Juan Ignacio Bertucci

In Situ Localization and Rhythmic Expression of Ghrelin and ghs-r1 Ghrelin Receptor in the Brain and Gastrointestinal Tract of Goldfish (Carassius auratus)

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.

Tissue-specific expression of ghrelinergic and NUCB2/nesfatin-1 systems in goldfish (Carassius auratus) is modulated by macronutrient composition of diets.

The macronutrient composition of diets is a very important factor in the regulation of body weight and metabolism. Several lines of research in mammals have shown that macronutrients differentially regulate metabolic hormones, including ghrelin and nesfatin-1 that have opposing effects on energy balance. This study aimed to determine whether macronutrients modulate the expression of ghrelin and the nucleobindin-2 (NUCB2) encoded nesfatin-1 in goldfish (Carassius auratus). Fish were fed once daily on control, high-carbohydrate, high-protein, high-fat and very high-fat diets for 7 (short-term) or 28 (long-term) days. The expression of preproghrelin, ghrelin O-acyl transferase (goat), growth hormone secretagogue receptor 1 (ghs-r1) and nucb2/nesfatin-1 mRNAs was quantified in the hypothalamus, pituitary, gut and liver. Short-term feeding with fat-enriched diets significantly increased nucb2 mRNA levels in hypothalamus and liver, preproghrelin, goat and ghs-r1 expression in pituitary, and ghs-r1 expression in gut. Fish fed on a high-protein diet exhibited a significant reduction in preproghrelin and ghs-r1 mRNAs in the liver. After long-term feeding, fish fed on high-carbohydrate and very high-fat diets had significantly increased preproghrelin, goat and ghs-r1 expression in pituitary. Feeding on a high-carbohydrate diet also upregulated goat and ghs-r1 transcripts in gut, while feeding on a high-fat diet elicited the same effect only for ghs-r1 in liver. Nucb2 expression increased in pituitary, while it decreased in gut after long-term feeding of a high-protein diet. Collectively, these results show for the first time in fish that macronutrients differentially regulate the expression of ghrelinergic and NUCB2/nesfatin-1 systems in central and peripheral tissues of goldfish.

Nesfatin-1-Like Peptide Encoded in Nucleobindin-1 in Goldfish is a Novel Anorexigen Modulated by Sex Steroids, Macronutrients and Daily Rhythm.

Nesfatin-1 is an 82 amino acid anorexigen encoded in a secreted precursor nucleobindin-2 (NUCB2). NUCB2 was named so due to its high sequence similarity with nucleobindin-1 (NUCB1). It was recently reported that NUCB1 encodes an insulinotropic nesfatin-1-like peptide (NLP) in mice. Here, we aimed to characterize NLP in fish. RT- qPCR showed NUCB1 expression in both central and peripheral tissues. Western blot analysis and/or fluorescence immunohistochemistry determined NUCB1/NLP in the brain, pituitary, testis, ovary and gut of goldfish. NUCB1 mRNA expression in goldfish pituitary and gut displayed a daily rhythmic pattern of expression. Pituitary NUCB1 mRNA expression was downregulated by estradiol, while testosterone upregulated its expression in female goldfish brain. High carbohydrate and fat suppressed NUCB1 mRNA expression in the brain and gut. Intraperitoneal injection of synthetic rat NLP and goldfish NLP at 10 and 100 ng/g body weight doses caused potent inhibition of food intake in goldfish. NLP injection also downregulated the expression of mRNAs encoding orexigens, preproghrelin and orexin-A, and upregulated anorexigen cocaine and amphetamine regulated transcript mRNA in goldfish brain. Collectively, these results provide the first set of results supporting the anorectic action of NLP, and the regulation of tissue specific expression of goldfish NUCB1.

Estradiol and testosterone modulate the tissue-specific expression of ghrelin, ghs-r, goat and nucb2 in goldfish.

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.

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.

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 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.

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)

Abstract 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 60 min 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. HighlightsGhrelin and GHS‐R1a colocalize CCK, PYY and GLP‐1 in goldfish intestinal cells.Ghrelin suppresses intestinal prepro‐cck, prepro‐pyy and proglucagon expression in vitro.PYY and GLP‐1 intestinal levels are downregulated by in vitro ghrelin treatment.GHS‐R1a is critical for ghrelin‐induced downregulation of prepro‐cck and prepro‐pyy.Ghrelin, coadministered with CCK, PYY or GLP‐1, is not able to induce goldfish appetite.

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.

Nesfatin-1 Regulates Feeding, Glucosensing and Lipid Metabolism in Rainbow Trout

Nesfatin-1 is an 82 amino acid peptide that has been involved in a wide variety of physiological functions in both mammals and fish. This study aimed to elucidate the role of nesfatin-1 on rainbow trout food intake, and its putative effects on glucose and fatty acid sensing systems. Intracerebroventricular administration of 25 ng/g nesfatin-1 resulted in a significant inhibition of appetite, likely mediated by the activation of central POMC and CART. Nesfatin-1 stimulated the glucosensing machinery (changes in sglt1, g6pase, gsase, and gnat3 mRNA expression) in the hindbrain and hypothalamus. Central fatty acid sensing mechanisms were unaltered by nesfatin-1, but this peptide altered the expression of mRNAs encoding factors regulating lipid metabolism (fat/cd36, acly, mcd, fas, lpl, pparα, and pparγ), suggesting that nesfatin-1 promotes lipid accumulation in neurons. In the liver, intracerebroventricular nesfatin-1 treatment resulted in decreased capacity for glucose use and lipogenesis, and increased the potential of fatty acid oxidation. Altogether, the present results demonstrate that nesfatin-1 is involved in the homeostatic regulation of food intake and metabolism in fish.