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Background Compensatory growth refers to the phenomenon in which organisms grow faster after the improvement of an adverse environment and is thought to be an adaptive evolution to cope with the alleviation of the hostile environment. Many fish have the capacity for compensatory growth, but the underlying cellular mechanisms remain unclear. In the present study, microarray and nontargeted metabolomics were performed to characterize the transcriptome and metabolome of zebrafish liver during compensatory growth. Results Zebrafish could regain the weight they lost during 3 weeks of fasting and reach a final weight similar to that of fish fed ad libitum when refed for 15 days. When refeeding for 3 days, the liver displayed hyperplasia accompanied with decreased triglyceride contents and increased glycogen contents. The microarray results showed that when food was resupplied for 3 days, the liver TCA cycle (Tricarboxylic acid cycle) and oxidative phosphorylation processes were upregulated, while DNA replication and repair, as well as proteasome assembly were also activated. Integration of transcriptome and metabolome data highlighted transcriptionally driven alterations in metabolism during compensatory growth, such as altered glycolysis and lipid metabolism activities. The metabolome data also implied the participation of amino acid metabolism during compensatory growth in zebrafish liver. Conclusion Our study provides a global resource for metabolic adaptations and their transcriptional regulation during refeeding in zebrafish liver. This study represents a first step towards understanding of the impact of metabolism on compensatory growth and will potentially aid in understanding the molecular mechanism associated with compensatory growth.

In the present study, four full-length cDNAs of somatostatin receptor (sstr) were cloned from the forebrain and pituitary of red-spotted grouper. The four full-length cDNAs were designated 2292, 1522, 1873 and 1789 bp and identified as sstr1, sstr2, sstr3, and sstr5 by BLAST analysis; the corresponding sizes of the open reading frames (ORFs) were 1155, 1113, 1467 and 1503 bp, which encoding 384, 370, 488 and 500 aa, respectively. The four receptors have seven transmembrane structures and contain the YANSCANPI/VLY sequence, which is the conserved amino acid sequence of the SSTR family. A tissue distribution study showed that the four sstrs had different expression patterns, suggesting that they may play different roles in regulating different physiological processes. The four receptors mediate ERK1/2 phosphorylation by SS-14 in HEK293 cells, and SS-14 promotes ATK and ERK1/2 phosphorylation in primary hepatocytes of red-spotted grouper. These results facilitate the study of SSTRs-mediated intracellular signaling pathways.

Neuropeptide Y is known to stimulate food intake in fish. In this study, we investigated tilapia NPY (tNPY) both for its effects on the growth of tilapia (Oreochromis niloticus, GIFT) in low fish meal and for its thermal stability. Three diets were formulated containing 0, 3 and 10 % fish meal (NF, LF and HF). From these diets, six experimental diets were prepared by spraying either tNPY solution (0.3 μg/g feed) or distilled water (DW) onto the surface of formulated feeds (NF + DW, NF + tNPY, LF + DW, LF + tNPY, HF + DW and HF + tNPY). Tilapia were fed the six experimental diets for 8 weeks. Fish in the NF + tNPY, LF + tNPY and HF + tNPY groups showed increasing trends in the weight gain rate and specific growth rate compared to its corresponding control group. The feed coefficient of group HF + tNPY was significantly lower than that of the control group. The growth performance of the LF + tNPY approached that of the HF + DW group. The mRNA levels of npy in NF + tNPY were significantly higher than those in NF + DW. A field experiment in which tNPY was sprayed in feeds by the vacuum spray method with doses of 0, 0.2 and 0.4 μg/g feed was performed for three months, and the FBW of tilapia receiving tNPY at 0.2 and 0.4 μg/g feed was higher than that of the control group although not significantly. The bioactivity of tNPY was confirmed by its ability to reduce cAMP levels and activate the ERK1/2 pathway. These results demonstrated that tNPY could promote tilapia growth with oral administration low fish meal diets.

As the close paralog of adiponectin, C1q/TNF-Related Protein 9 (CTRP9) has been reported to be involved in the regulation of glucose and fat metabolism, immunization and endothelial cell functions. However, information regarding the actions of Ctrp9 on reproduction is extremely limited in fish. As a first step, Ctrp9, adiponectin receptor 1 (Adipor1) and Adipor2 were identified from Nile tilapia. The open reading frame (ORF) of ctrp9 was 1020 bp which encoded a 339 amino acids. Moreover, the ORFs of adipor1 and adipor2 were 1131 bp and 1134 bp encoding 376 and 377 amino acids, respectively. Tissue distribution showed that ctrp9 mRNA levels were highest in the kidney in both sexes. And, the expression of adipor1 and adipor2 were widely distributed in all tissues examined, exhibiting high levels in the brain, gonad, gut and stomach. In addition, intraperitoneal (i.p.) injection of gCtrp9 (globular Ctrp9) suppressed the hypothalamic expression of gnrh2 (gonadotropin-releasing hormone 2) and gnrh3, as well as gthα (gonadotropic hormone α), fshβ (follicle-stimulating hormone β), lhβ (luteinizing hormone β), lhr (LH receptor) and fshr (FSH receptor) mRNA levels in the pituitary. The mRNA levels of adipor1, but not adipor2, in the gonads were also inhibited after injection. Moreover, the levels of serum E2 (estrogen) in female and T (testosterone) in male were significantly decreased after injection of gCtrp9. Overall, our data provides novel data indicating, for the first time, a regulatory effect of CTRP9 on teleost reproduction.

