Dietary chenodeoxycholic acid attenuates high-fat diet-induced growth retardation, lipid accumulation and bile acid metabolism disorder in the liver of yellow catfish Pelteobagrus fulvidraco.

This experiment was conducted to investigate whether dietary chenodeoxycholic acid (CDCA) could attenuate high-fat (HF) diet-induced growth retardation, lipid accumulation and bile acid (BA) metabolism disorder in the liver of yellow catfish Pelteobagrus fulvidraco. Yellow catfish (initial weight: 4·40 (sem 0·08) g) were fed four diets: the control (105·8 g/kg lipid), HF diet (HF group, 159·6 g/kg lipid), the control supplemented with 0·9 g/kg CDCA (CDCA group) and HF diet supplemented with 0·9 g/kg CDCA (HF + CDCA group). CDCA supplemented in the HF diet significantly improved growth performance and feed utilisation of yellow catfish (P < 0·05). CDCA alleviated HF-induced increment of hepatic lipid and cholesterol contents by down-regulating the expressions of lipogenesis-related genes and proteins and up-regulating the expressions of lipololysis-related genes and proteins. Compared with the control group, CDCA group significantly reduced cholesterol level (P < 0·05). CDCA significantly inhibited BA biosynthesis and changed BA profile by activating farnesoid X receptor (P < 0·05). The contents of CDCA, taurochenodeoxycholic acid and glycochenodeoxycholic acid were significantly increased with the supplementation of CDCA (P < 0·05). HF-induced elevation of cholic acid content was significantly attenuated by the supplementation of CDCA (P < 0·05). Supplementation of CDCA in the control and HF groups could improve the liver antioxidant capacity. This study proved that CDCA could improve growth retardation, lipid accumulation and BA metabolism disorder induced by HF diet, which provided new insight into understanding the physiological functions of BA in fish.

Enrofloxacin (ENR) exposure induces lipotoxicity by promoting mitochondrial fragmentation via dephosphorylation of DRP1 at S627 site.

Enrofloxacin (ENR) is a kind of widespread hazardous pollutant on aquatic ecosystems and causes toxic effects, such as disorders of metabolism, on aquatic animals. However, its potential mechanisms at an environmental concentration on metabolic disorders of aquatic organisms remain unclear. Herin, we found that hepatic lipotoxicity was induced by ENR exposure, which led to ENR accumulation, oxidative stress, mitochondrial fragmentation, and fatty acid transfer blockage from lipid droplets into fragmented mitochondria. ENR-induced lipotoxicity and mitochondrial ß-oxidation down-regulation were mediated by reactive oxygen species (ROS). Moreover, dynamin-like protein 1 (DRP1) mediated ENR-induced mitochondrial fragmentation and changes of lipid metabolism. Mechanistically, ENR induced increment of DRP1 mitochondrial localization via dephosphorylating DRP1 at S627 and promoted its interaction with mitochondrial fission factor (MFF), leading to mitochondria fragmentation. For the first time, our study provides an innovative mechanistic link between hepatic lipotoxicity and mitochondrial fragmentation under ENR exposure, and thus identifies previously unknown mechanisms for the direct relationship between environmental ENR concentration and lipotoxicity in aquatic animals. Our study provides innovative insights for toxicological mechanisms and environmental risk assessments of antibiotics in aquatic environment.

Selenium Ameliorated Oxidized Fish Oil-Induced Lipotoxicity via the Inhibition of Mitochondrial Oxidative Stress, Remodeling of Usp4-Mediated Deubiquitination, and Stabilization of Pparα.

