Cloning and characterization of a mammalian fatty acyl-CoA
elongase as a lipogenic enzyme regulated by SREBPs
Takashi Matsuzaka,* Hitoshi Shimano,1,* Naoya Yahagi,† Tomohiro Yoshikawa,†
Michiyo Amemiya-Kudo,† Alyssa H. Hasty,† Hiroaki Okazaki,† Yoshiaki Tamura,† Yoko Iizuka,†
Ken Ohashi,† Jun-ichi Osuga,† Akimitsu Takahashi,* Shigeru Yato,* Hirohito Sone,*
Shun Ishibashi,† and Nobuhiro Yamada*
Department of Internal Medicine,* Institute of Clinical Medicine, University of Tsukuba, Ibaraki 305-8575, Japan;
and Department of Metabolic Diseases,† University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan

Abstract The mammalian enzyme involved in the final
elongation of de novo fatty acid biosynthesis following thebuilding of fatty acids to 16 carbons by fatty acid synthasehas yet to be identified. In the process of searching for genes
activated by sterol regulatory element-binding protein 1(SREBP-1) by using DNA microarray, we identified and characterized a murine cDNA clone that is highly similar to afatty acyl-CoA elongase gene family such as Cig30, Sscs, andyeast ELOs. Studies on the cells overexpressing the fulllength cDNA indicate that the encoded protein, designatedfatty acyl-CoA elongase (FACE), has a FACE activity specificfor long-chains; C12-C16 saturated- and monosaturated-fatty
acids. Hepatic expression of this identified gene was consistently activated in the livers of transgenic mice overexpressing nuclear SREBP-1a, -1c, or -2. FACE mRNA levels aremarkedly induced in a refed state after fasting in the liver
and adipose tissue. This refeeding response is significantly
reduced in SREBP-1 deficient mice. Dietary PUFAs caused a
profound suppression of this gene expression, which could
be restored by SREBP-1c overexpression. Hepatic FACE expression was also highly up-regulated in leptin-deficient ob/
ob mice. Hepatic FACE mRNA was markedly increased byadministration of a pharmacological agonist of liver X-activated receptor (LXR), a dominant activator for SREBP-1c
expression. These data indicated that this elongase is a
new member of mammalian lipogenic enzymes regulated by
SREBP-1, playing an important role in de novo synthesis of
long-chain saturated and monosaturated fatty acids in conjunction with fatty acid synthase and stearoyl-CoA desaturase.—Matsuzaka, T., H. Shimano, N. Yahagi, T. Yoshikawa,
M. Amemiya-Kudo, A. H. Hasty, H. Okazaki, Y. Tamura, Y. Iizuka, K. Ohashi, J-i. Osuga, A. Takahashi, S. Yato, H. Sone, S.
Ishibashi, and N. Yamada. Cloning and characterization of a
mammalian fatty acyl-CoA elongase as an lipogenic enzyme
regulated by sterol regulatory element-binding proteins. J.
Lipid Res. 2002. 43: 911–920.
Supplementary key words
lipogenesis • nutrition

ids with lengths of 16–18 carbon atoms, constituting the
majority of total fatty acids in the cells, are major productsof de novo synthesis in most mammalian tissues. Theselong chain fatty acids play an important role in cellular biological functions, including: energy metabolism, membrane fluidity, and others. There appear to be several distinct metabolic pathways that produce long chain fatty
acids. Cytoplasmic fatty acid synthase (FAS) plays a majorrole in the de novo synthesis of fatty acids. However, theelongation of fatty acids by this enzyme terminates atpalmitic acid (C16:0). The end product of mammalian lipogenesis is usually oleic acid (C18:1n-9) or vaccenic acid
(C18:1n-7) (1, 2). Mammals have long been thought topossess a membrane bound enzyme that elongates and/or
desaturates saturated fatty acyl-CoAs produced by FAS or
derived from dietary resources (3). Stearoyl-CoA desaturase (SCD) has been shown to be committed to the desaturation; however, the gene catalyzing for the C2 elongationof the C16:0 and C16:1 has never been identified.
To date, several enzymes involved in the elongation of
long-chain fatty acids in non-mammalian cells have been
identified. One such family consists of the yeast ELO genes.
The yeast ELO1 gene is involved in the elongation of C14:0
to C16:0 (4). The ELO2 and ELO3 genes were identified
based on the homology to the ELO1 gene (5). ELO2 protein is involved in the elongation of saturated and monounsaturated fatty acids up to 24 carbons in length, while ELO3
elongates a broader group of saturated and monounsaturated fatty acids, and is essential for the conversion of
C24:0 into C26:0 (5). Another known elongase family,
Cig30, which was originally identified as a cold-induced
gene in brown fat, is the first mouse fatty acid elongase iden-sterol regulatory element-binding protein •Biosynthesis of fatty acids is the major function part oflipogenesis in its role as an energy storage system. Fatty acCopyright © 2002 by Lipid Research, Inc.
This paper is available online at http://www.jlr.org

