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In this study, we report the distribution of orexin A (OXA), orexin B (OXB), and orexin receptor (OX2R) immunoreactive (ir) cells in the hypothalamus and gastrointestinal tract of Oncorhynchus mykiss fed diets with different dietary fatty acid compositions. Trout were fed five iso-energetic experimental diets containing fish oil, or one of four different vegetable oils (olive, sunflower, linseed, and palm oils) as the added dietary lipid source for 12 weeks. OXA, OXB, and OX2R immunoreactive neurons and nervous fibers were identified in the lateral and ventro-medial hypothalamus. OXA, OXB, and OX2R ir cells were found in the mucosa and glands of the stomach and in the mucosa of both the pyloric cecae and intestine. OX2R ir cells were localized in the mucosa layer of both the pyloric cecae and intestine. These immunohistochemical (IHC) results were confirmed via Western blotting. Antibodies against preproorexin (PPO) crossreacted with a band of ∼16 kDa in the hypothalamus, stomach, pyloric cecae, and intestine. Antibodies against OX2R crossreacted with a band of ∼38 kDa in the hypothalamus, pyloric cecae, and intestine. The presence and distribution of OXA, OXB, and OX2R ir cells in the hypothalamus and gastrointestinal tract did not appear to be affected by dietary oils. The presence of orexin system immunoreactive cells in the stomach, pyloric cecae, and intestine of rainbow trout, but not in the enteric nervous system, could suggest a possible role of these peptides as signaling of gastric emptying or endocrine modulation, implying a main local action played by orexins.

This study provides a general approach to the presence and possible role of orexins and their receptors in the gut (three gastric chambers and intestine) of confined environment bottlenose dolphin. The expression of prepro-orexin, orexin A and B and orexin 1 and 2 receptors were investigated by single immunostaining and western blot analysis. The co-localization of vasoactive intestinal peptide and orexin 1 receptor in the enteric nervous system was examined by double immunostaining. Also, orexin A concentration were measured in plasma samples to assess the possible diurnal variation of the plasma level of peptide in this species. Our results showed that the orexin system is widely distributed in bottlenose dolphin enteric nervous system of the all gastrointestinal tract examined. They are very peculiar and partially differs from that of terrestrial mammals. Orexin peptides and prepro-orexin were expressed in the main stomach, pyloric stomach and proximal intestine; while orexin receptors were expressed in the all examined tracts, with the exception of main stomach where found no evidence of orexin 2 receptor. Co-localization of vasoactive intestinal peptide and orexin 1 receptor were more evident in the pyloric stomach and proximal intestine. These data could suggest a possible role of orexin system on the contractility of bottlenose dolphin gastrointestinal districts. Finally, in agreement with several reports, bottlenose dolphin orexin A plasma level was higher in the morning during fasting. Our results emphasize some common features between bottlenose dolphin and terrestrial mammals. Certainly, further functional investigations may help to better explain the role of the orexin system in the energy balance of bottlenose dolphin and the complex interaction between feeding and digestive physiology.

In this study, we report nesfatin-1 immunoreactivity in the gastrointestinal tract of Casertana breed pig. The newly discovered anorexigenic peptide nesfatin-1 has been shown to possess physiological relevance in regulating food intake and energy homeostasis at a central level, although evidence has been accumulating that it may also play important functions at a more local gastroenteric level. Nesfatin-1 immunoreactive endocrine cells have been detected in the gastric fundus and ileocecal valve. Nesfatin-1 immunopositive neurons and nerve fibers have been observed mainly in the enteric plexuses. Western blot analysis confirmed the immunohistochemical observations, showing immunoreactive bands in all analyzed gastrointestinal tracts with the exception of the rectum. Nesfatin-1 immunodetection in the swine digestive system reinforces the importance of the role played by nesfatin-1 at the gastrointestinal level and sustains the necessity to study the role of this peptide in the regulation of food intake in farm species for which weight gain is essential for optimizing production.

Summary The presence of neuroendocrine (NE) cells producing biogenic amines and hormonal peptides has been investigated in the vestibular glands of the genital tracts of cows and pigs using immunohistochemistry. NE cells containing chromogranin A-, serotonin-, cholecystokinin- and somatostatin-immunoreactive material were found in both major and minor vestibular glands. Such cells were numerous, scattered in the acini and excretory duct epithelium, small in size and rounded, triangular or bipolar in shape. The function of the NE vestibular cells has been related to the secretory activity of the glands and to a sexual climax induction mechanism involving the stimulation of 5HT 3 receptors of vestibular nociceptor nerve fibers. The role of NE cells in small cell carcinomas of the vestibular glands is a topic for further investigation owing to possible parallelism between this type of tumor and the small cell carcinoma of the human prostate.

