Highly Unsaturated Fatty Acid Synthesis Research Articles

Food quality upgrade of carbon from submerged macrophytes by flagellates via a heterotrophic pathway can stimulate growth of Daphnia magna.

Submerged macrophytes play crucial roles in maintaining the stability of clear-water states in shallow lakes. Recent stable isotope studies have shown that crustacean zooplankton can utilize submerged macrophyte carbon, but macrophytes alone cannot support the growth and reproduction of such grazers, being deficient in highly unsaturated fatty acids (HUFA). We hypothesized that flagellates feeding on macrophytes can synthesize HUFA and thereby support crustacean zooplankton. To test this hypothesis, we conducted a feeding experiment in which Daphnia magna were provided with a diet of submerged macrophyte Hydrilla verticillata detritus which had been degraded by lake microbes. The chlorophyte Scenedesmus bijuga and undegraded macrophyte detritus were used as controls for comparison of Daphnia's performance. Using biochemical analysis, we examined how the degradation process affected the food quality of the macrophyte. Flagellates were subsequently isolated from the degraded macrophyte and cultured heterotrophically to detect their HUFA synthesis. The 5-day degraded H. verticillata showed significantly higher HUFA concentrations than undegraded macrophyte detritus. They supported better Daphnia performance than undegraded macrophyte, being comparable with S. bijuga. Two isolated flagellates (SL-1 and SL-2), identified as Ochromonas sp. and Poterioochromonas sp., were found to contain HUFA when cultured heterotrophically without dietary sources of fatty acids, suggesting their HUFA synthesis ability. Our results demonstrate that submerged macrophytes may thus indirectly support crustacean zooplankton via flagellate mediation. As crustacean zooplanktons are of key importance for water quality in the grazer control of phytoplankton, this microbial facilitation may contribute to the maintenance of macrophyte clear-water conditions in shallow lakes.

Fads2b Plays a Dominant Role in ∆6/∆5 Desaturation Activities Compared with Fads2a in Common Carp (Cyprinus carpio).

Highly unsaturated fatty acids (HUFAs) are essential for mammalian health, development and growth. However, most mammals, including humans, are incapable of synthesizing n-6 and n-3 HUFAs. Fish can convert C18 unsaturated fatty acids into n-6 and n-3 HUFAs via fatty acid desaturase (Fads), in which Fads2 is a key enzyme in HUFA biosynthesis. The allo-tetraploid common carp theoretically encode two duplicated fads2 genes. The expression patterns and desaturase functions of these two homologous genes are still unknown. In this study, the full length of the fads2a and fads2b were identified in common carp (Cyprinus carpio). Expression analyses indicate that both genes were mainly expressed in the liver and the expression of fads2b is higher than fads2a at different developmental stages in carp embryos. Heterogenous expression and 3D docking analyses suggested that Fads2b demonstrated stronger ∆6 and ∆5 desaturase activities than Fads2a. The core promotor regions of fads2a and fads2b were characterized and found to have different potential transcriptional binding sites. These results revealed the same desaturase functions, but different activities of two homologues of fasd2 genes in common carp. The data showed that fads2b played a more important role in HUFA synthesis through both expression and functional analyses.

Polyunsaturated Fatty Acid Desaturation Is a Mechanism for Glycolytic NAD+ Recycling

The reactions catalyzed by the delta-5 and delta-6 desaturases (D5D/D6D), key enzymes responsible for highly unsaturated fatty acid (HUFA) synthesis, regenerate NAD+ from NADH. Here, we show that D5D/D6D provide a mechanism for glycolytic NAD+ recycling that permits ongoing glycolysis and cell viability when the cytosolic NAD+/NADH ratio is reduced, analogous to lactate fermentation. Although lesser in magnitude than lactate production, this desaturase-mediated NAD+ recycling is acutely adaptive when aerobic respiration is impaired invivo. Notably, inhibition of either HUFA synthesis or lactate fermentation increases the other, underscoring their interdependence. Consistent with this, a type 2 diabetes risk haplotype in SLC16A11 that reduces pyruvate transport (thus limiting lactate production) increases D5D/D6D activity invitro and in humans, demonstrating a chronic effect of desaturase-mediated NAD+ recycling. These findings highlight key biologic roles for D5D/D6D activity independent of their HUFA end products and expand the current paradigm of glycolytic NAD+ regeneration.

