Antipsychotic drug-induced behavioral abnormalities in common carp: The potential involvement of the gut microbiota-brain axis.

The effects of antipsychotic drugs on aquatic organisms have received widespread attention owing to their widespread use and continued release in aquatic environments. The toxicological effects of antipsychotics on aquatic organisms, particularly fish, are unexplored, and the underlying mechanisms remain unelucidated. This study aimed to use common carp to explore the effects of antipsychotics (olanzapine [OLA] and risperidone [RIS]) on behavior and the potential mechanisms driving these effects. The fish were exposed to OLA (0.1 and 10 µg/L) and RIS (0.03 and 3 µg/L) for 60 days. Behavioral tests and neurological indicators showed that exposure to antipsychotics could cause behavioral abnormalities and neurotoxicity in common carp. Further, 16 S rRNA sequencing revealed gut microbiota alteration and decreased relative abundance of some strains related to SCFA production after OLA and RIS exposure. Subsequently, a pseudo-sterile common carp model was successfully constructed, and transplantation of the gut microbiota from antipsychotic-exposed fish caused behavioral abnormalities and neurotoxicity in pseudo-sterile fish. Further, SCFA supplementation demonstrated that SCFAs ameliorated the behavioral abnormalities and neurological damage caused by antipsychotic exposure. To our knowledge, the present study is the first to investigate the effects of antipsychotics on various complex behaviors (swimming performance and social behavior) in common carp, highlighting the potential health risks associated with antipsychotic drug-induced neurotoxicity in fish. Although these results do not fully elucidate the mechanisms underlying the effects of antipsychotic drugs on fish behavior, they serve as a valuable initial investigation and form the basis for future research.

Amino-Acid-Encoded Bioinspired Supramolecular Self-Assembly of Multimorphological Nanocarriers.

Supramolecular self-assembly has emerged as an efficient tool to construct well-organized nanostructures for biomedical applications by small organic molecules. However, the physicochemical properties of self-assembled nanoarchitectures are greatly influenced by their morphologies, mechanical properties, and working mechanisms, making it challenging to design and screen ideal building blocks. Herein, using a biocompatible firefly-sourced click reaction between the cyano group of 2-cyano-benzothiazole (CBT) and the 1,2-aminothiol group of cysteine (Cys), an amino-acid-encoded supramolecular self-assembly platform Cys(SEt)-X-CBT (X represents any amino acid) is developed to incorporate both covalent and noncovalent interactions for building diverse morphologies of nanostructures with bioinspired response mechanism, providing a convenient and rapid strategy to construct site-specific nanocarriers for drug delivery, cell imaging, and enzyme encapsulation. Additionally, it is worth noting that the biodegradation of Cys(SEt)-X-CBT generated nanocarriers can be easily tracked via bioluminescence imaging. By caging either the thiol or amino groups in Cys with other stimulus-responsive sites and modifying X with probes or drugs, a variety of multi-morphological and multifunctional nanomedicines can be readily prepared for a wide range of biomedical applications.

Molecular characterization of the grass carp bscl2 gene and its expression response to lipid accumulation, nutritional status, insulin and glucagon.

Bscl2 plays a role in lipid metabolism of mammals, however its role in teleost fish remains unclear. Using the grass carp (Ctenopharyngodon idella) as a model, the bscl2 gene was isolated from the brain and characterized. Thereafter, the tissue distribution of the gene was examined, before expression was analyzed as a function of fasting, refeeding, oral glucose administration and overfeeding. In addition, bscl2 mRNA levels were evaluated in grass carp primary hepatocytes treated with glucagon, insulin, oleic acid, and glucose. Results showed that the cloned bscl2 gene was 1341 bp, encoding 446 amino acids, and was highly expressed in the brain, heart, and gonad. Following oral glucose administration, bscl2 expression increased. Expression of bscl2 decreased in fasted fish but increased following refeeding. Overfeeding, which resulted in elevated lipid accumulation, also stimulated bscl2 expression. In primary hepatocytes, bscl2 levels were increased by glucose, oleic acid, and insulin treatments, and reduced by glucagon treatment. These data suggest that bscl2 may play an important role in nutrient metabolism in teleost fish.

