RESUMO
Information on transgenerational effects of cadmium (Cd) and zinc (Zn) within hour of exposure is scarce. To the end, larvae of marine medaka Oryzias melastigma at 0 day-post-hatching (dph) were subjected to LC50 for 96-h of Cd or Zn for 0.5 and 6 h, and then transferred into clear water for 95 days until the generation of offspring larvae at 25 dph. Growth, antioxidant capacity and stress response in offspring larvae were examined. Exposure to Zn for 0.5 h or Cd for 0.5 h and 6 h promoted growth performance and reduced total antioxidant capacity (TAC) and activities of superoxide dismutase (SOD) and catalase (CAT). Malondialdehyde (MDA) and cortisol levels declined in larvae following Zn exposure for 0.5 h, whereas Cd exposure increased MDA content and did not affect cortisol levels. These physiological changes could be partially explained by transcription of genes in the hormone/insulin-like growth factor-I (GH/IGF) axis, NF-E2-related factor 2 (Nrf2) signaling, and hypothalamus-pituitary-interrenal (HPI) axis. For example, Zn exposure for 0.5 h up-regulated genes encoding growth hormone (gh) and insulin-like growth factor binding protein (igfbp1) and down-regulated mRNA levels of nrf2, Kelch-like-ECH-associated protein 1 gene (keap1a), keap1b, sod1, mineralocorticoid receptor (mr), corticotropin-releasing hormone receptor (crhr1), corticotropin-releasing hormone binding protein (crhbp), cytochrome P450 (cyp11a1, cyp17a1) and hydroxysteroid dehydrogenase (hsd3b1). Cd exposure for 0.5 and 6 h up-regulated growth hormone release hormone (ghrh) and igfbp1, down-regulated nrf2 and keap1a, and did not affect mRNA levels of HPI axis genes. Taken together, this study demonstrated that short-term metal exposure during larvae phase had positive and negative effects on offspring even after a long recovery.
Assuntos
Oryzias , Poluentes Químicos da Água , Animais , Antioxidantes/metabolismo , Zinco/toxicidade , Cádmio/toxicidade , Oryzias/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Hormônio Liberador da Corticotropina , Hidrocortisona , Poluentes Químicos da Água/toxicidade , Hormônio do Crescimento/genética , RNA MensageiroRESUMO
In this study, the transcriptional regulation of PI3KC3 by three transcription factors (PPARγ, PPARα, and STAT3) and the potential role of PI3KC3 in mediating lipid accumulation were determined in yellow catfish Pelteobagrus fulvidraco. The 5'-deletion assay, overexpression assay, site-mutation assay, and electrophoretic mobility shift assay suggested that PPARα, PPARγ, and STAT3 negatively regulated the promoter activity of pi3kc3. Moreover, the transcriptional inactivation of pi3kc3 was directly mediated by PPARα and PPARγ under fatty acid (FA) treatment. Using primary hepatocytes from yellow catfish, FA incubation significantly increased triacylglyceride (TG) content, non-esterified fatty acid (NEFA) content, and lipid drops (LDs) content, the mRNA level of pparα, pparγ, stat3, and dnmt3b, the protein level of PPARα, PPARγ, and STAT3, and the methylation level of pi3kc3, but significantly reduced the mRNA and protein level of PI3KC3. Our findings offer new insights into the mechanisms for transcriptional regulation of PI3KC3 and for PI3KC3-mediated lipid accumulation in fish.
Assuntos
Peixes-Gato , Animais , Peixes-Gato/genética , Peixes-Gato/metabolismo , Metabolismo dos Lipídeos , Lipídeos , Fígado/metabolismo , PPAR alfa/genética , PPAR gama/genética , RNA Mensageiro/metabolismoRESUMO
In the present study, two new SLC34 family members, named slc34a1b and slc34a2a, were isolated and characterized from grass carp Ctenopharyngodon idella. Topology, tissue distribution, and transcriptional response to phosphorus (Pi) and pH were compared among three members of SLC34 family (slc34a1b, slc34a2a, and slc34a2b) in grass carp. The length of validated cDNAs of grass carp slc34a1b and slc34a2a was 1494 bp and 1902 bp, and these two cDNAs encoded 497 and 633 amino acid residues, respectively. The domain analysis showed that three SLC34 members of grass carp contain architecture similar to that in mammals. Moreover, the mRNA of three slc34s was widely expressed in nine tissues (heart, brain, intestine, kidney, liver, muscle, gill, spleen, and skin), but at various levels. Our results revealed that 6 mM and 9 mM Pi incubation significantly reduced the mRNA expression of three slc34s in both CIK and L8824 cell lines from grass carp. The expression of slc34a1b was decreased in the CIK cells, but not in the L8824 cells after 3 mM Pi incubation. In CIK cells, 3 mM Pi incubation downregulated the expression of slc34a1b and slc34a2a, but not slc34a2b. In addition, the expression of three slc34s was significantly reduced at acidic pH in the CIK cells. Taken together, we characterized three SLC34 family members, revealed their specific distribution among different tissues, and elucidated their transcriptional responses to Pi and pH in two cell lines from grass carp. Our findings provide an insight into the physiological function of three SLC34s in fish.
