Comparative analysis of nutritional and transcriptional regulation of hacd1 in large yellow croaker (Larimichthys crocea) and rainbow trout (Oncorhynchus mykiss).

3-hydroxyacyl-CoA dehydratases 1 (Hacd1) is a critical enzyme in long-chain polyunsaturated fatty acids (LC-PUFA) biosynthesis. The difference in expression of hacd1 might account for the stronger capacity of LC-PUFA biosynthesis in freshwater fish than in marine fish, but little is known about fish hacd1. Therefore, this study compared the responses of large yellow croaker and rainbow trout hacd1 to different oil sources or fatty acids, and also examined transcriptional regulation of this gene. In this study, hacd1 was highly expressed in the liver of large yellow croaker and rainbow trout, which is the main organ for LC-PUFA biosynthesis. Therefore, we cloned the hacd1 coding sequence, with a phylogenetic analysis showing that this gene is evolutionarily conserved. Its localization to the endoplasmic reticulum (ER), likely also indicates a conserved structure and function. The expression of hacd1 in the liver was significantly decreased after the substitution of soybean oil (SO) for fish oil but was not significantly affected after palm oil (PO) substitution. Linoleic acid (LA) incubation significantly promoted hacd1 expression in primary hepatocytes of large yellow croaker and eicosapentaenoic acid (EPA) incubation significantly promoted hacd1 expression in primary hepatocytes of rainbow trout. Transcription factors STAT4, C/EBPα, C/EBPß, HNF1, HSF3 and FOXP3 were identified in both large yellow croaker and rainbow trout. HNF1 had a stronger activation effect in rainbow trout than in large yellow croaker. FOXP3 inhibited hacd1 promoter activity in large yellow croaker but had no effect in rainbow trout. Therefore, the differences between HNF1 and FOXP3 affected the expression of hacd1 in the liver thus being responsible for the high capacity of LC-PUFA biosynthesis in rainbow trout.

Environmental adaptation in fish induced changes in the regulatory region of fatty acid elongase gene, elovl5, involved in long-chain polyunsaturated fatty acid biosynthesis.

Fish are the main source of long-chain polyunsaturated fatty acids (LC-PUFA) for human consumption. In the process of evolution via natural selection, adaptation to distinct environments has likely driven changes in the endogenous capacity for LC-PUFA biosynthesis between marine and freshwater fishes. However, the molecular mechanisms underlying adaptive changes in this metabolic pathway are poorly understood. Here, we compared the transcriptional regulation of elongation of very long chain fatty acids protein 5 (Elovl5), which is one of the critical enzymes in LC-PUFA biosynthesis pathway, in marine large yellow croaker (Larimichthys crocea) and freshwater rainbow trout (Oncorhynchus mykiss). Comparative transcriptomic and absolute mRNA quantification analyses revealed that the expression of elovl5 in rainbow trout was markedly higher than that in large yellow croaker. Correspondingly, the number of chromatin accessible areas in the regulatory region of elovl5 in rainbow trout was higher than in large yellow croaker, which revealed that chromatin accessibility in the regulatory region of elovl5 in rainbow trout was higher. Furthermore, the differences in sequence and activity of the elovl5 promoter were observed between rainbow trout and large yellow croaker, and transcription factors including CCAAT/enhancer-binding protein ß (CEBPß), GATA binding protein 3 (GATA3) and upstream stimulatory factor 2 (USF2) displayed different regulatory roles on elovl5 expression between the two species. We propose that changes in the gene regulatory region driven by natural selection likely play a key role in differences in elovl5 expression and the activity of Elovl5, which may influence the LC-PUFA biosynthesis capacities of rainbow trout and large yellow croaker. These findings may also provide opportunities to improve the quality of aquatic products and, consequently, human health.

LPS Stimulation Induces Small Heterodimer Partner Expression Through the AMPK-NRF2 Pathway in Large Yellow Croaker (Larimichthys crocea).

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-1ß, 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.

High Fat Activates O-GlcNAcylation and Affects AMPK/ACC Pathway to Regulate Lipid Metabolism.

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.

Determinants of Tourism Stocks During the COVID-19: Evidence From the Deep Learning Models.

This paper examines the determinants of tourism stock returns in China from October 25, 2018, to October 21, 2020, including the COVID-19 era. We propose four deep learning prediction models based on the Back Propagation Neural Network (BPNN): Quantum Swarm Intelligence Algorithms (QSIA), Quantum Step Fruit-Fly Optimization Algorithm (QSFOA), Quantum Particle Swarm Optimization Algorithm (QPSO) and Quantum Genetic Algorithm (QGA). Firstly, the rough dataset is used to reduce the dimension of the indices. Secondly, the number of neurons in the multilayer of BPNN is optimized by QSIA, QSFOA, QPSO, and QGA, respectively. Finally, the deep learning models are then used to establish prediction models with the best number of neurons under these three algorithms for the non-linear real stock returns. The results indicate that the QSFOA-BPNN model has the highest prediction accuracy among all models, and it is defined as the most effective feasible method. This evidence is robust to different sub-periods.

Molecular Characterization, Nutritional and Insulin Regulation of Elovl6 in Rainbow Trout (Oncorhynchus mykiss).

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.

Molecular cloning and the involvement of IRE1α-XBP1s signaling pathway in palmitic acid induced - Inflammation in primary hepatocytes from large yellow croaker (Larimichthys crocea).

Apart from mitigating endoplasmic reticulum (ER) stress, vast studies have demonstrated the crucial role of inositol-requiring transmembrane kinase and endonuclease 1α (IRE1α) - spliced X-box binding protein 1 (XBP1s) signaling pathway in inflammatory response in mammals. In addition, palmitic acid (PA)-induced inflammation has been verified in large yellow croaker (Larimichthys crocea). However, whether the IRE1α-XBP1s signaling pathway is involved in inflammatory response caused by PA remains poorly studied in fish. The present study was aimed at elucidating the role of the IRE1α-XBP1s signaling pathway in inflammatory response induced by PA in primary hepatocytes from large yellow croaker. In the present study, the full-length cDNA of ire1α and xbp1s were cloned and comprised 3793 bp and 1789 bp with an open reading frame of 3279 bp and 1170 bp, encoding 1093 and 390 amino acids, respectively. IRE1α protein possessed a protein kinase and endoribonuclease domain and XBP1s protein possessed a basic-leucine zipper domain. The IRE1α protein and XBP1s protein located to the ER membrane and nucleus respectively. The ire1α and xbp1s were widely transcribed in various tissues with the higher level in intestine, liver, adipose and head kidney. The ER stress-inducing agent tunicamycin (Tm) and PA treatment significantly activated the IRE1α-XBP1s signaling pathway and increased the pro-inflammatory genes expression including tumor necrosis factor α (tnfα), interleukin 6 (il-6) and interleukin 1ß (il-1ß) (P < 0.05). When KIRA6, the IRE1α kinase inhibitor, was used to block the IRE1α-XBP1s signaling pathway, the Tm and PA-induced pro-inflammatory genes expression was significantly suppressed (P < 0.05). These data indicated that the IRE1α-XBP1s signaling pathway was involved in the PA-induced inflammatory response in large yellow croaker.

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.