Yundi Gao

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.

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.

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.

Cloning, distribution and effects of fasting status of melanocortin 4 receptor (MC4R) in Schizothorax prenanti

Melanocortin 4 receptor (MC4R) has an important role in the regulation of energy homeostasis in both mammals and fish. In this study, MC4R was characterized in S. prenanti (Schizothorax prenanti) and designated as SpMC4R. SpMC4R cDNA is composed of 1004 nucleotides with a 978 nucleotide open reading frame encoding a protein of 326 amino acids. The SpMC4R contained predicted regions that were structural features of MCR subtypes of vertebrates. In addition, phylogenetic analyses suggested that S. prenanti MC4R was closely related to fish MC4Rs. The SpMC4R mRNA was detected in embryos at developmental stages. Further, its mRNA was detectable in unfertilized eggs. Using real-time RT-PCR, MC4R is widely expressed, with highest levels of expression in brain and ovary. An experiment was conducted to determine the expression profile of MC4R during short-term and long-term fasting of the brain. The expression level of MC4R in unfed fish was significantly increased at 6, 9 and 24h post-fasting (hpf) and 14days fasting than in fed fish, this suggests that MC4R is conserved peptide that might be involved in the regulation of food intake and other physiological function in S. prenanti.

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.

Molecular cloning, expression analysis, and appetite regulatory effect of peptide YY in Siberian sturgeon (Acipenser baerii)

Peptide YY (PYY) is an anorectic brain-gut peptide involved in feeding regulation and well characterized in mammals. However, the functional role of PYY in the appetite regulatory of fish is not clear. In this study, we characterized a high conservation of PYY cDNA and found high expression levels of PYY mRNA in the brain and digestive tract of Siberian sturgeon. Then, we examined preprandial (pre- and post-feeding) changes of PYY mRNA expression in the brain that showed a significantly increased in 3h post-feeding, suggesting an anorectic possible function of PYY in Siberian sturgeon. Next, we examined the expression of PYY mRNA during 15 days fasting and refeed after fasting. The SsPYY mRNA expression of unfed fish had a significant 2.4, 1.7, 2.0, 2.2, and 2.1-fold decrease compared to 1-, 3-, 6-, 10- and 15-day ad libitum fed animals, respectively. After refeed, SsPYY mRNA significantly increased 1.9 and 4.1-fold above that of the 15-day fed and unfed fish control group (P<0.01). Furthermore, a single intraperitoneal injection of 10, 100 and 200 ng/g BW SsPYY1-36 caused a reduction in the next feeding and no significant reduction in food intake was observed in fish injected with a 1 ng/g BW. Overall, PYY has a potentially role in food intake attenuation of Siberian sturgeon.

Characterization, tissue distribution and regulation of neuropeptideY in Schizothorax prenanti.

In this study, the full-length neuropeptide Y (npy) complementary (c)DNA was cloned in ya fish Schizothorax prenanti. npy cDNA was composed of 789 nucleotides with a 288 nucleotide open reading frame encoding a protein of 96 amino acids. The deduced amino acid sequences contained a 28 amino acids signal peptide followed by a 36 amino acids mature neuropeptide Y (NPY). The npy mRNA was expressed mainly in the brain and eye as detected by real-time quantitative polymerase chain reaction RT-PCR (rt-qPCR). The S. prenanti NPY was detectable from blastulation to hatch, suggesting that npy might be involved in the late embryonic development of S. prenanti. An experiment was conducted to determine the expression profile of npy during feeding of a single meal and during long-term fasting. The expression level of npy in fed fish was significantly decreased at 0.5, 1.5, 3 and 9 h post-feeding (hpf) than in fasting fish. Fasting for 14 days induced an increase in npy messenger (m)RNA expression in the brain. Overall, the results suggest that NPY is a conserved peptide that might be involved in the regulation of feeding and other physiological function in S. prenanti.

Appetite regulation in Schizothorax prenanti by three CART genes.

In recent years, cocaine- and amphetamine-regulated transcript (CART) has received much attention as mediators of appetite regulation in mammals. However, the involvement of CART in the feeding behavior of teleosts has not been well understood. In this study, three distinct CARTs were cloned from the Schizothorax prenanti (S. prenanti). Real-time quantitative PCR were applied to characterize the tissue distribution and appetite regulatory effects of CARTs in S. prenanti. The S. prenanti CART-1, CART-2 and CART-3 full-length cDNA sequences were 597 bp, 694 bp and 749 bp in length, encoding the peptides of 125, 120 and 104 amino acid residues, respectively. All the S. prenanti CARTs consisted of three exons and two introns. Tissue distribution analysis showed that the high mRNA levels of S. prenanti CART-1 were observed in the telencephalon and eye, followed by the hypothalamus, myelencephalon, and mesencephalon. The S. prenanti CART-2 mRNA was mainly found in the mesencephalon, hypothalamus, telencephalon and myelencephalon. The S. prenanti CART-3 mRNA was widely distributed among the tissues, with the high levels in the hypothalamus and foregut. In the periprandial experiment, all three CARTs mRNA expressions in the hypothalamus were highly elevated after a meal, suggesting that CARTs are postprandial satiety signals. In the fasting experiment, all three CARTs mRNA expressions decreased after fasting and increased after refeeding, suggesting that CARTs might be involved in regulation of appetite in the S. prenanti.

Characterization of Schizothorax prenanti cgnrhII gene: fasting affects cgnrhII expression

In this study, the role of chicken gonadotropin-releasing hormone II (cgnrhII) in feeding regulation was investigated in Schizothorax prenanti. First, the full-length S. prenanti cgnrhII cDNA consisted of 693 bp with an open reading frame of 261 bp encoding a protein of 86 amino acids. Next, cgnrhII was widely expressed in the central and peripheral tissues. Last, there were significant changes in cgnrhII mRNA expression in the fasted group compared to the fed group in the S. prenanti hypothalamus during 24 h fasting (P < 0.05). Furthermore, the cgnrhII gene expression presented a significant decrease in the fasted group compared with the fed group (P < 0.05) on days 3, 5 and 7, after re-feeding, there was no significant changes in cgnrhII mRNA expression level between refed and fed group on day 9 (P > 0.05). Thus, the results suggest that cGnRH II expression is influenced by fasting and the gene may be involved in feeding regulation in S. prenanti.