Advertisement
J. Lipid Res.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1194/jlr.C200019-JLR200 on January 16, 2003

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
C200019-JLR200v1
44/3/450    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Guillou, H.
Right arrow Articles by Legrand, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Guillou, H.
Right arrow Articles by Legrand, P.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Journal of Lipid Research, Vol. 44, 450-454, March 2003
Copyright © 2003 by Lipid Research, Inc.

Rapid Communication

Conversion of hexadecanoic acid to hexadecenoic acid by rat {Delta}6-desaturase

Hervé Guillou*, Vincent Rioux*, Daniel Catheline*, Jean-Noël Thibault§, Monique Bouriel*, Sophie Jan*,{dagger}, Sabine D'Andrea* and Philippe Legrand1,*

* Laboratoire de Biochimie, INRA-ENSA, Rennes, France
{dagger} Université du Maine, Le Mans, France
§ UMR-VP INRA-ENSA, Rennes, France

Published, JLR Papers in Press, January 16, 2003. DOI 10.1194/jlr.C200019-JLR200

1 To whom correspondence should be addressed. e-mail: philippe.legrand{at}agrorennes.educagri.fr


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 {Delta}6-desaturase in mammals. To investigate the putative involvement of the known {Delta}6-desaturase (FADS2) in this process, COS-7 cells expressing rat {Delta}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 {Delta}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 {Delta}6-desaturation.

Abbreviations: FCS, fetal calf serum; GC, gas chromatography; MS, mass spectrometry; pCMV/{Delta}6, rat {Delta}6-desaturase open reading frame inserted in pCMV vector; pCDNA3/{Delta}9, rat {Delta}9-desaturase open reading frame inserted in pcDNA3 vector

Supplementary key words desaturation • saturated fatty acids • FADS2 • animal

The presence of a palmitoyl-CoA {Delta}6-desaturase has recently been described in mice (1). This unexpected {Delta}6-desaturase activity was found in mice deficient for stearoyl-CoA desaturase 1 (SCD1-/- mice). However, this palmitoyl-CoA {Delta}6-desaturase activity has not yet been ascribed to any known desaturase gene.

The {Delta}6-desaturase (FADS2) has been cloned in several mammalian species (2, 3). In a previous work (4), we reported that recombinant rat {Delta}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 {Delta}6-desaturase.

In the present work, we investigated the putative involvement of FADS2 protein in the {Delta}6-desaturation of palmitic acid. We established that C16:0 is desaturated to C16:1 n-10 by COS-7 cells when expressing rat {Delta}6-desaturase. Moreover, coexpression of both {Delta}6-desaturase and SCD1 resulted in both {Delta}6- and {Delta}9-desaturation of palmitic acid. Therefore, this study reports for the first time that a single gene encodes a mammalian {Delta}6-desaturase that acts on one saturated fatty acid in addition to act on PUFAs.


MATERIALS AND METHODS

Chemicals
Fetal 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 [1-14C]palmitic acid was purchased from Perkin Elmer Life Sciences (Paris, France). The anti-rat liver {Delta}9-desaturase serum was a generous gift from Dr J. Ozols (7).

Plasmid construction for expression of rat {Delta}6- and {Delta}9-desaturases
The plasmid constructed for expression of rat {Delta}6-desaturase in mammalian cells (referred to as pCMV/{Delta}6) has already been described (4). A plasmid coding for rat {Delta}9-desaturase (SCD1) was constructed for expression in mammalian cells and is referred to as pcDNA3/{Delta}9. From the published (8) rat SCD1 sequence (GenBank accession number J02585), oligonucleotide primers were designed to PCR amplify the entire coding sequence with its stop codon using the high fidelity Pfu polymerase from Promega (Lyon, France). The forward primer (5'-CAATGGATCCATGCCGGCCCACATGC-3') included the translation start codon (in bold) and BamHI restriction site (underlined). The reverse primer (5'-CGTGCTCGAGCTCAGCTACTCTTGTGGCT-3') contained the translation stop codon (in bold) and XhoI restriction site (underlined). The PCR product amplified from rat liver cDNA was treated with BamHI and XhoI before cloning into pcDNA3 (Invitrogen, San Diego, CA). The integrity of the construct was confirmed by DNA sequencing.

