Terrestrial Compound Protein Replacing Dietary Fishmeal Improved Digestive Enzyme Activity, Immune Response, Intestinal Microflora Composition, and Protein Metabolism of Golden Pompano (Trachinotus ovatus).

Terrestrial compound protein (Cpro) can be potentially used to replace fishmeal (FM) in the marine carnivorous teleost, golden pompano (Trachinotus ovatus). Four isonitrogenous (45%) and isolipidic (12%) diets named FM30, AP80, PP80, and CP80 were formulated. FM30 (control) contained 30% FM and 25% basic protein, while AP80, PP80, and CP80 only contained 6% FM, where 80% FM and 25% basic protein of control diet were completely replaced by animal protein, plant protein, and Cpro, respectively. After golden pompano juveniles (initial weight: 10.32 ± 0.09 g) were, respectively, fed the four diets in floating sea cages for 10 weeks, the growth performance, intestinal digestive enzyme activity, and immune responses, protein metabolism indices of the CP80 group were similar to or better than those of the FM30 group (P > 0.05), and significantly better than those of the AP80 and PP80 groups. Specifically, the weight gain (WG), feed conversion ratio (FCR), activity of alanine transaminase (ALT), growth hormone (GH), and insulin-like growth factor-1 (IGF-1) contents of serum, mRNA level of interleukin-10 (il-10), zonula occludens-2 (zo-2), claudin-3, claudin-12, and eukaryotic translation initiation factor 4G (eif4g) were significantly higher, and the activity of α-amylase (AMS), lipase (LPS) in the foregut and midgut, interleukin-8 (il-8) expression in the intestine was significantly lower than that in the CP80 group, compared with those in AP80 and PP80 groups (P < 0.05). Moreover, the intestinal microflora composition of golden pompano fed with the CP80 diet was improved. Specifically, at the phylum level, the relative abundance of harmful bacterial strains cyanobacteria and TM7 of CP80 group was similar to those of FM30 group (P > 0.05), but was significantly lower than those of AP80 and PP80 groups (P < 0.05). At the genus level, the beneficial bacterial strains Agrobacterium and Blantia of CP80 group were also similar to those of FM30 group (P < 0.05), which were significantly higher than those of AP80 and PP80 groups, but the beneficial bacterial strains Bifidobacterium and Devosia of CP80 group were significantly higher than that in the other groups (P < 0.05). Besides, in diet CP80, the contents of amino acids and anti-nutritional factor, as well as the in vitro digestion rate were comparable to those of FM30, and the anti-nutritional factor content was between AP80 and PP80; total essential amino acids (EAAs) and methionine contents were higher than those in AP80, the glycine content was higher than that in PP80. Taken together, these results indicated that the CP80 diet had better amino acid composition and relatively low content of anti-nutritional factors, as well as high-digestion rate, and thus leads to the fish fed CP80 displaying improved effects in digestive enzyme activity, immune response, protein metabolism, and intestinal microbiota composition, which may be the important reasons to explain why that 80% of FM can be replaced by Cpro in the diet of golden pompano.

GPR120-ERK1-Srebp1c signaling pathway regulates long-chain polyunsaturated fatty acids biosynthesis in marine teleost Siganus canaliculatus.