Aims: Studies demonstrated that oxidized fish oil (OFO) promoted oxidative stress and induced mitochondrial dysfunction and lipotoxicity, which attenuated beneficial effects of fish oil supplements in the treatment of nonalcoholic fatty liver disease (NAFLD). The current study was performed on yellow catfish, a good model to study NAFLD, and its hepatocytes to explore whether selenium (Se) could alleviate OFO-induced lipotoxicity via the inhibition of oxidative stress and determine its potential mechanism. Results: The analysis of triglycerides content, oxidative stress parameters, and histological and transmission electronic microscopy observation showed that high dietary Se supplementation alleviated OFO-induced lipotoxicity, oxidative stress, and mitochondrial injury and dysfunction. RNA-sequencing and immunoblotting analysis indicated that high dietary Se reduced OFO-induced decline of peroxisome-proliferator-activated receptor alpha (Pparα) and ubiquitin-specific protease 4 (Usp4) protein expression. High Se supplementation also alleviated OFO-induced reduction of thioredoxin reductase 2 (txnrd2) messenger RNA (mRNA) expression level and activity. The txnrd2 knockdown experiments revealed that txnrd2 mediated Se- and oxidized eicosapentaenoic acid (oxEPA)-induced changes of mitochondrial reactive oxygen species (mtROS) and further altered Usp4 mediated-deubiquitination and stabilization of Pparα, which, in turn, modulated mitochondrial fatty acid ß-oxidation and metabolism. Mechanistically, Usp4 deubiquitinated Pparα and ubiquitin-proteasome-mediated Pparα degradation contributed to oxidative stress-induced mitochondrial dysfunction. Innovation: These findings uncovered a previously unknown mechanism by which Se and OFO interacted to affect lipid metabolism via the Txnrd2-mtROS-Usp4-Pparα pathway, which provides the new target for NAFLD prevention and treatment. Conclusion: Se ameliorated OFO-induced lipotoxicity via the inhibition of mitochondrial oxidative stress, remodeling of Usp4-mediated deubiquitination, and stabilization of Pparα.

Functional analysis of selenok, selenot and selenop promoters and their regulation by selenium in yellow catfish Pelteobagrus fulvidraco.

The selenok, selenot and selenop are three key selenoproteins involved in stress response. Our study, using the yellow catfish Pelteobagrus fulvidraco as the experimental animal, obtained the 1993-bp, 2000-bp and 1959-bp sequences of selenok, selenot and selenop promoters, respectively, and predicted the binding sites of several transcriptional factors on their promoters, such as Forkhead box O 4 (FoxO4), activating transcription factor 4 (ATF4), Kruppel-like factor 4 (KLF4) and nuclear factor erythroid 2-related factor 2 (NRF2). Selenium (Se) increased the activities of the selenok, selenot and selenop promoters. FoxO4 and Nrf2 can directly bind with selenok promoter and controlled selenok promoter activities positively; KLF4 and Nrf2 can directly bind with selenot promoter and controlled selenot promoter activities positively; FoxO4 and ATF4 can directly bind to selenop promoter and regulated selenop promoter activities positively. Se promoted FoxO4 and Nrf2 binding to selenok promoter, KLF4 and Nrf2 binding to selenot promoter, and FoxO4 and ATF4 binding to selenop promoter. Thus, we provide the first evidence for FoxO4 and Nrf2 bindnig elements in selenok promoter, KLF4 and Nrf2 binding elements in selenot promoter, and FoxO4 and ATF4 binding elements in selenop promoter, and offer novel insight into regulatory mechanism of these selenoproteins induced by Se.

Characterization and tissue expression of twelve selenoproteins in yellow catfish Pelteobagrus fulvidraco fed diets varying in oxidized fish oil and selenium levels.

BACKGROUND: Selenium (Se) functions through selenoproteins and is essential to growth and metabolism of vertebrates. The present study was conducted to identify twelve selenoproteins genes (selenoe, selenof, selenoh, selneoi, selenom, selenok, selneon, selenoo, selenot, selenos, selenou and msrb1) from yellow catfish. Their mRNA expression patterns, as well as their response to dietary oxidized fish oils and Se addition were explored. METHODS: We use 3'and 5' RACE PCR to clone full-length cDNA sequence of twelve selenoprotein genes from yellow catfish. Their mRNA expression patterns were assessed via quantitative real-time PCR. Yellow catfish were fed diet adequate Se+ fresh fish oil, adequate Se+ oxidized fish oil, high Se+ fresh fish oil and high Se+ oxidized fish oil, respectively, for 10 weeks. Their kidney, heart, brain and testis were used to assess the mRNA expression of twelve selenoprotein. RESULTS: Twelve selenoprotein genes had similar domains with mammals and the other fish. Their mRNAs were expressed widely in eleven tissues but varied with the tissues. Dietary oxidized fish oils and Se addition influenced their mRNA abundances of twelve selenoproteins in a tissue-dependent manner. CONCLUSION: Our study demonstrated the characterization and expression of twelve selenoproteins, and elucidated their responses in yellow catfish fed diets varying in oxidized fish oils and Se addition, which increased our knowledge into the biological function and regulatory mechanism of Se and selenoproteins in fish.

