of its concentration during fasting

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Localization of ghrelin-like peptide in the gastrointestinal tract of the golden apple snail (Pomacea canaliculata) and changing of its concentration during fasting Apichart Ngernsoungnern, Piyada Ngernsoungnern ∗ School of Anatomy, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand

a r t i c l e

i n f o

Article history: Received 11 November 2015 Received in revised form 22 January 2016 Accepted 22 January 2016 Available online xxx Keywords: Pomacea canaliculata Golden apple snail Ghrelin Gastrointestinal tract

a b s t r a c t Ghrelin is an endogenous hormone detected in the gastrointestinal tracts (GI) of various species. In the present study, ghrelin-like peptide (ghrelin-LP) was identified in the GI tract of the golden apple snail, Pomacea canaliculata. Using immunohistochemistry, the result revealed an immunoreactivity (-ir) of ghrelin-LP in regions of the GI tract. The ghrelin-LP-ir was observed in both opened-type and closedtype cells of the esophagus, stomach, and small and large intestines. The highest density of ghrelin-LP immunoreactive cells was found in the esophagus and the least density was detected in the stomach. The highest percentages of the opened-type and closed-type cells were present in the esophagus and small intestine, respectively. In immunoblotting, the molecular weight of ghrelin-LP was related to the human ghrelin peptide (∼13 kDa). Moreover, the concentration of ghrelin-LP was significantly higher in snails that were fasted for 24 h compared with fed snails. The concentration decreased after refeeding. The present study could be useful for understanding the physiological role of ghrelin-LP in mollusk species. © 2016 Elsevier GmbH. All rights reserved.

1. Introduction The golden apple snail (Pomacea canaliculata, Lamarck 1822) is a freshwater snail (Gastropod, Prosobranchia) that is commonly found in South America. Since 1980, the snail has been brought to Asia for food consumption instead of abalone, which is more costly. The snail was also used to clean algae in fish aquariums (Mochida, 1991; Naylor, 1996; Hayes et al., 2008). However, they escaped and/or were released into the natural freshwater and agricultural wetlands. Because the snail is good at adaptation for survival and it has a high growth rate and short reproductive cycle, its population has since then increased rapidly. In addition, a female snail can produce more than 10,000 eggs/reproductive cycle (Estebenet and Martin, 2002; Carlsson et al., 2004). This species has thus become an important agricultural wetland pest, particularly in rice fields, where it significantly impacts rice production. Ghrelin (a 28-amino acid peptide) was firstly identified and isolated from the stomach of rats (Kojima et al., 1999). Later, the

∗ Corresponding author at: School of Anatomy, Institute of Science, Suranaree University of Technology, 111 University Ave., Nakhon Ratchasima 30000, Thailand. Fax: +66 44224185. E-mail addresses: [email protected], [email protected] (P. Ngernsoungnern).

chicken ghrelin peptide consisting of 26 amino acids was identified (Kaiya et al., 2002). Although ghrelin is different among species in terms of the numbers of amino acids, it contains a conserved region in which the third serine residue is n-octanoylated by ghrelin O-acyltransferase (GOAT). This modification is known to be essential for the ligand–receptor binding complex which regulates the release of growth hormone (GH) from the pituitary gland (Kojima et al., 1999; Ahmed and Harvey, 2002; Yamazaki et al., 2002). It is well known that the primary role of GH is promotion of overall body and cell growth. GH also regulates carbohydrate–protein–lipid metabolism (Sugino et al., 2004; Javorsky et al., 2011; Sperling, 2015). The pulsatality of GH secretion is controlled by regulatory hormones, including GH-releasing hormone, GH-inhibiting hormone, and also ghrelin. It was stated that ghrelin stimulated GH release through GH secretagogue receptor and subsequently caused an elevation of intracellular Ca2+ levels via IP3 signal transduction pathway (Khatib et al., 2014). A study thus suggested an involvement of ghrelin in metabolism and energy balance (Kamegai et al., 2000). Various studies have stated that ghrelin regulates food intake via the central and peripheral nervous system (Wren et al., 2000; Nakazato et al., 2001; Date et al., 2002; Saito et al., 2002; Sugino et al., 2004; Shousha et al., 2005). Ghrelin level was decreased in human obesity (Shiiya et al., 2000; Tschöp et al., 2001; Marzullo et al., 2006), and fasting plasma ghrelin was lower in a population with a very high prevalence of obesity (Tschöp et al.,

http://dx.doi.org/10.1016/j.acthis.2016.01.005 0065-1281/© 2016 Elsevier GmbH. All rights reserved.

