Background: Excessive dietary fat intake induces lipid deposition and contributes to the progress of nonalcoholic fatty liver disease (NAFLD). However, the underlying mechanisms are still unclear. Methods: Yellow catfish were given two experimental diets with dietary lipid levels of 11.3 and 15.4%, respectively, for 56 days, and the contents of triglyceride (TG), nonesterified free fatty acids (NEFA) and bile acid (BA), RNA-seq, enzymatic activities and mRNA expression were deteremined in the liver tissues. Hepatocytes from yellow catfish liver tissues were isolated and cultured. Fatty acids (FA) (palmitic acid: OA, oleic acid =1:1), pathway inhibitors (MA, autophagy inhibitor; guggulsterone, FXR inhibitor) and agonist (rapamyicn, autophagy agonist; GW4064, FXR agonist) were used to incubate the cells. TG and NEFA contents, ultrastructural observation, autophagic vesicles and intracellular LD,apoptosis,western blot and Co-IP, and Immunofluorescence analysis, enzymatic activities and Q-PCR were decided. Results: Using RNA sequencing, we found that high fat diets induced changes in expression of many genes associated with the pathways of lipid metabolism and autophagy. The mRNA profiles of the differentially expressed genes (DEG) indicated that high dietary fat-induced lipid deposition was predominantly influenced by the inhibition of autophagy. Using primary hepatocytes, we found that fatty acids (FA) suppressed autophagy, which in turn reduced cellular free FA level by decreasing triglyceride (TG) breakdown. Moreover, our study indicated that farnesoid X receptor (FXR)-cyclic AMP-responsive element-binding protein (CREB) axis was the pivotal physiological switch regulating FA-induced changes of autophagy and lipid metabolism, which represented cellular defenses against FA-induced lipotoxicity. Conclusion: This discovery may provide new targets for treating pathological changes involved in the dysfunction of autophagy and metabolism, including NAFLD. Graphical

This research was conducted to investigate the effects of four dietary zinc (Zn) sources on growth performance, Zn metabolism, antioxidant capacity, endoplasmic reticulum (ER) stress, and tight junctions in the intestine of grass carp Ctenopharyngodon idella. Four Zn sources consisted of Zn dioxide nanoparticles (ZnO NPs), Zn sulfate heptahydrate (ZnSO₄·7H₂O), Zn lactate (Zn-Lac), and Zn glycine chelate (Zn-Gly), respectively. Grass carp with an initial body weight of 3.54 g/fish were fed one of four experimental diets for 8 weeks. Compared to inorganic Zn (ZnSO₄·7H₂O), grass carp fed the ZnO NPs and Zn-Gly diets exhibited better growth performance. Furthermore, grass carp fed the organic Zn (Zn-Lac and Zn-Gly) diets displayed enhanced Zn transport activity, improved intestinal histology, and increased intestinal tight junction-related genes expression compared to other groups. In comparison to other Zn sources, dietary ZnO NPs caused increased Zn deposition and damaged antioxidation capacity by suppressing antioxidant enzymatic activities and related gene expression in the intestine. Grass cap fed the ZnO NPs diet also exhibited lower mRNA abundance of endoplasmic reticulum (ER) stress- and tight junction-associated genes. According to the above findings, it can be concluded that dietary organic Zn addition (Zn-Lac and Zn-Gly) is more beneficial for intestinal health in grass carp compared to inorganic and nanoform Zn sources. These findings provide valuable insights into the application of organic Zn sources, specifically Zn-Lac and Zn-Gly, in the diets for grass carp and potentially for other fish species.

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.

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 β-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. Antioxid. Redox Signal. 40, 433–452.

The autophagy-lysosome pathway, which involves many crucial genes and proteins, plays crucial roles in the maintenance of intracellular homeostasis by the degradation of damaged components. At present, some of these genes and proteins have been identified but their specific functions are largely unknown. This study was performed to clone and characterize the full-length cDNA sequences of nine key autolysosome-related genes (vps11, vps16, vps18, vps33b, vps41, lamp1, mcoln1, ctsd1 and tfeb) from yellow catfish Pelteobagrus fulvidraco. The expression of these genes and the transcriptional responses to a high-fat diet and fatty acids (FAs) (palmitic acid (PA) and oleic acid (OA)) were investigated. The mRNAs of these genes could be detected in heart, liver, muscle, spleen, brain, mesenteric adipose tissue, intestine, kidney and ovary, but varied with the tissues. In the liver, the mRNA levels of the nine autolysosome-related genes were lower in fish fed a high-fat diet than those fed the control, indicating that a high-fat diet inhibited formation of autolysosomes. Palmitic acid (a saturated FA) significantly inhibited the formation of autolysosomes at 12 h, 24 h and 48 h incubation. In contrast, oleic acid (an unsaturated FA) significantly induced the formation of autolysosomes at 12 h, but inhibited them at 24 h. At 48 h, the effects of OA incubation on autolysosomes were OA concentration-dependent in primary hepatocytes of P. fulvidraco. The results of flow cytometry and laser confocal observations confirmed these results. PA and OA incubation also increased intracellular non-esterified fatty acid (NEFA) concentration at 12 h, 24 h and 48 h, and influenced mRNA levels of fatty acid binding protein (fabp) and fatty acid transport protein 4 (fatp4) which facilitate FA transport in primary hepatocytes of P. fulvidraco. The present study demonstrated the molecular characterization of the nine autolysosome-related genes and their transcriptional responses to fat and FAs in fish, which provides the basis for further exploring their regulatory mechanism in vertebrates.