Trans-vaccenate is Δ13-desaturated by FADS3 in rodents

Abstract

Fatty acid desaturases play critical roles in regulating the biosynthesis of unsaturated fatty acids in all biological kingdoms. As opposed to plants, mammals are so far characterized by the absence of desaturases introducing additional double bonds at the methyl-end site of fatty acids. However, the function of the mammalian fatty acid desaturase 3 (FADS3) gene remains unknown. This gene is located within the FADS cluster and presents a high nucleotide sequence homology with FADS1 (Δ5-desaturase) and FADS2 (Δ6-desaturase). Here, we show that rat FADS3 displays no common Δ5-, Δ6- or Δ9-desaturase activity but is able to catalyze the unexpected Δ13-desaturation of trans-vaccenate. Although there is no standard for complete conclusive identification, structural characterization strongly suggests that the Δ11,13-conjugated linoleic acid (CLA) produced by FADS3 from trans-vaccenate is the trans11,cis13-CLA isomer. In rat hepatocytes, knockdown of FADS3 expression specifically reduces trans-vaccenate Δ13-desaturation. Evidence is presented that FADS3 is the first "methyl-end" fatty acid desaturase functionally characterized in mammals.

Keywords: FADS cluster; conjugated linoleic acid; desaturase activity; fatty acid desaturase; rumenic acid; trans-vaccenic acid; trans11,cis13-octadecadienoic acid.

Fig. 1.

Expression and desaturase activities of recombinant rat FADS1, FADS2, and FADS3 in COS-7 cells. (A) COS-7 cells were transfected with the following plasmids: pCMV empty (control cells), pCMV/FADS1, pCMV/FADS2, and pCMV/FADS3. Immunoblots were assessed on COS-7 cell extracts transiently expressing the recombinant FADS proteins. Recombinant FADS1, FADS2, and FADS3 were detected with polyclonal antibodies targeting specific antigenic peptides of these proteins. Quantitative estimation of protein loading was performed with an anti-actin antibody. Molecular weights are indicated in kDa. (B) GC-MS profiles of FAMEs extracted from COS-7 cells expressing rat FADS1, FADS2, or FADS3 and incubated with α-linolenic acid (18:3 n-3) (C) GC-MS profiles of FAMEs extracted from COS-7 cells expressing rat FADS1, FADS2, or FADS3 and incubated with eicosatrienoic acid (20:3 n-6). Control COS-7 cells and COS-7 cells transiently expressing the recombinant FADS proteins were incubated for 24 h with albumin-bound FA (200 µM). The identity of each important FA is indicated above its respective peak. Representative example of n = 5 distinct experiments.

Fig. 2.

GC-MS profiles of FAMEs extracted from COS-7 cells expressing rat FADS1, FADS2, or FADS3 and incubated with TVA. Control COS-7 cells and COS-7 cells transiently expressing the recombinant FADS proteins were cultivated for 24 h with albumin-bound TVA (200 µM). The identity of each important FA is indicated above its respective peak. Rumenic acid (R_t = 27.5 min) was detected in all the experimental conditions (control cells and transfected cells). Three different and unknown octadecadienoic acids (with FAME molecular ions at m/z 294) were produced in COS-7 cells expressing FADS1 (R_t = 21.9 min), FADS2 (R_t = 22.1 min,) and FADS3 (R_t = 28.2 min). Representative example of n = 5 distinct experiments.

Fig. 3.

GC-MS profiles of cellular FAMEs extracted from COS-7 cells expressing rat FADS1, FADS2, or FADS3 and incubated with rumenic acid (cis9,trans11-CLA). Control COS-7 cells and COS-7 cells transiently expressing the recombinant FADS proteins were cultivated for 24 h with albumin-bound rumenic acid (200 µM). The identity of each important FA is indicated above its respective peak. It shows that only the cells expressing FADS2 were able to use RA as a substrate. Indeed, detectable amounts of an octadecatrienoic acid (18:3) with FAME molecular ion at m/z 292 amu and R_t = 29.1 min were produced when compared with control cells. Although not fully characterized in the present study, this FA was identified as the cis6,cis9,trans11-18:3, the known Δ6-desaturated product of RA (–58), by using the NIST database. Representative example of n = 3 distinct experiments.

Fig. 4.

Identification of the unknown octadecadienoic acid produced from trans-vaccenate in cells expressing rat FADS3 as a Δ11,13-conjugated linoleic acid isomer. (A) Electron-impact mass spectrum of the DMOX derivative of the unknown ¹³C-octadecadienoic acid obtained after incubation of COS-7 cells expressing FADS3 with [1-¹³C]TVA. (B) GC-MS profile of the FAMEs extracted from COS-7 cells expressing FADS3 and incubated with TVA compared with commercial rumenic acid and a commercial mixture of cis11,trans13-18:2, cis11,cis13-18:2, and trans11,trans13-18:2 as FAME standards. (C) Electron-impact mass spectrum of the DMOX derivative of the commercial cis11,trans13-18:2.

Fig. 5.

