RESUMEN
In goldfish, circulating LH and growth hormone (GH) levels surge at the time of ovulation. In the present study, changes in gene expression of salmon gonadotropin-releasing hormone (sGnRH), chicken GnRH-II (cGnRH-II), somatostatin (SS) and pituitary adenylate cyclase activating polypeptide (PACAP) were analyzed during temperature- and spawning substrate-induced ovulation in goldfish. The results demonstrated that increases in PACAP gene expression during ovulation are best correlated with the GH secretion profile. These results suggest that PACAP, instead of GnRH, is involved in the control of GH secretion during ovulation. Increases of two of the SS transcripts during ovulation are interpreted as the activation of a negative feedback mechanism triggered by high GH levels. The results showed a differential regulation of sGnRH and cGnRH-II gene expression during ovulation, suggesting that sGnRH controls LH secretion, whereas cGnRH-II correlates best with spawning behavior. This conclusion is further supported by the finding that nonovulated fish induced to perform spawning behavior by prostaglandin F2alpha treatment increased cGnRH-II expression in both forebrain and midbrain, but decreased sGnRH expression in the forebrain.
Asunto(s)
Encéfalo/metabolismo , Proteínas de Peces/metabolismo , Carpa Dorada/metabolismo , Hormona Liberadora de Gonadotropina/metabolismo , Hormona del Crecimiento/metabolismo , Hormona Luteinizante/metabolismo , Ovulación , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/metabolismo , Somatostatina/metabolismo , Animales , Encéfalo/efectos de los fármacos , Dinoprost/farmacología , Femenino , Proteínas de Peces/genética , Carpa Dorada/genética , Hormona Liberadora de Gonadotropina/genética , Mesencéfalo/metabolismo , Oviposición/efectos de los fármacos , Ovulación/genética , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/genética , Prosencéfalo/metabolismo , ARN Mensajero/metabolismo , Conducta Sexual Animal/efectos de los fármacos , Somatostatina/genética , Temperatura , Factores de TiempoRESUMEN
In teleost fish, melanin-concentrating hormone (MCH) is a cyclic heptadecapeptide released from the pituitary during white background adaptation. In the periphery MCH concentrates melanin granules in melanophores thus lightening the body color of fish. Evidence from mammalian studies has demonstrated the involvement of MCH in the control of energy balance and the reproductive axis. Information about the hormonal regulation of MCH neurons in non-mammalian systems is scarce and nothing is known about its role in the regulation of the reproductive axis. We here report the molecular characterization of two MCH precursors in the goldfish. Both precursors are peripherally expressed and the expression in the central nervous system is restricted to the mediobasal hypothalamus. Hypothalamic MCH-mRNA production is upregulated during white background adaptation. Both testosterone and estradiol stimulate MCH mRNA expression in the hypothalamus in a sex-dependent manner, with females showing the greatest responsiveness. In addition, in vitro experiments demonstrated that graded doses of salmon MCH stimulate LH, but not GH, secretion from dispersed pituitary cells. Results suggest that hypothalamic MCH may participate in the steroid positive feedback loop on pituitary LH secretion.
Asunto(s)
Estradiol/farmacología , Hormonas Hipotalámicas/metabolismo , Hipotálamo/metabolismo , Hormona Luteinizante/metabolismo , Melaninas/metabolismo , Hormonas Hipofisarias/metabolismo , Testosterona/farmacología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Carpa Dorada , Hormona del Crecimiento/metabolismo , Hormonas Hipotalámicas/genética , Hipotálamo/citología , Masculino , Melaninas/genética , Datos de Secuencia Molecular , Hormonas Hipofisarias/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Caracteres SexualesRESUMEN
This study analyzes daily changes in the expression of somatostatin precursors PSS-I and PSS-III (structurally related to cortistatin) in the goldfish brain. The results indicate that PSS-I expression correlates with the light cycle only in optic tectum-thalamus (OT-Tha). PSS-III expression correlates with the light cycle in telencephalon-preoptic area (Tel-POA) and OT-Tha. In Tel-POA, PSS-III reaches a minimum level at the beginning of the active phase and a maximum level late in this phase. These results suggest that PSS-I in OT-Tha and PSS-III in Tel-POA and OT-Tha could be involved in the control of the activity cycles in goldfish.
