Effects And Mechanisms Of Copper On Lipid Metabolism In Yellow Catfish Pelteobagrus Fulvidraco And Javelin Goby Synechogobius Hasta

Copper(Cu), an essential microelement for fish, has numerous physiological functions. However, it is also a heavy-metal element and can be deleterious at excessive concentrations. Previous studies mainly concern about the effects of Cu on growth performance, tissue Cu accumulation, histological changes and oxidative stress in fish, but information is quite scarce on Cu influencing lipid deposition and metabolism. In this study, the c DNA sequence of genes involved in lipid metabolism from yellow catfish Pelteobagrus fulvidraco and javelin goby Synechogobius hasta were cloned and their m RNA tissue expression profiles were determined, and then the effects and mechanisms of waterborne Cu exposure and dietary Cu levels(deficiency and excess) on lipid metabolism in these two fish species were investigated. The main results of the present study are as follows: 1. Molecular cloning and tissue m RNA levels of genes involved in lipid metabolism from yellow catfish Pelteobagrus fulvidraco and javelin goby Synechogobius hastaTwo complete HSL c DNA sequences, designated HSL1 and HSL2, were amplified by RT-PCR and RACE approaches from P. fulvidraco using the degenerate primers. The validated c DNAs encoding for HSL1 and HSL2 were 2739 and 2629 bp 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 HSL1 and HSL2 were expressed in all tested tissues(including liver, brain, gill, heart, muscle, spleen, intestine, kidney and mesenteric fat) during different developmental stages(larvae, juvenile, sub-adult and adult), but the abundance of each HSL m RNA showed the tissue- and developmental stage-dependent expression patterns, probably suggesting that these two HSL isoforms had different physiological functions.By RT-PCR method with the degenerate primers, we obtained the partial c DNA sequence of 15 genes involved in lipid metabolism from S. hasta, including G6 PD, 6PGD, ME, ICDH, FAS, ACCα, ACCβ, LPL, ATGL, HSL1, HSL2, CPT IA, SREBP-1, PPARα and PPARγ. The amino acid sequence of each gene shared 54.6%–93.1% similarity with other species(Homo sapiens, Rattus norvegicus, Xenopus tropicalis, and Danio rerio). Phylogenetic analysis further identified these genes, and confirmed the classification and evolutionary status of S. hasta. m RNA of all genes was detected in the liver, spleen, muscle, gill, brain, intestine, and heart, but at varying levels. 2. Effects of waterborne Cu exposure on growth performance, tissue Cu accumulation, and hepatic lipid metabolism in javelin goby Synechogobius hastaThe present study was conducted to determine growth performance, tissue Cu accumulation and hepatic lipid metabolism in S. hasta exposed to waterborne Cu concentrations of control, 57, and 118 μg Cu/L, respectively, for 30 days. Growth decreased, but hepatosomatic index(HSI), viscerosomatic index(VSI), and hepatic lipid content increased with increasing waterborne Cu levels. Staining with oil red O showed extensive steatosis in the liver of Cu-exposed fish. Cu exposure increased hepatic 6PGD, G6 PD and ME activities, whereas FAS, ICDH and CPT I activities remained unaffected. In addition, Cu exposure increased Cu contents in liver, gill and gastrointestinal tract, but had no significant effect on Cu concentrations in muscle and spleen. This study indicated that waterborne Cu exposure could enhance the metabolism of lipid synthesis and consequently induce the increase of hepatic lipid deposition in S. hasta. 3. Effects of waterborne Cu exposure on lipid metabolism in yellow catfish Pelteobagrus fulvidracoIn order to investigate the effects and mechanisms of waterborne Cu exposure on lipid metabolism in liver and visceral adipose tissue(VAT) of P. fulvidraco, fish were exposed to four waterborne Cu concentrations(control, 24, 71, and 198 μg Cu/L, respectively) for 6 weeks. Waterborne Cu exposure had a negative effect on growth and several condition indices(viscerosomatic index, hepatosomatic index, visceral adipose index and condition factor). In liver, lipid content, activities of lipogenic enzymes(6PGD, G6 PD, ME, ICDH and FAS) as well as the m RNA levels of 6PGD, G6 PD, FAS and SREBP-1 genes decreased with increasing Cu concentrations. However, the activity and the m RNA level of LPL in liver increased. In VAT, G6 PD, ME and LPL activities as well as the m RNA levels of FAS, LPL and PPARγ decreased in fish exposed to higher Cu concentrations. Additionally, the differential Pearson correlations between transcription factors(SREBP-1 and PPARγ), and the activities and m RNA 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 m RNA 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. 