Polyunsaturated Fatty Acid Desaturase‐Mediated NAD+ Recycling Permits Ongoing Glycolysis and Cell Proliferation

The delta‐5 desaturase (D5D) and delta‐6 desaturase (D6D), encoded by FADS1 and FADS2 respectively, are required for the synthesis of highly unsaturated fatty acids (HUFAs). Genetic variants associated with FADS1 and FADS2 expression are associated with HUFA content in circulating lipids, as well as fasting glucose, height and weight, and risk of colon and laryngeal cancer. Why genetic variation in HUFA synthesis has such a broad impact on human metabolic and proliferative phenotypes is unknown. Here, we show that HUFA synthesis is a mechanism for cytosolic NAD+ recycling that permits ongoing glycolysis. Consistent with this, lowering the cytosolic NAD+/NADH ratio via inhibition of mitochondrial respiration increased D5D and D6D activity in vitro and in vivo. Conversely, increasing the cytosolic NAD+/NADH ratio via expression of an NADH oxidase or activation of lactate dehydrogenase decreased D5D and D6D activity. Increasing D5D and D6D expression increased cytosolic NAD+/NADH, whereas reduced expression or inhibition of D5D and D6D decreased cytosolic NAD+/NADH and increased lactate production. Inhibition of D5D and D6D also reduced cell proliferation, an effect that was reversed by boosting cytosolic NAD+ but not with HUFA end‐product supplementation. Finally, we show that the type 2 diabetes risk haplotype in SLC16A11, which reduces pyruvate transport, also increases D5D and D6D activity, demonstrating the relevance of our findings to human disease. These results outline a novel link between glycolysis and HUFA desaturation mediated by cytosolic NAD+, and provide new insight on how D5D and D6D activity may impact glycemic, growth, and cancer traits in humans.Support or Funding InformationThis work was supported by NIH U01 DK060990 and the Extramural Grant Program of Satellite Health Care, a not‐for‐profit renal care provider.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Two Elovl5-like elongase genes in Cyprinus carpio var. Jian: Gene characterization, mRNA expression, and nutritional regulation

Elovl5 elongase is a critical enzyme involved in the highly unsaturated fatty acid (HUFA) biosynthesis. There is very little information on the evolution and functional characterization of Elovl5-a and Elovl5-b genes in common carp (Cyprinus carpio var. Jian). In the present study, the genomic sequences and structures of two putative Elovl5-like elongase genes in the common carp genome were obtained. The mRNA expression patterns of Elovl5-a and Elovl5-b in tissues, hatching carp embryos, and juveniles under nutritional regulation were investigated. The results show that the two Elovl5 elongase genes have similar organization, coding 8 exons of high identity and introns of distinct size and sequence composition. They are not allelic variants of a single gene. Both Elovl5 elongase genes are highly expressed in liver, intestine (pyloric caeca) and brain. Elovl5-a and Elovl5-b mRNAs showed increased expression from newly hatched to 20 days after hatching. The regulation of Elovl5-a and Elovl5-b in response to dietary fatty acid composition was determined in liver, brain and intestine (pyloric caeca) of common carp fed with diets: (i) fish oil (FO) rich in n-3 HUFA, (ii) corn oil (CO, 18:2n-6) or (iii) linseed oil (LO, 18:3n-3). Also the differential expression of Elovl5-a and Elovl5-b genes in liver, brain and intestine in common carps fed with different oil sources was studied. Further work aimed at the determination of the mechanisms of differential expression of the Elovl5-a and Elovl5-b in different tissues and the roles of transcription factors in regulating HUFA synthesis is in progress.

Two Δ6-desaturase-like genes in common carp (Cyprinus carpio var. Jian): Structure characterization, mRNA expression, temperature and nutritional regulation