Cloning grass carp (Ctenopharyngodon idella) ccdc3 and its expression affected by nutrition state, insulin, and glucagon.

As an adipokine, coiled-coil domain-containing 3 (CCDC3) plays multiple physiological roles in fatty liver, lipid metabolism, and abdominal obesity. Grass carp was selected as the experimental animal in this study to investigate the roles of Ccdc3 in teleosts. Results showed that the open reading frame (ORF) of cloned ccdc3 was 831 bp and encoded 276 amino acids. Three N-glycosylation sites and a predicted coiled-coil domain motif were located in the identified Ccdc3. Moreover, a nuclear localization signal (NLS) was contained in the coiled-coil domain motif of the identified Ccdc3. The results on tissue distribution revealed that ccdc3 was highly detected in grass carp fat and brain tissue. In the oral glucose tolerance test (OGTT), the expression of ccdc3 increased remarkably in the brain, hypothalamus, and visceral fat in the glucose treatment group. In the fasting and refeeding experiment, the ccdc3 expression levels were remarkably reduced in the brain, hypothalamus, and visceral fat after 14 days of fasting. In the refeeding group, the ccdc3 expression levels were considerably elevated compared with those in the fasting group. In the induced overfeeding experiment, the ccdc3 expression increased remarkably in the hepatopancreas, brain, and visceral fat tissues. The ccdc3 expression in the primary hepatocytes was remarkably increased with glucose, oleic acid, and insulin treatment. However, ccdc3 expression was markedly decreased with glucagon treatment. In conclusion, these results indicate that Ccdc3 is involved in regulating glucose and lipid metabolism of teleosts.

The fusion gene hsf5-rnf43 in Nile tilapia: A potential regulator in the maintenance of testis function and sexual differentiation.

We identified that the genes heat shock transcription factor 5 (hsf5) and ring finger protein 43 (rnf43) happened fusion in Nile tilapia (Oreochromis niloticus), called hsf5-rnf43, and provided the characteristic and functional analysis of hsf5-rnf43 gene in fish for the first time. Analysis of spatiotemporal expression showed that hsf5-rnf43 was specifically expressed in the testis and located in primary spermatocytes of adult Nile tilapia and gradually increased during testis development from 5 to 180 days after hatching. We also found DNA methylation regulated sex-biased expression of hsf5-rnf43 in the early development of Nile tilapia, and was affected by high temperature during the thermosensitive period of Nile tilapia sex differentiation. Therefore, we first reported that the fusion gene hsf5-rnf43 was sex-biased expressed in the testis regulated by DNA methylation and affected by high temperature, which may be involved in the maintenance of testis function and sex differentiation of Nile tilapia.

Identification of hub genes in digestive system of mandarin fish (Siniperca chuatsi) fed with artificial diet by weighted gene co-expression network analysis.

Mandarin fish (Siniperca chuatsi) is a carnivorous freshwater fish and an economically important species. The digestive system (liver, stomach, intestine, pyloric caecum, esophagus, and gallbladder) is an important site for studying fish domestication. In our previous study, we found that mandarin fish undergoes adaptive changes in histological morphology and gene expression levels of the digestive system when subjected to artificial diet domestication. However, we are not clear which hub genes are highly associated with domestication. In this study, we performed WGCNA on the transcriptomes of 17 tissues and 9 developmental stages and combined differentially expressed genes analysis in the digestive system to identify the hub genes that may play important functions in the adaptation of mandarin fish to bait conversion. A total of 31,657 genes in 26 samples were classified into 23 color modules via WGCNA. The modules midnightblue, darkred, lightyellow, and darkgreen highly associated with the liver, stomach, esophagus, and gallbladder were extracted, respectively. Tan module was highly related to both intestine and pyloric caecum. The hub genes in liver were cp, vtgc, c1in, c9, lect2, and klkb1. The hub genes in stomach were ghrl, atp4a, gjb3, muc5ac, duox2, and chia2. The hub genes in esophagus were mybpc1, myl2, and tpm3. The hub genes in gallbladder were dyst, npy2r, slc13a1, and slc39a4. The hub genes in the intestine and pyloric caecum were slc15a1, cdhr5, btn3a1, anpep, slc34a2, cdhr2, and ace2. Through pathway analysis, modules highly related to the digestive system were mainly enriched in digestion and absorption, metabolism, and immune-related pathways. After domestication, the hub genes vtgc and lect2 were significantly upregulated in the liver. Chia2 was significantly downregulated in the stomach. Slc15a1, anpep, and slc34a2 were significantly upregulated in the intestine. This study identified the hub genes that may play an important role in the adaptation of the digestive system to artificial diet, which provided novel evidence and ideas for further research on the domestication of mandarin fish from molecular level.

