Dietary protein:lipid ratio modulates somatic growth and expression of genes involved in somatic growth, lipid metabolism and food intake in Pejerrey fry (Odontesthes bonariensis).

Pejerrey is a freshwater fish from South America with high potential for aquaculture. This study was designed to determine the effects of different dietary protein:lipid ratio on growth rate and the expression of growth, lipid metabolism and feeding-related genes of this species during early developmental stages. Pejerrey fry were fed for 60 days with four experimental diets containing low (400 g Kg-1) or high (500 g Kg-1) protein (LP or HP, respectively) and low (120 g Kg-1) or high (200 g Kg-1) lipid (LL or HL, respectively), in the combinations: LP-LL; LP-HL; HP-LL and HP-HL. Measurements of growth, lipid and fatty acid content of fry, expression of genes from the endocrine axis (gh, ghrs, igfs), fatty acid metabolism (∆6-desaturase), and food intake behavior (nucb2/nesfatin-1) were collected. Fry fed with diets LP-LL and HP-LL showed the highest growth rate and growth hormone (gh) mRNA expression levels. The gene expression of ∆6-desaturase was high in head of fry fed with diet LP-HL. The mRNA expression of nucb2/nesfatin-1 and gh followed the same patterns in head, and the inverse pattern in body. In conclusion, diets with LL ensure a higher growth of pejerrey fry compared to those that contain HL, without altering the final lipid amount nor the fatty acid profile on fry. In LL groups, the expression of genes from the GH-IGF axis is associated with the observed promotion of somatic growth. The expression of nucb2/nesfatin-1 indicates an effect of this peptide not related to food intake regulation, e.g., a negative regulatory role on GH expression, that would warrant future research.

Nesfatin-1 stimulates the hypothalamus-pituitary-interrenal axis hormones in goldfish.

Stress in vertebrates is mediated by the hypothalamus-pituitary-adrenal (in mammals)/interrenal (in fish) (HPA/I) axis, which produces the corticotropin-releasing factor (CRF), adrenocorticotropic hormone (ACTH), and corticosteroids, respectively. Nesfatin-1, a novel anorexigenic peptide encoded in the precursor nucleobindin-2 (NUCB2), is increasingly acknowledged as a peptide that influences the stress axis in mammals. The primary aim of this study was to characterize the putative effects of nesfatin-1 on the fish HPI axis, using goldfish (Carassius auratus) as an animal model. Our results demonstrated that nucb2/nesfatin-1 transcript abundance was detected in the HPI tissues of goldfish, with most abundant expression in the pituitary. NUCB2/nesfatin-1-like immunoreactivity was found in the goldfish hypothalamus, pituitary, and interrenal cells of the head kidney. GPCR12, a putative receptor for nesfatin-1, was also detected in the pituitary and interrenal cells. NUCB2/nesfatin-1-like immunoreactivity was observed in ACTH-expressing pituitary corticotrophs. Acute netting and restraint stress upregulated nucb2/nesfatin-1 mRNA levels in the forebrain, hypothalamus, and pituitary, as well as crf and crf-r1 expression in the forebrain and hypothalamus. Intraperitoneal and intracerebroventricular administration of nesfatin-1 increased cortisol release and hypothalamic crf mRNA levels, respectively. Finally, we found that nesfatin-1 significantly stimulated ACTH secretion from dispersed pituitary cells in vitro. Collectively, our data provide the first evidence showing that nesfatin-1 is a stress responsive peptide, which modulates the stress axis hormones in fish.

Leptin signalling in teleost fish with emphasis in food intake regulation.

Leptin, the product of the obese (ob or Lep) gene, was first cloned in teleost fish in 2005, more than a decade after its identification in mammals. This was because bony fish and mammalian leptins share a very low amino acid sequence identity, which suggests different functionality of the leptin system in fish compared to that of mammals. Indeed, major differences are evident between the mammalian and fish leptin system. Thus, for instance, mammalian leptin is synthesized and released by the adipose tissue in response to the amount of fat depots, while several tissues (mainly the liver) are the main sources of leptin in fish, whose determining factors of production are still unclear. In mammals, the main physiological role for leptin is its involvement in the maintenance of energy balance by decreasing food intake and increasing energy expenditure, although a wide variety of actions have been attributed to this hormone (e.g., regulation of lipid and carbohydrate metabolism, reproduction and immune functions). In fish, available literature also points towards a multifunctional nature for leptin, although knowledge on its functions is limited. In this review, we offer an overview of teleostean leptin structure and mechanism of action, and discuss the available knowledge on the role of this hormone in food intake regulation in teleost fish, aiming to provide a comparative overview between the functioning of the teleostean and mammalian leptin systems.