The rabbitfish Siganus canaliculatus is the first marine teleost reported to possess long-chain polyunsaturated fatty acids (LC-PUFA) biosynthetic ability; its regulatory mechanisms have been investigated at the transcriptional and posttranscriptional levels, but little is known about its regulation at the cellular signaling level. The present study investigated the regulatory role of the G-protein-coupled receptor 120 (GPR120) signaling pathway in LC-PUFA biosynthesis in rabbitfish. S. canaliculatus hepatocyte line (SCHL) cells treated with GRP120 agonists (TUG891 and GW9508) showed significantly lower docosahexaenoic acid (DHA) content and mRNA levels of the key genes involved in LC-PUFA biosynthesis, encoding Δ6/Δ5 Fads2, Elovl5, and transcriptional factor Srebp1c. Transcriptome analysis of the treated SCHL cells showed significantly lower mRNA levels of genes encoding extracellular signal-regulated kinase 1 (ERK1), AMP-activated protein kinase (AMPKα2), target of rapamycin (TORC2) and Srebp1c, suggesting that these proteins are potentially involved in the GRP120 signaling pathway. Moreover, treatment of SCHL cells with signaling chemicals of ERK1, AMPKα2, TORC2, and Srebp1c confirmed the involvement of the ERK1-Srebp1c signaling pathway in the regulation of LC-PUFA biosynthesis. The mRNA levels of Srebp1c, Δ6/Δ5 fads2 and elovl5 were significantly lower in cells treated with PUFAs (linoleic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, DHA) but higher in those treated with ERK1 inhibitors (U0126 and CI-1040). CI-1040-treated cells showed significantly higher DHA content, but the other treatment groups (except PD98059) showed significantly lower DHA content. These results indicate that the GPR120-ERK1-Srebp1c signaling pathway regulates rabbitfish LC-PUFA biosynthesis, representing a novel regulatory mechanism in vertebrates.

Insulin can up-regulate LC-PUFA biosynthesis with the involvement of Srebp-1c and stimulatory protein 1 (Sp1) in marine teleost Siganus canaliculatus.

The rabbitfish Siganus canaliculatus is the first marine teleost found to have the biosynthetic ability of long-chain polyunsaturated fatty acids (LC-PUFA) from C18 precursors catalyzed by fatty acyl desaturases (Δ6/Δ5 Fads, Δ4 Fads) and elongases of very long chain fatty acids (Elovls). Previously, we predicted the existence of insulin (INS) response elements (IREs) including nuclear factor Y (NF-Y) and sterol regulatory element (SRE) in the core promoter region of rabbitfish Δ6/Δ5 fads and Δ4 fads. To clarify the potential regulatory effect and mechanism of INS in LC-PUFA biosynthesis, INS responding region was identified at -456 bp to + 51 bp of Δ6/Δ5 fads core promoter, but not in Δ4 fads promoter. Moreover, a unique stimulatory protein 1 (Sp1) element was predicted in the INS responding region of Δ6/Δ5 fads. Subsequently, SRE, NF-Y and Sp1 elements were proved as IREs in Δ6/Δ5 fads promoter. The up-regulation of INS on gene expression of Srebp-1c, Sp1, Δ6/Δ5 fads and elovl5 as well as the LC-PUFA biosynthesis was further demonstrated in S. canaliculatus hepatocyte line (SCHL) cells, but no influence was detected on Δ4 fads. Besides, inhibitors of transcription factors Srebp-1c (Fatostatin, PF-429242) and Sp1 (Mithramycin) could inhibit the gene expression of Srebp-1c, Δ6/Δ5 fads and elovl5, and abolish the up-regulation of INS on these genes' expression and LC-PUFA biosynthesis. These results indicated that INS could up-regulate LC-PUFA biosynthesis with the involvement of Srebp-1c and Sp1 in rabbitfish S. canaliculatus, which is the first report in teleost.

Insulin activates LC-PUFA biosynthesis of hepatocytes by regulating the PI3K/Akt/mTOR/Srebp1 pathway in teleost Siganus canaliculatus.

Insulin is well known an important metabolic regulator in glucose and lipid metabolism. It has been proved to activate long-chain (≥ C20) polyunsaturated fatty acids (LC-PUFA) biosynthesis in mammals, but little is known about such a role in fish. To explore the effects and molecular mechanisms of insulin in fish LC-PUFA biosynthesis, we treated the rabbitfish S. canaliculatus hepatocyte line (SCHL) cells with 65 nM insulin for 12 h, and the results showed that the mRNA levels of genes encoding the key enzymes and transcription factor involved in rabbitfish LC-PUFA biosynthesis such as Δ6Δ5 fads2, elovl5 and srebp1, as well as those of PI3K pathway genes including pdk1, akt2 and mtor increased significantly. Moreover, SCHL cells treated with different PI3K/Akt pathway inhibitors (LY294002, Wortmannin, AKTi-1/2) alone or combined with insulin decreased the mRNA levels of PI3K/Akt/mTOR downstream signaling genes, including Δ6Δ5 fads2, Δ4 fads2, elovl5, elovl4 and srebp1. While PI3K/Akt agonists (740 Y-P, IGF-1, SC-79) had the opposite results. The results of fatty acid composition analysis of hepatocytes showed that insulin stimulation increased the Δ6Δ5 Fads2-dependent PUFA desaturation indexes, while Elovl5-dependent PUFA elongation indexes had upward trends, and consequently LC-PUFA contents increased. Taken together, these results indicated that insulin activated LC-PUFA biosynthesis probably through PI3K/Akt/mTOR/Srebp1 pathway in S. canaliculatus hepatocytes.