Expression of rat {Delta}6- and {Delta}9-desaturases in COS-7 cells
Rat {Delta}6- and {Delta}9-desaturases were expressed by transiently transforming COS-7 cells with pCMV/{Delta}6 and pcDNA3/{Delta}9. COS-7 cells were routinely maintained at about 50% confluence and were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% FCS, 50 IU/ml penicillin, and 50 µg/ml streptomycin. The cells were split 1 day before transfection to 30% confluence and transfected the following day by using the Easyject Plus electroporator (Equibio, Monchelsea, UK). Briefly, 106 COS-7 cells in 0.8 ml of DMEM were mixed with 30 µg of purified plasmid, electroporated at 250 V and 1,500 µF with unlimited resistance, and seeded on a 10 cm dish (Falcon, AES, Combourg, France) containing culture medium. For cotransfection of {Delta}6- and {Delta}9-desaturases, 30 µg of each plasmid was mixed before electroporation.

Western blotting
Reduced protein samples were analyzed by SDS-PAGE and blotted onto nitrocellulose (Schleicher and Schuell, Dassel, Germany). Membranes were probed with anti-rat {Delta}6-desaturase or anti-rat {Delta}9-desaturase sera as previously described (4, 7). Rabbit antibodies were revealed with horseradish-peroxidase-conjugated sheep anti-rabbit IgG (Sigma). Peroxidase activity was revealed by following the procedure provided for the ECL Plus reagent detection system kit (Amersham Biosciences, Les Ulis, France).

Incubation of transfected COS-7 cells with fatty acid albuminic complexes
The activity of the expressed protein was investigated by incubating the transfected COS-7 cells with different fatty acid albuminic complexes. Fatty acid albuminic complexes were prepared as previously described (4, 9). The final fatty acid concentration of the incubation medium was 0.1 mM. When incubations were carried out with radiolabeled palmitic acid, the specific radioactivity was 37 MBq/mmol. At 3 h post-transfection, the incubation of COS-7 cells was initiated by replacing the culture medium with 10 ml of the fatty acid-containing medium per dish. Incubation was carried out for 24 h at 37°C in 5% CO2 atmosphere.

Fatty acid analysis
COS-7 cells were washed twice with ice-cold PBS and scraped in PBS. Cellular lipids were extracted with hexane-isopropanol (3:2, v/v) as previously described (9). After saponification, fatty acids were either methylated or naphthacylated (4).

Fatty acid methyl esters were extracted with pentane and analyzed by gas chromatography (GC) using an Agilent Technologies 6890N (Bios Analytique, Toulouse, France) with a split injector (1:20) at 250°C and a bonded silica capillary column (30 m x 0.25 mm ID, BPX 70, SGE, Villeneuve-St-Georges, France) with a stationary phase of 70% cyanopropylpolysilphenylene-siloxane (0.25 µm film thickness). Helium was used as gas vector (average velocity 24 cm/s). The column temperature program started at 150°C, ramping at 2°C per min to 220°C and holding at 220°C for 10 min. The flame ionisation detector temperature was 250°C. Identification of the fatty acid methyl esters was based upon retention times obtained for methyl ester standards. A C16:1 n-10 methyl ester standard was prepared from Thunbergia alata seeds that contain 80% C16:1 n-10 (10). To confirm C16:1 n-10 identity, fatty acids extracted from COS-7 cells were converted to 4,4-dimethyloxazoline derivatives (11) and analyzed by GC-mass spectrometry (GC-MS) in electron impact ionization mode (GC 8060 chromatograph coupled to a VG Platform II, Fisons Instruments, Altrincham, England). The column and gas vector were similar to those used for GC analysis.

Fatty acid naphthacyl esters were extracted and analyzed by HPLC as described earlier (4).


RESULTS

Recombinant rat {Delta}6-desaturase and rat SCD1 expressed in COS-7 cells were analyzed by Western blot. Figure 1 shows the result of Western blot analysis of total homogenates obtained from COS-7 cells transfected with pCMV/{Delta}6 or both pcDNA3/{Delta}9 and pCMV/{Delta}6 versus nontransfected cells. A single band (45 kDa) was detected in COS-7 cells expressing rat {Delta}6-desaturase (Fig. 1, lanes 2 and 3). A major (42 kDa) band was detected in COS-7 cells expressing rat SCD1 (Fig. 1, lane 3). These bands were not revealed in nontransfected COS-7 cells (Fig. 1, lane 1).