Effects of Dietary Selenium and Oxidized Fish Oils on Intestinal Lipid Metabolism and Antioxidant Responses of Yellow Catfish Pelteobagrus fulvidraco.

Currently, the effect of selenium and oxidized fish oil interactions on the intestinal lipid metabolism and antioxidant responses of fish remains unknown. Herein, yellow catfish Pelteobagrus fulvidraco (weight: 3.99 ± 0.01 g) were used as experimental animals and were fed four diets: an adequate amount of selenium (0.25 mg kg-1) with fresh fish oil (A-Se+FFO), an adequate amount of selenium with oxidized fish oil (A-Se+OFO), a high amount of selenium (0.50 mg kg-1) with fresh fish oil (H-Se+FFO), and a high amount of selenium with oxidized fish oil (H-Se+OFO). The feeding experiment was conducted for 10 weeks. The results showed that selenium supplementation alleviated the intestinal tissue damage and reduced the lipid accumulation that was induced by oxidized fish oils. Meanwhile, we also found that 0.50 mg kg-1 selenium reduced the oxidative stress that is caused by oxidized fish oils through increasing the GSH and the activity and mRNA expression of antioxidant enzymes. Dietary selenium and oxidized fish oils also affected the mRNA expression of intestinal selenoproteins including selenow2a, selenop2, and selenot2. Mechanistically, Se and oxidized eicosapentaenoic acid (oxEPA) influenced the GSH content by affecting the DNA binding ability of activating transcription factor (ATF) 3 to the slc7a11 promoter. For the first time, our results suggested that selenium alleviated the oxidized fish oil-induced intestinal lipid deposition and the oxidative stress of the fish. We also elucidated the novel mechanism of selenium increasing the GSH content by affecting the interaction of ATF3 and the slc7a11 promoter.

Dietary choline prevents high fat-induced disorder of hepatic cholesterol metabolism through SREBP-2/HNF-4α/CYP7A1 pathway in a freshwater teleost yellow catfish Pelteobagrus fulvidraco.

Lipid overload-induced hepatic cholesterol accumulation is a major public health problem worldwide, and choline has been reported to ameliorate cholesterol accumulation, but its mechanism remains unclear. Our study found that choline prevented high-fat diet (HFD)-induced cholesterol metabolism disorder and enhanced choline uptake and phosphatidylcholine synthesis in the liver tissues; choline incubation prevented fatty acid (FA)-induced cholesterol accumulation and FA-induced inhibition of bile acid synthesis. Moreover, compared to single FA incubation, choline incubation or FA + choline co-incubation increased the mRNA abundances and protein levels of HNF4α and up-regulated the degradation of cholesterol into bile acids. Mechanistically, choline prevented the FA-induced accumulation of SREBP2 protein and the interaction between SREBP2 and HNF4α, thereby enhancing the DNA binding capacity of the HNF4α to the CYP7A1 promoter, and promoting the degradation of cholesterol into bile acids. Our study elucidated the novel regulatory mechanisms of choline preventing HFD-induced cholesterol accumulation and increasing bile acid synthesis by SREBP-2/HNF-4α/CYP7A1 pathway.

Iron increases lipid deposition via oxidative stress-mediated mitochondrial dysfunction and the HIF1α-PPARγ pathway.

Iron is an essential micro-element, involved in multiple biological activities in vertebrates. Excess iron accumulation has been identified as an important mediator of lipid deposition. However, the underlying mechanisms remain unknown. In the present study, we found that a high-iron diet significantly increased intestinal iron content and upregulated the mRNA expression of two iron transporters (zip14 and fpn1). Intestinal iron overload increased lipogenesis, reduced lipolysis and promoted oxidative stress and mitochondrial dysfunction. Iron-induced lipid accumulation was mediated by hypoxia-inducible factor-1 α (HIF1α), which was induced in response to mitochondrial oxidative stress following inhibition of prolyl hydroxylase 2 (PHD2). Mechanistically, iron promoted lipid deposition by enhancing the DNA binding capacity of HIF1α to the pparγ and fas promoters. Our results provide experimental evidence that oxidative stress, mitochondrial dysfunction and the HIF1α-PPARγ pathway are critical mediators of iron-induced lipid deposition.

Transcriptional Regulation and Protein Localization of Zip10, Zip13 and Zip14 Transporters of Freshwater Teleost Yellow Catfish Pelteobagrus fulvidraco Following Zn Exposure in a Heterologous HEK293T Model.