Electron-impact mass spectra of DMOX derivatives of the different ¹³C-octadecadienoic acids obtained after incubation of [1-¹³C]TVA with COS-7 cells expressing either rat FADS1 or FADS2. Molecular ions (M+) are m/z 334. (A) ¹³C-rumenic acid (R_t = 27.5 min) obtained after incubation of [1-¹³C]TVA with COS-7 cells in all the experimental conditions. (B) Unknown ¹³C-octadecadienoic acid (R_t = 21.9 min) obtained after incubation of [1-¹³C]TVA with COS-7 cells expressing rat FADS1. A gap of 12 amu was shown between ions containing 10 and 11 carbons (m/z 223 and 235 amu) locating this double bond in position 11-12, while the ion at m/z 154 amu was a diagnostic guide for a double bond in position 5-6 (59). This fatty acid was therefore identified as Δ5,11-18:2 (cis5,trans11-18:2) (C) Unknown ¹³C-octadecadienoic acid (R_t = 22.1 min) obtained after incubation of [1-¹³C]TVA with COS-7 cells expressing rat FADS2. A gap of 12 amu was shown between ions containing 10 and 11 carbons (m/z 223 and 235 amu) locating this double bond in position 11-12, while the fingerprint ions at m/z 153, 167, and 181 amu identified a double bond in position 6-7 (60). This fatty acid was therefore identified as Δ6,11-18:2 (cis6,trans11-18:2). Representative example of n= 3 distinct experiments.

Fig. 6.

GC-MS profile of cellular FAMEs extracted from COS-7 cells expressing rat FADS1, FADS2, or FADS3 and incubated with cis-vaccenate. Control COS-7 cells and COS-7 cells transiently expressing the recombinant FADS proteins were cultivated for 24 h with albumin-bound cis-vaccenate (200 µM). The identity of each important FA is indicated above its respective peak. It shows that only the cells expressing FADS1, but not FADS2 or FADS3, were able to desaturate CVA into an octadecadienoic acid (R_t = 22.4 min), which was identified as the cis5,cis11-18:2 by the NIST database. Representative example of n = 3 distinct experiments.

Fig. 7.

GC-MS profiles of FAMEs extracted from cultured rat hepatocytes incubated with TVA and [1-¹³C]TVA. Rat hepatocytes were incubated for 24 h with albumin-bound trans-vaccenate, [1-¹³C]trans-vaccenate, or no FA (control medium). The identity of each important FA is indicated above its respective peak. Representative example of n = 5 distinct experiments.

Fig. 8.

Electron-impact mass spectra of DMOX derivatives of the different octadecadienoic acids obtained after incubation of TVA with cultured rat hepatocytes. Molecular ions (M+) are m/z 333. (A) Octadecadienoic acid (R_t = 28.2 min) similar to the suspected trans11,cis13-C18:2 obtained in COS-7 cells expressing rat FADS3. (B) Rumenic acid (R_t = 27.5 min). (C) Octadecadienoic acid (R_t = 22.1 min) similar to the cis6,trans11-C18:2 obtained in COS-7 cells expressing rat FADS2.

Fig. 9.

Effect of native FADS3 extinction and recombinant FADS3 expression on the levels of FADS3 mRNA detected by qRT-PCR and FADS3 protein detected by immunoblotting in culture rat hepatocytes. Hepatocytes were transfected with siRNA control or siRNA against Fads3 for 24 h and incubated for a further 24 h with TVA. Similarly, hepatocytes were transfected with the pCMV empty or pCMV/FADS3 for 24 h before incubation with TVA. (A, B) The cells were collected and analyzed for Fads1, Fads2, Fads3, and Scd1 mRNA expression by qRT-PCR in each experimental condition. (C) Expression of the 48 kDa immunoreactive band of FADS3 in rat hepatocytes assessed by immunoblot in each of the four distinct experiments realized. (D, E) Gray level quantification of the immunoreactive band at 48 kDa relative to actin in each experimental condition. Data are presented as mean ± SD (n = 4).

Fig. 10.

Effect of native FADS3 extinction and recombinant FADS3 expression on the conversion of trans-vaccenate into the suspected trans11,cis13-CLA in rat hepatocytes. Hepatocytes were transfected with siRNA control or siRNA against Fads3 for 24 h and incubated for a further 24 h with TVA. Similarly, hepatocytes were transfected with the pCMV empty or pCMV/FADS3 for 24 h before incubation with TVA. (A) GC-MS profiles of FAMEs extracted from cultured rat hepatocytes in each experimental condition. For this set of experiments, the GC column, which was previously used for all the other GC-MS analyses, was changed, and isomers of eicosenoic acid (20:1) were eluted in the same region of the chromatogram as the suspected trans11,cis13-CLA. (B, C) Quantification (percentage of total FA) of the most interesting FAs (TVA, cis9,trans11-CLA, cis6,trans11-18:2, and the suspected trans11,cis13-CLA) after GC-MS analysis of cellular FAMEs extracted from rat hepatocytes in each experimental condition. Data are presented as mean ± SD (n = 4).

Fig. 11.

Proposed metabolic pathway of trans-vaccenate conversion into cis9,trans11-CLA catalyzed by the Δ9-desaturase and into trans11,cis13-CLA by the newly discovered FADS3-catalyzed Δ13-desaturase.

Fig. 12.

Alignment of the amino acid sequences of rat, mouse, and human FADS1, FADS2, and FADS3 deduced from their nucleotide sequences. In the N-terminal end, the cytochrome b5-like domain (present in FADS1, FADS2, and FADS3 sequences) is surrounded by the blue box and characterized by a HPGG motif (black box). In the carboxyl-terminal end, the desaturase domain is characterized by three histidine boxes (red) that are necessary for the catalytic activity, which are present in all the sequences shown.