Growth and Fatty Acid Composition of Black Soldier Fly Hermetia illucens (Diptera: Stratiomyidae) Larvae Are Influenced by Dietary Fat Sources and Levels.

A 16-day rearing trial was performed to investigate the influence of two supplemental levels (5% and 10%) of six dietary fat sources (linseed oil, peanut oil, coconut oil, soybean oil, lard oil and fish oil) on the growth, development and nutrient composition of black solider fly larvae. Our results demonstrated that the pre-pupa rate of larvae was linearly influenced by dietary C18:0, C18:3n-3 and C18:2n-6 content (pre-pupa rate = 0.927 × C18:0 content + 0.301 × C18:3n-3 content-0.258 × C18:2n-6 content p < 0.001)), while final body weight was linearly influenced by that of C16:0 (final body weight = 0.758 × C16:0 content, p = 0.004). Larval nutrient composition was significantly affected by dietary fat sources and levels, with crude protein, fat and ash content of larvae varying between 52.0 and 57.5, 15.0 and 23.8, and 5.6 and 7.2% dry matter. A higher level of C12:0 (17.4-28.5%), C14:0 (3.9-8.0%) and C16:1n-9 (1.3-4.3%) was determined in larvae fed the diets containing little of them. In comparison, C16:0, C18:1n-9, C18:2n-6 and C18:3n-3 proportions in larvae were linearly related with those in diets, with the slope of the linear equations varying from 0.39 to 0.60. It can be concluded that sufficient C16:0, C18:0 and C18:3n-3 supply is beneficial for larvae growth. Larvae could produce and retain C12:0, C14:0, and C16:1n-9 in vivo, but C16:0, C18:1n-9, C18:2n-6 and C18:3n-3 could only be partly incorporated from diets and the process may be enhanced by a higher amount of dietary fat. Based on the above observation, an accurately calculated amount of black soldier fly larvae could be formulated into aquafeed as the main source of saturated fatty acids and partial source of mono-unsaturated and poly-unsaturated fatty acids to save fish oil.

Regulation of long-chain polyunsaturated fatty acid biosynthesis in teleost fish.

Omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFA, C20-24), including eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), are involved in numerous biological processes and have a range of health benefits. Fish have long been considered as the main source of n-3 LC-PUFA in human diets. However, the capacity for endogenous biosynthesis of LC-PUFA from C18 PUFA varies in fish species based on the presence, expression and activity of key enzymes including fatty acyl desaturases (Fads) and elongation of very long-chain fatty acids (Elovl) proteins. In this article, we review progress on the identified Fads and Elovl, as well as the regulatory mechanisms of LC-PUFA biosynthesis both at transcriptional and post-transcriptional levels in teleosts. The most comprehensive advances have been obtained in rabbitfish Siganus canaliculatus, a marine teleost demonstrated to have the entire pathway for LC-PUFA biosynthesis, including the roles of transcription factors hepatocyte nuclear factor 4α (Hnf4α), liver X receptor alpha (Lxrα), sterol regulatory element-binding protein 1 (Srebp-1), peroxisome proliferator-activated receptor gamma (Pparγ) and stimulatory protein 1 (Sp1), as well as post-transcriptional regulation by individual microRNA (miRNA) or clusters. This research has, for the first time, demonstrated the involvement of Hnf4α, Pparγ and miRNA in the regulation of LC-PUFA biosynthesis in vertebrates. The present review provides readers with a relatively comprehensive overview of the progress made into understanding LC-PUFA biosynthetic systems in teleosts, and some insights into improving endogenous LC-PUFA biosynthesis capacity aimed at reducing the dependence of aquafeeds on fish oil while maintaining or increasing flesh LC-PUFA content and the nutritional quality of farmed fish.