Asunto(s)
Carpa Dorada/fisiología , Neuropéptidos/fisiología , Precursores de Proteínas/fisiología , Somatostatina/fisiología , Animales , Ritmo Circadiano/genética , Ritmo Circadiano/fisiología , Carpa Dorada/genética , Neuropéptidos/biosíntesis , Neuropéptidos/genética , Precursores de Proteínas/biosíntesis , Precursores de Proteínas/genética , Somatostatina/biosíntesis , Somatostatina/genética , Proteínas de Pez Cebra/biosíntesis , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/fisiologíaRESUMEN
In goldfish, growth hormone (GH) transiently rises 30 min after meals, returning to baseline at 1 h postmeal. Somatostatin (SRIF) is the major inhibitor of GH release. Three cDNAs encoding pre-pro-SRIF (PSS) have been previously cloned from goldfish brain: PSS-I, which encodes SRIF-14; PSS-II, which is potentially processed into gSRIF-28 that has [Glu(1),Tyr(7)(,)Gly(10)]SRIF-14 at the COOH terminus; and PSS-III, which encodes [Pro(2)]SRIF-14 at its COOH terminus. In goldfish, bombesin (BBS), mimicking the endogenous gastrin-releasing peptide (GRP), acutely suppresses food intake and also stimulates GH release. Ghrelin was recently characterized in goldfish as a GH secretagogue and an orexigen. In this paper, we studied the changes in SRIF mRNA levels during feeding and analyzed the influences of BBS and ghrelin peptides on forebrain PSS expression. The results showed a 60% reduction in PSS-II mRNA after meals, but no changes in the expression of PSS-I and PSS-III were found. Intraperitoneal injections of 100 ng/g body wt of BBS increased GH secretion and decreased PSS-I and PSS-II gene expression. Intraperitoneal injection of goldfish ghrelin (100 ng/g body wt) transiently increased the serum GH levels and increased PSS-I, while decreasing PSS-II mRNA levels. Ghrelin (50 ng/g body wt) blocked the effects of BBS (100 ng/g body wt) on PSS-I but not on PSS-II expression. Coadministration of BBS and ghrelin decreased only the PSS-II gene expression. We conclude that the interactions between BBS/GRP and ghrelin can account for the postprandial variations in serum GH levels and the forebrain expression of PSS-II. Furthermore, we demonstrate that intraperitoneal administration of BBS reduces the ghrelin expression levels in the gut. Thus the inhibition of production of ghrelin in the gut may contribute to the satiety effects of BBS/GRP peptides.
Asunto(s)
Péptido Liberador de Gastrina/metabolismo , Carpa Dorada/fisiología , Hormona del Crecimiento/metabolismo , Hormonas Peptídicas/fisiología , Periodo Posprandial , Somatostatina/fisiología , Animales , Bombesina/administración & dosificación , Bombesina/farmacología , Interacciones Farmacológicas , Expresión Génica/efectos de los fármacos , Ghrelina , Carpa Dorada/metabolismo , Inyecciones Intraperitoneales , Mucosa Intestinal/metabolismo , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/farmacología , Hormonas Peptídicas/administración & dosificación , Hormonas Peptídicas/farmacología , Prosencéfalo/metabolismo , Precursores de Proteínas/genética , ARN Mensajero/metabolismo , Somatostatina/genética , Factores de TiempoRESUMEN
One of the most successful chromatic adaptations in vertebrates is the dorsal-ventral pigment pattern in which the dorsal skin is darkly colored, whereas the ventrum is light. In fish, the latter pattern is achieved because a melanization inhibition factor inhibits melanoblast differentiation and supports iridophore proliferation in the ventrum. In rodents, the patterned pigmentation results from regional production of the agouti-signaling protein (ASP). This peptide controls the switch between production of eumelanin and pheomelanin by antagonizing alphaMSH effects on melanocortin receptor (MCR) 1 in the melanocytes. In addition, ASP inhibits the differentiation and proliferation of melanoblast. Thus, the mammalian ASP may be homologous to the poikilotherm melanization inhibition factor. By screening of a genomic library, we deduced the amino acid sequence of goldfish ASP. The ASP gene is a four-exon gene spanning 3097 bp that encodes a 125-amino acid precursor. Northern blot analysis identified two different ASP mRNAs in ventral skin of red- and black-pigmented and albino fish, but no expression levels were observed in the dorsal skin of the same fish. The dorsal-ventral expression polarity was also detected in both black dorsally pigmented fish and albino fish. Pharmacological studies demonstrate that goldfish ASP acts as a melanocortin antagonist at Fugu MC1R and goldfish MC4R. In addition, goldfish ASP inhibited Nle4, D-Phe7-MSH-stimulated pigment dispersion in medaka melanophores. Our studies support agouti signaling protein as the melanization inhibition factor, a key factor in the development of the dorsal-ventral pigment pattern in fish.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Péptidos y Proteínas de Señalización Intercelular/genética , Proteína de Señalización Agouti , Alelos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Unión Competitiva , Bioensayo , Northern Blotting , Southern Blotting , Diferenciación Celular , Línea Celular , Proliferación Celular , Clonación Molecular , Medios de Cultivo Condicionados/farmacología , AMP Cíclico/metabolismo , ADN/química , Etidio/farmacología , Femenino , Biblioteca de Genes , Vectores Genéticos , Carpa Dorada , Humanos , Ligandos , Masculino , Melaninas/metabolismo , Datos de Secuencia Molecular , Isoformas de Proteínas , ARN/química , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Transducción de Señal , Takifugu , Distribución Tisular , Transfección , alfa-MSH/metabolismoRESUMEN
