The role of miR-216a-mediated Nrf2 pathway in muscle oxidative stress of Siniperca chuatsi induced by cadmium.

Cadmium (Cd) is a toxic heavy metal pollutant in the environment. Excessive Cd in water has toxic effects on fish, endangering their healthy growth and ultimately affecting the quality and safety of aquatic products. To evaluate the toxicity of excessive Cd to fish through potential oxidative damage, Siniperca chuatsi was exposed to Cd in water for 15 days. It was found that Cd exposure significantly decreased the survival rate of S. chuatsi and Cd was detected in their muscle. Meanwhile, Cd disrupts the redox balance by reducing antioxidant enzyme activities, increasing reactive oxygen species (ROS) and malondialdehyde (MDA) levels in muscle, and promoting oxidative damage. Histomorphology showed that enlargement of muscle fiber gaps, cell swelling and vacuolar degeneration after Cd exposure. In addition, Cd toxicity induced up-regulating the expression of miR-216a, while down-regulation of Nrf2 protein and its downstream antioxidant enzyme genes expression. Further analysis revealed that miR-216a was significantly negatively correlated with the expression of Nrf2, and injection of miR-216a antagomir significantly enhanced the expression of Nrf2 and antioxidant enzyme genes, as well as the activity of antioxidant enzymes, thereby reducing the damage of Cd to fish. These results suggested that miR-216a-mediated Nrf2 signaling pathway plays an important role in Cd-induced oxidative stress of S. chuatsi muscle.

The circadian rhythm regulates branched-chain amino acids metabolism in fast muscle of Chinese perch (Siniperca chuatsi) during short-term fasting by Clock-KLF15-Bcat2 pathway.

As an internal time-keeping mechanism, circadian rhythm plays crucial role in maintaining homoeostasis when in response to nutrition change; meanwhile, branched-chain amino acids (BCAA) in skeletal muscle play an important role in preserving energy homoeostasis during fasting. Previous results from our laboratory suggested that fasting can influence peripheral circadian rhythm and BCAA metabolism in fish, but the relationship between circadian rhythm and BCAA metabolism, and whether circadian rhythm regulates BCAA metabolism to maintain physiological homoeostasis during fasting remains unclear. This study shows that the expression of fifteen core clock genes as well as KLF15 and Bcat2 is highly responsive to short-term fasting in fast muscle of Siniperca chuatsi, and the correlation coefficient between Clock and KLF15 expression is enhanced after fasting treatment. Furthermore, we demonstrate that the transcriptional expression of KLF15 is regulated by Clock, and the transcriptional expression of Bcat2 is regulated by KLF15 by using dual-luciferase reporter gene assay and Vivo-morpholinos-mediated gene knockdown technique. Therefore, fasting imposes a dynamic coordination of transcription between the circadian rhythm and BCAA metabolic pathways. The findings highlight the interaction between circadian rhythm and BCAA metabolism and suggest that fasting induces a switch in KLF15 expression through affecting the rhythmic expression of Clock, and then KLF15 promotes the transcription of Bcat2 to enhance the metabolism of BCAA, thus maintaining energy homoeostasis and providing energy for skeletal muscle as well as other tissues.

Effect of starvation and refeeding on reactive oxygen species, autophagy and oxidative stress in Chinese perch (Siniperca chuatsi) muscle growth.

In skeletal muscle, autophagy regulates the development and growth of muscle fibres and maintains the normal muscle metabolism. Under starvation and refeeding conditions, the effect of reactive oxygen species (ROS) levels on skeletal muscle autophagy is still unclear, although the excessive accumulation of ROS has been shown to increase autophagy in cells. The purpose of this study was to explore the effects of starvation and diet after starvation on the autophagy of adult Chinese perch muscle, and to determine the level of ROS in the muscle. We performed zero (Normal control), three and seven starvation treatments on adult Chinese perch, and returned to normal feeding for 3 days after starvation for 7 days. In the muscles of the adult Chinese perch muscle after 3 days of starvation, the autophagy marker protein LC3 and the number of autophagosomes remained basically the same as in the normal feeding situation. However, on starvation for 7 days, the mitochondrial autophagy was sensitive and the number of autophagosomes increased, but the antioxidant-related molecules (malondialdehyde, catalase, glutathione S-transferase, glutathione and anti-superoxide anion) decreased and the accumulation of ROS was obvious. In addition, the extended starvation time also increased the level of LC3 protein. However, by refeeding after starvation this nutritional stress resulted in a decrease in ROS levels and a partial restoration of antioxidant enzyme activity. Our data show that in the adult Chinese perch muscle, starvation could reduce the antioxidant activity through the accumulation of ROS, and that the number of autophagosomes continues to increase. Refeeding after starvation could effectively compensate for the level of ROS, and restore the mRNA abundance of antioxidant genes and the activity of antioxidant enzymes to reduce autophagy and improve feed efficiency. Further research should optimize starvation conditions to reduce autophagy in muscles and maintain normal muscle metabolism.

