Ghrelin is a gut-brain peptide hormone, which binds to the growth hormone secretagogue receptor (GHS-R) to regulate a wide variety of biological processes in fish. Despite these prominent physiological roles, no studies have reported the anatomical distribution of preproghrelin transcripts using in situ hybridization in a non-mammalian vertebrate, and its mapping within the different encephalic areas remains unknown. Similarly, no information is available on the possible 24-h variations in the expression of preproghrelin and its receptor in any vertebrate species. The first aim of this study was to investigate the anatomical distribution of ghrelin and GHS-R1a ghrelin receptor subtype in brain and gastrointestinal tract of goldfish (Carassius auratus) using immunohistochemistry and in situ hybridization. Our second aim was to characterize possible daily variations of preproghrelin and ghs-r1 mRNA expression in central and peripheral tissues using real-time reverse transcription-quantitative PCR. Results show ghrelin expression and immunoreactivity in the gastrointestinal tract, with the most abundant signal observed in the mucosal epithelium. These are in agreement with previous findings on mucosal cells as the primary synthesizing site of ghrelin in goldfish. Ghrelin receptor was observed mainly in the hypothalamus with low expression in telencephalon, pineal and cerebellum, and in the same gastrointestinal areas as ghrelin. Daily rhythms in mRNA expression were found for preproghrelin and ghs-r1 in hypothalamus and pituitary with the acrophase occurring at nighttime. Preproghrelin, but not ghs-r1a, displayed a similar daily expression rhythm in the gastrointestinal tract with an amplitude 3-fold higher than the rest of tissues. Together, these results described for the first time in fish the mapping of preproghrelin and ghrelin receptor ghs-r1a in brain and gastrointestinal tract of goldfish, and provide the first evidence for a daily regulation of both genes expression in such locations, suggesting a possible connection between the ghrelinergic and circadian systems in teleosts.

Ghrelin is the only known hormone posttranslationally modified with an acylation. This modification is crucial for most of ghrelin's physiological effects and is catalyzed by the polytopic enzyme ghrelin O-acyltransferase (GOAT). The aim of this study was to characterize GOAT in a teleost model, goldfish (Carassius auratus). First, the full-length cDNA sequence was obtained by RT-PCR and rapid amplification of cDNA ends methods. Two highly homologous cDNAs of 1491 and 1413 bp, respectively, named goat-V1 and goat-V2 were identified. Deduced protein sequences (393 and 367 amino acids, respectively) are predicted to present 11 and 9 transmembrane regions, respectively, and both contain two conserved key residues proposed to be involved in catalysis: asparagine 273 and histidine 304. RT-qPCR revealed that both forms of goat mRNAs show a similar widespread tissue distribution, with the highest expression in the gastrointestinal tract and gonads and less but considerable expression in brain, pituitary, liver and adipose tissue. Immunostaining of intestinal sections showed the presence of GOAT immunoreactive cells in the intestinal mucosa, some of which colocalize with ghrelin. Using an in vitro approach, we observed that acylated ghrelin downregulates GOAT gene and protein levels in cultured intestine in a time-dependent manner. Finally, we found a rhythmic oscillation of goat mRNA expression in the hypothalamus, pituitary and intestinal bulb of goldfish fed at midday, but not at midnight. Together, these findings report novel data characterizing GOAT, and offer new information about the ghrelinergic system in fish.

Glucose homeostasis is an important biological process that involves a variety of regulatory mechanisms. This study aimed to determine whether ghrelin, a multifunctional gut-brain hormone, modulates intestinal glucose transport in goldfish (Carassius auratus). Three intestinal glucose transporters, the facilitative glucose transporter 2 (GLUT2), and the sodium/glucose co-transporters 1 (SGLT1) and 2 (SGLT2), were studied. Immunostaining of intestinal sections found colocalization of ghrelin and GLUT2 and SGLT2 in mucosal cells. Some cells containing GLUT2, SGLT1 and SGLT2 coexpressed the ghrelin/growth hormone secretagogue receptor 1a (GHS-R1a). Intraperitoneal glucose administration led to a significant increase in serum ghrelin levels, as well as an upregulation of intestinal preproghrelin, ghrelin O-acyltransferase and ghs-r1 expression. In vivo and in vitro ghrelin treatment caused a concentration- and time-dependent modulation (mainly stimulatory) of GLUT2, SGLT1 and SGLT2. These effects were abolished by the GHS-R1a antagonist [D-Lys3]-GHRP-6 and the phospholipase C inhibitor U73122, suggesting that ghrelin actions on glucose transporters are mediated by GHS-R1a via the PLC/PKC signaling pathway. Finally, ghrelin stimulated the translocation of GLUT2 into the plasma membrane of goldfish primary intestinal cells. Overall, data reported here indicate an important role for ghrelin in the modulation of glucoregulatory machinery and glucose homeostasis in fish.

