| The endoplasmic reticulum(ER) is an important intracellular organelle responsible for protein and lipid synthesis, xenobiotic detoxification and cellular calcium storage. Conditions disrupting the ER homeostasis cause accumulation of unfolded and misfolded proteins in the ER lumen, create a state defined as ER Stress, and activate a complex signaling network termed unfolded protein response(UPR). During the last few years, in mammals, ER stress and UPR-related proteins have been extensively analyzed at the molecular level; the role and mechanism of ER Stress and UPR in hepatic lipid metabolism has also been under intense investigation. However, in fish, the study involved in the ER Stress and UPR was often scarce.Copper(Cu) is a cofactor of variety of enzymes and plays an important role in fish metabolism. Recent studies in our laboratory indicated that Cu exposure influenced hepatic lipid deposition and metabolism in the fish. However, the earlier study in our laboratory and other laboratories only determined the change of lipid metabolism-related genes expression and enzyme activities after Cu exposure. The molecular and regulatory mechanisms in the upstream pathway of hepatic lipid metabolism under Cu exposure have not been explored. Given the ER origin of lipid droplets and the close association of the UPR, we are interested in investigating the potential role of the ER stress and disturbed Ca2+ homeostasis in the regulation of hepatic lipid metabolism in fish. In this study, the c DNA sequence of genes involved in ER stress from yellow catfish Pelteobagrus fulvidraco and javelin goby Synechogobius hasta were cloned, and then the roles and mechanisms of ER stress and disturbed calcium homeostasis are involved in Cu-induced alteration in hepatic lipid metabolism in the two fish species were investigated. The main results are as follows: Part One: Molecular cloning and tissue m RNA levels of genes involved in ER Stress from yellow catfish Pelteobagrus fulvidraco and javelin goby Synechogobius hastaTwo ER molecular chaperones [glucose-regulated protein 78(GRP78) and calreticulin(CRT)] and three ER stress sensors [PKR-like ER kinase(PERK), inositol requiring enzyme(IRE)-1α, and activating transcription factor(ATF)-6α] complete c DNAs were firstly characterized using the degenerate primers by RACE approaches from P. fulvidraco and S. hasta. In P. fulvidraco, the GRP78, CRT, PERK, IRE-1α and ATF-6α genes were shown to be 2544 bp, 1678 bp, 3776 bp, 3440 bp and 2785 bp, respectively, encoding a protein of 652 amino acids, 418 amino acids, 1080 amino acids, 1038 amino acids and 657 amino acids, respectively; In S. hasta, the GRP78, CRT, PERK, IRE-1α and ATF-6α genes were shown to be 2477 bp, 1953 bp, 4050 bp, 4095 bp and 2845 bp, 2544 bp, 1678 bp, 3776 bp, 3440 bp and 2785 bp, respectively, encoding a protein of 652 amino acids, 419 amino acids, 1106 amino acids, 1027 amino acids and 645 amino acids, respectively. In P. fulvidraco, the amino acid sequence of each gene shared 40.4%–94.9% similarity with other species(Danio rerio, Oryzias latipe, Xenopus tropicalis, and Homo sapiens); In S.hasta, the amino acid sequence of each gene shared 41.5%–97.8% similarity with other species(Danio rerio, Rattus norvegicus, Xenopus tropicalis, and Homo sapiens). Phylogenetic analysis further confirmed the classification and evolutionary status of P. fulvidraco and S. hasta. The predicted amino acid sequences for the P. fulvidraco and S. hasta GRP78, CRT, PERK, IRE-1α and ATF-6α revealed that the proteins contained all of the structural features that were characteristic of the five genes in other species, including the KDEL motif, signal peptide, sensor domain and effector domain.The analysis of tissue distribution profiles by q PCR indicated that m RNAs of the five genes(GRP78, CRT, PERK, IRE-1α and ATF-6α) were expressed in various tissues of the P. fulvidraco and S. hasta, but their m RNA levels varied among tissues. The m RNA levels of five ER stress-related genes were relatively higher in the liver than those in other tissues. Part Two: Effects of Cu on ER Stress and the ER Stress-mediated signal pathway in yellow catfish Pelteobagrus fulvidraco and javelin goby Synechogobius hasta and their mechanism 2.1. Effects of waterborne Cu exposure on ER stress and the ER stress-mediated signal pathway in javelin goby Synechogobius hastaThe present