👤 Douglas R Tocher

Provision of long-chain polyunsaturated fatty acids (LC-PUFA) in vertebrates occurs through the diet or via endogenous production from C We demonstrate that functional Fads1Δ5 and Fads2∆6 arose from a tandem gene duplication in the ancestor of vertebrates, since they are present in the Arctic lamprey. Additionally, we show that a similar condition was retained in ray-finned fish such as the Senegal bichir and spotted gar, with the identification of fads1 genes in these lineages. Functional characterisation of the isolated desaturases reveals the first case of a Fads1 enzyme with ∆5 desaturase activity in the Teleostei lineage, the Elopomorpha. In contrast, in Osteoglossomorpha genomes, while no fads1 was identified, two separate fads2 duplicates with ∆6 and ∆5 desaturase activities respectively were uncovered. We conclude that, while the essential genetic components involved LC-PUFA biosynthesis evolved in the vertebrate ancestor, the full completion of the LC-PUFA biosynthesis pathway arose uniquely in gnathostomes. Show less

Rainbow trout, Oncorhynchus mykiss, are intensively cultured globally. Understanding their requirement for long-chain polyunsaturated fatty acids (LC-PUFA) and the biochemistry of the enzymes and biosynthetic pathways required for fatty acid synthesis is important and highly relevant in current aquaculture. Most gnathostome vertebrates have two fatty acid desaturase (fads) genes with known functions in LC-PUFA biosynthesis and termed fads1 and fads2. However, teleost fish have exclusively fads2 genes. In rainbow trout, a fads2 cDNA had been previously cloned and found to encode an enzyme with Δ6 desaturase activity. In the present study, a second fads2 cDNA was cloned from the liver of rainbow trout and termed fads2b. The full-length mRNA contained 1578 nucleotides with an open reading frame of 1365 nucleotides that encoded a 454 amino acid protein with a predicted molecular weight of 52.48 kDa. The predicted Fads2b protein had the characteristic traits of the microsomal Fads family, including an N-terminal cytochrome b5 domain containing the heme-binding motif (HPPG), histidine boxes (HDXGH, HFQHH and QIEHH) and three transmembrane regions. The fads2b was expressed predominantly in the brain, liver, intestine and pyloric caeca. Expression of the fasd2b in yeast generated a protein that was found to specifically convert eicosatetraenoic acid (20:4n-3) to eicosapentaenoic acid (20:5n-3), and therefore functioned as a Δ5 desaturase. Therefore, rainbow trout have two fads2 genes that encode proteins with Δ5 and Δ6 desaturase activities, respectively, which enable this species to perform all the desaturation steps required for the biosynthesis of LC-PUFA from C18 precursors. Show less

Fish currently supplies only 40% of the eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) required to allow all individuals globally to meet the minimum intake recommendation of 500 mg/d. Therefore, alternative sustainable sources are needed. The main objective was to investigate the ability of genetically engineered Camelina sativa (20% EPA) oil (CO) to enrich tissue EPA and DHA relative to an EPA-rich fish oil (FO) in mammals. Six-week-old male C57BL/6J mice were fed for 10 wk either a palm oil-containing control (C) diet or diets supplemented with EPA-CO or FO, with the C, low-EPA CO (COL), high-EPA CO (COH), low-EPA FO (FOL), and high-EPA FO (FOH) diets providing 0, 0.4, 3.4, 0.3, and 2.9 g EPA/kg diet, respectively. Liver, muscle, and brain were collected for fatty acid analysis, and blood glucose and serum lipids were quantified. The expression of selected hepatic genes involved in EPA and DHA biosynthesis and in modulating their cellular impact was determined. The oils were well tolerated, with significantly greater weight gain in the COH and FOH groups relative to the C group (P < 0.001). Significantly lower (36-38%) blood glucose concentrations were evident in the FOH and COH mice relative to C mice (P < 0.01). Hepatic EPA concentrations were higher in all EPA groups relative to the C group (P < 0.001), with concentrations of 0.0, 0.4, 2.9, 0.2, and 3.6 g/100 g liver total lipids in the C, COL, COH, FOL, and FOH groups, respectively. Comparable dose-independent enrichments of liver DHA were observed in mice fed CO and FO diets (P < 0.001). Relative to the C group, lower fatty acid desaturase 1 (Fads1) expression (P < 0.005) was observed in the COH and FOH groups. Higher fatty acid desaturase 2 (Fads2), peroxisome proliferator-activated receptor α (Ppara), and peroxisome proliferator-activated receptor γ (Pparg) (P < 0.005) expressions were induced by CO. No impact of treatment on liver X receptor α (Lxra) or sterol regulatory element-binding protein 1c (Srebp1c) was evident. Oil from transgenic Camelina is a bioavailable source of EPA in mice. These data provide support for the future assessment of this oil in a human feeding trial. Show less

