Transcriptional regulation of Znt family members znt4, znt5 and znt10 and their function in zinc transport in yellow catfish (Pelteobagrus fulvidraco).

The study characterized the transcriptionally regulatory mechanism and functions of three zinc (Zn) transporters (znt4, znt5 and znt10) in Zn2+ metabolism in yellow catfish (Pelteobagrus fulvidraco), commonly freshwater fish in China and other countries. We cloned the sequences of znt4 promoter, spanning from -1217 bp to +80 bp relative to TSS (1297 bp); znt5, spanning from -1783 bp to +49 bp relative to TSS (1832 bp) and znt10, spanning from -1923 bp to +190 bp relative to TSS (2113 bp). In addition, after conducting the experiments of sequential deletion of promoter region and mutation of potential binding site, we found that the Nrf2 binding site (-607/-621 bp) and Klf4 binding site (-5/-14 bp) were required on znt4 promoter, the Mtf-1 binding site (-1674/-1687 bp) and Atf4 binding site (-444/-456 bp) were required on znt5 promoter and the Atf4 binding site (-905/-918 bp) was required on znt10 promoter. Then, according to EMSA and ChIP, we found that Zn2+ incubation increased DNA affinity of Atf4 to znt5 or znt10 promoter, but decreased DNA affinity of Nrf2 to znt4 promoter, Klf4 to znt4 promoter and Mtf-1 to znt5 promoter. Using fluorescent microscopy, it was revealed that Znt4 and Znt10 were located in the lysosome and Golgi, and Znt5 was located in the Golgi. Finally, we found that znt4 knockdown reduced the zinc content of lysosome and Golgi in the control and zinc-treated group; znt5 knockdown reduced the zinc content of Golgi in the control and zinc-treated group and znt10 knockdown reduced the zinc content of Golgi in the zinc-treated group. High dietary zinc supplement up-regulated Znt4 and Znt5 protein expression. Above all, for the first time, we revealed that Klf4 and Nrf2 transcriptionally regulated the activities of znt4 promoter; Mtf-1 and Atf4 transcriptionally regulated the activities of znt5 promoter and Atf4 transcriptionally regulated the activities of znt10 promoter, which provided innovative regulatory mechanism of zinc transporting in yellow catfish. Our study also elucidated their subcellular location, and regulatory role of zinc homeostasis in yellow catfish.

Characterization and expression analysis of seven lipid metabolism-related genes in yellow catfish Pelteobagrus fulvidraco fed high fat and bile acid diet.

SREBPs, such as SREBP1 and SREBP2, were the key transcriptional factors regulating lipid metabolism. The processing of SREBPs involved many genes, such as scap, s1p, s2p, cideb. Here, we deciphered the full-length cDNA sequences of scap, srebp1, srebp2, s1p, s2p, cideb and cidec from yellow catfish Pelteobagrus fulvidraco. Their full-length cDNA sequences ranged from 1587 to 3884 bp, and their ORF length from 1191 to 2979 bp, encoding 396-992 amino acids. Some conservative domains were predicted, including the multiple transmembrane domains in SCAP, the bHLH-ZIP domain in SREBP1 and SREBP2, the ApoB binding region, ER targeting region and LD targeting region in CIDEb, the LD targeting region in the CIDEc, the conserved catalytic site and processing site in S1P, and the transmembrane helix domain in S2P. Their mRNA expression could be observed in the heart, spleen, liver, kidney, brain, muscle, intestine and adipose, but varied with tissues. The changes of their mRNA expression in responses to high-fat (HFD) and bile acid (BA) diets were also investigated in the brain, heart, intestine, kidney and spleen tissues. In the brain, HFD significantly increased the mRNA expression of seven genes (scap, srebp1, srebp2, s1p, s2p, cideb and cidec), and the BA attenuated the increase of scap, srebp1, srebp2, s1p, s2p, cideb and cidec mRNA expression induced by HFD. In the heart, HFD significantly increased the mRNA abundances of six genes (srebp1, srebp2, scap, s2p, cideb and cidec), and BA attenuated the increase of their mRNA abundances induced by HFD. In the intestine, HFD increased the cideb, s1p and s2p mRNA abundances, and BA attenuated the HFD-induced increment of their mRNA abundances. In the kidney, HFD significantly increased the scap, cidec and s1p mRNA expression, and BA diet attenuated the increment of their mRNA expression. In the spleen, HFD treatment increased the scap, srebp2, s1p and s2p mRNA expression, and BA diet attenuated HFD-induced increment of their mRNA expression. Taken together, our study elucidated the characterization, expression profiles and transcriptional response of seven lipid metabolic genes, which would serve as the good basis for the further exploration into their function and regulatory mechanism in fish.

