Dual regulation of mouse D5- and D6-desaturase
gene expression by SREBP-1 and PPARa
Takashi Matsuzaka,† Hitoshi Shimano,1,† Naoya Yahagi,* Michiyo Amemiya-Kudo,*
Tomohiro Yoshikawa,* Alyssa H. Hasty,* Yoshiaki Tamura,* Jun-ichi Osuga,* Hiroaki Okazaki,*
Yoko Iizuka,* Akimitsu Takahashi,† Hirohito Sone,† Takanari Gotoda,* Shun Ishibashi,*and Nobuhiro Yamada†
Department of Metabolic Diseases,* University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan;
and Department of Internal Medicine,† Institute of Clinical Medicine, University of Tsukuba, 1-1-1 Tennodai,
Tsukuba, Ibaraki, 305-8575, Japan

Abstract In the process of seeking sterol regulatory elementbinding protein 1a (SREBP-1a) target genes, we identified andcloned a cDNA clone encoding mouse D5-desaturase (D5D).
The hepatic expression of D5D as well as D6-desaturase (D6D)
was highly activated in transgenic mice overexpressing nuclear
SREBP-1a, -1c, and -2. Disruption of the SREBP-1 gene significantly reduced the expression of both desaturases in
the livers of SREBP-1-deficient mice refed after fasting.
The hepatic expression of both desaturases was downregulated by dietary PUFA, which were reported to suppress
SREBP-1c gene expression. Sustained expression of hepatic
nuclear SREBP-1c protein in the transgenic mice abolishedthe PUFA suppression of both desaturases. Although thesedata suggested that SREBP-1c regulates D5D and D6D expression, there was no difference in either the D5D or D6DmRNA level between fasted and refed normal mouse livers,
indicating a mechanism for fasting induction of both desaturases. Administration of fibrate, a pharmacological ligand
for peroxisome proliferator activating receptor a (PPARa),caused a significant increase in expression of both desaturases. The data suggested that D5D and D6D expressionis dually regulated by SREBP-1c and PPARa, two reciprocaltranscription factors for fatty acid metabolism, and could
be involved in lipogenic gene regulation by producing
PUFA. —Matsuzaka, T., H. Shimano, N. Yahagi, M. AmemiyaKudo, T. Yoshikawa, A. H. Hasty, Y. Tamura, J-i. Osuga, H.
Okazaki, Y. Iizuka, A. Takahashi, H. Sone, T. Gotoda, S. Ishibashi, and N. Yamada. Dual regulation of mouse D5- and D6desaturase gene expression by SREBP-1 and PPARa. J. Lipid
Res. 2002. 43: 107–114.
Supplementary key words lipogenesis • nutrition • polyunsaturated
fatty acids • sterol regulatory element-binding protein

PUFA such as arachidonic acid (AA, 20:4n-6), EPA(20:5n-3), and DHA (22:6n-3) play pivotal roles in a number of biological functions including brain development,
cognition, reproduction, inflammatory responses, and hemostasis. In addition to being crucial components of membrane phospholipids and functioning in key steps of cellsignaling, PUFA have a unique ability to suppress the transcription of hepatic genes encoding lipogenic enzymes(1–4). The availability of 20- and 22-carbon (n-3 and n-6)
PUFA is determined by the synthesis of g-linolenic acid
(18:3n-6) and stearidonic acid (18:4n-3) and by the subsequent elongation and desaturation of these fatty acids to
20:4n-6 and 22:6n-3 (5). One of two rate-limiting steps inthe production of these polyunsaturated fatty acids is thedesaturation of eicosatrienoic acid (20:3n-6) and eicosatetraenoic acid (20:4n-3) to 20:4n-6 and 20:5n-3, determined by the activity of D5-desaturase (D5D). The otherstep is the desaturation of linoleic acid (18:2n-6) and alinolenic acid (18:3n-3) to 18:3n-6 and 18:4n-3 catalyzed
by D6-desaturase (D6D). Studies based on enzymatic activity suggest that the liver is the primary site for 20-carbon
polyenoic fatty acid synthesis because the liver is an organwith a great amount of D6D and D5D activity (6). Both enzymes are coordinately regulated by various dietary conditions and hormonal levels in the liver. For instance, D6D
activity is induced by peroxisome proliferators and by the
supplementation of insulin to diabetic rats (7, 8). It is also
highly suppressed by dietary PUFA, indicating that these
enzymes are involved in feedback regulation in the production of AA, EPA, and DHA. Remarkable progress inunderstanding the molecular basis of the nutritional regulation of these desaturases has been made by cloning
mammalian D5D and D6D cDNAs (9, 10). The predicted
amino acid sequences were suggested to contain a cytochrome b5-like domain that might dispense with the presence of a separate cytochrome b5. Suppression of D5D and