Molecular Cloning, Characterization, and Expression Regulation of Acyl-CoA Synthetase 6 Gene and Promoter in Common Carp Cyprinus carpio.

Omega-3 long chain polyunsaturated fatty acids (n-3 LC-PUFA), particularly docosahexaenoic acids (22:6n-3, DHA), have positive effects on multiple biologic and pathologic processes. Fish are the major dietary source of n-3 LC-PUFA for humans. Growing evidence supports acyl-coenzyme A (acyl-CoA) synthetase 6 (acsl6) being involved in cellular DHA uptake and lipogenesis in mammals, while its molecular function and regulatory mechanism remain unknown in fish. The present study focused on investigating the molecular characterization and transcription regulation of the acsl6 gene in the freshwater teleost common carp (Cyprinus carpio). First, the full length of acsl6 cDNA contained a coding region of 2148 bp for 715 amino acids, which possessed all characteristic features of the acyl-CoA synthetase (ACSL) family. Its mRNA expression was the highest in the brain, followed by in the heart, liver, kidney, muscle, and eyes, but little expression was detected in the ovary and gills. Additionally, a candidate acsl6 promoter region of 2058 bp was cloned, and the sequence from -758 bp to -198 bp was determined as core a promoter by equal progressive deletion and electrophoretic mobility shift assay. The binding sites for important transcription factors (TFs), including stimulatory protein 1 (SP1), CCAAT enhancer-binding protein (C/EBPα), sterol-regulatory element binding protein 1c (SREBP1c), peroxisome proliferator activated receptor α (PPARα), and PPARγ were identified in the core promoter by site-directed mutation and functional assays. Furthermore, the intraperitoneal injection of PPARγ agonists (balaglitazone) increased the expression of acsl6 mRNA, coupling with an increased proportion of DHA in the muscle, while opposite results were obtained in the injection of the SREBP1c antagonist (betulin). However, the expression of acsl6 and DHA content in muscle were largely unchanged by PPARα agonist (fenofibrate) treatment. These results indicated that acsl6 may play an important role for the muscular DHA uptake and deposition in common carp, and PPARγ and SREBP-1c are the potential TFs involved in the transcriptional regulation of acsl6 gene. To our knowledge, this is the first report of the characterization of acsl6 gene and its promoter in teleosts.

Hepatocyte nuclear factor 4α (Hnf4α) is involved in transcriptional regulation of Δ6/Δ5 fatty acyl desaturase (Fad) gene expression in marine teleost Siganus canaliculatus.

As the first marine teleost demonstrated to biosynthesize long-chain polyunsaturated fatty acids (LC-PUFAs) from C18 precursors such as linoleic acid (LOA, 18:2n-6) and α-linolenic acid (ALA, 18:3n-3), the rabbitfish (Siganus canaliculatus) contains the complete enzymatic system for LC-PUFA biosynthesis, including Δ6/Δ5 fatty acid desaturase (Fad), Δ4 Fad, and elongase 5 (Elovl5). Previously, our group demonstrated that hepatocyte nuclear factor 4α (Hnf4α) is a transcription factor (TF) for rabbitfish Δ4 fad and elovl5, and interacts with the core promoter of Δ6/Δ5 fad. To fully clarify the role of Hnf4α in the regulation of LC-PUFA biosynthesis, the present study aimed to explore the regulatory role of Hnf4α on Δ6/Δ5 fad gene expression. First, Hnf4α overexpression and agonist assays identified the Hnf4α response region in the Δ6/Δ5 fad core promoter as -456 bp to +51 bp. Bioinformatic analysis predicted four potential Hnf4α binding elements in the core promoter, which were confirmed by site-directed mutation and functional assays in a dual luciferase assay system. Moreover, the mRNA expression levels of hnf4α, Δ6/Δ5 fad, and Δ4 fad were significantly increased in the S. canaliculatus hepatocyte line (SCHL) cells after treatment with Hnf4α agonists (Alverine and Benfluorex) or its mRNA overexpression. By contrast, the expression levels of these three genes were markedly decreased after hnf4a small interfering RNA (siRNA) transfection. The results indicated that Hnf4α has a regulatory effect on rabbitfish Δ6/Δ5 fad gene transcription, identifying Hnf4α as a TF of Δ6/Δ5 fad in vertebrates for the first time.

