Effects of feeding status on nucb1 and nucb2A mRNA expression in the hypothalamus of Schizothorax davidi.

NUCB1 and NUCB2, two novel nucleobindins, have attracted extensive attention for their role in the appetite regulation in mammals. However, little is known about the appetite regulation of NUCB1 and NUCB2 in fish species. Therefore, we investigated the role of these peptides in the regulation of feeding in Schizothorax davidi (S. davidi). In this study, full-length cDNA sequences of nucb1 and nucb2A of S. davidi were obtained for the first time. Additionally, the tissue distribution and the effects of different energy status on nucb1 and nucb2A mRNAs abundance were assessed, showing that nucb1 and nucb2A are widely distributed in 18 detected tissues, with the highest expression in the cerebellum. The abundances of nucb1 and nucb2A increased in the hypothalamus at 1 h and 3 h post-feeding. Furthermore, fasting and re-feeding experiments showed that the expressions of nucb1 and nucb2A in hypothalamus significantly decreased after fasting for 7 days, and returned to the control level after re-feeding for 3 or 5 days. In conclusion, the present study suggests that both NUCB1 and NUCB2A are involved in the short-term and long-term appetite regulation, as an anorexigenic factor, in S. davidi. These results can provide a basis for further investigation into the appetite regulatory role of NUCB family in teleost.

Cloning, expression profiling, and effects of fasting status on neuropeptide Y in Schizothorax davidi.

To better comprehend the mechanism that neuropeptide Y (npy) regulates feeding in Schizothorax davidi, we cloned and identified the full-length cDNA sequence of the npy gene in this species using RACE technology. Subsequently, we explored the npy mRNA distribution in 18 tissues and investigated the expression of npy mRNA at postprandial and fasting stages. We found that the npy full-length cDNA sequence is 803 bp. Moreover, npy mRNAs extensively expressed in all detected tissues, with the highest expression in hypothalamus. In postprandial study, the expression of npy mRNA in the hypothalamus was significantly decreased after eating (p < 0.01). In addition, the expression of the npy gene was significantly increased on the fifth day after fasting (p < 0.05). However, after refeeding, the expression of the npy gene was decreased significantly on days 9, 11, and 14 (p < 0.01). Our research suggest that npy may have an orexigenic role in S. davidi. PRACTICAL APPLICATIONS: S. davidi, a coldwater fish native to China, has high economic value, and it has gained great popularity. To date, there is still no large-scale breeding of S. davidi in China. How to strengthen the production performance of S. davidi is a hot research area. Neuropeptide Y (NPY), a 36-amino-acid single-chain polypeptide, is one of the main appetite regulation factors. However, to date, no studies have reported on the biological function of npy in the feeding of S. davidi. In our study, we revealed that the trend of hypothalamic npy expression during the postprandial and fasting stages. The results suggested that npy might be an appetite-promoting factor in this species. Overall, we provide the theoretical basis for how to strengthen the production performance of S. davidi through appetite regulation.

Evidence that ghrelin may be associated with the food intake of gibel carp (Carassius auratus gibelio).

Ghrelin, a non-amidated peptide hormone, is a potent anorectic neuropeptide implicated in feeding regulation in mammals and non-mammalian vertebrates. However, the involvement of ghrelin in the feeding behavior of teleosts has not been well understood. To better understand the role of ghrelin in the regulation of appetite in fish, in this study, we cloned the cDNAs encoding ghrelin and investigated their mRNA distributions in gibel carp tissues. We also assessed the effects of different nutritional status on ghrelin mRNA abundance. Ghrelin mRNAs were ubiquitously expressed in ten tissues (intestine, liver, brain, mesonephron, head kidney, spleen, skin, heart, muscle, gill and pituitary gland), and relatively high expression levels were detected in the gut. Postprandial studies analysis revealed a significant postprandial decrease in ghrelin mRNA expression in the gut (1 and 3 h after the regular feeding time). In addition, ghrelin mRNA expression in the gut significantly increased at day 7 after fasting and declined sharply after refeeding, which suggested that ghrelin might be involved in the regulation of appetite in gibel carp. Overall, our result provides basis for further investigation into the regulation of feeding in gibel carp.

Molecular characterization of melanin-concentrating hormone (MCH) in Schizothorax prenanti: cloning, tissue distribution and role in food intake regulation.