Abbreviations: AA, arachidonic acid; D5D, D5-desaturase; D6D, D6desaturase; PPAR, peroxisome proliferator activating receptor; SRE,
sterol regulatory element; SREBP, sterol regulatory element-binding
protein.
1 To whom correspondence should be addressed.
e-mail: shimano-tky@umin.ac.jp

Journal of Lipid Research Volume 43, 2002

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D6D activities by dietary unsaturated fatty acids was shown
to be associated with decreased D5D and D6D mRNA levels(9, 10).
Sterol regulatory element-binding proteins (SREBP)
are membrane-bound transcription factors that belong to
the basic helix-loop-helix leucine zipper family (11 –13).
A sterol-regulated cleavage is required for SREBP to enter
the nucleus and activate the transcription of genes involved in cholesterol and fatty acid synthesis by binding
to a sterol regulatory element (SRE) or its related sequences, including E-boxes, within their promoter regions (14, 15). There are three forms of SREBP that havebeen characterized: SREBP-1a and -1c (also known as
ADD1) and SREBP-2 (16 –18). Most organs, including
the liver and adipose tissue, express predominantly
SREBP-2 and the c isoform of SREBP-1 (19). Lipogenic
enzymes, which are involved in energy storage throughthe synthesis of fatty acids and triglycerides, are coordinately regulated at the transcriptional level during different metabolic states. 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 oroverexpression of SREBP-1 on hepatic lipogenic gene expression (20 –22), as well as physiological changes of
SREBP-1c protein in normal mice after dietary manipulation, such as placement on high carbohydrate diets, polyunsaturated fatty acid-enriched diets, and fasting-refeeding
regimens (23 –27). 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, and the oxysterol liver
X receptor-retinoid X receptor (28, 29). The mechanism
for PUFA suppression of the SREBP-1c promoter is underinvestigation.
Both D5D and D6D play crucial roles in the productionof PUFA, which in turn suppresses SREBP-1. In addition,
D5D, D6D, and SREBP-1c are nutritionally regulated.
These lines of evidence suggest that D5D and D6D expression might be related to SREBP-1 activity. In thescreening of SREBP-activated genes, we cloned and identified a mouse D5D gene. Our current studies suggestthat nutritional expression of D5D and D6D is regulated
by both SREBP-1 and peroxisome proliferator activating
receptor a (PPARa).

EXPERIMENTAL PROCEDURES
Materials and general methods
We purchased 20:3n-6 from Sigma (St. Louis, MO), Redivue[a-32P]dCTP (6,000 Ci/mmol) from Amersham Pharmacia Biotech (Piscataway, NJ), and restriction enzymes from New England BioLabs (Beverly, MA).
Standard molecular biology techniques were used. DNA sequencing was performed with a CEQTM dye terminator cycle
sequencing kit and CEQ2000 DNA analysis system (Beckman
Coulter, Fullerton, CA).

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Preparation of SREBP-1a transgenic liver cDNA library
An expression cDNA library of SREBP-1a transgenic liver (21)
was prepared as previously described for construction of a cDNA
library of SREBP-1-deficient mouse adipose tissue, except thatpoly(A)1 RNA was prepared from livers of SREBP-1a transgenic
mice (29).

Cloning of mouse D5D cDNA
From a DNA microarray system using hepatic poly(A) 1 RNA
of SREBP-1a transgenic and nontransgenic littermate mice, we
identified an expressed sequence tag (EST) clone (GenBank accession number AA068575) that was activated 8-fold in SREBP-1atransgenic liver as compared with wild-type liver, and resembled
the mouse D6D. Using this sequence information, an [ a-32P]dCTPlabeled DNA probe was prepared and used in the screening
from an SREBP-1a transgenic mouse liver cDNA librar y by colony hybridization. Positive clones were sequenced and theclones containing an open reading frame were used for expression experiments.