Orexin receptor 1 (OX₁R) and orexin receptor 2 (OX₂R) are two G-protein-coupled receptors that bind their ligands, orexin A (OXA) and B (OXB), with different affinities. The male genital system represents an important target for OXA, which appears to play a role in the control of steroidogenesis and germ cell development in the testis. It is known that among domestic breeding animals, in the boar the number of Leydig cells is very high and OXA appears to have stimulatory activity on testosterone production. In this study, we aimed to evaluate the presence of OX₁R in the boar testis in order to extend our knowledge concerning the distribution and a potential functional role of the orexinergic system in the male reproductive tract of farm animals. The presence of OX₁R immunopositive cells in seminiferous tubules of the boar testis enables us to hypothesize a possible role of OXA on male germ cells cycle in pig. Further investigations, involving functional and ultrastructural analysis, may contribute to our understanding of the role of orexins in the boar genital system.

Calpains are Ca2+-dependent proteases able to cleave a large number of proteins involved in many biological functions. Particularly, in skeletal muscle they are involved in meat tenderizing during post mortem storage. In this report we analyzed the presence and expression of µ- and m-calpains in two skeletal muscles of the Marchigiana cattle soon after slaughter, using immunocytochemical and immunohistochemical techniques, Western blotting analysis and Casein Zymography. Therefore, the presence and the activity of these proteases was investigated until 15th day post-mortem during normal process of meat tenderizing. The results showed m- and µ-calpain immunosignals in the cytoplasm both along the Z disk/I band regions and in the form of intracellular stores. Moreover, the expression level of µ-calpain but not m-calpain decreased after 10 days of storage. Such a decrease in µ-calpain was accompanied by a gradual reduction of activity. On the contrary, m-calpain activity persisted up to 15 days of post-mortem storage. Such data indicate that expression and activity of both µ-calpain and m-calpain analyzed in the Marchigiana cattle persist longer than reported in literature for other bovines and may be related to both the type of muscle and breed examined.

Expression and distribution of leptin and ghrelin in the digestive ap-paratus of DIO (diet-induced obesity) zebrafish Maruccio L, Russo F, Arcamone N, Mania M, Randazzo B, Ciriaco E In this report we analyzed the expression and distribution of leptin and ghrelin in the digestive apparatus of DIO zebrafish. Leptin is anorexigenic peptide while ghrelin is orexigenic one. They act as antagonists. In this study in order to develop an obesity phenotype adult zebrafish were divided into two dietary groups. The con-trol group was fed with Artemia (5 mg cysts/fish/day) once per day. The overfeeding group was fed three times per day with Artemia (60 mg cysts/fish/day). For calorie restriction, the zebrafish was fed with Artemia (2.5 mg cysts/fish/day) for 2 weeks after being overfed for 8 weeks. At the end of this period, after 24h of fasting, the zebrafish digestive apparatus were sampled for immunohistochemistry, western blotting and qRT-PCR techniques. Leptin and ghrelin immunoreactivity were found in the enteric nervous system and neuroen-docrine cells in overfeeding and control zebrafish intestine. The number of immunopositive cells is greater in DIO zebrafish than in control ones. In both groups leptin immunoreactive nervous fibers were found around ducts and vessels of liver. The presence of proteins were confirmed by western blotting analysis. By qRT-PCR, leptin and ghrelin mRNA levels are higher in the overfeeding zebrafish intestine and in control zebrafish liver. The immunological detection of ghrelin and leptin in control zebrafish are in agreement with literature data. In DIO zebrafish the results are similar to those found for obese mammals. In conclusion, this study represent a starting point to investigate the mechanisms involved in the regulation of appetite and energy balance in DIO zebrafish.