Δ6-Desaturase is the rate-limiting enzyme involved in highly unsaturated fatty acid (HUFA) biosynthesis. There is very little information on the evolution and functional characterization of Δ6Fad-a and Δ6Fad-b in common carp (Cyprinus carpio var. Jian). In the present study, the genomic sequences and structures of two putative Δ6-desaturase-like genes in common carp genome were obtained. We investigated the mRNA expression patterns of Δ6Fad-a and Δ6Fad-b in tissue, hatching carp embryos, larvae by temperature shock and juveniles under nutritional regulation. Our results showed that the two Δ6Fad genes had identical coding exon structures, being comprised of 12 coding exons, and with introns of distinct size and sequence composition. They were not allelic variants of a single gene. Both Δ6Fad genes were highly expressed in liver, intestine (pyloric caeca) and brain. The Δ6Fad-a and Δ6Fad-b mRNAs showed an increase in expression from newly hatched to 25days after hatching. The expression levels of Δ6Fad-a were obviously regulated by temperature, whereas Δ6Fad-b was not affected by temperature. The regulation of Δ6Fad-a and Δ6Fad-b in response to dietary fatty acid composition was determined in liver, brain and intestine (pyloric caeca) of common carp fed with diets: diet1with fish oil (FO) rich in n−3 HUFA, diet2 with corn oil (CO, 18:2n−6) and diet3 with linseed oil (LO, 18:3n−3). The differential expression of Δ6Fad-a and Δ6Fad-b genes in liver, brain and intestine in common carps was fed with different oil sources, respectively. Further work is in progress to determine the mechanism of differential expression of the Δ6Fad-a and Δ6Fad-b genes in different tissues and the roles of transcription factors in regulating HUFA synthesis.

Investigation of highly unsaturated fatty acid metabolism in the Asian sea bass, Lates calcarifer

Lates calcarifer, commonly known as the Asian sea bass or barramundi, is an interesting species that has great aquaculture potential in Asia including Malaysia and also Australia. We have investigated essential fatty acid metabolism in this species, focusing on the endogenous highly unsaturated fatty acid (HUFA) synthesis pathway using both biochemical and molecular biological approaches. Fatty acyl desaturase (Fad) and elongase (Elovl) cDNAs were cloned and functional characterization identified them as ∆6 Fad and Elovl5 elongase enzymes, respectively. The ∆6 Fad was equally active toward 18:3n-3 and 18:2n-6, and Elovl5 exhibited elongation activity for C18-20 and C20-22 elongation and a trace of C22-24 activity. The tissue profile of gene expression for ∆6 fad and elovl5 genes, showed brain to have the highest expression of both genes compared to all other tissues. The results of tissue fatty acid analysis showed that the brain contained more docosahexaenoic acid (DHA, 22:6n-3) than flesh, liver and intestine. The HUFA synthesis activity in isolated hepatocytes and enterocytes using [1-(14)C]18:3n-3 as substrate was very low with the only desaturated product detected being 18:4n-3. These findings indicate that L. calcarifer display an essential fatty acid pattern similar to other marine fish in that they appear unable to synthesize HUFA from C18 substrates. High expression of ∆6 fad and elovl5 genes in brain may indicate a role for these enzymes in maintaining high DHA levels in neural tissues through conversion of 20:5n-3.

Upregulated mRNA expression of desaturase and elongase, two enzymes involved in highly unsaturated fatty acids biosynthesis pathways during follicle maturation in zebrafish

BackgroundAlthough unsaturated fatty acids such as eicosapentaenoic acid (EPA, C20:5n-3), docosahexaenoic acid (DHA, C22:6n-3) and arachidonic acid (ARA, C20:4n-6), collectively known as the highly unsaturated fatty acids (HUFA), play pivotal roles in vertebrate reproduction, very little is known about their synthesis in the ovary. The zebrafish (Danio rerio) display capability to synthesize all three HUFA via pathways involving desaturation and elongation of two precursors, the linoleic acid (LA, C18:2n-6) and linolenic acid (LNA, C18:3n-3). As a prerequisite to gain full understanding on the importance and regulation of ovarian HUFA synthesis, we described here the mRNA expression pattern of two enzymes; desaturase (fadsd6) and elongase (elovl5), involved in HUFA biosynthesis pathway, in different zebrafish ovarian follicle stages. Concurrently, the fatty acid profile of each follicle stage was also analyzed.MethodsmRNA levels of fadsd6 and elovl5 in different ovarian follicle stages were determined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) assays. For analysis of the ovarian follicular fatty acid composition, gas chromatography was used.ResultsOur results have shown that desaturase displayed significant upregulation in expression during the oocyte maturation stage. Expression of elongase was significantly highest in pre-vitellogenic follicles, followed by maturation stage. Fatty acid composition analysis of different ovarian follicle stages also showed that ARA level was significantly highest in pre-vitellogenic and matured follicles. DHA level was highest in both late vitellogenic and maturation stage.ConclusionCollectively, our findings seem to suggest the existence of a HUFA synthesis system, which could be responsible for the synthesis of HUFA to promote oocyte maturation and possibly ovulation processes. The many advantages of zebrafish as model system to understand folliculogenesis will be useful platform to further elucidate the regulatory and mechanism aspects of ovarian HUFA synthesis.