Dietary Bacillus velezensis R-71003 and sodium gluconate improve antioxidant capacity, immune response and resistance against Aeromonas hydrophila in common carp.

This study aimed to evaluate the effects of dietary supplementation with Bacillus velezensis R-71003 combined with sodium gluconate on antioxidant capacity, immune response and resistance against Aeromonas hydrophila in common carp. In addition, the biocontrol potential of the secondary metabolites of B. velezensis R-71003 was also evaluated to analyze the possible mechanism of B. velezensis R-71003 against A. hydrophila. The results indicated that the antibacterial crude extract of B. velezensis R-71003 can destroy the cell wall of A. hydrophila. Moreover, the results showed that dietary B. velezensis R-71003 could promote antioxidant capacity, which significantly increased the activities of CAT and SOD and decreased the content of MDA. Additionally, B. velezensis R-71003 supplementation significantly enhanced the immunity of common carp, as measured by the mRNA expression levels of cytokine-related genes (TNF-α, TGF-ß, IL-1ß and IL-10). In addition, dietary B. velezensis R-71003 exhibited an upregulation of IL-10 and a downregulation of IL-1ß, coupled with higher survival rates when challenged with A. hydrophila compared to the positive group. Furthermore, compared to prechallenge, the mRNA expression levels of TLR-4, MyD88, IRAK1, TRAF6, TRIF and NF-κB in the head kidney of common carp were significantly increased after challenge. The fish fed the B. velezensis R-71003 diet showed lower expression of TLR-4, MyD88, IRAK1, TRAF6, TRIF and NF-κB after the challenge than those fed the control diet. Thus, this study revealed that B. velezensis R-71003 can improve the resistance of common carp to pathogenic bacteria by destroying bacterial cell walls and improving fish immunity by activating the TLR4 signaling pathway. Importantly, this study indicated that sodium gluconate has a positive effect on B. velezensis R-71003 in enhancing the anti-infection ability of common carp. The results of this study will lay the foundation for the application of B. velezensis R-71003 in combination with sodium gluconate as an alternative to antibiotics in aquaculture.

The antipsychotic drug olanzapine altered lipid metabolism in the common carp (Cyprinus carpio L.): Insight from the gut microbiota-SCFAs-liver axis.

Olanzapine (OLA) is a common drug used to treat schizophrenia and has recently come under increasing scrutiny as an emerging contaminant. However, its impact on lipid metabolism in fish and its mechanisms of action are not well understood. In this study, common carp were exposed to 0, 10, 100, and 250 µM OLA for 60 days. The results indicated that OLA exposure increased weight gain, total cholesterol (TC), low-density lipoprotein (LDL), and triglycerides (TG) and decreased high-density lipoprotein (HDL). In addition, lipids accumulated in the liver of the common carp. To explore the underlying mechanisms of action, gut microbiota, short-chain fatty acids (SCFAs), liver transcripts, and genes related to lipid metabolism were measured. It was discovered that OLA exposure altered the common carp gut microbiota composition and increased the abundance of SCFA-producing bacteria. Correspondingly, this study showed that OLA exposure increased the levels of SCFAs, which are highly relevant to the development of lipid accumulation. Transcriptome sequencing results indicated that OLA exposure could change lipid metabolism signalling pathways, including steroid biosynthesis, the PPAR signalling pathway, asglycerophospholipid metabolism, glycerolipid metabolism, and fatty acid metabolic pathways of the common carp. Additionally, OLA exposure interrupted lipid metabolism by means of significant upregulation of lipid synthesis-related genes, including pparγ, srebp1, and fas. OLA exposure also resulted in significant lipolysis-related gene downregulation, including cpt, lpl, hsl, and pparα. The results of this study indicated that contamination of aquatic environments with OLA alters lipid metabolism in common carp. In addition, the underlying mechanism might be due in part to the modulation of the gut microbiota-SCFA-PPAR signalling pathway.