The miRNA expression profile directly reflects the energy metabolic differences between slow and fast muscle with nutritional regulation of the Chinese perch (Siniperca chuatsi).

Fish skeletal muscles are composed of two distinct types, slow and fast muscles, and they play important roles in maintaining the body's movement and energy metabolism. The two types of muscle are easy to separate, so they are often used as the model system for studies on their physiological and functional characteristics. In this study, we revealed that the carbohydrate and lipid metabolic KEGG pathways are different between slow and fast muscles of Chinese perch with transcriptome analysis. In fast muscle, glucose metabolism was catabolic with higher glycolysis capacity, while in slow muscle, glucose metabolism was anabolic with more glycogen synthesis. In addition, oxidative metabolism in slow muscle was stronger than that in fast muscle. By analyzing the expression levels of 40 miRNAs involved in metabolism in the muscles of Chinese perch, 18 miRNAs were significantly upregulated and 7 were significantly downregulated in slow muscle compared with fast muscle. Based on functional enrichment analysis of their target genes, the differential expression levels of 17 miRNAs in slow and fast muscles were reflected in their carbohydrate and lipid metabolism. Among these, 15 miRNAs were associated with carbohydrate metabolism, and 6 miRNAs were associated with lipid metabolism. After 3 days of starvation, the expression levels of 15 miRNAs involved in glucose metabolism in fast and slow muscles increased. However, after 7 days of starvation, the mRNA levels of miR-22a, miR-23a, miR-133a-3p, miR-139, miR-143, miR-144, miR-181a and miR-206 decreased to basal levels. Our data suggest that the possible reason for the difference in glucose and lipid metabolism is that more miRNAs inhibit the expression of target genes in slow muscle.

Exposure to waterborne cadmium induce oxidative stress, autophagy and mitochondrial dysfunction in the liver of Procypris merus.

The present study was performed to determine the effect of waterborne cadmium (Cd) exposure on oxidative stress, autophagy and mitochondrial dysfunction, and to explore the mechanism of Cd-induced liver damage in freshwater teleost Procypris merus. To this end, P. merus were exposed to waterborne 0, 0.25 and 0.5 mg/L Cd for 30 days (equal to 0, 2.22 and 4.45 µmol Cd/l). The waterborne Cd exposure significantly increased hepatic Cd accumulation and impaired histological structure of the liver of P. merus. both low and high-dose waterborne Cd exposure induced oxidative stress in the liver of P. merus, through increases Malondialdehyde (MDA) and reactive oxide species (ROS) accumulation in the liver. The Cd-induced oxidative stress in liver may result from reduction of enzyme activities (superoxide dismutases (SOD), catalases (CAT), GSH-S-transferases (GST)) and transcriptional expression of antioxidant related genes (gpx1, gpx2, cata, gsta1, sod1). Furthermore, the present study showed that waterborne Cd exposure decreased the transcriptional factor (nrf2) expression, which might lead to the down-regulation of antioxidant gene expression. Transmission electron microscopy (TEM) observations demonstrated that waterborne Cd exposure induced autophagy in the liver of P. merus. Gene expression analysis showed that waterborne Cd exposure also induced mRNA expression of a set of genes (beclin1, ulk1, atg5, lc3a, atg4b, atg9a, and p62) involved in the autophagy process, indicating that the influence of Cd on autophagy involved transcription regulation of autophagy gene expression. Waterborne Cd exposure induced a sharp decrease in ATP content in the liver of P. merus. In addition, the expression of mitochondrial function genes (sdha, cox4i1, cox1, atp5f1, and mt-cyb) are significantly decreased in the liver of P. merus in Cd treated groups, manifesting the suppression of Cd on mitochondrial energy metabolism. Taken together, our experiments demonstrate that waterborne Cd exposure induced oxidative stress, autophagy and mitochondrial dysfunction in the liver of P. merus. These results may contribute to the understanding of mechanisms that hepatotoxicity of Cd in teleost.

Nr1d1 affects autophagy in the skeletal muscles of juvenile Nile tilapia by regulating the rhythmic expression of autophagy-related genes.