Expression of mouse D5D
HEK-293 cells were grown at 37 8C in an atmosphere of 5%
CO2 in DMEM containing 25 mM glucose, penicillin (100 U/ml), and streptomycin sulfate (100 mg/ml) supplemented with
10% FCS on 100-mm culture plates. At 80% confluence, themouse D5D expression plasmid or the basic plasmid CMV7
alone (10 mg) was transfected into cells, using SuperFect transfection reagent (Qiagen, Valencia, CA) according to the manufacturer protocol. After transfection, cells were treated witheither 100 mM 20:3n-6 diluted in ethanol or ethanol alone for
24 h and then used for fatty acid analysis.

Fatty acid analysis
Total fatty acid extracted from HEK-293 cells transfected withthe mouse D5D expression plasmid or CMV7, in the presence of
100 mM 20:3n-6, was analyzed by gas chromatography of methyl
ester derivatives. Lipids were extracted and transmethylated withmethanolic HCl, and the fatty acid methyl esters were analyzed
as described previously (10).

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. Before sacrifice, each group of animals
was fed a diet containing the indicated fatty acids and drugs for 7
days. Fasting and refeeding regimens for wild-type and SREBP-1
knockout mice were previously described (22). Transgenic miceoverexpressing the nuclear form of SREBP-1a, -1c, and -2 in theliver under the control of the phospho enolpyruvate carboxykinase promoter were previously described (21, 30, 31). SREBP-1c
and -2 transgenic mice were homozygous for the transgene (32).
These three transgenic lines and wild-type controls (nontransgenic littermates of SREBP-1a transgenic mice) were put on ahigh protein/low carbohydrate diet to induce transgenic micefor 2 weeks, and were fasted for 12 h before sacrifice. For fasting
and refeeding treatment, SREBP-1-deficient and wild-type mice
were fasted for 24 h and fed a high sucrose/fat-free diet for 12 h.

Total RNA preparation and Northern blotting
Total RNA was extracted from mouse livers and various tissues
with TRIzol reagent (Life Technologies, Rockville, MD). RNA
samples were run on a 1% agarose gel containing formaldehyde
and transferred a nylon membrane (Hybond-N; Amersham Pharmacia Biotech). The probes used were labeled with [ a-32P]dCTP,

using the Megaprime DNA labeling system kit (Amersham Pharmacia Biotech). The cDNA probe for mouse D5D was preparedby digesting the cloned cDNA with NotI and EcoRI. The cDNA
probes for mouse D6D and ribosomal phosphoprotein P0(36B4) were prepared as described previously (10, 22). The
membranes were hybridized with the radiolabeled probe in
Rapid-Hyb buffer (Amersham Pharmacia Biotech) at 65 8C andwashed in 0.13 SSC, 0.1% SDS at 65 8C. The resulting bands
were quantified by exposure of the filters to BAS2000 with
BASstation software (Fuji Photo Film, Tokyo, Japan).

RESULTS
Cloning mouse D5D as an SREBP-activated gene
DNA microarray analysis identified an EST clone whose
expression was increased 7-fold in the livers of SREBP-1atransgenic as compared with wild-type mice. This clone(GenBank accession number AA068575) was originallynamed an EST clone slightly similar to mouse D6D in the
database. Mouse D6D was cloned and reported (10). The
sequences suggested that this clone should encode something similar, but not identical, to D6D. Using this EST
clone as a probe, we cloned the cDNA clone that contains
an open reading frame (ORF) from an SREBP-1a transgenic liver cDNA library. The DNA sequence analysis revealed that the predicted amino acid sequence was highly
similar to human and rat D5D (9). Alignment of the predicted amino acid sequences for mouse and human D5D
is shown in Fig. 1. Eighty-eight percent of the amino acidsequence for the mouse and human homologs was identical. The high homology includes the presence of a cytochrome b5 domain, transmembrane domains, and histidine-rich regions, all of which were reported for human
D5D and mouse D6D.