View larger version (11K):
[in this window]
[in a new window]
  		Fig. 1. Detection of rat {Delta}6- and {Delta}9-desaturases in transfected COS-7 cells by Western blot analysis. COS-7 cells were not transfected (lane 1), transfected with rat {Delta}6-desaturase opening frame inserted in pCMV vector (pCMV/{Delta}6) (lane 2) or transfected with both pCMV/{Delta}6 and rat {Delta}9-desaturase opening frame inserted in pcDNA3 vector (pcDNA3/{Delta}9) (lane 3). COS-7 total cell homogenates (2 µg of protein) were resolved by SDS-PAGE and blotted. The blot was immunoprobed with anti-rat {Delta}6-desaturase sera or anti-rat {Delta}9-desaturase sera. Detection was performed using chemifluorescence (ECL Plus detection system).

 
In order to investigate the putative involvement of rat FADS2 protein in the {Delta}6-desaturation of palmitic acid, COS-7 cells transfected with pCMV/{Delta}6 were grown for 24 h in culture medium supplemented with palmitic acid (C16:0). COS-7 cells transfected with pCMV/{Delta}6 produced a detectable amount of an unknown fatty acid (Fig. 2A) when compared with nontransfected COS-7 cells (Fig. 2B). GC-MS analysis allowed the identification of this fatty acid as C16:1 n-10, the {Delta}6-desaturated product of C16:0 (Fig. 2C). We therefore established that rat {Delta}6-desaturase expressed in COS-7 cells confers to these cells the ability to synthesize hexadecenoic acid (C16:1 n-10). Similarly, when radiolabeled palmitic acid was incubated with COS-7 cells expressing rat {Delta}6-desaturase, radiolabeled C16:1 n-10 was synthesized (Fig. 3) .



View larger version (22K):
[in this window]
[in a new window]
  		Fig. 2. Gas chromatography (GC) analysis of cellular fatty acid methyl esters from COS-7 cells incubated with C16:0 and expressing rat {Delta}6-desaturase (A) versus control COS-7 cells (B). GC-mass spectrometry (MS) identification of C16:1 n-10 (C). COS-7 cells transfected with pCMV/{Delta}6 (A) or nontransfected (B) were cultivated for 24 h with albumin-bound C16:0 (100 µM). The identity of each fatty acid peak is indicated above the respective peak. The unidentified peak was confirmed to be C16:1 n-10 by GC-MS analysis of 4,4-dimethyloxazoline derivatives (C).

 


View larger version (15K):
[in this window]
[in a new window]
  		Fig. 3. HPLC analysis of cellular fatty acid naphthacyl esters from COS-7 cells incubated with [1-14C]C16:0 and expressing rat {Delta}6-desaturase versus control COS-7 cells. COS-7 cells transfected with pCMV/{Delta}6 (large line) or nontransfected (dotted line) were cultivated for 24 h with albumin-bound [1-14C]C16:0 (100 µM, 37 MBq/mmol). The identity of each fatty acid peak is indicated above the respective peak.

 
When COS-7 cells were cotransfected with both pCMV/{Delta}6 and pcDNA3/{Delta}9 and grown for 24 h in culture medium supplemented with palmitic acid (C16:0), the amount of both C16:1 n-10 (2% of total fatty acids) and C16:1 n-7 was higher than in nontransfected (0.9% of total fatty acids) (Fig. 4) . However, the C16:1 n-10 production was lower than in cells only transfected with pCMV/{Delta}6 (3.7% of total fatty acids) (Fig. 4).



View larger version (19K):
[in this window]
[in a new window]
  		Fig. 4. GC analysis of cellular fatty acid methyl esters from COS-7 cells incubated with C16:0 and expressing both rat {Delta}6-desaturase and {Delta}9-desaturase (A), or only rat {Delta}6-desaturase (B), versus control COS-7 cells (C). COS-7 cells transfected with both pCMV/{Delta}6 and pcDNA3/{Delta}9 (A), with pCMV/{Delta}6 only (B), or nontransfected (C) were cultivated for 24 h with albumin-bound C16:0 (100 µM). The identity of each fatty acid peak is indicated above the respective peak. C17:0 was added as an internal standard.