Autophagy is an important evolutionary conserved process in eukaryotic organisms for the turnover of intracellular substances. Recent studies revealed that autophagy displays circadian rhythms in mice and zebrafish. To date, there is no report focused on the rhythmic changes of autophagy in fish skeletal muscles upon nutritional deprivation. In this study, we examined the circadian rhythms of 158 functional genes in tilapia muscle in response to starvation. We found that 12 genes were involved in autophagy changed their rhythm after starvation. Among these genes, Atg4c, Bnip3la, Lc3a, Lc3b, Lc3c, and Ulk1a exhibited a daily rhythmicity in tilapia muscle, and Atg4b, becn1, bnip3la, bnip3lb, Lc3a, and ulk1b were significantly upregulated in response to starvation. The number of autophagosomes was dramatically increased in fasted fish, indicating that nutritional signals affect not only the muscular clock system but also its autophagy behavior. Administration of GSK4112, an activator of Nr1d1, altered rhythmic expression of both circadian clock genes and autophagy genes in tilapia muscle. Taken together, these findings provide evidence that nutritional deficiency affects both circadian regulation and autophagy activities in skeletal muscle.

Zinc reduces hepatic lipid deposition and activates lipophagy via Zn2+/MTF-1/PPARα and Ca2+/CaMKKß/AMPK pathways.

Zinc (Zn) deficiency is the most consistently discovered nutritional manifestations of fatty liver disease. Although Zn is known to stimulate hepatic lipid oxidation, little is known about its underlying mechanism of action in lipolysis. Given the potential role of lipophagy in lipid metabolism, the purpose of this study was to test the hypothesis that Zn attenuates hepatic lipid accumulation by modulating lipophagy. The present study indicated that Zn is a potent promoter of lipophagy. Zn administration significantly alleviated hepatocellular lipid accumulation and increased the release of free fatty acids in association with enhanced fatty acid oxidation and inhibited lipogenesis, which was accompanied by activation of autophagy. Moreover, Zn reduced lipid accumulation and stimulated lipolysis by autophagy-mediated lipophagy. Zn-induced up-regulation of autophagy and lipid depletion is free Zn2+-dependent in the cytosols. Zn-induced autophagy and lipid turnover involved up-regulation of the calcium/calmodulin-dependent protein kinase kinase-ß (Ca2+/CaMKKß)/AMPK pathway. Meanwhile, Zn2+-activated autophagy and lipid depletion were via enhancing metal response element-binding transcription factor (MTF)-1 DNA binding at PPARα promoter region, which in turn induced transcriptional activation of the key genes related to autophagy and lipolysis. Zn activated the pathways of Zn2+/MTF-1/ Peroxisome proliferator-activated receptor (PPAR)α and Ca2+/CaMKKß/AMPK, resulting in the up-regulation of lipophagy and accordingly reduced hepatic lipid accumulation. Our study, for the first time, provided innovative evidence of the direct relationship between metal elements (Zn) and lipid metabolism. The present study also indicated the novel mechanism for Zn-induced lipolysis by the activation of Zn2+/MTF-1/PPARα and Ca2+/CaMKKß/AMPK pathways, which induced the occurrence of lipophagy. These results provide new insight into Zn nutrition and its potential beneficial effects on the prevention of fatty liver disease in vertebrates.-Wei, C.-C., Luo, Z., Hogstrand, C., Xu, Y.-H., Wu, L.-X., Chen, G.-H., Pan, Y.-X., Song, Y.-F. Zinc reduces hepatic lipid deposition and activates lipophagy via Zn2+/MTF-1/PPARα and Ca2+/CaMKKß/AMPK pathways.

Six indicator genes for zinc (Zn) homeostasis in freshwater teleost yellow catfish Pelteobagrus fulvidraco: molecular characterization, mRNA tissue expression and transcriptional changes to Zn exposure.

Excessive Zn in the aquatic environment can be toxic and causes dysfunction in Zn homeostasis for fish, which ultimately influences the function of various biological processes. Zn homeostasis is controlled by Zn transporters. This study cloned and characterized the full-length cDNA sequences of six Zn transport-relevant genes (ZnT1, ZnT5, ZnT7, ZIP4, ZIP5 and MTF-1) from yellow catfish Pelteobagrus fulvidraco. The six genes share similar domains to their corresponding members of mammals. Their mRNA amounts were widely existent across eight tissues (intestine, liver, brain, heart, gill, muscle, spleen and mesenteric fat), but relatively predominant in the liver and intestine. On day 28, Zn exposure tended to increase transcript levels of ZnT1, ZnT5 and MTF-1, decrease hepatic ZIP5 expression, but did not significantly affect the expression of ZnT7 and ZIP4. On day 56, Zn exposure tended to increase transcript levels of ZnT1 and MTF-1, down-regulate hepatic mRNA amounts of ZIP4 and ZIP5; among three Zn treatments, ZnT5 expression in the 0.5 mg Zn/L group and ZnT7 expression in the 0.25 mg Zn/L group were the highest. The mRNA abundances of these genes showed Zn concentration- and exposure time-dependent manners. For the first time, we characterized the full-length cDNA sequences of six Zn transport-relevant genes in fish, explored their tissue expression profiles and transcriptional responses to Zn exposure. Our study built good basis for further investigating their physiological functions of these genes and provided new insights into the regulatory mechanisms of Zn homeostasis in fish.