Sp1 is Involved in Vertebrate LC-PUFA Biosynthesis by Upregulating the Expression of Liver Desaturase and Elongase Genes.

The rabbitfish Siganus canaliculatus was the first marine teleost demonstrated to have the ability for the biosynthesis of long-chain (≥C20) polyunsaturated fatty acids (LC-PUFA) from C18 PUFA precursors, and all the catalytic enzymes including two fatty acyl desaturase 2 (Δ4 Fads2 and Δ6/Δ5 Fads2) and two elongases (Elovl4 and Elovl5) have been identified, providing a good model for studying the regulatory mechanisms of LC-PUFA biosynthesis in fish. Stimulatory protein 1 (Sp1) has been speculated to be a vital transcription factor in determining the promoter activity of Fads-like genes in fish, however its regulatory effects on gene expression and LC-PUFA biosynthesis have not been demonstrated. Bioinformatic analysis predicted potential Sp1 binding sites in the promoters of the rabbitfish Δ6/Δ5 fads2 and elovl5, but not in Δ4 fads2 promoter. Here we cloned full-length cDNA of the rabbitfish sp1 gene, which encoded a putative protein of 701 amino acids, and was expressed in all tissues studied with highest levels in gill and eyes. The dual luciferase reporter assay in HepG2 line cells demonstrated the importance of the Sp1 binding site for the promoter activities of both Δ6/Δ5 fads2 and elovl5. Moreover, the electrophoretic mobility shift assay confirmed the direct interaction of Sp1 with the two promoters. Insertion of the Sp1 binding site of Δ6/Δ5 fads2 promoter into the corresponding region of the Δ4 fads2 promoter significantly increased activity of the latter. In the Siganus canaliculatus hepatocyte line (SCHL) cells, mRNA levels of Δ6/Δ5 fads2 and elovl5 were positively correlated with the expression of sp1 when sp1 was overexpressed or knocked-down by RNAi or antagonist (mithramycin) treatment. Moreover, overexpression of sp1 also led to a higher conversion of 18:2n-6 to 18:3n-6, 18:2n-6 to 20:2n-6, and 18:3n-3 to 20:3n-3, which related to the functions of Δ6/Δ5 Fads2 and Elovl5, respectively. These results indicated that Sp1 is involved in the transcriptional regulation of LC-PUFA biosynthesis by directly targeting Δ6/Δ5 fads2 and elovl5 in rabbitfish, which is the first report of Sp1 involvement in the regulation of LC-PUFA biosynthesis in vertebrates.

Pparγ Is Involved in the Transcriptional Regulation of Liver LC-PUFA Biosynthesis by Targeting the Δ6Δ5 Fatty Acyl Desaturase Gene in the Marine Teleost Siganus canaliculatus.

