The fatty acid desaturase 2 (FADS2) gene product catalyzes Δ4 desaturation to yield n-3 docosahexaenoic acid and n-6 docosapentaenoic acid in human cells

Abstract

Docosahexaenoic acid (DHA) is a Δ4-desaturated C22 fatty acid and the limiting highly unsaturated fatty acid (HUFA) in neural tissue. The biosynthesis of Δ4-desaturated docosanoid fatty acids 22:6n-3 and 22:5n-6 are believed to proceed via a circuitous biochemical pathway requiring repeated use of a fatty acid desaturase 2 (FADS2) protein to perform Δ6 desaturation on C24 fatty acids in the endoplasmic reticulum followed by 1 round of β-oxidation in the peroxisomes. We demonstrate here that the FADS2 gene product can directly Δ4-desaturate 22:5n-3→22:6n-3 (DHA) and 22:4n-6→22:5n-6. Human MCF-7 cells lacking functional FADS2-mediated Δ6-desaturase were stably transformed with FADS2, FADS1, or empty vector. When incubated with 22:5n-3 or 22:4n-6, FADS2 stable cells produce 22:6n-3 or 22:5n-6, respectively. Similarly, FADS2 stable cells when incubated with d5-18:3n-3 show synthesis of d5-22:6n-3 with no labeling of 24:5n-3 or 24:6n-3 at 24 h. Further, both C24 fatty acids are shown to be products of the respective C22 fatty acids via elongation. Our results demonstrate that the FADS2 classical transcript mediates direct Δ4 desaturation to yield 22:6n-3 and 22:5n-6 in human cells, as has been widely shown previously for desaturation by fish and many other organisms.

Keywords: metabolism; nutrition; polyunsaturated fatty acids; Δ6 desaturation.

Figure 1.

Kinetics of C22→C24 PUFAs. A) 22:5n-6 incubations. FADS2, FADS1, and control stable MCF-7 cells were incubated with 22:5n-6 at 50 μM and sampled at 3, 6, 12, 18, and 24 h. In all cells, 24:5n-6 starts to appear at 3 h and increases to 24 h, rising in parallel with 22:5n-6 (right y axis) showing that 24:5n-6 is a product of 22:5n-6. B) 22:6n-3 incubations. FADS2, FADS1, and control stable MCF-7 cells were incubated with 22:6n-3 at 50 μM. Analogous to (A), the time-dependent increase in 24:6n-3 is seen in all 3 cell types, showing that 24:6n-3 is a product of 22:6n-3.

Figure 2.

22:4n-6 incubations. FADS2, FADS1, and control stable MCF-7 cells were incubated with 22:4n-6 at 100 μM for 1 h, then incubated with medium. FADS2 cells synthesize 22:5n-6; putative intermediate 24:5n-6 appears only at 12 h. Both FADS1 and control cells elongate 22:4n-6 to 24:4n-6; no synthesis of 22:5n-6 is detected. Points labeled with different letters are significantly different at P < 0.05.

Figure 3.

22:5n-3 incubations. FADS2, FADS1, and control stable MCF-7 cells incubated with 22:5n-3 at 10 μM and sampled at 3, 6, 12, 18, and 24 h. FADS2 cells show an increase in 22:6n-3 to 24 h, whereas 24:6n-3 plateaus at 3 h. Both FADS1 and control cells elongate 22:5n-3 to 24:5n-3, which plateaus by 12 h; no change in 22:6n-3 is evident.

Figure 4.

d5-18:3n-3 metabolism. A) FADS2, FADS1, and control stable MCF-7 cells were incubated with 100 μM of d5-18:3n-3 and sampled at 24 and 60 h. No labeled intermediates or C22 products are seen in FADS1 and control cells. FADS2 cells show labeling in all n-3 intermediates and 22:6n-3; no labeled C24 products were detected at 24 h. B) d5-18:3n-3 kinetics for FADS2 cells. C24 products (d5-24:5n-3 and d5-24:6n-3) are detected at 36 h and plateau between 36 and 72 h. C22 (d5-22:6n-3) continues to rise at 72 h.

Figure 5.

FADS2 subcellular localization. SK-N-SH cells transfected with control or FADS2 were stained with MitoTracker Red (Mt) or ER-tracker Blue-White DPX (ER), then visualized with an inverted meta confocal microscope (A, B). C) Western blot analysis using GFP, COX IV, and β-actin antibodies. No visible bands were seen with PDI antibody.