Fatty Acid ß-Oxidation Is Essential in Leptin-Mediated Oocytes Maturation of Yellow Catfish Pelteobagrus fulvidraco.

Although several studies have been conducted to study leptin function, information is very scarce on the molecular mechanism of leptin in fatty acid ß-oxidation and oocytes maturation in fish. In this study, we investigated the potential role of fatty acid ß-oxidation in leptin-mediated oocytes maturation in Pelteobagrus fulvidraco. Exp. 1 investigated the transcriptomic profiles of ovary and the differential expression of genes involved in ß-oxidation and oocytes maturation following rt-hLEP injection; rt-hLEP injection was associated with significant changes in the expression of genes, including twenty-five up-regulated genes (CPT1, Acsl, Acadl, Acadm, Hadhb, Echsl, Hsd17b4, Acca, PPARα, CYP8B1, ACOX1, ACBP, MAPK, RINGO, Cdc2, MEK1, IGF-1R, APC/C, Cdk2, GnRHR, STAG3, SMC1, FSHß and C-Myc) and ten down-regulated gene (PPARγ, FATCD36, UBC, PDK1, Acads, Raf, Fizzy, C3H-4, Raf and PKC), involved in fatty acid ß-oxidation and oocytes maturation. In Exp. 2, rt-hLEP and specific inhibitors AG490 (JAK-STAT inhibitor) were used to explore whether leptin induced oocytes maturation, and found that leptin incubation increased the diameters of oocytes and percentage of germinal vesicle breakdown (GVBD)-MII oocytes, up-regulated mRNA levels of genes involved in oocytes maturation and that leptin-induced oocyte maturation was related to activation of JAK-STAT pathway. In Exp. 3, primary oocytes of P. fulvidraco were treated with (R)-(+)-etomoxir (an inhibitor of ß-oxidation) or l-carnitine (an enhancer of ß-oxidation) for 48 h under rt-hLEP incubation. Exp. 3 indicated that the inhibition of fatty acid ß-oxidation resulted in the down-regulation of gene expression involved in oocytes maturation, and repressed the leptin-induced up-regulation of these gene expression. Activation of fatty acid ß-oxidation improved the maturation rate and mean diameter of oocytes, and up-regulated gene expression involved in oocytes maturation. Leptin is one of the main factors that links fatty acid ß-oxidation with oocyte maturation; ß-oxidation is essential for leptin-mediated oocyte maturation in fish.

Functional Analysis of Promoters from Three Subtypes of the PI3K Family and Their Roles in the Regulation of Lipid Metabolism by Insulin in Yellow Catfish Pelteobagrus fulvidraco.

In the present study, the length of 360, 1848 and 367 bp sequences of promoters from three subtypes of PI3K family (PI3KCa, PI3KC2b and PI3KC3) of yellow catfish Pelteobagrus fulvidraco were cloned and characterized. Bioinformatics analysis revealed that PI3KCa, PI3KC2b and PI3KC3 had different structures in their core promoter regions. The promoter regions of PI3KCa and PI3KC2b had CpG islands but no CAAT and TATA box. In contrast, the promoter of PI3KC3 had the canonical TATA and CAAT box but no CpG island. The binding sites of several transcription factors, such as HNF1, STAT and NF-κB, were predicted on PI3KCa promoter. The binding sites of transcription factors, such as FOXO1, PPAR-RXR, STAT, IK1, HNF6 and HNF3, were predicted on PI3KC2b promoter and the binding sites of FOXO1 and STAT transcription factors were predicted on PI3KC3 promoter. Deletion analysis indicated that these transcriptional factors were the potential regulators to mediate the activities of their promoters. Subsequent mutation analysis and electrophoretic mobility-shift assay (EMSA) demonstrated that HNF1 and IK1 directly bound with PI3KCa and PI3KC2b promoters and negatively regulated the activities of PI3KCa and PI3KC2b promoters, respectively. Conversely, FOXO1 directly bound with the PI3KC2b and PI3KC3 promoters and positively regulated their promoter activities. In addition, AS1842856 (AS, a potential FOXO1 inhibitor) incubation significantly reduced the relative luciferase activities of several plasmids of PI3KC2b and PI3KC3 but did not significantly influence the relative luciferase activities of the PI3KCa plasmids. Moreover, by using primary hepatocytes from yellow catfish, AS incubation significantly down-regulated the mRNA levels of PI3KCa, PI3KC2b and PI3KC3 and reduced triacylglyceride (TG) accumulation and insulin-induced TG accumulation, as well as the activities and the mRNA levels of several genes involved in lipid metabolism. Thus, the present study offers new insights into the mechanisms for transcriptional regulation of PI3Ks and for PI3Ks-mediated regulation of lipid metabolism by insulin in fish.