Background: Myostatin (Mstn), a member of the TGF-β superfamily, is a negative regulator of skeletal muscle mass in mammals. Precise regulation of Mstn expression is important for somite growth in fish. MicroRNA (miRNA), a type of small non-coding RNA, regulates gene expression at the post-transcriptional level and participates in various physiological functions. A growing amount of evidence has emphasized the importance of miRNA in the development of skeletal muscle. Aims: This study aims to study how miRNAs regulate myostatin b (mstnb) post-transcriptionally in tilapia. Methods/Results: Mstnb 3′ UTR sequences were obtained, and the results of tissue distribution showed that mstnb was expressed in several tissues, including brain, white muscle, gut, and adipose tissue. A total of 1,992 miRNAs were predicted to target mstnb in tilapia using bioinformatics, and a dual-luciferase reporter experiment confirmed that miR-181a/b-5p, miR-30-3p, miR-200a, and miR-27a were the target miRNAs of mstnb. Mutagenesis of the miR-181b-5p binding sites of mstnb significantly increased the luciferase signal compared to the wild-type mstnb. In tilapia primary muscle cells, overexpression of miR-181b-5p led to the downregulation of MSTNb expression, and the inhibitory effect of MSTNb on the downstream genes was dismissed, while inhibition of miR-181b-5p could reverse these phenomena. Conclusion: Taken together, our results suggested that miR-181b-5p could promote the growth of skeletal muscle by decreasing the MSTNb protein level in tilapia.

Compensatory growth (CG) is defined as a phase of accelerated growth when the disadvantageous environment is improved, accompanied by metabolic adjustment. Here, we report that hepatic oxidative phosphorylation (OXPHOS) activity was enhanced during compensatory growth in zebrafish. Mitochondrial metabolism enabled the generation of reactive oxygen species (ROS), which activated the nrf2 (nuclear factor-erythroid 2-related factor 2) signaling pathway, as well as the mTOR signaling pathway. Tempol (a superoxide dismutase mimetic) treatment blocked ROS signaling in the liver as well as CG in zebrafish. These results demonstrated that mitochondrial ROS signaling are essential for the occurrence of compensatory growth in zebrafish.

CTRP9 is a member of the C1q/TNF-related protein (CTRP) superfamily and has been studied in mammals, whereas the comparative studies of CTRP9 in non-mammalian species are still absent. In this study, ctrp9 was isolated and characterized from the orange-spotted grouper (Epinephelus coioides). The full-length cDNA of ctrp9 was 1378 bp in size with an ORF (open reading frame) of 1020 bp that encodes a 339 amino acid pre–pro hormone. The mRNA expression of ctrp9 showed a rather high level in the kidney and brain, but a low level in other tissues. Furthermore, the mRNA expression of ctrp9 decreased significantly in the liver after fasting for 7 days and restored to the normal levels after refeeding. In contrast, the ctrp9 mRNA level increased in the hypothalamus after fasting. The recombinant gCtrp9 (globular Ctrp9) was prepared using the Pichia pastoris expression system and was verified by Western blot as well as mass spectrometry assays. In the primary hepatocytes culture, the recombinant gCtrp9 could inhibit the glucose production after 12-h treatment. After i.p. (intraperitoneal) injection with recombinant gCtrp9, in hypothalamus, mRNA expression levels of npy and orexin (orexigenic factors) decreased, whereas the expression levels of crh and pomc (anorexigenic factors) increased. Moreover, i.p. injection with the recombinant gCtrp9 could reduce the serum concentrations of glucose, TG and low-density lipoprotein cholesterol but increase the content of high-density lipoprotein cholesterol. Our studies for the first time unveil the structure of Ctrp9 and its potential role as a regulatory factor of metabolism and food intake in teleost.