Please cite this article in press as: Ngernsoungnern, A., Ngernsoungnern, P., Localization of ghrelin-like peptide in the gastrointestinal tract of the golden apple snail (Pomacea canaliculata) and changing of its concentration during fasting. Acta Histochemica (2016), http://dx.doi.org/10.1016/j.acthis.2016.01.005

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2001). This might be a compensatory response to a sustained positive energy balance (Cummings and Shannon, 2003; Dostálová and Haluzík, 2009). In addition, accumulating evidence suggests that ghrelin plays a role in gastrointestinal (GI) tract functions, for example motility and secretion (Masuda et al., 2000; Kojima and Kangawa, 2005; Kitazawa et al., 2009). Immunoreactivity (-ir) of ghrelin was identified in the GI tract and accessory organs of various species, for example, in the stomach, small intestine, large intestine, and pancreas of mammals (Hayashida et al., 2002; Sakata et al., 2002; Wierup et al., 2002; Kageyama et al., 2005; Zhao and Sakai, 2008; Kasacka et al., 2014). In avians, ghrelin producing cells were found in the proventiculus, gizzard, and all parts of the intestine (Wada et al., 2003; Neglia et al., 2005; Wang et al., 2009; Shao et al., 2010). Moreover, evidence has revealed the presence of ghrelin immunoreactive cells in stomach and intestinal epithelia of teleosts, gold fish and Mediterranean sea bass (Arcamone et al., 2009). As previously mentioned, P. canaliculata has been brought to Thailand for over many decades and the snail has become a cause of major problems in agriculture as a wetland pest. However, at present, knowledge on the snail’s biology is limited, especially its endocrinology. In addition to growth hormone, the high growth rate and high food consumption of the species could be related to its high level of ghrelin which is also known as an appetite-stimulating hormone. Because ghrelin is highly conserved among species (Kaiya et al., 2011), the present study, thus, attempts to identify the localization and distribution of ghrelin-like peptide (ghrelin-LP) in the GI tract of P. canaliculata and its function on feeding. 2. Materials and methods 2.1. Animal and tissue preparations Golden apple snails were collected from rice fields in the Nakhon Ratchasima, Thailand. The snails with operculum widths ranging from 4.5 to 5.6 cm were selected according to Youens and Burks (2008). They were housed in 20-l bowls containing dechlorinated tap water (25–28 ◦ C). The water was changed daily. The snails were fed once a day with morning glories. Female snails were used because they consumed more food. 2.2. Immunoperoxidase The snails were anesthetized at –20 ◦ C. Parts of the GI tract (esophagus, stomach, small intestine, and large intestine) were dissected out separately. The tissues were immediately immersed in Bouin’s fixative overnight and subsequently processed conventionally for paraffin sectioning. The 5-␮m thick sections were cleared, rehydrated, and then immersed in 1% H2 O2 and 1% LiCO3 in 70% ethanol to remove endogenous peroxidase and residual picric acid, respectively. The sections were blocked for non-specific binding with 4% bovine serum albumin (BSA) in phosphate buffer saline (PBS) for 60 min and subsequently incubated with rabbit antihuman ghrelin antibody (Santa Cruz Biotechnology, Dallas, TX, USA, Catalog number sc-50297) (1:100 in PBS), overnight at 4 ◦ C. The antibody was raised against amino acids 21–60 mapping at the N-terminus of ghrelin of human origin. It is recommended for detection of precursor and mature ghrelin of mouse, rat, and human origin by Western blotting, immunoprecipitation, immunofluorescence, and solid phase enzyme-linked immunosorbant assay (ELISA) (information from the manufacturer). The antibody reacted with human ghrelin (Fischer et al., 2008; O’Brien et al., 2010) and cross-reacted with rat ghrelin (Zhao et al., 2013). Negative controls were performed by omitting the primary antibody or by preabsorption of the primary antibody with human ghrelin pep-

