Elongase reactions as control points in long-chain polyunsaturated fatty acid synthesis

Abstract

Background: Δ6-Desaturase (Fads2) is widely regarded as rate-limiting in the conversion of dietary α-linolenic acid (18:3n-3; ALA) to the long-chain omega-3 polyunsaturated fatty acid docosahexaenoic acid (22:6n-3; DHA). However, increasing dietary ALA or the direct Fads2 product, stearidonic acid (18:4n-3; SDA), increases tissue levels of eicosapentaenoic acid (20:5n-3; EPA) and docosapentaenoic acid (22:5n-3; DPA), but not DHA. These observations suggest that one or more control points must exist beyond ALA metabolism by Fads2. One possible control point is a second reaction involving Fads2 itself, since this enzyme catalyses desaturation of 24:5n-3 to 24:6n-3, as well as ALA to SDA. However, metabolism of EPA and DPA both require elongation reactions. This study examined the activities of two elongase enzymes as well as the second reaction of Fads2 in order to concentrate on the metabolism of EPA to DHA.

Methodology/principal findings: The substrate selectivities, competitive substrate interactions and dose response curves of the rat elongases, Elovl2 and Elovl5 were determined after expression of the enzymes in yeast. The competitive substrate interactions for rat Fads2 were also examined. Rat Elovl2 was active with C(20) and C(22) polyunsaturated fatty acids and this single enzyme catalysed the sequential elongation reactions of EPA→DPA→24:5n-3. The second reaction DPA→24:5n-3 appeared to be saturated at substrate concentrations not saturating for the first reaction EPA→DPA. ALA dose-dependently inhibited Fads2 conversion of 24:5n-3 to 24:6n-3.

Conclusions: The competition between ALA and 24:5n-3 for Fads2 may explain the decrease in DHA levels observed after certain intakes of dietary ALA have been exceeded. In addition, the apparent saturation of the second Elovl2 reaction, DPA→24:5n-3, provides further explanations for the accumulation of DPA when ALA, SDA or EPA is provided in the diet. This study suggests that Elovl2 will be critical in understanding if DHA synthesis can be increased by dietary means.

Figure 1. Fgure 1. Comparison of the rat Elovl5 and Elovl2 substrate specificities.

Recombinant S. cerevisiae expressing Elovl5 (A) or Elovl2 (B) were grown in the presence of 100 µM of various C₁₈, C₂₀ and C₂₂ PUFA substrates. Fatty acids were extracted from the recombinant S. cerevisiae and the amount of each fatty acid was expressed as a percentage of the total amount of all fatty acids. The proportion of substrate fatty acid converted to longer chain fatty acid product(s) was calculated as [product(s)/(product(s) + substrate)]×100. The product was 2 carbons longer than the substrate. ^§ denotes that the conversion includes the 4 carbon elongation product. The results are the means ± S.D. of triplicate incubations. Values with different symbols are significantly different from each other. ND, not detected.

Figure 2. Competition between n-3 PUFA substrates for elongation by Elovl5.

Recombinant S. cerevisiae expressing Elovl5 were grown in the presence of 100 µM of SDA and 0–300 µM EPA (A) or 100 µM of EPA and 0–300 µM SDA (B). Recombinant S. cerevisiae containing the empty pYES2 vector were grown in the presence of 0–300 µM EPA or SDA. Fatty acids were extracted from the recombinant S. cerevisiae and the amount of each fatty acid was quantified in µg g⁻¹ of S. cerevisiae or expressed as a percentage of the total amount of all fatty acids. The proportion of substrate fatty acid converted to longer chain fatty acid product(s) was calculated as [product(s)/(product(s) + substrate)]×100. The conversion of SDA includes the 4 carbon elongation product 22:4n-3. The results are the means ± S.D. of triplicate incubations. Values with different symbols are significantly different from each other.

Figure 3. Examination of product/substrate relationships for elongation by Elovl5 and Elovl2.

Recombinant S. cerevisiae expressing empty pYES2 vector or Elovl5 were grown in the presence of EPA (A) or recombinant S. cerevisiae expressing empty pYES2 vector or Elovl2 were grown in the presence of EPA (B) or DPA (C). Fatty acids were extracted from the recombinant S. cerevisiae and the amount of each fatty acid was quantified in µg g⁻¹ of S. cerevisiae. The results are the means ± S.D. of triplicate incubations. Values with different symbols are significantly different from each other.

Figure 4. Comparison of the absolute number of Elovl2 and Elovl5 mRNA template copies / ng of total RNA in rat liver and heart.

Elovl2 was detected in heart at <10 copies / ng of total RNA. All measurements were performed in duplicate and the results are the mean ± S.E.M. (n = 3).

Figure 5. The rat Fads2 substrate specificity.

Recombinant S. cerevisiae expressing Fads2 were grown in the presence of 100 µM of various PUFA substrates. Fatty acids were extracted from the recombinant S. cerevisiae and the amount of each fatty acid was expressed as a percentage of the total amount of all fatty acids. The proportion of substrate fatty acid converted to desaturation product was calculated as [product/(product + substrate)]×100. The product contained one more double bond than the substrate. The results are the means ± S.D. of triplicate incubations. Values with different symbols are significantly different from each other.

Figure 6. Examination of product/substrate relationships for desaturation by Fads2.

Recombinant S. cerevisiae expressing empty pYES2 vector or Fads2 were grown in the presence of ALA (A) or 24:5n-3 (B). Fatty acids were extracted from the recombinant S. cerevisiae and the amount of each fatty acid was quantified in µg g⁻¹ of S. cerevisiae. The results are the means ± S.D. of triplicate incubations. Values with different symbols are significantly different from each other.

Figure 7. Competition between n-3 PUFA substrates for desaturation by Fads2.

Recombinant S. cerevisiae expressing Fads2 were grown in the presence of 25 µM of 24:5n-3 and 0–400 µM ALA. Fatty acids were extracted from the recombinant S. cerevisiae and the amount of each fatty acid was expressed as a percentage of the total amount of all fatty acids. The proportion of substrate fatty acid converted to desaturation product was calculated as [product/(product + substrate)]×100. The results are the means ± S.D. of triplicate incubations. Values with different symbols are significantly different from each other.

Figure 8. N-3 long-chain polyunsaturated fatty acid synthesis showing two-stages of Δ6-desaturase involvement and the individual elongase involvement.

? denotes that Elovl5 or Elovl2 can catalyse this reaction.