Comparison of effects of vegetable oils blended with southern hemisphere fish oil and decontaminated northern hemisphere fish oil on growth performance, composition and gene expression in Atlantic salmon (Salmo salar L.)

Replacement of fish oil with sustainable alternatives, such as vegetable oil, in aquaculture diets has to be achieved without compromising the nutritional quality, in terms of n-3 highly unsaturated fatty acid (HUFA) content, of the product. This may be possible if the level of replacement is not too high and oil blends are chosen carefully but, if high levels of fish oil are substituted, a fish oil finishing diet prior to harvest would be required to restore n-3HUFA. However, a decontaminated fish oil would be required to avoid increasing undesirable contaminants. Here we test the hypotheses that blending of rapeseed and soybean oils with southern hemisphere fish oil will have a low impact upon tissue n-3HUFA levels, and that decontamination of fish oil will have no major effect on the nutritional quality of fish oil as a feed ingredient for Atlantic salmon. Salmon (initial weight ~0.8 kg) were fed for 10 weeks with diets in which 60% of fish oil was replaced with blends of soybean, rapeseed and southern hemisphere fish oil (SVO) or 100% decontaminated northern fish oil (DFO) in comparison with a standard northern fish oil diet (FO). Decontamination of the oil was a two-step procedure that included treatment with activated carbon followed by thin film deodorisation. Growth performance and feed efficiency were unaffected by either the SVO or DFO diets despite these having lower gross nutrient and fatty acid digestibilities than the FO diet. There were also no effects on the gross composition of the fish. Liver and, to a lesser extent flesh, lipid levels were lower in fish fed the SVO blends, due to lower proportions of neutral lipids, specifically triacylglycerol. Tissue lipid levels were not affected in fish fed the DFO diet. Reflecting the diet, flesh eicosapentaenoic acid (EPA) and total n-3 fatty acids were higher, and 18:1n-9 lower, in fish fed DFO than FO, whereas there were no differences in liver fatty acid compositions. Flesh EPA levels were only slightly reduced from about 6% to 5% although docosahexaenoic acid (DHA) was reduced more severely from around 13% to about 7% in fish fed the SVO diets. In contrast, the liver fatty acid compositions showed higher levels of n-3 HUFA, with DHA only reduced from 21% to about 18% and EPA increased from under 8% to 9–10% in fish fed the SVO diets. The evidence suggested that increased liver EPA (and arachidonic acid) was not simply retention, but also conversion of dietary 18:3n-3 and 18:2n-6. Increased HUFA synthesis was supported by increased hepatic expression of fatty acyl desaturases in fish fed the SVO diets. Flesh n-3HUFA levels and desaturase expression was significantly higher in fish fed soybean oil than in fish fed rapeseed oil. In conclusion, partial replacement of fish oil with blends of vegetable oils and southern hemisphere fish oil had minimal impact on HUFA levels in liver, but a greater effect on flesh HUFA levels. Despite lower apparent digestibility, decontamination of fish oil did not significantly impact its nutritional quality for salmon.

Nutritional and environmental regulation of the synthesis of highly unsaturated fatty acids and of fatty-acid oxidation in Atlantic salmon (Salmo salar L.) enterocytes and hepatocytes