TABLE 1. Fatty acid profiles of HEK-293 cells transfectedwith mouse D5-desaturase
1100 mM 20:3n-6
Fatty Acid

CMV7

C14:0
C16:0
C16:1n-7
C18:0
C18:1n-9
C18:2n-6
C20:3n-6
C20:4n-6
C22:4n-6
C22:5n-3
C22:6n-32.80 6 0.16
25.63 6 0.28
3.24 6 0.03
11.23 6 0.36
15.10 6 0.24
2.69 6 0.12
26.55 6 0.82
5.69 6 0.24
2.20 6 0.13
1.79 6 0.08
1.84 6 0.11

D5D

mol% of fatty acids
2.80 6 0.14
26.58 6 0.78
3.26 6 0.08
11.58 6 0.51
15.39 6 0.08
2.58 6 0.08
20.65 6 0.88a
8.98 6 0.10b4.43 6 0.35c
1.88 6 0.25
1.78 6 0.08

Fatty acid composition of HEK-293 cells transfected with themouse D5-desaturase and CMV7, grown in the presence of 100 mM
20:3n-6. Results are expressed as the mol percentage of total fatty acids
and as means 6 SEM for three independent cultures. Statistical significance was assessed by unpaired t-test.
a P , 0.01 versus CMV7.
b P , 0.0005 versus CMV7.
c P , 0.005 versus CMV7.

Expression of mouse D5D
The high similarity of the predicted amino acid sequence from the ORF of the cloned mouse cDNA to thehuman D5D amino acid sequence suggested that the cDNA
encodes the mouse D5D. To confirm this, the cDNA was expressed in HEK-293 cells supplemented with C20:3n-6. As
shown in Table 1, gas chromatography analysis of the cellular fatty acids revealed that overexpression of this putative D5D clone increased the relative amounts of C20:4n-6
and C22:4n-6, accompanying a decrease in C20:3n-6 in thecells as compared with mock-transfected cells. The data suggested that the cells transfected with the cDNA were capableof synthesizing 20:3n-6 from eicosadienoic acid (20:2n-6),
indicating that the cDNA product has a D5D activity.
Tissue distribution of mouse D5D mRNA
Expression of D5D and D6D was examined in various
tissues as estimated by Northern blot analysis. Mouse D5D
is a single transcript that is approximately 4.5 kb in size
(Fig. 2). The expression was high in the adrenal gland,
liver, brain, and testis, tissues where lipogenesis and steroidogenesis are active. The tissue distribution pattern ofmouse D6D expression was similar, suggesting that D5D
and D6D cooperatively produce polyenoic fatty acids.

Fig. 1. Predicted amino acid sequence and homology analysis ofmouse and human D5D. The cytochrome b5 domain is underlined.
Transmembrane domains are shown as dashed lines, and threehistidine-rich regions are in boxes.

Activation of D5D and D6D expression
in liver of SREBP transgenic mice
We performed Northern blot analysis of liver RNA from
nuclear SREBP-1a, -1c, and -2 transgenic and wild-type miceto estimate the effects of nuclear SREBP on the expressionof D5D as well as D6D. As shown in Fig. 3, the hepatic
mRNA level of D5D was robustly increased by overexpression of nuclear SREBP-1a, -1c, and -2 at this order of magnitude. Hepatic D6D mRNA levels were also induced by
SREBP overexpression in a pattern similar to that of D5D
mRNA. A shorter mouse D6D transcript was barely detect-

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109

Fig. 3. Northern blot analysis of D5D and D6D mRNA in livers
from wild-type (WT) mice and transgenic (Tg) mice overexpressing the nuclear form of SREBP-1a, -1c, and -2. Total RNA (15 mg)
pooled equally from each group was subjected to Northern blotting, followed by hybridization with the D5D and D6D cDNA
probes. A cDNA probe for 36B4 (acidic ribosomal phosphoproteinP0) was used to confirm equal loading.

Fig. 2. D5D and D6D mRNA levels in mouse tissues. Northernblotting analysis was performed with 10 mg of total RNA isolated
from a variety of mouse tissues with the indicated cDNA probes.
WAT, White adipose tissue.