Characterization of fifteen key genes involved in iron metabolism and their responses to dietary iron sources in yellow catfish Pelteobagrus fulvidraco.

BACKGROUND: Iron is an essential metal element for organisms, whose metabolism is regulated by many genes and also dietary iron sources. However, the characterization, distribution and the responses of iron metabolism-related genes to different iron sources were not clear in fish. METHODS: The full-length cDNA sequences of fifteen iron metabolism-relevant genes (tf, tfr1, hp, fpn1, ho1, ho2, tfr2, hjv, hepcidin, fth, ftl, ftm, irp1, irp2 and hif2α.) were obtained via 3' and 5' RACE PCR from yellow catfish, a widely distributed freshwater teleost in China and other Asian countries. Their molecular characterizations were analyzed via the bioinformatic methods. Real-time quantitative PCR was used to explore their mRNA distribution in nine tissues. Their mRNA expression responses in four tissues (heart, brain, kidney and gill) were explored in yellow catfish fed diets with five iron sources, including ferrous sulfate (FeSO4), ferrous bisglycinate (Fe-Gly), ferrous chloride (FeCl2), ferric citrate (Fe-CA) and ferric oxide nanoparticles (Fe2O3NPs). RESULTS: Compared with mammals and other teleost, these members shared similar domains. Their mRNAs were expressed in nine tested tissues, but mRNA levels varied. Yellow catfish fed the diets containing Fe-Gly and Fe2O3NPs had higher iron contents in heart, brain, kidney and gill. Meantime, different dietary iron sources addition affected their mRNA expression differentially in brain, heart, kidney and gill. It should be pointed out that only three biological replicate tanks were used in the present feeding treatment, and more biological replicate tanks (more than five) should be emphasized in further researches. CONCLUSION: Taken together, our study identified fifteen iron metabolism-relevant genes, explored their mRNA expression in nine tissues, and their mRNA expression in the responses to different dietary iron sources in four tissues, indicating their important regulatory function in iron metabolism and homeostasis.

Effects of Different Dietary Zinc (Zn) Sources on Growth Performance, Zn Metabolism, and Intestinal Health of Grass Carp.

This research was conducted to investigate the effects of four dietary zinc (Zn) sources on growth performance, Zn metabolism, antioxidant capacity, endoplasmic reticulum (ER) stress, and tight junctions in the intestine of grass carp Ctenopharyngodon idella. Four Zn sources consisted of Zn dioxide nanoparticles (ZnO NPs), Zn sulfate heptahydrate (ZnSO4·7H2O), Zn lactate (Zn-Lac), and Zn glycine chelate (Zn-Gly), respectively. Grass carp with an initial body weight of 3.54 g/fish were fed one of four experimental diets for 8 weeks. Compared to inorganic Zn (ZnSO4·7H2O), grass carp fed the ZnO NPs and Zn-Gly diets exhibited better growth performance. Furthermore, grass carp fed the organic Zn (Zn-Lac and Zn-Gly) diets displayed enhanced Zn transport activity, improved intestinal histology, and increased intestinal tight junction-related genes expression compared to other groups. In comparison to other Zn sources, dietary ZnO NPs caused increased Zn deposition and damaged antioxidation capacity by suppressing antioxidant enzymatic activities and related gene expression in the intestine. Grass cap fed the ZnO NPs diet also exhibited lower mRNA abundance of endoplasmic reticulum (ER) stress- and tight junction-associated genes. According to the above findings, it can be concluded that dietary organic Zn addition (Zn-Lac and Zn-Gly) is more beneficial for intestinal health in grass carp compared to inorganic and nanoform Zn sources. These findings provide valuable insights into the application of organic Zn sources, specifically Zn-Lac and Zn-Gly, in the diets for grass carp and potentially for other fish species.

Enrofloxacin (ENR) exposure induces lipotoxicity by promoting mitochondrial fragmentation via dephosphorylation of DRP1 at S627 site.