The gut-brain axis in vertebrates: implications for food intake regulation.

The gut and brain are constantly communicating and influencing each other through neural, endocrine and immune signals in an interaction referred to as the gut-brain axis. Within this communication system, the gastrointestinal tract, including the gut microbiota, sends information on energy status to the brain, which, after integrating these and other inputs, transmits feedback to the gastrointestinal tract. This allows the regulation of food intake and other physiological processes occurring in the gastrointestinal tract, including motility, secretion, digestion and absorption. Although extensive literature is available on the mechanisms governing the communication between the gut and the brain in mammals, studies on this axis in other vertebrates are scarce and often limited to a single species, which may not be representative for obtaining conclusions for an entire group. This Review aims to compile the available information on the gut-brain axis in birds, reptiles, amphibians and fish, with a special focus on its involvement in food intake regulation and, to a lesser extent, in digestive processes. Additionally, we will identify gaps of knowledge that need to be filled in order to better understand the functioning and physiological significance of such an axis in non-mammalian vertebrates.

Feeding and food availability modulate brain-derived neurotrophic factor, an orexigen with metabolic roles 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.

FGF21 Mimics a Fasting-Induced Metabolic State and Increases Appetite in Zebrafish.

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.

Phoenixin-20 suppresses food intake, modulates glucoregulatory enzymes, and enhances glycolysis in zebrafish.

Phoenixin is a 20-amino acid peptide (PNX-20) cleaved from the small integral membrane protein 20 (SMIM20), with multiple biological roles in mammals. However, its role in nonmammalian vertebrates is poorly understood. This research aimed to determine whether PNX-20 influences feeding and metabolism in zebrafish. The mRNAs encoding SMIM20 and its putative receptor, super conserved receptor expressed in brain 3 (SREB3), are present in both central and peripheral tissues of zebrafish. Immunohistochemical analysis confirmed the presence of PNX-like immunoreactivity in the gut and in zebrafish liver (ZFL) cell line. We also found that short-term fasting (7 days) significantly decreased smim20 mRNA expression in the brain, gut, liver, gonads, and muscle, which suggests a role for PNX-20 in food intake regulation. Indeed, single intraperitoneal injection of 1,000 ng/g body wt PNX-20 reduced feeding in both male and female zebrafish, likely in part by enhancing hypothalamic cart and reducing hypothalamic/gut preproghrelin mRNAs. Furthermore, the present results demonstrated that PNX-20 modulates the expression of genes involved in glucose transport and metabolism in ZFL cells. In general terms, such PNX-induced modulation of gene expression was characterized by the upregulation of glycolytic genes and the downregulation of gluconeogenic genes. A kinetic study of the ATP production rate from both glycolytic and mitochondrial pathways demonstrated that PNX-20-treated ZFL cells exhibited significantly higher ATP production rate associated with glycolysis than control cells. This confirms a positive role for PNX-20 on glycolysis. Together, these results indicate that PNX-20 is an anorexigen with important metabolic roles in zebrafish.

Goldfish adipocytes are pancreatic beta cell-like, glucose-responsive insulin-producing cells.

Glucose homeostasis plays a key role in maintaining stable physiological conditions, and its dysfunction causes severe chronic health issues including diabetes. In this study, we have characterized goldfish adipocytes as cells with properties similar to that of pancreatic ß-cells: they express considerable high levels of preproinsulin mRNAs, possess the necessary machinery for processing preproinsulin (prohormone convertases 1 and 2, carboxypeptidase E and trypsin) and responding to extracellular glucose (glucokinase and the glucose transporters 1, 2, and 4), produce insulin in a glucose-responsive manner and express key transcription factors typically involved in pancreas development (Pdx1, Neurogenin3, Nkx2.2, Pax6, and FOXO1A). These findings reinforce the feature of fish adipocytes as alternate sources of active insulin, holding the promise that they could eventually be developed as transplantable sources of this vital hormone.

Growth differentiation factor 15 (GDF-15) is a novel orexigen in fish.