Assuntos
Carpas , Doenças dos Peixes , Animais , Carpas/genética , Carpas/metabolismo , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Concentração de Íons de Hidrogênio , RNA Mensageiro , Distribuição TecidualRESUMO
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.
Assuntos
Peixes-Gato/genética , Regulação da Expressão Gênica/genética , Regiões Promotoras Genéticas , Esteroide 17-alfa-Hidroxilase/genética , Fator Esteroidogênico 1/genética , Animais , Sítios de Ligação , Peixes-Gato/metabolismo , Clonagem Molecular , Genes Reporter , Células HEK293 , Humanos , Leptina/metabolismo , Luciferases/metabolismo , Mutação , PPAR alfa/genética , PPAR alfa/metabolismo , PPAR gama/genética , PPAR gama/metabolismo , Ligação Proteica , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Esteroide 17-alfa-Hidroxilase/metabolismo , Fator Esteroidogênico 1/metabolismo , Regulação para CimaRESUMO
It is important to explore the regulatory mechanism of phosphorus homeostasis in fish, which help avoid the risk of P toxicity and prevent P pollution in aquatic environment. The present study obtained the full-length cDNA sequences and the promoters of three SLC20 members (slc20a1a, slc20a1b and slc20a2) from grass carp Ctenopharyngodon idella, and explored their responses to inorganic phosphorus (Pi). Grass carp SLC20s proteins possessed conservative domains and amino acid sites relevant with phosphorus transport. The mRNAs of three slc20s appeared in the nine tissues, but their expression levels were tissue-dependent. The binding sites of three transcription factors (SREBP1, NRF2 and VDR) were predicted on the slc20s promoters. The mutation and EMSA analysis indicated that: (1) SREBP1 binding site (-783/-771 bp) negatively but VDR (-260/-253 bp) binding site positively regulated the activities of slc20a1a promoter; (2) SREBP1 (-1187/-1178 bp), NRF2 (-572/-561 bp) and VDR(615/-609 bp) binding sites positively regulated the activities of slc20a1b promoter; (3) SREBP1 (-987/-977 bp), NRF2 (-1469/-1459 bp) and VDR (-1124/-1117 bp) binding sites positively regulated the activities of the slc20a2 promoter. Moreover, Pi incubation significantly reduced the activities of three slc20s promoters, and Pi-induced transcriptional inactivation of slc20s promoters abolished after the mutation of the VDR element but not SREBP1 and NRF2 elements. Pi incubation down-regulated the mRNA levels of three slc20s. For the first time, our study elucidated the transcriptional regulatory mechanisms of SLC20s and their responses to Pi, which offered new insights into the Pi homeostatic regulation and provided the basis for reducing phosphorus discharge into the waters.