Long-chain polyunsaturated fatty acids (LC-PUFAs) such as arachidonic (ARA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids are essential components of biomembranes, particularly in neural tissues. Endogenous synthesis of ARA, EPA and DHA occurs from precursor dietary essential fatty acids such as linoleic and α-linolenic acid through elongation and Δ5 and Δ6 desaturations. With respect to desaturation activities some noteworthy differences have been noted in vertebrate classes. In mammals, the Δ5 activity is allocated to the Fads1 gene, while Fads2 is a Δ6 desaturase. In contrast, teleosts show distinct combinations of desaturase activities (e.g. bifunctional or separate Δ5 and Δ6 desaturases) apparently allocated to Fads2-type genes. To determine the timing of Fads1-Δ5 and Fads2-Δ6 evolution in vertebrates we used a combination of comparative and functional genomics with the analysis of key phylogenetic species. Our data show that Fads1 and Fads2 genes with Δ5 and Δ6 activities respectively, evolved before gnathostome radiation, since the catshark Scyliorhinus canicula has functional orthologues of both gene families. Consequently, the loss of Fads1 in teleosts is a secondary episode, while the existence of Δ5 activities in the same group most likely occurred through independent mutations into Fads2 type genes. Unexpectedly, we also establish that events of Fads1 gene expansion have taken place in birds and reptiles. Finally, a fourth Fads gene (Fads4) was found with an exclusive occurrence in mammalian genomes. Our findings enlighten the history of a crucially important gene family in vertebrate fatty acid metabolism and physiology and provide an explanation of how observed lineage-specific gene duplications, losses and diversifications might be linked to habitat-specific food web structures in different environments and over geological timescales. Show less

Fish are important sources of high quality protein, essential minerals such as iodine and selenium, vitamins including A, D and E, and omega-3 fatty acids in the human diet. With declining fisheries worldwide, farmed fish constitute an ever-increasing proportion of fish in the food basket. Sustainable development of aquaculture dictates that diets will have to contain increasing levels of plant products that are devoid of cholesterol, but contain phytosterols that are known to have physiological effects in mammals. Liver X receptors (LXR) are transcription factors whose activity is modulated by sterols, with activation inducing cholesterol catabolism and de novo fatty acid biosynthesis in liver. Transcriptomic analysis has shown that substitution of fish meal and oil with plant products induces genes of cholesterol and fatty acid metabolism in salmonids. Here we report the cloning of LXR cDNAs from two species of salmonid fish that are important in aquaculture. The full-length cDNA (mRNA) of LXR obtained from salmon was shown to be 3766 bp, which included a 5'-untranslated region (UTR) of 412 bp and a 3'-UTR of 1960 bp and an open reading frame (ORF) of 1394 bp, which specified a protein of 462 amino acids. The trout LXR full-length cDNA was 2056 bp, including 5'- and 3'-UTRs of 219 and 547 bp, respectively, and an ORF of 1290 bp, which specified a protein of 427 amino acids. The protein sequences included characteristic features of mammalian LXRs, including the DNA binding (DBD), containing P-box, ligand binding (LBD) and activation function-2 (AF-2) domains, D-box, D (hinge) region, and eight cysteines that belong to the two zinc fingers. Phylogenetic analysis clustered the salmonid LXRs together, more closely with zebrafish and more distantly from medaka and stickleback. A pair-wise comparison among vertebrate LXR sequences showed the amino acid sequence predicted by the salmon LXR ORF showed greatest identity to that of trout 97%, and 97%, 87% and 81% identity to LXRs of zebrafish, frog and human (LXRalpha). The trout LXR ORF showed 96%, 92% and 82% identity to LXRs of zebrafish, frog and human (LXRalpha). Surprisingly, the expression of LXR was lowest in liver of all tissues examined and in salmon the greatest expression was observed in pyloric caeca with liver showing intermediate expression. It is likely that tissue expression was affected by the physiological status of the sampled animals. Certainly, nutritional, environmental and/or developmental regulation was evident in salmon, where the expression of LXR in liver was higher in fish in seawater than in freshwater, and higher in fish fed fish oil compared to fish fed vegetable oil in adult salmon. Show less