Zip family proteins are involved in the control of zinc (Zn) ion homeostasis. The present study cloned the promoters and investigated the transcription responses and protein subcellular localizations of three LIV-1 subfamily members (zip10, zip13, and zip14) from common freshwater teleost yellow catfish, Pelteobagrus fulvidraco, using in vitro cultured HEK293T model cells. The 2278 bp, 1917 bp, and 1989 bp sequences of zip10, zip13, and zip14 promoters, respectively, were subcloned into pGL3-Basic plasmid for promoter activity analysis. The pcDNA3.1 plasmid coding EGFP tagged pfZip10, pfZip13, and pfZip14 were generated for subsequent confocal microscope analysis. Several potential transcription factors' binding sites were predicted within the promoters. In vitro promoter analysis in the HEK293T cells showed that high Zn administration significantly reduced the transcriptional activities of the zip10, zip13, and zip14 promoters. The -2017 bp/-2004 bp MRE in the zip10 promoter, the -360 bp/-345 bp MRE in the zip13 promoter, and the -1457 bp/-1442 bp MRE in the zip14 promoter were functional loci that were involved in the regulation of the three zips. The -606 bp/-594 bp KLF4 binding site in the zip13 promoter was a functional locus responsible for zinc-responsive regulation of zip13. The -1383 bp/-1375 bp STAT3 binding site in the zip14 promoter was a functional locus responsible for zinc-responsive regulation of zip14. Moreover, confocal microscope analysis indicated that zinc incubation significantly reduced the fluorescence intensity of pfZip10-EGFP and pfZip14-EGFP but had no significant influence on pfZip13-EGFP fluorescence intensity. Further investigation found that pfZip10 localizes on cell membranes, pfZip14 colocalized with both cell membranes and lysosome, and pfZip13 colocalized with intracellular ER and Golgi. Our research illustrated the transcription regulation of zip10, zip13, and zip14 from P. fulvidraco under zinc administration, which provided a reference value for the mechanisms involved in Zip-family-mediated control of zinc homeostasis in vertebrates.

Sirt3-Sod2-mROS-Mediated Manganese Triggered Hepatic Mitochondrial Dysfunction and Lipotoxicity in a Freshwater Teleost.

Exposure to excessive manganese (Mn) is toxic to humans and animals. However, the toxic effects and mechanisms of excessive Mn influencing the vertebrates have been highly overlooked. In the present study, dietary Mn overload significantly increased hepatic lipid and Mn contents, decreased superoxide dismutase 2 (Sod2) activity, increased the Sod2 acetylation level, and induced mitochondrial dysfunction; Mn induced mitochondrial dysfunction through Mtf1/sirtuin 3 (Sirt3)-mediated acetylation of Sod2 at the sites K55 and K70. Meanwhile, mitochondrial oxidative stress was involved in Mn-induced lipotoxicity. Mechanistically, Mn-induced lipotoxicity was via oxidative stress-induced Hsf1 nucleus translocation and its DNA binding capacity to the regions of a peroxisome proliferator-activated receptor g (pparg) promoter, which in turn induced the transcription of lipogenic-related target genes. For the first time, our study demonstrated that Mn-induced hepatic lipotoxicity via a mitochondrial oxidative stress-dependent Hsf1/Pparg pathway and Mtf1/sirt3-mediated Sod2 acetylation participated in mitochondrial dysfunction. Considering that lipid metabolism and lipotoxicity are widely used as the biomarkers for environmental assessments of pollutants, our study provided innovative and important insights into Mn toxicological and environmental evaluation in aquatic environments.

Selenoprotein F (SELENOF)-mediated AKT1-FOXO3a-PYGL axis contributes to selenium supranutrition-induced glycogenolysis and lipogenesis.

Mounting evidence showed that excess selenium (10.0-15.0-fold of adequate Se) intake caused severe hepatic lipid deposition in the vertebrate. However, the underlying mechanism remains unclear. The study was performed to elucidate the mechanism of Se supranutrition mediated-changes of lipid deposition and metabolism. We found that dietary excessive Se addition increased hepatic TGs and glucose contents, up-regulated lipogenic enzyme activities and reduced hepatic glycogen contents. Transcriptomic and immunoblotting analysis showed that Se supranutrition significantly influenced serine/threonine kinase 1 (AKT1)-forkhead box O3a (FOXO3a)-PYGL signaling and protein levels of SELENOF. Knockdown of SELENOF and PYGL by RNA interference revealed that the AKT1-FOXO3a-PYGL axis was critical for Se supranutrition-induced lipid accumulation. Moreover, Se supranutrition-induced lipid accumulation was via the increased DNA binding capacity of FOXO3a to PYGL promoter, which increased glycogenolysis, and accordingly promoted lipogenesis and lipid accumulation. Our finding provides new insight into the mechanism of Se supranutrition-induced lipid accumulation and suggests that SELENOF may be a therapeutic target for Se supranutrition induced-lipid disorders in the vertebrates.