 
Finally, when COS-7 cells transfected with pCMV/{Delta}6 were incubated with both palmitic acid and {alpha}-linolenic acid, the production of C16:1 n-10 was still observed while C18:3 n-3 was converted to C18:4 n-3 as well (Fig. 5) .



View larger version (17K):
[in this window]
[in a new window]
  		Fig. 5. GC analysis of cellular fatty acid methyl esters from COS-7 cells incubated with both C16:0 and C18:3 n-3 and expressing rat {Delta}6-desaturase (A) versus control COS-7 cells (B). COS-7 cells transfected with pCMV/{Delta}6 (A) or nontransfected (B) were cultivated for 24 h with albumin-bound fatty acids: 80 µM C16:0, 20 µM C18:3n-3. The identity of each fatty acid peak is indicated above the respective peak. C17:0 was added as an internal standard.

 

DISCUSSION

This study was designed to address the possible role of the characterized rat {Delta}6-desaturase (2, 4) in the conversion of palmitic acid to hexadecenoic acid (C16:1 n-10). We provide evidence that the expression of rat {Delta}6-desaturase in COS-7 cells (Fig. 1) leads to a significant conversion of C16:0 to C16:1 n-10 when cells were incubated with C16:0 (Fig. 2, 3).

Our results show that the biosynthesis of C16:1 n-10 was limited when COS-7 cells expressed both rat {Delta}6- and {Delta}9-desaturases as compared with COS-7 cells expressing {Delta}6-desaturase only (Fig. 4). The presence of high amounts of C16:1 n-10 has initially been reported in the preputial gland of SCD1-/- mice (1). Consistent with this study, our present results suggest that C16:1 n-10 biosynthesis may occur in tissues with high {Delta}6-desaturase expression and low SCD1 expression.

In the presence of both C16:0 and C18:3 n-3, the latter being a well-characterized substrate for rat {Delta}6-desaturase (4), the {Delta}6-desaturation of palmitic acid was not abolished, while conversion of C18:3 n-3 to C18:4 n-3 was also observed (Fig. 5). This result assesses that a high level of {Delta}6-desaturase expression simultaneously allows the {Delta}6-desaturation of C16:0 and C18:3 n-3. However, Miyazaki and coworkers (1) suggested that the conversion of C16:0 to C16:1 n-10 could be performed by an enzyme distinct from FADS2. From our study, it appears unlikely that an additional enzyme desaturates C16:0 to C16:1 n-10. Indeed, in the FADS gene cluster (12) identified in mammals, FADS1 is a {Delta}5-desaturase (5, 13). Moreover, we recently isolated FADS3 gene cDNA from rat (GenBank accession number AJ494720). When transfection studies were conducted for expression of rat FADS3 in COS-7 cells, the protein encoded by this cDNA was not able to desaturate C16:0 to C16:1 n-10 (data not shown).

Therefore, we show that FADS2 acts as a palmitoyl-CoA {Delta}6-desaturase and present the first evidence that a mammalian gene (FADS2) encodes a desaturase that acts on both PUFAs and one saturated fatty acid.


ACKNOWLEDGMENTS

The antibody to rat liver microsomal stearoyl-CoA desaturase 1 was a generous gift from Dr. Juris Ozols (University of Connecticut Health Center, Farmington, CT). The authors thank A. Leborgne and K. L. Cung for able technical assistance.

Manuscript received November 22, 2002 and in revised form December 23, 2002.