AKTs/PKBs: molecular characterization, tissue expression and transcriptional responses to insulin and/or wortmannin in yellow catfish Pelteobagrus fulvidraco.

In the present study, four AKT isoforms termed AKT1, AKT2, AKT3a and AKT3b were isolated and characterized from yellow catfish. Their molecular characterizations, tissue expressions and transcriptional responses to insulin and/or wortmannin were determined. The validated complementary DNA (cDNA) of yellow catfish AKT1, AKT2, AKT3a and AKT3b were 1422, 1431, 1389 and 1440 bp in length, encoding the peptide of 472, 475, 462 and 479 amino acid residues, respectively. The amino acid sequences of yellow catfish AKTs possessed all the characteristics of AKTs in other species. AKT1, AKT2 and AKT3b contained a conserved domain structure including a specific PH domain, a central catalytic domain and a C-terminal regulatory domain, while AKT3a lacked the C-terminal regulatory domain. All mRNAs of AKTs were expressed at the highest levels in the ovary. Among other tissues, the messenger RNA (mRNA) of AKT1 was widely distributed in all tested tissues, and AKT2 mRNA was more abundant in the muscle, liver and fat and lowest in other tested tissues, while AKT3a mRNA was predominant in the brain and showed no significant difference among other tested tissues, and AKT3b mRNA was highly expressed in the ovary, followed by the brain, muscle and fat and was relatively low in other tissues. Intraperitoneal insulin injection and incubation increased the mRNA expression of AKT1 and AKT2, but not that of AKT3a and AKT3b in the liver and hepatocytes of yellow catfish. Wortmannin reduced the mRNA level of all AKT isoforms and also alleviated the insulin-induced changes of AKT2 expression. The present study cloned full-length cDNA sequences of four AKTs in fish and determined their tissue expression profiles and studied their transcriptional responses to insulin and/or wortmannin, which serves to increase our understanding of their physiological function in lipid metabolism in fish.

IRS1 and IRS2: molecular characterization, tissue expression and transcriptional regulation by insulin in yellow catfish Pelteobagrus fulvidraco.

The insulin receptor substrate (IRS) proteins, in particular, IRS1 and IRS2, are the key downstream players of insulin signaling pathway and the regulation of lipid metabolism. In the present study, two genes of IRS (IRS1 and IRS2) were isolated and characterized from yellow catfish Pelteobagrus fulvidraco. Their molecular characterizations, tissue expressions, and transcriptional levels by insulin both in vivo and in vitro were determined. The validated complementary DNAs encoding for IRS1 and IRS2 were 3693 and 3177 bp in length, encoding proteins of 1230 and 1058 amino acid residues, respectively. Similarly to mammals, amino acid sequence alignment revealed that IRSs contained an N-terminal pleckstrin homology (PH) domain, a phosphotyrosine-binding (PTB) domain, and several C-terminal multiple sites of tyrosine phosphorylation. Both IRS1 and IRS2 were widely expressed across the ten tissues (liver, white muscle, spleen, brain, gill, mesenteric fat, anterior intestine, heart, mid-kidney, and ovary), but at the variable levels. Insulin injection at 1 µg/g in vivo significantly stimulated the messenger RNA (mRNA) expression of IRS2, but not IRS1 mRNA expression levels in the liver of yellow catfish after 48 h. In hepatocytes of yellow catfish, insulin incubation significantly stimulated the IRS1 (at a 1000 nM insulin group) and IRS2 (at both 100 and 1000 nM insulin groups) mRNA expressions, which indicated that IRS2 was more sensitive than IRS1 to insulin stimulation in the liver of yellow catfish, and IRS2 played a more important role in mediating insulin's effects on the liver metabolism. The present study serves to increase our understanding into the function of IRS in fish.

Cloning, mRNA expression and transcriptional regulation of five retinoid X receptor subtypes in yellow catfish Pelteobagrus fulvidraco by insulin.

