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Abstract Triglyceride (TAG) deposition in the liver is associated with metabolic disorders. In lower vertebrate, the propensity to accumulate hepatic TAG varies widely among fish species. Diacylglycerol acyltransferases (DGAT1 and DGAT2) are major enzymes for TAG synthesis. Here we show that large yellow croaker (Larimichthys crocea) has significantly higher hepatic TAG level than that in rainbow trout (Oncorhynchus mykiss) fed with same diet. Hepatic expression of DGATs genes in croaker is markedly higher compared with trout under physiological condition. Meanwhile, DGAT1 and DGAT2 in both croaker and trout are required for TAG synthesis and lipid droplet formation in vitro. Furthermore, oleic acid treatment increases DGAT1 expression in croaker hepatocytes rather than in trout and has no significant difference in DGAT2 expression in two fish species. Finally, effects of various transcription factors on croaker and trout DGAT1 promoter are studied. We find that DGAT1 is a target gene of the transcription factor CREBH in croaker rather than in trout. Overall, hepatic expression and transcriptional regulation of DGATs display significant species differences between croaker and trout with distinct hepatic triglyceride deposition, which bring new perspectives on the use of fish models for studying hepatic TAG deposition.

The development of host-derived probiotics offers an alternative nutritional strategy for aquaculture. Turbot (Scophthalmus maximus L.) is an important cultured fish in the world. However, research on its host-associated probiotics is limited. The present study evaluates the probiotic potential of Bacillus velezensis T20 (T20) isolated from the intestine of turbot. In the experiment, turbots were fed with two diets: the control diet (CON) and CON supplemented with 1 × 108 CFU/g T20 for 8 weeks. Results showed that the addition of T20 did not affect the growth performance of turbot, but significantly decreased the mortality of turbot challenged by Edwardsiella tarda. Moreover, T20 could survive in the intestine of turbot, it might contribute to the improved intestinal antioxidant capacity, physical barrier and digestive enzymes activities of turbot. In terms of intestinal microbiota, T20 modulated intestinal microbiota communities. Specifically, T20 increased the relative abundances of Bacillus and B. velezensis in both intestinal mucosa and digesta, and decreased potential pathogen such as Mycoplasma, Staphylococcus and Weissella in intestinal digesta. In conclusion, the present study showed that dietary B. velezensis T20 could enhance the disease resistance, improve the communities of intestinal microbiota and promote the intestinal health of turbot.

A feeding study lasting 30 days was carried out to determine the effects of dietary Oryzanol (Ory) on the survival, growth, antioxidant capacity, peptic enzymes, as well as lipid metabolism of Larimichthys crocea larvae (11.87 ± 0.59 mg) using four different Ory concentrations in microfeed formulations (0, 20, 40, and 80 mg/kg), all preserving isolipidic (18.25% crude lipid) and isonitrogenous (52.08% crude protein) conditions. Results revealed that larvae given 40 and 80 mg/kg Ory revealed markedly higher survival rates; in particular, the 80 mg/kg Ory larvae exhibited a considerably higher specific growth rate than the control. Furthermore, the 80 mg/kg dietary Ory resulted in increased peptic enzyme activity, indicating heightened digestive capabilities of larvae. Meanwhile, Ory supplementation at 20, 40, and 80 mg/kg also increased antioxidant enzyme activities and reduced malondialdehyde levels, indicating an enhanced antioxidant capacity. Moreover, the incorporation of 20 and 40 mg/kg Ory demonstrated efficacy in enhancing the lipid metabolism of the larvae. This was evident in the reduction of triglyceride and total cholesterol levels in the larval visceral mass, attributed to the downregulation of genes that participate in lipid synthesis, and the upregulation of genes associated with lipid catabolism. Overall, the study suggests that the addition of Ory (ranging from 40 to 80 mg/kg) significantly improves both survival and developmental aspects, possibly mediated by enhanced digestion and antioxidative effects, alongside the induction of lipid metabolism in Larimichthys crocea larvae.

Vitamin D3 (VD3) is a steroid hormone that plays pivotal roles in pathophysiology, and 1,25(OH)2D3 is the most active form of VD3. In the current study, the crucial role of VD3 in maintaining energy homeostasis under short-term fasting conditions was investigated. Our results confirmed that glucose-depriving pathways were inhibited while glucose-producing pathways were strengthened in zebrafish after fasting for 24 or 48 h. Moreover, VD3 anabolism in zebrafish was significantly suppressed in a time-dependent manner under short-fasting conditions. After fasting for 24 or 48 h, zebrafish fed with VD3 displayed a higher gluconeogenesis level and lower glycolysis level in the liver, and the serum glucose was maintained at higher levels, compared to those fed without VD3. Additionally, VD3 augmented the expression of fatty acids (FAs) transporter cd36 and lipogenesis in the liver, while enhancing lipolysis in the dorsal muscle. Similar results were obtained in cyp2r1−/− zebrafish, in which VD3 metabolism is obstructed. Importantly, it was observed that VD3 induced the production of gut GLP-1, which is considered to possess a potent gluconeogenic function in zebrafish. Meanwhile, the gene expression of proprotein convertase subtilisin/kexin type 1 (pcsk1), a GLP-1 processing enzyme, was also induced in the intestine of short-term fasted zebrafish. Notably, gut microbiota and its metabolite acetate were involved in VD3-regulated pcsk1 expression and GLP-1 production under short-term fasting conditions. In summary, our study demonstrated that VD3 regulated GLP-1 production in zebrafish by influencing gut microbiota and its metabolite, contributing to energy homeostasis and ameliorating hypoglycemia under short-term fasting conditions.

ABSTRACTAlthough evidence has shown that vitamin D (VD) influences gut homeostasis, limited knowledge is available how VD regulates intestinal immunity against bacterial infection. In the present study, cyp2r1 mutant zebrafish, lacking the capacity to metabolize VD, and zebrafish fed a diet devoid of VD, were utilized as VD-deficient animal models. Our results confirmed that the expression of antimicrobial peptides (AMPs) and IL-22 was restrained and the susceptibility to bacterial infection was increased in VD-deficient zebrafish. Furthermore, VD induced AMP expression in zebrafish intestine by activating IL-22 signaling, which was dependent on the microbiota. Further analysis uncovered that the abundance of the acetate-producer Cetobacterium in VD-deficient zebrafish was reduced compared to WT fish. Unexpectedly, VD promoted the growth and acetate production of Cetobacterium somerae under culture in vitro. Importantly, acetate treatment rescued the suppressed expression of β-defensins in VD-deficient zebrafish. Finally, neutrophils contributed to VD-induced AMP expression in zebrafish. In conclusion, our study elucidated that VD modulated gut microbiota composition and production of short-chain fatty acids (SCFAs) in zebrafish intestine, leading to enhanced immunity.

Summary: Chromatin remodeling plays an important role in regulating gene transcription, in which chromatin remodeling complex is a crucial aspect. Brg1/Brm-associated factor 60c (BAF60c) subunit forms a bridge between chromatin remodeling complexes and transcription factors in mammals; hence, it has received extensive attention. However, the roles of BAF60c in fish remain largely unexplored. In this study, we identified BAF60c-interacting proteins by using HIS-pull-down and LC-MS/MS analysis in fish. Subsequently, the RNA-seq analysis was performed to identify the overall effects of BAF60c. Then, the function of BAF60c was verified through BAF60c knockdown and overexpression experiments. We demonstrated for the first time that BAF60c interacts with glucose-regulated protein 78 (GRP78) and regulates lipid metabolism, endoplasmic reticulum (ER) stress, and inflammation. Knockdown of BAF60c reduces fatty acid biosynthesis, ER stress, and inflammation. In conclusion, the results enriched BAF60c-interacting protein network and explored the function of BAF60c in lipid metabolism and inflammation in fish.

