Vitamin D promotes the folate transport and metabolism in zebrafish (Danio rerio).

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.NEW & NOTEWORTHY Our study has identified that VD influences intestinal uptake and transport of folate in zebrafish while also altering hepatic folate metabolism and storage. Interestingly, the regulatory effects of VD on folate transport and metabolism diminished after the gut flora was interrupted by antibiotic treatment, suggesting that the regulatory effects of VD on folate metabolism in zebrafish are most likely dependent on the intestinal flora.

Sterol Regulatory Element Binding Protein 1: A Mediator for High-Fat Diet-Induced Hepatic Gluconeogenesis and Glucose Intolerance in Fish.

BACKGROUND: Sterol regulatory element binding protein (SREBP) 1 is considered to be a crucial regulator for lipid synthesis in vertebrates. However, whether SREBP1 could regulate hepatic gluconeogenesis under high-fat diet (HFD) condition is still unknown, and the underlying mechanism is also unclear. OBJECTIVES: This study aimed to determine gluconeogenesis-related gene and protein expressions in response to HFD in large yellow croaker and explore the role and mechanism of SREBP1 in regulating the related transcription and signaling. METHODS: Croakers (mean weight, 15.61 ± 0.10 g) were fed with diets containing 12% crude lipid [control diet (ND)] or 18% crude lipid (HFD) for 10 weeks. The glucose tolerance, insulin tolerance, hepatic gluconeogenesis-related genes, and proteins expressions were determined. To explore the role of SREBP1 in HFD-induced gluconeogenesis, SREBP1 was inhibited by pharmacologic inhibitor (fatostatin) or genetic knockdown in croaker hepatocytes under palmitic acid (PA) condition. To explore the underlying mechanism, luciferase reporter and chromatin immunoprecipitation assays were conducted in HEK293T cells. Data were analyzed using analysis of variance or Student t test. RESULTS: Compared with ND, HFD increased the mRNA expressions of gluconeogenesis genes (2.40-fold to 2.60-fold) (P < 0.05) and reduced protein kinase B (AKT) phosphorylation levels (0.28-fold to 0.34-fold) (P < 0.05) in croakers. However, inhibition of SREBP1 by fatostatin addition or SREBP1 knockdown reduced the mRNA expressions of gluconeogenesis genes (P < 0.05) and increased AKT phosphorylation levels (P < 0.05) in hepatocytes, compared with that by PA treatment. Moreover, fatostatin addition or SREBP1 knockdown also increased the mRNA expressions of irs1 (P < 0.05) and reduced serine phosphorylation of IRS1 (P < 0.05). Furthermore, SREBP1 inhibited IRS1 transcriptions by binding to its promoter and induced IRS1 serine phosphorylation by activating diacylglycerol-protein kinase Cε signaling. CONCLUSIONS: This study reveals the role of SREBP1 in hepatic gluconeogenesis under HFD condition in croakers, which may provide a potential strategy for improving HFD-induced glucose intolerance.

Dietary palm oil enhances Sterol regulatory element-binding protein 2-mediated cholesterol biosynthesis through inducing endoplasmic reticulum stress in muscle of large yellow croaker (Larimichthys crocea).

Sterol regulatory element-binding protein 2 (SREBP2) is considered to be a major regulator to control cholesterol homoeostasis in mammals. However, the role of SREBP2 in teleost remains poorly understand. Here, we explored the molecular characterisation of SREBP2 and identified SREBP2 as a key modulator for 3-hydroxy-3-methylglutaryl-coenzyme A reductase and 7-dehydrocholesterol reductase, which were rate-limiting enzymes of cholesterol biosynthesis. Moreover, dietary palm oil in vivo or palmitic acid (PA) treatment in vitro elevated cholesterol content through triggering SREBP2-mediated cholesterol biosynthesis in large yellow croaker. Furthermore, our results also found that PA-induced activation of SREBP2 was dependent on the stimulating of endoplasmic reticulum stress (ERS) in croaker myocytes and inhibition of ERS by 4-Phenylbutyric acid alleviated PA-induced SREBP2 activation and cholesterol biosynthesis. In summary, our findings reveal a novel insight for understanding the role of SREBP2 in the regulation of cholesterol metabolism in fish and may deepen the link between dietary fatty acid and cholesterol biosynthesis.

Dietary methanotroph bacteria meal alleviates soybean meal-induced enteritis by improving immune tolerance and intestinal flora profile of juvenile turbot (Scophthalmus maximus L.).

