The AMPK and AKT/GSK3ß pathways are involved in recombinant proteins fibroblast growth factor 1 (rFGF1 and rFGF1a) improving glycolipid metabolism in rainbow trout (Oncorhynchus mykiss) fed a high carbohydrate diet.

Fibroblast growth factor 1 (FGF1) regulates vertebrate cell growth, proliferation and differentiation, and energy metabolism. In this study, we cloned rainbow trout (Oncorhynchus mykiss) fgf1 and fgf1a, prepared their recombinant proteins (rFGF1 and rFGF1a), and described the molecular mechanisms by which they improve glycolipid metabolism in carnivorous fish. A 31-d feeding trial was conducted to investigate whether they could enhance glycolipid metabolism in rainbow trout on high-carbohydrate diets (HCD). A total of 720 rainbow trout (8.9 ± 0.5 g) were equally divided into 4 groups: the chow diet (CD) group injected with PBS, the HCD group injected with PBS, the HCD group injected with rFGF1 (400 ng/g body weight), and the HCD group injected with rFGF1a (400 ng/g body weight). The results showed that short-term HCD had a significant positive effect on the specific growth rate (SGR) of rainbow trout (P < 0.05). However, it led to an increase in crude fat, serum triglyceride (TG) and glucose content, as well as serum glutamic pyruvic transaminase (GPT) and glutamic oxalacetic transaminase (GOT) contents (P < 0.05), suggesting a negative health effect of HCD. Nevertheless, rFGF1 and rFGF1a showed beneficial therapeutic effects. They significantly reduced the crude fat content of the liver, serum TG, GOT, and GPT contents caused by HCD (P < 0.05). The upregulation in atgl, hsl, and acc2 mRNAs implied the promotion of TG catabolism. Moreover, rFGF1 and rFGF1a contributed to promoting lipolysis by activating the AMPK pathway and reducing lipid accumulation in the liver caused by HCD. In addition, the rFGF1 and rFGF1a-treated groups significantly reduced serum glucose levels and elevated hepatic glycogen content under HCD, and increased glucose uptake by hepatocytes. We observed a decrease in mRNA levels for pepck, g6pase, and pygl, along with an increase in mRNA levels for gys, glut2, and gk in the liver. Furthermore, these proteins regulated hepatic gluconeogenesis and glycogen synthesis by increasing the phosphorylation level of AKT, ultimately leading to an increase in GSK3ß phosphorylation. In conclusion, this study demonstrates that rFGF1 and rFGF1a can enhance lipolysis and glucose utilization in rainbow trout by activating the AMPK pathway and AKT/GSK3ß axis.

Structural and pharmacological characterization of a medium-chain fatty acid receptor GPR84 in common carp (Cyprinus carpio).

The medium-chain fatty acid receptor GPR84, a member of the G protein-coupled receptor family, is mainly expressed in macrophages and microglia, and is involved in the regulation of inflammatory responses and retinal development in mammals and amphibians. However, structure, tissue distribution, and pharmacology of this receptor have rarely been reported in fish. In this study, we cloned the coding sequence (CDS) of common carp GPR84 (ccGPR84), examined its tissue distribution, and explored its cellular signaling function. The results showed that the CDS of ccGPR84 is 1191 bp and encodes a putative protein with 396 amino acids. Phylogenetic and chromosomal synteny analyses revealed that ccGPR84 was evolutionarily conserved with Cyprinids. Real-time quantitative PCR (qPCR) indicated that ccGPR84 was predominantly expressed in the intestine and spleen. Luciferase reporter assay demonstrated that nonanoic acid, capric acid (decanoic acid), undecanoic acid and lauric acid could inhibit cAMP signaling pathway and activate MAPK/ERK signaling pathway, while the potencies of these four fatty acids on the two signaling pathways were different. Lauric acid has the highest inhibitory potency on cAMP signaling pathway, followed by undecanoic acid, nonanoic acid, and capric acid. While for MAPK/ERK signaling pathway, nonanoic acid has the highest activation potency, followed by undecanoic acid, capric acid, and lauric acid. These findings lay the foundation for revealing the roles of different medium-chain fatty acids in the inflammatory response of common carp.

FGF21 Improves Glycolipid Metabolism in Rainbow Trout (Oncorhynchus mykiss) Fed a High-Carbohydrate Diet by Inhibiting Inflammatory Responses and Activating Autophagy.