Dietary high soybean oil (SO) levels might cause hepatic lipid deposition, induce oxidative stress and inflammatory response in aquatic animals, while octanoate (OCT) is beneficial to metabolism and health in mammals. However, the effect of OCT has been studied rarely in aquatic animals. In this study, a 10-week feeding trial was conducted to investigate the effect of supplemental OCT on hepatic lipid metabolism, serum biochemical indexes, antioxidant capacity and inflammatory response of large yellow croaker (Larimichthys crocea) fed with high SO levels diet. The negative control diet contained 7% fish oil (FO), while the positive control diet contained 7% SO. The other four experimental diets were supplemented with 0.7, 2.1, 6.3 and 18.9 g/kg sodium octanoate (OCT) based on the positive control diet. Results showed that OCT supplementation effectively reduced the hepatic crude lipid, triglyceride (TG), total cholesterol (TC) and non-esterified free fatty acids contents, and alleviated lipid accumulation caused by the SO diet. Meanwhile, OCT supplementation decreased the serum TG, TC, alanine transaminase, aspartate transaminase and low-density lipoprotein cholesterol levels, increased the serum high-density lipoprotein cholesterol level, improved the serum lipid profiles and alleviated hepatic injury. Furthermore, with the supplementation of OCT, the mRNA expression of genes related to lipogenesis (acc1, scd1, fas, srebp1, dgat1 and cebpα) and fatty acid (FA) transport (fabp3, fatp and cd36) were down-regulated, while the mRNA expression of genes related to lipolysis (atgl, hsl and lpl) and FA β-oxidation (cpt1 and mcad) were up-regulated. Besides that, dietary OCT increased the total antioxidant capacity, activities of peroxidase, catalase and superoxide dismutase and the content of reduced glutathione, decreased the content of 8-hydroxy-deoxyguanosine and malondialdehyde and relieved hepatic oxidative stress. Supplementation of 0.7 and 2.1 g/kg OCT down-regulated the mRNA expression of genes related to pro-inflammatory cytokines (tnfα, il1β and ifnγ), and suppressed hepatic inflammatory response. In conclusion, supplementation with 0.7-2.1 g/kg OCT could reduce hepatic lipid accumulation, relieve oxidative stress and regulate inflammatory response in large yellow croaker fed the diet with high SO levels, providing a new way to alleviate the hepatic fat deposition in aquatic animals.

A 30-day feeding trial was designed to evaluate the effect of supplemental fulvic acid (FA) on survival, growth performance, digestive ability and immunity of large yellow croaker (Larimichthys crocea) larvae (initial body weight 11.33 ± 0.57 mg). Four isonitrogenous and isolipids diets containing 0.00%, 0.01%, 0.02% and 0.04% FA were formulated, respectively. Results showed that the supplementation of 0.04% FA significantly improved survival rate of large yellow croaker larvae. Meanwhile, supplemental FA significantly increased final body weight and specific growth rate. Based on the specific growth rate, the optimal supplementation was 0.0135% FA. Larvae fed the diet with 0.01% FA had significantly higher villus height than the control. The supplementation of 0.01%–0.02% FA significantly increased the muscular thickness of intestine. Moreover, supplementation of FA significantly increased mRNA expression of intestinal epithelial proliferation and barrier genes (pcna, zo-1 and zo-2). Diets supplemented with 0.02%–0.04% FA significantly increased the activity of trypsin in the intestinal segment, while 0.01%–0.02% FA significantly increased the activity of trypsin in the pancreatic segment. Compared with the control, supplementation of FA remarkably increased activities of alkaline phosphatase and leucine aminopeptidase in the brush border membrane of intestine. Larvae fed the diet with 0.01% FA significantly increased activities of lysozyme and total nitric oxide synthase. Furthermore, the supplementation of 0.01% to 0.02% FA significantly decreased the mRNA expression of pro-inflammatory cytokines (tnf-α and il-6). Concurrently, supplemental FA significantly increased anti-inflammatory cytokine (il-10) mRNA expression level. In conclusion, this study indicated that the supplementation of FA could improve the survival rate and growth performance of larvae by promoting intestinal development, digestive enzymes activities and innate immunity.

Now the replacement of fish oil (FO) with vegetable oils (VOs) has been broadly applied in aquatic feed, but studies reported that there were negative effects on growth, skin color, flavor and muscle texture. A 10-week feeding trial was conducted to investigate the effects of dietary mixed bile acids (BA) on growth performance, body composition, digestive enzyme activities, skin color and flesh quality of juvenile large yellow croaker (initial weight, 13.10 ± 0.18 g). Four iso-nitrogenous and iso-lipidic experimental diets were formulated and designated as soybean oil (SO), SO supplemented with 300 (BA300), 600 (BA600), and 1200 (BA1200) mg/kg bile acids. Two hundred and forty fish were randomly allocated into 12 floating net cages (1 m × 1 m × 1.5 m) that were located at marine fishing rafts. Each diet was randomly distributed in triplicate (three replicates per treatment, 20 fish per replicate). Fish were hand-fed to apparent satiation twice daily (05:30 and 17:30) for 10 weeks. Results showed that specific growth rate presented quadratic pattern with supplemental bile acids level, peaking at BA600 group. Meanwhile, feed conversion rate of cultured fish was significantly improved in BA600 group (P < 0.05). The redness (a*) of dorsal and lateral line skin and the yellowness (b*) of abdominal skin showed significantly quadratic pattern with the increase of supplemental BA level (P < 0.05), peaking at BA600 group. In terms of dorsal muscle texture, springiness showed a decreasing trend in significantly linear pattern with the increase of supplemental BA level, bottoming at BA600 group. However, cohesiveness and gumminess were significantly linear increased with the increase of supplemental BA level (P < 0.05). No significant differences were observed in lipase, amylase, and trypsin activities (P > 0.05). Total proportion of muscle n-3LCPUFA showed a linearly increasing trend with the increase of supplemental BA level. Muscle TG content was linearly increased with the increase of supplemental BA level (P < 0.05), and significant differences occurred as compared to fish fed diets with SO when supplemental BA level were reached 600 mg/kg and 1200 mg/kg (P < 0.05). The gene expression of acyl-CoA oxidase (aco) was significantly decreased in fish fed diets supplemented with 600 mg/kg bile acids compared with 300 mg/kg other than those fed diets with SO. In conclusion, these results suggested that mixed bile acids supplemented to SO-based diets could improve feed conversion ratio, skin color and flesh quality to some extent.

An 8-week growth experiment was conducted to investigate effects of tributyrin (TB) supplementation on growth performance, intestinal digestive enzyme activity, antioxidant capacity, and inflammation-related gene expression of juvenile large yellow croaker (Larimichthys crocea) (initial weight of 12.90±0.02 g) fed diets with high level of Clostridium autoethanogenum protein (CAP). In the negative control diet, 40% fish meal was used as the major source of protein (named as FM), while 45% fish meal protein of FM was substituted with CAP (named as FC) to form a positive control diet. Based on the FC diet, grade levels of 0.05%, 0.1%, 0.2%, 0.4%, and 0.8% tributyrin were added to formulate other five experimental diets. Results showed that fish fed diets with high levels of CAP significantly decreased the weight gain rate (WGR) and specific growth rate (SGR) compared with fish fed the FM diet (P

Unfolded protein responses from endoplasmic reticulum (ER) stress have been implicated in inflammatory signaling. The vicious cycle of ER stress and inflammation makes regulation even more difficult. This study examined effects of farnesoid X receptor (FXR) in ER-stress regulation in large yellow croakers. The soybean-oil-diet-induced expression of ER stress markers was decreased in fish with FXR activated. In croaker macrophages, FXR activation or overexpression significantly reduced inflammation and ER stress caused by tunicamycin (TM), which was exacerbated by FXR knockdown. Further investigation showed that the TM-induced phosphorylation of PERK and EIF2α was inhibited by the overexpression of croaker FXR, and it was increased by FXR knockdown. Croaker NCK1 was then confirmed to be a regulator of PERK, and its expression in macrophages is increased by FXR overexpression and decreased by FXR knockdown. The promoter activity of croaker NCK1 was inhibited by yin-yang 1 (YY1). Furthermore, the results show that croaker FXR overexpression could suppress the P65-induced promoter activity of YY1 in HEK293t cells and decrease the TM-induced expression of yy1 in macrophages. These results indicate that FXR could suppress P65-induced yy1 expression and then increase NCK1 expression, thereby inhibiting the PERK pathway. This study may benefit the understanding of ER stress regulation in fish, demonstrating that FXR can be used in large yellow croakers as an effective target for regulating ER stress and inflammation.

Fish have limited ability in endogenous biosynthesis of arginine. Arginine is an indispensable amino acid for fish, and the arginine requirement varies with fish species and fish size. Recent studies on fish have demonstrated that arginine influences nutrient metabolism, stimulates insulin release, is involved in nonspecific immune responses and antioxidant responses, and elevates disease resistance. Specifically, arginine can regulate energy homeostasis via modulating the adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) pathway, and also regulate protein synthesis via activating the target of rapamycin (TOR) signaling pathway. The present article reviews pertinent knowledge of arginine in fish, including dietary quantitative requirements, endogenous anabolism and catabolism, regulation of the endocrine and metabolic systems, and immune-regulatory functions under pathogenic challenge. Our findings showed that further data about the distribution of arginine after intake into specific cells, its sub-cellular sensor to initiate downstream signaling pathways, and its effects on fish mucosal immunity, especially the adaptive immune response against pathogenic infection in different species, are urgently needed.