Abbreviations: ACC, acetyl-CoA carboxylase; ACBP, acyl-CoA binding protein; ACS, acyl-CoA synthetase; FACE, fatty acyl-CoA elongase;
FAS, fatty acid synthase; LXR, liver X-activated receptor; RXR, retinoic
acid receptor; SCD, stearoyl-CoA desaturase; SREBP, sterol regulatory
element-binding protein; WAT, white adipose tissue.
1To whom correspondence should be addressed.
e-mail: shimano-tky@umin.ac.jp

Journal of Lipid Research Volume 43, 2002

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tified (6). Ssc1 and Ssc2 were cloned based on the homology to Cig30 (7). Complementation studies in yeast mutants
indicated that Cig30 and SSC1 are functionally orthologous
to ELO2 and ELO3, respectively. The specific activity of Ssc2
has not been identified. Elovl4, a retinal photoreceptor-specific gene, plays a role in the elongation of very long chain
fatty acids and has been reported to be a causative gene forinherited macular degeneration (8). HELO1, as a memberof HELO family cloned by homology to ELO2, is involved in
the elongation of PUFAs and monounsaturated fatty acids,
whereas the specific activity of HELO2 has not been not
identified (9). These elongase could consist of a family thatshare a common structure of at least five membrane-spanning regions and a single histidine-box motif.
Sterol regulatory element-binding proteins (SREBPs) are
membrane-bound transcription factors that belong to thebasic helix-loop-helix leucine zipper family (10–12). SREBPs
have been established as a regulator for biosynthesis of bothcholesterol and fatty acids. To exert transcriptional activity
on SRE-containing SREBP target genes, SREBPs have to undergo proteolytic cleavage in a complex with a sterol-sensing
cofactor, SREBP-cleavage activating protein, that escorts
SREBP for a rER-Golgi trafficking, a key step for regulationof cellular cholesterol biosynthesis. There are three isoforms of SREBP that have been characterized, SREBP-1aand -1c (also known as ADD1), and SREBP-2 (13–15). Lipogenic enzymes, which are involved in energy storage
through synthesis of fatty acids and triglycerides, are coordinately regulated at the transcriptional level during different
metabolic states. Recent in vivo studies demonstrated that
SREBP-1c plays a crucial role in the dietary regulation ofmost hepatic lipogenic genes, whereas SREBP-2 is actively
involved in the transcription of cholesterogenic enzymes.
These include studies of the effects of the absence or overexpression of SREBP-1 on hepatic lipogenic gene expression (16–18), as well as physiological changes of SREBP-1c
protein in normal mice after dietary manipulation, such asplacement on high carbohydrate diets, PUFA-enriched diets, and fasting-refeeding regimens (19–22). Previous reports on the regulation of SREBP-1c have all demonstrated
the induction to be at the mRNA level. Promoter analysis revealed that the expression of the SREBP-1c gene is regulated by two factors: SREBP itself, forming an autoloop, andliver X-activated receptor/retinoic acid receptor (LXR/
RXR) oxysterol receptor (23–25, 35).
In the screening of SREBP-activated genes, we cloned a
mammalian fatty acid elongase, designated fatty acyl-CoA
elongase (FACE). Our current data suggest that the enzyme activity of this clone explains the missing identity stepin the conversion of C16 to C18 fatty acids. In addition, thenutritional regulation of this SREBP-regulated FACE expression is consistent with its roles as a lipogenic enzyme.

MATERIALS AND METHODS
Materials and general methods
We purchased fatty acids from Sigma, restriction enzymes
from New England Biolabs, redivue [-32P]dCTP (6,000 Ci/

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Journal of Lipid Research Volume 43, 2002mmol) from Amersham Pharmacia, and radioactive [2-14C]malonyl-CoA (51 mCi/mmol) from New England Nuclear. Standard
molecular biology techniques were used. DNA sequencing was
performed with the CEQ™ dye terminator cycle sequencing kit
and CEQ2000 DNA Analysis System (Beckman Coulter).