Feeding tea polysaccharides affects lipid metabolism, antioxidant capacity and immunity of common carp (Cyprinus carpio L.).

Tea polysaccharides plays a role in lipid metabolism, antioxidant capacity and immunity of mammals. To investigate the functions of tea polysaccharides on fish, the common carp (Cyprinus carpio L.) was selected as the animal model in this study. In our study, the common carp (45±0.71g) were randomly divided into four groups and were fed fodder with 50% carbohydrate. The common carp were orally administrated with 0 mg/kg BW (control group), 200 mg/kg BW (low-dose group), 400 mg/kg BW (medium-dose group) and 800 mg/kg BW (high-dose group) tea polysaccharide for two week. At the end of experiment, the serum glucose, TG, MDA contents and antioxidase activities were measured by commercial kits. The serum immune factors levels were tested by ELISA. The genes expression levels related to antioxidant capacity, metabolism and immunity were measured by real-time PCR. The results showed that the glucose, TG and MDA contents in serum were significantly decreased by tea polysaccharides treatment. The serum activities of SOD were significantly increased by low-dose tea polysaccharides treatment. The serum activities of GPX were significantly increased by medium-dose tea polysaccharides treatment. The serum levels of IL-1ß and TNFα were significantly decreased in the tea polysaccharides treatment group. In the high-dose treatment group, the serum level of TGFß was significantly increased, and the serum level of IL-12 was markedly decreased. In the hepatopancreas, the expression of acc1, fas, srebp1c, lpl, gys and pparγ were significantly reduced, and the expression of pygl, cat, mnsod, ho-1 and gr were significantly up-regulated in the tea polysaccharides group. In the intestine, the expression of zo-1, occ and gip was significantly up-regulated in the high-dose treatment group. Moreover, the expression of glut2 and sglt1 were significantly down regulated. In the spleen, the expression of il-12, tnfα and il-6 were significantly decreased, and the expression of il-10 and tgfß was significantly increased by the tea polysaccharides. In the spleen cells, the tea polysaccharides could relieve the LPS-induced immune damage. In conclusion, tea polysaccharides can improve antioxidant capacity, lipid metabolism and immunity of common carp.

Neurosecretory protein GL in GIFT tilapia (Oreochromis niloticus): cDNA cloning, tissue distribution and effects of feeding on its expression.

Neurosecretory protein GL (NPGL), a novel neuropeptide, has been identified in the hypothalamus of chicks and rodents. NPGL plays a crucial role in monitoring energetic status via the regulation of feeding and metabolism. However, no study on NPGL has been reported in fish thus far. In the present study, the full-length cDNA of NPGL was identified from the hypothalamus of GIFT tilapia (Oreochromis niloticus). The ORF of tilapia NPGL is 471 bp and encodes a precursor peptide with a size of 156 a.a, consisting of a 26 a.a signal peptide and an 82 a.a mature peptide. Tissue distribution profiles of npgl in tilapia were acquired using semiquantitative PCR and in situ hybridization (ISH). The results showed that the highest npgl mRNA is expressed in the telencephalic-preoptic complex, which comprises both the telencephalon and the anterior preoptic area (POA) of male tilapia, and in the ovary of female tilapia. In addition, in male tilapia, the ISH results showed that the cells containing npgl mRNA were distributed exclusively in the anterior periventricular pretectal nucleus (Ppa) of the POA. FISH results demonstrated that npgl mRNA is also expressed in the lateral tuberal nucleus of the hypothalamus (NLT). Real-time PCR showed that npgl mRNA significantly increased in the telencephalic-preoptic complex of male tilapia that were fasted for 24 h and then fed a full diet for 20 min compared with the unfed group. Results of the FISH study showed that parvocellular cells containing npgl mRNA in the Ppa of fed fish were apparently more abundant than those of the unfed group. Few npgl positive signals also appeared in the NLT after full feeding, where pomc mRNA is highly expressed. These results indicate that NPGL may be a short-term satiety factor in fish and that the coexpression of NPGL and POMC may be present in the hypothalamus of male tilapia.