tide (Abcam, Cambridge, MA, USA), overnight at 4 ◦ C (50 ␮g/0.1 ml antibody at working dilution). The sections were washed twice with PBS containing 0.1% Tween-20 (PBST) and then incubated with horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG (Invitrogen, Carlsbad, CA, USA, Catalog number 65–6120) (1:500 in PBS) for 60 min at room temperature. After washing with PBST, antigen-antibody complex was visualized by incubating the sections with a 3,3 -diaminobenzidine-tetrachloride (DAB) kit (Vector Laboratories, Burlingame, CA, USA), followed by dehydration, clearing, and mounting. Ghrelin-LP-ir was identified under a light microscope (80i, Nikon, Japan). The experiments were repeated using tissues from 6 different individuals. 2.3. Determining of immunoreactive cell densities Densities of ghrelin-LP immunoreactive cells from parts of the GI tract were determined. Ten immunoperoxidase sections were selected from each region of the GI tract. From each section, 10 microscopic fields were photographed. The immunoreactive cells were counted and presented as the numbers of positive cells per mm2 (a microscopic field area equals 0.75 mm2 at 40× magnification) using a computerized image analysis program (Cell∧ D software). Moreover, percentages of opened-type and closed-type cells counted from each region of the GI tract were also calculated. The results were expressed as mean ± S.D. 2.4. Immunoblotting Parts of the GI tract were quickly removed, immersed in liquid nitrogen, and kept at −80 ◦ C until used. The tissues were then homogenized in a cold lysis buffer (10 mM Tris–HCl, 150 mM NaCl, 0.5% Triton X-100, 1 mM EDTA, and 100 mM PMSF, pH 7.2). The homogenates were then centrifuged at 15,000 × g for 15 min at 4 ◦ C and the supernatants were collected. Concentrations of the protein lysates were measured using a Coomassie protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). The protein lysates (40 ␮g, each) and human ghrelin peptide (1 ␮g) were separated in 15% SDSPAGE, and then electro-transferred onto a 0.45 ␮m nitrocellulose membrane (GE healthcare, Piscataway, NJ, USA). The membrane was blocked with 5% skimmed milk in PBS for 60 min at room temperature and subsequently immerged in rabbit anti-human ghrelin antibody (Santa Cruz Biotechnology) (1:1000 in PBS), overnight at 4 ◦ C. The membrane was washed twice with PBST and incubated with HRP conjugated goat anti-rabbit IgG (Invitrogen) (1:3000 in PBS) for 60 min at room temperature, followed by washing with PBST. The antigen-antibody complex was obtained by adding a DAB kit (Vector Laboratories). Analysis of intensities of ghrelin-LP immunoreactive bands in the parts of the GI tract was performed using ImageJ software under the same pixel area. The experiments were repeated using tissues from 6 different individuals. 2.5. Quantitation of secreted ghrelin-LP concentrations Concentrations of secreted ghrelin-LP were determined by ELISA. The snails were acclimatized for 2 weeks and supplied with food once daily. The snails were then divided into 2 groups; fed (control) and fasted groups (n = 8, each). The snails were fasted for 24 h and hemolymph samples were collected according to the method described by Sminia (1972). The fasted snails were then provided with food, and hemolymph samples were collected at 3 h after refeeding. The samples were subsequently centrifuged at 10,000 × g for 10 min at 4 ◦ C. Supernatants were collected, diluted with ELISA coating buffer, coated in duplicate onto 96-well plates (Nunc, Roskilde, Denmark), and left overnight at 4 ◦ C. Human ghrelin peptide was used as a standard. The plates were blocked for non-specific binding with 0.25% BSA in 0.01 M PBS for 1 h at