Ghrelin, and nesfatin-1 (encoded by nucleobindin2/nucb2) are two metabolic peptides with multiple biological effects in vertebrates. While sex steroids are known to regulate endogenous ghrelin and NUCB2 in mammals, such actions by steroids in fish remain unknown. This study aimed to determine whether estradiol (E2) and testosterone (T) affects the expression of preproghrelin, ghrelin/growth hormone secretagogue receptor (GHS-R), ghrelin O-acyl transferase (GOAT) and NUCB2 in goldfish (Carassius auratus). First, a dose-response assay was performed in which fish were intraperitoneally (ip) implanted with pellets containing 25, 50 or 100 μg/g body weight (BW) of E2 or T. It was found that sex steroids (100 μg/g BW) administered for 2.5 days achieved the highest E2 or T in circulation. In a second experiment, fish were ip implanted with pellets containing 100 μg/g BW of E2, T or without hormone (control). RT-qPCR analyses at 2.5 days post-administration show that gut preproghrelin and GOAT expression was upregulated by both E2 and T treatments, while the same effect was observed for GHS-R only in the pituitary. Both treatments also reduced hypothalamic preproghrelin mRNA expression. NUCB2 expression was increased in the forebrain of T treated group and reduced in the gut and pituitary under both treatments. These results show for the first time a modulation of preproghrelin and nucb2/nesfatin-1 by sex steroids in fish. The interaction between sex steroids and genes implicated in both metabolism and reproduction might help meeting the reproduction dependent energy demands in fish.

Ghrelin is an important gut-derived hormone with an appetite stimulatory role, while most of the intestinal hormones, including cholecystokinin (CCK), peptide YY (PYY) and glucagon-like peptide-1 (GLP-1), are appetite-inhibitors. Whether these important peptides with opposing roles on food intake interact to regulate energy balance in fish is currently unknown. The aim of this study was to characterize the putative crosstalk between ghrelin and CCK, PYY and GLP-1 in goldfish (Carassius auratus). We first determined the localization of CCK, PYY and GLP-1 in relation to ghrelin and its main receptor GHS-R1a (growth hormone secretagogue 1a) in the goldfish intestine by immunohistochemistry. Colocalization of ghrelin/GHS-R1a and CCK/PYY/GLP-1 was found primarily in the luminal border of the intestinal mucosa. In an intestinal explant culture, a significant decrease in prepro-cck, prepro-pyy and proglucagon transcript levels was observed after 60min of incubation with ghrelin, which was abolished by preincubation with the GHS-R1a ghrelin receptor antagonist [D-Lys3]-GHRP-6 (except for proglucagon). The protein expression of PYY and GLP-1 was also downregulated by ghrelin. Finally, intraperitoneal co-administration of CCK, PYY or GLP-1 with ghrelin results in no modification of food intake in goldfish. Overall, results of the present study show for the first time in fish that ghrelin exerts repressive effects on enteric anorexigens. It is likely that these interactions mediate the stimulatory effects of ghrelin on feeding and metabolism in fish.

Ghrelin, a multifunctional gut-brain hormone, is involved in the regulation of gastric functions in mammals. This study aimed to determine whether ghrelin modulates digestive enzymes in goldfish (Carassius auratus). Immunofluorescence microscopy found colocalization of ghrelin, GHS-R1a and the digestive enzymes sucrase-isomaltase, aminopeptidase A, trypsin and lipoprotein lipase in intestinal and hepatopancreatic cells. In vitro ghrelin treatment in intestinal and hepatopancreas explant culture led to a concentration- and time-dependent modulation (mainly stimulatory) of most of the digestive enzymes tested. The ghrelin-induced upregulations of digestive enzyme expression were all abolished by preincubation with the GHS-R1a ghrelin receptor antagonist [D-Lys3]-GHRP-6, and most of them by the phospholipase C inhibitor U73122 or the protein kinase A inhibitor H89. This indicates that ghrelin effects on digestive enzymes are mediated by GHS-R1a, partly by triggering the PLC/PKC and AC/PKA intracellular signaling pathways. These data suggest a role for ghrelin on digestive processes in fish.

