RESUMEN
As in mammals, fatty acid (FA) metabolism plays diverse and vital roles in regulating food intake in fish. Multiple lines of evidence suggest that the effect of FA metabolism on food intake is linked to changes in the level of neuropeptide Y (NPY) in the hypothalamus of the rainbow trout. In mammals, the evidence suggests that FA metabolism regulates feeding via hypothalamic NPY. NPY is therefore considered an important factor that mediates the modulation of food intake by FA metabolism in vertebrates. The stimulatory effect of NPY on food intake is well known. However, to the best of our knowledge, the effect of NPY on FA metabolism in the hypothalamus has not been examined. In this study, we cloned the cDNA of four key enzymes involved in FA metabolism and assessed the effect of energy status and NPY on their mRNA expression in the hypothalamus of grouper. The full-length cDNAs of UCP2 and CPT1a and the partial coding sequence (CDS) of ACC1 and FAS were isolated from the grouper hypothalamus. These genes are expressed in the hypothalamus and during the organogenetic stage of embryogenesis. A feeding rhythm study showed that the hypothalamic expression level of NPY and CPT1a was highly correlated with feeding rhythm. Long-term fasting was found to significantly induce the hypothalamic mRNA expression of NPY, CPT1a and UCP2. An in vitro study demonstrated that NPY strongly stimulated CPT1a and UCP2 mRNA expression in a time- and dose-dependent manner. Collectively, these results suggest that these four genes related to FA metabolism may play a role in regulating food intake in grouper and, that NPY modulates FA metabolism in the grouper hypothalamus. This study showed, for the first time in vertebrates, the effect of NPY on the gene expression of FA metabolism-related enzymes.
Asunto(s)
Lubina/genética , Ingestión de Alimentos/efectos de los fármacos , Proteínas de Peces/genética , Hipotálamo/efectos de los fármacos , Metabolismo de los Lípidos/efectos de los fármacos , Neuropéptido Y/farmacología , ARN Mensajero/genética , Acetil-CoA Carboxilasa/genética , Acetil-CoA Carboxilasa/metabolismo , Secuencia de Aminoácidos , Animales , Lubina/metabolismo , Carnitina O-Palmitoiltransferasa/genética , Carnitina O-Palmitoiltransferasa/metabolismo , Clonación Molecular , Cartilla de ADN , Ingestión de Alimentos/genética , Ayuno , Ácido Graso Sintasas/genética , Ácido Graso Sintasas/metabolismo , Ácidos Grasos/metabolismo , Proteínas de Peces/metabolismo , Regulación de la Expresión Génica , Hipotálamo/metabolismo , Canales Iónicos/genética , Canales Iónicos/metabolismo , Metabolismo de los Lípidos/genética , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Datos de Secuencia Molecular , Neuropéptido Y/genética , Neuropéptido Y/metabolismo , Especificidad de Órganos , ARN Mensajero/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Proteína Desacopladora 2RESUMEN
Orexin-A and -B, collectively called orexins, are hypothalamic neuropeptides involved in the regulation of food intake, sleep and energy balance. In this study, the full-length cDNA of prepro-orexin was isolated from the hypothalamus of orange-spotted grouper (Epinephelus coioides) using RT-PCR and RACE. The grouper prepro-orexin cDNA is 711 bp in length and encodes a 149-amino acid precursor protein that contains a 46-amino acid signal peptide, a 43-amino acid mature orexin-A peptide, a 27-amino acid mature orexin-B peptide and a 33-amino acid C terminus of unknown function. The tissue distribution and ontogeny of prepro-orexin were examined by quantitative real-time PCR. We found that the prepro-orexin mRNA is widely expressed in brain and peripheral tissues, with abundant expression in the hypothalamus. During the embryonic development, prepro-orexin mRNA was first detected in neurula stage embryos, and its expression gradually increased during the remainder of embryogenesis. Our analysis of grouper hypothalamic prepro-orexin expression showed that prepro-orexin mRNA levels were greater in the light phase than in the dark phase and increased significantly at meal-time. Intraperitoneal injection of orexin-A caused a dose-related increase in hypothalamus NPY mRNA expression level after 4h. Orexin-A also increased NPY mRNA expression level from static hypothalamic fragments incubation. Our results imply that orexin may be involved in feeding in the orange-spotted grouper and orexin-A is a stimulator of NPY mRNA expression in vivo and in vitro.
Asunto(s)
Lubina/metabolismo , Conducta Alimentaria/efectos de los fármacos , Proteínas de Peces/metabolismo , Regulación del Desarrollo de la Expresión Génica , Hipotálamo/fisiología , Péptidos y Proteínas de Señalización Intracelular , Neuropéptido Y/metabolismo , Neuropéptidos , Precursores de Proteínas , Secuencia de Aminoácidos , Animales , Lubina/embriología , Lubina/genética , Ritmo Circadiano/efectos de los fármacos , Ritmo Circadiano/fisiología , Clonación Molecular , ADN Complementario/análisis , Conducta Alimentaria/fisiología , Femenino , Proteínas de Peces/genética , Expresión Génica , Hipotálamo/embriología , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Datos de Secuencia Molecular , Neuropéptido Y/genética , Neuropéptidos/genética , Neuropéptidos/metabolismo , Orexinas , Filogenia , Plásmidos , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Técnicas de Cultivo de TejidosRESUMEN
GH secretagogue receptor (GHSR) is the receptor of ghrelin, a circulating GH-releasing and appetite-inducing hormone. In this paper, two Ghsr cDNAs, gpGhsr1a and gpGhsr1b, were identified and characterized in a teleost, the orange-spotted grouper (Epinephelus coioides). The gpGHSR1a is 1512 bp in length with an open reading frame (ORF) that encodes a protein of 383 amino acids with seven transmembrane (TM) domains, while the 1703 bp gpGHSR1b contains an ORF encoding for 303 amino acids with five TM domains. Comparison between cDNA and gene sequences showed that the two transcripts are two alternative splicing forms of a single gpGhsr gene. Tissue distribution and ontogeny of two gpGhsr mRNAs were examined by RT-PCR. The gpGHSR1a is mainly expressed in brain and pituitary gland, when compared with a more widespread expression of gpGHSR1b. During embryonic and larval development, the gpGhsr1b mRNA appears before the gpGhsr1a mRNA. Furthermore, quantitative real-time PCR performed on brain showed that both transcripts have the highest expression level in the pituitary gland. The expression level of gpGHSR1a was generally higher than that of gpGHSR1b. GHSR expressing cells were also detected widely in grouper brain by in situ hybridization, with a broader distribution than previous reports in mammals. Finally, an in vitro study showed that expression of both gpGHSR transcripts in pituitary and hypothalamus is downregulated by GH and ghrelin but not by des-acyl ghrelin, and this suggests that feedback regulation of GHSR also exists in teleostean fishes.