Please cite this article in press as: Ngernsoungnern, A., Ngernsoungnern, P., Localization of ghrelin-like peptide in the gastrointestinal tract of the golden apple snail (Pomacea canaliculata) and changing of its concentration during fasting. Acta Histochemica (2016), http://dx.doi.org/10.1016/j.acthis.2016.01.005

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37 ◦ C and then incubated with rabbit anti-human ghrelin antibody (Santa Cruz Biotechnology) (1:3000 in PBS). The antibody showed a specificity of 92.08% to human ghrelin peptide. The plates were washed twice with PBST and then incubated with HRP conjugated goat anti-rabbit IgG (Invitrogen) (1:3000 in PBS) for 1 h at 37 ◦ C. Negative controls were performed by omitting the primary antibody or using the preabsorbed one. Finally, tetramethylbenzidine substrate (Sigma–Aldrich, St. Louis, MO, USA) was added, followed by 2 M H2 SO4 . The color was then read spectrophotometrically (BioRad, Hercules, CA, USA) at 450 nm. The assay showed a sensitivity of 0.125 ng/0.1 ml. The intra-assay and inter-assay coefficients of variation were 3.89% and 6.56%, respectively. 2.6. Statistical analysis Differences among numbers of the ghrelin-LP immunoreactive cells in the GI tract, percentages of opened-type and closed-type cells, intensities of ghrelin-LP immunoreactive bands, and concentrations of secreted ghrelin-LP compared between fed and fasted snails were statistically analyzed using one-way analysis of variance (ANOVA) followed by a Tukey post hoc multiple comparison. A probability value of less than 0.05 (P < 0.05) was used to indicate a significant difference. 3. Results 3.1. Localization of ghrelin-LP immunoreactive cells Ghrelin-LP immunoreactive cells were identified in parts of the GI tract (Fig. 1). Two types of the immunoreactive cells were found; opened-type cells with elongated staining and closed-type cells with round shaped staining. Both opened-type and closed-type cells were observed in the esophagus (Fig. 1A), stomach (Fig. 1B), small intestine (Fig. 1C), and large intestine (Fig. 1D). No ghrelinLP-ir was shown in negative control sections (Fig. 1E). 3.2. Densities of ghrelin-LP immunoreactive cells Densities of ghrelin-LP immunoreactive cells (both opened-type and closed-type cells) in parts of the GI tract are shown in Fig. 2A. The highest density of ghrelin-LP immunoreactive cells was found in the esophagus (51.07 ± 13.16 cells/mm2 ). Lesser densities were observed in the large intestine and small intestine (45.98 ± 11.45 and 26.81 ± 13.49 cells/mm2 , respectively). The least density was found in the stomach (8.46 ± 2.70 cells/mm2 ) which was significantly lower than those of the esophagus, small intestine, and large intestine (P < 0.05). Percentages of the opened-type and closed-type cells from parts of the GI tract are shown in Fig. 2B. The highest percentage of the opened-type cells was found in the esophagus (73.56 ± 8.41%) which was significantly higher than those of other regions (P < 0.05). Percentages of the opened-type cells decreased from the large intestine to the stomach, and then the small intestine (33.11 ± 4.35, 29.99 ± 3.31, 23.04 ± 5.56%, respectively). In contrast, the small intestine showed the significantly highest percentage (P < 0.05) of the closed-type cells (76.96 ± 5.56%). Percentages of the close-type cells decreased from the stomach (70.01 ± 3.31%) to the large intestine (66.89 ± 4.35%). The lowest percentage of the closed-type cells was observed in the esophagus (26.44 ± 8.41%). 3.3. Expression of ghrelin-LP Ghrelin-LP in all parts of the GI tract were identified at ∼13 kDa which were the same as that of the molecular weight of human ghrelin peptide (Fig. 3A). The peptide was not detected with preabsorbed antibody (Fig. 3B).