Preparation of SREBP-1a transgenic liver cDNA library
An expression cDNA library of SREBP-1a transgenic liver (16)was prepared as previously described for construction of a cDNA
library of SREBP-1 deficient mouse adipose tissue, except thatpoly(A) RNA was prepared from livers of SREBP-1a transgenic
mice (25).

cDNA cloning and sequencing
From a DNA microarray system using hepatic poly(A) RNA
of SREBP-1a transgenic and non-transgenic littermate mice
(GenomeIncyte), we identified an EST clone (GenBank ID number AA239254) that was activated 19.5-fold in SREBP-1a transgenic liver as compared with wild-type liver. Using this sequence
information, a [-32P]dCTP labeled DNA probe was preparedand used in the screening of an SREBP-1a transgenic mouse liver
cDNA library by colony hybridization. Positive clones were sequenced; however, the clones were the 3-fragment of the cDNA.
To isolate the 5 ends of the clones, the 5-RACE method wasused. Poly(A) RNA was isolated using oligo-dT Latex (TaKaRa)
from the liver of a SREBP-1a transgenic mouse and was used for
cDNA synthesis and amplification with the 5-Full RACE Core Set(TaKaRa). The cDNA sequence was subjected to BLAST searchof mouse EST database and UniGene mouse database. The
search revealed a cluster of mouse EST sequences (UniGene
cluster ID number Mm. 26171) which contained a single open
reading frame of 822 bp with similarity to Cig30, another known
mouse fatty acid elongase. The putative elongase gene was tentatively designated FACE. Primers TMBSP1 (5-TGG ATG CGG
ACG CTG GGA GG–3) and TMBAP1 (5-AGT TGC ACT CAGCGA GTC CT-3) were designed based on the putative FACE sequence and used to amplify the full length FACE cDNA fromSREBP-1a transgenic mouse liver cDNA. The 1.1 kb PCR amplified product was subcloned into pGEM-T easy vector (Promega)and sequenced. The FACE cording sequence was isolated fromthis plasmid by NotI digestion and blunt ended using DNA Blunting Kit (TaKaRa), and inserted into SmaI site of expression
CMV7 vector.

Preparation of microsomes from transfected HEK-293 cells
HEK-293 cells were grown at 37C in an atmosphere of 5% CO2
in DMEM containing 25 mM glucose, 100 U/ml penicillin, and100 g/ml streptomycin sulfate supplemented with 10% FBS on
100 mm culture plates. At 80% confluency, the mouse FACE expression plasmid or the empty plasmid CMV7 (10 g) was transfected into cells using SuperFect Transfection Reagent (Qiagen)according to the manufacturer’s protocol. After transfection, cells
were incubated with DMEM plus 10% FBS for 24 h. The microsomal fractions from the cells were prepared as previously described, with some modification (26). Twenty four hours after thetransfection, cells were washed with PBS and scraped in 5 ml of
ice-cold 0.25 M sucrose, 0.02 M HEPES, pH7.5. The cells were
washed and resuspended in 3 ml of ice-cold sucrose/HEPES anddounce-homogenized. The homogenate was centrifuged 1,000 g
for 7 min at 4C. The pellet was resuspended in 1 ml of sucrose/
HEPES, dounce-homogenized, and the suspension was centrifuged at 1,000 g for 7 min at 4C. The supernatants were combined and re-centrifuged at 2,000 g for 30 min at 4C. Supernatant from this centrifugation was centrifuged at 105,000 g for 60
min at 4C. The resultant pellets were resuspended in 100 l of
0.1 M Tris-HCl, pH 7.4 and used for fatty acid elongation assay.

Fatty acid elongation assay
Microsomal fatty acid elongation activity was assayed by themeasurement of [2-14C]malonyl-CoA incorporation into exogenous acyl-CoAs as described previously (27), with some modification. The assay mixtures (0.25 ml total, including protein addition) contained 100 M Tris-HCl, pH 7.4, 60 M palmitoyl-CoA,
500 M NADPH, and 30 g of freshly obtained microsomal protein. After 2 min of preincubation at 37C, the reaction was initiated by the addition of 60 M malonyl-CoA (containing 0.037
Ci of [2-14C]malonyl-CoA) and carried out for 5 min at 37C.
The incubation was terminated by addition of 0.5 ml of 15%
KOH in methanol and saponified at 65C for 45 min. Then thesamples were cooled and acidified with 0.5 ml of ice-cold 5 N
HCl. Free fatty acids were extracted from the mixture three times
with 1 ml of hexane (total vol 3 ml). The pooled hexane fractions were dried under vacuum, and after addition of 3 ml ofscintillation mixture, the radioactivity incorporated was counted(BECKMAN LS6500). Blanks were carried out in parallel reactions incubated without microsomal fractions.