An 8-week growth trial was performed to investigate the protective effects of methanotroph bacteria meal (MBM) produced from methane against soybean meal-induced enteritis (SBMIE) in juvenile turbot (Scophthalmus maximus L.). Five isonitrogenous and isolipidic diets were formulated: fishmeal-based diet (FM, the control group); FM with approximate 50% of fishmeal substituted by 399.4 g/kg soybean meal (SBM); SBM supplemented with 63.6, 127.2 and 190.8 g/kg MBM (named MBM1, MBM2 and MBM3), each diet was randomly assigned to triplicate fibreglass tanks. Results showed that fish fed with SBM exhibited enteritis, identified by reduced relative weight of intestine (RWI), as well as expanded lamina propria width and up-regulated gene expression of pro-inflammatory cytokines (tnf-α, il-6 and il-8) in intestine. While the above symptoms were reversed when diet SBM supplemented with MBM at the levels of 63.6 and 127.2 g/kg, as well as characterized by up-regulated gene expression of anti-inflammatory cytokines (tgf-ß and il-10) and tight junction protein (claudin3, claudin4 and claudin7) in intestine. Intestinal transcriptome analysis showed that the differentially expressed genes between groups FM and SBM predominantly enriched in the JAK-STAT signaling pathway, and the enrichment of differentially expressed genes between groups SBM and SBM supplemented with 63.6 g/kg MBM was in the inflammatory bowel disease (IBD) and JAK-STAT signaling pathway. To be specific, the expression of jak1, jak2b, stat1 and stat5a was significantly up-regulated when fish fed with SBM, suggested the activation of JAK-STAT signaling pathway, while the expression of these above genes was depressed by providing MBM to diet SBM, and the gene expression of toll-like receptors tlr2 and tlr5b showed a similar pattern. Moreover, intestinal flora analysis showed that community richness and abundance of beneficial bacteria (Cetobacterium and acillus_coagulans) were improved when fish fed with SBM supplemented with 63.6 g/kg MBM. Overall, methanotroph bacteria meal may alleviate SBMIE by regulating the expression of tight junction protein, toll-like receptors and JAK-STAT signaling pathway, as well as improving intestinal flora profile, which would be beneficial for enhancing the immune tolerance and utilization efficiency of turbot to dietary soybean meal.

Dietary Clostridium autoethanogenum protein has dose-dependent influence on the gut microbiota, immunity, inflammation and disease resistance of abalone Haliotis discus hannai.

Clostridium autoethanogenum protein (CAP) is an eco-friendly protein source and has great application potential in aquafeeds. The present study aimed to investigate the effects of dietary CAP inclusion on the anti-oxidation, immunity, inflammation, disease resistance and gut microbiota of abalone Haliotis discus hannai after a 110-day feeding trial. Three isonitrogenous and isolipidic diets were formulated by adding 0 % (control), 4.10 % (CAP4.10) and 16.25 % (CAP16.25) of CAP, respectively. A total of 540 abalones with an initial mean body weight of 22.05 ± 0.19 g were randomly distributed in three groups with three replicates per group and 60 abalones per replicate. Results showed that the activities of superoxide dismutase and glutathione peroxidase in the cell-free hemolymph (CFH) were significantly decreased and the content of malondialdehyde in CFH was significantly increased in the CAP16.25 group. The diet with 4.1 % of CAP significantly increased the activities of lysozyme and acid phosphatase in CFH. The expressions of pro-inflammatory genes such as tumor necrosis factor-α (tnf-α), nuclear factor-κb (nf-κb) and toll-like receptor 4 (tlr4) in digestive gland were downregulated, and the expressions of anti-inflammatory genes such as ß-defensin and mytimacin 6 in digestive gland were upregulated in the CAP4.10 group. Dietary CAP inclusion significantly decreased the cumulative mortality of abalone after the challenge test with Vibrio parahaemolyticus for 7 days. Dietary CAP inclusion changed the composition of gut microbiota of abalone. Besides, the balance of the ecological interaction network of bacterial genera in the intestine of abalone was enhanced by dietary CAP. The association analysis showed that two bacterial genera Ruegeria and Bacteroides were closely correlated with the inflammatory genes. In conclusion, the 4.10 % of dietary CAP enhanced the immunity and disease resistance as well as inhibited the inflammation of abalone. The 16.25 % of dietary CAP decreased the anti-oxidative capacity of abalone. The structure of the gut microbiota of abalone changed with dietary CAP levels.

Loss of Gcn2 exacerbates gossypol induced oxidative stress, apoptosis and inflammation in zebrafish.