Retinoid X receptors (RXRs) are members of the nuclear receptor superfamily and mediate development, reproduction, homeostasis and cell differentiation processes in vertebrates. In this study, full-length cDNA sequences of five rxr subtypes from yellow catfish Pelteobagrus fulvidraco were cloned. Their mRNA expression patterns in different tissues and transcriptional regulation by insulin were determined. Five P. fulvidraco rxr (Pf-rxr) subtypes differed in the length of cDNA sequence and the open reading frame, but shared the similar domain structures as in typical nuclear receptors. Phylogenetic analysis revealed that the five Pf-rxr subtypes were paralogous genes, and that Pf-rxrßa and Pf-rxrßb had arisen during a teleost-specific genome duplication event. Five subtypes of Pf-rxr were detected in all the tested tissues. Overlapping and distinct expression patterns were found for different Pf-rxr subtypes, suggesting functional redundancy and divergence of these duplicates. Intraperitoneal insulin injection and incubation reduced the mRNA expression of Pf-rxrgb, but not other subtypes, in the liver and hepatocytes of P. fulvidraco, respectively, suggesting that Pf-rxrgb is the dominant rxr subtype involved in the insulin signaling pathway in P. fulvidraco.

JAK and STAT members of yellow catfish Pelteobagrus fulvidraco and their roles in leptin affecting lipid metabolism.

The present study clones and characterizes the full-length cDNA sequences of members in JAK-STAT pathway, explores their mRNA tissue expression and the biological role in leptin influencing lipid metabolism in yellow catfish Pelteobagrus fulvidraco. Full-length cDNA sequences of five JAKs and seven STAT members, including some splicing variants, were obtained from yellow catfish. Compared to mammals, more members of the JAKs and STATs family were found in yellow catfish, which provided evidence that the JAK and STAT family members had arisen by the whole genome duplications during vertebrate evolution. All of these members were widely expressed across the eleven tissues (liver, white muscle, spleen, brain, gill, mesenteric fat, anterior intestine, heart, mid-kidney, testis and ovary) but at the variable levels. Intraperitoneal injection in vivo and incubation in vitro of recombinant human leptin changed triglyceride content and mRNA expression of several JAKs and STATs members, and genes involved in lipid metabolism. AG490, a specific inhibitor of JAK2-STAT pathway, partially reversed leptin-induced effects, indicating that the JAK2a/b-STAT3 pathway exerts main regulating actions of leptin on lipid metabolism at transcriptional level. Meanwhile, the different splicing variants were differentially regulated by leptin incubation. Thus, our data suggest that leptin activated the JAK/STAT pathway and increases the expression of target genes, which partially accounts for the leptin-induced changes in lipid metabolism in yellow catfish.

Effect and mechanism of waterborne prolonged Zn exposure influencing hepatic lipid metabolism in javelin goby Synechogobius hasta.

The present study was conducted to determine the effect and mechanism of waterborne Zn exposure influencing hepatic lipid deposition and metabolism in javelin goby Synechogobius hasta. S. hasta were exposed to four waterborne Zn concentrations (Zn 0.005 [control], 0.18, 0.36 and 0.55 mg l(-1) , respectively) for 60 days. Sampling occurred at days 20, 40 and 60, respectively. Zn exposure increased Zn content, declined hepatic lipid content and reduced viscerosomatic and hepatosomatic indices and lipogenic enzyme activities, including 6-phosphogluconate dehydrogenase (6PGD), glucose-6-phosphate dehydrogenase (G6PD), malic enzyme (ME) and fatty acid synthase (FAS). At days 20 and 60, Zn exposure decreased hepatic mRNA levels of 6PGD, G6PD, ME, FAS, acetyl-CoA carboxylase (ACC)α, ACCß, hormone-sensitive lipase (HSL)a, HSLb, sterol-regulator element-binding protein (SREBP)-1, peroxisome proliferators-activated receptor (PPAR)α and PPARγ. However, the mRNA levels of CPT 1 and adipose triglyceride lipase increased following Zn exposure. On day 40, Zn exposure reduced hepatic mRNA expression of 6PGD, G6PD, ME, FAS, ACCα, ACCß, HSLa, HSLb, SREBP-1 and PPARγ but increased mRNA expression of CPT 1, adipose triglyceride lipase and PPARα. General speaking, Zn exposure reduced hepatic lipid content by inhibiting lipogenesis and stimulating lipolysis. For the first time, the present study provided evidence that chronic Zn exposure differentially influenced mRNA expression and activities of genes and enzymes involved in lipogenic and lipolytic metabolism in a duration-dependent manner, and provided new insight into the relationship between metal elements and lipid metabolism. Copyright © 2015 John Wiley & Sons, Ltd.