Manganese-Induced Oxidative Stress Contributes to Intestinal Lipid Deposition via the Deacetylation of PPARγ at K339 by SIRT1.

Aims: Excessive manganese (Mn) exposure is toxic, and induces lipid deposition, but the underlying mechanisms remain elusive. Herein, we explored how dietary Mn supplementation affects lipid deposition and metabolism in the intestine of vertebrates using the yellow catfish Pelteobagrus fulvidraco as the model. Results: High-Mn (H-Mn) diet increased intestinal Mn content, promoted lipid accumulation and lipogenesis, and inhibited lipolysis. In addition, it induced oxidative stress, upregulated metal-response element-binding transcription factor-1 (MTF-1), and peroxisome proliferator-activated receptor gamma (PPARγ) protein expression in the nucleus, induced PPARγ acetylation, and the interaction between PPARγ and retinoid X receptor alpha (RXRα), while it downregulated sirtuin 1 (SIRT1) expression and activity. Mechanistically, Mn activated the MTF-1/divalent metal transporter 1 (DMT1) pathway, increased Mn accumulation in the mitochondria, and induced oxidative stress. This in turn promoted lipid deposition via deacetylation of PPARγ at K339 by SIRT1. Subsequently, PPARγ mediated Mn-induced lipid accumulation through transcriptionally activating fatty acid translocase, stearoyl-CoA desaturase 1, and perilipin 2 promoters. Innovation: These studies uncover a previously unknown mechanism by which Mn induces lipid deposition in the intestine via the oxidative stress-SIRT1-PPARγ pathway. Conclusion: High dietary Mn intake activates MTF-1/DMT1 and oxidative stress pathways. Oxidative stress-mediated PPARγ deacetylation at K339 site contributes to increased lipid accumulation. Our results provided a direct link between Mn and lipid metabolism via the oxidative stress-SIRT1-PPARγ axis. Antioxid. Redox Signal. 37, 417-436.

HSF1-SELENOS pathway mediated dietary inorganic Se-induced lipogenesis via the up-regulation of PPARγ expression in yellow catfish.

At present, studies involved in the effects of dietary Se sources on lipid metabolism were very scarce and the underlying mechanism remains unknown. Previous studies reported that dietary Se sources differentially affected selenoprotein S (SELENOS) expression and SELENOS affected lipid metabolism via the inositol-requiring enzyme 1α (IRE1α)- spliced X-box binding protein 1 (XBP1s) pathway. Thus, we used yellow catfish as an experimental model to explore whether dietary selenium sources affected the hepatic lipid metabolism, and further determined the role of SELENOS-IRE1α-XBP1s pathway in dietary selenium sources affecting hepatic lipid metabolism. Compared with the selenomethionine (S-M) group, sodium selenite (SS) group possessed higher liver triglycerides (TGs) (34.7%), lipogenic enzyme activities (57.9-70.6%), and lower antioxidant enzyme activities (23.3-35.5%), increased protein levels of heat shock transcription factor 1 (HSF1) and SELENOS (1.17-fold and 47.4%, respectively), and XBP1s- peroxisome proliferators-activated receptor γ (PPARγ) pathway. Blocking SELENOS and PPARγ by RNA interference demonstrated that the SELENOS/XBP1s/PPARγ axis was critical for S-S-induced lipid accumulation. Moreover, S-S-induced upregulation of SELENOS was via the increased DNA binding capacity of HSF1 to SELENOS promoter, which activated the XBP1s/PPARγ pathway and promoted lipogenesis and lipid accumulation. XBP1s is required for S-S-induced upregulation of PPARγ expression. Our finding elucidated the mechanism of dietary Se sources affecting the lipid metabolism in the liver of yellow catfish and demonstrated novel function of SELENOS in metabolic regulation. Our study also suggested that seleno-methionine was a better Se source than selenite against abnormal lipid deposition in the liver of yellow catfish.