Enrofloxacin (ENR) is a kind of widespread hazardous pollutant on aquatic ecosystems and causes toxic effects, such as disorders of metabolism, on aquatic animals. However, its potential mechanisms at an environmental concentration on metabolic disorders of aquatic organisms remain unclear. Herin, we found that hepatic lipotoxicity was induced by ENR exposure, which led to ENR accumulation, oxidative stress, mitochondrial fragmentation, and fatty acid transfer blockage from lipid droplets into fragmented mitochondria. ENR-induced lipotoxicity and mitochondrial ß-oxidation down-regulation were mediated by reactive oxygen species (ROS). Moreover, dynamin-like protein 1 (DRP1) mediated ENR-induced mitochondrial fragmentation and changes of lipid metabolism. Mechanistically, ENR induced increment of DRP1 mitochondrial localization via dephosphorylating DRP1 at S627 and promoted its interaction with mitochondrial fission factor (MFF), leading to mitochondria fragmentation. For the first time, our study provides an innovative mechanistic link between hepatic lipotoxicity and mitochondrial fragmentation under ENR exposure, and thus identifies previously unknown mechanisms for the direct relationship between environmental ENR concentration and lipotoxicity in aquatic animals. Our study provides innovative insights for toxicological mechanisms and environmental risk assessments of antibiotics in aquatic environment.

Novel mechanism for zinc inducing hepatic lipolysis via the HDAC3-mediated deacetylation of ß-catenin at lysine 311.

Zinc (Zn) is a multipurpose trace element indispensable for vertebrates and possesses essential regulatory roles in lipid metabolism, but the fundamental mechanism remains largely unknown. In the current study, we found that a high-Zn diet significantly increased hepatic Zn content and influenced the expression of Zn transport-relevant genes. Dietary Zn addition facilitated lipolysis, inhibited lipogenesis, and controlled ß-catenin signal; Zn also promoted T-cell factor 7-like 2 (TCF7L2) to interact with ß-catenin and regulating its transcriptional activity, thereby inducing lipolysis and inhibiting lipogenesis; Zn-induced lipid degradation was mediated by histone deacetylase 3 (HDAC3) which was responsible for ß-catenin deacetylation and the regulation of ß-catenin signal under the Zn treatment. Mechanistically, Zn promoted lipid degradation via stimulating HDAC3-mediated deacetylation of ß-catenin at lysine 311 (K311), which enhanced the interaction between ß-catenin and TCF7L2 and then transcriptionally inhibited fatty acid synthase (FAS), 2-acylglycerol O-acyltransferase 2 (MOGAT2), and sterol regulatory element-binding protein 1 (SREBP1) expression, but elevated the mRNA abundance of adipose triglyceride lipase (ATGL), hormone-sensitive lipase a (HSLA) and carnitine palmitoyltransferase 1a1b (CPT1A1B). Overall, our research reveals a novel mechanism into the important roles of HDAC3/ß-catenin pathway in Zn promoting lipolysis and inhibiting lipogenesis, and highlights the essential roles of K311 deacetylation in ß-catenin actions and lipolytic metabolism, and accordingly provides novel insight into the prevention and treatment of steatosis in the vertebrates.

Copper induces liver lipotoxicity disease by up-regulating Nrf2 expression via the activation of MTF-1 and inhibition of SP1/Fyn pathway.

Excessive copper (Cu) intake leads to hepatic lipotoxicity disease, which has adverse effects on health, but the underlying mechanism is unclear. We found that Cu increased lipotoxicity by promoting Nrf2 recruitment to the ARE site in the promoters of five lipogenic genes (g6pd, 6pgd, me, icdh and pparγ). We also found that Cu affected the Nrf2 expression via different pathways: metal regulatory transcription factor 1 (MTF-1) mediated the Cu-induced Nrf2 transcriptional activation; Cu also enhanced the expression of Nrf2 by inhibiting the SP1 expression, which was achieved by inhibiting the negative regulator Fyn of Nrf2. These promoted the enrichment of Nrf2 in the nucleus and ultimately affected lipotoxicity. Thus, for the first time, we elucidated that Cu induced liver lipotoxicity disease by up-regulating Nrf2 expression via the MTF-1 activation and the inhibition of SP1/Fyn pathway. Our study elucidates the Cu-associated obesity and NAFLD for fish and possibly humans.