Vitamin D3 (VD3) participated widely in the nuclear factor-κB (NF-κB)-mediated inflammation, apoptosis, and autophagy through the vitamin D receptor (VDR). However, the molecular mechanisms remain not understood in teleost. The present study investigated the functions of VD3/VDR on intestinal inflammation, autophagy, and apoptosis of turbot in vivo and in vitro. Triple replicates of 30 fish were fed with each of three diets with graded levels of 32.0 (D0), 1012.6 (D1), and 3978.2 (D2) IU/kg VD3. Obvious intestinal enteritis was observed in the D0 group and followed with dysfunction of intestinal mucosal barriers. The intestinal inflammatory response induced by VD3 deficiency was regulated by the NF-κB/inflammasome signalling. The promotion of intestinal apoptosis and suppression of intestinal autophagy were also observed in the D0 group. Similarly, VD3 deficiency in vitro induced more intense inflammation regulated by NF-κB/inflammasome signalling. The mutually exclusive apoptosis and autophagy were also observed in the group without 1,25(OH)2D3in vitro, accompanied by similar changes in apoptosis and autophagy increased apoptosis. The gene expression of VDRs was significantly increased with the increasing VD3 supplementation both in vivo and in vitro. Moreover, VDR knockdown in turbot resulted in intestinal inflammation, and this process relied on the activation of inflammasome mediated by NF-κB signalling. Simultaneously, intestinal apoptosis was promoted, whereas intestinal autophagy was inhibited. In conclusion, VD3 deficiency could induce intestinal inflammation via activation of the NF-κB/inflammasome pathway, intestinal apoptosis, and autophagy formed a mutually exclusive relation in teleost. And VDR is the critical molecule in those processes.

Clostridium autoethanogenum meal (CAM) is a novel single-cell protein, which is produced from bacteria using carbon monoxide (CO) as sole carbon source. To evaluate the efficiency of CAM as an alternative for dietary fish meal, a 56-days growth experiment was performed on juvenile turbot (Scophthalmus maximus L.) with initial average weight of 9.13 ± 0.02 g. Six iso-nitrogenous (crude protein, 51.0%) and iso-lipidic (crude lipid, 11.5%) diets were formulated with 0%, 15%, 30%, 45%, 60% and 80% dietary fish meal protein substituted by CAM protein, which were designated as CAM0 (the control group), CAM15, CAM30, CAM45, CAM60 and CAM80, respectively. Results showed that no significant differences were observed in survival rate (over 97.50%) among different dietary treatments (p > 0.05). The specific growth rate (SGR) was not significantly affected when replacement levels of dietary fish meal with CAM were less than 45% (p > 0.05). The feed intake (FI) was significantly linear reduced with increasing dietary CAM (p < 0.05), whereas no significant differences were observed in feed efficiency ratio (FER), protein efficiency ratio (PER) and protein retention (PR) among different dietary treatments (p > 0.05). With increasing dietary CAM, lipid retention (LR) and carcass lipid tended to be increased in both significantly linear and quadratic patterns (p < 0.05). The apparent digestibility coefficient (ADC) of crude protein and some essential amino acids, including threonine, valine, lysine, histidine and arginine, showed significantly linear increase with increasing dietary CAM (p < 0.05). Furthermore, with the increase of dietary CAM, the gene expression of intestinal peptide and amino acids transporters was first up-regulated and then down-regulated with significantly quadratic pattern (p < 0.05), peaking in fish fed with diets CAM30 or CAM45, which was similar to the expression of genes related protein degradation in muscle. For genes related to protein metabolism in liver and muscle, the expression of mammalian target of rapamycin (mtor) was not significantly affected by dietary CAM, while the general control nonderepressible 2 (gcn2) tended to be first up-regulated and then down-regulated with significantly quadratic pattern (p < 0.05). Apart from that, the lipid metabolism of turbot was also affected by high dietary CAM, evidenced by increased expression of hepatic genes related to lipogenesis as well as reduced expression of genes related to lipid oxidation and lipid transport. In conclusion, CAM can replace up to 45% fish meal protein in diet for juvenile turbot without significantly adverse effects on growth performance. But excessive dietary CAM would result in significant growth reduction, and excessive lipid deposition may also occur in fish fed diets with high levels of CAM.

In the 21st century, intestinal homeostatic imbalance has emerged as a growing health challenge worldwide. Accumulating evidence reveals that excessive intake of saturated fatty acid (SFA) induces intestinal homeostatic imbalance. However, the potential molecular mechanism is still unclear. In the present study, we found that palm oil or palmitic acid (PA) treatment disturbed lipid metabolism homeostasis and triggered endoplasmic reticulum (ER) stress and inflammation in the intestine or intestinal cells of large yellow croaker (Larimichthys crocea). Interestingly, PA treatment significantly decreased phosphatidylethanolamine (PE) content in the intestinal cells. PE supplementation decreased triglyceride content in the intestinal cells induced by PA treatment by inhibiting fatty acid uptake and lipogenesis. PE supplementation suppressed ER stress. Meanwhile, PE supplementation alleviated inflammatory response through p38 MAPK-p65 pathway, reducing the damage of intestinal cells caused by PA treatment to some extent. Our work revealed that intestinal homeostatic imbalance caused by PA treatment was partly due to the decrease of PE content. PE consumption might be a nutritional strategy to regulate intestinal homeostasis in fish and even human beings.

Summary: LDLR, as the uptake receptor of low-density lipoprotein, plays a crucial role in lipid metabolism. However, the detailed mechanism by which LDLR affects hepatic triglyceride (TG) accumulation has rarely been reported. Here, we found that knockdown of LDLR effectively mitigated PA-induced TG accumulation. Further analysis revealed that the expression of LDLR was controlled by SREBP2 directly and indirectly. On one hand, transcription factor SREBP2 activated the transcription of LDLR directly. On the other hand, SREBP2 indirectly regulated LDLR by increasing the transcription of lncRNA LDLR-AS in fish. Mechanism analysis found that LDLR-AS functioned as an RNA scaffold to recruit heterogeneous nuclear ribonucleoprotein R (hnRNPR) to the 5′ UTR region of LDLR mRNA, which stabilized LDLR mRNA at the post-transcription level. In conclusion, our study demonstrates that increased LDLR transcription and mRNA stability is regulated by SREBP2 directly or indirectly, and promotes hepatic TG accumulation by endocytosing LDL in fish.

Octanoate is a type of classical medium-chain fatty acids, which is widely used to treat neurological and metabolic syndrome. However, the specific role of octanoate in repairing intestinal health impairment is currently unknown. Therefore, we investigated whether dietary octanoate repaired the intestinal damage induced by surplus soybean oil in Larimichthys crocea. In this study, dietary octanoate alleviated abnormal morphology of the intestine and enhanced expression of ZO-1 and ZO-2 to improve intestinal physical barrier. Further, dietary octanoate increased antioxidant enzymic activities and decreased the level of ROS to alleviate the intestinal oxidative stress. Dietary octanoate also attenuated the expression of proinflammatory cytokines and the polarity of macrophage to reduce the intestinal inflammatory response. Moreover, the result of intestinal microbial 16S rRNA sequence showed that dietary octanoate repaired the intestinal mucosal microbial dysbiosis, and increased the relative abundance of Lactobacillus. Dietary octanoate supplementation also increased the level of acetic acid in intestinal content and serum through increasing the abundance of acetate-producing strains. Overall, in Larimichthys crocea, dietary octanoate might alleviated oxidative stress, inflammatory response and microbial dysbiosis to repair the intestinal damage induced by surplus soybean oil. This work provides vital insights into the underlying mechanisms and treatment strategies for intestinal damage in vertebrates.

Abstract Soybean meal-induced enteropathy (SBMIE) is prevalent in aquaculture. The aim of this study is to evaluate the role of daidzein on SBMIE of juvenile turbot (Scophthalmus maximus L.) by feeding with fish meal diet (FM), soybean meal diet (SBM, 40% fish meal protein in FM replaced by soybean meal protein) and daidzein diet (DAID, 40 mg/kg daidzein supplemented to SBM) for 12 weeks. We found that daidzein supplementation elevated the gene expression of anti-inflammatory cytokine TGF-β, decreased gene expression of pro-inflammatory cytokines TNF-α and signal molecules p38, JNK and NF-κB. SBM up-regulated the genes expression related to oxidative stress and apoptosis, but dietary daidzein restored it to the similar level with that in FM group. Moreover, dietary daidzein up-regulated gene expression of tight junction protein, and modified the intestinal microbial profiles with boosted relative abundance of phylum Proteobacteria and Deinococcus–Thermus, genera Sphingomonas and Thermus, species Lactococcus lactis, and decreased abundance of some potential pathogenic bacteria. In conclusion, dietary daidzein could ameliorate SBM-induced intestinal inflammatory response, oxidative stress, mucosal barrier injury and microbiota community disorder of turbot. Moreover, p38, JNK and NF-κB signaling might be involved in the anti-inflammatory process of daidzein, and daidzein itself might act as an antioxidant to resist SBM-induced oxidative damage.