In the present study the brain distribution of three somatostatin (SRIF)-encoding genes, PSS-I, PSS-II, and PSS-III, was analyzed by in situ hybridization (ISH) in the goldfish. The PSS-I mRNA showed the widest distribution throughout the brain, whereas PSS-II transcripts were restricted to some hypothalamic nuclei. On the other hand, PSS-III presents an intermediate distribution pattern. All SRIF encoding genes are expressed in hypophysiotropic nuclei supporting the idea that, in addition to SRIF-14, [Pro(2)] SRIF-14, and gSRIF-28 have pituitary-controlling functions. Moreover, each of the genes is expressed in nuclei directly associated with feeding behavior, suggesting a role for SRIF peptides in the central control of food intake and energy balance. Alternatively, they might have a role in processing sensory information related with feeding behavior, since PSS genes are expressed in the main gustatory, olfactory, and visual centers, which project to the hypothalamic feeding center in teleost fish.
Asunto(s)
Mapeo Encefálico , Encéfalo/metabolismo , Precursores de Proteínas/metabolismo , Somatostatina/metabolismo , Animales , Encéfalo/anatomía & histología , Química Encefálica , Carpa Dorada , Hibridación in Situ/métodos , Precursores de Proteínas/genética , Procesamiento de Señales Asistido por Computador , Somatostatina/genéticaRESUMEN
The gram-negative bacteria-derived endotoxin lipopolysaccharide (LPS) is known to play an important role in immune and neurological manifestations during bacterial infections. In mammals, peripheral or brain administration of LPS induces anorexia and is thought to exert its effects through activation of pro-inflammatory cytokines. In this study, we investigated the effects of peripheral (intraperitoneal, IP) and central (intracerebroventricular, ICV) injections of LPS on food intake of goldfish. Fish treated IP with 10, 25, 50, 100 or 250 ng/g LPS or ICV with 1, 10 and 100 ng/g LPS showed a significant dose-dependent decrease in food intake, compared to the saline-treated fish. We also examined the brain mRNA expression of several hypothalamic appetite-related neuropeptides in response to the administration of LPS. IP injections of LPS (100 ng/g) induced a decrease in NPY expression and an increase in CCK, CRF and CART expression. These results indicate that LPS is a potent anorexigenic factor in goldfish and that this endotoxin induces a reduction in appetite, at least in part, by influencing gene expression of appetite-related neuropeptides.
Asunto(s)
Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Carpa Dorada/fisiología , Lipopolisacáridos/administración & dosificación , Neuropéptidos/efectos de los fármacos , Animales , Colecistoquinina/biosíntesis , Colecistoquinina/efectos de los fármacos , Hormona Liberadora de Corticotropina/biosíntesis , Hormona Liberadora de Corticotropina/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Conducta Alimentaria , Expresión Génica , Inyecciones Intraperitoneales , Inyecciones Intraventriculares , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/efectos de los fármacos , Neuropéptido Y/biosíntesis , Neuropéptido Y/efectos de los fármacos , Neuropéptidos/biosíntesis , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisisRESUMEN
Posttranscriptional processing of proopiomelanocortin (POMC) yields melanocortin peptides, which are involved in the regulation of energy balance in mammals. The sequence preservation of the main brain melanocortin, alpha-melanocyte-stimulating hormone (alpha-MSH), suggests a conserved function throughout vertebrate evolution. We studied the involvement of the central melanocortin system in the control of food intake in the goldfish. In situ hybridization studies done following molecular cloning of POMC mRNA demonstrated positive POMC mRNA cell bodies exclusively expressed within the mediobasal hypothalamus, in the anterior, posterior and inferior part of the lateral tuberal nucleus and the medial region of the lateral recess nucleus. POMC expression is localized in brain areas appropriate for involvement in food intake and neuroendocrine regulation. Progressive fasting did not affect POMC mRNA expression levels. Intracerebroventricular administration of [Nle(4), D-Phe(7)]-alpha-MSH (NDP-alpha-MSH), a universal melanocortin agonist, within nanomolar range, dose-dependently inhibited food intake 2 h after treatment. The results show for the first time a functional melanocortin system in fishes that participates in central regulation of food intake. The conserved central expression pattern of POMC mRNA and role of MSH peptides in physiological regulation of food intake suggests that melanocortin functions were gained early in vertebrate evolution.