Animal experiments
All mice were housed in a controlled environment with a 12-h
light/dark cycle and free access to water and diet. For fatty acidor drug experiments, 7-week-old male C57BL/6J mice (21–23 g)
were purchased from CLEA (Tokyo, Japan) and adapted to theenvironment for 1 week. Prior to sacrifice, each group of animals
was fed a diet containing the indicated fatty acids and drugs for 7
days. SREBP-1a, and -1c, and -2 transgenic mice (16, 18, 28) andwild-type controls (non-transgenic littermates of SREBP-1a transgenic mice) were put on a high protein/low carbohydrate dietfor 2 weeks to induce the transgene expression, and were fasted
for 12 h prior sacrifice. For fasting and refeeding treatment,
SREBP-1 deficient (29) and wild-type mice were fasted for 24 h
and fed a high sucrose/fat free diet for 12 h. Ob/ mice on a
C57Bl/6 background were purchased from Jackson Laboratories. Ob/ mice were crossed to obtain leptin deficient ob/ob
mice and wild-type mice. At 12 weeks old, ob/ob and wild-typemice were sacrificed in the early light phase following a 2 h fast.

Total RNA preparation and Northern blotting
Total RNA was extracted from mouse livers, white adipose tissue (WAT), and various tissues using TRIZOL Reagent (LifeTechnologies, Inc.). RNA samples were run on a 1% agarose gel
containing formaldehyde and transferred to a nylon membrane(Hybond-N, Amersham Pharmacia Biotech). The probes usedwere labeled with [-32P]dCTP using the Megaprime DNA Labeling System kit (Amersham Pharmacia Biotech). The cDNA
probe for mouse FACE was prepared by digesting the cloned
cDNA with NotI. The cDNA probes for mouse SREBP-1 and ribosomal phosphoprotein PO (36B4) were prepared as described
previously (17). The membranes were hybridized with the radiolabeled probe in Rapid-hyb Buffer (Amersham Pharmacia Biotech) at 65C and washed in 0.1  SSC, 0.1% SDS at 65C. The
resulting bands were quantified by exposure of the filters to
BAS2000 with BAS station software (Fuji Photo Film Co., Ltd).

RESULTS
Cloning of FACE as an SREBP-activated gene
DNA microarray analysis identified an EST clone whose
expression was increased 19.5-fold in the livers of SREBP1a transgenic mice as compared with wild-type mice. Using this EST clone (GenBank ID number AA239254) as aprobe, we obtained the full-length 6 kb cDNA by screening an SREBP-1a transgenic liver cDNA library, followedby 5 RACE. As shown in Fig. 1A, nucleotide sequence of
this clone revealed that it encodes a putative protein of
267 amino acid residues with a theoretical molecular massof 31.6 kDa and very basic pI of 9.38. We found a humanhomolog (GenBank ID number AK027031) of mouse
FACE after searching BLAST database. The predicted
amino acid sequence revealed that 96% identical and
97% similarity in mouse and human homologs (Fig. 1B).
Hydropathy analyses by the Kyte-Doolittle algorithm (30)suggest that this predicted protein contains five transmembrane regions, typical for members of the elongase
family (Fig. 1C). An HXXHH motif, often present indesaturase/hydroxylase enzymes containing di-iron-oxocluster (Fe-O-Fe) proteins (31), was found between predicted transmembrane regions II and III, at amino acidpositions 141–145, and could function to receive electrons
from either cytochrome b5 or a cytochrome b5-like domain in an NAD(P)H-dependent way (Fig. 1B, C). The
COOH terminus of the FACE polypeptide contains alysine residue in position 3 (KKXX-like motif), suggesting that the predicted protein is located in the endoplasmic reticulum (ER) membrane (Fig. 1B) (32). The predicted amino acid sequence of mouse FACE showed aconsiderable similarity to Cig30 (44% identical), Ssc1
(29%), Ssc2 (26%), and Elovl4 (26%), all of which are
known to be involved in the elongation of fatty acids by
two carbon atoms, and they all contain 100% conserved
HXXHH motif characteristic of this protein family (Fig. 2).
Expression of FACE in HEK-293 cells and substrate specificity of FACE activity
This cDNA clone is highly likely to encode a mammalian
fatty acid elongase, and is tentatively designated FACE.
When the FACE cDNA was over-expressed in HEK293 cells
supplemented with various fatty acids, gas chromatography
analysis of the cellular fatty acids showed a trend that overexpression of FACE caused a slight increase in the relative
amounts of stearic acid (C18:0) and oleic acid (C18:1n-9),accompanying a decrease in palmitic acid (C16:0) andmyristic acid (C14:0) in the cells as compared with mocktransfected cells (CMV7) (data not shown). These datasuggested that the cells transfected with FACE were capable of synthesizing C18:0 from C16:0, indicating that FACE
is involved in the elongation of C16:0 and that FACE may
have an elongase acitivity for C14:0.
To verify that FACE plays a role in the conversion of
C16:0 to C18:0, we performed in vitro microsomal fatty acidelongation assays. Microsomal fatty acid elongation activity
was assayed by the measurement of [2-14C]malonyl-CoA incorporation into exogenously added acyl-CoA esters of saturated and mono-unsaturated fatty acids with chain length
12 to 18, namely lauroyl-CoA (C12:0), myristoyl-CoA(C14:0), palmitoyl-CoA (C16:0), palmitoleoyl-CoA (C16:1n7), stearoyl-CoA (C18:0), and oleoyl-CoA (C18:1n-9). Compared with control cells that had been transfected withempty plasmid CMV7, the transfection of the FACE cDNA
resulted in two 5-fold increases in elongase activity when