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The highest intensity of ghrelin-LP immunoreactive band was observed in the esophagus (89.67 ± 5.04 arbitrary units (AU)) which was significantly higher (P < 0.05) than those of the small intestine (61.01 ± 3.80 AU) and stomach (40.92 ± 1.31 AU) (Fig. 3C). Similar band intensity to that of the esophagus was found in the large intestine (78.02 ± 3.08 AU). 3.4. Changing of ghrelin-LP concentrations during fasting and refeeding After fasting for 24 h, concentration of secreted ghrelin-LP was elevated to 2.48 ± 0.48 ng/0.1 ml which was significantly higher than that of the control group (0.84 ± 0.14 ng/0.1 ml) (Fig. 4). The concentration of secreted ghrelin-LP of the fasted group was approximately 3 folds higher than that of the fed group. After refeeding, concentration of secreted ghrelin-LP in the fasted group decreased to a similar level (0.95 ± 0.63 ng/0.1 ml) compared to that of the control group (0.91 ± 0.21 ng/0.1 ml). 4. Discussion Ghrelin is present in a variety of tissues, for example hypothalamus, pituitary gland, pancreas, spleen, liver, kidney, and GI tract (Wang et al., 2002; Sato et al., 2012; Al Massadi et al., 2015). In the present study, ghrelin-LP-ir was found located in regions of the GI tract; esophagus, stomach, small intestine, and large intestine. This finding corresponds to the previous studies of two teleost species (Carassius auratus and Dicentrachus labrax) which showed the immunopositive cells localized in all regions of the alimentary canal, especially in the intestinal epithelium (Arcamone et al., 2009). Ghrelin-ir was also found in the stomach of frogs (Galas et al., 2002), fresh water eels (Kaiya et al., 2006), rats (Date et al., 2000; Ahmed and Harvey 2002; Zhao et al., 2012), and humans (Date et al., 2000). Moreover, ghrelin immunoreactive cells were identified in the GI tract of various avian species, such as, domestic fowl, white leghorn chicken, African ostrich chick, and Peking duck (Ahmed and Harvey 2002; Wada et al., 2003; Neglia et al., 2004, 2005; Wang et al., 2009; Shao et al., 2010). Our study demonstrated that ghrelin-LP-ir was detected in both opened-type and closed-type cells. This result is in an agreement with previous studies in fish, chickens, ducks, ostriches, rats, and amphioxi (Sakata et al., 2002; Wada et al., 2003; Neglia et al., 2005; Zhao and Sakai, 2008; Weng et al., 2008; Wang et al., 2009; Shao et al., 2010; Kasacka et al., 2014). The opened-type and closed-type cells are known to be involved in various physiological functions (Sakata et al., 2002). The opened-type cells responded to food or pH in lumen of the GI tract. It is likely that food induces ghrelin release from the GI tract and also modulates the GI motility (Neglia et al., 2005). On the other hand, the closed-type cells were stimulated by hormones, local factors, neuronal stimulation, and/or mechanical stress (Solcia et al., 2000; Neglia et al., 2005). Thus, the existence of 2 types of ghrelin-LP immunoreactive cells in the GI tract of P. canaliculata suggests that the release of ghrelin-LP from these cell types could also be regulated by different factors. Although ghrelin is also known as a stomach hormone (Kojima et al., 1999; Wang et al., 2002; Sato et al., 2012; Al Massadi et al., 2015), the highest density of ghrelin-LP immunoreactive cells was observed in the snail esophagus. In contrast, medium densities of the cells were found in the small intestine and large intestine, and the least density was found in the stomach. This observation suggests that the main production site of ghrelinLP in P. canaliculata could differ from that of vertebrate species. Like those of other species, the mollusk’s esophagus is not only a food passage. Epithelial cells lining the esophagus also play the role as secreting cells. Previous studies identified various types of

Please cite this article in press as: Ngernsoungnern, A., Ngernsoungnern, P., Localization of ghrelin-like peptide in the gastrointestinal tract of the golden apple snail (Pomacea canaliculata) and changing of its concentration during fasting. Acta Histochemica (2016), http://dx.doi.org/10.1016/j.acthis.2016.01.005