Assuntos
Carpas/genética , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/genética , Proteínas Cotransportadoras de Sódio-Fosfato/genética , Animais , Carpas/metabolismo , Clonagem Molecular , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Homeostase/genética , Redes e Vias Metabólicas/genética , Fósforo/metabolismo , Fósforo/farmacologia , Regiões Promotoras Genéticas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Elementos de Resposta/genética , Análise de Sequência de DNA , Proteínas Cotransportadoras de Sódio-Fosfato/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/metabolismoRESUMO
Early molecular events after the exposure of heavy metals, such as aberrant DNA methylation, suggest that DNA methylation was important in regulating physiological processes for animals and accordingly could be used as environmental biomarkers. In the present study, we found that copper (Cu) exposure increased lipid content and induced the DNA hypermethylation at the whole genome level. Especially, Cu induced hypermethylation of glucose-regulated protein 78 (grp78) and peroxisome proliferator-activated receptor gamma coactivator-1α (pgc1α). CCAAT/enhancer binding protein α (C/EBPα) could bind to the methylated sequence of grp78, whereas C/EBPß could not bind to the methylated sequence of grp78. These synergistically influenced grp78 expression and increased lipogenesis. In contrast, DNA methylation of PGC1α blocked the specific protein 1 (SP1) binding and interfered mitochondrial function. Moreover, Cu increased reactive oxygen species (ROS) production, activated endoplasmic reticulum (ER) stress and damaged mitochondrial function, and accordingly increased lipid deposition. Notably, we found a new toxicological mechanism for Cu-induced lipid deposition at DNA methylation level. The measurement of DNA methylation facilitated the use of these epigenetic biomarkers for the evaluation of environmental risk.
Assuntos
Carpas/fisiologia , Cobre/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Carpas/metabolismo , Cobre/metabolismo , Estresse do Retículo Endoplasmático , Glucose/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Lipídeos , Metilação , Mitocôndrias/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Ativação Transcricional , Regulação para CimaRESUMO
Excessive fat deposition in the hepatocytes, associated with excess dietary fat intake, was related to the occurrence of fatty livers in fish. miR-101b plays the important roles in controlling lipid metabolism, but the underlying mechanism at the post-transcriptional level remains unclear. The purpose of this study is to explore the roles and mechanism of miR-101b-mediating lipid deposition and metabolism in yellow catfish Pelteobagrus fulvidraco. We found that miR-101b directly targeted fatty acid translocase (cd36), caspase9 (casp9) and autophagy-related gene 4A (atg4a). Furthermore, using palmitic acid (PA) or oleic acid (OA) to incubate the primary hepatocytes of yellow catfish, we demonstrated that miR-101b inversely regulated cd36, casp9, and atg4a expression at the transcriptional level; the inhibition of miR-101b aggravated fatty acids (FAs, PA or OA)-induced lipid accumulation, indicating that miR-101b mediated FAs-induced variations of lipid metabolism in yellow catfish. Taken together, our study gave novel insight into the regulatory mechanism of lipid deposition and metabolism and might provide potential targets for the prevention and treatment of fatty livers in fish.
Assuntos
Peixes-Gato/metabolismo , Ácidos Graxos/metabolismo , Proteínas de Peixes/metabolismo , Hepatócitos/metabolismo , Metabolismo dos Lipídeos/genética , MicroRNAs/genética , Animais , Autofagia , Peixes-Gato/genética , Proteínas de Peixes/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The present study was conducted to explore the mechanism of nano-Zn absorption and its influence on lipid metabolism in the intestine of yellow catfish Pelteobagrus fulvidraco. Compared to ZnSO4, dietary nano-Zn addition increased the triglyceride (TG) content, enzymatic activities of malic enzyme (ME) and fatty acid synthase (FAS), and up-regulated mRNA levels of 6pgd, fas, acca, dgat1, pparγ, and fatp4. Using primary intestinal epithelial cells of yellow catfish, compared to the ZnSO4 group, nano-Zn incubation increased the contents of TG and free fatty acids (FFA), the activities of glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6GPD), ME, and FAS, up-regulated mRNA levels of lipogenic genes (6pgd, g6pd, fas, dgat1, and pparγ), genes of lipid transport (fatp4 and ifabp), and Zn transport genes (znt5, znt7, mt, and mtf1), and increased the protein expression of fatty acid transport protein 4 (FATP4) and peroxisome proliferator activated receptor gamma (PPARγ). Further studies found that nano-Zn absorption was via the clathrin-dependent endocytic mechanism. PPARγ mediated the nano-Zn-induced increase in TG, and nano-Zn increased Zn accumulation and induced TG accumulation by activating the PPARγ pathway and up-regulating lipogenesis.