Endoplasmic reticulum (ER) homeostasis plays a vital role in cell physiological functions. Various factors can destroy the homeostasis of the ER and cause ER stress. Moreover, ER stress is often related to inflammation. Glucose-regulated protein 78 (GRP78) is an ER chaperone, which plays a vital role in maintaining cellular homeostasis. Nevertheless, the potential effects of GRP78 on ER stress and inflammation is still not fully elucidated in fish. In the present study, ER stress and inflammation was induced by tunicamycin (TM) or palmitic acid (PA) in the macrophages of large yellow croakers. GRP78 was treated with an agonist/inhibitor before or after the TM/PA treatment. The results showed that the TM/PA treatment could significantly induce ER stress and an inflammatory response in the macrophages of large yellow croakers whereas the incubation of the GRP78 agonist could reduce TM/PA-induced ER stress and an inflammatory response. Moreover, the incubation of the GRP78 inhibitor could further induce TM/PA-induced ER stress and an inflammatory response. These results provide an innovative idea to explain the relationship between GRP78 and TM/PA-induced ER stress or inflammation in large yellow croakers.

A 30-day feeding trial was conducted to determine the effects of dietary glycyrrhizin (GL) on survival, growth performance, expression of feeding-related genes, activities of digestive enzymes, antioxidant capacity, and expression of inflammatory factors of large yellow croaker larvae with an initial weight of 3.78±0.27 mg. Four 53.80% crude protein and 16.40% crude lipid diets were formulated with supplementation of 0%, 0.005%, 0.01%, and 0.02% GL, respectively. Results indicated that larvae fed diets with GL had higher survival rate and specific growth rate than the control (P

A 70-day feeding experiment was carried out to assess the replacement of dietary fishmeal (FM) protein with degossypolized cottonseed protein (DCP) on large yellow croaker (Larimichthys crocea) with initial body weight (13.09±0.50 g). Five isonitrogenous and isolipidic diets replaced fishmeal protein with 0%, 20%, 40%, 60%, and 80% DCP were formulated and named as FM (the control group), DCP20, DCP40, DCP60, and DCP80, respectively. Results displayed that weight gain rate (WGR) and specific growth rate (SGR) in the DCP20 group (263.91% and 1.85% d-1) were significantly increased compared with the control group (194.79% and 1.54% d-1) (P

The study is aimed to investigate the protective effect and potential mechanisms of sodium butyrate (NaBT) on soyasaponins (SA) induced intestinal epithelial cells (IECs) injury in vitro. The primary IECs of turbot were developed and treated with 0.4, 1 and 4 mM NaBT in the presence of 0.4 mg/mL SA for 6 h to explore the protective effects of NaBT. The results showed that the addition of NaBT significantly down-regulated gene expression of inflammatory cytokine TNF-α, IL-1β and IL-8, pro-apoptosis relevant gene BAX, caspase-3, caspase-7 and caspase-9 induced by SA, while up-regulated anti-apoptosis gene Bcl-2. SA stimulation did not induce reactive oxygen species production, but elevated gene expression of antioxidant enzyme heme oxygenase-1 and superoxide dismutase. Moreover, the gene expression of those antioxidant enzyme was further up-regulated in NaBT groups. Furthermore, NaBT supplementation decreased the acid phosphatase and alkaline phosphatase activities and suppressed phosphorylation of p38 and c-Jun N-terminal kinase (JNK). In conclusion, NaBT could mitigate SA-induced inflammation and apoptosis and elevate gene expression of antioxidant enzymes on IECs of turbot and p38 and JNK signaling pathway participated in those processes.

The mall heterodimer partner (SHP) plays an important regulatory role in mammal inflammation. The main objective of this study was to investigate the response of SHP to inflammatory stimulation and its underlying mechanism. The shp gene from large yellow croakers, was cloned, and this gene is mainly expressed in the liver and intestine. Lipopolysaccharide (LPS) stimulation induced the mRNA expression and protein level of SHP in macrophages of large yellow croakers. Overexpression of SHP significantly decreased mRNA expression of tnfα, il-1β, il-6 and cox2 induced by LPS treatment in macrophages. LPS stimulation increased the phosphorylation level of Adenosine 5’-monophosphate (AMP)-activated protein kinase (AMPK) in macrophages. AMPK inhibitor treatment significantly decreased the expression of SHP induced by LPS while AMPK activator significantly increased the expression of SHP. The nuclear factor-erythroid 2-related factor 2 (NRF2) increased the promoter activity of SHP in large yellow croakers and the level of nuclear NRF2 was increased by LPS stimulation and AMPK activation. NRF2 inhibitor treatment significantly decreased mRNA expression of shp induced by LPS and AMPK activator. In conclusion, LPS can induce SHP expression by activating the AMPK-NRF2 pathway while SHP could negatively regulate LPS-induced inflammation in large yellow croakers. This study may be benefit to the development of immunology of marine fish and provide new ideas for inflammation-related diseases.

Summary: Acetylation coordinates many biological processes to ensure cells respond appropriately to nutrients. However, how acetylation regulates lipid surplus-induced inflammation remains poorly understood. Here, we found that a high-fat diet (HFD) enhanced mitochondrial fatty acid β-oxidation, which enhanced acetyl-CoA levels in the liver of the large yellow croaker. The HFD activated ACLY to govern the “citrate transport” to transfer acetyl-CoA from the mitochondria to the nucleus. Elevated acetyl-CoA activated CBP to increase p65 acetylation and then aggravated inflammation. SIRT1 was deactivated with a decline in NAD+/NADH, which further aggravated inflammation. Therefore, acetylation-dependent regulation of transcription factor activity is an adaptation to proinflammatory stimuli under nutrient stress, which was also confirmed in AML12 hepatocytes. In vitro octanoate stimulation further verified that acetyl-CoA derived from fatty acid β-oxidation mediated acetylation homeostasis in the nucleus. The broad therapeutic prospects of intermediate metabolites and acetyltransferases/deacetylases might provide critical insights for the treatment of metabolic diseases in vertebrates.

The study was conducted to investigate the effects of DL-Methionyl-DL-Methionine (Met-Met) (a dipeptide of two DL-methionine molecules) on growth performance, immune and antioxidative responses for Litopenaeus vannamei fed low fishmeal diet. Tested treatments consisted of positive control diet (PC) with 20 % fishmeal or 7 % fishmeal (basal diet) diets supplemented with different levels of Met-Met (0.00 %, 0.1 %, 0.15 %, 0.20 %, 0.25 % and 0.3 %). A total of 1680 shrimp (0.071 ± 0.001 g) were randomly distributed into 42 tanks (6 repetitions / each diet, 40 shrimp / tank) and fed for 91 days. Results showed that reduction of fishmeal from 20 % down to 7 % did not affect the survival rate of shrimp. Weight gain rate and feed efficiency were significantly decreased in the basal group compared to the PC group, while these were significantly increased with the increasing of Met-Met into the diets up to 0.15 % compared to the basal group. Malondialdehyde in 0.15 % Met-Met and PC groups were significantly lower than the basal group. Shrimp fed the diet with 0.15 % Met-Met had the highest phenol oxidase content and peroxidase and total antioxidant capacity. The highest values of intestinal wall thickness were observed in 0.15 % Met-Met and PC groups. The similar morphology of the hepatopancreas was observed between 0.15 % and PC groups. Overall, the present study indicated that Met-Met could improve adverse effects of Met deficiency on growth caused by low fishmeal. Low fishmeal diet with 0.15 % Met-Met (0.65 %–0.66 % Met and 1.14 % Met + Cys) is beneficial to the health of intestine and hepatopancreas, improves immunity and antioxidative capacity and promotes the growth of L. Vannamei.

Dietary intake of omega-3 fatty acids found in fish has been reported to reduce the risk of Alzheimer’s Disease (AD). Stearidonic acid (SDA), a plant-based omega-3 fatty acid, has been targeted as a potential surrogate for fish-based fatty acids. However, its role in neuronal degeneration is unknown. This study was designed to evaluate effects of SDA on Amyloid-β(A-β)-induced neurotoxicity in rat hippocampal cells. Results showed that SDA effectively converted to eicosapentaenoic acid (EPA) in hippocampal cells. Aβ-induced apoptosis in H19-7 cells was protected by SDA pretreatment as evidenced by its regulation on the expression of relevant pro- and anti-apoptotic genes, as well as the inhibition on caspase activation. SDA also protected H19-7 cells from Aβ-induced oxidative stress by regulating the expression of relevant pro- and anti-oxidative genes, as well as the improvement in activity of catalase. As for Aβ/LPS-induced neuronal inflammation, SDA pretreatment reduced the release of IL-1β and TNFα. Further, we found that the anti-Aβ effect of SDA involves its inhibition on the expression of amyloid precursor protein and the regulation on MAPK signaling. These results demonstrated that SDAs have neuroprotective effect in Aβ-induced H19-7 hippocampal cells. This beneficial effect of SDA was attributed to its antiapoptotic, antioxidant, and anti-inflammatory properties.