This study provides an immunohistochemical approach to the expression of leptin in the gastrointestinal tract of the monogastric South American sea lion (Otaria flavescens), and the poligastric bottlenose dolphin (Tursiops truncatus). The specific organization of the gastrointestinal tract is examined in relation to the neuroendocrine regulation of the gut exerted by leptin. In the South American sea lion some leptin-like-immunoreactive (ir) cells, and endocrine type cells, were found in the pit of gastric mucosal folds and in the epithelium of duodenum as well as numerous neurons were detected in the submucosal and myenteric plexuses of the stomach. In the bottlenose dolphin, many leptin-like-ir cells, and exocrine type cells, were identified in the mucosal layer of the main stomach as well as several neurons and nervous fibers were detected in nervous plexuses of main stomach, pyloric stomach, proximal, and middle intestine. Our data suggest that the distribution of leptin-like peptides is similar in the two species, notwithstanding the different anatomical organization of the gastrointestinal apparatus of South American sea lion and bottlenose dolphin. These findings "suggest" the presence of a basal plan in the regulation of food intake, body weight, energy balance and of the gastrointestinal functions in general also in marine mammals with different and specific feeding habits.

Leptin is a modulator of food intake and energy homeostasis both in mammals and in some species of nonmammals vertebrates. In this study, we reported for the first time, using an immunohistochemical and immunochemical approach, the presence and distribution of immunoreactivity to leptin-like protein in the gastroenteric tract of Dicentrarchus labrax (bass) and Carassius auratus (goldfish), two teleostean species with different feeding and different adaptative morphological organization of the gastroenteric tract. Bass stomach showed intense immunoreactivity to leptin-like protein in all regions, with immunoreactive cells located at the base of the mucosal plicae and at the apical margin of the gastric crypts. Immunoreactive fibers and neuronal cells were observed close to vascular structures in the pyloric region. In bass and goldfish intestine, rare immunoreactive cells were observed along the mucosal epithelium mostly at the base or the apex of intestinal folds in the proximal and medium intestine; numerous immunoreactive nerve fibers in the circular and longitudinal layers of the tunica muscolaris as well as in the myenteric plexus were observed. Western blot analysis recognized a ∼15 kDa signal with a similar expression pattern for goldfish and sea bass. Our results could contribute to confirm the evolutive conservation of leptin-like proteins and their probably precocious functional diversification in fish. Microsc. Res. Tech., 2011. © 2010 Wiley-Liss, Inc.

KEY WORDS ghrelin; Carassius auratus; Dicentrarchus labrax; immunohistochemistry;gastrointestinal tractABSTRACT The occurrence and localization of ghrelin peptide in the gastrointestinal tract ofCarassius auratus and Dicentrarchus labrax, two fish species that exhibit different feeding behav-ior, different habitats, and different anatomical organizations of the gastroenteric tract, wereexamined by immunohistochemical methods and western blotting analysis. All of the gastrointesti-nal segments studied displayed immunohistochemical localizations of ghrelin peptide. Numeroussingle or clustered immunoreactive cells were found along the gastric folds, particularly in the py-loric region of Dicentrarcus labrax, whereas scattered ghrelin immunoreactive cells were observedin the intestinal epithelium of both fish species. Double immunolabeling PGP 9.5/ghrelin demon-strated the localization of ghrelin peptide also in nerve fibers and neuronal cells of the submucosaland myenteric plexuses, often in association with vascular structures. Western blotting analysisconfirmed the presence of ghrelin peptide in the gatrointestinal tract of both species studied, whosemolecular weight was similar to that of the corresponding mammalian prepro-ghrelin. The findingscould support the hypothesis that this peptide is an important appetite regulator in fish and couldconfirm the presence of the ghrelin peptide, starting from its precursor proteins, in the gastrointes-tinal tract of the goldfish and the sea bass. Microsc. Res. Tech. 00:000–000, 2009.

Orexin A (oxA) and orexin B are recently discovered peptides derived from the proteolytic cleavage of the common precursor prepro-orexin. They bind two G protein-coupled receptors, defined orexin 1 (ox1R) and orexin 2 receptor. Both peptides are highly expressed in the lateral hypothalamic area of the brain and are involved in the regulation of many functions of the body, the best investigated of which is food intake. Recent data described the presence of orexins in peripheral organs such as the adrenal glands, stomach, bowel, pancreas, and testis. Here, we report the detection of oxA and ox1R in the exocrine and endocrine cytotypes of the cattle urethroprostatic complex by using immunohistochemistry. The expression of prepro-orexin and ox1R mRNA transcripts in the prostatic tissue was assessed by reverse-transcriptase polymerase chain reaction, while the presence of both the proteins in the tissue was confirmed by Western blotting analysis. Our findings provide the first evidence for the presence of oxA and ox1R in the urethroprostatic complex of the cattle and demonstrate that both proteins are locally synthesized, thus suggesting a role for oxA on both physiological and pathological functioning of the complex.