4. Effects of waterborne Cu exposure on carnitine concentrations, kinetics and m RNA levels of CPT I in yellow catfish Pelteobagrus fulvidracoIn order to further clarify the mechanisms of waterborne Cu exposure affecting lipid metabolism in P. fulvidraco, fish were exposed to four waterborne Cu concentrations(control, 24, 71, and 198 μg Cu/L, respectively) for 6 weeks. Afterwards, carnitine concentrations, kinetics, and the m RNA levels of CPT I in the liver and muscle were analyzed. In the liver, waterborne Cu exposure increased maximal reaction rates(Vmax), Michaelis–Menten constants(Km) and catalytic efficiency(Vmax/Km); while in the muscle, Vmax in the 198 μg Cu/L group was higher than that in the control, but Km and Vmax/Km showed no significant differences among the treatments. For carnitine status, total carnitine(TC) and acylcarnitine(AC) contents as well as AC/FC ratio increased with increasing waterborne Cu concentrations in the liver, but free carnitine(FC) content showed no significant differences among the treatments; in the muscle, FC content showed a decreasing trend but AC content showed an increasing trend with the increase of Cu levels, and consequently increased the AC/FC ratio with increase in Cu doses. For the expression of CPT I isoforms, fish exposed to 198 μg Cu/L significantly up-regulated the m RNA levels of CPT Iα1a, CPT Iα1b and CPT Iα2a in the liver, as well as CPT Iα1a, CPT Iα1b and CPT Iβ in the muscle. Additionally, some correlations were observed among CPT I activities, m RNA levels and Km for carnitine. Thus, the present study indicated that waterborne Cu exposure influenced CPT I activity by changing carnitine composition, CPT I kinetics and the m RNA levels of CPT I isoforms, thereby affecting lipid metabolism and deposition in P. fulvidraco. 5. Effects of dietary Cu deficiency and excess on lipid metabolism in yellow catfish Pelteobagrus fulvidracoIn order to investigate the effects and mechanisms of dietary Cu deficiency and excess on lipid metabolism in liver, muscle and visceral adipose tissue(VAT) of P. fulvidraco, fish were fed 0.76(Cu deficiency), 4.18(adequate Cu) and 92.45(Cu excess) mg Cu/kg diet, respectively, for 8 weeks. Weight gain and specific growth rate 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, G6 PD and FAS, and the m RNA levels of 6PGD, G6 PD, FAS, ACCα, PPARγ, LXR, HSL, PPARα and ATGL; Cu excess induced Cu accumulation, reduced the lipid content, FAS activity as well as the m RNA levels of 6PGD, G6 PD, 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 m RNA levels of the most tested genes, including of 6PGD, G6 PD, FAS, SREBP-1, PPARγ, HSL and LPL. In VAT, Cu and lipid contents, FAS activity, and the m RNA levels of 6PGD, G6 PD, 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 expressions related to lipid metabolism. Different response patterns of enzymatic activities and gene expressions in various tissues following dietary Cu levels indicated the tissue-specific regulatory effect by Cu. 6. Effects of dietary Cu deficiency and excess on carnitine concentrations, kinetics and m RNA levels of CPT I in yellow catfish Pelteobagrus fulvidracoIn order to further clarify the mechanisms of dietary Cu deficiency and excess affecting lipid metabolism in P. fulvidraco, fish were fed 0.76(Cu deficiency), 4.18(adequate Cu) and 92.45(Cu excess) mg Cu/kg diet, respectively, for 8 weeks. Afterwards, carnitine concentrations, kinetics, and m RNA levels of CPT I in the liver and muscle were analyzed. In the liver, Cu deficiency did not significantly affect the contents of FC, TC, and AC as well as the ratio of AC/FC; while Cu excess reduced FC, TC and AC contents, and the ratio of AC/FC. In the muscle, dietary Cu levels showed no significant effects on the contents of FC, TC and AC, but Cu excess significantly increased the ratio of AC/FC. Compared with the adequate Cu group, dietary Cu deficiency did not significantly affect the Vmax, Km and Vmax/Km in the liver and muscle; but Cu excess decreased Vmax and Vmax/Km in the liver, and increased Vmax in the muscle. The m RNA expression of four CPT I isoforms(CPT Iα1a, CPT Iα1b, CPT Iα2a and CPT Iβ) in the liver and muscle were differentially influenced by dietary Cu levels. Additionally, some correlations were observed among CPT I activities, m RNA levels and Km for carnitine. The present study indicated that dietary Cu deficiency and excess differentially influenced carnitine status, kinetics and expression profiles of CPT I in the liver and muscle of P. fulvidraco, which consequently affected lipid deposition and metabolism.