The present study was conducted to investigate the effects of dietary vegetable oils (VOs) replacing fish oil (FO) on fatty acid composition, lipid metabolism and inflammatory response in adipose tissue (AT) of large yellow croaker (Larimichthys crocea). The initial body weight of a large yellow croaker was 10.07 ± 0.13 g. Three iso-nitrogenous and iso-lipidic diets were formulated by replacing FO with 0% (the control group), 100% soybean oil (SO) and 100% linseed oil (LO). Results showed that the contents of C18:2n-6 and C18:3n-3 were significantly increased in AT of fish fed the SO and LO diets compared with the FO diet, respectively. The proportion of n-6 polyunsaturated fatty acid (PUFA) was increased in SO and LO diets, while the proportions of saturated fatty acid and n-3 LC-PUFA were decreased. Moreover, dietary SO and LO significantly induced excess fat accumulation of AT by increasing the triglyceride content and the hypertrophy of adipocytes. Dietary SO and LO significantly increased lipogenesis-related gene expressions (dagt2, fabp10, srebp1, cebpα and pparγ), while decreasing the gene expression of lpl. Meanwhile, dietary SO increased the expression of genes related to fatty acid β-oxidation (cpt1 and aco), while LO showed no differences. Furthermore, dietary SO and LO increased the pro-inflammatory gene expressions and decreased the anti-inflammatory gene il10 expression. The phosphorylation levels of p38 MAPK and NF-κB were significantly upregulated by dietary SO and LO. In addition, there was a significant increase in macrophage infiltration and M1 polarization in AT of fish fed SO and LO diets. In conclusion, the present study revealed that dietary SO and LO replacing FO affected fatty acid composition and induced lipid dysmetabolism and inflammatory response in the adipose tissue of large yellow croaker.

The early life period is considered an essential period for gut microbial colonization. Manipulating gut microbiota interventions during early life periods has been proven to be a promising method to boost healthy growth. Therefore, the aim of the present study was to investigate the effects of dietary fucoidan (Fuc) on the growth, digestive tract maturation, and gut microbiota of large yellow croaker (Larimichthys crocea) larvae. Four diets were formulated with different levels of Fuc (0.00%, 0.50%, 1.00%, and 2.00%). Results showed that dietary Fuc significantly improved the growth performance of larvae. Meanwhile, dietary Fuc promoted digestive tract maturation. Dietary 1.00% Fuc significantly improved intestinal morphology. Dietary Fuc upregulated the expression of intestinal cell proliferation and differentiation related-genes and intestinal barrier related-genes. Dietary 2.00% Fuc significantly increased the activities of brush border membranes enzymes and lipase while inhibiting α-amylase. Furthermore, dietary Fuc maintained healthy intestinal micro-ecology. In detail, dietary 1.00% and 2.00% Fuc altered the overall structure of the gut microbiota and increased the relative abundance of Bacteroidetes while decreasing the relative abundance of opportunistic pathogens and facultative anaerobe. In conclusion, appropriate dietary Fuc (1.00–2.00%) could improve the growth of large yellow croaker larvae by promoting digestive tract maturation and maintaining an ideal intestinal micro-ecology.

The small intestine is crucial for lipid homeostasis and immune regulation of the whole body. Endoplasmic reticulum (ER) stress may affect lipid metabolism and inflammation in the intestine, but the potential mechanism is not completely understood. In the present study, intraperitoneal injection of tunicamycin (TM) induced ER stress in the intestine of large yellow croaker (Larimichthys crocea). ER stress induced excessive accumulation of triglyceride (TG) in the intestine by promoting lipid synthesis. However, it also enhanced lipid secretion and fatty acid β-oxidation. In addition, ER stress augmented inflammation in the intestine by promoting p65 into the nucleus and increasing proinflammatory genes expression. In the isolated intestinal cells, the obtained results showed that TM treatment significantly upregulated the mRNA expression of lipid synthesis and inflammatory response genes, which were consistent with those in vivo. Moreover, overexpression of unfolded protein response (UPR) sensors significantly upregulated promoter activities of lipid synthesis and proinflammatory genes. In conclusion, the results suggested that ER stress disturbed lipid metabolism and augmented inflammation in the intestine and isolated intestinal cells of large yellow croaker, which may contribute to finding novel therapies to tackle lipid dysregulation and inflammation in the intestine of fish and human beings.

Free fatty acid receptor 4 (FFAR4) plays a key role in regulating the inflammatory response in mammals. The present study aimed to investigate the function of large yellow croaker FFAR4 on inflammation. In the present study, ffar4 was widely expressed in 10 tissues of large yellow croaker including gill, head kidney and spleen. Further studies showed that treatment of head kidney macrophages with agonists (TUG891 or GSK137647A) or overexpression of ffar4 reduced the mRNA expression of pro-inflammatory genes induced by LPS, and increased the expression of pparγ. Treatment of macrophages with antagonist AH7614 increased the mRNA expression of pro-inflammatory genes induced by LPS, and decreased the mRNA expression of pparγ. In order to verify the immunomodulatory effect of PPARγ, PPARγ was overexpressed in macrophages which significantly reduced the mRNA expression of pro-inflammatory genes il6, il1β, il8, tnfα and cox2. Moreover, results of dual-luciferase assays showed that PPARγ downregulated the transcriptional activity of il6 and il1β promoters. In conclusion, FFAR4 showed anti-inflammatory effects on LPS-induced inflammation in large yellow croaker.

High levels of soybean oil (SO) in fish diets enriched with linoleic acid (LA, 18:2n-6) could induce strong inflammation. However, the molecular mechanism underlying LA-induced inflammation in the liver of large yellow croaker (Larimichthys crocea) has not been elucidated. Based on previous research, autophagy has been considered a new pathway to relieve inflammation. Therefore, the present study was performed to investigate the role of autophagy in regulating LA-induced inflammation in the liver of large yellow croaker in vivo and in vitro. The results of the present study showed that activation of autophagy in liver or hepatocytes could significantly reduce the gene expression of proinflammatory factors, such as tumor necrosis factor α (TNFα) and interleukin 1β (IL1β). The results of the present study also showed that inhibition of autophagy could upregulate the gene expression of proinflammatory factors and downregulate the gene expression of anti-inflammatory factors in vivo and in vitro. Furthermore, autophagy could alleviate LA-induced inflammatory cytokine gene expression in vivo and in vitro, while inhibition of autophagy obtained the opposite results. In conclusion, our study shows that autophagy could regulate inflammation and alleviate LA-induced inflammation in the liver of large yellow croaker in vivo and in vitro for the first time, which may offer considerable benefits to the aquaculture industry and human health.

Aiming to optimize soymeal-based diets for Japanese seabass (Lateolabrax japonicas), a 105-day feeding trial was conducted to evaluate the effects of functional additives, including antioxidants (ethoxyquin, thymol and carvacrol) and chelated trace elements (Cu, Mn and Zn), on the growth, immunity, antioxidant capacity and disease resistance of fish fed diets with conventional soybean meal replacing 50% of fishmeal. Three isonitrogenous (45%) and isolipidic (11%) diets were formulated: (1) standard reference diet (FM, 42% fishmeal); (2) soymeal-based diet (SBM, 21% fishmeal and 30% conventional soybean meal); (3) SBM diet supplemented 0.0665% functional additives (FAS). Each experimental diet was randomly fed to quadruplicate groups of forty feed-trained Japanese seabass (initial average body weight = 125.6 ± 0.6 g) stocked in a saltwater floating cage. Upon the conclusion of the feeding trial, lower feed intake was observed in fish fed SBM compared to those fed FM and FAS. Fish fed FM showed the highest growth performance, estimated as the weight gain rate. Notably, FAS supported faster growth of fish than those fed SBM, indicating the optimal growth performance of dietary functional additives. The feed conversion rate showed the opposite trend among dietary treatments, with the highest value in fish fed SBM. Regarding immunity, fish fed soymeal-based diets suppressed the serum alternative complement pathway activities compared to FM, whereas the respiratory burst activity in macrophages of head kidneys showed a similar picture, but no statistical differences were observed. Further, fish fed soymeal-based diets had lower serum Cu-Zn SOD, CAT and GPx activities as well as liver vitamin E levels and scavenging rates of hydroxyl radical but higher liver MDA contents compared to the FM-fed group. Fish fed FAS had higher serum Cu-Zn SOD and GPx activities and liver vitamin E levels than those fed SBM, suggesting the enhancement of antioxidant capacity of dietary functional additives. For the disease resistance against Vibrio harveyi infection, fish fed SBM had the highest cumulative mortality, followed by the FAS and FM groups. Additionally, the biomarkers related to the immune and antioxidant capacities had a positive correlation with the relative abundance of Paracoccus and Pseudomonas, while liver MDA levels had a negative correlation with the relative abundance of Pseudomonas and Psychrobacter. Collectively, soymeal replacing 50% of fishmeal suppressed the growth, immunity, antioxidant capacity and disease resistance of Japanese seabass, while dietary supplementation of antioxidants and chelated trace elements could mitigate soymeal-induced adverse effects on growth and disease resistance through the improvement in antioxidant capacity and regulation of gut microbiota.