Molecular Characterization of Grass Carp GIPR and Effect of Nutrition States, Insulin, and Glucagon on Its Expression.

GIP plays an important regulatory role in glucose and lipid metabolism. As the specific receptor, GIPR is involved in this physiological process. To assess the roles of GIPR in teleost, the GIPR gene was cloned from grass carp. The ORF of cloned GIPR gene was 1560 bp, encoding 519 amino acids. The grass carp GIPR was the G-protein-coupled receptor which contains seven predicted transmembrane domains. In addition, two predicted glycosylation sites were contained in the grass carp GIPR. The grass carp GIPR expression is in multiple tissues and is highly expressed in the kidney, brain regions, and visceral fat tissue. In the OGTT experiment, the GIPR expression is markedly decreased in the kidney, visceral fat, and brain by treatment with glucose for 1 and 3 h. In the fast and refeeding experiment, the GIPR expression in the kidney and visceral fat tissue was significantly induced in the fast groups. In addition, the GIPR expression levels were markedly decreased in the refeeding groups. In the present study, the visceral fat accumulation of grass carp was induced by overfed. The GIPR expression was significantly decreased in the brain, kidney, and visceral fat tissue of overfed grass carp. In primary hepatocytes, the GIPR expression was promoted by treatment with oleic acid and insulin. The GIPR mRNA levels were significantly reduced by treatment with glucose and glucagon in the grass carp primary hepatocytes. To our knowledge, this is the first time the biological role of GIPR is unveiled in teleost.

Bacillus coagulans SCC-19 maintains intestinal health in cadmium-exposed common carp (Cyprinus carpio L.) by strengthening the gut barriers, relieving oxidative stress and modulating the intestinal microflora.

Heavy metal cadmium (Cd) pollution is a serious problem affecting the sustainable development of aquaculture and the safety of aquatic foods. Research about the use of probiotics to attenuate toxic damage caused by Cd2+ in aquatic animals has received widespread attention. Bacillus coagulans (B. coagulans), a kind of probiotics commonly used in aquaculture, has been shown to adsorb Cd2+ both in vivo and vitro. Here, we aimed to determine the effects of B. coagulans on Cd2+ bioaccumulation, gut barrier function, oxidative stress and gut microbiota in common carp following Cd2+ exposure. The fish were exposure to Cd2+ at 0 and 0.5 mg/L and/or fed a B. coagulans-containing diet at 107, 108 and 109 CFU/g for 8 weeks. The results indicated that B. coagulans can maintain gut barrier function in Cd2+-exposed fish by reducing Cd2+ bioaccumulation, increasing the mRNA levels of tight junction protein genes (occludin, claudin-2 and zonula occludens-1), and decreasing the levels of diamine oxidase and D-lactic acid. In addition, B. coagulans could relieve oxidative stress in Cd2+-exposed fish by restoring the activities of glutathione peroxidase, catalase and superoxide dismutase. Moreover, Cd2+ exposure decreased the intestinal microbiota diversity and changed the intestinal microbiota compositions in common carp. However, supplementation with B. coagulans could reverse the altered intestinal microbiota diversity and composition after Cd2+ exposure, decrease the abundance of some pathogens (Shewanella and Vibrio), and increase the abundance of probiotics (Bacillus and Lactobacillus). These results indicate that B. coagulans may serve as a potential antidote for alleviating Cd2+ toxicity.

Molecular identification of glucose transporter 4: The responsiveness to starvation, glucose, insulin and glucagon on glucose transporter 4 in common carp (Cyprinus carpio L.).