able from livers of fasted wild-type mice, but became
prominent in SREBP transgenic livers. This minor, shorttranscript was identified in human D6D and was reported
to be nutritionally regulated as well as the major transcript. The data indicate that every isoform of SREBP can
activate the expression of both desaturase genes.
Nutritional regulation of D5D and D6D
expression in SREBP-1-deficient mice
Nutritional regulation of hepatic D5D and D6D mRNAlevels was estimated in wild-type and SREBP-1 deficientmice. As shown in Fig. 4, these mice were fasted and thenrefed with a high sucrose/fat-free diet. Interestingly, thewild-type mice did not show a marked change in D5D or
D6D mRNA level between fasting and refeeding. In thefasted state, the SREBP-1-deficient mice showed hepatic
expression levels of both D5D and D6D similar to those ofwild-type mice. After refeeding, there was a considerable
reduction in the expression of both desaturases in SREBP-1
null mice, showing a much lower level than in the refedwild-type mice. Expression of PPARa was induced by fasting and suppressed by refeeding, whereas the SREBP-1c
mRNA level showed the opposite response. These data
demonstrate that the hepatic expression of D5D and D6D
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Journal of Lipid Research Volume 43, 2002

is not upregulated by refeeding, but that SREBP-1 contributes strongly to their expression in the refed state. The
nutritional regulation of these enzymes is distinct fromthat of regular lipogenic enzymes, which are suppressedby fasting and are markedly induced by refeeding, although both these desaturases and lipogenic enzymes are
regulated by SREBP-1.
Effects of PUFAs on D5D and D6D expression
Numerous dietary studies indicate that the hepatic activity and expression of both desaturases are induced bydiets low in essential fatty acids and suppressed by dietsrich in PUFA such as vegetable or marine oils. We compared mouse hepatic D5D and D6D expression levels inmice fed a fat-free high carbohydrate diet with various
kinds of fatty acids. Figure 5 showed that stearate (C18:0)
or oleate (C18:1) did not affect the expression of D5D
and D6D. However, when a high carbohydrate diet was
supplemented with linoleate or EPA, the expression ofboth desaturases was considerably suppressed, and the reduction was more marked with fish oils rich in EPA andDHA. These data were consistent with previous reports on
the expression of human and rat desaturase genes. We
also tested PUFA suppression of their expression in
SREBP-1c transgenic mice (Fig. 6). This line of transgenic
mice expresses amounts of nuclear SREBP-1c protein, derived from the transgene, similar to the physiological levelof nuclear SREBP-1c in the livers of normal refed mice(27). The sustained level of nuclear SREBP-1c completely
abolished PUFA suppression of D5D and D6D mRNA levels. These data suggest that endogenous SREBP-1c couldFig. 4. Northern blot analysis of D5D and D6D mRNA in livers
from fasted or refed wild-type (WT) and SREBP-1 knockout (KO)mice. Total RNA was extracted from the livers of the mice in eachtreatment group. Equal aliquots of 15 mg were pooled and subjected to electrophoresis and blot hybridization with the D5D, D6D,
SREBP-1, and PPARa 32P-labeled cDNA probes. A cDNA probe for
36B4 (acidic ribosomal phosphoprotein P0) was used to confirm
equal loading. Note that SREBP-1c mRNA signal from refed KO
mice was an aberrant transcript from the disrupted gene and its
product is inactive (22).

be involved in maintaining D5D and D6D expression in a
fed state.
Effects of PPARa ligands on D5D and D6D expression
As shown in Fig. 4, fasting treatment induces expressionof PPARa, crucial for fatty acid oxidation. To estimate thepossible involvement of PPARa in D5D and D6D expression, we challenged mice with fibrate, a pharmacological
PPAR ligand. Addition of fibrate to the diet increased
both D5D and D6D mRNA levels (Fig. 7) whereas troglitazone, which induces PPARg, had no effect, indicating thatboth desaturases can be induced by PPARa and not by
PPARg. These results are consistent with the previous report that peroxisome proliferators enhanced linoleic acidmetabolism in rat liver (8).

DISCUSSION
D5D and D6D as potential SREBP-1 targets
The current study clearly demonstrates that SREBP activate the expression of both D5D and D6D in the liver.
Both hepatic D5D and D6D mRNA levels were markedly
increased by overexpression of SREBP, with preference
for SREBP-1 relative to SREBP-2. Furthermore, the ab-

Fig. 5. Northern blot analysis of D5D and D6D mRNA from liversof mice fed a diet containing various fatty acids. Mice (three male
C57BL/6J, 8 weeks old) were fed the indicated diet for 7 days andkilled in a nonfasted state. Diets were as follows. Lane 1, a high carbohydrate fat-free diet; lane 2, a high carbohydrate diet with 20%
tristearin (18:0); lane 3, 20% triolein (18:1); 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. Total RNA (10 mg) pooled
equally from three mice was subjected to Northern blotting, followed by hybridization with the D5D and D6D cDNA probes. A
cDNA probe for 36B4 (acidic ribosomal phosphoprotein P0) wasused to confirm equal loading.