Asunto(s)
Anticarcinógenos/farmacología , Encéfalo/metabolismo , Ingestión de Alimentos/efectos de los fármacos , Carpa Dorada/fisiología , Proopiomelanocortina/genética , alfa-MSH/análogos & derivados , alfa-MSH/farmacología , Secuencia de Aminoácidos , Animales , Anticarcinógenos/metabolismo , Secuencia de Bases , Clonación Molecular , Sondas de ADN , ADN Complementario/genética , Ayuno , Femenino , Hibridación in Situ , Inyecciones Intraventriculares , Masculino , Datos de Secuencia Molecular , Proopiomelanocortina/metabolismo , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , alfa-MSH/metabolismoRESUMEN
Agouti-related protein (AGRP) is a naturally occurring antagonist of melanocortin. In mammals, central AGRP expression is restricted to the arcuate nucleus in which it plays a key role in the control of energy balance by antagonizing melanocortin effects at melanocortin 4 receptors. In goldfish, melanocortin 4 receptor is profusely expressed within the main brain areas for the control of energy balance, and central administration of agonist or antagonist analogs inhibits or stimulates food intake, respectively. Here we demonstrate that the goldfish genome has a homologous gene to mammalian AGRP. Detailed brain mapping by in situ hybridization shows that AGRP is exclusively expressed in the ventrobasal hypothalamic lateral tuberal nucleus, the teleostean homolog of the arcuate nucleus. Fasting up-regulates its mRNA levels in the lateral tuberal nucleus. In the periphery, AGRP is expressed in several tissues including ovary, muscle, and ventral skin, suggesting that AGRP might regulate peripheral actions of melanocortin peptides. The results provide the first evidence for an endogenous melanocortin antagonist in nontetrapod species and suggest that hypothalamic overexpression during fasting might regulate the inhibitory effects of melanocortin peptides on food intake in goldfish.
Asunto(s)
Encéfalo/metabolismo , Ayuno/fisiología , Carpa Dorada/metabolismo , Proteínas/genética , Proteínas/fisiología , alfa-MSH/antagonistas & inhibidores , Proteína Relacionada con Agouti , Secuencia de Aminoácidos/genética , Animales , Secuencia de Bases/genética , Clonación Molecular , Metabolismo Energético/fisiología , Femenino , Expresión Génica , Péptidos y Proteínas de Señalización Intercelular , Masculino , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Distribución TisularRESUMEN
We report cloning, pharmacological characterization, tissue distribution, detailed brain mapping, and role in control of food intake of melanocortin 4 receptor in goldfish (gMC4R). The gMC4R protein has 68% identity with the human ortholog and is conserved in important functional domains. Pharmacological profiling showed similar affinities and potency order to hMC4R for MSH peptides, whereas MTII and HS024 were identified as high-affinity agonist and antagonist analogs, respectively. The gMC4R-mRNA was found in brain and some peripheral tissues including the ovary, gill, and spleen. Detailed MC4R-mRNA mapping showed expression in main neuroendocrine and food intake-controlling areas. High expression levels were found in the telencephalon, preoptic area, ventral thalamus, tuberal hypothalamus, and hypothalamic inferior lobe. By RT-PCR, low levels were also detected in the cerebellum, medulla, and spinal cord. Intracerebroventricular MTII administration inhibited food intake in 24-h fasted animals in a dose-dependent manner, whereas HS024 stimulated food intake in fed animals, suggesting that melanocortins exert a tonic inhibitory effect on food intake, which is mediated through central MC4R signaling. The conserved central expression pattern and physiological role in regulation of food intake for the MC4R suggests that neuronal pathways of the melanocortin system may be important for regulation of energy homeostasis in most vertebrates.