Growth differentiation factor 15 (GDF-15), an anti-inflammatory and anti-tumorigenic cytokine, has been emerging as a regulator of appetite and energy homeostasis in mammals. In fish, the physiological role of this peptide remains to be elucidated. This research aimed to determine the possible role of GDF-15 on food intake in goldfish (Carassius auratus). To achieve our objectives, we first obtained a 595 bp gdf-15 cDNA sequence from goldfish tissues, and examined the tissue expression profile of mRNAs encoding both GDF-15 and its receptor (GFRAL). Both mRNAs were detected in several goldfish tissues, including the hypothalamus, foregut and liver (main tissues regulating appetite and energy balance). Food deprivation for 3 and 7 days significantly upregulated gdf-15 mRNAs in the foregut, but downregulated them in the liver. Our in vivo study using diets with varying amounts of carbohydrates, proteins and fats, and our in vitro study exposing goldfish tissues to different macronutrients revealed that gdf-15 mRNAs are importantly modulated by macronutrients. In general terms, we found an increase in gdf-15 mRNA levels in the goldfish foregut and liver in response to all macronutrients tested. Finally, our in vivo study testing the effects of GDF-15 on appetite levels demonstrated an important dose-dependent orexigenic role for this peptide in goldfish. Results from this study described GDF-15 as a novel regulator of appetite in fish, importantly modulated by food availability and diet composition.

Brain transcriptome profile after CRISPR-induced ghrelin mutations in zebrafish.

Ghrelin (GRL) is a gut-brain hormone with a role in a wide variety of physiological functions in mammals and fish, which points out the ghrelinergic system as a key element for the appropriate biological functioning of the organism. However, many aspects of the multifunctional nature of GRL remain to be better explored, especially in fish. In this study, we used the CRISPR/Cas9 genome editing technique to generate F0 zebrafish in which the expression of grl is compromised. Then, we employed high-throughput mRNA sequencing (RNA-seq) to explore changes in the brain transcriptome landscape associated with the silencing of grl. The CRISPR/Cas9 technique successfully edited the genome of F0 zebrafish resulting in individuals with considerably lower levels of GRL mRNAs and protein and ghrelin O-acyl transferase (goat) mRNAs in the brain, intestine, and liver compared to wild-type (WT) zebrafish. Analysis of brain transcriptome revealed a total of 1360 differentially expressed genes (DEGs) between the grl knockdown (KD) and WT zebrafish, with 664 up- and 696 downregulated DEGs in the KD group. Functional enrichment analysis revealed that DEGs are highly enriched for terms related to morphogenesis, metabolism (especially of lipids), entrainment of circadian clocks, oxygen transport, apoptosis, and response to stimulus. The present study offers valuable information on the central genes and pathways implicated in functions of GRL, and points out the possible involvement of this peptide in some novel functions in fish, such as apoptosis and oxygen transport.

Nesfatin-1 regulates glucoregulatory genes in rainbow trout (Oncorhynchus mykiss).

The aim of this work was to determine if the anorexigen nesfatin-1 modulates the expression of genes involved in glucoregulation in rainbow trout. First, the nesfatin-1 sequence from trout was confirmed. Second, the effects of 0.1, 1 and 10 nM nesfatin-1 on insulin, glucagon, igf-I, igf-II, glut1, glut2, glut4 and sglt1 expression were tested in cultured liver, gut, muscle and adipose tissue. In liver, the expression of insulin and glucagon isoforms X1 increased after 2 h of incubation with 0.1 nM nesfatin-1, while insulin and glucagon X2 expression increased after 4 h with 1 nM treatment. All nesfatin-1 doses tested decreased glut2 expression after 4 h. In adipose tissue, all nesfatin-1 concentrations reduced insulin X1 expression at 30 min, and 1 nM nesfatin-1 increased insulin X2 expression at 4 h. In gut, 0.1, 1 and 10 nM nesfatin-1 decreased glut2 and sglt1 mRNA levels after 240 min of incubation. In muscle, 0.1 nM nesfatin-1 increased the expression of igf-I after 240 min. The expression of igf-II in muscle increased after 30 min of incubation with 1 and 10 nM nesfatin-1 and after 120 min of incubation with 0.1 and 1 nM nesfatin-1. Expression of glut1 and sglt1 in muscle increased after 240 min of incubation with 0.1 nM nesfatin-1 and after 120 min with 0.1 and 10 nM nesfatin-1, respectively. These results suggest that nesfatin-1 could decrease the gut intake of dietary glucose, and increase its uptake in glucoregulatory tissues such as liver and muscle of rainbow trout.