In this study, the coding region of rainbow trout fgf21 was cloned and sequenced to synthesize a recombinant protein (rFGF21) and investigate its potential role in improving glycolipid metabolism. Acute injection of rFGF21 into rainbow trout effectively reduced serum glucose levels. To investigate the effect of rFGF21 on high-carbohydrate diet (HCD)-induced metabolic disorders in rainbow trout, a 31-day feeding experiment was conducted. At the end of the third week, fish were injected with either PBS or rFGF21. The results showed that the final body weight (FBW) significantly increased in rainbow trout on an HCD (P < 0.05), but there were potential risks including disturbances in glycolipid metabolism and increased inflammatory responses. However, these effects were altered by rFGF21 treatment. In addition, rFGF21 promotes glucose uptake by increasing the phosphorylation levels of AKT (protein kinase B) and GSK3ß (glycogen synthase kinase 3ß), increasing hepatic glycogen, thereby lowering serum glucose. Notably, the rFGF21 did not exacerbate the inflammatory response but downregulated the expression of inflammatory factors. Interestingly, the activation of autophagy and the AMPK pathway may contribute to the positive effect of rFGF21, where rFGF21 injection significantly increased the levels of LC3I/II protein and phosphorylate AMPKα (P < 0.05).

Molecular Cloning, Tissue Distribution, and Pharmacological Characterization of GPR84 in Grass Carp (Ctenopharyngodon Idella).

The G-protein-coupled receptor GPR84, activated by medium-chain fatty acids, primarily expressed in macrophages and microglia, is involved in inflammatory responses and retinal development in mammals and amphibians. However, our understanding of its structure, function, tissue expression, and signaling pathways in fish is limited. In this study, we cloned and characterized the coding sequence of GPR84 (ciGPR84) in grass carp. A phylogenetic analysis revealed its close relationship with bony fishes. High expression levels of GPR84 were observed in the liver and spleen. The transfection of HEK293T cells with ciGPR84 demonstrated its responsiveness to medium-chain fatty acids and diindolylmethane (DIM). Capric acid, undecanoic acid, and lauric acid activated ERK and inhibited cAMP signaling. Lauric acid showed the highest efficiency in activating the ERK pathway, while capric acid was the most effective in inhibiting cAMP signaling. Notably, DIM did not activate GPR84 in grass carp, unlike in mammals. These findings provide valuable insights for mitigating chronic inflammation in grass carp farming and warrant further exploration of the role of medium-chain fatty acids in inflammation regulation in this species.

Molecular cloning, tissues distribution, and function analysis of thioredoxin-like protein-1 (TXNL1) in Chinese giant salamanders Andrias davidianus.

Thioredoxin-like protein-1 (TXNL1) is the member of thioredoxin superfamily, a family of thiol oxidoreductases. TXNL1 plays an important role in scavenging ROS and the maintenance of cellular redox balance. However, its physiological functions in Andrias davidianus have not been well understood. In the present study, the full-length cDNA encoding thioredoxin-like protein-1 (AdTXNL1) of A. davidianus was cloned, the mRNA tissue distribution was analyzed, and the function was characterized. The Adtxnl1 cDNA contained an open reading frame (ORF) of 870 bp encoding a polypeptide of 289 amino acids with the N-terminal TRX domain, a Cys34-Ala35-Pro36-Cys37 (CAPC) motif, and the C-terminal proteasome-interacting thioredoxin domain (PITH). The mRNA of AdTXNL1 was expressed in a wide range of tissues, with the highest level in the liver. The transcript level of AdTXNL1 was significantly up-regulated post Aeromonas hydrophila challenge in liver tissue. Moreover, the recombinant AdTXNL1 protein was produced and purified, and used to investigate the antioxidant activity. In the insulin disulfide reduction assay, rAdTXNL1 exhibited strong antioxidant capability. Altogether, the thioredoxin-like protein-1 may be involved in reduction/oxidation (redox) balance and as an important immunological gene in A. davidianus.

Molecular cloning and functional characterization of melanocortin-3 receptor in grass carp (Ctenopharyngodon idella).