Δ6 fatty acyl desaturase (Δ6Fads2) is regarded as the first rate-limiting desaturase that catalyzes the biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFA) from 18-carbon fatty acid in vertebrates, but the underlying regulatory mechanism of fads2 has not been comprehensively understood. This study aimed to investigate the regulation role of fads2 subjected to fatty acid in large yellow croaker and rainbow trout. In vivo, large yellow croaker and rainbow trout were fed a fish oil (FO) diet, a soybean oil (SO) diet or a linseed oil (LO) diet for 10 weeks. The results show that LO and SO can significantly increase fads2 expression (p < 0.05). In vitro experiments were conducted in HEK293T cells or primary hepatocytes to determine the transcriptional regulation of fads2. The results show that CCAAT/enhancer-binding protein α (C/EBPα) can up-regulate fads2 expression. GATA binding protein 3 (GATA3) can up-regulate fads2 expression in rainbow trout but showed opposite effect in large yellow croaker. Furthermore, C/EBPα protein levels were significantly increased by LO and SO (p < 0.05), gata3 expression was increased in rainbow trout by LO but decreased in large yellow croaker by LO and SO. In conclusion, we revealed that FO replaced by LO and SO increased fads2 expression through a C/EBPα and GATA3 dependent mechanism in large yellow croaker and rainbow trout. This study might provide critical insights into the regulatory mechanisms of fads2 expression and LC-PUFA biosynthesis.

Marine fish larvae are vulnerable during the early life period. The early intervention using probiotics may be a promising method to improve growth of fish larvae. In this study, a 30-day feeding trial was conducted to evaluate the effects of early life intervention using probiotic Clostridium butyricum (CB) on growth performance, intestinal development, immune response and gut microbiota of large yellow croaker (Larimichthys crocea) larvae. Four isonitrogenous and isolipidic diets were formulated with the supplementation of four different levels of CB (5 × 109 CFU g−1), 0.00% (Control), 0.10% (CB1), 0.20% (CB2), and 0.40% (CB3). Results showed that larvae fed diets with CB had significant higher final length than the control group. Meanwhile, larvae fed the diet with 0.10% CB had significant higher final weight and specific growth rate (SGR) than the control group. However, no significant difference in survival rate was observed among dietary treatments. CB supplementation significantly increased the height of intestinal villus and the length of intestinal enterocyte. Similarly, CB supplementation significantly increased the expression of tight zonula occludens-2 (zo-2) and ornithine decarboxylase (odc) than the control group. Larvae fed the diet with 0.20% CB had significant higher lipase and leucine-aminopeptidase (LAP) activity than the control group. Moreover, CB supplementation significantly improved immune enzyme activities than the control group. Sequencing of bacterial 16S rRNA V4-5 region indicated that dietary CB altered intestinal microbiota profile and decreased intestinal microbial diversities of larvae. CB supplementation could effectively increase the abundance of CB, and decrease the abundance of some potential pathogenic bacteria in larval gut. These results revealed that early life intervention using 0.10–0.20% CB could promote growth of large yellow croaker larvae probably through promoting intestinal development, improving immune enzyme activities and modulating gut microbiota.

A growth experiment was conducted to evaluate the effects of dietary fish oil (FO) replaced by linseed oil (LO) on the growth performance, antioxidant capacity, hepatic lipid metabolism, and expression of inflammatory genes in large yellow croaker (Larimichthys crocea). Fish (initial weight: 15.88 ± 0.14 g) were fed four experimental diets with 0% (the control), 33.3%, 66.7%, and 100% of FO replaced by LO. Each diet was randomly attributed to triplicate seawater floating cages (1.0 × 1.0 × 2.0 m) with 60 fish in each cage. Results showed that the growth performance of fish fed the diet with 100% LO was markedly decreased compared with the control group (P < 0.05), while no remarkable difference was observed in the growth performance of fish fed diets within 66.7% LO (P > 0.05). The percentage of 18:3n-3 was the highest in the liver and muscle of fish fed the diet with 100% LO among the four treatments. When dietary FO was entirely replaced by LO, fish had a markedly higher total cholesterol, total triglyceride, low-density lipoprotein cholesterol content, and alanine transaminase activity in the serum than the control group (P < 0.05). The concentration of malondialdehyde was markedly higher, while the activity of catalase was markedly lower in fish fed the diet with 100% LO than the control group (P < 0.05). When dietary FO was entirely replaced by LO, hepatic lipid content, transcriptional levels of fatp1 and cd36, and CD36 protein expression were significantly higher, while transcriptional level of cpt-1 and CPT-1 protein expression were significantly lower than the control group (P < 0.05). As for the gene expression of cytokines, fish fed the diet with 100% LO had markedly higher transcriptional levels of il-1β, tnfα, and il-6 than the control group (P < 0.05). In conclusion, the substitution of 66.7% FO with LO had no significant effects on the growth performance of fish, while 100% LO decreased the growth performance and increased the inflammation and hepatic lipid content of fish. The increase of hepatic lipid content was probably due to the increased fatty acid uptake and decreased fatty acid oxidation in fish.

This study aimed to investigate the benefit of dietary cecropin AD (CAD) on the intestinal health of turbot (Scophthalmus maximus) fed diets with a high level of soybean meal. A 12-week feeding trial was conducted with four isonitrogenous and isolipidic diets: a fishmeal-based diet (FM), a diet replacing 40% fish meal protein of FM diet with soybean meal protein (SBM), and the SBM diet supplemented with 0.5 g/kg (C1) and 1.0 g/kg (C2) CAD, respectively. The results of histology of distal intestine (DI) showed that turbots fed the SBM diet exhibited distinct symptoms of enteritis. However, fish fed diets with CAD supplementation kept the normal appearance of the DI which was similar to that in FM group. Compared with the SBM group, diet with CAD supplementation significantly decreased the gene expression of intestinal pro-inflammatory cytokines tumor necrosis factor-α (tnf-α), interleukin-1β (il-1β), interferon-gamma (ifn-γ), and nuclear factor-kappa B p65 (nf-κb p65), while up-regulated the gene expression of intestinal tight junction proteins claudin-3, claudin-4, occludin, and zonula occludens-1 (zo-1). Besides, diet C1 shaped the intestinal microbiota profile toward an anti-inflammatory phenotype represented by the increased abundance of Blutia, Firmicutes/Bacteroides ratio, and decreased Prevotellaceae. In conclusion, dietary CAD could positively modulate the intestinal health of turbot from the impairment induced by soybean meal, which expands its application to help fish better adapt to the increasing plant protein level in aquafeed.

A 30-day feeding experiment was conducted to investigate effects of dietary allicin on survival, growth, antioxidant capacity, innate immunity and expression of inflammatory and appetite related genes in large yellow croaker larvae. Four iso-nitrogenous (53% crude protein) and iso-lipidic (19% crude lipid) diets were formulated via supplementing graded levels of allicin (0.0 (the control), 0.005, 0.01, and 0.02% dry diet, respectively). Results showed that, among dietary treatments, larvae fed the diet with 0.005% allicin had the highest survival rate (SR) (P < 0.05), while larvae fed the diet with 0.01% allicin had the highest specific growth rate (SGR) (P < 0.05). Activities of α-amylase in both pancreatic (PS) and intestine segments (IS) of larvae fed the diet with 0.01% allicin were significantly lower than that in the control (P < 0.05). On the other hand, the supplementation of 0.01% allicin in diets significantly increased activities of alkaline phosphatase (AKP) and leucine aminopeptidase (LAP) in the intestinal brush border membrane (BBM) of larvae than the control (P < 0.05), indicating the promoting roles of allicin on fish larval intestinal development. Moreover, compared to the control, both the nitric oxide (NO) content and the activity of nitric oxide synthase (NOS) were significantly up-regulated in larvae fed the diet with 0.005% allicin, and catalase (CAT) were significantly upregulated in larvae fed the diet with 0.02% allicin (P < 0.05). Transcriptional levels of pro-inflammatory genes including cyclooxygenase-2 (cox-2), interleukin-1β (il-1β) and interleukin-6 (il-6) significantly decreased with increasing allicin, compared to the control. The expression of appetite genes including npy, ghrelin and leptin significantly increased with the prolonged fasting period, and dietary allicin supplementation significantly increased the transcriptional level of neuropeptide Y (npy) at 0.01%, while increased the transcriptional level of leptin in larvae at 0.02% dosages (P < 0.05). These results showed that the supplementation of 0.005% – 0.01% allicin in diets could improve the survival and growth of large yellow croaker larvae probably by promoting intestinal development, alleviating inflammation and enhancing appetite.