Nutrient Regulation of Endocrine Factors Influencing Feeding and Growth in Fish.

Endocrine factors regulate food intake and growth, two interlinked physiological processes critical for the proper development of organisms. Somatic growth is mainly regulated by growth hormone (GH) and insulin-like growth factors I and II (IGF-I and IGF-II) that act on target tissues, including muscle, and bones. Peptidyl hormones produced from the brain and peripheral tissues regulate feeding to meet metabolic demands. The GH-IGF system and hormones regulating appetite are regulated by both internal (indicating the metabolic status of the organism) and external (environmental) signals. Among the external signals, the most notable are diet availability and diet composition. Macronutrients and micronutrients act on several hormone-producing tissues to regulate the synthesis and secretion of appetite-regulating hormones and hormones of the GH-IGF system, eventually modulating growth and food intake. A comprehensive understanding of how nutrients regulate hormones is essential to design diet formulations that better modulate endogenous factors for the benefit of aquaculture to increase yield. This review will discuss the current knowledge on nutritional regulation of hormones modulating growth and food intake in fish.

Ghrelin and NUCB2/Nesfatin-1 Co-Localization With Digestive Enzymes in the Intestine of Pejerrey (Odontesthes bonariensis).

Ghrelin (orexigenic) and nesfatin-1 (anorexigenic) are two peptides with opposing actions on food intake regulation and are mainly expressed in the hypothalamus and gut of mammals and fish. Both are involved in the regulation of a wide range of physiological processes in vertebrates, including metabolism, growth, and reproduction. However, the anatomical relationship between these peptides and the nutrient assimilation processes are not well understood. Thus, the aim of this work was to determine the localization of ghrelin, nesfatin-1, and several enzymes involved in the digestive process (lipoprotein lipase, aminopeptidase A, trypsin, and sucrase-isomaltase) in the intestine of pejerrey (Odontesthes bonariensis), a species with commercial importance in South America. We observed co-localization of ghrelin and nesfatin-1 in enteroendocrine cells, absorptive cells, and in cells of the lamina propia. Approximately half of the cells displaying ghrelin-like immunoreactivity co-localized the NUCB2/nesfatin-1-like signal. In addition, both peptides showed co-localization with lipoprotein lipase, aminopeptidase A, trypsin, or sucrase-isomaltase. All digestive enzymes except for aminopeptidase A and trypsin, showed high co-localization (68-88%) with both ghrelin-like and NUCB2/nesfatin-1-like signals in absorptive, enteroendocrine, and lamina propria cells. Together, our results provide immunohistochemical evidence supporting a role for both ghrelin and NUCB2/nesfatin-1 in the regulation of nutrient assimilation in fish. Anat Rec, 302:973-982, 2019. © 2018 Wiley Periodicals, Inc.

Galanin decreases spontaneous resting contractions and potentiates acetyl choline-induced contractions of goldfish gut.

Galanin (GAL) is a 29 amino acid peptide, first identified from the porcine intestine and widely distributed within the brain and peripheral tissues. Among GAL biological functions, its role as a potent appetite-stimulating peptide is probably the most studied. With galanin's established role in the modulation of food intake in fish, this study aims to evaluate the effects of GAL on the intestinal motility of the goldfish, Carassius auratus, using an organ bath system. Our results found that application of GAL to the organ bath causes a significant concentration-dependent decrease in the amplitude of spontaneous contractions of goldfish gut. Preincubations of intestinal strips with acetylcholine (ACh) and GAL showed that GAL increases the force of ACh-induced contractions of the goldfish gut. These results provide the first evidence for a role of GAL in gut motility in goldfish. This also suggests a crosstalk between the effects of GAL and ACh in such functions, thus pointing to a putative joint role between the two molecules. These findings offer novel information that strengthens the role of the galaninergic system in fish feeding.

Why goldfish? Merits and challenges in employing goldfish as a model organism in comparative endocrinology research.