Glucose transporter 4 (GLUT4) is comprehensively investigated in mammals, while the comparative research of GLUT4 in common carp is deficient. To investigate the function of GLUT4, carp glut4 was first isolated. The open reading frame of carp glut4 was 1518 bp in length, encoding 505 amino acids. A high-sequence homology was identified in carp and teleost, and the phylogenetic tree displayed that the carp GLUT4 was clustered with the teleost. A high level of glut4 mRNA was analysed in fat, red muscle and white muscle. After fasting treatment, glut4 mRNA expression was increased significantly in muscle. In the oral glucose tolerance test experiment, glut4 mRNA was also significantly elevated in muscle, gut and fat. Furthermore, intraperitoneal injection of insulin resulted in the upregulation of glut4 gene expression significantly in white muscle, gut and fat. On the contrary, the glut4 mRNA level in the white muscle, gut and fat was markedly downregulated after glucagon injection. These results suggest that GLUT4 might play important roles in food intake and could be regulated by nutrient condition, insulin and glucagon in common carp. Our study is the first to report on GLUT4 in common carp. These data provide a basis for further study on fish GLUT4.

Molecular identification of FNDC5 and effect of irisin on the glucose metabolism in common carp (Cyprinus carpio L.).

Irisin, encoded by fibronectin type III domain-containing protein 5 (FNDC5) gene, plays a role in energy expenditure and insulin sensitivity in mice. In fish, the function of irisin related to glucose metabolism is less reported. It may increase glucose utilization in fish. The aim of the present study was to characterize the regulatory role of irisin in glucose metabolism in common carp (Cyprinus carpio L.). In this study, FNDC5a and FNDC5b were isolated from common carp. The cDNA of FNDC5a and FNDC5b were 722 bp and 714 bp, encoding 221 and 207 amino acids, respectively. FNDC5a was abundantly expressed in the brain and gonad. FNDC5b was mainly expressed in brain. Different expression pattern of FNDC5a and FNDC5b under fasting/refeeding and OGTT experiment were identified. The recombinant common carp irisinA and irisinB were prepared by prokaryotic expression system. Glucose concentration was decreased in treatment with irisinA or irisinB in the in vitro and in vivo experiments. The mRNA expression levels of gluconeogenesis-related genes were significantly down-regulated, while the mRNA expression of glycolysis-related genes were significantly up-regulated after treatment with recombinant irisinA or irisinB in liver in vivo and in primary hepatocytes in vitro. Our research shows that irisin inhibits hepatic gluconeogenesis and promotes hepatic glycolysis. Taken together, this study for the first time revealed the two subtypes of FNDC5 and explored the function and mechanisms of irisinA and irisinB in fish glucose homeostasis.

The Regulatory Role of Apelin on the Appetite and Growth of Common Carp (Cyprinus Carpio L.).

Apelin, a kind of active polypeptide, has many biological functions, such as promoting food intake, enhancing immunity, and regulating energy balance. In mammals, studies have indicated that apelin is involved in regulating food intake. However, there are relatively few studies about the regulatory effect of apelin on fish feeding, and the specific mechanism is not clear. Therefore, the purpose of this study was to preliminarily investigate the regulatory effects of apelin on key genes of feeding and growth in common carp (Cyprinus Carpio L.) through in vitro and in vivo experiments. In the present study, after incubation with different concentrations of Pyr-apelin-13 (0, 10, 100, and 1000 nM) in hypothalamic fragments, the expressions of Neuropeptide Y (NPY) and Agouti related peptide (AgRP) mRNA were significantly up-regulated at 12 and 3 h, respectively, and the significant down-regulation of Cocaine and amphetamine-related transcript (CART) mRNA expression was observed at 1 and 3 h. In vivo, after Pyr-apelin-13 oral administration (0, 1, 10, and 100 pmol/g), the orexin mRNA level in the hypothalamus of common carp was significantly increased at 1, 6, and 12 h, while CART/(Proopiomelanocortin) POMC mRNA levels in the hypothalamus of common carp were significantly down-regulated. Following incubation with different concentrations of Pyr-apelin-13 (0, 10, 100, and 1000 nM) in primary hepatocytes, GHR (Growth hormone receptor), IGF2 (Insulin-like growth factor 2), IGFBP2 (Insulin like growth factor binding protein 2), and IGFBP3 (Insulin like growth factor binding protein 3) mRNA levels were significantly increased at 3 h. In vivo, the levels of IGF1 (Insulin-like growth factor 1), IGF2, IGFBP2 (Insulin like growth factor binding protein 2), and IGFBP3 mRNA were significantly increased after the oral administration of Pyr-apelin-13 in the hepatopancreas, in a time and dose-dependent manner. These results support the hypothesis that Pyr-apelin-13 might regulate the feeding and growth of common carp through mediating the expressions of appetite- and growth-related genes. Overall, apelin, which is an orexigenic peptide, improves food intake and is involved in the growth of common carp.