Assuntos
Peixes-Gato/metabolismo , Mucosa Intestinal/metabolismo , Lipogênese/efeitos dos fármacos , Nanopartículas Metálicas/química , PPAR gama/metabolismo , Triglicerídeos/metabolismo , Zinco/metabolismo , Animais , Peixes-Gato/crescimento & desenvolvimento , Sobrevivência Celular/efeitos dos fármacos , Clorpromazina/farmacologia , Dieta , Endocitose/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Mucosa Intestinal/enzimologia , Lipogênese/genética , Malato Desidrogenase/metabolismo , PPAR gama/genéticaRESUMO
We characterized the promoters of target genes of the signal transducer and activator of transcription 3, STAT3 (carnitine palmitoyltransferase I, CPT Iα1b, acetyl-CoA carboxylase alpha, ACCα; fatty acid synthase, FAS; and peroxisome proliferator-activated receptor gamma, PPARγ) in a teleost Pelteobagrus fulvidraco. Binding sites of STAT3 were predicted on these promoters, indicating that STAT3 probably mediated their transcriptional activities. Leptin had no effect on the activity of ACCα and PPARγ promoters, but increased CPT Iα1b promoter activity and decreased FAS promoter activity. The −979/−997 STAT3 binding site of CPT Iα1b and the −794/−812 STAT3 binding site of FAS were functional binding loci responsible for leptin-induced transcriptional activation. The study provided direct evidence that STAT3 regulated the expression of CPT Iα1b and FAS at the transcription level, and determined the STAT3 response element on promoters of CPT Iα1b and FAS under leptin signal.
RESUMO
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.
Assuntos
Peixes-Gato/genética , Regulação da Expressão Gênica , Insulina/metabolismo , Metabolismo dos Lipídeos/genética , Família Multigênica , Fosfatidilinositol 3-Quinases/genética , Regiões Promotoras Genéticas , Animais , Sequência de Bases , Sítios de Ligação/genética , Clonagem Molecular , Ilhas de CpG/genética , Luciferases/metabolismo , Mutagênese Sítio-Dirigida , Plasmídeos/metabolismo , Ligação Proteica , RNA Antissenso/metabolismo , Análise de Sequência de DNA , Deleção de Sequência , Fatores de Transcrição/metabolismo , Triglicerídeos/metabolismoRESUMO
The present working hypothesis is that absorption of dietary Cu is related to mRNA expressions of genes involved in Cu uptake and transport of the intestine in fish. To this end, the full-length cDNA sequences of eight Cu uptake related genes, including two isoforms of copper transporter genes (ctr1 and ctr2), three copper chaperone genes (atox1, ccs and cox17), two Cu-ATPase genes (atp7a and atp7b) and divalent metal ion transporter 1 (dmt1), were cloned and characterized in yellow catfish P. fulvidraco, respectively. Their mRNA tissue expression and transcriptional responses to dietborne Cu exposure were investigated. Compared to the corresponding members of mammals, all of these members in P. fulvidraco shared the similar conserved domain structures. Their mRNAs were expressed in a wide range of tissues (including liver, muscle, spleen, brain, gill, intestine, heart and kidney), but at variable levels. In anterior intestine, mRNA levels of ctr1, cox17, dmt1 and atp7a declined with increasing dietary Cu levels. The mRNA levels of ctr2 and mt were the highest for excess dietary Cu group and showed no significant differences between other two treatments. Atox1 mRNA levels were the highest for Cu-deficient group and showed no significant differences between other two treatments. The mRNA levels of ccs were the highest for Cu-deficient group, followed by Cu-excess group and the lowest for adequate-Cu group. In contrast, atp7b mRNA levels were the highest for Cu-excess group and the lowest for adequate Cu group. In the mid-intestine, mRNA levels of ctr1, ctr2, atox1, ccs, cox17, dmt1 and atp7a declined with increasing dietary Cu levels. Atp7b mRNA levels were the lowest for adequate Cu group and showed no significant differences between other two treatments. Mt mRNA levels were the lowest for adequate Cu group and highest for Cu-excess group. For the first time, our study cloned and characterized ctr1, ctr2, atox1, ccs, cox17, atp7a, atp7b and dmt1 genes in P. fulvidraco and determined their tissue-specific expression, and transcriptional responses in the anterior and mid-intestine of yellow catfish under dietborne Cu exposure, which shed new light on the Cu uptake system and help to understand the molecular mechanisms of Cu homeostasis in fish.