Gossypol, a naturally occurring compound found in cottonseed meal, shows promising therapeutic potential for human diseases. However, within the aquaculture industry, it is considered an antinutritional factor. The incorporation of cottonseed meal into fish feed introduces gossypol, which induces intracellular stresses and hinders overall health of farmed fish. The aim of this study is to determine the role of General control nonderepressible 2 (gcn2), a sensor for intracellular stresses in gossypol-induced stress responses in fish. In the present study, we established two gcn2 knockout zebrafish lines. A feeding trial was conducted to assess the growth-inhibitory effect of gossypol in both wild type and gcn2 knockout zebrafish. The results showed that in the absence of gcn2, zebrafish exhibited increased oxidative stress and apoptosis when exposed to gossypol, resulting in higher mortality rates. In feeding trial, dietary gossypol intensified liver inflammation in gcn2-/- zebrafish, diminishing their growth and feed conversion. Remarkably, administering the antioxidant N-acetylcysteine (NAC) was effective in reversing the gossypol induced oxidative stress and apoptosis, thereby increasing the gossypol tolerance of gcn2-/- zebrafish. Exposure to gossypol induces more severe mitochondrial stress in gcn2-/- zebrafish, thereby inducing metabolic disorders. These results reveal that gcn2 plays a protective role in reducing gossypol-induced oxidative stress and apoptosis, attenuating inflammation responses, and enhancing the survivability of zebrafish in gossypol-challenged conditions. Therefore, maintaining appropriate activation of Gcn2 may be beneficial for fish fed diets containing gossypol.

Effects of dietary supplementation with heat-killed Lactobacillus acidophilus on growth performance, digestive enzyme activity, antioxidant capacity, and inflammatory response of juvenile large yellow croaker (Larimichthys crocea).

A ten-week culture trial in juvenile large yellow croaker (Larimichthys crocea) (10.80 ± 0.10 g) was conducted to assess the impact of supplementing heat-killed Lactobacillus acidophilus (HLA) on growth performance, intestinal digestive enzyme activity, antioxidant capacity and inflammatory response. Five iso-nitrogenous (42 % crude protein) and iso-lipidic (12 % crude lipid) experimental feeds with different levels of HLA (0.0 %, 0.1 %, 0.2 %, 0.4 %, or 0.8 %) were prepared. They were named FO (control group), HLA0.1, HLA0.2, HLA0.4 and HLA0.8, respectively. The results indicated that HLA addition had no impact on survival (P > 0.05). In this experiment, the final body weight, weight gain rate and specific growth rate showed a quadratic regression trend, initially increasing and subsequently decreasing with the increasing in HLA levels, and attained the peak value at 0.2 % HLA supplemental level (P < 0.05). In contrast to the control group, in terms of digestive ability, amylase, lipase and trypsin exhibited a notable linear and quadratic pattern, demonstrating a substantial increase when 0.1% 0.2 % HLA was added in the diets (P < 0.05). Notably, elevated levels of catalase (CAT) activity, superoxide dismutase (SOD) activity, and total antioxidant capacity (T-AOC) were observed in the liver when adding 0.1%-0.2 % HLA, and the level of malondialdehyde (MDA) was significantly decreased and the liver exhibited a notable upregulation in the mRNA expression levels of nrf2, cat, sod2, and sod3 (P < 0.05). Additionally, the mRNA levels of genes associated with tight junctions in the intestines (zo-1, zo-2 and occludin) exhibited a significant upregulation when 0.2 % HLA was added in the feed (P < 0.05). Furthermore, the levels of mRNA expression for proinflammatory genes in the intestines including tnf-α, il-1ß, il-6 and il-8 exhibited a quadratic regression trend, characterized by an initial decline followed by subsequent growth (P < 0.05). Meanwhile, the levels of mRNA expression for genes linked to anti-inflammatory responses in the intestines (including il-10, tgf-ß, and arg1) exhibited a quadratic regression pattern, initially increasing and subsequently decreasing (P < 0.05). Compare with the control group, the levels of tnf-α, il-1ß and il-8 expression were notably downregulated in all HLA addition groups (P < 0.05). When 0.2 % HLA was added, the expression levels of il-10, tgf-ß and arg1 in the intestinal tract were markedly increased (P < 0.05). Overall, the supplementation of 0.2 % HLA in the feed has been shown to enhance the growth performance. The enhancement was attributed to HLA's capacity to improve antioxidant function, intestinal barrier integrity, and mitigate inflammatory responses. This research offers a scientific foundation for the utilization of HLA in aquaculture.

