Xiao-Ying Tan

Differential effects of acute and chronic zinc (Zn) exposure on hepatic lipid deposition and metabolism in yellow catfish Pelteobagrus fulvidraco

The present study is conducted to determine the potential mechanisms of Zn on hepatic lipid deposition and metabolism for yellow catfish Pelteobagrus fulvidraco with 8-week chronic exposure to low Zn levels (Zn levels: 0.05, 0.35 and 0.86mg/l Zn, respectively) and 96-h acute exposure to a high Zn level (Zn level: 4.71mg/l Zn, respectively). For that purpose, hepatic lipid deposition and Zn accumulation, hepatic carnitine palmitoyltransferase I (CPT I) and lipoprotein lipase (LPL) activities, and the hepatic mRNA expression of ten genes involved in lipid metabolism are determined. Chronic (8 weeks) exposure to low Zn levels apparently increases hepatic lipid content, hepatosomatic index (HSI) (P<0.05) and LPL activity, and reduces hepatic CPT I activity. In contrast, the acute (96h) exposure to high Zn level reduces hepatic lipid content, HSI and LPL activity, and increases CPT I activity. The change of mRNA levels of genes related to lipid metabolism is Zn concentration-dependent. Pearson correlations among mRNA expression levels, lipid content, CPT I and LPL activities in liver are also observed in yellow catfish with the 8-week chronic Zn exposure. For the first time, our study demonstrates the effect of waterborne Zn exposure on lipid metabolism at the molecular levels in fish, which may contribute to understanding the mechanism of Zn-induced hepatic toxicity in fish.

Characterization and tissue distribution of leptin, leptin receptor and leptin receptor overlapping transcript genes in yellow catfish Pelteobagrus fulvidraco.

In the present study, full-length cDNA sequences of leptin (LEP), leptin receptor (LEPR) and leptin receptor overlapping transcript (LEPROT) were cloned from yellow catfish Pelteobagrus fulvidraco, and their tissue distribution profiles were determined. The validated cDNA of yellow catfish leptin (ycLEP), leptin receptor (ycLEPR) and LEPROT were 1119, 4195 and 827bp in length, encoding the peptide of 172, 1086 and 130 amino acid residues, respectively. The phylogenetic analysis revealed that fish LEP, LEPR and LEPROT were separated from tetrapod, and also ycLEPS were separated from other fish species. The ycLEP mRNA expression levels were highest in liver, followed by ovary, mesenteric fat and spleen, and lowest in intestine, heart, muscle, pituitary and testis. The ycLEPR mRNA levels were highest in pituitary, intermediate in mesenteric fat, liver, ovary, muscle and spleen, and lowest in heart, intestine and testis. The ycLEPROT mRNA levels were highest in pituitary, followed by spleen, mesenteric fat, heart, ovary, liver, muscle, testis and intestine. Identification and tissue distribution of yellow catfish LEP, LEPR and LEPROT genes provided initial step towards understanding their biological roles in yellow catfish.

Molecular cloning and expression pattern of 11 genes involved in lipid metabolism in yellow catfish Pelteobagrus fulvidraco

11 genes involved in lipid metabolism were cloned from liver of yellow catfish Pelteobagrus fulvidraco, including CPT 1A, CPT 1B, PPARα, PPARγ, SREBP-1, G6PD, 6PGD, FAS, acetyl-CoA ACCa, ACCb, and LPL. Phylogenetic analysis further identified these genes, and confirmed the classification and evolutionary status of yellow catfish. mRNA of all eleven genes was present in liver, muscle, mesenteric adipose, ovary and heart, but at varying levels. The present study will facilitate further studies on the regulation of lipid metabolism at the molecular level for the fish species.

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.

