Mei-Qin Zhuo

Differential induction of enzymes and genes involved in lipid metabolism in liver and visceral adipose tissue of juvenile yellow catfish Pelteobagrus fulvidraco exposed to copper.

The present study was conducted to determine the mechanism of waterborne Cu exposure influencing lipid metabolism in liver and visceral adipose tissue (VAT) of juvenile 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 6 weeks. Waterborne Cu exposure had a negative effect on growth and several condition indices (condition factor, viscerosomatic index, hepatosomatic index and visceral adipose index). In liver, lipid content, activities of lipogenic enzymes (6-phosphogluconate dehydrogenase (6PGD), glucose-6-phosphate dehydrogenase (G6PD), malic enzyme (ME), isocitrate dehydrogenase (ICDH), and fatty acid synthase (FAS)) as well as mRNA levels of 6PGD, G6PD, FAS and sterol-regulator element-binding protein-1 (SREBP-1) genes decreased with increasing Cu concentrations. However, activity and mRNA level of lipoprotein lipase (LPL) gene in liver increased. In VAT, G6PD, ME and LPL activities as well as the mRNA levels of FAS, LPL and PPARγ genes decreased in fish exposed to higher Cu concentrations. The differential Pearson correlations between transcription factors (SREBP-1 and peroxisome proliferators-activated receptor-γ (PPARγ)), and the activities and mRNA expression of lipogenic enzymes and their genes were observed between liver and VAT. Thus, our study indicated that reduced lipid contents in liver and VAT after Cu exposure were attributable to the reduced activities and mRNA expression of lipogenic enzymes and their genes in these tissues. Different response patterns of several tested enzymes and genes to waterborne Cu exposure indicated the tissue-specific regulatory effect of lipid metabolism following waterborne Cu exposure. To our knowledge, the present study provides, for the first time, evidence that waterborne chronic Cu exposure can disturb the normal processes of lipid metabolism at both the enzymatic and molecular levels, and in two tissues (the liver and adipose tissue), which serves to increase our understanding of the mechanisms underlying lipid metabolism during Cu exposure.

Regulation of insulin on lipid metabolism in freshly isolated hepatocytes from yellow catfish (Pelteobagrus fulvidraco)

Although the metabolic actions of insulin in fish have been investigated widely in the past years, the regulatory effect of insulin on lipid metabolism has received little attention, especially in primary hepatocytes of fish. In the present study, freshly hepatocytes were isolated from yellow catfish, cultured and subjected to different insulin levels (0, 10, 100 and 1000nM) for 0h, 24h and 48h. Triglyceride (TG) content, activity and expression of several key enzymes involved in lipid metabolism, as well as mRNA levels of key transcription factors related to lipid metabolism, were assessed at 0h, 24h and 48h, respectively. Insulin incubation tended to increase the activities and expression of several lipogenic enzymes (such as FAS, G6PD, 6PGD). However, reduced CPT I gene expression was observed in hepatocytes following incubation treatment. Insulin administration also tended to up-regulate SREBP-1 expression but down-regulate PPARα mRNA levels. Insulin incubation enhanced lipogenesis and reduced lipolysis of freshly isolated hepatocytes of yellow catfish, in coincidence with increased TG content. Pearson correlations between expression of SREBP-1 and PPARα, and expression and activity of several enzymes were also observed, especially at 48-h insulin incubation. To the best of our knowledge, this is the first to study the effects of insulin on lipogenesis and lipolysis at both transcriptional and enzymatic levels using primary hepatocytes culture model in fish, which will help to understand the regulation of lipid metabolism by insulin in vivo, and will give us new insight into the insulin role in nutrient metabolism in fish.