The objective of this work was to determine whether highly unsaturated fatty acid (HUFA) synthesis and fatty-acid oxidation in Atlantic salmon (Salmo salar L.) intestine was under environmental and/or seasonal regulation. Triplicate groups of salmon were grown through a full two-year cycle on two diets containing either fish oil (FO) or a diet with 75% of the FO replaced by a vegetable oil (VO) blend containing rapeseed, palm, and linseed oils. At key points in the life cycle fatty acyl desaturation/elongation (HUFA synthesis) and oxidation activity were determined in enterocytes and hepatocytes using [1−14C]18:3n−3 as substrate. As observed previously, HUFA synthesis in hepatocytes reached a peak at seawater transfer and declined thereafter, with activity consistently greater in fish fed the VO diet. In fish fed FO, HUFA synthesis in enterocytes in the freshwater stage was at a level similar to that in hepatocytes. HUFA synthesis in enterocytes increased rapidly after seawater transfer, however, and remained high for some months after transfer before decreasing to levels that were again similar to those observed in hepatocytes. Enterocyte synthesis of HUFA was usually higher in fish fed the VO diet than in those fed the FO diet. Oxidation of [1−14C]18:3n−3 in hepatocytes from fish fed FO tended to decrease during the freshwater phase but then increased steeply, peaking just after transfer before decreasing during the remaining seawater phase. At the peak in oxidation activity around seawater transfer, activity was significantly lower in fish fed VO than in fish fed FO. In enterocytes, oxidation of [1−14C]18:3 in fish fed FO reached a peak in activity just before seawater transfer. In fish fed VO, except for high activity at nine months the pattern was similar to that obtained in enterocytes from fish fed FO, with high activity around seawater transfer and declining activity in seawater. In conclusion, fatty acid metabolism in intestinal cells seemed to be under dual nutritional and environmental or seasonal regulation. Temporal patterns of oxidation of fatty acids were usually similar in the two cell types, but HUFA synthesis in enterocytes peaked over the summer seawater phase rather than at transfer, as with hepatocytes, suggesting the possibility of different regulatory cues.

Effect of diets enriched in Δ6 desaturated fatty acids (18:3 n − 6 and 18:4 n − 3), on growth, fatty acid composition and highly unsaturated fatty acid synthesis in two populations of Arctic charr ( Salvelinus alpinus L.)

This study aimed to test the hypothesis that diets containing relatively high amounts of the Δ6 desaturated fatty acids stearidonic acid (STA, 18:4 n − 3) and γ-linolenic acid (GLA, 18:3 n − 6), may be beneficial in salmonid culture. The rationale being that STA and GLA would be better substrates for highly unsaturated fatty acid (HUFA) synthesis as their conversion does not require the activity of the reputed rate-limiting enzyme, fatty acid Δ6 desaturase. Duplicate groups of two Arctic charr ( Salvelinus alpinus L.) populations with different feeding habits, that had been reported previously to show differences in HUFA biosynthetic capacity, were fed for 16 weeks on two fish meal based diets containing 47% protein and 21% lipid differing only in the added lipid component, which was either fish oil (FO) or echium oil (EO). Dietary EO had no detrimental effect on growth performance and feed efficiency, mortalities, or liver and flesh lipid contents in either population. The proportions of 18:2 n − 6, 18:3 n − 3, 18:3 n − 6, 18:4 n − 3, 20:3 n − 6 and 20:4 n − 3 in total lipid in both liver and flesh were increased by dietary EO in both populations. However, the percentages of 20:5 n − 3 and 22:6 n − 3 were reduced by EO in both liver and flesh in both strains, whereas 20:4 n − 6 was only significantly reduced in flesh. In fish fed FO, HUFA synthesis from both [1- 14C]18:3 n − 3 and [1- 14C]20:5 n − 3 was significantly higher in the planktonivorous Coulin charr compared to the demersal, piscivorous Rannoch charr morph. However, HUFA synthesis was increased by EO in Rannoch charr, but not in Coulin charr. In conclusion, dietary EO had differential effects in the two populations of charr, with HUFA synthesis only stimulated by EO in the piscivorous Rannoch morph, which showed lower activities in fish fed FO. However, the hypothesis was not proved as, irrespective of the activity of the HUFA synthesis pathway in either population, feeding EO resulted in decreased tissue levels of n − 3HUFA and 20:4 n − 6. This has been observed previously in salmonids fed vegetable oils, and thus the increased levels of Δ6 desaturated fatty acids in EO did not effectively compensate for the lack of dietary HUFA.

Peroxisome proliferator-activated receptor α is required for feedback regulation of highly unsaturated fatty acid synthesis