EPA and DHA promote cell proliferation and enhance activity of the Akt-TOR-S6K anabolic signaling pathway in primary muscle cells of turbot (Scophthalmus maximus L.).

Fish growth and health are predominantly governed by dietary nutrient supply. Although the beneficial effects of omega-3 polyunsaturated fatty acids supplementation have been shown in a number of fish species, the underlying mechanisms are still mostly unknown. In this study, we conducted an investigation into the effects of EPA and DHA on cell proliferation, nutrient sensing signaling, and branched-chain amino acids (BCAA) transporting in primary turbot muscle cells. The findings revealed that EPA and DHA could stimulate cell proliferation, promote protein synthesis and inhibit protein degradation through activation of target of rapamycin (TOR) signaling pathway, a pivotal nutrient-sensing signaling cascade. While downregulating the expression of myogenin and myostatin, EPA and DHA increased the level of myogenic regulatory factors, such as myoD and follistatin. Furthermore, we observed a significant increase in the concentrations of intracellular BCAAs following treatment with EPA or DHA, accompanied by an upregulation of the associated amino acid transporters. Our study providing valuable insights into the mechanisms underlying the growth-promoting effects of omega-3 fatty acids in fish.

Vitamin D regulates insulin pathway and glucose metabolism in zebrafish (Danio rerio).

1,25-dihydroxyvitamin D3 [1,25(OH)2 D3 ], the most active vitamin D (VD) metabolite, is a steroid hormone playing an important role in many physiological functions in addition to maintaining mineral homeostasis. In this study, we explored the mechanism that the VD regulated insulin pathway and glucose metabolism in zebrafish in vitro and in vivo. Our results show that 1,25(OH)2 D3  significantly enhances the expression of insulin receptor a (insra), insulin receptor substrate 1 (irs1) and glucose transporter 2 (glut2), and promotes glycolysis and glycogenesis, while suppressing gluconeogenesis in zebrafish liver cell line (ZFL) under the condition of high glucose (20 mM), instead of the normal glucose (10 mM). Moreover, consistent results were obtained from the zebrafish fed with VD3 -deficient diet, as well as the cyp2r1-/- zebrafish, in which endogenous VD metabolism is blocked. Furthermore, results from dual-luciferase reporting system exhibited that 1,25(OH)2 D3 directly activated the transcription of insra, rather than insrb in zebrafish by binding to vitamin D response element (VDRE) located at -181 to -167 bp in the promoter region of insra. Importantly, the 1,25(OH)2 D3 treatment significantly alleviated the symptoms of hyperglycemia in diabetic zebrafish. In conclusion, our study demonstrated that VD activates VDRE located in the promoter area of insra in zebrafish to promote insulin/insra signaling pathway, thereby contributing to the maintenance of glucose homeostasis.

LPS stimulation stabilizes HIF-1α by enhancing HIF-1α acetylation via the PARP1-SIRT1 and ACLY-Tip60 pathways in macrophages.

Hypoxia and inflammatory mediators stabilize hypoxia-inducible factor (HIF)-1α through posttranslational modifications, such as phosphorylation and succinylation. Here, we identified sirtuin 1 (SIRT1) and 60 kDa Tat-interactive protein (Tip60)-mediated acetylation as another critical posttranslational modification that regulates HIF-1α protein stability under lipopolysaccharide (LPS) stimulation. Mechanistically, DNA damage induced by excessive reactive oxygen species (ROS) activated poly (ADP-ribose) polymerase 1 (PARP1) to consume oxidized nicotinamide adenine dinucleotide (NAD+ ). Correspondingly, SIRT1 activity was decreased with the decline in NAD+ levels, resulting in increased HIF-1α acetylation. LPS also activated the ATP-citrate lyase (ACLY)-Tip60 pathway to further enhance HIF-1α acetylation. Acetylation contributed to HIF-1α stability and exacerbated LPS-induced inflammation. Thus, inhibiting HIF-1α stability by decreasing its acetylation could partly alleviate LPS-induced inflammation. In conclusion, we revealed the mechanism by which LPS stabilized HIF-1α by increasing its acetylation via the PARP1-SIRT1 and ACLY-Tip60 pathways in fish macrophages. This study may provide novel insights for manipulation of HIF-1α acetylation as a therapeutic strategy against inflammation from the perspective of acetylation in vertebrates.

Oleic and palmitic acids induce hepatic angiopoietin-like 4 expression predominantly via PPAR-γ in Larimichthys crocea.