Molecular identification, tissue distribution and in vitro functional analysis of growth hormone and its receptors in red-spotted grouper (Epinephelus akaara).

Red-spotted grouper (Epinephelus akaara) is one of the high economic value grouper species, however, the knowledge regarding its growth is limited. In this study, full-length cDNAs of growth hormone (gh) and its receptors (ghr1 and ghr2) were cloned from the pituitary and liver of red-spotted grouper, respectively. Tissue distribution analysis showed that gh mRNA was predominantly expressed in the pituitary. ghr1 mRNA was highly expressed in the liver, muscle, fat and gonad, while ghr2 mRNA expression was ubiquitously high in the peripheral tissues. However, the mRNA expression of both ghr isoforms was relatively low in the central nervous system. Secretory recombinant grouper GH (rgGH) was expressed in yeast Pichia pastoris and verified. HEK293T cells transiently transfected with the GHR isoforms were used to elucidate the receptor-mediated signaling pathways related to growth regulation. rgGH activated rapid phosphorylation of Janus kinase 2, signal transducer and activator of transcription 5 (STAT5) and extracellular signal-regulated protein kinase 1/2 through GHR1, but only STAT5 was phosphorylated via GHR2. rgGH strongly activated STAT5 phosphorylation and significantly stimulated ghr1, ghr2 and insulin-like growth factor (igf1, igf2) mRNA expression in primary cultured hepatocytes. Data showed that the recombinant protein rgGH played effects on igf1/2 mRNA expression via GHR-mediated signaling pathways. Our findings provide essential information about GH and GHRs characteristics in red-spotted grouper.

Grape seed proanthocyanidin extract ameliorates hepatic lipid accumulation and inflammation in grass carp (Ctenopharyngodon idella).

Hepatic lipid metabolism disorder due to excessive fat accumulation in fish is a significant problem in aquaculture. Studies have shown that grape seed procyanidin extract (GSPE) can regulate fish lipid metabolism and improve fish immunity. However, the mechanism is unclear. In this study, we used grass carp that stores excess fat in the liver as a model. In vitro, GSPE treatment of hepatocytes for 3 h significantly decreased TG content, accompanied with decreased expression of SREBP-1c, FAS, and ACC and increased expression of PPARα, ATGL, and LPL. GSPE treatment for 1 h significantly decreased expression of pro-inflammatory cytokines (TNFα, IL-6, IL-1ß, and NF-κB) and increased the expression of anti-inflammatory cytokines (IL-10 and TGF-ß1). In vivo, the administration of GSPE significantly reduced high-fat diet-induced increase of serum CHOL, TG, and HDL, but increased LDL content. GSPE treatment for 3 h increased expression of ATGL and LPL, and significantly decreased the expression of HFD-fed-induced SREBP-1c, ACC, FAS, PPARγ, PPARα, and H-FABP. GSPE treatment for 3 h also significantly decreased the expression of pro-inflammatory cytokines (TNFα, IL-6, and IL-1ß) and increased the expression of the anti-inflammatory cytokine IL-10. The expression levels of the lipogenic miRNAs, miR-33, and miR-122, were suppressed both in vivo and in vitro by GSPE. In summary, GSPE had hypolipidemic and potential anti-inflammatory effects in the liver, potentially mediated by miR-33 and miR-122.

Molecular identification of grass carp igfbp2 and the effect of glucose, insulin, and glucagon on igfbp2 mRNA expression.