Delta6 desaturase (D6D), the rate-limiting enzyme for highly unsaturated fatty acid (HUFA) synthesis, is induced by essential fatty acid-deficient diets. Sterol regulatory element-binding protein-1c (SREBP-1c) in part mediates this induction. Paradoxically, D6D is also induced by ligands of peroxisome proliferator-activated receptor alpha (PPARalpha). Here, we report a novel physiological role of PPARalpha in the induction of genes specific for HUFA synthesis by essential fatty acid-deficient diets. D6D mRNA induction by essential fatty acid-deficient diets in wild-type mice was diminished in PPARalpha-null mice. This impaired D6D induction in PPARalpha-null mice was not attributable to feedback suppression by tissue HUFAs because PPARalpha-null mice had lower HUFAs in liver phospholipids than did wild-type mice. Furthermore, PPARalpha-responsive genes were induced in wild-type mice under essential fatty acid deficiency, suggesting the generation of endogenous PPARalpha ligand(s). Contrary to genes for HUFA synthesis, the induction of other lipogenic genes under essential fatty acid deficiency was higher in PPARalpha-null mice than in wild-type mice even though mature SREBP-1c protein did not differ between the genotypes. The expression of PPARgamma was markedly increased in PPARalpha-null mice and might have contributed to the induction of genes for de novo lipogenesis. Our study suggests that PPARalpha, together with SREBP-1c, senses HUFA status and confers pathway-specific induction of HUFA synthesis by essential fatty acid-deficient diets.

Influence of dietary palm oil on growth, tissue fatty acid compositions, and fatty acid metabolism in liver and intestine in rainbow trout (Oncorhynchus mykiss)

This study aimed to investigate the effects of dietary crude palm oil (CPO) on fatty acid metabolism in liver and intestine of rainbow trout. Triplicate groups of rainbow trout for 10 weeks at 13 °C were fed on diets in which CPO replaced fish oil (FO) in a graded manner (0–100%). At the end of the trial, fatty acid compositions of flesh, liver and pyloric caeca were determined and highly unsaturated fatty acid (HUFA) synthesis and fatty acid oxidation were estimated in isolated hepatocytes and caecal enterocytes using [1-14C]18:3n-3 as substrate. Growth performance and feed efficiency were unaffected by dietary CPO. Fatty acid compositions of selected tissues reflected the dietary fatty acid composition with increasing CPO resulting in increased proportions of 18:1n-9 and 18:2n-6 and decreased proportions of n-3HUFA, 20:5n-3 and 22:6n-3. Palmitic acid, 16:0, was also increased in flesh and pyloric caeca, but not in liver. The capacity of HUFA synthesis from 18:3n-3 increased by up to threefold in both hepatocytes and enterocytes in response to graded increases in dietary CPO. In contrast, oxidation of 18:3n-3 was unaffected by dietary CPO in hepatocytes and reduced by high levels of dietary CPO in enterocytes. The results of this study suggest that CPO can be used at least to partially replace FO in diets for rainbow trout in terms of permitting similar growth and feed conversion, and having no major detrimental effects on lipid and fatty acid metabolism, although flesh fatty acid compositions are significantly affected at an inclusion level above 50%, with n-3HUFA reduced by up to 40%.

Highly unsaturated fatty acid synthesis in vertebrates: New insights with the cloning and characterization of a Δ6 desaturase of Atlantic salmon

Fish are an important source of the n-3 highly unsaturated fatty acids (HUFA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids that are crucial to the health of higher vertebrates. The synthesis of HUFA involves enzyme-mediated desaturation, and a delta5 fatty acyl desaturase cDNA has been cloned from Atlantic salmon (Salmo salar) and functionally characterized previously. Here we report cloning and functional characterization of a delta6 fatty acyl desaturase of Atlantic salmon and describe its genomic structure, tissue expression, and nutritional regulation. A salmon genomic library was screened with a salmon delta5 desaturase cDNA and positive recombinant phage isolated and subcloned. The full-length cDNA for the putative fatty acyl desaturase was shown to comprise 2106 bp containing an open reading frame of 1365 bp specifying a protein of 454 amino acids (GenBank accession no. AY458652). The protein sequence included three histidine boxes, two transmembrane regions, and an N-terminal cytochrome b5 domain containing the heme-binding motif HPGG, all of which are characteristic of microsomal fatty acid desaturases. Functional expression showed that this gene possessed predominantly delta6 desaturase activity. Screening and sequence analysis of the genomic DNA of a single fish revealed that the delta6 desaturase gene constituted 13 exons in 7965 bp of genomic DNA. Quantitative real-time PCR assay of gene expression in Atlantic salmon showed that both delta6 and delta5 fatty acyl desaturase genes, and a fatty acyl elongase gene, were highly expressed in intestine, liver, and brain, and less so in kidney, heart, gill, adipose tissue, muscle, and spleen. Furthermore, expression of both delta6 and delta5 fatty acyl desaturase genes in intestine, liver, red muscle, and adipose tissue was higher in salmon fed a diet containing vegetable oil than in fish fed a diet containing fish oil.