Angiopoietin-like 4 (ANGPTL4) is a potent regulator of TAG metabolism, but knowledge of the mechanisms underlying ANGPTL4 transcription in response to fatty acids is still limited in teleost. In the current study, we explored the molecular characterisation of ANGPTL4 and regulatory mechanisms of ANGPTL4 in response to fatty acids in large yellow croaker (Larimichthys crocea). Here, croaker angptl4 contained a 1416 bp open reading frame encoding a protein of 471 amino acids with highly conserved 12-amino acid consensus motif. Angptl4 was widely expressed in croaker, with the highest expression in the liver. In vitro, oleic and palmitic acids (OA and PA) treatments strongly increased angptl4 mRNA expression in croaker hepatocytes. Moreover, angptl4 expression was positively regulated by PPAR family (PPAR-α, ß and γ), and expression of PPARγ was also significantly increased in response to OA and PA. Moreover, inhibition of PPARγ abrogated OA- or PA-induced angptl4 mRNA expression. Beyond that, PA might increase angptl4 expression partly via the insulin signalling. Overall, the expression of ANGPTL4 is strongly upregulated by OA and PA via PPARγ in the liver of croaker, which contributes to improve the understanding of the regulatory mechanisms of ANGPTL4 in fish.

Dietary l-carnitine regulates liver lipid metabolism via simultaneously activating fatty acid ß-oxidation and suppressing endoplasmic reticulum stress in large yellow croaker fed with high-fat diets.

Dietary l-carnitine (LC) is a nutritional factor that reduces liver lipid content. However, whether dietary LC can improve lipid metabolism via simultaneous activation of mitochondrial fatty acid (FA) ß-oxidation and suppression of endoplasmic reticulum (ER) stress is still unknown. Large yellow croaker were fed with a high-fat diet (HFD) supplemented with dietary LC at 0, 1·2 or 2·4 ‰ for 10 weeks. The results indicated that a HFD supplemented with LC reduced the liver total lipid and TAG content and improved serum lipid profiles. LC supplementation administered to this fish increased the liver antioxidant capacity by decreasing serum and liver malondialdehyde levels and enhancing the liver antioxidant capacity, which then relieved the liver damage. Dietary LC increased the ATP dynamic process and mitochondrial number, decreased mitochondrial DNA damage and enhanced the protein expression of mitochondrial ß-oxidation, biogenesis and mitophagy. Furthermore, dietary LC supplementation increased the expression of genes and proteins related to peroxisomal ß-oxidation and biogenesis. Interestingly, feeding fish with LC-enriched diets decreased the protein levels indicative of ER stress, such as glucose-regulated protein 78, p-eukaryotic translational initiation factor 2a and activating transcription factor 6. Dietary LC supplementation downregulated mRNA expression relative to FA synthesis, reduced liver lipid and relieved liver damage through regulating ß-oxidation and biogenesis of mitochondria and peroxisomes, as well as the ER stress pathway in fish fed with HFD. The present study provides the first evidence that dietary LC can improve lipid metabolism via simultaneously promoting FA ß-oxidation capability and suppressing the ER stress pathway in fish.

Organic copper promoted copper accumulation and transport, enhanced low temperature tolerance and physiological health of white shrimp (Litopenaeus vannamei Boone, 1931).

This study was conducted to assess the effects of dietary copper source and level on hematological parameters, copper accumulation and transport, resistance to low temperature, antioxidant capacity and immune response of white shrimp (Litopenaeus vannamei Boone, 1931). Seven experimental diets with different copper sources and levels were formulated: C, no copper supplementation; S, 30 mg/kg copper in the form of CuSO4·5H2O; SO, 15 mg/kg copper in CuSO4·5H2O + 7.5 mg/kg copper in Cu-proteinate; O1, O2, O3 and O4, 10, 20, 30 and 40 mg/kg copper in the form of Cu-proteinate, respectively. A total of 840 shrimp (5.30 ± 0.04 g) were randomly distributed to 21 tanks (3 tanks/diet, 40 shrimp/tank). An 8-week feeding trial was conducted. The results showed that there was no significant difference in growth performance and whole shrimp chemical compositions among all groups. Compared with inorganic copper, dietary organic copper (O2 and O3) increased total protein, albumin, and glucose content of plasma, while decreased triglyceride and total cholesterol of plasma. Copper concentration in plasma and muscle and gene expression of metallothionein and copper-transporting ATPase 2 like in hepatopancreas were higher in shrimp fed organic copper (SO, O2, O3 and O4). The lowest mortality after low temperature (10 °C) challenge test was observed in the O2 and O3 groups. Organic copper (SO, O2, O3 and O4) significantly enhanced the antioxidant capacity (in terms of higher activities of total superoxide dismutase, copper zinc superoxide dismutase, catalase, glutathione peroxidase and total antioxidant capacity, lower malondialdehyde concentration of plasma, and up-regulated gene expression of superoxide dismutase, copper zinc superoxide dismutase, catalase and glutathione peroxidase of hepatopancreas). Organic copper (SO, O2, O3 and O4) enhanced the immune response (in terms of higher number of total hemocytes, higher activities of acid phosphatase, alkaline phosphatase, phenoloxidase, hemocyanin and lysozyme in plasma, and higher gene expressions of alkaline phosphatase, lysozyme and hemocyanin in hepatopancreas). Inorganic copper (Diet S) also had positive effects on white shrimp compared with the C diet, but the SO, O2, O3 and O4 diets resulted in better results, among which the O2 diet appeared to be the best one. In conclusion, organic copper was more beneficial to shrimp health than copper sulfate.