De novo characterization of the liver transcriptome of javelin goby Synechogobius hasta and analysis of its transcriptomic profile following waterborne copper exposure.

Previous studies have investigated the physiological responses to chronic copper (Cu) exposure in the liver of Synechogobius hasta; however, little information is available on the underlying molecular mechanisms. In an effort to better understand the mechanisms of Cu toxicity and to illuminate global gene expression patterns modulated by Cu exposure, we obtained the liver transcriptome information of S. hasta by RNA sequencing (RNA-seq) technology and also investigated the differential expression of genes following waterborne Cu exposure. Using the Illumina sequencing platform, as many as 60,217 unigenes were generated, with 815 bp of average length and 1298 bp of unigene N50 after filtering and assembly. For functional annotation analysis, 34,860, 31,526, 31,576, 25,808, 11,542, and 21,721 unigenes were annotated to the NR, NT, Swiss-Prot, KEGG, COG, and GO databases, respectively, and total annotation unigenes were 37,764. After 30 days of exposure to 55 µg Cu/l, a total of 292 and 1076 genes were significantly up- and down-regulated, respectively. By KEGG analysis, 660 had a specific pathway annotation. Subsequent bioinformatics analysis revealed that the differentially expressed genes were mainly related to lipid metabolism, immune system, apoptosis, and signal transduction, suggesting that these signaling pathways may be regulated by Cu exposure. The present study provides comprehensive sequence information for subsequent gene expression studies regarding S. hasta, and the transcriptome profiling after Cu exposure is also expected to improve our understanding of the molecular toxicology of Cu.

Effect of waterborne zinc exposure on lipid deposition and metabolism in hepatopancreas and muscle of grass carp Ctenopharyngodon idella.

The aim of the present study was to explore the effect of waterborne zinc (control, 0.85, 2.20, 3.10 mg/l, respectively) exposure on lipid deposition and metabolism in the hepatopancreas and muscle of grass carp Ctenopharyngodon idella. The lipid content, Zn accumulation, and the activities and expression levels of several enzymes involved in lipid metabolism were determined in hepatopancreas and muscle. Waterborne Zn exposure reduced growth performance and increased Zn accumulation in both tested tissues. In hepatopancreas, Zn exposure increased lipid content, the activities of lipogenic enzymes, such as 6PGD, G6PD, ME, ICDH and FAS, as well as the mRNA expression level of G6PD, 6PGD, ICDH, FAS and SREBP-1. But the activity of CPT I and the mRNA expression of HSL, CPT Iα1a, CPT Iα2a and PPARα were down-regulated by Zn exposure. In contrast, in muscle, waterborne Zn exposure decreased lipid deposition, activities of 6GPD, ICDH and ME, as well as the mRNA expression level of G6PD, ICDH, ME, FAS and SREBP-1. However, the activity of CPT I as well as the mRNA expression level of PPARα, HSL, CPT Iα2a, CPT Iα1b and CPT Iß were up-regulated by Zn exposure. Our results indicate that waterborne Zn increases lipid content by up-regulating lipogenesis and down-regulating lipolysis in hepatopancreas. But, in muscle, waterborne Zn reduces lipid accumulation by up-regulating lipolysis and down-regulating lipogenesis. Differential patterns of lipid deposition, enzymatic activities and genes' expression indicate the tissue-specific regulatory mechanism in fish.

Effects of insulin and its related signaling pathways on lipid metabolism in the yellow catfish Pelteobagrus fulvidraco.

The influence of insulin on hepatic metabolism in fish is not well understood. The present study was therefore conducted to investigate the effects of insulin on lipid metabolism, and the related signaling pathways, in the yellow catfish Pelteobagrus fulvidraco. Hepatic lipid and intracellular triglyceride (TG) content, the activity and expression levels of several enzymes and the mRNA expression of transcription factors (PPARα and PPARγ) involved in lipid metabolism were determined. Troglitazone, GW6471, fenofibrate and wortmannin were used to explore the signaling pathways by which insulin influences lipid metabolism. Insulin tended to increase hepatic lipid accumulation, the activity of lipogenic enzymes (6PGD, G6PD, ME, ICDH and FAS) and mRNA levels of FAS, G6PD, 6PGD, CPT IA and PPARγ, but down-regulated PPARα mRNA level. The insulin-induced effect could be stimulated by the specific PPARγ activator troglitazone or reversed by the PI3 kinase/Akt inhibitor wortmannin, demonstrating that signaling pathways of PPARγ and PI3 kinase/Akt were involved in the insulin-induced alteration of lipid metabolism. The specific PPARα pathway activator fenofibrate reduced insulin-induced TG accumulation, down-regulated the mRNA levels of FAS, G6PD and 6PGD, and up-regulated mRNA levels of CPT IA, PPARα and PPARγ. The specific PPARα pathway inhibitor GW6471 reduced insulin-induced changes in the expression of all the tested genes, indicating that PPARα mediated the insulin-induced changes of lipid metabolism. The present results contribute new knowledge on the regulatory role of insulin in hepatic metabolism in fish.