Nesfatin-1 (NESF) and NESF-like peptide (NLP), encoded in nucleobindin 2 and 1 (NUCB2 and NUCB1), respectively, are orphan ligands and metabolic factors. We hypothesized that NESF and NLP suppress growth hormone (GH) synthesis, and aimed to determine whether mammalian somatotrophs are a source and site of action of these peptides. Using immortalized rat somatotrophs (GH3 cells), NUCB expression was determined by qPCR, immunofluorescence and Western blot. NESF and NLP binding to GH3 cells was tested using fluorescence imaging. Both time- and concentration-dependent studies were performed to test whether NESF and NLP affect GH. Moreover, the ability of these peptides to modulate the effects of ghrelin, and cell-signaling pathways were studied. GH3 cells express NUCB mRNAs and protein. Labeled NESF and NLP bind to the surface of GH3 cells, and incubation with either NESF or NLP decreased GH mRNA and protein expression, downregulated pit-1 mRNA, and blocked the GH stimulatory effects of ghrelin. Pre-incubation with either of these peptides reduced CREB phosphorylation by an AC-activator, but not when PKA was directly activated by a cAMP analog. Our results indicate that rat somatotrophs are a source of NUCBs, and that NESF and NLP downregulate GH synthesis through the AC/PKA/CREB signaling pathway.

Nesfatin-1 is secreted, meal-responsive anorexigenic peptide encoded in the precursor nucleobindin-2 [NUCB2]. Circulating nesfatin-1 increases post-prandially, but the dietary components that modulate NUCB2/nesfatin-1 remain unknown. We hypothesized that carbohydrate, fat and protein differentially regulate tissue specific expression of nesfatin-1. NUCB2, prohormone convertases and nesfatin-1 were detected in mouse stomach ghrelinoma [MGN3-1] cells. NUCB2 mRNA and protein were also detected in mouse liver, and small and large intestines. MGN3-1 cells were treated with glucose, fatty acids or amino acids. Male C57BL/6 mice were chronically fed high fat, high carbohydrate and high protein diets for 17 weeks. Quantitative PCR and nesfatin-1 assays were used to determine nesfatin-1 at mRNA and protein levels. Glucose stimulated NUCB2 mRNA expression in MGN3-1 cells. L-Tryptophan also increased NUCB2 mRNA expression and ghrelin mRNA expression, and nesfatin-1 secretion. Oleic acid inhibited NUCB2 mRNA expression, while ghrelin mRNA expression and secretion was enhanced. NUCB2 mRNA expression was significantly lower in the liver of mice fed a high protein diet compared to mice fed other diets. Chronic intake of high fat diet caused a significant reduction in NUCB2 mRNA in the stomach, while high protein and high fat diet caused similar suppression of NUCB2 mRNA in the large intestine. No differences in serum nesfatin-1 levels were found in mice at 7 a.m, at the commencement of the light phase. High carbohydrate diet fed mice showed significantly elevated nesfatin-1 levels at 1 p.m. Serum nesfatin-1 was significantly lower in mice fed high fat, protein or carbohydrate compared to the controls at 7 p.m, just prior to the dark phase. Mice that received a bolus of high fat had significantly elevated nesfatin-1/NUCB2 at all time points tested post-gavage, compared to control mice and mice fed other diets. Our results for the first time indicate that nesfatin-1 is modulated by nutrients.

Fibroblast growth factor 21 (FGF21) is a member of the FGF superfamily that acts in an endocrine manner. FGF21 is a key regulator of energy balance and metabolism in mammals, and has emerged as a therapeutic potential for treating obesity and diabetes. Here, we report that mRNAs encoding FGF21 and its receptors are widely distributed within the zebrafish tissues and are importantly modulated by fasting (decreased in brain and liver, and increased in gut). FGF21 stimulates food intake in zebrafish, likely in part by modulating brain npy/agrp and nucb2/nesfatin-1 and gut ghrelin and cck mRNA expression. In accordance with this orexigenic role, the expression of FGF21 and its receptors were observed to increase preprandially and decrease post-feeding in the foregut and/or liver. Finally, we found important evidence in favor of a role for FGF21 in regulating glucose and lipid metabolism in the zebrafish liver in a way that mimics a fasting metabolic state.