Glycophagy mediated glucose-induced changes of hepatic glycogen metabolism via OGT1-AKT1-FOXO1Ser238 pathway.

Glycophagy is the autophagy degradation of glycogen. However, the regulatory mechanisms for glycophagy and glucose metabolism remain unexplored. Herein, we demonstrated that high-carbohydrate diet (HCD) and high glucose (HG) incubation induced glycogen accumulation, protein kinase B (AKT)1 expression and AKT1-dependent phosphorylation of forkhead transcription factor O1 (FOXO1) at Ser238 in the liver tissues and hepatocytes. The glucose-induced FOXO1 phosphorylation at Ser238 prevents FOXO1 entry into the nucleus and the recruitment to the GABA(A) receptor-associated protein like 1 (gabarapl1) promoter, reduces the gabarapl1 promoter activity, and inhibits glycophagy and glucose production. The glucose-dependent O-GlcNAcylation of AKT1 by O-GlcNAc transferase (OGT1) enhances the stability of AKT1 protein and promotes its binding with FOXO1. Moreover, the glycosylation of AKT1 is crucial for promoting FOXO1 nuclear translocation and inhibiting glycophagy. Our studies elucidate a novel mechanism for glycophagy inhibition by high carbohydrate and glucose via OGT1-AKT1-FOXO1Ser238 pathway in the liver tissues and hepatocytes, which provides critical insights into potential intervention strategies for glycogen storage disorders in vertebrates, as well as human beings.

Phosphorus Overload Promotes Hepatic Lipolysis by Suppressing GSK3ß-Dependent Phosphorylation of PPARα at Ser84 and Thr265 in a Freshwater Teleost.

Excessive phosphorus (Pi) contributes to eutrophication in an aquatic environment, which threatens human and fish health. However, the mechanisms by which Pi overload influences aquatic animals remain largely unexplored. In the present study, Pi supplementation increased the Pi content, inhibited lipid accumulation and lipogenesis, and stimulated lipolysis in the liver. Pi supplementation increased the phosphorylation of glycogen synthase kinase-3 ß (GSK3ß) at serine 9 (S9) but inhibited the phosphorylation of GSK3α at tyrosine 279 (Y279), GSK3ß at tyrosine 216 (Y216), and peroxisome proliferator-activated receptor α (PPARα) at serine 84 (S84) and threonine 265 (T265). Pi supplementation also upregulated PPARα protein expression and stimulated its transcriptional activity, thereby inducing lipolysis. Pi suppressed GSK3ß activity and prevented GSK3ß, but not GSK3α, from interacting with PPARα, which in turn alleviated PPARα phosphorylation. GSK3ß-induced phosphorylation of PPARα was dependent on GSK3ß S9 dephosphorylation rather than Y216 phosphorylation. Mechanistically, underphosphorylation of PPARα mediated Pi-induced lipid degradation through transcriptionally activating adipose triglyceride lipase (atgl) and very long-chain-specific acyl-CoA dehydrogenase (acadvl). Collectively, our findings uncovered a new mechanism by which Pi facilitates lipolysis via the GSK3ß-PPARα pathway and highlighted the importance of S84 and T265 phosphorylation in PPARα action.

Klf4-Sirt3/Pparα-Lcad pathway contributes to high phosphate-induced lipid degradation.