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Journal of Lipid Research Volume 43, 2002

Fig. 1. Sequence and homology analysis of fatty acyl-CoA elongase (FACE) cDNA and protein. A: Nucleotide sequence of mouse FACE
cDNA. Predicted amino acid sequence is shown below. Open reading frame and putative transmembrane domains are underlined andnumbered with Roman numerals. Polyadenylation signal consensus sequences are double-underlined. Sequence data have been deposited
with the GenBank accession number AB072039. B: Amino acid sequence comparison of mouse and human FACE. The dioxy iron-binding
HXXHH motif and COOH-terminal lysine signal responsible for retention in endoplasmic reticulum are indicated above protein sequence. C: Hydropathy profile of mouse FACE protein. Roman numerals indicate five regions above the horizontal bars predicted to span
the membrane according to the algorithm of Kyte and Doolittle. The region containing the histidine motif is indicated H.

added lauroyl-CoA, myristoyl-CoA, palmitoyl-CoA and palmitoleoyl-CoA (Fig. 3). When stearoyl-CoA and oleoyl-CoA
were used as a substrate, there were no significant changes
in the elongation activity. These data demonstrated that
FACE possesses elongase activity specific to C12–C16 saturated and monounsaturated fatty acids.
Tissue distribution of mouse FACE mRNA
Expression of FACE was examined in various tissues asestimated by Northern blot analysis. Whereas the dominant transcript of mouse FACE is approximately 6.0 kb insize, mouse tissues also contain a minor transcript that is
approximately 2.5 kb in size, (Fig. 4), presumably due tothe presence of two (poly)A signal sequences in the 3UTR (Fig. 1A). The expression was high in adrenal gland,
liver, WAT, brain, testis, and skin where lipogenesis andsteroidogenesis are active. Lower expression was found in
kidney, heart, skeletal muscle, lung, and spleen, whereas
the dominant transcript in testis is 2.5 kb.

Activation of FACE expression in livers from SREBPtransgenic mice
To confirm SREBP-1a activation of FACE expression
and to estimate the effect of other nuclear SREBP isoforms, Northern blot analysis was performed on liver
RNAs from nuclear SREBP-1a, -1c, and -2 transgenic andwild-type mice. As shown in Fig. 5, hepatic mRNA levels of
FACE were robustly increased by over-expression of nuclear SREBP-1a, -2, and -1c in this order of magnitude.
The shorter FACE transcript was barely detectable fromlivers of fasted wild-type mice, but became prominent in
SREBP transgenic livers. This minor short transcript wasalso nutritionally regulated in the same manner as the major transcript, consistent with the notion that the dualtranscripts are due to different (poly)A signals. The data
indicate that every isoform of SREBP can activate the expression of FACE gene, with the SREBP-1a isoform being
the most effective.