Differential effects of dietary copper deficiency and excess on lipid metabolism in yellow catfish Pelteobagrus fulvidraco

The present study was conducted to investigate the effects and mechanism of dietary Cu deficiency and excess on lipid metabolism in the liver, muscle and VAT of juvenile Pelteobagrus fulvidraco. To this end, yellow catfish were fed 0.76 (Cu deficiency), 4.18 (adequate Cu) and 92.45 (Cu excess) mg Cu kg(-1) diet, respectively, for 8 weeks. WG and SGR in the adequate Cu group were significantly higher than those in Cu deficiency and excess groups. In liver, Cu deficiency showed no significant effect on Cu and lipid contents, the activities of 6PGD, G6PD and FAS, and the mRNA levels of many tested genes, including 6PGD, G6PD, FAS, ACCα, PPARγ, LXR, HSL, PPARα and ATGL. Cu excess induced Cu accumulation, reduced the lipid content, FAS activity as well as the mRNA levels of 6PGD, G6PD, FAS, ACCα, PPARγ, HSL and ATGL. In muscle, dietary Cu levels showed no significant effects on lipid content, the activities of lipogenic enzymes and the mRNA levels of the most tested genes, including of 6PGD, G6PD, FAS, SREBP-1, PPARγ, HSL and LPL. In VAT, Cu and lipid contents, FAS activity, and the mRNA levels of 6PGD, G6PD, FAS, SREBP-1, LXR, PPARα and LPL were not significantly influenced by dietary Cu levels. Thus, the change of lipid contents among tissues could be related to the enzymatic activities and gene expression related to lipid metabolism. Different response patterns of enzymatic activities and gene expression in various tissues following dietary Cu levels indicated the tissue-specific regulatory effect by Cu.

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 proteins 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.

Time-dependent effects of waterborne copper exposure influencing hepatic lipid deposition and metabolism in javelin goby Synechogobius hasta and their mechanism

The present study was conducted to determine the time-course of waterborne chronic copper (Cu) exposure effects influencing hepatic lipid deposition and metabolism in javelin goby Synechogobius hasta and their mechanisms. S. hasta were exposed to four waterborne Cu concentrations (2 (control), 18, 38 and 55 μg Cu/l) for 60 days. Sampling occurred on day 30 and day 60, respectively. Survival decreased and hepatic Cu content increased with increasing Cu levels. On day 30, Cu exposure increased hepatic lipid content, viscerosomatic index (VSI) and hepatosomatic index (HSI), and activities of lipogenic enzymes (6PGD, G6PD, ME, ICDH and FAS) as well as the mRNA levels of 6PGD, G6PD, ME, FAS, ACCα, LPL, PPARγ and SREBP-1 in the liver. However, the mRNA levels of ATGL, HSL and PPARα declined following Cu exposure. On day 60, Cu exposure reduced hepatic lipid content, HSI, VSI, activities of G6PD, ME, ICDH and FAS, and the mRNA expression of 6PGD, G6PD, ME, FAS and SREBP-1, but increased mRNA expression of CPT 1, HSL and PPARα. The differential Pearson correlation between transcriptional changes of genes encoding transcription factors (PPARα, PPARγ and SREBP-1), and the activities and mRNA expression of enzymes involved in lipogenesis and lipolysis were observed on day 30 and day 60, respectively. Cu exposure for 30 days induced hepatic lipid accumulation by stimulating lipogenesis and inhibiting lipolysis. However, 60-day Cu exposure reduced hepatic lipid content by inhibiting lipogenesis and stimulating lipolysis. To our knowledge, for the first time, the present study provided experimental evidence that waterborne chronic Cu exposure differentially influenced genes involved in lipogenic and lipolytic metabolic pathway and the enzymes encoded in a duration-dependent manner in fish, and provided new insight into the relationship between metal toxicity and lipid metabolism.

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.

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

ABSTRACT 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. Summary: Insulin plays a regulatory role in hepatic metabolism in yellow catfish, increasing lipid and triglyceride accumulation. These changes are mediated by the modulation of PPARα, PPARγ and PI3K signaling pathways.

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.

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.

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.