As the first marine teleost demonstrated to have the ability of long-chain polyunsaturated fatty acids (LC-PUFA) biosynthesis from C18 PUFA precursors, the rabbitfish Siganus canaliculatus provides us a unique model for clarifying the regulatory mechanisms of LC-PUFA biosynthesis in teleosts aiming at the replacement of dietary fish oil (rich in LC-PUFA) with vegetable oils (rich in C18 PUFA precursors but devoid of LC-PUFA). In the study of transcription regulation of gene encoding the Δ6Δ5 fatty acyl desaturase (Δ6Δ5 Fads), a rate-limiting enzyme catalyzing the first step of LC-PUFA biosynthesis in rabbitfish, a binding site for the transcription factor (TF), peroxisome proliferator-activated receptor γ (Pparγ), was predicted in Δ6Δ5 fads2 promoter by bioinformatics analysis, and thus the present study focused on the regulatory roles of Pparγ on Δ6Δ5 fads2. First, the activity of the Δ6Δ5 fads2 promoter was proved to be downregulated by pparγ overexpression and upregulated by treatment of Pparγ antagonist (GW9662), respectively, in HEK 293T cells with the dual luciferase reporter assay. Pparγ was further confirmed to interact with the promoter by electrophoretic mobility shift assay. Moreover, in S. canaliculatus hepatocyte line (SCHL) cells, GW9662 decreased the expression of pparγ together with increase of Δ6Δ5 fads2 mRNA. Besides, Δ6Δ5 fads2 expression was increased by pparγ RNAi knockdown and reduced by its mRNA overexpression. Furthermore, knockdown of pparγ induced a high conversion of 18:3n-3 to 18:4n-3 and 18:2n-6 to 18:3n-6, while pparγ mRNA overexpression led to a lower conversion of that, and finally a significant decrease of 20:4n-6(ARA), 20:5n-3(EPA), and 22:6n-3(DHA) production. The results indicate that Pparγ is involved in the transcriptional regulation of liver LC-PUFA biosynthesis by targeting Δ6Δ5 fads2 in rabbitfish, which is the first report of Pparγ involvement in the regulation of LC-PUFA biosynthesis in teleosts.

Hnf4α Is Involved in LC-PUFA Biosynthesis by Up-Regulating Gene Transcription of Elongase in Marine Teleost Siganus canaliculatus.

The rabbitfish Siganus canaliculatus is the first marine teleost shown to be able to biosynthesize long-chain polyunsaturated fatty acids (LC-PUFA) from C18 PUFA precursors catalyzed by two fatty acyl desaturases (fad) including Δ4 Fad and Δ6/Δ5 Fad as well as two elongases (Elovl4 and Elovl5). Previously, hepatocyte nuclear factor 4α (Hnf4α) was demonstrated to be predominant in the transcriptional regulation of two fads. To clarify the regulatory mechanisms involved in rabbitfish lipogenesis, the present study focused on the regulatory role of Hnf4α to elovl5 expression and LC-PUFA biosynthesis. Bioinformatics analysis predicted two potential Hnf4α elements in elovl5 promoter, one binding site was confirmed to interact with Hnf4α by gel shift assays. Moreover, overexpression of hnf4α caused a remarkable increase both in elovl5 promoter activity and mRNA contents, while knock-down of hnf4α in S. canaliculatus hepatocyte line (SCHL) resulted in a significant decrease of elovl5 gene expression. Meanwhile, hnf4α overexpression enhanced LC-PUFA biosynthesis in SCHL cell, and intraperitoneal injection to rabbitfish juveniles with Hnf4α agonists (Alverine and Benfluorex) increased the expression of hnf4α, elvol5 and Δ4 fad, coupled with an increased proportion of total LC-PUFA in liver. The results demonstrated that Hnf4α is involved in LC-PUFA biosynthesis by up-regulating the transcription of the elovl5 gene in rabbitfish, which is the first report of Hnf4α as a transcription factor of the elovl5 gene in vertebrates.

Cloning and characterization of ∆6/∆5 fatty acyl desaturase (Fad) gene promoter in the marine teleost Siganus canaliculatus.