Melanin-concentrating hormone (MCH) is a crucial neuropeptide involved in various biological functions in both mammals and fish. In this study, the full-length MCH cDNA was obtained from Schizothorax prenanti by rapid amplification of cDNA ends polymerase chain reaction. The full-length MCH cDNA contained 589 nucleotides including an open reading frame of 375 nucleotides encoding 256 amino acids. MCH mRNA was highly expressed in the brain by real-time quantitative PCR analysis. Within the brain, expression of MCH mRNA was preponderantly detected in the hypothalamus. In addition, the MCH mRNA expression in the S. prenanti hypothalamus of fed group was significantly decreased compared with the fasted group at 1 and 3 h post-feeding, respectively. Furthermore, the MCH gene expression presented significant increase in the hypothalamus of fasted group compared with the fed group during long-term fasting. After re-feeding, there was a dramatic decrease in MCH mRNA expression in the hypothalamus of S. prenanti. The results indicate that the expression of MCH is affected by feeding status. Taken together, our results suggest that MCH may be involved in food intake regulation in S. prenanti.

Molecular and physiological evidences for the role in appetite regulation of apelin and its receptor APJ in Ya-fish (Schizothorax prenanti).

Apelin is a recently discovered peptide produced by several tissues with diverse physiological actions mediated by its receptor APJ. In order to better understand the role of apelin in the regulation of appetite in fish, we cloned the cDNAs encoding apelin and APJ, and investigated their mRNA distributions in Ya-fish (Schizothorax prenanti) tissues. We also assessed the effects of different nutritional status on apelin and APJ mRNAs abundance. Apelin and APJ mRNAs were ubiquitously expressed in all tissues tested, relatively high expression levels were detected in the heart, spleen, hypothalamus and kidney. Short-term fasting significant increased APJ mRNA expression, but no significant difference between fasted fish and fed control on 5- and 7-day. Meanwhile, apelin mRNA expression consistently increased during the 7-day food deprivation. In order to further characterize apelin in fish, we performed intraperitoneal (i.p.) injection of apelin-13 and examined food intake of the injected fish. Apelin injected at a dose of 100 ng/g body weight induced a significant increase in food intake compared to saline injected fish. Our results suggest that apelin acts as an orexigenic factor in Ya-fish. Their widespread distributions also suggest that apelin and APJ might play multiple physiological regulating roles in fish.

Leptin and cholecystokinin in Schizothorax prenanti: molecular cloning, tissue expression, and mRNA expression responses to periprandial changes and fasting.

In the present study, full-length cDNA sequences of leptin and cholecystokinin (CCK) were cloned from Schizothorax prenanti (S. prenanti), and applied real-time quantitative PCR to characterize the tissue distribution, and appetite regulatory effects of leptin and CCK in S. prenanti. The S. prenanti leptin and CCK full-length cDNA sequences were 1121 bp and 776 bp in length, encoding the peptide of 171 and 123 amino acid residues, respectively. Tissue distribution analysis showed that leptin mRNA was mainly expressed in the liver of S. prenanti. CCK was widely expressed, with the highest levels of expression in the hypothalamus, myelencephalon, telencephalon and foregut of S. prenanti. The CCK mRNA expression was highly elevated after feeding, whereas the leptin mRNA expression was not affected by single meal. These results suggested that CCK is a postprandial satiety signal in S. prenanti, but leptin might not be. In present study, leptin and CCK gene expression were both decreased after fasting and increased after refeeding, which suggested leptin and CCK might be involved in regulation of appetite in S. prenanti. This study provides an essential groundwork to further elucidate the appetite regulatory systems of leptin and CCK in S. prenanti as well as in other teleosts.

Molecular characterization and tissue expression of peptide YY in Schizothorax prenanti: effects of periprandial changes and fasting on expression in the hypothalamus.

Peptide YY (PYY) is a potent anorectic neuropeptide implicated in feeding regulation in mammals. However, the involvement of PYY in the feeding behavior of teleosts has not been well understood. In this study, we employed molecular, real-time quantitative PCR and physiological studies to characterize the structure, distribution, and appetite regulatory effects of PYY in Schizothorax prenanti (S. prenanti). A very high conservation in PYY sequences was found in teleosts. PYY is widely expressed, with the highest levels of expression in telencephalon, medulla oblongata, pituitary and hypothalamus of S. prenanti. The PYY mRNA expression in the hypothalamus was highly elevated after a meal, suggesting a satiety signal role for PYY in S. prenanti. In addition, PYY gene expression in the hypothalamus was decreased after fasting and increased sharply after refeeding, which suggested that PYY might be involved in the central regulation of appetite in S. prenanti. Overall, our result provides basis for further investigation into the regulation of feeding in S. prenanti.

Schizothorax prenanti corticotropin-releasing hormone (CRH): molecular cloning, tissue expression, and the function of feeding regulation.