Matsuzaka et al. A fatty acyl-CoA elongase regulated by SREBPs

915

Fig. 3. Microsomal fatty acyl-CoA chain elongation activity from
HEK-293 cells transfected with FACE or CMV7. Total fatty acidelongation activities in the presence of NADPH were determined in
microsomal fractions prepared from HEK-293 cells transfected with
FACE or empty vector CMV7. Specific activity was expressed aspmol of radioactive malonyl-CoA incorporated into hydrophobic
long chain fatty acid fractions by 1 mg of microsomal protein in 1
min. Values represent the mean SEM (n 3).

mice was low and essentially similar to those of fasted normal mice. However, the SREBP-1/ mice had a lower induction of FACE upon refeeding compared with wild-type
mice. These data demonstrate that the hepatic expressionof FACE is at least partly under the control of the SREBP-1.
In addition to liver, adipose tissue is another organ inwhich lipogenic enzymes are thought to respond to fasting-refeeding at the transcription level. As shown in Fig.
6B, fasting-refeeding changes in mRNA levels of FACE
from adipose tissues of wild-type mice were observed similar to those in the liver. SREBP-1/ adipose tissues exhibited modestly impaired induction of FACE in both fasted
and refed states. These data demonstrate that FACE
mRNA level in lipogenic organs are nutritionally regulated in a lipogenic fashion, and are regulated by SREBP-1,

Fig. 2. Amino acid sequence alignment of mouse FACE homologs. Amino acid positions conserved in the homologs are indicated under the protein sequence. The HXXHH motif, characteristic of desaturase/hydroxylase enzymes containing a diiron-oxocluster (Fe-O-Fe) is underlined.

Nutritional regulation of FACE expression in SREBP-1
deficient mice
Nutritional regulation of hepatic FACE mRNA levels
was estimated in wild-type and SREBP-1 deficient mice.
These mice were fasted and then refed a high sucrose/fat
free diet. As shown in Fig. 6A, the mRNA levels of FACE
were suppressed in livers of fasted wild-type mice andmarkedly activated by refeeding in the wild-type mice,
showing nutritional regulation of FACE as a lipogenic enzyme. The mRNA level of FACE in the fasted SREBP-1/
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Fig. 4. FACE mRNA levels in mouse tissues. Northern blotting
analysis was performed with 10 g of total RNA isolated from variety of mouse tissues with the indicated cDNA probes. WAT, white
adipose tissue.

Fig. 5. Northern blots analysis of FACE mRNA in livers fromtransgenic (Tg) mice that overexpress each nuclear form of
SREBP-1a, -1c, and -2. Total RNA (15 g) pooled equally fromthree mice was subjected to Northern blotting, followed by hybridization with the FACE cDNA probe. A cDNA probe for 36B4 (acidic
ribosomal phosphoprotein P0) was used to confirm equal loading.

although SREBP-1-independent expression is also involved in FACE expression.
Effects of PUFAs on FACE expression
Numerous dietary studies indicate that hepatic activity
and expression of lipogenic enzymes are suppressed by diets rich in PUFAs such as vegetable or marine oils (20–22). We used Northern blot analysis to compare mouse
hepatic FACE expression levels in mice fed fat-free highcarbohydrate diet with the addition of various fatty acids

Fig. 6. Northern blots analysis of FACE mRNA in livers (A) andWAT (B) from fasted or refed wild-type (WT) and SREBP-1 knockout (KO) mice. Total RNA was extracted from the livers of the micein each treatment group. Equal aliquots of 15 g were pooled andsubjected to electrophoresis and blot hybridization with the FACE
32P-labeled cDNA probe. A cDNA probe for 36B4 (acidic ribosomal
phosphoprotein P0) was used to confirm equal loading.

Fig. 7. Northern blot analysis of FACE mRNA from livers of wildtype (A) or TgSREBP-1c (B) mice fed a diet containing various fatty
acids. A: mice (three male C57BL/6J, 8 weeks old) were fed the indicated diet for 7 days and sacrificed in a non-fasted state. Diets
were as follows: lane 1, a high carbohydrate fat-free diet; lane 2, ahigh carbohydrate diet with 20% tristearin (18:0); lane 3, 20% triolein; lane 4, 5% linoleate ethyl ester (18:2); lane 5, 5% EPA ethyl
ester (20:5); lane 6, 20% sardine fish oil; lane 7, 20% tuna fish oil.
B: Wild-type mice were fed a high carbohydrate fat-free diet (lane1), and homozygous TgSREBP-1c mice were fed a high protein dietwith 20% triolein (lane 2), 5% EPA ethyl ester plus 20% triolein(lane 3), or 20% sardine fish oil (lane 4) for 7 days and sacrificed ina nonfasted state. Total RNA (10 g) pooled equally from livers ofeach group was subjected to Northern blotting, followed by hybridization with the FACE cDNA probe. A cDNA probe for 36B4 (acidic
ribosomal phosphoprotein P0) was used to confirm equal loading.