Assuntos
Peixes-Gato/genética , Cobre/metabolismo , Dieta , Proteínas de Peixes/genética , Perfilação da Expressão Gênica , Transcrição Gênica , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar/genética , Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica , Humanos , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
In the present study, seven phosphoinositide 3-kinase (PI3K) members (PI3KCa, PI3KCb, PI3KCd, PI3KCg, PI3KC2a, PI3KC2b and PI3KC3, respectively) were isolated and characterized from yellow catfish Pelteobagrus fulvidraco, and their roles in insulin-induced changes of protein metabolism were determined. These seven PI3Ks can be divided into three classes, class I (including PI3KCa, PI3KCb, PI3KCd and PI3KCg), class II (including PI3KC2a and PI3KC2b) and class III (only including PI3KC3). Compared with mammals, all of these members share similar domain structure. Their mRNAs were widely expressed across ten tested tissues (liver, white muscle, spleen, brain, gill, mesenteric fat, intestine, heart, kidney and ovary), but at variable levels. In the in vivo study, insulin treatment significantly increased hepatic protein content at 3h, accompanied with reduced plasma total amino acid contents and liver ALT activity, and with increased total RNA content and the mRNA levels of PI3KCb, PI3KC2a, AKT2, mTORC1 and S6K1 in liver. At 6h and 12h, insulin injection showed no significant effect on liver protein content and plasma total amino acid, but reduced liver ALT activity and increased liver total RNA and the mRNA levels of AKT2, mTORC1 and S6K1 in liver at 6h. In the in vitro study, insulin incubation also tended to increase protein content of hepatocytes, accompanied with reduced cell medium total amino acid contents and hepatocytes ALT activity, and increased total RNA content and the mRNA levels of PI3KCb, PI3KC2a, AKT2, mTORC1 and S6K1 in hepatocytes. However, insulin treatment showed no significant effect on GDH activity and mRNA expression of PI3KCa, PI3KCd, PI3KCg, PI3KC2b, PI3KC3 and eEF2 both in the in vivo and in vitro studies. Effects of insulin on the mRNA levels of eIF-4E and 4E-BP1 were different between the in vivo and in vitro studies, and also time-dependent. Compared to single insulin group, insulin+wortmannin group increased ALT activity at 6h but reduced T-RNA content at 6 and 12h. AKT2 and S6K1 mRNA levels at 6 and 12h, mRNA levels of mTORC1, 4E-BP1 and eEF2 at 3 and 6h, and EIF-4E mRNA levels at 3 and 12h, PI3KCb and PI3KC2a mRNA levels were significantly lower in insulin+wortmannin group than those in single insulin group. Thus, our study demonstrated that among seven PI3K members, PI3KCb and PI3KC2a were more sensitive to the insulin signaling pathway, and insulin stimulated hepatic protein synthesis in yellow catfish through PI3K signaling pathway.
Assuntos
Peixes-Gato/metabolismo , Insulina/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Sequência de Aminoácidos , Aminoácidos/sangue , Androstadienos/farmacologia , Animais , Sequência de Bases , Peixes-Gato/sangue , Peixes-Gato/genética , DNA Complementar/genética , Feminino , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/genética , Domínios Proteicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/efeitos dos fármacos , WortmaninaRESUMO
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.
Assuntos
Peixes-Gato/genética , Proteínas de Peixes/genética , Proteínas Substratos do Receptor de Insulina/genética , Insulina/farmacologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , RNA Mensageiro/metabolismoRESUMO
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.
Assuntos
Androstadienos/farmacologia , Peixes-Gato/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Insulina/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sequência de Aminoácidos , Androstadienos/administração & dosagem , Animais , Sequência de Bases , DNA Complementar/genética , Feminino , Hepatócitos/efeitos dos fármacos , Insulina/administração & dosagem , Metabolismo dos Lipídeos , Masculino , Filogenia , Isoformas de Proteínas , Proteínas Proto-Oncogênicas c-akt/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Distribuição Tecidual , WortmaninaRESUMO
Signal transducers and activators of transcription proteins (STATs) act as important mediators in multiple biological processes induced by a large number of cytokines. In the present study, full-length cDNA sequences of seven STAT members, including some splicing variants different from those in mammals, were obtained from Synechogobius hasta. The phylogenetic analysis revealed that the seven STAT members were derived from paralogous genes that might have arisen by whole genome duplication (WGD) events during vertebrate evolution. All of these members share similar domain structure compared with those of mammals, and were widely expressed across the tested tissues (brain, gill, heart, intestine, liver, muscle and spleen), but at variable levels. Incubation in vitro of recombinant human leptin changed the intracellular triglyceride (TG) content and mRNA levels of several STATs members, as well as expressions and activities of genes involved in lipid metabolism. Furthermore, Tyrphostin B42 (AG490), a specific inhibitor of the Janus Kinase 2(JAK2)-STAT pathway, partially reversed leptin-induced change on STAT3 and its two spliced isoforms expression, as well as expressions and activities of genes involved in lipid metabolism. As a consequence, the decrease of TG content was also reversed. Thus, our study suggests that STAT3 is the requisite for the leptin signal and the activation of the STAT3 member may account for the leptin-induced changes in lipid metabolism in S. hasta.