Effects of dietary chloroquine on fish growth, hepatic intermediary metabolism, antioxidant and inflammatory responses in turbot.

Autophagy is a conserved cellular self-digestion process and is essential for individual growth, cellular metabolism and inflammatory responses. It was responsive to starvation, pathogens infection and environmental stress. However, the information on the regulation of autophagy in fish hepatic intermediary metabolism, antioxidant system, and immune responses were limited. In the present study, turbot with inhibited autophagy flux was built by dietary chloroquine. The hepatic metabolic response, antioxidant enzymes and immune responses were explored. Results showed that dietary chloroquine induced the expression of Beclin 1, SQSTM and LC-3II, and effectively inhibited autophagy flux. Autophagy dysfunction depressed fish growth and feed utilization, while it induced clusters of liver lipid droplets. The genes involved in lipolysis and fatty acid ß-oxidation, as well as the lipogenesis-related genes in chloroquine group were depressed. The phosphorylation of AMPK was activated in chloroquine group, and the genes involved in glycolysis were induced. The hepatic content of malonyldialdehyde and the activities of SOD and CAT were induced when autophagy was inhibited. The content of Complement 3, Complement 4 and Immunoglobulin M, as well as the activity of lysozyme in plasma were depressed in chloroquine group. Dietary chloroquine induced the expression of toll-like receptors and stimulated the expression of myd88 and nf-κb p65, as well as the pro-inflammatory cytokines, such as tnf-α and il-1ß. The expression of anti-inflammatory cytokine tgf-ß was depressed in the chloroquine group. Our results would extend the knowledge on the role of autophagy in teleost and assist in improving fishery production.

Comprehensive study on the effect of dietary leucine supplementation on intestinal physiology, TOR signaling and microbiota in juvenile turbot (Scophthalmus maximus L.).

Intestinal damage and inflammation are major health and welfare issues in aquaculture. Considerable efforts have been devoted to enhancing intestinal health, with a specific emphasis on dietary additives. Branch chain amino acids, particularly leucine, have been reported to enhance growth performance in various studies. However, few studies have focused on the effect of leucine on the intestinal function and its underlying molecular mechanism is far from fully illuminated. In the present study, we comprehensively evaluated the effect of dietary leucine supplementation on intestinal physiology, signaling transduction and microbiota in fish. Juvenile turbot (Scophthalmus maximus L.) (10.13 ± 0.01g) were fed with control diet (Con diet) and leucine supplementation diet (Leu diet) for 10 weeks. The findings revealed significant improvements in intestinal morphology and function in the turbot fed with Leu diet. Leucine supplementation also resulted in a significant increase in mRNA expression levels of mucosal barrier genes, indicating enhanced intestinal integrity. The transcriptional levels of pro-inflammatory factors il-1ß, tnf-α and irf-1 was decreased in response to leucine supplementation. Conversely, the level of anti-inflammatory factors tgf-ß, il-10 and nf-κb were up-regulated by leucine supplementation. Dietary leucine supplementation led to an increase in intestinal complement (C3 and C4) and immunoglobulin M (IgM) levels, along with elevated antioxidant activity. Moreover, dietary leucine supplementation significantly enhanced the postprandial phosphorylation level of the target of rapamycin (TOR) signaling pathway in the intestine. Finally, intestinal bacterial richness and diversity were modified and intestinal bacterial composition was re-shaped by leucine supplementation. Overall, these results provide new insights into the beneficial role of leucine supplementation in promoting intestinal health in turbot, offering potential implications for the use of leucine as a nutritional supplement in aquaculture practices.