Irisin is a myokine encoded in fibronectin type III domain containing 5 (FNDC5). FNDC5 forms an integral part of the muscle post-exercise, and causes an increase in energy expenditure in mammals. Irisin is abundantly expressed in cardiac and skeletal muscles and is secreted upon activation of peroxisome proliferator-activated receptor gamma coactivator-1 (PGC-1 alpha). Irisin regulates feeding behaviour and cardiovascular function in mammals. More recently, irisin has gained importance as a potential biomarker for myocardial infarction due to its abundance in cardiac muscle. The goal of this research was to determine whether irisin influences feeding, and regulates appetite regulatory peptides in zebrafish. Intraperitoneal injection of irisin [0.1, 1, 10 and 100ng/g body weight (BW)] did not affect feeding, but its knockdown using siRNA (10ng/g BW) caused a significant reduction in food intake. Knockdown of irisin reduced ghrelin and orexin-A mRNA expression, and increased cocaine and amphetamine regulated transcript mRNA expression in zebrafish brain and gut. siRNA mediated knockdown of irisin also downregulated brain derived neurotrophic factor mRNA in zebrafish. The role of endogenous irisin on food intake is likely mediated by its actions on other metabolic peptides. Collectively, these results indicate that unaltered endogenous irisin is required to maintain food intake in zebrafish.

Emerging findings point to a role for brain-derived neurotrophic factor (BDNF) on feeding in mammals. However, its role on energy balance is unclear. Moreover, whether BDNF regulates energy homeostasis in non-mammals remain unknown. This research aimed to determine whether BDNF is a metabolic peptide in zebrafish. Our results demonstrate that BDNF mRNAs and protein, as well as mRNAs encoding its receptors trkb2, p75ntra and p75ntrb, are detectable in the zebrafish brain, foregut and liver. Intraperitoneal injection of BDNF increased food intake at 1, 2 and 6 h post-administration, and caused an upregulation of brain npy, agrp and orexin, foregut ghrelin, and hepatic leptin mRNAs, and a reduction in brain nucb2. Fasting for 7 days increased bdnf and p75ntrb mRNAs in the foregut, while decreased bdnf, trkb2, p75ntra and p75ntrb mRNAs in the brain and liver. Additionally, the expression of bdnf and its receptors increased preprandially, and decreased after a meal in the foregut and liver. Finally, we observed BDNF-induced changes in the expression and/or activity of enzymes involved in glucose and lipid metabolism in the liver. Overall, present results indicate that BDNF is a novel regulator of appetite and metabolism in fish, which is modulated by energy intake and food availability.

Nucleobindin-1 has high sequence similarity to nucleobindin-2, which encodes the anorectic and metabolic peptide, nesfatin-1. We previously reported a nesfatin-1-like peptide (NLP), anorectic in fish and insulinotropic in mice islet beta-like cells. The main objective of this research was to determine whether NLP is a metabolic regulator in male Wistar rats. A single intraperitoneal (IP) injection of NLP (100 μg/kg BW) decreased food intake and increased ambulatory movement, without causing any change in total activity or energy expenditure when compared to saline-treated rats. Continuous subcutaneous infusion of NLP (100 μg/kg BW) using osmotic mini-pumps for 7 days caused a reduction in food intake on days 3 and 4. Similarly, water intake was also reduced for two days (days 3 and 4) with the effect being observed during the dark phase. This was accompanied by an increased RER and energy expenditure. However, decreased whole-body fat oxidation, and total activity were observed during the long-term treatment (7 days). Body weight gain was not significantly different between control and NLP infused rats. The expression of mRNAs encoding adiponectin, resistin, ghrelin, cholecystokinin and uncoupling protein 1 (UCP1) were significantly upregulated, while leptin and peptide YY mRNA expression was downregulated in NLP-treated rats. These findings indicate that administration of NLP at 100 μg/kg BW reduces food intake and modulates whole body energy balance. In summary, NLP is a novel metabolic peptide in rats.