Assuntos
Proteínas de Peixes/metabolismo , Leptina/metabolismo , Metabolismo dos Lipídeos , Fatores de Transcrição STAT/metabolismo , Animais , Proteínas de Peixes/genética , Humanos , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/metabolismo , Perciformes , Fatores de Transcrição STAT/genética , Transdução de SinaisRESUMO
Liver X Receptor (LXR) plays a pivotal role in metabolic regulation in mammals, but little is known about its function in fish. In this study, two lxra isoforms, namely lxra1 and lxra2, were isolated. Their molecular characterization, tissues distribution and transcriptional regulation by insulin in vivo and in vitro were determined. lxrα1 and lxrα2 cDNA covered 2775bp and 3093bp, encoding 446 and 515 amino acid residues, respectively. The protein sequence of yellow catfish lxra included characteristic feature of mammalian lxrs, including the DNA binding (DBD) (containing P-box), ligand binding (LBD) and activation function-2 (AF-2) domains, D-box, and D (hinge) region. Phylogenetic analysis revealed that yellow catfish lxra grouped with lxra of zebrafish but was distant from those of medaka and stickleback. lxrß clades was absent in teleosts in phylogenetic tree, proving gene loss of lxrß in teleosts during evolution. The two lxra isoforms (lxra1 and lxra2) mRNAs were ubiquitously expressed in 11 tested tissues. Compared to lxra2, lxra1 mRNA expression was predominant in all tested tissues. The expression of lxrα1 was the highest in testis, then in liver, and the lowest in other tissues. lxrα2 expression was the highest in liver, then in testis, and the lowest in ovary. Insulin significantly stimulated the mRNA expression of lxra1 in vitro and in vivo, while the expression of lxra2 remained unchanged after insulin treatment. The present study serves to increase our understanding into the function of lxra in fish.
Assuntos
Peixes-Gato/genética , Proteínas de Peixes/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Insulina/farmacologia , Receptores Nucleares Órfãos/genética , Transcrição Gênica/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Bovinos , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Receptores X do Fígado , Dados de Sequência Molecular , Receptores Nucleares Órfãos/química , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
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.
Assuntos
Peixes-Gato/metabolismo , Insulina/metabolismo , Metabolismo dos Lipídeos , Animais , Regulação da Expressão Gênica , Insulina/farmacologia , Fígado/metabolismo , PPAR alfa/metabolismo , PPAR gama/metabolismo , RNA Mensageiro/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Triglicerídeos/metabolismoRESUMO
The present study was conducted to determine the effect of waterborne copper (Cu) exposure on carnitine concentration, carnitine palmitoyltransferases I (CPT I) kinetics, and expression levels of four CPT I isoforms in the liver, muscle and heart of yellow catfish Pelteobagrus fulvidraco. Yellow catfish were exposed to four waterborne copper (Cu) concentrations (2 (control), 24 (low), 71 (medium), 198 (high) µg Cu/l, respectively) for 6weeks. Waterborne Cu exposure increased maximal reaction rates (Vmax) in the liver and muscle, but not in the heart. Michaelis-Menten constants (Km) tended to increase in the liver, but decreased in the heart after Cu exposure. The contents of total carnitine (TC) and acylcarnitine (AC) in the liver, and free carnitine (FC) in the muscle increased with increasing waterborne Cu concentrations, while FC content in the muscle declined with the increase of Cu levels. Waterborne Cu exposure also significantly influenced carnitine composition and profiles in heart. The mRNA expression of CPT Iα1a, CPT Iα1b and CPT Iα2a in the liver, and CPT Iα1a, CPT Iα1b and CPT Iß in the muscle as well as CPT Iα1a in the heart were up-regulated by Cu exposure. Additionally, correlations were observed in the expression levels of CPT I isoforms and Km for carnitine, and between CPT I isoform expression and CPT I activity. To our knowledge, for the first time, the present study provided evidence that waterborne Cu exposure could influence carnitine composition, CPT I kinetics and mRNA levels of four CPT I isoforms in yellow catfish, which served to increase our understanding of the mechanisms underlying lipid catabolism during Cu exposure.