The GH (growth hormone)/IGFs (insulin-like growth factors) system has an important function in the regulation of growth. In this system, IGFBPs play a crucial regulatory role in IGF functions. As a member of the IGFBP family, IGFBP2 can bind to IGF and regulate IGF functions to regulate development and growth. In addition, IGFBP2 shows key regulatory functions in cell proliferation and metabolism. In this study, the igfbp2 gene was cloned from grass carp (Ctenopharyngodon idellus) liver. The ORF of grass carp igfbp2 is 834 bp long and encodes 277 amino acids. The tissue distribution results showed that igfbp2 is expressed in multiple tissues in grass carp and has a high expression level in the liver. In the OGTT, igfbp2 expression was significantly decreased in the liver and brain after 6 h of treatment with glucose. In vitro, igfbp2 expression in grass carp's primary hepatocytes was significantly suppressed by insulin after treatment for 6 and 12 h. Moreover, igfbp2 expression was markedly increased in a dose-dependent manner with glucagon incubation in grass carp's primary hepatocytes. To the best of our knowledge, this is the first report about Igfbp2 in grass carp. These results will provide a basis for the in-depth study of grass carp Igfbp2.

Glucose transporter 2 in common carp (Cyprinus carpio L.): molecular cloning, tissue expression, and the responsiveness to glucose, insulin, and glucagon.

Glucose transporter 2 (glut2) has been studied in mammals, aves, and several fish, while the comparative studies of glut2 in common carp are still lacking. In this study, glut2 was firstly isolated and characterized from the liver of common carp. The full-length cDNA of glut2 was 2351 bp with an open reading frame (ORF) of 1512 bp, encoding 503 amino acids. Alignment of glut2 amino acid sequences from different species revealed that common carp glut2 showed higher sequence identity with teleosts, and lower homology with mammals and amphibians. Tissue distribution demonstrated that glut2 mRNA level was mainly expressed in liver, foregut, and midgut. To investigate the actions of glut2 on glucose metabolism, the level of glut2 mRNA was detected after intraperitoneal injection of glucose, human insulin and glucagon (100 ng/g), respectively. Following glucose administration, glut2 gene expression was significantly upregulated at 3 h in the foregut. However, no change was found in hepatic glut2 mRNA level, indicating that glut2 may have a role in intestinal glucose uptake rather than in the liver. Following insulin treatment, the expression of glut2 was markedly downregulated at 3 h and 6 h in the liver, and at 3 h in the foregut, respectively. Furthermore, glut2 mRNA expression was unaffected by glucagon injection in the liver and foregut. These results suggested that the expression of glut2 regulated by pancreatic hormones was different. Taken together, our studies firstly revealed the structure of the glut2 gene and its potential functions in glucose metabolism of common carp.

Expression of glut2 in response to glucose load, insulin and glucagon in grass carp (Ctenophcuyngodon idellus).

Generally, fish are thought to have a limited ability to utilize carbohydrate. Postprandial blood glucose is cleared sluggishly in fish, resulting in prolonged hyperglycemia. Facilitative glucose transporters (GLUTs) play an important role in glucose utilization. In the present study, the expression levels of glut2 in different tissues were detected in grass carp. Furthermore, the effects of oral glucose administration on glut2 mRNA expression in the liver, intestine and kidney were investigated, and we also evaluated the response of glut2 mRNA to insulin and glucagon in the primary hepatocytes of grass carp. The expression level of glut2 mRNA was highest in the liver, followed by the intestine and kidney, but lower in other tissues. The result of glucose tolerance test (GTT) showed that serum glucose reached the highest level at 3 h after GTT and recovered to the basic level at 6 h. The glut2 mRNA in the intestine was up-regulated at 1 h after GTT. However, the glut2 mRNA expression in the liver of grass carp was unchanged after GTT for 1, 3, 6 h, and even decreased at 12 h after GTT. In addition, the expression of glut2 mRNA in the primary hepatocytes was enhanced by insulin and glucagon at 3 h post treatment. These results suggested that glut2 expression in the liver of grass carp was sensitive to insulin and glucagon, but not blood glucose. The up-regulation of glut2 by these hormones might be involved in the bi-directional transportation of glucose in the liver.