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Fig. 1. Ghrelin-LP-ir in the GI tract of P. canaliculata. The ghrelin-LP-ir was observed in opened-type (arrowheads) and closed-type (arrows) cells of the esophagus (A), stomach (B), small intestine (C), and large intestine (D). Negative control section from the esophagus showed no ghrelin-LP-ir (E). Scale bars = 500 ␮m.

secretory granules including enzymatic granules in the esophageal epithelial cells (Bowen, 1970; Roldan and Garcia-Corrales, 1988; Dimitriadis, 2001; Martin et al., 2010). Moreover, activities of various enzymes, for example, phosphatase, hydrolase, and glycosidase were detected in the esophagus of the giant key hole limpet, Megathura crenulata (Martin et al., 2011). In P. canaliculata, the function of ghrelin-LP might relate to either motility or the secretory function of the esophagus. However, this hypothesis cannot be concluded from the results of the present study. Ghrelin-LP-ir and expression were observed in parts of the GI tract. The molecular weight of the ghrelin-LP was approximately 13 kDa. The same antibody was reported to react with pro-ghrelin with molecular weights of 15 kDa in human myometrium (O’Brien et al., 2010) and 13 kDa in the gastric mucosa of rats (Zhao et al., 2013). This could be because there are some differences in the structure of ghrelin among species. However, the molecular weight of ghrelin-LP obtained in the present study was related to that of the positive control (human ghrelin peptide). Moreover, the ghrelinLP-ir was abolished by the pre-absorbed the antibody with human ghrelin peptide. This suggests that ghrelin-LP in P. canaliculata could be structurally similar to that of the human ghrelin peptide. In the present study, concentration of secreted ghrelin-LP increased in fasted snails and decreased after refeeding. It was

shown that levels of plasma ghrelin were elevated in fasted rats (Bagnasco et al., 2002; Lee et al., 2002), quails (Shousha et al., 2005), and cats (Ida et al., 2007), and the changes were inverted with refeeding (Bagnasco et al., 2002; Lee et al., 2002; Shousha et al., 2005). Similarly, ghrelin mRNA in fish (Feng et al., 2013; Ji et al., 2015) and rats (Zhao and Sakai, 2008) increased after starvation and decreased after refeeding. Ghrelin is known to stimulate food intake in various species. Administration of ghrelin increases food intake and also adiposity (Asakawa et al., 2001; Nakazato et al., 2001; Wren et al., 2001; Peeters, 2005; Volkoff et al., 2005; Valassi et al., 2008; Peric´ Mataruga et al., 2009; Date and Kangawa, 2012; Müller et al., 2015). Circulating ghrelin level increases before a meal indicating the role of ghrelin as a hunger signal to stimulate food consumption. Ghrelin levels then fall after feeding (Sugino et al., 2004; Natalucci et al., 2005). It is stated that ghrelin is more potent than any other orexigenic peptide, except neuropeptide Y (NYP) (Wang et al., 2002) and ghrelin may directly reach the hypothalamus and stimulate NYP and agouti-related protein, thereby inducing feeding. The present study suggests that ghrelinLP may act as the orexigenic signal in P. canaliculata as well as in other species. Although various investigations reported the function of ghrelin as an appetite-regulating peptide, a study revealed that ghrelin-null

Please cite this article in press as: Ngernsoungnern, A., Ngernsoungnern, P., Localization of ghrelin-like peptide in the gastrointestinal tract of the golden apple snail (Pomacea canaliculata) and changing of its concentration during fasting. Acta Histochemica (2016), http://dx.doi.org/10.1016/j.acthis.2016.01.005

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Fig. 2. (A) Densities of ghrelin-LP-immunoreactive cells in parts of the GI tract. Data is presented as mean ± SD. (n = 6). Different letters indicate significant differences between groups (P < 0.05). (B) Percentages of opened-type and closed-type cells in parts of the GI tract. Data is presented as mean ± SD (n = 6). Asterisks indicate significant differences between groups (P < 0.05) compared between the same type of cells.