The rabbitfish Siganus canaliculatus was the first marine teleost demonstrated to have the ability of biosynthesizing long-chain polyunsaturated fatty acids (LC-PUFA) from C18 PUFA precursors, and all genes encoding the key enzymes for LC-PUFA biosynthesis have been cloned and functionally characterized, which provides us a potential model to study the regulatory mechanisms of LC-PUFA biosynthesis in teleosts. As the primary step to clarify such mechanisms, present research focused on promoter analysis of gene encoding ∆6/∆5 fatty acyl desaturase (Fad), a rate-limiting enzyme catalyzing the first step in the conversion of C18 PUFA to LC-PUFA. First, 2044 bp promoter sequence was cloned by genome walking, and the sequence from -456 bp to +51 bp was determined as core promoter by progressive deletion mutation. Moreover, binding sites of transcription factors (TF) such as CCAAT enhancer binding protein (C/EBP), nuclear factor 1 (NF-1), stimulatory protein 1 (Sp1), nuclear factor Y (NF-Y), activated protein 1 (AP1), sterol regulatory element (SRE), hepatocyte nuclear factor 4α (HNF4α) and peroxisome proliferator activated receptor γ (PPARγ) were identified in the core promoter by site-directed mutation and functional assays. Moreover, NF-1 and HNF4α were confirmed to interact with the core promoter region by gel shift assay and mass spectrometry. This is the first report of the promoter structure of a ∆6/∆5 Fad in a marine teleost, and a novel discovery of NF-1 and HNF4α binding to the ∆6/∆5 Fad promoter.

Hnf4α is involved in the regulation of vertebrate LC-PUFA biosynthesis: insights into the regulatory role of Hnf4α on expression of liver fatty acyl desaturases in the marine teleost Siganus canaliculatus.

Long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis is an important metabolic pathway in vertebrates, especially fish, considering they are the major source of n-3 LC-PUFA in the human diet. However, most fish have only limited capability for biosynthesis of LC-PUFA. The rabbitfish (Siganus canaliculatus) is able to synthesize LC-PUFA as it has all the key enzyme activities required including Δ6Δ5 Fads2, Δ4 Fads2, Elovl5, and Elovl4. We previously reported a direct interaction between the transcription factor Hnf4α and the promoter regions of Δ4 and Δ6Δ5 Fads2, which suggested that Hnf4α was involved in the transcriptional regulation of fads2 in rabbitfish. For functionally investigating it further, a full-length cDNA of 1736-bp-encoding rabbitfish Hnf4α with 454 amino acids was cloned, which was highly expressed in intestine, followed by liver and eyes. Similar to the expression characteristics of its target genes Δ4 and Δ6Δ5 fads2, levels of hnf4α mRNA in liver and eyes were higher in fish reared at low salinity than those reared in high salinity. After the rabbitfish primary hepatocytes were, respectively, incubated with alverine, benfluorex or BI6015, which were anticipated agonists or antagonist for Hnf4α, the mRNA level of Δ6Δ5 and Δ4 fads2 displayed a similar change tendency with that of hnf4α mRNA. Furthermore, when the mRNA level of hhf4α was knocked down using siRNA, the expression of Δ6Δ5 and Δ4 fads2 also decreased. Together, these data suggest that Hnf4α is involved in the transcriptional regulation of LC-PUFA biosynthesis, specifically, by targeting Δ4 and Δ6Δ5 fads2 in rabbitfish.

Cloning and expression characterization of peroxisome proliferator-activated receptors (PPARs) with their agonists, dietary lipids, and ambient salinity in rabbitfish Siganus canaliculatus.

Rabbitfish Siganus canaliculatus is the first marine teleost reported to have the ability of biosynthesizing C20-22 long-chain polyunsaturated fatty acids (LC-PUFA) from C18 precursors, and thus provides a model for studying the regulatory mechanisms of LC-PUFA biosynthesis in teleosts. To investigate the possible roles of peroxisome proliferator-activated receptors (PPARs), critical transcription factors involved in the regulation of lipid metabolism, in the regulation of LC-PUFA biosynthesis in rabbitfish, the PPAR genes were cloned and their expression characterization with PPAR agonists, dietary lipid resource, and ambient salinity were examined. Three cDNA sequences respectively encoding 477, 516 and 519 amino acids of PPARα, PPARß, and PPARγ isoforms were obtained. PPARα exhibited a wide tissue expression with its highest levels in the heart and brain; PPARß was predominantly expressed in the gills, while PPARγ was highly expressed in the intestine and gills. In rabbitfish primary hepatocytes, both the PPAR agonists 2-bromopalmitate (2-Bro) and fenofibrate (FF) increased the expression of PPARγ, SREBP1c and Elovl5, whereas FF depressed the expression of Δ6/Δ5 Fad. Moreover, a higher hepatic PPARß expression was observed in fish fed diets with vegetable oils (VO) than that with fish oil (FO), in the former the expression of PPARα, PPARß, and PPARγ were increased at the low ambient salinity (10ppt), where an increasing expression of Δ5/Δ6 Fad, Δ4 Fad and Elovl5 genes was previously reported. These results suggest that PPARs might be involved in the upregulation of LC-PUFA biosynthesis with dietary VO and low ambient salinity in rabbitfish.