Effects of dietary bile acids levels on growth performance, anti-oxidative capacity, immunity and intestinal microbiota of abalone Haliotis discus hannai.

Abalone Haliotis discus hannai (initial weight: 38.79 ± 0.70 g) was used as the experimental animal in a 105-day feeding trial to investigate the influence of dietary bile acids levels on the growth, anti-oxidation, immune response and intestinal microbiota. Six isonitrogenous and isolipidic diets were prepared by adding 0 (control group), 15, 30, 60, 120 and 240 mg/kg of bile acids, respectively (named BA0, BA15, BA30, BA60, BA120 and BA240, respectively). It was found that survival of abalone between groups had no significant difference (P > 0.05). Compared to the control, significant improvements in weight gain rate (WGR) were observed in the groups of BA30 and BA60 (P < 0.05). Based on WGR, the broken line regression model analysis showed that the optimum demand for dietary bile acids for abalone was 35.47 mg/kg. Dietary bile acids increased the total anti-oxidative capacity and activities of catalase, superoxide dismutase, lysozyme and alkaline phosphatase, meanwhile decreased the content of malondialdehyde, alanine aminotransferase and aspartate aminotransferase activities in the cell-free hemolymph (P < 0.05). When bile acids were added to the diets, mRNA levels of genes related to pro-inflammatory factors and apoptosis in the digestive gland were down-regulated (P < 0.05). In contrast, the expression of genes related to anti-oxidation was significantly up-regulated (P < 0.05). The Firmicutes, Actinobacteriota and Proteobacteria were the most abundant phyla in intestine. And dietary bile acids significantly decreased the abundance of Actinobacteria and increased the abundance of Firmicutes (P < 0.05). In conclusion, supplementation of dietary bile acids within 120 mg/kg significantly increased the growth of abalone. The 34.62 mg/kg of dietary bile acids significantly increased the anti-oxidative capacity of abalone. Appropriate levels of dietary bile acids (34.62-61.75 mg/kg) promote the immunity of abalone. Application of appropriate levels of bile acids in diets (34.62 mg/kg) changed the intestinal microbiota and promoted the intestinal health of abalone.

Vitamin D3 enhances the antibacterial ability in head-kidney macrophages of turbot (Scophthalmus maximus L.) through C-type lectin receptors.

It has been known that vitamin D3 (VD3) not only plays an important role in regulating calcium and phosphorus metabolism in animals, but also has extensive effects on immune functions. In this study, the mechanism how VD3 influences bactericidal ability in turbot was explored. The transcriptomic analysis identified that dietary VD3 significantly upregulated the gene expression of C-type lectin receptors (CLRs), including mannose receptors (mrc1, mrc2, pla2r1) and collectins (collectin 11 and collectin 12) in turbot intestine. Further results obtained from in vitro experiments confirmed that the gene expression of mannose receptors and collectins in head-kidney macrophages (HKMs) of turbot was induced after the cells were incubated with different concentrations of VD3 (0, 1, 10 nM) or 1,25(OH)2D3 (0, 10, 100 pM). Meanwhile, both phagocytosis and bactericidal functions of HKMs were significantly improved in VD3 or 1,25(OH)2D3-incubated HKMs. Furthermore, phagocytosis and bacterial killing of HKMs decreased after collectin 11 was knocked down. Moreover, VD3-enhanced antibacterial activities diminished in collectin 11-interfered cells. Interestingly, the evidence was provided in the present study that inactive VD3 could be metabolized into active 1,25(OH)2D3 via hydroxylases encoded by cyp27a1 and cyp27b1 in fish macrophages. In conclusion, VD3 could be metabolized to 1,25(OH)2D3 in HKMs, which promoted the expression of CLRs in macrophages, leading to enhanced bacterial clearance.

Butyrate-induced IL-22 expression in fish macrophages contributes to bacterial clearance.

