Novel insights for PI3KC3 in mediating lipid accumulation in yellow catfish Pelteobagrus fulvidraco.

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.

Topology, tissue distribution, and transcriptional level of SLC34s in response to Pi and pH in grass carp Ctenopharyngodon idella.

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.

Waterborne copper exposure up-regulated lipid deposition through the methylation of GRP78 and PGC1α of grass carp Ctenopharyngodon idella.

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.

Isolation and Characterization of Three Sodium-Phosphate Cotransporter Genes and Their Transcriptional Regulation in the Grass Carp Ctenopharyngodon idella.

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.

Nano-Zn Increased Zn Accumulation and Triglyceride Content by Up-Regulating Lipogenesis in Freshwater Teleost, Yellow Catfish Pelteobagrus fulvidraco.

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.

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.

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.

Characterization and mechanism of phosphoinositide 3-kinases (PI3Ks) members in insulin-induced changes of protein metabolism in yellow catfish Pelteobagrus fulvidraco.

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.

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.

Isolation and Expression Analysis of STAT Members from Synechogobius hasta and Their Roles in Leptin Affecting Lipid Metabolism.

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.

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.

Regulation and mechanism of leptin on lipid metabolism in ovarian follicle cells from yellow catfish Pelteobagrus fulvidraco.

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.

Dietary methimazole-induced hypothyroidism reduces hepatic lipid deposition by down-regulating lipogenesis and up-regulating lipolysis in Pelteobagrus fulvidraco.

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.

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.

In vitro effects of selenium on copper-induced changes in lipid metabolism of grass carp (Ctenopharyngodon idellus) hepatocytes.

The present study was performed to evaluate the in vitro effects of selenium (Se) supplementation to prevent copper (Cu)-induced changes in lipid metabolism of hepatocytes from grass carp (Ctenopharyngodon idellus). Four groups (control and 100 µM Cu in combination with 0, 5, and 10 µM Se, respectively) were chosen. Compared with the control, activities of glucose 6-phosphatedehydrogenase, 6-phosphogluconate dehydrogenase, malic enzyme, and carnitine palmitoyltransferase I (CPT I) of all three Cu-exposed groups at 24 and 48 h were significantly greater. However, among three Cu-exposed groups, increasing Se concentration tended to increase activities of G6PD and ME at 24 h and 6PGD activity at 24 and 48 h but decreased CPT I activity at 24 h. Compared with the control, Cu exposure alone, or in combination with Se, downregulated mRNA levels of sterol regulatory element-binding protein-1 (SREBP-1c), fatty acid synthase (FAS), acetyl-CoA carboxylase, peroxisome proliferator activated receptor alpha (PPARα), CPT I, and hormone-sensitive lipase (HSL) at 24 h as well as SREBP-1c, FAS, and ACC mRNA levels at 48 h. However, upregulated mRNA levels of PPARα, CPT I, and HSL, as well as decreased triglyceride content, were recorded at 48 h. Thus, although toxic at greater levels, lower levels of Se provided significant protection against Cu-induced changes in lipid metabolism. For the first time, our study indicates the dose- and time-dependent effects of Se addition on changes in lipid metabolism induced by Cu in fish hepatocytes and provides new insights into Se-Cu interaction at both enzymatic and molecular levels.

In vitro exposure to copper influences lipid metabolism in hepatocytes from grass carp (Ctenopharyngodon idellus).

In the present study, three different copper (Cu) concentrations (control, 10 and 100 lM, respectively) and three incubation times (24, 48 and 96 h) were chosen to assess in vitro effect of Cu on lipid metabolism in hepatocytes of grass carp Ctenopharyngodon idellus. Increased glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and carnitine palmitoyltransferase I activities were observed in hepatocytes with increasing Cu concentration and exposure duration. Cu decreased mRNA levels of several lipogenic and lipolytic genes at 24 h. However, at 48 h, Cu down-regulated the process of lipogenesis but up-regulated that of lipolysis. The Cudriven up-regulation of lipolytic genes was maintained after 96 h and accompanied by a decreased intracellular triglyceride accumulation, while no effect on lipogenic genes was shown. Thus, 96-h Cu exposure induced lipid depletion, possibly due to the upregulation of lipolysis. Although in this process, lipogenesis might be up-regulated, it was not enough to compensate lipid consumption. Our study represents the first approach to concentration- and time-dependent in vitro effects of Cu on lipid metabolism of fish hepatocytes and provides new insights into Cu toxicity in fish at both enzymatic and molecular levels.