Polyunsaturated fatty acids (PUFAs) not only serve as essential nutrients but also function as modulators of the immune response in marine fish. However, their immunomodulatory mechanism is poorly understood given that the underlying regulation of the innate immune response in fish has not been fully elucidated. Hence, study of the innate immunity of fish could help elucidate the mechanism by which PUFAs affect the fish immune response. Here, we used combined transcriptome analysis and in vitro experimentation to study the mechanism of LPS-induced inflammation. Transcriptome profiling indicated that LPS elicited strong pro-inflammatory responses featuring high expression levels of pathogen recognition receptors (PRRs) and cytokines along with the activation of NF-κB and MAPK signaling pathways. The transcription factor p65 alone could increase the transcription of IL1β by binding to the promoter of IL1β, and this promoting effect disappeared after mutation or deletion of its binding sites. We then examined the effects of PUFAs on the levels of gene expression and the abundance of proteins of critical kinases associated with LPS-induced inflammation. We found that LA exerts pro-inflammatory response while ALA, EPA, and DHA induced anti-inflammatory effects by modulating the expression of PRRs, phosphorylation of IKK and p38, and the nuclear translocation of p65. Overall, this study advances our understanding of the regulatory mechanisms by which PUFAs regulate LPS-induced inflammation in a non-model fish species.

Palmitic acid (PA) is a saturated fatty acid (SFA) that can cause an inflammatory response, while docosahexaenoic acid (DHA) is always used as a nutritional modulator due to its anti-inflammatory properties. However, the potential molecular mechanism is still not completely elucidated in fish. Herein, the PA treatment induced an inflammatory response in macrophages of large yellow croaker (Larimichthys crocea). Meanwhile, the mRNA expression of Toll-like receptor (TLR)-related genes, especially tlr22, and the phosphorylation of the mitogen-activated protein kinase (MAPK) pathway were significantly upregulated by PA. Further investigation found that the PA-induced inflammatory response was suppressed by tlr22 knockdown and MAPK inhibitors. Moreover, the results of the peroxisome proliferator-activated receptor γ (PPARγ) agonist and inhibitor treatment proved that PPARγ was involved in the PA-induced inflammation. PA treatment decreased the protein expression of PPARγ, while tlr22 knockdown and MAPK inhibitors recovered the decreased expression. Besides, the PA-induced activation of Nrf2 was regulated by p38 MAPK. Furthermore, DHA-executed anti-inflammatory effects by regulating the phosphorylation of the MAPK pathway and expressions of PPARγ and Nrf2. Overall, the present study revealed that DHA alleviated PA-induced inflammation in macrophages via the TLR22-MAPK-PPARγ/Nrf2 pathway. These results could advance the understanding of the molecular mechanism of the SFA-induced inflammatory response and provide nutritional mitigative strategies.

TIR domain-containing adaptor-inducing interferon-β (TRIF), a cytosolic adaptor protein, plays a key role in the mammalian toll-like receptor-mediated signaling pathway. However, the role of TRIF in large yellow croaker (LcTRIF) remains poorly understood. The main objective of this study was to explore the role of LcTRIF in triggering antiviral immune responses and the potential function of LcTRIF in regulating lipid metabolism. In the present study, the full-length coding sequence of TRIF from large yellow croaker was cloned and characterized. In vivo, upon poly (I:C) stimulation, the transcriptional levels of LcTRIF were rapidly elevated in immune-related tissues at the early stage of injection. In vitro, the MRNA expression of LcTRIF was significantly but not dramatically upregulated in macrophages treated with poly (I:C). Activation of LcTRIF by poly (I:C) significantly increased the transcription of genes involved in inflammatory responses, and this induction was blocked by knockdown of LcTRIF. Moreover, the ability of LcTRIF to induce inflammatory responses may partially be attributed to the promotion of mRNA expression of IFNh and NF-κB pathway genes. In addition, activation of the LcTRIF-mediated pathway inhibited the increase in hepatic stearoyl-coenzyme A (CoA) desaturase 1 induced by palmitic acid and subsequently alleviated lipid accumulation in hepatocytes. These results revealed the crucial role of LcTRIF in triggering antiviral immune responses and the unconventional metabolic function of LcTRIF in regulating hepatic lipogenesis in large yellow croaker.

High-fat diets induced abnormal lipid accumulation in the liver of cultured fish that caused body damage and diseases. The purpose of this research was to investigate the role and mechanism of farnesoid X receptor (FXR) in regulating lipid metabolism and to determine how high-fat diets affect FXR expression in large yellow croakers. The results showed that ligand-meditated FXR-activation could prevent abnormal lipid accumulation in the liver and hepatocytes of large yellow croakers. FXR activation increased the expression of lipid catabolism-related genes while decreasing the expression of lipogenesis-related genes. Further investigation found that the promoter activity of proliferator-activated receptor α (PPARα) could be increased by croaker FXR. Through the influence of SHP on LXR, FXR indirectly decreased the promoter activity of sterol regulatory element binding protein 1 (SREBP1) in large yellow croakers. Furthermore, the findings revealed that endoplasmic reticulum (ER)-stress-induced-activation of JNK and P38 MAPK participated in the reduction of FXR induced by high-fat diets. Then, hepatocyte nuclear factor 1α (HNF1α) was confirmed to be an FXR regulator in large yellow croaker, and it was reduced by high-fat diets and ER stress. In addition, co-expression of c-Jun with HNF1α inhibited the effect of HNF1α on FXR promoter, and suppression of P38 MAPK could relieve the HNF1α expression reduction caused by ER stress activation. In summary, the present study showed that FXR mediated lipid metabolism can prevent abnormal lipid accumulation through regulating PPARα and SREBP1 in large yellow croakers, while high-fat diets can suppress FXR expression by ER stress mediated-activation of JNK and P38 MAPK pathways. This research could benefit the study of FXR functions in vertebrate evolution and the development of therapy or preventative methods for nutrition-related disorders.

A high-fat diet often leads to excessive fat deposition and adversely affects the organism. However, the mechanism of liver fat deposition induced by high fat is still unclear. Therefore, this study aimed at acetyl-CoA carboxylase (ACC) to explore the mechanism of excessive liver deposition induced by high fat. In the present study, the ORF of ACC1 and ACC2 were cloned and characterized. Meanwhile, the mRNA and protein of ACC1 and ACC2 were increased in liver fed with a high-fat diet (HFD) or in hepatocytes incubated with oleic acid (OA). The phosphorylation of ACC was also decreased in hepatocytes incubated with OA. Moreover, AICAR dramatically improved the phosphorylation of ACC, and OA significantly inhibited the phosphorylation of the AMPK/ACC pathway. Further experiments showed that OA increased global O-GlcNAcylation and agonist of O-GlcNAcylation significantly inhibited the phosphorylation of AMPK and ACC. Importantly, the disorder of lipid metabolism caused by HFD or OA could be rescued by treating CP-640186, the dual inhibitor of ACC1 and ACC2. These observations suggested that high fat may activate O-GlcNAcylation and affect the AMPK/ACC pathway to regulate lipid synthesis, and also emphasized the importance of the role of ACC in lipid homeostasis.

Abstract In the present study, two elongases, Elovl4 and Elovl5, were functionally characterized and their transcriptional regulation in response to n-3 LC-PUFA administration were investigated in vivo and in vitro. We previously described the molecular characterization of croaker elovl5. Here, we report the full-length cDNA sequence of croaker elovl4, which contained 1794 bp (excluding the polyA tail), including 909 bp of coding region that encoded a polypeptide of 302 amino acids possessing all the characteristic features of Elovl proteins. Functional studies showed that croaker Elovl5, displayed high elongation activity towards C18 and C20 PUFA, with only low activity towards C22 PUFA. In contrast, croaker Elovl4 could effectively convert both C20 and C22 PUFA to longer polyenoic products up to C34. n-3 LC-PUFA suppressed transcription of the two elongase genes, as well as srebp-1 and lxrα, major regulators of hepatic lipid metabolism. The results of dual-luciferase reporter assays and in vitro studies both indicated that the transcriptions of elovl5 and elovl4 elongases could be regulated by Lxrα. Moreover, Lxrα could mediate the transcription of elovl4 directly or indirectly through regulating the transcription of srebp-1. The above findings contribute further insight and understanding of the mechanisms regulating LC-PUFA biosynthesis in marine fish species.

Elongation of very long-chain fatty acids protein 6 (Elovl6) is a crucial enzyme in the synthesis of endogenous fatty acids, which participates in the energy balance and metabolic diseases. The main objective of this study was to explore the molecular characterization of Elovl6 and the regulation of elovl6 expression in response to dietary fatty acids and insulin. In the present study, the ORF (open reading frame) of Elovl6 from rainbow trout was cloned and characterized, which showed a high identity (87%) with mammals and other teleost. The results of quantitative PCR showed that the transcriptional levels of elovl6 from rainbow trout that were fed diets containing soybean oil (enriched with 18:2n-6, linoleic acid (LA)) or linseed oil (enriched with 18:3n-3, α-linolenic acid (ALA)) were lower than those in the group that were fed diets containing fish oil (enriched with 20:5n-3, eicosapentaenoic acid (EPA) and 22:6n-3, docosahexaenoic acid (DHA)). Correspondingly, mRNA expression of elovl6 in hepatocytes treated with DHA was dramatically higher than that in LA and ALA groups. The transcriptional expression of elovl6 in hepatocytes treated with insulin was also significantly increased. Moreover, the dual luciferase assay showed the transcription factor CREB1 dramatically up-regulated the promoter activity of elovl6, while FOXO1 significantly down-regulated the elovl6 promoter activity in rainbow trout. The differences in transcriptional expression of crbe1 and foxo1 may contribute to the increase or decrease of elovl6 expression in rainbow trout in response to fatty acids or insulin. These findings revealed the molecular characterization of elovl6 and the regulation of elovl6 expression by CREB1 and FOXO1 in rainbow trout in response to dietary fatty acids or insulin.