Growth hormone is a hormone secreted from the pituitary and is involved in the regulation of most major physiological processes such as growth, development and metabolism. Therefore, an accurate and sensitive detection method is needed for the detection of tilapia serum Gh level. Phage display technology is widely used in the expression of antibody fragments, in which fragments of antibodies are expressed as a fusion with phage proteins and are displayed on the phage surface for easy screening. Time-resolved fluorescence immunoassay (TRFIA) is a microanalysis method developed nearly two decades ago and is one of the most sensitive analytical techniques. With the use of a special lanthanide, the detection background can be distinguished, which can greatly improve the sensitivity of detection. In this report, we cloned the VH and VL DNA fragments from the lymphocytes of rabbits immunized with recombinant Gh and assembled them with a linker to form a single-chain variable fragment (scFv) gene pool. Using phage display technology, we isolated scFv DNA fragments from the pool, which encode a protein that specifically binds to tilapia Gh. We then established Eu-DTTA-based TRFIA for measuring plasma Gh in tilapia. The sensitivity of double antibody sandwich Gh-TRFIA was 0.225 ng/ml, and the linear range of the standard curve was 0.225–250 ng/ml. The intra- and interassay coefficients of variation (CVs) were In conclusion, Gh-TRFIA is a safe (no concerns about radioactive isotopes), economical, and efficient detection method for the quantification of plasma Gh. Thus, the application of phage display technology for antibody screening and the use of TRFIA for tilapia Gh detection are conducive to research in the field of fish endocrinology.

Growth in vertebrates is mainly mediated by the growth hormone (GH)-insulin-like growth factor (IGF) axis, and somatostatin (SRIF) inhibits growth by decreasing GH release at the pituitary level and antagonizing the release and action of GHRH in the hypothalamus. However, the effects of SRIF on the regulation of growth at levels other than GH release from the pituitary gland are less well known. In the present study, we comprehensively examined the pituitary and peripheral actions of SRIF on the GH-IGF axis in grouper using a primary pituitary and hepatocyte cell culture system. Our results showed that SRIF inhibited GH release at the pituitary level, but had no influence on GH mRNA expression. Basal hepatic GH receptor 1 (GHR1), IGF-I and IGF-II mRNA levels declined over time, whereas GHR2 mRNA levels remained stable throughout the culture period. GH stimulated the hepatic expression of GHR and IGF mRNAs in a dose-dependent manner, while SRIF suppressed both basal and GH-stimulated expression of GHR and IGF mRNAs in primary cultured hepatocytes. The inhibition of GHR and IGF mRNA levels by SRIF was not attributed to the rate of mRNA degradation. To the best of our knowledge, we demonstrated the effects of SRIF on basal and GH-stimulated IGF-II mRNA levels in teleosts for the first time. These results indicate that SRIF regulates growth at the level of the pituitary and peripheral liver.

Leptin actions at the pituitary level have been extensively investigated in mammalian species, but remain insufficiently characterized in lower vertebrates, especially in teleost fish. Prolactin (PRL) is a pituitary hormone of central importance to osmoregulation in fish. Using goldfish as a model, we examined the global and brain-pituitary distribution of a leptin receptor (lepR) and examined the relationship between expression of lepR and major pituitary hormones in different pituitary regions. The effects of recombinant goldfish leptin-AI and leptin-AII on PRL mRNA expression in the pituitary were further analysed, and the mechanisms underlying signal transduction for leptin-induced PRL expression were determined by pharmacological approaches. Our results showed that goldfish lepR is abundantly expressed in the brain-pituitary regions, with highly overlapping PRL transcripts within the pituitary. Recombinant goldfish leptin-AI and leptin-AII proteins could stimulate PRL mRNA expression in dose- and time-dependent manners in the goldfish pituitary, by both intraperitoneal injection and primary cell incubation approaches. Moreover, the PI3K/Akt/mTOR, MKK3/6/p38MAPK, and MEK1/2/ERK1/2-but not JAK2/STAT 1, 3 and 5 cascades-were involved in leptin-induced PRL mRNA expression in the goldfish pituitary.

CTRP9 is a member of the C1q/TNF-related protein (CTRP) superfamily and has been studied in mammals, whereas the comparative studies of CTRP9 in non-mammalian species are still absent. In this study

Adiponectin is an abundantly secreted adipokine from adipose tissue in mammals, which plays important roles in the regulation of glucose and lipid metabolism. The biological function of adiponectin is mediated by at least two receptors (AdipoR1 and AdipoR2). Although both of them were identified in mammals, there are few researches about adiponectin and its receptors in teleosts. In this study, two types of adiponectin receptors have been isolated and characterized in the orange-spotted grouper (Epinephelus coioides). The cDNAs of grouper AdipoR1 and AdipoR2 are 1444 and 2034 bp in length, encoding proteins of 376 amino acids and 375 amino acids, respectively. Multiple alignment results showed that there was a variable region at the N-terminal of AdipoR1/R2, which has never been reported. Both AdipoR1 and AdipoR2 were found to be widely expressed in various tissues of grouper. Compared to AdipoR2, AdipoR1 expressed at higher levels in the nervous system and pituitary gland, but at lower levels in some peripheral tissues, including heart, liver, adipose tissue, stomach, intestine and especially gonad. Fasting and refeeding experiments showed that the mRNA expressions of AdipoR1/R2 were up-regulated by fasting in the muscle and adipose tissue of grouper, and restored rapidly to normal levels after refeeding. However, the mRNA expressions of AdipoR1/R2 in the hypothalamus and liver of grouper were insensitive to fasting. By indirect immunofluorescence, we demonstrated that grouper AdipoR1/R2 were integral membrane proteins; the C-terminals were extracellular, while the N-terminals were intracellular.

