Elongation of long-chain fatty acids in rabbitfish Siganus canaliculatus: Cloning, functional characterisation and tissue distribution of Elovl5- and Elovl4-like elongases

Elongases of very long-chain fatty acids (Elovl) catalyse the rate-limiting step of the elongation pathway that results in net 2 C elongation of pre-existing fatty acyl chains. As the biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFA) is particularly relevant in fish, Elovl involved in the pathway have been investigated in various studies. Here we report the molecular cloning, functional characterisation and tissue distribution of two distinct elovl-like cDNAs isolated from the herbivorous marine teleost Siganus canaliculatus. Unlike the carnivorous marine fish previously investigated, we hypothesise that the rabbitfish has an enhanced LC-PUFA biosynthetic capability as previously anticipated in a former study on fatty acyl desaturases (Fad). The results of the present study showed that rabbitfish expresses at least two elovl cDNAs, which have high homology in sequence and function to Elovl5 and Elovl4 elongases that have been investigated previously in other fish species. Furthermore, the results confirm that the activities of the Elovl5 and Elovl4 enzymes enable rabbitfish to perform all the elongation reactions required for the biosynthesis of the physiologically essential C20-22 LC-PUFA including eicosapentaenoic (20:5n-3), arachidonic (20:4n-6) and docosahexaenoic (22:6n-3, DHA) acids, as well as the less common very long-chain fatty acids (greater than C24). Rabbitfish is thus the first marine teleost in which genes encoding Fad and Elovl enzymes, with all the activities required for the production of DHA from C18 PUFA, have been characterised. Highlights Rabbitfish possess at least two fatty acid elongases. The elongases were functionally characterised as Elovl5 and Elovl4 types.  The elongases possess all the activities necessary for the biosynthesis of DHA.  Elovl4 is capable of synthesising very long-chain fatty acids up to C36 in length.  Rabbitfish represent a marine fish not dependent upon dietary EPA and DH

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

Characteristics of the fads2 gene promoter in marine teleost Epinephelus coioides and role of Sp1-binding site in determining promoter activity

Δ6 fatty acyl desaturase (Fads2) is a rate-limiting enzyme in long-chain polyunsaturated fatty acids (LC-PUFA) biosynthesis. Comparative analysis of gene promoters of Fads2 between salmonids and carnivorous marine sh suggested that the lack of binding site for stimulatory protein 1 (Sp1) was responsible for the low expression of fads2 gene of carnivorous marine species. To con rm this speculation, the fads2 candidate promoter (2646 bp) was cloned from carnivorous marine teleost Epinephelus coioides, and 330 bp core regulatory region was identi ed. Several binding sites for transcriptional factors such as nuclear factor 1, nuclear factorY, sterol regulatory element and hepatocyte nuclear factor 4γ were identi ed, while that for Sp1 was shown to be absent in the promoter by both bioinformatic analysis and site-directed mutation. Moreover, after the Sp1-binding site from the fads2 promoter of herbivorous Siganus canaliculatus, the rst marine teleost demonstrated to have LC-PUFA biosynthetic ability, was inserted into the corresponding region of E. coioides fads2 promoter, activity was signi cantly increased. The results provided direct data for the importance of the Sp1- binding site in determining fads2 promoter activity, and indicated that its lack may be a reason for low expression of fads2 and poor LC-PUFA biosynthetic ability in E. coioides

Investigating long-chain polyunsaturated fatty acid biosynthesis in teleost fish: Functional characterization of fatty acyl desaturase (Fads2) and Elovl5 elongase in the catadromous species, Japanese eel Anguilla japonica