Dietary selenium sources differentially regulate selenium concentration, mRNA and protein expression of representative selenoproteins in various tissues of yellow catfish Pelteobagrus fulvidraco.

The study was conducted to determine the effects of three dietary Se sources, such as sodium-selenite (S-S), seleno-yeast (S-Y) and seleno-methionine (S-M), on Se concentration, glutathione peroxidase (GPX) and TXNRD activities, and mRNA expression of fifteen representative selenoproteins, and protein expression of four endoplasmic reticulum-resided selenoproteins in a wide range of tissues of yellow catfish. Compared with S-S and S-M groups, dietary S-Y significantly decreased growth performance and feed utilisation of yellow catfish. Dietary Se sources significantly influenced Se contents in the spleen, dorsal muscle and the kidney, GPX activities in spleen, kidney, intestine, muscle and mesenteric fat, and TXNRD activities in the heart, intestine and mesenteric fat. Among ten tested tissues, dietary Se sources influenced mRNA expression of GPX4 and SELENOK in three tissues; GPX3, SELENOS and TXNRD2 in four tissues; SELENOF, SELENON and DIO2 in five tissues; SELENOM, GPX1/2 and TXNRD3 in six tissues; SELENOW in seven tissue and SELENOP and SELENOT in eight tissues. Based on these observations above, S-S and S-M seem to be suitable Se sources for improving growth performance and feed utilisation of yellow catfish. Dietary Se sources differentially influence the expression of selenoproteins in various tissues of yellow catfish. For the first time, we determined the expression of selenoproteins in fish in responses to dietary Se sources, which contributes to a better understanding of the functions and regulatory mechanisms of selenoporteins.

Oxidized fish oils increased lipid deposition via oxidative stress-mediated mitochondrial dysfunction and the CREB1-Bcl2-Beclin1 pathway in the liver tissues and hepatocytes of yellow catfish.

At present, the harmful effects and relevant mechanism of oxidized fish oils on fish and fish cells remain unknown. Our study found that oxidized fish oils increased lipogenesis, and reduced lipolysis, activated oxidative stress by decreasing glutathione peroxidase (GPX) activity, increasing malondialdhyde (MDA) content and damaging mitochondrial structure, and activated autophagy in the liver of yellow catfish; oxidized eicosapentaenoic acid (oxEPA) induced oxidative stress in yellow catfish hepatocytes. Oxidative stress, mitochondrial dysfunction and lipophagy mediated oxEPA induced-variations in lipid metabolism. Our further investigation indicated that oxEPA-activated lipophagy was via inhibiting the DNA binding capacity of the cAMP-response element binding protein (CREB)-1 to the region of Bcl-2 promoter, which in turn suppressed the binding activity of Bcl-2 to Beclin1 and promoted autophagosome formation. For the first time, our study elucidated the mechanisms of oxidized fish oils-induced lipid deposition by the oxidative stress, mitochondrial dysfunction and CREB1-Bcl-2-Beclin1 pathway in fish.

Dietary Marginal and Excess Selenium Increased Triglycerides Deposition, Induced Endoplasmic Reticulum Stress and Differentially Influenced Selenoproteins Expression in the Anterior and Middle Intestines of Yellow Catfish Pelteobagrus fulvidraco.