Elongation of very long chain fatty acids protein 6 (Elovl6) is a key enzyme in fatty acid synthesis, which participates in converting palmitate (C16:0) to stearate (C18:0). Although studies of Elovl6 have been carried out in mammals, the nutritional regulation of elovl6 in fish remains poorly understood. In the present study, the cloning and nutritional regulation of elovl6 were determined in large yellow croaker. Sequence and phylogenetic analysis revealed that the full-length cDNA of elovl6 was 1360 bp, including an open reading frame of 810 bp encoding a putative protein of 269 amino acid that possesses the characteristic features of Elovl proteins. The transcript level of elovl6 was significantly increased in the liver of croaker fed the diets with soybean oil (enriched with 18: 2n-6, LA) or linseed oil (enriched with 18: 3n-3, ALA) than that in croaker fed the diet with fish oil (enriched with 20: 5n-3 and 22: 6n-3). Correspondingly, the elovl6 expression in croaker’s hepatocytes treated with ALA or LA was remarkably increased compared to the controls. Furthermore, the transcription factors including hepatocyte nuclear factor 1α (HNF1α), CCAAT-enhancer-binding protein β (CEBPβ), retinoid X receptor α (RXRα), and cAMP response element-binding protein 1 (CREB1) greatly enhanced promoter activity of elovl6 in large yellow croaker, and the expression of transcription factors is consistent with the changes of elovl6 expression in response to fatty acids in vivo and in vitro. In conclusion, this study revealed that elovl6 expression in large yellow croaker could be upregulated by dietary ALA or LA via the increased transcriptional expression of transcription factors including hnf1α, cebpβ, rxrα, and creb1.

The enzymes involved in the biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFAs) are widely studied in fish species, as fish are the main source of n-3 LC-PUFAs for human beings. In the present study, a putative gene for elovl5, which encodes a key enzyme involved in LC-PUFA synthesis, was cloned and functionally characterized, and its transcription in response to dietary n-3 LC-PUFA exposure was investigated. Moreover, cell transfection and luciferase assays were used to explore the mechanism underlying the regulation of elovl5. The full-length cDNA of elovl5 was 1242 bp (excluding the polyA tail), including an 885 bp coding region encoding a 295 amino acid protein that possesses all of the characteristic features of elovl proteins. Functional characterization of heterologously expressed grouper Elovl5 indicated that it effectively elongates both C18 (18:2n-6, 18:3n-3, 18:3n-6 and 18:4n-3) and C20 (20:4n-6 and C20:5n-3) PUFAs, but not the C22 substrates. The expression of elovl5 was significantly affected by dietary n-3 LC-PUFA exposure: a high n-3 LC-PUFA level repressed the expression of elovl5 by slightly down-regulating the expression of sterol regulatory element-binding protein (SREBP)-1 and liver X receptor (LXR) α, which are major regulators of hepatic lipid metabolism. Promoter studies showed that grouper elovl5 reporter activity was induced by over-expression of LXRα but not SREBP-1. This finding suggests that elovl5 is a direct target of LXRα, which is involved in the biosynthesis of PUFAs via transcriptional regulation of elovl5. These findings may contribute to a further understanding of the mechanism underlying the regulation of LC-PUFA biosynthesis in marine fish species.

Ectopic lipid accumulation has been observed in fish fed a high-lipid diet. However, no information is available on the mechanism by which dietary lipid levels comprehensively regulate lipid transport, uptake, synthesis and catabolism in fish. Therefore, the present study aimed to gain further insight into how dietary lipids affect lipid deposition in the liver of large yellow croaker(Larimichthys crocea). Fish (150.00±4.95 g) were fed a diet with a low (6%), moderate (12%, the control diet) or high (18%) crude lipid content for 10 weeks. Growth performance, plasma biochemical indexes, lipid contents and gene expression related to lipid deposition, including lipoprotein assembly and clearance, fatty acid uptake and triacylglycerol synthesis and catabolism, were assessed. Growth performance was not significantly affected. However, the hepato-somatic and viscera-somatic indexes as well as plasma triacylglycerol, non-esterified fatty acids and LDL-cholesterol levels were significantly increased in fish fed the high-lipid diet. In the livers of fish fed the high-lipid diet, the expression of genes related to lipoprotein clearance (LDLR) and fatty acid uptake (FABP11) was significantly up-regulated, whereas the expression of genes involved in lipoprotein assembly (apoB100), triacylglycerol synthesis and catabolism (DGAT2, CPT I) was significantly down-regulated compared with fish fed the control diet, and hepatic lipid deposition increased. In fish fed the low-lipid diet, the expression of genes associated with lipoprotein assembly and clearance (apoB100, LDLR, LRP-1), fatty acid uptake (CD36, FATP1, FABP3) and triacylglycerol synthesis (FAS) was significantly increased, whereas the expression of triacylglycerol catabolism related genes (ATGL, CPT I) was reduced compared with fish fed the control diet. However, hepatic lipid content in fish fed the low-lipid diet decreased mainly due to low dietary lipid intake. In summary, findings of this study provide molecular insight into the role of lipid deposition in the liver in response to different dietary lipid contents.

The present study was conducted to evaluate the influences of different dietary fatty acid profiles on the tissue content and biosynthesis of LC-PUFA in a euryhaline species Japanese seabass reared in seawater. Six diets were prepared, each with a characteristic fatty acid: Diet PA: Palmitic acid (C16:0); Diet SA: Stearic acid (C18:0); Diet OA: Oleic acid (C18:1n-9); Diet LNA: α-linolenic acid (C18:3n-3); Diet N-3 LC-PUFA: n-3 LC-PUFA (DHA+EPA); Diet FO: the fish oil control. A 10-week feeding trial was conducted using juvenile fish (29.53 ± 0.86 g). The results showed that Japanese seabass had limited capacity to synthesize LC-PUFA and fish fed PA, SA, OA and LNA showed significantly lower tissue n-3 LC-PUFA contents compared to fish fed N-3 LC-PUFA and FO. The putative gene promoter and full-length cDNA of FADS2 was cloned and characterized. The protein sequence was confirmed to be homologous to FADS2s of marine teleosts and possessed all the characteristic features of microsomal fatty acid desaturases. The FADS2 transcript levels in liver of fish fed N-3 LC-PUFA and FO were significantly lower than those in fish fed other diets except LNA while Diet PA significantly up-regulated the FADS2 gene expression compared to Diet LNA, N-3 LC-PUFA and FO. Inversely, fish fed N-3 LC-PUFA and FO showed significantly higher promoter methylation rates of FADS2 gene compared to fish fed the LC-PUFA deficient diets. These results suggested that Japanese seabass had low LC-PUFA synthesis capacity and LC-PUFA deficient diets caused significantly reduced tissue n-3 LC-PUFA contents. The liver gene expression of FADS2 was up-regulated in groups enriched in C16:0, C18:0 and C18:1n-9 respectively but not in the group enriched in C18:3n-3 compared to groups with high n-3 LC-PUFA contents. The FADS2 gene expression regulated by dietary fatty acids was significantly negatively correlated with the methylation rate of putative FADS2 gene promoter.

Vitamin D (VD) is a fat-soluble sterol that possesses a wide range of physiological functions. The present study aimed to evaluate the effects of VD on folate metabolism in zebrafish and further investigated the underlying mechanism. Wild-type (WT) zebrafish were fed with a diet containing 0 IU/kg VD3 or 800 IU/kg VD3 for 3 wk. Meanwhile, cyp2r1 mutant zebrafish with impaired VD metabolism was used as another model of VD deficiency. Our results showed that VD deficiency in zebrafish suppressed the gene expression of folate transporters, including reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT) in the intestine. Moreover, VD influenced the gene expression of several enzymes related to cellular folate metabolism in the intestine and liver of zebrafish. Importantly, VD-deficient zebrafish contained a remarkably lower level of folate content in the liver. Notably, VD was incapable of altering folate metabolism in zebrafish when gut microbiota was depleted by antibiotic treatment. Further studies proved that gut commensals from VD-deficient fish displayed a lower capacity to produce folate than those from WT fish. Our study revealed the potential correlation between VD and folate metabolism in zebrafish, and gut microbiota played a key role in VD-regulated folate metabolism in zebrafish. [ABSTRACT FROM AUTHOR]