BACKGROUND: Phosphorus commonly reduces lipid deposition in the vertebrates. However, the underlying mechanisms involved in the process remain unclear. METHODS: Yellow catfish were given three experimental diets with dietary phosphate levels of 3.22, 6.47 and 7.99 g Pi kg- 1, respectively, for 8 weeks. The contents of triglyceride, non-esterified free fatty acids, adenosine triphosphate, nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide, enzymatic activities, mRNA and protein expression were determined in the intestinal tissues. Hematoxylin and eosin, Oil Red O staining, and transmission electron microscope were performed for intestinal tissues. Primary intestinal epithelial cells were isolated from yellow catfish intestine. Western blot analysis, Immunoprecipitation assays, Immunofluorescence staining, and RNA extraction and quantitative real-time PCR were decided. Luciferase reporter assays and electrophoretic mobility shift assay were used to evaluate the function of Sirt3, PPARα and Lcad promoters. RESULTS: High dietary phosphate intake activated intestinal phosphate absorption and excretion, and reduced lipid deposition through increasing lipolysis in the intestine. Moreover, phosphate incubation increased the mRNA and protein expression of krüppel like factor 4 (klf4), silent mating-type information regulation 2 homolog 3 (sirt3), peroxisome proliferator activated receptor alpha (pparα) and long chain acyl-CoA dehydrogenase (lcad) in the intestinal epithelial cells (IECs), and klf4 knockdown attenuated the phosphate-induced increase of protein levels of Sirt3, Pparα and Lcad. Further investigation found that Klf4 overexpression increased the activity of sirt3 and pparα promoters, which in turn reduced the acetylation and protein level of Lcad. CONCLUSION: Dietary Pi excess induced lipid degradation by the activation of the Klf4-Sirt3/Pparα-Lcad pathway in the intestine and primary IECs. Video Abstract.

Effects of Dietary Selenium and Oxidized Fish Oils on Intestinal Lipid Metabolism and Antioxidant Responses of Yellow Catfish Pelteobagrus fulvidraco.

Currently, the effect of selenium and oxidized fish oil interactions on the intestinal lipid metabolism and antioxidant responses of fish remains unknown. Herein, yellow catfish Pelteobagrus fulvidraco (weight: 3.99 ± 0.01 g) were used as experimental animals and were fed four diets: an adequate amount of selenium (0.25 mg kg-1) with fresh fish oil (A-Se+FFO), an adequate amount of selenium with oxidized fish oil (A-Se+OFO), a high amount of selenium (0.50 mg kg-1) with fresh fish oil (H-Se+FFO), and a high amount of selenium with oxidized fish oil (H-Se+OFO). The feeding experiment was conducted for 10 weeks. The results showed that selenium supplementation alleviated the intestinal tissue damage and reduced the lipid accumulation that was induced by oxidized fish oils. Meanwhile, we also found that 0.50 mg kg-1 selenium reduced the oxidative stress that is caused by oxidized fish oils through increasing the GSH and the activity and mRNA expression of antioxidant enzymes. Dietary selenium and oxidized fish oils also affected the mRNA expression of intestinal selenoproteins including selenow2a, selenop2, and selenot2. Mechanistically, Se and oxidized eicosapentaenoic acid (oxEPA) influenced the GSH content by affecting the DNA binding ability of activating transcription factor (ATF) 3 to the slc7a11 promoter. For the first time, our results suggested that selenium alleviated the oxidized fish oil-induced intestinal lipid deposition and the oxidative stress of the fish. We also elucidated the novel mechanism of selenium increasing the GSH content by affecting the interaction of ATF3 and the slc7a11 promoter.

Dietary choline prevents high fat-induced disorder of hepatic cholesterol metabolism through SREBP-2/HNF-4α/CYP7A1 pathway in a freshwater teleost yellow catfish Pelteobagrus fulvidraco.

Lipid overload-induced hepatic cholesterol accumulation is a major public health problem worldwide, and choline has been reported to ameliorate cholesterol accumulation, but its mechanism remains unclear. Our study found that choline prevented high-fat diet (HFD)-induced cholesterol metabolism disorder and enhanced choline uptake and phosphatidylcholine synthesis in the liver tissues; choline incubation prevented fatty acid (FA)-induced cholesterol accumulation and FA-induced inhibition of bile acid synthesis. Moreover, compared to single FA incubation, choline incubation or FA + choline co-incubation increased the mRNA abundances and protein levels of HNF4α and up-regulated the degradation of cholesterol into bile acids. Mechanistically, choline prevented the FA-induced accumulation of SREBP2 protein and the interaction between SREBP2 and HNF4α, thereby enhancing the DNA binding capacity of the HNF4α to the CYP7A1 promoter, and promoting the degradation of cholesterol into bile acids. Our study elucidated the novel regulatory mechanisms of choline preventing HFD-induced cholesterol accumulation and increasing bile acid synthesis by SREBP-2/HNF-4α/CYP7A1 pathway.

Transcriptional Regulation and Protein Localization of Zip10, Zip13 and Zip14 Transporters of Freshwater Teleost Yellow Catfish Pelteobagrus fulvidraco Following Zn Exposure in a Heterologous HEK293T Model.