The capacity for endogenous production of LC-PUFA from PUFA in euryhaline or diadromous fish is largely unknown other than for Atlantic salmon (Salmo salar), an anadromous species, which displays a freshwater pattern. The aim of the present study was to characterize the enzymes of the LC-PUFA pathway in Japanese eel (Anguilla japonica), the most important catadromous species currently being farmed. cDNAs of two key genes were cloned and functional assays showed they encoded a desaturase (Fads2) with D6 and D8 activity and an elongase (Elovl5) with activity towards C18 and C20 PUFA, with activities similar to marine fish and an D6/D8 activity ratio similar to Atlantic salmon. Furthermore, tissue distribution of the mRNA showed a clear marine pattern with highest expression in brain and eye. Phylogenetic analysis placed the eel cDNAs in line with classical taxonomy. The data suggest that diadromous species display a pattern of LC-PUFA biosynthesis capacity that likely reflects the environmental and nutritional influence of their early life stages rather than those of adult fish. Future studies aim to establish the full range of PUFA desaturases and elongases in Japanese eel and to provide further insight to the importance and relevance of LC-PUFA biosynthesis in fish species and the influence of diadromy

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 C18PUFA precursors, rabbitfishSiganus canaliculatusprovides 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 ofsrebp1, 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 twofad, 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 facilitatingfadexpression, probably through targetinginsig1. To our knowledge, this is the first report of the participation of miR-33 in LC-PUFA biosynthesis in vertebrates

Total Replacement of Dietary Fish Oil with a Blend of Vegetable Oils in the Marine Herbivorous Teleost, Siganus canaliculatus

To investigate the feasibility of total replacement of dietary fish oil with vegetable oils (VO) and the optimal dietary polyunsaturated fatty acid (PUFA) level in the marine herbivorous teleost Siganus canaliculatus, six isonitrogenous (32 %) and isolipidic (8 %) diets were formulated. Control diet (FO) used fish oil as lipid source, whereas diets VO1-VO5 contained various blends of palm, soybean, rapeseed and linseed oils, in which the dietary PUFA levels were 42.0 %, 38.2 %, 33.8 %, 29.9 % and 27.1 %, respectively. After S. canaliculatus juveniles were fed with the diets for 9 weeks, their growth performance exhibited no significant difference among the dietary groups. The tissue fatty acid profiles in liver and fillet generally reflected the dietary fatty acid compositions, and showed no significant difference among the VO dietary groups. The results suggested that dietary fish oil can be replaced completely by VO without affecting their growth performance. Concerning the effects of the dietary FA profile on the survival rate, HSI and VSI, and PUFA composition in fillets, diets VO1 and VO2 were more favorable compared with diets VO3–VO5. Considering the availability and cost of the VOs, diet VO2 was recommended for practical use in S. canaliculatus

Long-chain polyunsaturated fatty acid biosynthesis in the euryhaline herbivorous teleost Scatophagus argus: Functional characterization, tissue expression and nutritional regulation of two fatty acyl elongases

Both the spotted scat Scatophagus argus and rabbitfish Siganus canaliculatus belong to the few cultured herbivorous marine teleost, however, their fatty acyl desaturase (Fad) system involved in long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis is different. The S. argus has a △6 Fad, while the rabbitfish has △4 and △6/△5 Fads, which were the first report in vertebrate and marine teleost, respectively. In order to compare the characteristics of elongases of very long-chain fatty acids (Elovl) between them, two Elovl cDNAs were cloned from S. argus in the present study. One has 885bp of open read fragment (ORF) encoding a protein with 294 amino acid (aa) showing Elovl5 activity functionally characterized by heterologous expression in yeast, which was primarily active for the elongation of C18 and C20 PUFAs. The other has 915bp of ORF coding for a 305 aa protein showing Elovl4 activity, which was more efficient in the elongation of C20 and C22 PUFAs. Tissue distribution analyses by RT-PCR showed that elovl5 was highly expressed in the liver compared to other tissues determined, whereas elovl4 transcripts were only detected in the eye. The expression of elovl5 and elovl4 were significantly affected by dietary fatty acid composition, with highest expression of mRNA in the liver and eye of fish fed a diet with an 18:3n−3/18:2n−6 ratio of 1.7:1. These results indicated that the S. argus has a similar Elovl system in the LC-PUFA biosynthetic pathway to that of rabbitfish although their Fad system was different, suggesting that the diversification of fish LC-PUFA biosynthesis specificities is more associated with its Fad system. These new insights expand our knowledge and understanding of the molecular basis and regulation of LC-PUFA biosynthesis in fish

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 further functionally investigating it, 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 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 + 51bp 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

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