Selenium (Se) is an essential micro-mineral and plays important roles in antioxidant responses, and also influences lipid metabolism and selenoprotein expression in vertebrates, but the effects and mechanism remain unknown. The study was undertaken to decipher the insights into dietary Se influencing lipid metabolism and selenoprotein expression in the anterior and middle intestine (AI and MI) of yellow catfish Pelteobagrus fulvidraco. Yellow catfish (weight: 8.27 ± 0.03 g) were fed a 0.03- (M-Se), 0.25- (A-Se), or 6.39- (E-Se) mg Se/kg diet for 12 wk. AI and MI were analyzed for triglycerides (TGs) and Se concentrations, histochemistry and immunofluorescence, enzyme activities, and gene and protein levelsassociated with antioxidant responses, lipid metabolism, endoplasmic reticulum (ER) stress, and selenoproteome. Compared to the A-Se group, M-Se and E-Se diets significantly decreased weight gain (WG) and increased TGs concentration in the AI and MI. In the AI, compared with A-Se group, M-Se and E-Se diets significantly increased activities of fatty acid synthase, expression of lipogenic genes, and suppressed lipolysis. In the MI, compared to the A-Se group, M-Se and E-Se diets significantly increased activities of lipogenesis and expression of lipogenic genes. Compared with A-Se group, E-Se diet significantly increased glutathione peroxidase (GPX) activities in the AI and MI, and M-Se diet did not significantly reduce GPX activities in the AI and MI. Compared with the A- Se group, E-Se diet significantly increased glutathione peroxidase (GPX) activities in the plasma and liver, and M-Se diet significantly reduced GPX activities in the plasma and liver. Compared with the A-Se group, M-Se and E-Se groups also increased glucose-regulated protein 78 (GRP78, ER stress marker) protein expression of the intestine. Dietary Se supplementation also differentially influenced the expression of the 28 selenoproteins in the AI and MI, many of which possessed antioxidant characteristics. Compared with the A-Se group, the M-Se group significantly decreased mRNA levels of txnrd2 and txnrd3, but made no difference on mRNA levels of these seven GPX proteins in the MI. Moreover, we characterized sterol regulatory element binding protein 1c (SREBP1c) binding sites of three ER-resident proteins (selenom, selenon, and selenos) promoters, and found that Se positively controlled selenom, selenon, and selenos expression via SREBP1c binding to the selenom, selenon, and selenos promoter. Thus, dietary marginal and excess Se increased TGs deposition of yellow catfish P. fulvidraco, which might be mediated by ER-resident selenoproteins expression and ER stress.

miR-101b Regulates Lipid Deposition and Metabolism of Primary Hepatocytes in Teleost Yellow Catfish Pelteobagrus fulvidraco.

Excessive fat deposition in the hepatocytes, associated with excess dietary fat intake, was related to the occurrence of fatty livers in fish. miR-101b plays the important roles in controlling lipid metabolism, but the underlying mechanism at the post-transcriptional level remains unclear. The purpose of this study is to explore the roles and mechanism of miR-101b-mediating lipid deposition and metabolism in yellow catfish Pelteobagrus fulvidraco. We found that miR-101b directly targeted fatty acid translocase (cd36), caspase9 (casp9) and autophagy-related gene 4A (atg4a). Furthermore, using palmitic acid (PA) or oleic acid (OA) to incubate the primary hepatocytes of yellow catfish, we demonstrated that miR-101b inversely regulated cd36, casp9, and atg4a expression at the transcriptional level; the inhibition of miR-101b aggravated fatty acids (FAs, PA or OA)-induced lipid accumulation, indicating that miR-101b mediated FAs-induced variations of lipid metabolism in yellow catfish. Taken together, our study gave novel insight into the regulatory mechanism of lipid deposition and metabolism and might provide potential targets for the prevention and treatment of fatty livers in fish.

Characterization and expression analysis of seven selenoprotein genes in yellow catfish Pelteobagrus fulvidraco to dietary selenium levels.

BACKGROUND: Selenium (Se) appears in the selenoproteins in the form of selenocysteine (Sec) and is important for the growth and development of vertebrates. The present study characterized seven selenoproteins, consisting of the GPX1, GPX3, GPX4, SELENOW, SELENOP, TXNRD2 and TXNRD3 cDNAs in various tissues of yellow catfish, explored their regulation to dietary Se addition. METHODS: 3' and 5' RACE PCR were used to clone full-length cDNA sequences of seven selenoprotein genes (GPX1, GPX3, GPX4, SELENOW, SELENOP, TXNRD2 and TXNRD3). Their molecular characterizations were analyzed, including conservative motifs and the SECIS elements. The phylogenetic trees were generated through neighbor-joining (NJ) method with MEGA 6.0 with 1000 bootstrap replications. Quantitative real-time PCR was used to explore their mRNA tissue distribution in the heart, anterior intestine, dorsal muscle, head kidney, gill, liver, brain, spleen and mesenteric fat. Yellow catfish (mixed sex) were fed diets with dietary Se contents at 0.03 (low Se), 0.25 (adequate Se) and 6.39 (high Se) mg Se/kg, respectively, for 12 weeks, and their spleen, kidney, testis and brain were used for the determination of the mRNA levels of the seven selenoproteins. RESULTS: The seven selenoproteins had similar domains to their corresponding members of other vertebrates. They were widely expressed in nine tissues, including heart, liver, brain, spleen, head kidney, dorsal muscle, mesenteric fat, anterior intestine and gill, but showed tissue-dependent expression patterns. Dietary Se addition affected the expression of the seven genes in spleen, kidney, testis and brain tissues of yellow catfish. CONCLUSION: Taken together, our study demonstrated the characterization, expression and regulation of seven selenoproteins, which increased our understanding of the biological functions of Se and selenoproteins in fish.