Zip family proteins are involved in the control of zinc (Zn) ion homeostasis. The present study cloned the promoters and investigated the transcription responses and protein subcellular localizations of three LIV-1 subfamily members (zip10, zip13, and zip14) from common freshwater teleost yellow catfish, Pelteobagrus fulvidraco, using in vitro cultured HEK293T model cells. The 2278 bp, 1917 bp, and 1989 bp sequences of zip10, zip13, and zip14 promoters, respectively, were subcloned into pGL3-Basic plasmid for promoter activity analysis. The pcDNA3.1 plasmid coding EGFP tagged pfZip10, pfZip13, and pfZip14 were generated for subsequent confocal microscope analysis. Several potential transcription factors' binding sites were predicted within the promoters. In vitro promoter analysis in the HEK293T cells showed that high Zn administration significantly reduced the transcriptional activities of the zip10, zip13, and zip14 promoters. The -2017 bp/-2004 bp MRE in the zip10 promoter, the -360 bp/-345 bp MRE in the zip13 promoter, and the -1457 bp/-1442 bp MRE in the zip14 promoter were functional loci that were involved in the regulation of the three zips. The -606 bp/-594 bp KLF4 binding site in the zip13 promoter was a functional locus responsible for zinc-responsive regulation of zip13. The -1383 bp/-1375 bp STAT3 binding site in the zip14 promoter was a functional locus responsible for zinc-responsive regulation of zip14. Moreover, confocal microscope analysis indicated that zinc incubation significantly reduced the fluorescence intensity of pfZip10-EGFP and pfZip14-EGFP but had no significant influence on pfZip13-EGFP fluorescence intensity. Further investigation found that pfZip10 localizes on cell membranes, pfZip14 colocalized with both cell membranes and lysosome, and pfZip13 colocalized with intracellular ER and Golgi. Our research illustrated the transcription regulation of zip10, zip13, and zip14 from P. fulvidraco under zinc administration, which provided a reference value for the mechanisms involved in Zip-family-mediated control of zinc homeostasis in vertebrates.

Fish Meal Replacement by Mixed Plant Protein in the Diets for Juvenile Yellow Catfish Pelteobagrus fulvidraco: Effects on Growth Performance and Health Status.

Increasing dietary replacement levels of fish meal by alternative plant proteins are of value for aquaculture. Here, a 10-week feeding experiment was undertaken to explore the effects of fish meal replacement by mixed plant protein (at a 2 : 3 ratio of cottonseed meal to rapeseed meal) on growth performance, oxidative and inflammatory responses, and mTOR pathway of yellow catfish Pelteobagrus fulvidraco. Yellow catfish (2.38 ± 0.1 g, mean ± SEM) were randomly divided into 15 indoors fiberglass tanks, 30 fish each tank, and fed five isonitrogenous (44% crude protein) and isolipidic (9% crude fat) diets with fish meal replaced by mixed plant protein at 0% (the control), 10% (RM10), 20% (RM20), 30% (RM30), and 40% (RM40), respectively. Among five groups, fish fed the control, and RM10 diets tended to have higher growth performance, higher protein content, and lower lipid content in livers. Dietary mixed plant protein substitute increased hepatic free gossypol content and damaged liver histology and reduced the serum total essential amino acids, total nonessential amino acids, and total amino acid contents. Yellow catfish fed the control, and RM10 diets tended to have higher antioxidant capacity. Dietary mixed plant protein replacement tended to promote proinflammatory responses and inhibited mTOR pathway. Based on the second regression analysis of SGR against mixed plant protein substitutes, the optimal replacement level of fish meal by mixed plant protein was 8.7%.

Physiological and transcriptomic analyses reveal the toxicological mechanism and risk assessment of environmentally-relevant waterborne tetracycline exposure on the gills of tilapia (Oreochromis niloticus).