Assuntos
Carnitina O-Palmitoiltransferase/metabolismo , Carnitina/análogos & derivados , Peixes-Gato/metabolismo , Cobre/toxicidade , Monitoramento Ambiental/métodos , Poluentes Químicos da Água/toxicidade , Animais , Carnitina/análise , Carnitina/metabolismo , Carnitina O-Palmitoiltransferase/genética , Peixes-Gato/genética , Cobre/análise , Isoenzimas/genética , Cinética , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Músculos/efeitos dos fármacos , Músculos/metabolismo , RNA Mensageiro/metabolismo , Poluentes Químicos da Água/análiseRESUMO
The present study was conducted to determine the effect of leptin on lipid metabolism in ovarian follicle cells of yellow catfish Pelteobagrus fulvidraco. For that purpose, primary ovarian follicle cells were isolated from yellow catfish, cultured and subjected to different treatments (control, 0.1% DMSO, 500ng/ml leptin, 500ng/ml leptin plus 100µM wortmannin, 500ng/ml leptin plus 50nM AG490, respectively) for 48h. Intracellular triglyceride (TG) content, the activities (CPT I, FAS, G6PD, and 6PGD) and/or expression level of several enzymes (CPT I, FAS, G6PD, 6PGD, ACCa and ACCb), as well as the mRNA expression of transcription factors (PPARα, PPARγ and SREBP-1) involved in lipid metabolism were determined. Recombinant human leptin (rt-hLEP) incubation significantly reduced intracellular TG content, activities and mRNA levels of FAS, G6PD and 6PGD, SREBP-1 and PPARγ, but enhanced activity and mRNA level of CPT I, PPARα and ACCa. Specific inhibitors AG490 and wortmannin of JAK-STAT and IRS-PI3K signaling pathways prevented leptin-induced changes, indicating that JAK-STAT and IRS-PI3K signaling pathways were involved in the process of leptin-induced changes of lipid metabolism. Based on these observations above, for the first time, our study indicated that leptin reduced lipid deposition by activating lipolysis and suppressing lipogenesis in ovarian follicles of yellow catfish, and both JAK-STAT and IRS-PI3K signaling pathways were involved in the changes of leptin-induced lipid metabolism.
Assuntos
Peixes-Gato/metabolismo , Leptina/metabolismo , Metabolismo dos Lipídeos/fisiologia , Folículo Ovariano/metabolismo , Animais , FemininoRESUMO
The present study was conducted to investigate the effects and mechanisms of hypothyroidism, induced by administration of 0.2% methimazole through the food, on lipid metabolism in the liver of juvenile yellow catfish Pelteobagrus fulvidraco. To this end, yellow catfish were fed diets containing either 0 or 2g methimazole per kg of diet for 8weeks, respectively. The results showed that fish fed diet containing methimazole had a significant reduction in growth performance, plasma THs levels and hepatic lipid content. Meanwhile, methimazole treatment inhibited the activities of lipogenic enzymes (6-phosphogluconate dehydrogenase, glucose 6-phosphate dehydrogenase, malic enzyme, isocitrate dehydrogenase and fatty acid synthase) and the mRNA levels of genes involved in lipogenesis (6-phosphogluconate dehydrogenase, glucose 6-phosphate dehydrogenase, fatty acid synthase, acetyl-CoA carboxylase α, sterol-regulator element-binding protein-1 and liver X receptor), but increased lipolytic enzyme (carnitine palmitoyltransferase 1) activity and the expression of genes involved in lipolysis (carnitine palmitoyltransferase 1a, hormone-sensitive lipase and peroxisome proliferators-activated receptor α). Thus, our study indicated that dietary methimazole-induced hypothyroidism could disturb the normal processes of lipid metabolism at the enzymatic and molecular levels in yellow catfish, and the reduced hepatic lipid content by hypothyroidism was attributable to the down-regulation of lipogenesis and up-regulation of lipolysis.