SREBP-1 and LXRα pathways mediated Cu-induced hepatic lipid metabolism in zebrafish Danio rerio.

The present study was performed to explore the underlying molecular mechanism of Cu-induced disorder of lipid metabolism in fish. To this end, adult zebrafish were exposed to three waterborne Cu concentrations (0 (control), 8 and 16 µg Cu/L, respectively) for 60 days. Hepatic Cu content and hepatosomatic index increased after waterborne Cu exposure. H&E and oil red O stainings showed extensive steatosis in the liver of Cu-exposed fish. Cu exposure up-regulated lipogenic enzymes activities of ME, ICDH, 6PGD, G6PD and FAS, but down-regulated CPTI activities. Transcriptomic analysis indicated that lipid metabolism related pathways were significantly enriched in both low-dose and high-dose Cu exposure group. Genes involved in lipogenic process from fatty acid biosynthesis, fatty acid elongation, fatty acid desaturation to glycerolipid biosynthesis were up-regulated by Cu. To elucidate the mechanism, LXRα inhibitor SR9243 and SREBP1 inhibitor fatostatin were used to verify the role of LXRα and SREBP1 in Cu-induced disorder of lipid metabolism. Both SR9243 and fatostatin significantly attenuated the Cu-induced increase of TG accumulation of hepatocytes. Meanwhile, SR9243 significantly attenuated the Cu-induced up-regulation of expression of lipogenic genes (acaca, fas, icdh, dgat1, moat2 and moat3), and fatostatin significantly attenuated the up-regulation of expression of acaca, fas, g6pd, dgat1 and moat2. Enzymes analysis showed both SR9243 and fatostatin blocked the Cu-induced increase of lipogenic enzymes activities. Taken together, our findings highlight the importance of LXRα and SREBP1 in Cu-induced hepatic lipid deposition, which proposed a novel mechanism for elucidating metal element exposure inducing the disorder of lipid metabolism in aquatic vertebrates.

Identification of apoptosis-related genes Bcl2 and Bax from yellow catfish Pelteobagrus fulvidraco and their transcriptional responses to waterborne and dietborne zinc exposure.

Apoptosis plays a key role in the physiology of multicellular organisms, and has been well studied in mammals, but not in teleosts. Zinc (Zn) has been shown to be an important regulator of apoptosis and apoptosis involves in the regulation of lipid metabolism. Moreover, our recent study indicated that waterborne and dietborne Zn exposure differently influenced lipid metabolism in Pelteobagrus fulvidraco, but further mechanism remained unknown. The hypothesis of the present study is that apoptosis mediated the Zn-induced changes of lipid metabolism of P. fulvidraco subjected to different exposure pathways. To this end, we cloned full-length cDNA sequences of Bcl2 and three Bax subtypes involved in apoptosis in P. fulvidraco, explored their mRNA expressions in responses to different Zn exposure pathways. Bcl2 and three Bax subtypes shared similar domain structure as typical pro- and anti-apoptotic Bcl2 family members. Their mRNAs were widely expressed among various tissues, but at variable levels. Waterborne Zn exposure down-regulated mRNA levels of Baxg and ratios of Baxa/Bcl2, and Baxg/Bcl2, but showed no significant effects on mRNA abundances of Bcl2, Baxa and Baxb, and the ratio of Baxb/Bcl2. In contrast, dietborne Zn exposure up-regulated mRNA levels of Bcl2, Baxa, Baxb and Baxg, but reduced the ratios of Baxa/Bcl2, Baxb/Bcl2, and Baxg/Bcl2. Considering their important roles of these genes in apoptosis induced by Zn, apoptosis may mediate the Zn-induced changes of hepatic lipid metabolism of Pelteobagrus fulvidraco under different Zn exposure pathways. For the first time, we characterized the full-length cDNA sequences of Bcl2 and three Bax subtypes, determined their expression profiles and transcriptional responses to different Zn exposure pathways, which would contribute to our understanding of the molecular basis of apoptosis, and also provide new insights into physiological responses to different Zn exposure pathways.

Identification of autophagy related genes LC3 and ATG4 from yellow catfish Pelteobagrus fulvidraco and their transcriptional responses to waterborne and dietborne zinc exposure.