Molecular cloning and mRNA tissue expression of thyroid hormone receptors in yellow catfish Pelteobagrus fulvidraco and Javelin goby Synechogobius hasta

Thyroid hormones (THs) play a pivotal role in many physiological functions in vertebrates, including fish. Their effects are mediated by thyroid hormone receptors (TRs), which are members of the nuclear hormone receptor superfamily. In this study, full-length cDNA sequences of TRs from yellow catfish Pelteobagrus fulvidraco and Javelin goby Synechogobius hasta were cloned and their mRNA tissue expression profiles were determined. In P. fulvidraco, the validated cDNAs encoding for TRα and TRβ were 1789 and 1848 bp in length, encoding peptides of 401 and 378 amino acid residues, respectively. In addition, a TRβ spliced variant (named P. fulvidraco-TRβv), containing a 60-bp insertion, was detected. In S. hasta, cDNAs encoding for TRαA, TRαB and TRβ were 1827, 2295 and 2258 bp in length, encoding peptides of 401, 409 and 393 amino acid residues, respectively. The phylogenetic analysis revealed that TRα and TRβ cDNAs grouped into two separate clusters with other vertebrate counterparts and two TRα sequences grouped separately, suggesting that the two TRαs derived from paralogous genes that might arise during a teleost-specific genome duplication event. All TR mRNAs were detected in various tissues sampled. The mRNA levels of both TRα and TRβ from P. fulvidraco were the highest in brain, followed by liver, and lowest in heart, intestine, muscle, gill and spleen. However, in S. hasta, TRαA, TRαB and TRβ showed the highest mRNA levels in brain and lowest in muscle. Identification and mRNA tissue expression of TR genes from P. fulvidraco and S. hasta provide an initial step towards understanding their biological roles in the two fish species.

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.

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.

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.

JAK family members: Molecular cloning, expression profiles and their roles in leptin influencing lipid metabolism in Synechogobius hasta

Janus kinase (JAK) is a family of non-receptor tyrosine kinases that participate in transducing cytokine signals from the external environment to the nucleus in various biological processes. Currently, information about their genes structure and evolutionary history has been extensively studied in mammals as well as in several fish species. By contrast, limited reports have addressed potential role of diverse JAK in signaling responses to leptin in fish. In this study, we identified and characterized five JAK members of Synechogobius hasta. Compared to mammals, more members of the JAK family were found in S. hasta, which provided evidence that the JAK family members had arisen by the whole genome duplications during vertebrate evolution. For protein structure, all of these members possessed similar domains compared with those of mammals. Their mRNAs were expressed in a wide range of tissues, but at the different levels. Incubation in vitro of freshly isolated hepatocytes of S. hasta with different concentrations of recombinant human leptin decreased the intracellular triglyceride content and lipogenic genes expression, and increased mRNA expression of several JAK and lipolytic genes. AG490, a specific inhibitor of JAK, reversed leptin-induced effects on TG content and JAK2a, JAK2b, hormone-sensitive lipase (HSL2) and acetyl-CoA carboxylase (ACCa), indicating that the JAK2a/b may have mediated the actions of leptin on lipid metabolism at transcriptional level.

MiR-205 Mediated Cu-Induced Lipid Accumulation in Yellow Catfish Pelteobagrus fulvidraco

The present working hypothesis is that the Cu-induced changes in lipid metabolism may be mediated by miRNAs. Here, we describe the miRNA profile of the liver tissues of yellow catfish exposed to waterborne Cu, based on larger-scale sequencing of small RNA libraries. We identified a total of 172 distinct miRNAs. Among these miRNAs, compared to the control, mRNA expression levels of 16 miRNAs (miR-203a, 205, 1788-3p, 375, 31, 196a, 203b-3p, 2187-5p, 196d, 459-3p, 153a and miR-725, and two novel-miRNAs: chr4-1432, chr-7684) were down-regulated, and mRNA levels of miR-212 and chr20-5274 were up-regulated in Cu-exposed group. The functions of their target genes mainly involved ether lipid metabolism, glycerophospholipid metabolism, linoleic acid metabolism and α-linolenic acid metabolism. Cu exposure inhibited the expression of miR-205, whose predicted target genes were enriched in the pathway of lipid metabolism, including fas, lxrα, ddit3, lamp2, casp3a and baxa. These potential target genes were further verified by Dual-luciferase reporter gene assay. Using primary hepatocytes of yellow catfish, Cu incubation down-regulated miR-205 expression, and increased TG contents and FAS activity. LXR antagonist effectively ameliorate the Cu-induced change of TG content and FAS activity. These data suggest that down-regulation of the miRNA-205 may be an important step in Cu-induced changes in lipid metabolism in yellow catfish.