study was conducted to determine the effects of waterborne Cu exposure on ER ultrastructure, ER stress and the ER stress-mediated signal pathway in P. fulvidraco exposed to waterborne Cu concentrations of control, 30, and 60 μg Cu/L, respectively, for 56 days. Sampling occurred on day 28 and day 56, respectively. On day 28, the expression of hepatic GRP78, GRP94 and CRT were significantly increased in Cu-exposed group, indicating the occurrence of ER stress. The phenomenon was further confirmed by ER ultrastructural alterations in hepatocytes of P. fulvidraco after Cu exposure. On 28 day, Cu exposure significantly upregulated m RNA levels of hepatic PERK, e IF2α,IRE-1α and XBP-1, but not ATF-6 m RNA level, indicating the activation of PERK–e IF2α and IRE1α–XBP1 pathways. On day 56, the expression of ER Stress marker genes(GRP78/Bi P, GRP94 and CRT) showed no significant differences among the treatments, and the m RNA levels of hepatic e IF2α, IRE-1αand XBP-1 were significantly decreased in yellow catfish exposed to Cu.In addition, the ER is the most important intracellular calcium store, and plays a crucial role in calcium homeostasis. Disturbance of intracellular calcium homeostasis triggers the ER stress response, and maintaining ER calcium stores is critical for the quality and efficiency of protein folding. In the present study, using the primary hepatocytes of P. fulvidraco, 2-APB and dantrolene(specific inhibitors of the ER Ca2+-release channels IP3 and Ry R receptor, respectively), were used to examine whether the Ca2+ release from ER was involved in the Cu-induced ER stress by Fluo-4/AM. Compared to the DMSO treatment control, Cu alone significantly increased intracellular Ca2+ concentration. Dantrolene and 2-APB prevented Cu-induced intracellular Ca2+ elevation, which demonstrated that the release of Ca2+ from the ER was mediated by both Ry R and IP3 R. 2.2. Effects of dietary Cu deficiency and excess on ER Stress and the ER Stress-mediated signal pathway in yellow catfish Pelteobagrus fulvidracoIn order to determine the effects and mechanisms of dietary Cu deficiency and excess on ER stress and the ER stress-mediated signal pathway in liver and muscle of P. fulvidraco, fish were fed Cu deficiency(0.76 mg Cu/kg diet), adequate Cu(4.18 mg Cu/kg diet) and Cu excess(92.45 mg Cu/kg diet), respectively, for 8 weeks. In liver, compared to dietary adequate Cu group, dietary Cu excess significantly upregulated m RNA expression levels of GRP78, CRT, PERK, e IF2α, IRE-1α and XBP-1, and dietary Cu deficiency only significantly upregulated m RNA expression levels of CRT but showed no significant effects on other tested genes’ expression. In muscle, compared to dietary adequate Cu group, dietary Cu excess significantly upregulated m RNA levels of GRP78, CRT and XBP-1, but showed no significant effects on m RNA expression of PERK, e IF2α, IRE-1α, XBP-1 and ATF-6α. m RNA levels of all seven tested genes showed no significant differences between dietary Cu deficiency group and dietary adequate Cu groups in skeletal muscle. The ER stress plays important roles in lipid homeostasis, the ER stress and UPR may be the fundamental reaction to the dietary Cu exposure. 2.3. Effects of waterborne Cu exposure on ER Stress and the ER Stress-mediated signal pathway in javelin goby Synechogobius hastaThe present study was conducted to determine effects of waterborne Cu exposure on ER stress and the ER stress-mediated signal pathway. To this end, S. hasta were exposed to four waterborne Cu concentrations control, 18μg Cu/L, 38 μg Cu/L and 55μg Cu/L. Sampling occurred on day 30 and day 60, respectively. On day 30, the expression of hepatic GRP78 and CRT were significantly increased in Cu-exposed group, indicating the occurrence of ER stress. Waterborne Cu exposure also significantly up-regulated m RNA levels of hepatic PERK, e IF2α, IRE-1α, XBP-1 and ATF-6α, indicating the activation of UPR pathways. On day 60, the m RNA level of hepatic GRP78, CRT, PERK, e IF2α, IRE-1α, XBP-1 and ATF-6α were significantly increased in liver of S. hasta exposed to 19 μg Cu/l and 38 μg Cu/l. However, the m RNA levels of GRP78, CRT, PERK, e IF2α, IRE-1α, XBP-1 and ATF-6α from the 55 μg Cu/l groups were significantly lower than those from the control group, whereas the opposite result was observed on day 30. The present study indicated Cu exposure induces ER Stress and UPR in a time- and concentration-course change.To investigate whether the Ca2+ release from ER is involved in the Cu-induced ER stress, primary hepatocytes from S. hasta were treated with Cu exposure pretreated with 2-APB and dantrolene, respectively. Compared to the DMSO treatment control, Cu alone significantly increased intracellular Ca2+ concentration. 