Characterization and comparison of fatty acyl Δ6 desaturase cDNAs from freshwater and marine teleost fish species

Fish are the most important dietary source of the n-3 highly unsaturated fatty acids (HUFA), eicosapentaenoic (EPA) and docosahexaenoic acid (DHA), that have particularly important roles in human nutrition reflecting their roles in critical physiological processes. The objective of the study described here was to clone, functionally characterize and compare expressed fatty acid desaturase genes involved in the production of EPA and DHA in freshwater and marine teleost fish species. Putative fatty acid desaturase cDNAs were isolated and cloned from common carp ( Cyprinus carpio) and turbot ( Psetta maximus). The enzymic activities of the products of these cDNAs, together with those of cDNAs previously cloned from rainbow trout ( Oncorhynchus mykiss) and gilthead sea bream ( Sparus aurata), were determined by heterologous expression in the yeast Saccharomyces cerevisiae. The carp and turbot desaturase cDNAs included open reading frames (ORFs) of 1335 and 1338 base pairs, respectively, specifying proteins of 444 and 445 amino acids. The protein sequences possessed all the characteristic features of microsomal fatty acid desaturases, including three histidine boxes, two transmembrane regions, and N-terminal cytochrome b 5 domains containing the haem-binding motif, HPGG. Functional expression showed all four fish cDNAs encode basically unifunctional Δ6 fatty acid desaturase enzymes responsible for the first and rate-limiting step in the biosynthesis of HUFA from 18:3n-3 and 18:2n-6. All the fish desaturases were more active towards the n-3 substrate with 59.5%, 31.5%, 23.1% and 7.0% of 18:3n-3 being converted to 18:4n-3 in the case of turbot, trout, sea bream and carp, respectively. The enzymes also showed very low, probably physiologically insignificant, levels of Δ5 desaturase activity, but none of the products showed Δ4 desaturase activity. The cloning and characterization of desaturases from these fish is an important advance, as they are species in which there is a relative wealth of data on the nutritional regulation of fatty acid desaturation and HUFA synthesis, and between which substantive differences occur.

Essential fatty acid synthesis and its regulation in mammals

The tissue content of highly unsaturated fatty acids (HUFA) such as arachidonic acid and docosahexaenoic acid is maintained in a narrow range by feedback regulation of synthesis. Delta-6 desaturase (D6D) catalyzes the first and rate-limiting step of the HUFA synthesis. Recent identification of a human case of D6D deficiency underscores the importance of this pathway. Sterol regulatory element binding protein-1c (SREBP-1c) is a key transcription factor that activates transcription of genes involved with fatty acid synthesis. We recently identified sterol regulatory element (SRE) that is required for activation of the human D6D gene by SREBP-1c. Moreover, the same SRE also mediates the suppression of the D6D gene by HUFA. The identification of SREBP-1c as a key regulator of D6D suggests that the major physiological function of SREBP-1c in liver may be the regulation of phospholipid synthesis rather than triglyceride synthesis. Peroxisome proliferators (PP) induce fatty acid oxidation enzymes and desaturases in rodent liver. However, the induction of desaturases by PP is slower than the induction of oxidation enzymes. This delayed induction may be a compensatory reaction to the increased demand of HUFA caused by increased HUFA oxidation and peroxisome proliferation in PP administration. Recent studies have demonstrated a critical role of peroxisomal β-oxidation in DHA synthesis, and identified acyl CoA oxidase and D-bifunctional protein as the key enzymes.

Effects of dietary lipid level and vegetable oil on fatty acid metabolism in Atlantic salmon (Salmo salar L.) over the whole production cycle