for 7 days. Figure 7A shows that stearate (C18:0, lane 2)
and oleate (C18:1n-9, lane 3) did not affect the expressionof FACE. However, when the high carbohydrate diet was
supplemented with linoleates (lane 4) or eicosapentaenoic acid (EPA) (lane 5) the expression of FACE wasconsiderably suppressed, and the reduction was moremarked with fish oils rich in EPA and docosahexaenoic
acid (DHA) (lanes 6, 7). This specificity to PUFA corresponded to the pattern of suppression observed for otherlipogenic enzymes that are regulated by SREBP-1 (20).
To examine whether the PUFA suppression of FACE
was ascribed to the decrease in mature SREBP-1, we also
tested PUFA suppression of FACE expression in SREBP-1ctransgenic mice (Fig. 7B). This line of transgenic mice express similar amounts of nuclear SREBP-1c protein, derived from the transgene, to the physiological level of nuclear SREBP-1c in the livers from normal refed mice (20).
The sustained level of nuclear SREBP-1c completely abolished PUFA suppression of FACE mRNA levels. These
data suggest that endogenous SREBP-1c is involved inmaintaining FACE expression in a fed state.

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Fig. 8. Northern blot analysis of FACE mRNA from livers (A) andWAT (B) of wild-type or ob/ob mice. Mice were fed chow diet andsacrificed in a non-fasted state of early light phase following a 2 hfast. Total RNA (10 g) pooled equally from three mice was subjected to Northern blotting, followed by hybridization with the
FACE or SREBP-1 cDNA probe. A cDNA probe for 36B4 (acidic ribosomal phosphoprotein P0) was used to confirm equal loading.

Activation of FACE expression in livers from ob/ob mice
Leptin deficient mice (ob/ob) are an excellent murine
model for obesity, insulin resistance, and diabetes. Previous studies indicate that the mRNA level of SREBP-1 and
SREBP target genes involved in fatty acid biosynthesis weresignificantly elevated in livers from ob/ob mice comparedwith wild-type mice, but decreased in WAT (33, 34). We examined FACE mRNA levels in livers and WAT by Northernblot analysis. As shown in Fig. 8A, hepatic mRNA levels of
FACE and SREBP-1 from ob/ob mice fed ad libitum were
elevated compared with their respective wild-type controls.
In contrast, the mRNA levels of FACE and SREBP-1 did notshow a marked change in adipose tissues from ob/ob andwild-type mice (Fig. 8B). These data are consistent with theway lipogenic enzymes are regulated in ob/ob mice further supporting FACE regulation by SREBP-1c.
Effects of LXR agonist and PPAR ligands on
FACE expression
The oxysterol receptors, LXR and LXR , have beenshown to activate SREBP-1c promoter and expression ofits downstream lipogenic enzyme genes (24, 25, 35). As
shown in Fig. 9, administration of T0901317, a pharmacological LXR agonist (35), significantly increased hepatic
FACE mRNA levels, whereas the PPAR agonist Wy-14,643
and the PPAR agonist pioglitazone had no effect. These
data again suggest that endogenous SREBP-1c is involvedin the expression of FACE. The FACE expression level was
not highly induced by the LXR ligand in adipose tissue;
the mechanism is currently unknown.

DISCUSSION
In the current study, we have described the cloning andcharacterization of a cDNA that corresponds to a mamma918

Journal of Lipid Research Volume 43, 2002

Fig. 9. Northern blot analysis of FACE mRNA from livers (A) andWAT (B) of mice administered T0901317, Wy-14,643, or pioglitazone. T0901317, a pharmacological agonist for liver X-activated receptor (LXR) (T, 50 mg/kg), Wy-14,643 for PPAR (Wy, 50 mg/kg), pioglitazone for PPAR (Pio, 50 mg/kg), or vehicle (Veh, 0.5%
carboxymethyl-cellulose) was administered orally to the mice thatwere fasted and sacrificed 18 h after the treatment. Some mice were
fasted for 24 h and refed (R) a chow diet for 12 h. Total RNA (10g) pooled equally from three mice was subjected to Northernblotting followed by hybridization with the FACE cDNA probe. A
cDNA probe for 36B4 (acidic ribosomal phosphoprotein P0) wasused to confirm equal loading.

lian fatty acid elongase, which we have termed FACE.
Mouse FACE cDNA encodes 267 amino acid residues andcontains five transmembrane regions, a histidine-rich motif (HXXHH), and ER retention signal (KKXX-like), alltypical for members of fatty acid elongase family (6–8).
Difficulty in identification of the enzyme responsible forthis elongation could be due to its hydrophobicity. Expression studies suggest that FACE has specific activity for theelongation of C16 fatty acids C16:0 and C16:1n-7, a stepthat FAS cannot commit. Now it can be speculated in thecellular fatty acid synthesis that C16:0 produced by fatty
acid synthase in the cytosol is transferred to ER membranes, where ACS, ACBP, FACE, and SCD are sequentially involved to produce the main final product, oleylCoA, as schematized in Fig. 10. It well fits the fact that
oleate is a good endogenous substrate for triglyceride andcholesterol ester formation. In the process of preparing
this manuscript, cloning of the same gene from the similar strategy and detailed characterization of the enzyme
activity was reported (36). It was shown to have elongase