Asunto(s)
Química Encefálica/fisiología , Ingestión de Alimentos/fisiología , Carpa Dorada/fisiología , Receptores de Corticotropina/genética , alfa-MSH/análogos & derivados , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Unión Competitiva , Mapeo Encefálico , Clonación Molecular , Femenino , Expresión Génica/fisiología , Inyecciones Intraventriculares , Masculino , Datos de Secuencia Molecular , Péptidos Cíclicos/metabolismo , Péptidos Cíclicos/farmacología , ARN Mensajero/análisis , Receptor de Melanocortina Tipo 4 , Receptores de Corticotropina/metabolismo , Especificidad de la Especie , alfa-MSH/metabolismo , alfa-MSH/farmacología , beta-MSH/metabolismo , beta-MSH/farmacología , gamma-MSH/metabolismo , gamma-MSH/farmacologíaRESUMEN
To assess the role of leptin on food intake regulation in goldfish, we examined the effects of central (intracerebroventricular, ICV) and peripheral (intraperitoneal, IP) injections of recombinant murine leptin on feeding behavior. Centrally (100 ng/g) and peripherally (300 ng/g) injected leptin both caused a significant decrease in food intake, compared to the saline-treated controls. To test the hypothesis that leptin influenced orexigenic neuropeptide systems in goldfish, fish were co-injected with neuropeptide Y (NPY) or orexin A and leptin. Both NPY (5 ng/g) and orexin A (10 ng/g) significantly increased food intake. Fish co-injected ICV with NPY (5 ng/g) or orexin A (10 ng/g) and leptin (1 or 10 ng/g) had a food intake lower than that of fish treated with NPY or orexin A alone. NPY mRNA expression in goldfish brain was reduced 2 and 6 h following central injection of leptin. To test the hypothesis that the cholecystokinin (CCK) mediates the effects of leptin in goldfish, fish were simultaneously injected ICV with an ineffective dose of leptin (10 ng/g) and either ICV or IP with an ineffective doses of CCK (1 ng/g ICV or 25 ng/g IP). These fish had a food intake lower than vehicle-treated fish, suggesting that leptin potentiates the satiety actions of CCK. CCK hypothalamic mRNA expression was increased 2 h following central treatment with leptin. The CCK receptor antagonist proglumide blocked both central and peripheral CCK satiety effects. Blockade of CCK brain receptors by proglumide resulted in an inhibition of the leptin-induced decrease in food intake and an attenuation of the inhibiting action of leptin on both NPY- and orexin A-induced feeding. These data suggests that CCK has a role in mediating the effects of leptin on food intake. Fasting potentiated the actions of leptin and attenuated the effects of CCK. Whereas fasting had no effects on the brain mRNA expression of CCK, it increased the brain mRNA expression of NPY and decreased the expression of CART. These changes in neuropeptide expression were partially reversed when fish were treated ICV with leptin. These results provide strong evidence that, in goldfish, leptin influences food intake, in part by modulating the orexigenic effects of NPY and orexin and that its actions are mediated, at least in part, by CCK.
Asunto(s)
Proteínas Portadoras/metabolismo , Colecistoquinina/metabolismo , Ayuno/fisiología , Conducta Alimentaria/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular , Leptina/farmacología , Neuropéptido Y/metabolismo , Neuropéptidos/metabolismo , Animales , Conducta Animal , Colecistoquinina/genética , Sondas de ADN/metabolismo , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Ingestión de Alimentos/efectos de los fármacos , Conducta Alimentaria/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Carpa Dorada , Inyecciones Intraperitoneales/métodos , Inyecciones Intraventriculares/métodos , Proteínas del Tejido Nervioso/genética , Neuropéptido Y/genética , Receptores de Orexina , Orexinas , Proglumida/farmacología , ARN Mensajero/biosíntesis , ARN Mensajero/efectos de los fármacos , Receptores de Colecistoquinina/antagonistas & inhibidores , Receptores Acoplados a Proteínas G , Receptores de Neuropéptido , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Factores de TiempoRESUMEN
The melanocortin 5 receptor (MC5R) is activated by melanocyte-stimulating hormones (MSHs) and has a widespread tissue distribution, while its detailed central expression pattern and brain functions are fairly unknown. We report cloning, pharmacological characterization, tissue distribution and detailed brain mapping of melanocortin 5 receptor in goldfish (gMC5R). The goldfish orthologue protein is 69% identical to human MC5R and is conserved in important functional domains. The gMC5R showed similar potency to alpha-, beta- and gamma-MSH peptides in radioligand binding as the mammalian orthologues, while MTII and HS024 were both agonists at this receptor. The gMC5R-mRNA was found in the peripheral tissues including kidney, spleen, skin and retina, with low expression levels in the intestine, fat, muscle, gill, pituitary and ovary. In situ hybridization studies demonstrated that gMC5R transcripts are widely distributed in the goldfish brain. The gMC5R expression was found in ventral telencephalon, pre-optic area, dorsal and ventral thalamus, infundibular hypothalamus, posterior tuberculum, tectum and tegmentum mesencephali, reticular formation, vagal and facial lobes and spinal cord. The cloning and characterization of this receptor provides an important tool to elucidate its participation in neuroendocrine and behavioural control.