Changes in fatty acid metabolism in Atlantic salmon (Salmo salar) induced by vegetable oil (VO) replacement of fish oil (FO) and high dietary oil in aquaculture diets can have negative impacts on the nutritional quality of the product for the human consumer, including altered flesh fatty acid composition and lipid content. A dietary trial was designed to investigate the twin problems of FO replacement and high energy diets in salmon throughout the entire production cycle. Salmon were grown from first feeding to around 2 kg on diets in which FO was completely replaced by a 1:1 blend of linseed and rapeseed oils at low (14–17%) and high (25–35%) dietary oil levels. This paper reports specifically on the influence of diet on various aspects of fatty acid metabolism. Fatty acid compositions of liver, intestinal tissue and gill were altered by the diets with increased proportions of C18 polyunsaturated fatty acids and decreased proportions of n-3 highly unsaturated fatty acids (HUFA) in fish fed VO compared to fish fed FO. HUFA synthesis in hepatocytes and enterocytes was significantly higher in fish fed VO, whereas β-oxidation was unaltered by either dietary oil content or type. Over the entire production cycle, HUFA synthesis in hepatocytes showed a decreasing trend with age interrupted by a large peak in activity at seawater transfer. Gill cell prostaglandin (PG) production showed a possible seasonal trend, with peak activities in winter and low activities in summer and at seawater transfer. PG production in seawater was lower in fish fed the high oil diets with the lowest PG production generally observed in fish fed high VO. The changes in fatty acid metabolism induced by high dietary oil and VO replacement contribute to altered flesh lipid content and fatty acid compositions, and so merit continued investigation to minimize any negative impacts that sustainable, environmentally-friendly and cost-effective aquaculture diets could have in the future. Abbreviations: FO - fish oil; HUFA - highly unsaturated fatty acids acids (carbon chain length ≥C 20 with ≥3 double bonds); LO - linseed oil; RO - rapeseed oil; VO - vegetable oil.

Delayed induction of Δ-6 and Δ-5 desaturases by a peroxisome proliferator

Δ-6 Desaturase (D6D) is the key enzyme for the synthesis of highly unsaturated fatty acids (HUFA) such as arachidonic acid (AA) and docosahexaenoic acid (DHA) in mammals. Transcription of D6D gene is activated by both sterol regulatory element binding protein-1c (SREBP-1c) and peroxisome proliferators (PP). This response of D6D is paradoxical because SREBP-1c transactivates genes for fatty acid synthesis in liver, while PP induce enzymes for fatty acid oxidation. We hypothesized that the induction of D6D gene by PP is a compensatory response to the increased HUFA demand caused by peroxisome proliferation and induction of fatty acid oxidation. We investigated the time-course effects of a PP, Wy14643, on the induction of HUFA metabolizing genes and HUFA profile in rat liver. The mRNA of fatty acid oxidation enzymes in the Wy14643 fed group became significantly higher than controls at 4 h and reached maximum within 28 h. In contrast, the mRNA of Δ-6 and Δ-5 desaturases in the Wy14643 group was not significantly higher than control at 4 h and took >28 h to reach the maximum. Despite the induction of HUFA synthetic pathway, the concentration of end products (AA and DHA) remained unchanged throughout the 4-day period in liver phospholipids and non-esterified fatty acids. Taken together, this study supports our hypothesis and suggests that peroxisome proliferation and induction of fatty acid oxidation enzymes are the major mechanisms of the induction of HUFA synthesis by PP.

Metabolism and functions of highly unsaturated fatty acids: an update.

This review briefly examines the recent progress in knowledge about the synthesis and degradation of highly unsaturated fatty acids (HUFA) and their functions. Following the cloning of mammalian Delta6-desaturase (D6D), the D6D mRNA was found in many tissues, including adult brain, maternal organs, and fetal tissue, suggesting an active synthesis of HUFA in these tissues. The cloning also confirmed the long-postulated hypothesis that the same pathway is followed in n-6 and n-3 HUFA synthesis. Dietary n-6 and n-3 HUFA both induce fatty acid oxidation enzymes in peroxisomes when compared to their respective precursor polyunsaturated fatty acids. This suggests that peroxisomes may be the primary site of HUFA degradation when HUFA are supplied in excess from the diet. Peroxisome proliferators strongly induce the enzymes for the HUFA synthesis. The mechanism of this induction is currently unknown. Recent studies revealed new HUFA functions that are not mediated by eicosanoids. These functions include endocytosis/exocytosis, ion-channel modulation, DNA polymerase inhibition, and regulation of gene expression. These new discoveries will enable us to re-examine the underlying mechanisms for the classical symptoms of essential fatty acid deficiency as well as vitamin E deficiency. Progress has also been made in understanding the mechanism by which dietary HUFA reduce body fat deposition. One mechanism is induction of genes for fatty acid oxidation, which is mediated by peroxisome proliferator-activated receptor-alpha. Another likely mechanism is that HUFA suppress genes for fatty acid synthesis by reducing both mRNA and protein maturation of sterol regulatory element binding protein-1.