Fig. 3. (A) Expression of ghrelin-LP in parts of the GI tract. Human ghrelin peptide was used as a positive control. (B) The ghrelin-LP-ir was abolished using the pre-absorbed primary antibody. (C) Intensities of ghrelin-LP-immunoreactive bands in parts of the GI tract. Data is presented as mean ± SD (n = 6). Different letters indicate significant differences between groups (P < 0.05).

Please cite this article in press as: Ngernsoungnern, A., Ngernsoungnern, P., Localization of ghrelin-like peptide in the gastrointestinal tract of the golden apple snail (Pomacea canaliculata) and changing of its concentration during fasting. Acta Histochemica (2016), http://dx.doi.org/10.1016/j.acthis.2016.01.005

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Fig. 4. Concentrations of ghrelin-LP of fasted and refed groups compared to fed group (control). Data is presented as mean ± SD (n = 8, each). Asterisk indicates a significant difference compared to the control (P < 0.05).

mice were indistinguishable from wild-type mice in term of food consumption (Sun et al., 2003). No differences in food intake and meal pattern were observed compared between ghrelin/ghrelin receptor double knockout, ghrelin-deficient, ghrelin receptordeficient, and wild-type control mice (Pfluger et al., 2008). It was stated that stimulation of food consumption was carried by acylghrelin (Neary et al., 2006; Al Massadi et al., 2015). However, a study reported that GOAT null mice exhibited the same food intake as their wild-type ones (Kirchner et al., 2009). These data suggested that lacking of ghrelin or its activating enzyme did not lead to a reduction in appetite. Another study indicated that endogenous ghrelin was not an essential regulator of food intake, but played a prominent role in determining the type of metabolic substrate that was used for maintenance of energy balance, particularly under conditions of high fat intake (Wortley et al., 2004). In addition, various studies reported the function of ghrelin as regulators for various metabolic pathways (Kola et al., 2005; Kang et al., 2011; Dezaki, 2013). Other functions of ghrelin have been reported. Ghrelin is known to be a neuropeptide involved in gastrointestinal hypothalamicpituitary axis (Ahmed and Harvey, 2002; Wang et al., 2002). Ghrelin contributes the regulation of GH secretion (Kojima et al., 1999; Ahmed and Harvey, 2002; Kaiya et al., 2003; Neglia et al., 2005; Nass et al., 2011; Müller et al., 2015) with 2–3 times greater stimulatory effect than GH-releasing hormone (Sato et al., 2012). Ghrelin also plays other roles, including stimulating GI tract motility, gastric and pancreatic secretions, and gastric emptying (Masuda et al., 2000; Asakawa et al., 2001; Kojima and Kangawa, 2005; Kitazawa et al., 2009, 2011). It is known that ghrelin stimulates gastric acid secretion and motility via vagal control (Masuda et al., 2000; Dornonville de la Cour et al., 2004). Ghrelin is also known to be involved in locomotor activity, cardiovascular function, reproduction, cellular proliferation, and immunomodulation (Kojima and Kangawa, 2005; Hosoda et al., 2006; Jerlhag et al., 2006; Soares and LeiteMoreira, 2008; Kaiya et al., 2009; Kang et al., 2011). Moreover, ghrelin was reported to have a positive influence on frass elimination, body mass, nutritional indices, digestive enzyme activities, and mass of midgut of a caterpillar (Peric´ Mataruga et al., 2009, 2012). In summary, we demonstrate that ghrelin-LP-ir was detected in the GI tract of the golden apple snail. Moreover, the concentration

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Please cite this article in press as: Ngernsoungnern, A., Ngernsoungnern, P., Localization of ghrelin-like peptide in the gastrointestinal tract of the golden apple snail (Pomacea canaliculata) and changing of its concentration during fasting. Acta Histochemica (2016), http://dx.doi.org/10.1016/j.acthis.2016.01.005