The melanocortin-3-receptor (MC3R) plays an important role in mammals' food intake and energy homeostasis. However, its physiological role in bony fishes, such as grass carp, has not been well understood. This study reports the molecular cloning, tissue distribution, and pharmacological characterization of grass carp melanocortin-3-receptor (ciMC3R). Phylogenetic and chromosomal synteny analyses indicated that ciMC3R was closest to cyprinid fishes in evolution. Quantitative PCR experiments revealed that the mRNA of ciMC3R was highly expressed in the brain of grass carp. The cytological function of ciMC3R was investigated by the co-transfection of pcDNA3.1-ciMC3R and the signal-pathway-specific luciferase into the HEK293T cells. Results revealed that the four agonists, α-MSH, ß-MSH, ACTH, and NDP-MSH, potentiate the activation of ciMC3R and further increase the production of cAMP and upregulate the MAPK/ERK signaling, respectively. Our study will provide basic data for exploring the physiological functions of grass carp MC3R, especially in energy homeostasis and food intake.

Molecular cloning, tissue distribution, and pharmacologic function of melanocortin-3 receptor in common carp (Cyprinus carpio).

Melanocortin-3 receptor (MC3R) not only regulates energy homeostasis in animals, but also is an important regulator of inflammation. As one of the most widely farmed freshwater fish, common carp has attracted great interest for its feeding and inflammation regulation. In this study, we cloned the coding sequence (CDS) of common carp Mc3r (ccMc3r), examined its tissue expression profile, and investigated the function of this receptor in mediating downstream signaling pathways. The results showed that the CDS of ccMc3r was 975 bp, encoding a putative protein of 324 amino acids. Homology, phylogeny, and chromosomal synteny analyses revealed that ccMc3r is evolutionarily close to the orthologs of cyprinids. Quantitative real-time PCR (qPCR) indicated that ccMc3r was highly expressed in the brain and intestine. The luciferase reporter systems showed that four ligands, ACTH (1-24), α-MSH, ß-MSH, and NDP-MSH, were able to activate the cAMP and MAPK/ERK signaling pathways downstream of ccMc3r with different potencies. For the cAMP signaling pathway, ACTH (1-24) had the highest activation potency; while for the MAPK/ERK signaling pathway, ß-MSH had the greatest activation effect. In addition, we found that the four agonists were able to inhibit TNF-α-induced NF-κB signaling in approximately the same order of potency as cAMP signaling activation. This study may facilitate future studies on the role of Mc3r in common carp feed efficiency and immune regulation.

The interaction of MC3R and MC4R with MRAP2a in rainbow trout (Oncorhynchus mykiss).

Melanocortin 3 and 4 receptors are two important neural G protein-coupled receptors that regulate energy homeostasis in vertebrates. Melanocortin receptor accessory protein 2 (MRAP2) is also involved in the regulation of food intake and body weight as a variable regulator of melanocortin receptors. Rainbow trout (Oncorhynchus mykiss) is a valuable cold-water fish cultured worldwide. In the rainbow trout model, we cloned and identified mrap2a, a paralog of mrap2. Rainbow trout mrap2a consisted of a 690 bp ORF and was expected to encode a putative protein of 229 amino acids. The qPCR results showed that rainbow trout mrap2a was expressed at high levels in brain tissue similar to mc3r and mc4r. In addition, co-immunoprecipitation verified that MRAP2a interacts with MC3R and MC4R in vitro and that MRAP2a is involved in and regulates the constitutive activity and signaling of MC3R and MC4R. MRAP2a reduced constitutive and agonist-stimulated cAMP levels of MC3R; furthermore, MRAP2a increased constitutive ERK1/2 activation but reduced ligand-induced stimulation at high levels of expression. For MC4R, MRAP2a showed decreased cAMP basal activity but increased agonist-stimulated cAMP signaling and increased ACTH ligand sensitivity. However, MRAP2a failed to affect MC4R constitutive activity and agonist-induced ERK1/2 signaling. Undoubtedly, our study will have great significance for revealing the conserved role of MC4R and MC3R signaling in teleost fish, especially in cold-water fish growth and energy homeostasis.

Molecular characterization and functional exploration of GPR84 in Chinese Giant Salamander (Andrias davidianus).