With the increasing application of tetracycline (TC) in medical treatment, animal husbandry and aquaculture in recent decades, high quantities of TC have been frequently detected in the aquatic environment, and accordingly TC-related toxicity and environmental pollution have become a global concern. The present study was performed to explore the toxicological influences of TC exposure at its environmentally relevant concentrations on the gills of tilapia Oreochromis niloticus, based on the alteration in histopathology, oxidative stress, inflammatory response, cell cycle, mitochondrial function, apoptosis, and transcriptomic analysis. Our findings revealed that TC exposure damaged the structure and function, induced oxidative stress, affected inflammatory responses, and reduced Na+/K+-ATPase (NKA) activity in the gills. TC also caused the inhibition in cell cycle, resulted in mitochondrial dysfunction and activated apoptosis. Further transcriptomic analysis indicated the extensive influences of TC exposure on the gill function, and immune system was the main target to waterborne TC exposure. These results elucidated that environmental TC had more complex toxicological effects on gills of fish than previously assessed, and provided novel insight into molecular toxicology of TC on fish and good basis for assessing the environmental risk of TC.

Molecular Characterization and Functional Analysis of Two Steroidogenic Genes TSPO and SMAD4 in Yellow Catfish.

The steroid hormones are required for gonadal development in fish. The present study was undertaken to characterize the cDNA and promoter sequences of TSPO and SMAD4 genes in yellow catfish Pelteobagrus fulvidraco, explored the mRNA tissue expression and deciphered their promoter regions. Yellow catfish TSPO and SMAD4 shared the similar domains to the corresponding genes from other vertebrates. The TSPO and SMAD4 mRNAs were widely expressed in the detected tissues, but at different levels. Several transcription factors were predicted, such as Sp, GATA, AP1, SOX1, SRY, STAT, HNF4α, PPARγ, Pu.1 and FOXL2. PPARγ overexpression increased but STAT3 overexpression reduced TSPO promoter activity, and FOXL2 overexpression inhibited the promoter activity of TSPO and SMAD4. The site mutation and EMSA analysis indicated that TSPO promoter possessed STAT3 and FOXL2 sites. Overall, our provided the novel understanding into the transcriptionally regulatory mechanisms of TSPO and SMAD4 in fish.

Functional Analysis of Steroidogenic Factor 1 (sf-1) and 17α-Hydroxylase/Lyase (cyp17α) Promoters in Yellow Catfish Pelteobagrus fulvidraco.

The present study was performed to clone and characterize the structures and functions of steroidogenic factor 1 (sf-1) and 17α-hydroxylase/lyase (cyp17α) promoters in yellow catfish Pelteobagrus fulvidraco, a widely distributed freshwater teleost. We successfully obtained 1981 and 2034 bp sequences of sf-1 and cyp17α promoters, and predicted the putative binding sites of several transcription factors, such as Peroxisome proliferator-activated receptor alpha (PPARα), Peroxisome proliferator-activated receptor gamma (PPARγ) and Signal transducer and activator of transcription 3 (STAT3), on sf-1 and cyp17α promoter regions, respectively. Overexpression of PPARγ significantly increased the activities of sf-1 and cyp17α promoters, but overexpression of PPARα significantly decreased the promoter activities of sf-1 and cyp17α. Overexpression of STAT3 reduced the activity of the sf-1 promoter but increased the activity of the cyp17α promoter. The analysis of site-mutation and electrophoretic mobility shift assay suggested that the sf-1 promoter possessed the STAT3 binding site, but did not the PPARα or PPARγ binding sites. In contrast, only the PPARγ site, not PPARα or STAT3 sites, was functional with the cyp17α promoter. Leptin significantly increased sf-1 promoter activity, but the mutation of STAT3 and PPARγ sites decreased leptin-induced activation of sf-1 promoter. Our findings offered the novel insights into the transcriptional regulation of sf-1 and cyp17α and suggested leptin regulated sf-1 promoter activity through STAT3 site in yellow catfish.

Effects of dietary carbohydrate sources on lipid metabolism and SUMOylation modification in the liver tissues of yellow catfish.