IL-22 has been characterized as a critical cytokine in maintaining barrier integrity and host immunity. So far, it has been known that IL-22 is mainly produced by lymphoid lineage cells. In the present study, we have thoroughly investigated butyrate-induced production and function of IL-22 in fish macrophages. Our results demonstrated that short-chain fatty acids (SCFAs), major microbiota-derived metabolites, promoted the expression of IL-22 in head kidney macrophages (HKMs) of turbot (Scophthalmus maximus L.). Interestingly, butyrate-mediated intracellular bacterial killing in HKMs diminished when IL-22 expression was interfered. Furthermore, the turbot fed the diet containing sodium butyrate (NaB) exhibited significantly lower mortality after bacterial infection, compared to the fish fed a basal diet. At the meantime, a higher level of IL-22 expression and bactericidal activity was detected in HKMs from the turbot fed NaB-supplemented diet. In addition, NaB treatment promoted the expression of antimicrobial peptides (AMPs) ß-defensins in zebrafish (Danio rerio). However, butyrate-induced expression of AMPs was reduced in IL-22 mutant zebrafish compared to wild-type (WT) fish. Meanwhile, NaB treatment was incapable to protect IL-22 mutant fish from bacterial infection as it did in WT zebrafish. Importantly, our results demonstrated that IL-22 expression was remarkably suppressed in macrophage-depleted zebrafish, indicating that macrophage might be a cell source of IL-22 production in vivo. In conclusion, all these findings collectively revealed that SCFAs regulated the production and function of IL-22 in fish macrophages, which facilitated host resistance to bacterial invasion.

Transcription factor EB (TFEB) participates in antiviral immune responses independent of mTORC1 in macrophage of large yellow croaker (Larimichthys crocea).

Transcription factor EB (TFEB) plays an integral role in the production of proinflammatory cytokines and chemokines in response to pathogen stimulation in mammals. However, the role of TFEB in antiviral immune responses and the potential regulatory mechanisms in fish remain poorly understood. Here, we cloned and characterized Larimichthys crocea TFEB (LcTFEB) with 524 amino acids and a typical basic helix-loop-helix-leucine zipper domain. LcTFEB could translocate into the nucleus upon starvation and had a comparatively high expression in immune tissues. Similar to the expression of antiviral immune genes, the transcriptional expression and activity of LcTFEB showed a trend of increasing and then decreasing with the prolongation of stimulation. Inhibition of LcTFEB using siRNA dramatically increased the polyinosinic-polycytidylic acid (poly (I:C))-induced interferon response and pro-inflammatory cytokines mRNA expression levels, whereas pharmacological activation and overexpression of LcTFEB exhibited the reverse effects. Mechanically, LcTFEB might promote the expression of IFNh as negative feedback to limit the virus-induced inflammatory responses. Notably, although inhibition of mTORC1 exacerbated poly (I:C)-triggered inflammatory responses, the effects of LcTFEB were independent of mTORC1. Overall, this study revealed an unidentified critical role of LcTFEB in the regulation of antiviral immune responses and promoted the understanding of TFEB in the antiviral immunity of fish macrophages.

Glycerol monolaurate improved intestinal barrier, antioxidant capacity, inflammatory response and microbiota dysbiosis in large yellow croaker (Larimichthys crocea) fed with high soybean oil diets.

Glycerol monolaurate (GML) is a potential candidate for regulating metabolic syndrome and inflammatory response. However, the role of GML in modulating intestinal health in fish has not been well determined. In this study, a 70-d feeding trial was conducted to evaluate the effect of GML on intestinal barrier, antioxidant capacity, inflammatory response and microbiota community of large yellow croaker (13.05 ± 0.09 g) fed with high level soybean oil (SO) diets. Two basic diets with fish oil (FO) or SO were formulated. Based on the SO group diet, three different levels of GML 0.02% (SO0.02), 0.04% (SO0.04) and 0.08% (SO0.08) were supplemented respectively. Results showed that intestinal villus height and perimeter ratio were increased in SO0.04 treatment compared with the SO group. The mRNA expressions of intestinal physical barrier-related gene odc and claudin-11 were significantly up-regulated in different addition of GML treatments compared with the SO group. Fish fed SO diet with 0.04% GML addition showed higher activities of acid phosphatase and lysozyme compared with the SO group. The content of malonaldehyde was significantly decreased and activities of catalase and superoxide dismutase were significantly increased in 0.02% and 0.04% GML groups compared with those in the SO group. The mRNA transcriptional levels of inflammatory response-related genes (il-1ß, il-6, tnf-α and cox-2) in 0.04% GML treatment were notably lower than those in the SO group. Meanwhile, sequencing analysis of bacterial 16S rRNA V4-V5 region showed that GML addition changed gut microbiota structure and increased alpha diversity of large yellow croaker fed diets with a high level of SO. The correlation analysis results indicated that the change of intestinal microbiota relative abundance strongly correlated with intestinal health indexes. In conclusion, these results demonstrated that 0.02%-0.04% GML addition could improve intestinal morphology, physical barrier, antioxidant capacity, inflammatory response and microbiota dysbiosis of large yellow croaker fed diets with a high percentage of SO.