Urotensin II (UII) is a cyclic peptide that was originally extracted from the caudal neurosecretory system (CNSS) of fish. UII is well known to exhibit cardiovascular, ventilatory, and motor effects in vertebrates. Studies have reported that UII exerts mitogenic effects and can act as an autocrine/paracrine growth factor in mammals. However, similar information in fish is limited. In this study, the full-length cDNAs of UII and its receptor (UT) were cloned and characterized in the orange-spotted grouper. UII and UT were expressed ubiquitously in various tissues in grouper, and particularly high levels were observed in the CNSS, CNS, and ovary. A functional study showed that UT was coupled with intracellular Ca2+ mobilization in HEK293 cells. Studies carried out using i.p. injections of UII in grouper showed the following: i) in the hypothalamus, UII can significantly stimulate the mRNA expression of ghrh and simultaneously inhibit the mRNA expression of somatostatin 1 (ss1) and ss2 3 h after injection; ii) in the pituitary, UII also significantly induced the mRNA expression of gh 6 and 12 h after injection; and iii) in the liver, the mRNA expression levels of ghr1/ghr2 and igf1/igf2 were markedly increased 12 and 3 h after the i.p. injection of UII respectively. These results collectively indicate that the UII/UT system may play a role in the promotion of the growth of the orange-spotted grouper.

In vertebrates, the neuropeptide Y (NPY) family peptides have been recognized as key players in food intake regulation. NPY centrally promotes feeding, while peptide YY (PYY) and pancreatic polypeptide (PP) mediate satiety. The teleost tetraploidization is well-known to generate duplicates of both NPY and PYY; however, the functional diversification between the duplicate genes, especially in the regulation of food intake, remains unknown. In this study, we identified the two duplicates of NPY and PYY in Nile tilapia (Oreochromis niloticus). Both NPYa and NPYb were primarily expressed in the central nervous system (CNS), but the mRNA levels of NPYb were markedly lower than those of NPYa. Hypothalamic mRNA expression of NPYa, but not NPYb, decreased after feeding and increased after 7-days of fasting. However, both NPYa and NPYb caused a significant increase in food intake after an intracranial injection of 50 ng/g body weight dose. PYYb, one of the duplicates of PYY, had an extremely high expression in the foregut and midgut, whereas another form of duplicate PYYa showed only moderate expression in the CNS. Both hypothalamic PYYa and foregut PYYb mRNA expression increased after feeding and decreased after 7-days of fasting. Furthermore, the intracranial injection of PYYb decreased food intake, but PYYa had no significant effect. Our results suggested that although the mature peptides of NPYa and NPYb can both stimulate food intake, NPYa is the main endogenous functional NPY for feeding regulation. A functional division has been identified in the duplicates of PYY, which deems PYYb as a gut-derived anorexigenic peptide and PYYa as a CNS-specific PYY in Nile tilapia.

Somatostatin is the most effective inhibitor of GH release, and GHRH was recently identified as one of the primary GH-releasing factors in teleosts. In this study, we analyzed the possible intracellular transduction pathways that are involved in the mechanisms induced by SRIF and GHRH to regulate GH release. Using a pharmacological approach, the blockade of the PLC/IP/PKC pathway reversed the SRIF-induced inhibition of GH release but did not affect the GHRH-induced stimulation of GH release. Furthermore, SRIF reduced the GH release induced by two PKC activators. Inhibitors of the AC/cAMP/PKA pathway reversed both the SRIF- and GHRH-induced effects on GH release. Moreover, the GH release evoked by forskolin and 8-Br-cAMP were completely abolished by SRIF. The blockade of the NOS/NO pathway attenuated the GHRH-induced GH release but had minimal effects on the inhibitory actions of SRIF. In addition, inhibitors of the sGC/cGMP pathway did not modify the SRIF- or GHRH-induced regulation of GH release. Taken together, these findings indicate that the SRIF-induced inhibition of GH release is mediated by both the PLC/IP/PKC and the AC/cAMP/PKA pathways and not by the NOS/NO/sGC/cGMP pathway. In contrast, the GHRH-induced stimulation of GH secretion is mediated by both the AC/cAMP/PKA and the NOS/NO pathways and is independent of the sGC/cGMP pathway and the PLC/IP/PKC system.