Effects of recombinant human leptin administration on hepatic lipid metabolism in yellow catfish Pelteobagrus fulvidraco: in vivo and in vitro studies.

The present study was conducted to investigate the effects and mechanism of leptin influencing lipid metabolism in yellow catfish Pelteobagrus fulvidraco. To this end, hepatic lipid (in vivo experiment) and intracellular triglyceride (TG) (in vitro experiment) content, the activities and/or expression level of several enzymes (CPT-1, 6PGD, G6PD, FAS, ME and ICDH) as well as the mRNA expression of transcription factors (PPARα, PPARγ and SREBP-1) involved in lipid metabolism were determined. Using the primary hepatocytes of yellow catfish, specific inhibitors AG490 (JAK-STAT inhibitor) and wortmannin (IRS-PI3K inhibitor) were used to explore the signaling pathways of leptin effects on lipid metabolism. Intraperitoneal injection of recombinant human leptin (rt-hLEP) significantly reduced hepatic lipid content, activities of lipogenic enzymes (6PGD, G6PD, ME, ICDH and FAS) as well as mRNA levels of 6PGD, G6PD, FAS, PPARγ and SREBP-1 genes, but up-regulated activity and mRNA level of CPT-1 and PPARα. Using primary hepatocytes, rt-hLEP incubation also reduced intracellular TG content, mRNA levels of G6PD and PPARγ genes, but enhanced mRNA levels of PPARα, CPT-1 and SREBP-1. Leptin-induced effects could partially be reversed by specific inhibitors AG490, suggesting that JAK-STAT signaling pathways played important roles in the process of leptin-induced changes in lipid metabolism. Wortmannin significantly suppressed the decrease of TG content induced by leptin, reflecting that IRS-PI3K was involved in the leptin-mediate changes as well. To our knowledge, the present study provides, for the first time, evidence that rt-hLEP can increase lipolysis and reduce lipogenesis at the both enzymatic and molecular levels in fish with the combination of in vivo with in vitro studies, which serves to increase our understanding into the roles and mechanisms of leptin regulating lipid metabolism in fish.

Peroxisome proliferator-activated receptor gamma (PPARγ) in yellow catfish Pelteobagrus fulvidraco: molecular characterization, mRNA expression and transcriptional regulation by insulin in vivo and in vitro.

Peroxisome proliferator-activated receptor gamma (PPARγ) is ligand-inducible transcription factor and has important roles in lipid metabolism, cell proliferation and inflammation. In the present study, yellow catfish Pelteobagrus fulvidraco PPARγ cDNA was isolated from liver by RT-PCR and RACE, and its molecular characterization and transcriptional regulation by insulin in vivo and in vitro were determined. The generation of PPARγ1 and PPARγ2 was due to alternative promoter of PPARγ gene. PPARγ1 and PPARγ2 mRNA covered 2426 bp and 2537 bp, respectively, with an open reading frame (ORF) of 1584 bp encoding 527 amino acid residues. Yellow catfish PPARγ gene was organized in a manner similar to that of their mammalian homologs, implying a modular organization of the protein's domains. A comparison between the yellow catfish PPARγ amino acid sequence and the correspondent sequences of several other species revealed the identity of 55-76.2%. Two PPARγ transcripts (PPARγ1 and PPARγ2) mRNAs were expressed in a wide range of tissues, but the abundance of each PPARγ mRNA showed the tissue- and developmental stage-dependent expression patterns. Intraperitoneal injection of insulin in vivo significantly stimulated the mRNA expression of total PPARγ and PPARγ1, but not PPARγ2 in the liver of yellow catfish. In contrast, incubation of hepatocytes with insulin in vitro increased the mRNA levels of PPARγ1, PPARγ2 and total PPARγ. To our knowledge, for the first time, the present study provides evidence that PPARγ1 and PPARγ2 are differentially expressed with and among tissues during different developmental stages and also regulated by insulin both in vivo and in vitro, which serves to increase our understanding on PPARγ physiological function in fish.