Dysregulation in hepatic lipid synthesis by excess dietary carbohydrate intake is often relevant with the occurrence of fatty liver; therefore, the thorough understanding of the regulation of lipid deposition and metabolism seems crucial to search for potential regulatory targets. In the present study, we examined TAG accumulation, lipid metabolism-related gene expression, the enzyme activities of lipogenesis-related enzymes, the protein levels of transcription factors or genes involving lipogenesis in the livers of yellow catfish fed five dietary carbohydrate sources, such as glucose, maize starch, sucrose, potato starch and dextrin, respectively. Generally speaking, compared with other carbohydrate sources, dietary glucose promoted TAG accumulation, up-regulated lipogenic enzyme activities and gene expressions, and down-regulated mRNA expression of genes involved in lipolysis and small ubiquitin-related modifier (SUMO) modification pathways. Further studies found that sterol regulatory element binding protein 1 (SREBP1), a key transcriptional factor relevant to lipogenic regulation, was modified by SUMO1. Mutational analyses found two important sites for SUMOylation modification (K254R and K264R) in SREBP1. Mutant SREBP lacking lysine 264 up-regulated the transactivation capacity on an SREBP-responsive promoter. Glucose reduced the SUMOylation level of SREBP1 and promoted the protein expression of SREBP1 and its target gene stearoyl-CoA desaturase 1 (SCD1), indicating that SUMOylation of SREBP1 mediated glucose-induced hepatic lipid metabolism. Our study elucidated the molecular mechanism of dietary glucose increasing hepatic lipid deposition and found that the SREBP-dependent transactivation was regulated by SUMO1 modification, which served as a new target for the transcriptional programmes governing lipid metabolism.

Novel insights for SREBP-1 as a key transcription factor in regulating lipogenesis in a freshwater teleost, grass carp Ctenopharyngodon idella.

Disturbances in lipid metabolism are at the core of several health issues facing modern society, including fatty liver and obesity. The sterol regulatory element-binding protein 1 (SREBP-1) is one important transcription factor regulating lipid metabolism, but the relevant mechanism still remains unknown. The present study determined the transcriptional regulation of SREBP-1 and its target genes (including acetyl-CoA carboxylase α (accα), fatty acid synthase (fas) and stearoyl-CoA desaturase 1 (scd1)) in a freshwater teleost, grass carp Ctenopharyngodon idella. We cloned and characterised the 1988 bp, 2043 bp, 1632 bp and 1889 bp sequences of srebp-1, accα, scd1 and fas promoters, respectively. A cluster of putative binding sites of transcription factors, such as specific protein, yin yang 1, nuclear factor Y, sterol response elements (SRE) and enhancer box (E-box) element, were predicted on their promoter regions. Overexpression of nSREBP-1 reduced srebp-1 promoter activity, increased scd1 and fas promoter activity but did not influence accα promoter activity. The site-mutation and electrophoretic mobility shift assay analysis indicated that srebp-1, fas and scd1 promoters, but not accα promoter, possessed SRE. In Ctenopharyngodon idella kidney (CIK) cells of grass carp, nSREBP-1 overexpression significantly reduced srebp-1 mRNA expression and up-regulated miR-29 mRNA expression. The 3'UTR of srebp-1 possessed the potential miR-29 binding site and miR-29 up-regulated the luciferase activity of srebp-1 3'UTR and srebp-1 mRNA expression, implying a self-activating loop of SREBP-1 and miR-29 in grass carp. Based on the above-mentioned results, we found two novel transcriptional mechanisms for SREBP-1 in grass carp: (1) the auto-regulation sited on the SREBP-1 promoter regions was suppressive and (2) there was a self-activating loop of SREBP-1 and miR-29.

Molecular characterization of nine suppressors of cytokine signaling (SOCS) genes from yellow catfish Pelteobagrus fulvidraco and their changes in mRNA expression to dietary carbohydrate levels.

Suppressors of cytokine signaling (SOCS) are important molecules that mediates the regulation of glucose homeostasis. Here, we cloned and characterized the full-length cDNA sequences of nine genes of the SOCS family (SOCS1, 2, 3, 3b, 5, 5b, 6, 7 and CISH) from yellow catfish P. fulvidraco, explored their mRNA abundance across the tissues and their mRNA changes to dietary carbohydrate levels. Structural analysis indicated that the nine members shared conserved functional domains to the orthologues of the mammalian SOCS members, such as SRC homology 2 and the SOCS domains. Their mRNAs were constitutively expressed in various tissues but changed among the tissues. Their mRNA expression in response to dietary carbohydrate levels were explored in the liver, muscle, intestine, testis and ovary. Dietary carbohydrate addition showed significant effects on the mRNA levels of the nine SOCS members. Moreover, their mRNA expressions in response to dietary carbohydrate levels were also tissue-dependent. These indicated that SOCS members potentially mediated the utilization of dietary carbohydrate in yellow catfish.