The G protein-coupled receptor 84 (GPR84) is a putative medium-chain fatty acids (MCFAs) receptor involved in immune regulation and other metabolic processes. Most available studies focused on the GPR84 characterization from mammals, neglecting vital information that could be obtained from other levels of life, such as amphibians, necessary for an apt evolutionary understanding of the orphan GPR84. Hence, this study molecularly characterized and functionally explored the GPR84 from the Chinese Giant Salamander (Andrias davidianus). Therefore, we report that the Chinese Giant Salamander (CGS), one of the world's largest amphibians, expresses a GPR84 protein having 376 amino acids, with about 70% homologous to other amphibians and around 50% to human GPR84. Investigating the relative localized expression of gpr84 mRNA in CGS using quantitative PCR revealed the highest expression in the kidney and liver. Furthermore, four medium-chain fatty acids (MCFAs) at micromolar levels activated CGS-GPR84 transfected and expressed in HEK293 cells. In HEK293 cells, four different concentrations of MCFAs inhibited forskolin-induced cAMP accumulation and resulted in a dose-dependent increase in extracellular signal-regulated kinases 1 and 2 (ERK1/2). Interestingly, MCFAs activation of GPR84 concomitantly led to the upregulation of inflammatory mediators such as Nuclear Factor Kappa B (NF-κB) and IL-6. Conclusively, this study successfully elucidated the intriguing molecular and functional properties of CGS GPR84, particularly as an immune modulator, and has positioned the findings within the existing body of knowledge for a better overall understanding of GPR84, especially in amphibians.

Functional characterization of melanocortin-3 receptor in rainbow trout (Oncorhynchus mykiss).

The melanocortin-3 receptor (MC3R) is an important regulator of energy homeostasis and inflammation in mammals. However, its function in teleost fish needs to be further explored. In this study, we characterized rainbow trout MC3R (rtMC3R), which encoded a putative protein of 331 amino acids. Phylogenetic and chromosomal synteny analyses showed that rtMC3R was closely related to bony fishes. Quantitative PCR (qPCR) revealed that the transcripts of rtMC3R were highly expressed in the brain and muscle. The cellular function of rtMC3R was further verified by the signal-pathway-specific luciferase reporter assays. Four agonists such as α-MSH, ß-MSH, ACTH (1-24), and NDP-MSH can active rtMC3R, increasing the production of intracellular cAMP and upregulating MAPK/ERK signals. Moreover, we found that rtMC3R stimulated with α-MSH and NDP-MSH can significantly inhibit the NF-κB signaling pathway. This research will be helpful for further studies on the function of MC3R in rainbow trout, especially the role of energy metabolism and immune regulation.

Functional Characterization of Melanocortin-3 Receptor in a Hibernating Cavefish Onychostoma macrolepis.

Melanocortin-3 receptor (MC3R) plays an important role in the energy homeostasis of animals under different nutritional conditions. Onychostoma macrolepis is a hibernating cavefish found in the northern part of the Yangtze River, and its adaptation to a nutrient-poor environment has attracted growing interest. In this study, we characterized the protein structure of Onychostoma macrolepis Mc3r (omMc3r), examined its tissue distribution, and investigated its function in mediating cellular signaling. We showed that the CDS of omMc3r is 978 bp, encoding a putative protein of 325 amino acids. Homology and phylogenetic analyses indicated that omMc3r is evolutionary close to cyprinids. Real-time quantitative PCR (RT-qPCR) revealed that omMc3r was highly expressed in the liver and brain. The functions of omMc3r to mediate ligands activating downstream signaling have also been confirmed by using signal pathway-specific reporters. The four agonists α-MSH, ß-MSH, NDP-MSH, and ACTH (1-24) can all activate the cAMP and MAPK/ERK signaling pathway, albeit with different potency orders. The "primitive" ligand ACTH (1-24) had the highest potency on the cAMP signaling pathway, while the synthetic ligand NDP-MSH had the highest activation effect on the MAPK/ERK signaling pathway. This research will lay the foundation for studying the energy regulation mechanism of cavefish in an oligotrophic environment.

The complete mitochondrial genome of Homatula berezowskii (Cypriniformes, Nemacheilidae, Nemachilinae).

Homatula berezowskii (Günther 1896), used to be recognized as a synonym of Homatula variegate, was now identified as a valid species. However, these morphological studies lack genetic evidence to support. In the present study, we determined the first complete mitochondrial genome of H. berezowskii by Sanger dideoxy sequencing. The genome size is 16,570 bp and it contains 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and a non-coding control region (D-loop). Phylogenetic analyses based on the complete mitochondrial genome indicated the H. berezowskii was clustered with H. potanini first and then with the H. variegate. This work may be helpful to clarify the taxonomic status of H. berezowskii.