Yu-Feng Song

Hormone-sensitive lipase in yellow catfish Pelteobagrus fulvidraco: molecular characterization, mRNA tissue expression and transcriptional regulation by leptin in vivo and in vitro.

Hormone-sensitive lipase (hsl) plays a pivotal role in regulation of lipolysis in mammals, but information is very scarce about its gene structure and function in fish. In this study, two distinct hsl cDNAs, designated hsl1 and hsl2, were firstly isolated and characterized from yellow catfish Pelteobagrus fulvidraco. The validated cDNAs encoding for hsl1 and hsl2 were 2739 and 2629bp in length, encoding peptides of 679 and 813 amino acid residues, respectively, and shared 57.7% amino acid identity. The phylogenetic analysis revealed that hsl1 and hsl2 derived from paralogous genes that might have arisen during a teleost-specific genome duplication event. Both hsl mRNAs were expressed in a wide range of tissues, but the abundance of each hsl mRNA showed the tissue- and developmental stage-dependent expression patterns. Intraperitoneal injection in vivo and incubation in vitro of recombinant human leptin (rb-hLEP) stimulated the mRNA expression of hsl2, but not hsl1, in the liver and hepatocytes of P. fulvidraco, respectively, suggesting that two hsl isoforms might serve different roles in lipid metabolism. To our knowledge, for the first time, the present study provides evidence that two hsl mRNAs are differentially expressed with and among tissues during different developmental stages and also differentially regulated by leptin both in vivo and in vitro, which serves to increase our understanding on hsl physiological function 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.

Dietary l -carnitine supplementation increases lipid deposition in the liver and muscle of yellow catfish ( Pelteobagrus fulvidraco ) through changes in lipid metabolism

Carnitine has been reported to improve growth performance and reduce body lipid content in fish. Thus, we hypothesised that carnitine supplementation can improve growth performance and reduce lipid content in the liver and muscle of yellow catfish (Pelteobagrus fulvidraco), a commonly cultured freshwater fish in inland China, and tested this hypothesis in the present study. Diets containing l-carnitine at three different concentrations of 47 mg/kg (control, without extra carnitine addition), 331 mg/kg (low carnitine) and 3495 mg/kg (high carnitine) diet were fed to yellow catfish for 8 weeks. The low-carnitine diet significantly improved weight gain (WG) and reduced the feed conversion ratio (FCR). In contrast, the high-carnitine diet did not affect WG and FCR. Compared with the control diet, the low-carnitine and high-carnitine diets increased lipid and carnitine contents in the liver and muscle. The increased lipid content in the liver could be attributed to the up-regulation of the mRNA levels of SREBP, PPARγ, fatty acid synthase (FAS) and ACCa and the increased activities of lipogenic enzymes (such as FAS, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and malic enzyme) and to the down-regulation of the mRNA levels of the lipolytic gene CPT1A. The increased lipid content in muscle could be attributed to the down-regulation of the mRNA levels of the lipolytic genes CPT1A and ATGL and the increased activity of lipoprotein lipase. In conclusion, in contrast to our hypothesis, dietary carnitine supplementation increased body lipid content in yellow catfish.

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.

Endoplasmic reticulum stress and disturbed calcium homeostasis are involved in copper-induced alteration in hepatic lipid metabolism in yellow catfish Pelteobagrus fulvidraco.

The present study was conducted to investigate the effect of Cu exposure on ER stress and Ca(2+) homeostasis, and explore the underlying mechanism of the ER stress and disturbed Ca(2+) homeostasis in the regulation of hepatic lipid metabolism in yellow catfish Pelteobagrus fulvidraco. To this end, three experiments were conducted. In experiment 1, P. fulvidraco were exposed to three waterborne Cu concentrations for 56 days. Waterborne Cu exposure evoked ER stress and SREBP-1c activation and resulted in dysregulation of hepatic lipid metabolism in liver of P. fulvidraco in a time-dependent manner. In experiment 2, specific inhibitors 2-APB (IP3 receptor inhibitor) and dantrolene (RyR receptor inhibitor) were used to explore whether Ca(2+) release from ER was involved in the Cu-induced ER stress change. Dantrolene and 2-APB prevented Cu-induced intracellular Ca(2+) elevation, demonstrating that the release of Ca(2+) from the ER, mediated by both RyR and IP3R, contributed to dysregulation of lipid metabolism. In experiment 3, a chemical chaperone (PBA) was used to demonstrate whether Cu-induced alteration in lipid metabolism was suppressed through the attenuation of ER stress. PBA attenuated the Cu-induced elevation of mRNA expression of SREBP-1c, SCAP, ACC, FAS, GRP78/BiP, GRP94, CRT, eIF2α and XBP-1, and alleviated the Cu-induced downregulation of Insig-1. Based on these observations, these results reveal a link between ER stress and the change of lipid metabolism induced by Cu, which will help to understand the Cu-induced toxicity on cellular and molecular level, and provide some novel insights into the regulation of lipid metabolism in fish.

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

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

Different effect of dietborne and waterborne Zn exposure on lipid deposition and metabolism in juvenile yellow catfish Pelteobagrus fulvidraco

Juvenile yellow catfish Pelteobagrus fulvidraco were exposed to 0.04 or 0.35 mg l(-1) waterborne Zn, 27.25 or 213.84 mg kg(-1) dietary Zn, singly or in combination for 42 days. Growth and lipid metabolism in juvenile yellow catfish were investigated. Growth and survival were significantly inhibited by single waterborne Zn exposure but not by dietary Zn exposure. Dietary Zn addition reduced but waterborne Zn exposure increased hepatic lipid content. In contrast, muscle lipid content was reduced by waterborne Zn exposure but not by dietborne Zn exposure. The single exposure also affected several lipogenic enzymatic activities and expression of genes (in this article gene expression is taken synonymous to mRNA expression) related to lipogenesis and lipolysis. Pearson correlations among lipid content, enzymatic activities and mRNA expression levels were also observed, suggesting that changes at molecular and enzymatic levels may underlie the patterns of lipid metabolism and accordingly affect lipid deposition. For the first time, our study demonstrates the differential effect of different Zn exposure pathways on lipid metabolism at the molecular level in fish, indicating that the exposure route is critical to lipid deposition and metabolism.

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.

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

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