The miR-33 gene is identified in a marine teleost: a potential role in regulation of LC-PUFA biosynthesis in Siganus canaliculatus.

As the first marine teleost demonstrated to have the ability to biosynthesize long-chain polyunsaturated fatty acids (LC-PUFA) from C18 PUFA precursors, rabbitfish Siganus canaliculatus provides a good model for studying the regulatory mechanisms of LC-PUFA biosynthesis in teleosts. Here the potential roles of miR-33 in such regulation were investigated. The miR-33 gene was identified within intron 16 of the gene encoding sterol regulatory element-binding protein 1 (Srebp1), an activator of LC-PUFA biosynthesis. Expression of miR-33 in rabbitfish tissues correlated with that of srebp1, while its expression in liver was highly responsive to ambient salinities and PUFA components, factors affecting LC-PUFA biosynthesis. Srebp1 activation promoted the expression of Δ4 and Δ6 Δ5 fatty acyl desaturases (Fad), key enzymes for LC-PUFA biosynthesis, accompanied by elevated miR-33 abundance in rabbitfish hepatocytes. miR-33 overexpression induced the expression of the two fad, but suppressed that of insulin-induced gene 1 (insig1), which encodes a repressor blocking Srebp proteolytic activation and has targeting sites of miR-33. These results indicated that miR-33, cooperating with Srebp1, may be involved in regulation of LC-PUFA biosynthesis by facilitating fad expression, probably through targeting insig1. To our knowledge, this is the first report of the participation of miR-33 in LC-PUFA biosynthesis in vertebrates.

Hepatocyte Nuclear Factor 4α (HNF4α) Is a Transcription Factor of Vertebrate Fatty Acyl Desaturase Gene as Identified in Marine Teleost Siganus canaliculatus.

Rabbitfish Siganus canaliculatus was the first marine teleost demonstrated to have the capability of biosynthesizing long-chain polyunsaturated fatty acids (LC-PUFA) from C18 precursors, and to possess a Δ4 fatty acyl desaturase (Δ4 Fad) which was the first report in vertebrates, and is a good model for studying the regulatory mechanisms of LC-PUFA biosynthesis in teleosts. In order to understand regulatory mechanisms of transcription of Δ4 Fad, the gene promoter was cloned and characterized in the present study. An upstream sequence of 1859 bp from the initiation codon ATG was cloned as the promoter candidate. On the basis of bioinformatic analysis, several binding sites of transcription factors (TF) including GATA binding protein 2 (GATA-2), CCAAT enhancer binding protein (C/EBP), nuclear factor 1 (NF-1), nuclear factor Y (NF-Y), hepatocyte nuclear factor 4α (HNF4α) and sterol regulatory element (SRE), were identified in the promoter by site-directed mutation and functional assays. HNF4α and NF-1 were confirmed to interact with the core promoter of Δ4 Fad by gel shift assay and mass spectrometry. Moreover, over-expression of HNF4α increased promoter activity in HEK 293T cells and mRNA level of Δ4 Fad in rabbitfish primary hepatocytes, respectively. The results indicated that HNF4α is a TF of rabbitfish Δ4 Fad. To our knowledge, this is the first report on promoter structure of a Δ4 Fad, and also the first demonstration of HNF4α as a TF of vertebrate Fad gene involved in transcription regulation of LC-PUFA biosynthesis.