Conversion of hexadecanoic acid to hexadecenoic acid by rat Δ6-desaturase

Abstract

A higher content of C16:1 n-10 has recently been reported in the preputial gland of mice with a targeted disruption of the gene encoding stearoyl-CoA desaturase 1 (SCD1 ؊ / ؊ mice) when compared with wild-type mice. This result has provided the first physiological evidence for the presence and regulation of a palmitoyl-CoA ⌬ 6-desaturase in mammals. To investigate the putative involvement of the known ⌬ 6-desaturase (FADS2) in this process, COS-7 cells expressing rat ⌬ 6-desaturase were incubated with C16:0. Transfected cells were able to synthesize C16:1 n-10, while nontransfected cells did not produce any C16:1 n-10. Evidence is therefore presented that the rat ⌬ 6-desaturase, which acts on the 18-and 24-carbon fatty acids of the n-6 and n-3 series, is also able to catalyze palmitic acid ⌬ 6-desaturation. The presence of a palmitoyl-CoA ⌬ 6-desaturase has recently been described in mice (1). This unexpected ⌬ 6-desaturase activity was found in mice deficient for stearoyl-CoA desaturase 1 (SCD1 Ϫ / Ϫ mice). However, this palmitoyl-CoA ⌬ 6-desaturase activity has not yet been ascribed to any known desaturase gene.The ⌬ 6-desaturase (FADS2) has been cloned in several mammalian species (2, 3). In a previous work (4), we reported that recombinant rat ⌬ 6-desaturase expressed in COS-7 cells acts on 18-and 24-carbon fatty acids of the n-3 and n-6 series in polyunsaturated fatty acid (PUFA) biosynthesis. This result, consistent with experiments performed in yeast (5) and the PUFA biosynthetic pathway initially proposed by Sprecher and coworkers (6), illustrated the broad chain length specificity of the ⌬ 6-desaturase.In the present work, we investigated the putative involvement of FADS2 protein in the ⌬ 6-desaturation of palmitic acid. We established that C16:0 is desaturated to C16:1 n-10 by COS-7 cells when expressing rat ⌬ 6-desaturase. Moreover, coexpression of both ⌬ 6-desaturase and SCD1 resulted in both ⌬ 6-and ⌬ 9-desaturation of palmitic acid. Therefore, this study reports for the first time that a single gene encodes a mammalian ⌬ 6-desaturase that acts on one saturated fatty acid in addition to act on PUFAs. MATERIALS AND METHODS ChemicalsFetal calf serum (FCS) was purchased from Perbio (Bezons, France). Solvents were purchased from Fischer Scientific (Elancourt, France). Fatty acids and other reagents were from Sigma (St Quentin Fallavier, France). Radiolabeled [1-14 C]palmitic acid was purchased from Perkin Elmer Life Sciences (Paris, France). The anti-rat liver ⌬ 9-desaturase serum was a generous gift from Dr J. Ozols (7). Plasmid construction for expression of rat ⌬ 6-and ⌬ 9-desaturasesThe plasmid constructed for expression of rat ⌬ 6-desaturase in mammalian cells (referred to as pCMV/ ⌬ 6) has already been described (4). A plasmid coding for rat ⌬ 9-desaturase (SCD1) was constructed for expression in mammalian cells and is referred to as pcDNA3/ ⌬ 9. From the published (8) rat SCD1 sequence (GenBank accession number J02585), oligonucleotide primers were designed to PCR amplify the entire codi...

Trans-vaccenate is Δ13-desaturated by FADS3 in rodents

“…1 and data not shown). These results are in accordance with previous studies that have used this recombinant model to demonstrate the surprising diversity of ⌬ 6-desaturase substrates ( 34 ), including the conversion of 24:5 n-3 into 24:6 n-3, 24:4 n-6 into 24:5 n-6 ( 24 ), and 16:0 into 16:1 n-10 ( 29 ).…”

“…1C ). These results validated that our recombinant model was consistent with the known in vivo ⌬ 5-and ⌬ 6-desaturase activities and FA substrates of FADS1 and FADS2 ( 24,29 ) and showed that FADS3 was not functionally redundant with FADS1 and FADS2. Similar experiments carried out with saturated FA (12:0, 14:0, 16:0, and 18:0) also showed that FADS3 displayed no ⌬ 9-desaturase activity (data not shown).…”

“…For positional analysis of carbon double bonds, FAMEs were converted to 4,4-dimethyloxazoline (DMOX) derivatives ( 29,30 ) and analyzed by GC-MS in electron impact ionization mode. The column, gas vector, and detector were similar to those used for the analysis of FAMEs (see above).…”

Improvement of Atopic Dermatitis by Human Sebaceous Fatty Acids and Related Lipids

“…Human and rat fatty acid desaturase 2 (FADS2) -encoding delta 6-desaturases recognize a saturated fatty acid as a substrate under some conditions (Ge et al, 2003;Guillou et al, 2003), and a human delta 6-desaturase gene has been reported to be expressed only in the sebaceous gland. Regarding microbial delta 6-desaturases, industrial producers of unsaturated fatty acids have focused mainly on fungi of the Mortierella spp., especially Mortierella alpina and Mortierella cincinelloides.…”

Identification of amino acid residues that determine the substrate specificity of mammalian membrane-bound front-end fatty acid desaturases

“…The histidine residue is indispensable for electron transfer from NADH-dependent cytochrome b 5 reductase during the redox reaction ( 5, 6 ). Both this motif and that of diffused cytochrome b 5 are necessary to fully express desaturase activity ( 7,8 ). The other hydrophilic regions contain three histidine clusters (HX 3-4 H, HX 2-3 HH, and QX 2-3 HH) that form a catalytic center by coordinating nonheme diiron centers, and all of these histidine residues and the glutamine residue are essential for enzymatic activity ( 9, 10 ).…”