EPA,DHA, and Lipoic Acid Differentially Modulate the n-3 Fatty Acid Biosynthetic Pathway in Atlantic Salmon Hepatocytes

The aim of the present study was to investigate how EPA, DHA, and lipoic acid (LA) influence the different metabolic steps in the n‐3 fatty acid (FA) biosynthetic pathway in hepatocytes from Atlantic salmon fed four dietary levels (0, 0.5, 1.0 and 2.0%) of EPA, DHA or a 1:1 mixture of these FA. The hepatocytes were incubated with [1‐¹⁴C] 18:3n‐3 in the presence or absence of LA (0.2 mM). Increased endogenous levels of EPA and/or DHA and LA exposure both led to similar responses in cells with reduced desaturation and elongation of [1‐¹⁴C] 18:3n‐3 to 18:4n‐3, 20:4n‐3, and EPA, in agreement with reduced expression of the Δ6 desaturase gene involved in the first step of conversion. DHA production, on the other hand, was maintained even in groups with high endogenous levels of DHA, possibly due to a more complex regulation of this last step in the n‐3 metabolic pathway. Inhibition of the Δ6 desaturase pathway led to increased direct elongation to 20:3n‐3 by both DHA and LA. Possibly the route by 20:3n‐3 and then Δ8 desaturation to 20:4n‐3, bypassing the first Δ6 desaturase step, can partly explain the maintained or even increased levels of DHA production. LA increased DHA production in the phospholipid fraction of hepatocytes isolated from fish fed 0 and 0.5% EPA and/or DHA, indicating that LA has the potential to further increase the production of this health‐beneficial FA in fish fed diets with low levels of EPA and/or DHA.     

Cell Proliferation and Long Chain Polyunsaturated Fatty Acid Metabolism in a Cell Line From Southern Bluefin Tuna (Thunnus maccoyii)

Southern bluefin tuna (SBT, Thunnus maccoyii) aquaculture is a highly valuable industry, but research on these fish is hampered by strict catch quotas and the limited success of captive breeding. To address these limitations, we have developed a SBT cell line (SBT-E1) and here we report on fatty acid metabolism in this cell line. The SBT-E1 cells proliferated well in standard Leibovitz’s L-15 cell culture medium containing fetal bovine serum (FBS) as the source of fatty acids. Decreasing the FBS concentration decreased the cell proliferation. Addition of the C₁₈ polyunsaturated fatty acids (PUFA) α-linolenic acid (ALA, 18:3n-3) or linoleic acid (LNA, 18:2n-6) to the cell culture medium had little effect on the proliferation of the cells, whereas addition of the long-chain PUFA (LC-PUFA) arachidonic acid (ARA, 20:4n-6), eicosapentaenoic acid (EPA, 20:5n-3) or docosahexaenoic acid (DHA, 22:6n-3) significantly reduced the proliferation of the cells, especially at higher concentrations and especially for DHA. Addition of vitamin E to the culture medium overcame this effect, suggesting that it was due to oxidative stress. The fatty acid profiles of the total lipid from the cells reflected those of the respective culture media with little evidence for desaturation or elongation of any of the fatty acids. The only exceptions were EPA and ARA, which showed substantial elongation to 22:5n-3 and 22:4n-6, respectively, and DHA, which was significantly enriched in the cells compared with the culture medium. The results are discussed in light of the dietary PUFA requirements of SBT in the wild and in aquaculture.     

Determination of Substrate Preferences for Desaturases and Elongases for Production of Docosahexaenoic Acid from Oleic Acid in Engineered Canola

Production of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in plant seed oils has been pursued to improve availability of these omega‐3 fatty acids that provide important human health benefits. Canola (Brassica napus), through the introduction of 10 enzymes, can convert oleic acid (OLA) into EPA and ultimately DHA through a pathway consisting of two elongation and five desaturation steps. Herein we present an assessment of the substrate specificity of the seven desaturases and three elongases that were introduced into canola by expressing individual proteins in yeast. In vivo feeding experiments were conducted with 14 potential fatty acid intermediates in an OLA to DHA pathway to determine the fatty acid substrate profiles for each enzyme. Membrane fractions were prepared from yeast expression strains and shown to contain active enzymes. The elongases, as expected, extended acyl‐CoA substrates in the presence of malonyl‐CoA. To distinguish between enzymes that desaturate CoA‐ and phosphatidylcholine‐linked fatty acid substrates, we developed a novel in vitro method. We show that a delta‐12 desaturase from Phytophthora sojae, an omega‐3 desaturase from Phytophthora infestans and a delta‐4 desaturase from Thraustochytrium sp., all prefer phosphatidylcholine‐linked acyl substrates with comparatively low use of acyl‐CoA substrates. To further validate our method, a delta‐9 desaturase from Saccharomyces cerevisiae was confirmed to use acyl‐CoA as substrate, but could not use phosphatidylcholine‐linked substrates. The results and the assay methods presented herein will be useful in efforts to improve modeling of fatty acid metabolism and production of EPA and DHA in plants.