Corticotropin-releasing hormone (CRH) is a potent mediator of endocrine, autonomic, behavioral, and immune responses to stress. For a better understanding of the structure and function of the CRH gene and to study its effect on feeding regulation in cyprinid fish, the cDNA of the CRH gene from the brain of Schizothorax prenanti was cloned and sequenced. The full-length CRH cDNA consisted of 1,046 bp with an open reading frame of 489 bp encoding a protein of 162 amino acids. Real-time quantitative PCR analyses revealed that CRH was widely expressed in central and peripheral tissues. In particular, high expression level of CRH was detected in brain. Furthermore, CRH mRNA expression was examined in different brain regions, especially high in hypothalamus. In addition, there was no significant change in CRH mRNA expression in fed group compared with the fasted group in the S. prenanti hypothalamus during short-term fasting. However, CRH gene expression presented significant decrease in the hypothalamus in fasted group compared with the fed group (P < 0.05) on day 7; thereafter, re-feeding could lead to a significant increase in CRH mRNA expression in fasted group on day 9. The results suggest that the CRH may play a critical role in feeding regulation in S. prenanti.

Molecular characterization, tissue distribution and feeding related changes of NUCB2A/nesfatin-1 in Ya-fish (Schizothorax prenanti).

The protein nucleobindin-2 (NUCB2) was identified over a decade ago and recently raised great interest as its derived peptide nesfatin-1 was shown to reduce food intake and body weight in rodents. However, the involvement of NUCB2 in feeding behavior has not well been studied in fish. In the present study, we characterized the structure, distribution, and meal responsive of NUCB2A/nesfatin-1 in Ya-fish (Schizothorax prenanti) for the first time. The full length cDNA of Ya-fish was 2140base pair (bp), which encoded a polypeptide of 487 amino acid residues including a 23 amino acid signal peptide. A high conservation in NUCB2 sequences was found in vertebrates, however the proposed propeptide cleavage site (Arg-Arg) conserved among other species is not present in Ya-fish NUCB2A sequence. Tissue distribution analysis revealed that Ya-fish NUCB2A mRNA was ubiquitously expressed in all test tissues, and abundant expression was detected in several regions including the hypothalamus, hepatopancreas, ovary and intestines. NUCB2A mRNA expression respond to feeding status change may vary and be tissue specific. NUCB2A mRNA levels significantly increased (P<0.05) in the hypothalamus and intestines after feeding and substantially decreased (P<0.01) during a week food deprivation in the hypothalamus. Meanwhile, NUCB2A mRNA in the hepatopancreas was significantly elevated (P<0.001) during food deprivation, and a similar increase was also found after short-time fasting. This points toward a potential hepatopancreas specific local role for NUCB2A in the regulation of metabolism during food deprivation. Collectively, these results provide the molecular and functional evidence to support potential anorectic and metabolic roles for NUCB2A in Ya-fish.

Schizothorax davidi ghrelin: cDNA cloning, tissue distribution and indication for its stimulatory character in food intake.

Ghrelin is a gut/brain hormone with a unique acyl modification and various biological functions in fish and mammals. The objectives of this project were to identify ghrelin gene organization, study tissue specific ghrelin mRNA expression and investigate the short- (0, 0.5, 1.5, 3, 6, 9, 12h post-fasting) and long- (1, 3, 5, 7 days) term fasting as well as refeeding after a 7 day fasting induced changes in the expression of ghrelin mRNA in Schizothorax davidi. Our reverse transcription polymerase chain reaction analysis confirmed the predicted ghrelin sequence available in the GenBank and identified ghrelin mRNA expression in several tissues including the gut, liver, brain, heart, spleen, head kidney, gill and muscle. Quantitative PCR studies indicated that the expression level of ghrelin mRNA presented ascendant trend in short-term fasting group compared to the fed group, but it did not reach the significant level on statistics, while there is a significant increase in ghrelin mRNA expression in the gut of Schizothorax davidi fasted for 3, 5 and 7 days when compared to the expression in ad libitum fed fish. Refeeding after a 7 day fasting caused a significant and dramatic decrease in ghrelin mRNA expression in the gut of Schizothorax davidi. An increase in the expression of ghrelin mRNA during fasting, and its decrease following refeeding suggests an orexigenic role for ghrelin in Schizothorax davidi. Overall, our results provide evidence for a highly conserved structure and biological actions of ghrelin during evolution.

Characterization, tissue distribution and regulation of agouti-related protein (AgRP) in a cyprinid fish (Schizothorax prenanti).

Agouti-related protein (AgRP) is an important neuropeptide involved in the regulation of feeding in both mammals and fish. In this study, we have cloned the full-length cDNA sequence for AgRP in a cyprinid fish (Schizothorax prenanti). The AgRP gene, encoding 126-amino acids, was strongly expressed in the brain. The AgRP gene was detected in embryos at developmental stages. Further, its mRNA was detectable in unfertilized eggs. An experiment was conducted to determine the expression profile of AgRP during short-term and long-term fasting of the hypothalamus. The expression level of AgRP in unfed fish was significantly increased at 3 and 4h post-fasting than in fed fish but did not affect AgRP mRNA expression after 14 days fasting. Overall, our results suggest that AgRP is a conserved peptide that might be involved in the regulation of short-term feeding and other physiological function in Schizothorax prenanti.