Fig. 10. Enzymes involved in mammalian fatty acid elongation.
ACC, acetyl-CoA carboxylase; FAS, fatty acid synthase; SCD,
stearoyl-CoA desaturase; ACS, acyl-CoA synthetase; ACBP, Acyl-CoA
binding protein; FACE, fatty acyl-CoA elongase.

activity for C12–C16 fatty acids. Our study demonstrates
that FACE also has an elongase activity for conversion of
C16:1n-7 to C18:1n-7. It was reported consistently that
C18:1n-7 as well as C18:1n-9 is increased in the fatty compositions of liver of rats in a refed state (1).
The FACE cDNA contains an unusually long 3-untranslated region (UTR) of about 5 kb, which is similar to SCD(37, 38). The similarity of SCD and FACE in both protein
and gene structures and the coordinating functions ofboth proteins suggests that these genes could be derived
from a common ancestral gene. The role of such a long
3-UTR is currently unknown; however, FACE responds tomany stimuli such as nutrient intake and hormones, a dynamic response that may require additional post-transcriptional control in addition to transcriptional control
by SREBP-1. Indeed, several RNA structural motifs (e.g.,
AUUUA) characteristic of mRNA destabilization sequences
are seen in the 3-end of the non-coding region. These
AU-rich elements (ARE) may play active roles in the selective degradation of several mRNAs in response to various
factors (39).
The SREBP-activation as a clue to the cloning of thisgene and subsequent studies on in vivo regulation of
FACE in mice clearly demonstrate that expression of
FACE is controlled by SREBP-1 as lipogenic gene. C18:1 isthe major end-product of de novo synthesis of fatty acids
in mammalian livers. Consistently, the major fatty acidconstituent of livers of SREBP-1a transgenic mice was oleate, suggesting that SREBP activates the whole reactions of
this pathway (40). Both FAS and SCD are well-established
SREBP targets. Therefore, it is reasonable that FACE is
also regulated by SREBPs. Transgenic mouse studies suggest that any isoform of SREBPs can activate FACE expression. Therefore, FACE could also be under sterol-regulation by SREBP-2 in addition to lipogenic regulation by
SREBP-1. To establish FACE as an SREBP target gene, detailed promoter analysis on the FACE gene is needed, although the presence of SRE-like and NF-Y sites in the 5

flanking sequence convincingly suggests that FACE is a direct target of SREBP (36). Remaining FACE mRNA level,although decreased, in lipogenic organs of SREBP-1 nullmice could be due to the compensatory activation of
SREBP-2 (29), or some unknown SREBP-independent
mechanism. Since FACE is expressed in essentially all tissues tested here, it could play a role in a basal cellularfunction in addition to lipogenesis as an energy storage
system.
Our current study established that FACE is a new member of the lipogenic enzyme family, based upon its fatty
acyl elongation activity, SREBP activation, and lipogenic
regulation in numerous diet studies, such as overshooting
induction at refeeding and PUFA suppression. Theoretically, the role of FACE in lipogenesis seems crucial because no other known enzymes have been reported toshow the same activity as observed in this enzyme. However, the importance of this gene in lipogenesis as an energy storage system or in more basal cellular functions
awaits analysis of effects of the FACE gene disruption,such as characterization of gene knockout mice.
BLAST analysis of the genome resources by NCBI revealed that human and FACE is mapped to chromosome 4(4q25) and mouse FACE is mapped to chromosome 3. It isinteresting that the location of FACE gene is very close to alocus that has been reported to be linked to human exceptional longevity by a genome-wide scan (41). It was reported that elongase activity estimated by C18:0-C16:0 ratio
in the muscle is significantly related to adiposity in humans(42), suggesting that FACE could be related to human obesity. Further studies might unveil clinical relevance of FACE
to human diseases or pathophysiological states.
The authors thank Drs. Michael S. Brown and Joseph L. Goldstein for continuous support for the project. This study was
supported by the Promotion of Fundamental Studies in Health
Science of the Organization for Pharmaceutical Safety and Research (OPSR).
Manuscript received 20 January 2002 and in revised form 8 March 2002.

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