Purification and characterization of glucose 6-phosphate dehydrogenase (G6PD) from grass carp (Ctenopharyngodon idella) and inhibition effects of several metal ions on G6PD activity in vitro.

Glucose 6-phosphate dehydrogenase (G6PD) is a key enzyme catalyzing the first step of the pentose phosphate pathway which generates NADPH for anabolic pathways and protection systems in various organisms, including fish. In the present study, G6PD was purified from grass carp (Ctenopharyngodon idella) hepatopancreas using the methods of 2',5'-ADP-Sepharose 4B affinity chromatography followed by DEAE Sepharose Fast Flow ion exchange chromatography. The characterization of G6PD and inhibition effects of several metal ions on G6PD activity in vitro were also determined. Grass carp hepatopancreas G6PD, with a specific activity of 18 U/mg protein, was purified 1,066-fold with a yield of 19.5 % and Mr of 71.85 kDa. The enzyme had a temperature optimum of 42 °C, pH optimum of 7.5 and 9.0. The K(m) values for G6-P and NADP(+) were determined to be 0.026, 0.0068 mM, respectively. The V(max) values for G6-P and NADP(+) were 2.20 and 2.27 µM min(-1) mg protein(-1), respectively. The catalytic efficiency for G6-P and NADP as the substrates was 0.085 and 0.334 × 10(-6) min(-1) mg protein(-1), respectively. Inhibition effects of metal ions on the purified G6PD activity indicated that IC50 values of Zn(+2), Mn(+2), Al(+3), Cu(+2), and Cd(+2) were 0.42, 0.54, 0.94, 1.20, and 4.17 mM, respectively. The Ki constants of Zn(+2), Al(+3), Cu(+2), and Cd(+2) were 0.52, 1.12, 0.26, and 4.8 mM, respectively. Zn(+2), Al(+3), and Cd(+2) showed competitive inhibition, while Cu(+2) inhibited the G6PD in a noncompetitive inhibition manner. Our study provided important information about the control of the grass carp liver PPP, the biosynthesis of several important related biomolecules, and the status of detoxification systems in grass carp liver in relation to metabolism.

Purification and characterization of 6-phosphogluconate dehydrogenase (6-PGD) from grass carp (Ctenopharyngodon idella) hepatopancreas.

6-Phosphogluconate dehydrogenase (6-PGD, E.C.: 1.1.1.44) was purified and characterized from the hepatopancreas of grass carp (Ctenopharyngodon idella) for the first time. Grass carp represents the second largest aquaculture industry in the world after silver carp, constituting 14.7% of the world aquaculture production, with an average annual increase of 14% in China, mainly as a source of food. The purification procedure involved a single 2', 5'-ADP-Sepharose 4B affinity chromatographic step by using different elution buffers. The enzyme was purified 309-fold with a specific activity of 5.259 U/mg protein and yield of 68%. The purity and subunit molecular weights of the 6-PGD were checked on SDS-PAGE and purified enzyme showed a single band on the gel. The subunit molecular mass was 57 kDa, with an optimum pH, temperature and ionic strength at 7.96, 50 degrees C and 100 mM Tris-HCl, respectively. The Km values of 6-PGA and NADP+ were 0.019 and 0.0052 mM, respectively, while Vm of 6-PGA and NADP+ was 0.69 U/ml. Dissociation constants (Ki) for 6-PGA and NADP+ were 2.05 and 0.12 mM, respectively. NADPH inhibited the enzyme in a competitive manner and its Ki value was 0.032 mM. The Cu2+, Zn2+, Cd2+ and Al3+ showed inhibitory effects on the enzyme with IC50 values of 0.293, 0.099, 0.045 and 1.526 mM, respectively. All tested metals inhibited the enzyme in a competitive manner, indicating that these metals might be toxic even at low concentrations for the 6-PGD. As the fish is one of valuable foodstuff of animal sources for human consumption, under certain environmental conditions, metal ions accumulated in fish up to a lethal concentration may be harmful for human health. Therefore, it is impending to reduce the concentration of metal ions in contaminated lakes and rivers for fishery and also for human health.