Autophagy mediates the regulation of lipid metabolism. Moreover, our recent study indicated that waterborne and dietborne zinc (Zn) exposure differentially influenced lipid metabolism in a fish species of significance for aquaculture, yellow catfish Pelteobagrus fulvidraco, but further mechanism remained unknown. The hypothesis of the present study is that autophagy mediated the Zn-induced changes of lipid metabolism of yellow catfish subjected to different exposure pathways. To this end, we cloned key genes involved in autophagy in yellow catfish, explored their mRNA expressions in responses to different Zn exposure pathways. Full-length cDNA sequences of two LC3 subtypes and six ATG4 isoforms were isolated from yellow catfish. More ATG4 members were firstly identified in fish that might have arisen by teleost-specific whole genome duplication events. All of these members shared similar domain structure to their orthologous genes of vertebrates. Their mRNAs were widely expressed in various tissues, but at variable levels. Extra Zn addition in water or diets induced (P < 0.05) mRNA expression of ATG4Da, ATG4Db and LC3B. Considering their important roles of these genes in lipid metabolism, ATG4Da, ATG4Db and LC3B may mediate the changes of Zn-induced hepatic lipid metabolism of yellow catfish under different Zn exposure pathways. For the first time, we characterized the full-length cDNA sequences of six ATG4 isoforms and two LC3 subtypes, determined their tissue expression profiles and transcriptional responses to different Zn exposure pathways, which would contribute to our understanding of the molecular basis of autophagy, and also provide new insights into physiological responses to different Zn exposure pathways.

Effects and mechanisms of waterborne copper exposure influencing ovary development and related hormones secretion in yellow catfish Pelteobagrus fulvidraco.

The present study was conducted to determine the effects and mechanism of waterborne copper (Cu) exposure influencing ovary development and related hormones secretion in yellow catfish Pelteobagrus fulvidraco. To this end, two experiments were conducted. In Exp. 1, the partial cDNA sequences of three steroidogenesis-related genes (androgen receptor (ar), steroidogenic factor 1 (sf-1) and steroidogenic acute regulatory protein (star)) were firstly characterized from P. fulvidraco. The predicted amino acid sequences for the P. fulvidraco ar, sf-1 and star contained the main structural features characteristic in other species. In Exp. 2, P. fulvidraco were exposed to three waterborne Cu concentrations (control, 30µg/l and 60µg/l, respectively) for 56days. Sampling occurred on day 28 and day 56, respectively. On day 28, the levels of serum sex-steroid hormones (FSH and LH) and the mRNA levels of steroidogenesis-related genes (3ß-hsd, cyp11a1, cyp17, cyp19a, sf-1 and star) were significantly increased in ovary of P. fulvidraco exposed to 30µg Cu/l. The immunohistochemical analysis showed the positive reaction of ER, VTG and aromatase in low dose exposure group. These indicated that in low dose and relative short-term exposure, Cu was beneficial. In contrast, 60µg Cu/l exposure significantly reduced the levels of serum FSH, LH, E2 and P, and the mRNA levels of ovarian 20ß-hsd, cyp19a and erα in P. fulvidraco. On day 56, waterborne Cu concentration exposure reduced the levels of serum gonadotropins and sex hormones, and down-regulated the mRNA levels of steroidogenesis-related genes, indicating long-term Cu exposure had toxic effect on the secretion of sex-steroid hormone in P. fulvidraco. For the first time, our study cloned cDNA sequences of ar, sf-1 and star in P. fulvidraco, and demonstrated the effects and mechanism of waterborne Cu exposure influencing hormones secretion and synthesis in dose- and time-dependent manner in P. fulvidraco, which will help to understand the Cu-induced reproductive toxicity at both protein and transcriptional levels in fish.

Endoplasmic reticulum stress and dysregulation of calcium homeostasis mediate Cu-induced alteration in hepatic lipid metabolism of javelin goby Synechogobius hasta.

The present study was conducted to investigate the effect of Cu exposure on endoplasmic reticulum (ER) stress and Ca(2+) homeostasis, and also explore the underlying mechanism of the ER stress and Ca(2+) homeostasis in the Cu-induced change of hepatic lipid metabolism in javelin goby Synechogobius hasta. To this end, four experiments were conducted. In experiment 1, the full-length cDNA sequences of two 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] cDNAs were firstly characterized from S. hasta. The predicted amino acid sequences for the S. hasta GRP78, CRT, PERK, IRE-1α and ATF-6α revealed that the proteins contained all of the structural features characteristic in other species. mRNAs of the five genes were expressed in various tissues, but their mRNA levels varied among tissues. In experiment 2, S. hasta were exposed to four waterborne Cu concentrations (control, 19µg/l, 38µg/l, and 57µg/l, respectively) for 60days. Cu exposure evoked ER stress in liver of S. hasta in a time- and concentration-course change. In experiment 3, specific inhibitors, 2-aminoethyldiphenyl borate (2-APB) and dantrolene, 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, which demonstrated the release of Ca(2+) from the ER was mediated by both RyR and IP3R. In experiment 4, a chemical chaperone, 4-phenyl butyric acid (4-PBA), was used to demonstrate whether Cu-induced alteration in lipid metabolism was suppressed through the attenuation of ER stress. Cu exposure evoked ER stress and sterol-regulator element-binding protein-1c (SREBP-1c) activation in hepatocytes of S. hasta, resulting in dysregulation of hepatic lipid metabolism. 4-PBA attenuated the Cu-induced elevation of mRNA expression of ER stress-related genes. For the first time, our study cloned GRP78, CRT, PERK, IRE-1α and ATF-6α genes in S. hasta and demonstrated their differential expression among tissues. Moreover, the study demonstrated the molecular mechanism by which ER stress might underlie the change of lipid metabolism induced by Cu in S. hasta.

