Global climate change increases the impact of pollutant mixtures in the model species Paracentrotus lividus.

The goal of the present work is to study whether ocean- acidification (OA) and -warming (OW) could increase the toxicity of pollutants on P. lividus. We studied how model pollutants such as chlorpyrifos (CPF) and microplastics (MP), alone or in combination, impact the fertilisation process, and the development of larvae under conditions of OA (dissolved inorganic carbon increase of 126 × 10-6 mol per kg of sea water) and OW (temperature increase of 4 °C) predicted by FAO (Food and Agriculture Organization) for the next 50 years. Fertilisation was determined by microscopic examination after 1 h. Growth, morphology, and alteration level were measured after 48 h of incubation. Results showed that CPF has a marked effect on the growth of larvae, but less on the fertilisation rate. When larvae are exposed to both MP and CPF, the effect on fertilisation and growth is higher than when CPF is added alone. Larvae exposed to CPF tend to adopt a rounded shape which is detrimental to their buoyancy and the combination with other stressors aggravate this situation. The variables most influenced by CPF or its mixtures are those related to body length, body width, and higher levels of body abnormalities, which is consistent with the degenerative effects caused by CPF on sea urchin larvae. The PCA analysis showed that temperature has more influence when embryos or larvae are exposed to a combination of stressors, demonstrating that global climate change drastically increase the impact of CPF on aquatic ecosystems. Overall, in this work we demonstrated that global climate change conditions increase the sensitivity of embryos to MP and CPF. Our findings support the idea that global change conditions could have a severe impact on marine life, increasing the negative effect of toxic agents commonly present in the sea and their mixtures.

The effect of environmental stressors on growth in fish and its endocrine control.

Fish body growth is a trait of major importance for individual survival and reproduction. It has implications in population, ecology, and evolution. Somatic growth is controlled by the GH/IGF endocrine axis and is influenced by nutrition, feeding, and reproductive-regulating hormones as well as abiotic factors such as temperature, oxygen levels, and salinity. Global climate change and anthropogenic pollutants will modify environmental conditions affecting directly or indirectly fish growth performance. In the present review, we offer an overview of somatic growth and its interplay with the feeding regulatory axis and summarize the effects of global warming and the main anthropogenic pollutants on these endocrine axes.

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.

Nutrient regulation of somatic growth in teleost fish. The interaction between somatic growth, feeding and metabolism.

This review covers the current knowledge on the regulation of the somatic growth axis and its interaction with metabolism and feeding regulation. The main endocrine and neuroendocrine factors regulating both the growth axis and feeding behavior will be briefly summarized. Recently discovered neuropeptides and peptide hormones will be mentioned in relation to feeding control as well as growth hormone regulation. In addition, the influence of nutrient and nutrient sensing mechanisms on growth axis will be highlighted. We expect that in this process gaps of knowledge will be exposed, stimulating future research in those areas.

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.

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.

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.

Glucose, amino acids and fatty acids directly regulate ghrelin and NUCB2/nesfatin-1 in the intestine and hepatopancreas of goldfish (Carassius auratus) in vitro.

Ghrelin and nesfatin-1 are two peptidyl hormones primarily involved in food intake regulation. We previously reported that the amount of dietary carbohydrates, protein and lipids modulates the expression of these peptides in goldfish in vivo. In the present work, we aimed to characterize the effects of single nutrients on ghrelin and nesfatin-1 in the intestine and hepatopancreas. First, immunolocalization of ghrelin and NUCB2/nesfatin-1 in goldfish hepatopancreas cells was studied by immunohistochemistry. Second, the effects of 2 and 4hour-long exposures of cultured intestine and hepatopancreas sections to glucose, l-tryptophan, oleic acid, linolenic acid (LNA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on ghrelin and nesfatin-1 gene and protein expression were studied. Co-localization of ghrelin and NUCB2/nesfatin-1 in the cytoplasm of goldfish hepatocytes was found. Exposure to glucose led to an upregulation of preproghrelin and a downregulation of nucb2/nesfatin-1 in the intestine. l-Tryptophan mainly decreased the expression of both peptides in the intestine and hepatopancreas. Fatty acids, in general, downregulated NUCB2/nesfatin-1 in the intestine, but only the longer and highly unsaturated fatty acids inhibited preproghrelin. EPA exposure led to a decrease in preproghrelin, and an increase in nucb2/nesfatin-1 expression in hepatopancreas after 2h. These results show that macronutrients exert a dose- and time-dependent, direct regulation of ghrelin and nesfatin-1 in the intestine and hepatopancreas, and suggest a role for these hormones in the digestive process and nutrient metabolism.

Ghrelin modulates gene and protein expression of digestive enzymes in the intestine and hepatopancreas of goldfish (Carassius auratus) via the GHS-R1a: Possible roles of PLC/PKC and AC/PKA intracellular signaling pathways.

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.