Nano-Zn Increased Zn Accumulation and Triglyceride Content by Up-Regulating Lipogenesis in Freshwater Teleost, Yellow Catfish Pelteobagrus fulvidraco.

The present study was conducted to explore the mechanism of nano-Zn absorption and its influence on lipid metabolism in the intestine of yellow catfish Pelteobagrus fulvidraco. Compared to ZnSO4, dietary nano-Zn addition increased the triglyceride (TG) content, enzymatic activities of malic enzyme (ME) and fatty acid synthase (FAS), and up-regulated mRNA levels of 6pgd, fas, acca, dgat1, pparγ, and fatp4. Using primary intestinal epithelial cells of yellow catfish, compared to the ZnSO4 group, nano-Zn incubation increased the contents of TG and free fatty acids (FFA), the activities of glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6GPD), ME, and FAS, up-regulated mRNA levels of lipogenic genes (6pgd, g6pd, fas, dgat1, and pparγ), genes of lipid transport (fatp4 and ifabp), and Zn transport genes (znt5, znt7, mt, and mtf1), and increased the protein expression of fatty acid transport protein 4 (FATP4) and peroxisome proliferator activated receptor gamma (PPARγ). Further studies found that nano-Zn absorption was via the clathrin-dependent endocytic mechanism. PPARγ mediated the nano-Zn-induced increase in TG, and nano-Zn increased Zn accumulation and induced TG accumulation by activating the PPARγ pathway and up-regulating lipogenesis.

Endoplasmic Reticulum Stress-Mediated Autophagy and Apoptosis Alleviate Dietary Fat-Induced Triglyceride Accumulation in the Intestine and in Isolated Intestinal Epithelial Cells of Yellow Catfish.

BACKGROUND: The intestine is the main organ for absorbing dietary fat. High dietary lipid intake leads to fat deposition in the intestine and adversely influences fat absorption and health, but the underlying mechanism is unknown. OBJECTIVES: We used yellow catfish and their isolated intestinal epithelial cells to test the hypothesis that endoplasmic reticulum (ER) stress, autophagy, and apoptosis mediate fat-induced changes in lipid metabolism. METHODS: Male and female yellow catfish (weight: 3.79 ± 0.16 g; age: 3 mo) were fed diets containing lipid at 6.98% (low-fat diet; LFD), 11.3% (middle-fat diet; MFD), or 15.4% (high-fat diet; HFD) (by weight) for 8 wk. Each dietary group had 3 replicates, 30 fish per replicate. Their intestinal epithelial cells were isolated and incubated for 24 h in control solution or various concentrations of fatty acids (FAs) with or without 2-h pretreatment with an inhibitor [3-methyladenine (3-MA), 4-phenyl butyric acid (4-PBA), or Ac-DVED-CHO (AC)]. Triglyceride (TG) contents, genes, and enzymes involved in lipid metabolism, ER stress, autophagy, and apoptosis were determined in intestinal tissue and cells; immunoblotting, BODIPY 493/503 staining, ultrastructural observation, and the detection of autophagic and apoptotic vesicles were performed on intestinal cells. RESULTS: Compared with the LFD and MFD, the HFD increased intestinal TG content by 120-226%, activities of lipogenic enzymes by 19.0-245%, expression of genes related to lipogenesis (0.77-8.4-fold), lipolysis (0.36-6.0-fold), FA transport proteins (0.79-1.7-fold), ER stress (0.55-7.5-fold), autophagy (0.56-4.2-fold), and apoptosis (0.80-5.2-fold). Using isolated intestinal epithelial cells and inhibitors (4-PBA, 3-MA, and AC), we found that ER stress mediated FA-induced activation of autophagy (11.0-50.1%) and apoptosis (10.4-32.0%), and lipophagy and apoptosis mediated FA-induced lipolysis (3.40-41.6%). CONCLUSIONS: An HFD upregulated lipogenesis, lipolysis, and FA transport, induced ER stress, and activated autophagy and apoptosis. ER stress, autophagy, and apoptosis play important regulatory roles in fat-induced changes in lipid metabolism in the intestine and intestinal epithelial cells of yellow catfish.