Optimal dietary lipid levels alleviated adverse effects of high temperature on growth, lipid metabolism, antioxidant and immune responses in juvenile turbot (Scophthalmus maximus L.).

Fish physiological health is often negatively impacted by high-temperature environments and there are few studies on how dietary lipids affect fish growth and physiology when exposed to heat stress. The main objective of this research was to examine the impact of dietary lipid levels on growth and physiological status of juvenile turbot (Scophthalmus maximus L.) and determine if dietary lipid concentration could alleviate the possible adverse effects of heat stress. Five diets containing 6.81%, 9.35%, 12.03%, 14.74%, and 17.08% lipid, respectively, were formulated and fed to turbot (initial weight 5.13 ± 0.02 g) under high-temperature conditions (24.0-25.0 °C). Meanwhile, the diet with 12.03% lipid (considered by prior work to be an optimal dietary lipid level) was fed to turbot of the same size at normal temperature. Results suggested that, among the different dietary lipid levels under high-temperature conditions, fish fed the optimal lipid (12.03%) exhibited better growth compared to non-optimal lipid groups, as evidenced by higher weight gain and specific growth rate. Simultaneously, the optimal lipid diet may better maintain lipid homeostasis, as attested by lower liver and serum lipid, along with higher liver mRNA levels of lipolysis-related genes (pgc1α, lipin1, pparα, lpl and hl) and lower levels of synthesis-related genes (lxr, fas, scd1, pparγ, dgat1 and dgat2). Also, the optimal lipid diet might mitigate oxidative damage by improving antioxidant enzyme activity, decreasing malondialdehyde levels, and up-regulating oxidation-related genes (sod1, sod2, cat, gpx and ho-1). Furthermore, the optimal lipid may enhance fish immunity, as suggested by the decrease in serum glutamic-oxalacetic/pyruvic transaminase activities, down-regulation of pro-inflammatory genes and up-regulation of anti-inflammation genes. Correspondingly, the optimal lipid level suppressed MAPK signaling pathway via decreased phosphorylation levels of p38, JNK and ERK proteins in liver. In summary, the optimal dietary lipid level facilitated better growth and physiological status in turbot under thermal stress.

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.

Activation of farnesoid X receptor suppresses ER stress and inflammation via the YY1/NCK1/PERK pathway in large yellow croaker (Larimichthys crocea).

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.

Regulation of Δ6Fads2 Gene Involved in LC-PUFA Biosynthesis Subjected to Fatty Acid in Large Yellow Croaker (Larimichthys crocea) and Rainbow Trout (Oncorhynchus mykiss).

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.

Activation of the Farnesoid X Receptor (FXR) Suppresses Linoleic Acid-Induced Inflammation in the Large Yellow Croaker (Larimichthys crocea).

BACKGROUND: High linoleic acid (LA) intake leads to inflammation that adversely influences health in fish. However, whether the farnesoid X receptor (FXR) could be an effective target for regulating LA-induced inflammation remains unknown. OBJECTIVE: The purpose of this study was to investigate the role of FXR in the regulation of LA-induced inflammation in large yellow croakers. METHODS: Large yellow croakers (initial weight of 10.03 ± 0.02 g) were allocated to 4 groups and fed a fish oil diet (6% FO), a soybean oil diet (6% SO), or the SO diet supplemented with 300 or 900 mg chenodeoxycholic acid (CDCA)/kg for 10 wk. The cultured kidney cell line PCK and primary hepatocytes from large yellow croakers were stimulated by LA (50 µM) after pretreatment with an FXR ligand (GW4064 or CDCA) or transfection with fxr-small interfering RNA (siFXR). mRNA expression of proinflammatory genes in the head kidney and liver tissues, PCK cells, and primary hepatocytes was determined by qPCR. The luciferase reporter assay, electrophoretic mobility shift assay, and immunoprecipitation assay were conducted in HEK 293T cells to determine the transcriptional activity of P65 and protein interactions between P65 and FXR or the small heterodimer partner (SHP). RESULTS: Proinflammatory genes were 93-1180% higher in the SO group compared with the FO group. CDCA supplementation decreased mRNA expression of proinflammatory genes by 17-87% while increasing fxr and shp expression by 120-460%. In PCK cells and primary hepatocytes, ligand-mediated activation of FXR decreased the LA-induced expression of proinflammatory genes by 18-67%, whereas siRNA-mediated knockdown of FXR increased the LA-induced expression of proinflammatory genes by 64-96%. FXR bound to the promoter of shp and regulated its mRNA expression. Both FXR and SHP could bind to P65 to suppress the transcriptional activity of P65. CONCLUSIONS: These results indicate that FXR has anti-inflammatory properties in large yellow croakers by directly and indirectly suppressing NFκB activity.

Molecular Cloning, Characterization, and Nutritional Regulation of Elovl6 in Large Yellow Croaker (Larimichthys crocea).

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 ß (CEBPß), 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, cebpß, rxrα, and creb1.