Goldfish has been used as an unconventional model organism to study a number of biological processes. For example, goldfish is a well-characterized and widely used model in comparative endocrinology, especially in neuroendocrinology. Several decades of research has established and validated an array of tools to study hormones in goldfish. The detailed brain atlas of goldfish, together with the stereotaxic apparatus, are invaluable tools for the neuroanatomic localization and central administration of endocrine factors. In vitro techniques, such as organ and primary cell cultures, have been developed using goldfish. In vivo approaches using goldfish were used to measure endogenous hormonal milieu, feeding, behaviour and stress. While there are many benefits in using goldfish as a model organism in research, there are also challenges associated with it. One example is its tetraploid genome that results in the existence of multiple isoforms of endocrine factors. The presence of extra endogenous forms of peptides and its receptors adds further complexity to the already redundant multifactorial endocrine milieu. This review will attempt to discuss the importance of goldfish as a model organism in comparative endocrinology. It will highlight some of the merits and challenges in employing goldfish as an animal model for hormone research in the post-genomic era.

Ghrelin suppresses cholecystokinin (CCK), peptide YY (PYY) and glucagon-like peptide-1 (GLP-1) in the intestine, and attenuates the anorectic effects of CCK, PYY and GLP-1 in goldfish (Carassius auratus).

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.

Influence of water salinity on genes implicated in somatic growth, lipid metabolism and food intake in Pejerrey (Odontesthes bonariensis).

Pejerrey, Odontesthes bonariensis, is an euryhaline fish of commercial importance in Argentina. This work aimed to determine if water salinity affects the expression of genes involved in somatic growth (gh; ghr-I; ghr-II; igf-I), lipid metabolism (Δ6-desaturase) and food intake (nucb2/nesfatin-1). First, we identified the full-length cDNA sequences of Δ6-desaturase (involved in lipid metabolism) and nesfatin-1 (an anorexigen). Then, pejerrey juveniles were reared during 8weeks in three different water salinity conditions: 2.5g/L (S2.5), 15g/L (S15) and 30g/L (S30) of NaCl. Brain, pituitary, liver and muscle samples were collected in order to analyze mRNA expression. The expression of gh and ghr-II mRNAs increased in the pituitary of fish reared at S2.5 and S30 compared with the S15 group. The expression of ghr-I was higher in the liver of S30 group compared to S2.5 and S15. Igf-I mRNA expression in liver increased with the increment of water salinity, while it decreased in the muscle of S15 and S30 groups. Δ6-desaturase expression increased in S2.5 group compared to S15 in both liver and muscle. S30 caused a decrease in the Δ6-desaturase expression in liver compared to S15. The S30 treatment produced an increase in nucb2/nesfatin-1 mRNA expression in the brain and liver compared to S2.5 and S15. The changes in gene expression observed could help pejerrey perform better during salinity challenges. The S30 condition would likely promote pejerrey somatic growth in the long term.

Direct actions of macronutrient components on goldfish hepatopancreas in vitro to modulate the expression of ghr-I, ghr-II, igf-I and igf-II mRNAs.

In mammals and fish, somatic growth and metabolism are coordinated by the GH-IGF axis, composed of growth hormone (GH), growth hormone receptors I and II (GHR-I and GHR-II), and the insulin-like growth factors I and II (IGF-I and IGF-II). In order to determine if dietary macronutrients regulate the hepatopancreatic expression of ghr-I, ghr-II, igf-I and igf-II independently of circulating GH, organ culture experiments were conducted. Briefly, goldfish hepatopancreas sections were incubated with different doses of glucose; L-tryptophan; oleic acid; linolenic acid (LNA); eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). After two and four hours of treatment, the expression of ghr-I, ghr-II, igf-I and igf-II mRNAs was quantified. We found that glucose and L-tryptophan globally upregulate the mRNA expression of ghr-I; ghr-II; igf-I and igf-II. Duration of exposure, and unsaturation level of fatty acids differentially modulate ghr-I, ghr-II and igf-II mRNA expression. Additionally, we found that fatty acids increase the expression of igf-I depending on incubation time and fatty acid class. In conclusion, here we present evidence for GH-independent, direct effects exerted by dietary macronutrients on GHR and IGF in goldfish hepatopancreas.

Ghrelin Facilitates GLUT2-, SGLT1- and SGLT2-mediated Intestinal Glucose Transport in Goldfish (Carassius auratus).

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.

Characterization of Ghrelin O-Acyltransferase (GOAT) in goldfish (Carassius auratus).

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.