REFERENCES

  1. Miyazaki, M., F. E. Gomez, and J. M. Ntambi. 2002. Lack of stearoyl-CoA desaturase-1 function induces a palmitoyl-CoA {delta}6 desaturase and represses the stearoyl-CoA desaturase-3 gene in the preputial glands of the mouse. J. Lipid Res. 43: 2146–2154.[Abstract/Free Full Text]

  2. Aki, T., Y. Shimada, K. Inagaki, H. Higashimoto, S. Kawamoto, S. Shigeta, K. Ono, and O. Suzuki. 1999. Molecular cloning and functional characterization of rat delta-6 fatty acid desaturase. Biochem. Biophys. Res. Commun. 255: 575–579.[CrossRef][Medline]

  3. Cho, H. P., M. T. Nakamura, and S. D. Clarke. 1999. Cloning, expression, and nutritional regulation of the mammalian Delta- 6 desaturase. J. Biol. Chem. 274: 471–477.[Abstract/Free Full Text]

  4. D'Andrea, S., H. Guillou, S. Jan, D. Catheline, J-N. Thibault, M. Bouriel, V. Rioux, and P. Legrand. 2002. The same rat {Delta}6-desaturase not only acts on 18- but also on 24-carbon fatty acids in very-long-chain polyunsaturated fatty acid biosynthesis. Biochem. J. 364: 49–55.[Medline]

  5. de Antueno, R. J., L. C. Knickle, H. Smith, M. L. Elliot, S. J. Allen, S. Nwaka, and M. D. Winther. 2001. Activity of human {Delta}5 and {Delta}6 desaturases on multiple n-3 and n-6 polyunsaturated fatty acids. FEBS Lett. 509: 77–80.[CrossRef][Medline]

  6. Sprecher, H. 2000. Metabolism of highly unsaturated n-3 and n-6 fatty acids. Biochim. Biophys. Acta. 1486: 219–231.[Medline]

  7. Ozols, J. 1997. Degradation of hepatic stearyl CoA {Delta}9-desaturase. Mol. Biol. Cell. 8: 2281–2290.[Abstract/Free Full Text]

  8. Thiede, M. A., J. Ozols, and P. Strittmatter. 1986. Construction and sequence of cDNA for rat liver stearyl Coenzyme A desaturase. J. Biol. Chem. 28: 13230–13235.

  9. Rioux, V., P. Lemarchal, and P. Legrand. 2001. Myristic acid, unlike palmitic acid, is rapidly metabolized in cultured rat hepatocytes. J. Nutr. Biochem. 11: 198–207.

  10. Spencer, G. F., R. Kleiman, R. W. Miller, and F. R. Earle. 1971. Occurrence of cis-hexadecenoic acid as the major component of Thunbergia alata seed oil. Lipids. 6: 712–714.[CrossRef]

  11. Luthria, D. L., and H. Sprecher. 1993. 2-Alkenyl-4,4-dimethyloxazolines as derivatives for the structural elucidation of isomeric unsaturated fatty acids. Lipids. 28: 561–564.[CrossRef][Medline]

  12. Marquardt, A., H. Stohr, K. White, and B. H. Weber. 2000. cDNA cloning, genomic structure, and chromosomal localization of three members of the human fatty acid desaturase family. Genomics. 66: 175–183.[CrossRef][Medline]

  13. Cho, H. P., M. Nakamura, and S. D. Clarke. 1999. Cloning, expression, and fatty acid regulation of the human delta-5 desaturase. J. Biol. Chem. 274: 37335–37339.[Abstract/Free Full Text]


Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Lipid Res.Home page
W. J. Park, K. S. D. Kothapalli, P. Lawrence, C. Tyburczy, and J. T. Brenna
An alternate pathway to long-chain polyunsaturates: the FADS2 gene product {Delta}8-desaturates 20:2n-6 and 20:3n-3
J. Lipid Res., June 1, 2009; 50(6): 1195 - 1202.
[Abstract] [Full Text] [PDF]

Home page
 J. Lipid Res.Home page
H. Guillou, S. D'Andrea, V. Rioux, R. Barnouin, S. Dalaine, F. Pedrono, S. Jan, and P. Legrand
Distinct roles of endoplasmic reticulum cytochrome b5 and fused cytochrome b5-like domain for rat {Delta}6-desaturase activity
J. Lipid Res., January 1, 2004; 45(1): 32 - 40.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
C200019-JLR200v1
44/3/450    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Guillou, H.
Right arrow Articles by Legrand, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Guillou, H.
Right arrow Articles by Legrand, P.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Journal of Biological Chemistry 
 Molecular and Cellular Proteomics   ASBMB Today 
Advertisement
spacer
Advertisement
Advertisement