De novo characterization of the liver transcriptome of javelin goby Synechogobius hasta and analysis of its transcriptomic profile following waterborne copper exposure.

Previous studies have investigated the physiological responses to chronic copper (Cu) exposure in the liver of Synechogobius hasta; however, little information is available on the underlying molecular mechanisms. In an effort to better understand the mechanisms of Cu toxicity and to illuminate global gene expression patterns modulated by Cu exposure, we obtained the liver transcriptome information of S. hasta by RNA sequencing (RNA-seq) technology and also investigated the differential expression of genes following waterborne Cu exposure. Using the Illumina sequencing platform, as many as 60,217 unigenes were generated, with 815 bp of average length and 1298 bp of unigene N50 after filtering and assembly. For functional annotation analysis, 34,860, 31,526, 31,576, 25,808, 11,542, and 21,721 unigenes were annotated to the NR, NT, Swiss-Prot, KEGG, COG, and GO databases, respectively, and total annotation unigenes were 37,764. After 30 days of exposure to 55 µg Cu/l, a total of 292 and 1076 genes were significantly up- and down-regulated, respectively. By KEGG analysis, 660 had a specific pathway annotation. Subsequent bioinformatics analysis revealed that the differentially expressed genes were mainly related to lipid metabolism, immune system, apoptosis, and signal transduction, suggesting that these signaling pathways may be regulated by Cu exposure. The present study provides comprehensive sequence information for subsequent gene expression studies regarding S. hasta, and the transcriptome profiling after Cu exposure is also expected to improve our understanding of the molecular toxicology of Cu.

Endoplasmic Reticulum Stress-Related Genes in Yellow Catfish Pelteobagrus fulvidraco: Molecular Characterization, Tissue Expression, and Expression Responses to Dietary Copper Deficiency and Excess.

Two endoplasmic reticulum (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] cDNAs were first characterized from yellow catfish, Pelteobagrus fulvidraco. The predicted amino acid sequences for the yellow catfish 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. mRNAs of the five genes mentioned above were expressed in various tissues, but their mRNA levels varied among tissues. Dietary Cu excess, but not Cu deficiency, activated the chaperones (grp78 and crt) and folding sensors in ER, and the UPR signaling pathways (i.e., perk-eif2α and the ire1-xbp1) in a tissue-specific manner. For the first time, our study cloned grp78, crt, perk, ire-1α, and atf-6α genes in yellow catfish and demonstrated their differential expression among tissues. Moreover, the present study also indicated differential regulation of these ER stress-related genes by dietary Cu deficiency and excess, which will be beneficial for us to evaluate effects of dietary Cu levels in fish at the molecular level, based on the upstream pathway of lipid metabolism (the ER) and thus provide novel insights regarding the nutrition of Cu in fish.

Dietary fenofibrate reduces hepatic lipid deposition by regulating lipid metabolism in yellow catfish Pelteobagrus fulvidraco exposed to waterborne Zn.

Fenofibrate is known to possess lipid-lowering effects by regulation of gene transcription involved in lipid metabolism. Waterborne Zn exposure induces lipid deposition in yellow catfish Pelteobagrus fulvidraco. Thus, the present working hypothesis is that dietary fenofibrate addition will reduce hepatic lipids in yellow catfish exposed to waterborne Zn. To this end, juvenile yellow catfish were exposed to 0.04 (control), 0.35 mg/L waterborne Zn, 0.15% dietary fenofibrate, and 0.35 mg Zn/l + 0.15% dietary fenofibrate for 8 weeks. Growth performance, lipid deposition and metabolism in the liver were determined. Dietary fenofibrate promoted growth performance and reduced hepatic lipid content of yellow catfish exposed to waterborne Zn. However, these effects did not appear in fish in normal water. The lipid-lowering effect of fenofibrate on fish exposed to waterborne Zn was associated with increased lipolysis, as indicated by increased CPT I activities and expression of lipolytic genes PPARα, CPT IA, ATGL and HSL, and with reduced lipogenesis as indicated by reduced activities of G6PD, 6PGD, ME and ICDH. Dietary fenofibrate significantly increased mRNA levels of FAS, LPL and ACCα, but reduced mRNA levels of ACCß and PPARγ in fish exposed to waterborne Zn. Pearson correlations between transcriptional factors expression, and activities and expression of several enzymes were observed, indicating that changes at the molecular and enzymatic levels may underlie the patterns of lipid metabolism and accordingly affect hepatic fat storage. Taken together, our results suggest that the lipid-lowering effect of fenofibrate was attributed, in part, to the down-regulation of lipogenesis and up-regulation of fatty acid oxidation.

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.