2-APB, which inhibits the release of Ca2+ through ER IP3 receptor, reduced to about three-fourths of Ca2+ level. Dantrolene, which inhibits Ry R, also reduced the intracellular Ca2+ levels by one-third. Thus, the present study indicated that Cu induced ER Stress, at least in part, via Ca2+ release from ER. Part Three: Effects of ER Stress and the ER Stress-mediated signal pathway in Cu-induced alterations in hepatic lipid deposition and metabolism in yellow catfish Pelteobagrus fulvidraco and javelin goby Synechogobius hasta and their mechanism 3.1. Effects of ER Stress and the ER Stress-mediated signal pathway in Cu-induced alterations in hepatic lipid deposition and SREBP-1c activation in yellow catfish Pelteobagrus fulvidraco and their mechanismIn order to investigate the effects and mechanisms of waterborne Cu exposure on hepatic lipid deposition and SREBP-1c activation, P. fulvidraco exposed to waterborne Cu concentrations of control, 30, and 60 μg Cu/L, respectively, for 56 days. Sampling occurred on day 28 and day 56, respectively. On day 28, the m RNA level of SREBP-1c was significantly increased in Cu exposure groups; Cu exposure increased m RNA expression of ACC and FAS(SREBP-1c targeting enzymes). However, on day 56, Cu exposure significantly decreased the m RNA level of SREBP-1c, ACC and FAS. Cu exposure did not influence the m RNA levels of LXRα and PPARγ on day 28 and day 56, indicating that only SREBP-1c, not LXRα and PPARγ, mediated the Cu-induced changes of lipogenesis. To study how Cu exposure mediated hepatic SREBP-1c activation, the m RNA levels of Insig-1 and SCAP were analyzed. On day 28, Cu exposure significantly down-regulated the m RNA level of hepatic Insig-1, and up-regulated the SCAP m RNA level. However, on day 56, Cu exposure significantly reduced the m RNA level of hepatic Insig-1. The m RNA level of SCAP showed no significant differences on day 56 among the treatments. The suppressive effects of PBA on hepatic SREBP-1c activation were analyzed. PBA markedly attenuated the Cu-induced elevation of m RNA expression of hepatic SREBP-1c, SCAP, ACC and FAS, and downregulation of Insig-1. At the same time, an obvious hepatic lipid accumulation was observed with increasing Cu levels on day 28, whereas the opposite result was observed on day 56. Our study clearly indicated that Cu exposure evoked three typical ER Stress markers and activated two UPR branches, which played an important role on Cu-evoked hepatic SREBP-1c activation and dysregulation of lipid metabolism. 3.2. Effects of ER Stress and the ER Stress-mediated signal pathway in Cu-induced SREBP-1c activation in javelin goby Synechogobius hasta and their mechanismIn order to investigate whether Cu-induced SREBP-1c activation is suppressed through the attenuation of ER stress, primary hepatocytes of S. hasta were used to examine the effects of the chemical chaperone(4-PBA) on Cu-induced ER stress in hepatic lipid metabolism. 4-PBA significantly attenuated the Cu-evoked up-regulation of GRP78, CRT, PERK, e IF2α, IRE-1α, XBP-1 and ATF-6α m RNA levels. The m RNA levels of SREBP-1c and SCAP were significantly increased in Cu-treated groups; Cu increased m RNA expression of ACCα, SCD-1 and FAS, which are SREBP-1c target genes. Cu did not influence the m RNA level of LXRα and PPARγ, indicating that only SREBP-1c, not LXRα and PPARγ, mediated the Cu-induced changes of lipogenesis. In addition, FAS activity increased in Cu-treated groups, in parallel with the increase of hepatic TG content. The suppressive effects of 4-PBA on hepatic SREBP-1c activation were analyzed. 4-PBA markedly attenuated the Cu-induced elevation of m RNA expression of hepatic ACCα and FAS, and downregulation of Insig-1. Furthermore, 4-PBA significantly suppressed the Cu-induced reduction of FAS activity and TG content. The present study indicated ER stress and the ER stress-mediated signal pathway are involved in Cu-induced SREBP-1c activation in javelin goby Synechogobius hasta. |
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