sence of SREBP-1 markedly impaired the hepatic expression of these desaturases in a refed state. All these datasuggest that D5D and D6D are highly potential SREBP target genes and that SREBP-1 plays a pivotal role in the hepatic expression of these desaturases, at least under lipogenic conditions. The physiological relevance of SREBP
activation of these desaturases is currently unknown.
Under lipogenic conditions, when SREBP-1 is activated,
newly synthesized fatty acids could be dominated by saturated or monounsaturated fatty acids, and availability ofessential fatty acids might be limited. In this situation, activation of D5D and D6D might be necessary to meet cellular requirements for AA, EPA, and DHA. Cumulative evidence suggests that the cellular availability of PUFA is
crucial for the regulation of lipogenesis. On the assumption that the PUFA that these desaturases synthesize canbe used for inhibition of lipogenesis, SREBP-1 inductionof these desaturases would contribute to efficient suppression of lipogenesis whenever essential fatty acids such as
C18:2n-6 become available. It is consistent with the previous
report that D6D activity is important for PUFA suppression of hepatic lipogenic gene expression. This is the firstobservation that there could be a feedback system for thehepatic SREBP-1c-lipogenesis system, which was thought
to depend essentially on the supply of carbohydrates as
discussed in the autoloop activation of the SREBP-1c pro-

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111

Fig. 6. Northern blot of D5D and D6D mRNA from livers of wildtype or TgSREBP-1c mice fed PUFA diets. Wild-type mice were fed
a high carbohydrate fat-free diet (lane 1), and homozygous TgSREBP-1c mice were fed a high protein diet with 20% triolein (lane2), 5% EPA ethyl ester plus 20% triolein (lane 3), or 20% sardine
fish oil (lane 4) for 7 days and killed in a nonfasted state. Total RNA(10 mg) pooled equally from livers of each group was subjected to
Northern blotting, followed by hybridization with the D5D and
D6D cDNA probes. A cDNA probe for 36B4 (acidic ribosomal phosphoprotein P0) was used to confirm equal loading.

moter underlying the overshooting phenomenon in arefed state (28).
A mechanism for the feedback suppressionof D5D and D6D by PUFA
Dietary PUFA have been reported to decrease both theactivity and expression of D5D and D6D (9, 10), a finding
supported by our current observations. PUFA were reported to inhibit SREBP-1 activity by multiple mechanisms(24–27, 33, 34). Considering our new finding that D5D
and D6D are potential SREBP targets, it is likely that downregulation of D5D and D6D expression by PUFA could be
mediated through suppression of SREBP-1. This hypothesis is further supported by the observation that PUFA suppression of D5D and D6D was completely abolished by thesustained amount of nuclear SREBP-1c protein forcibly
expressed in SREBP-1c transgenic mice.
Dual regulation of D5D and D6D expression
by SREBP-1 and PPARa
This unique fashion of nutritional regulation (activation by SREBP-1c, but no change by a fasting-refeeding
treatment) places D5D and D6D in a position distinct
from regular lipogenic enzymes. In particular, in SREBP-1
knockout mice, D5D and D6D mRNA levels are considerably higher in fasted liver than in refed liver. These observations can be explained by the hypothesis that, in addi112

Journal of Lipid Research Volume 43, 2002

Fig. 7. Northern blot analysis of D5D and D6D mRNA from liversof mice administered fenofibrate or troglitazone. Mice (three male
C57BL/6J, 8 weeks old) were fed a high carbohydrate fat-free dietwithout (lane 1) or with 0.5% fenofibrate (lane 2) or 0.1% troglitazone (lane 3) for 7 days. The mice were killed in a nonfasted state.
Total RNA (10 mg) pooled equally from three mice was subjected to
Northern blotting, followed by hybridization with the D5D and
D6D cDNA probes. A cDNA probe for 36B4 (acidic ribosomal phosphoprotein P0) was used to confirm equal loading.

tion to SREBP-1c-mediated induction as observed in therefed state of normal mice, there is another regulatory
mechanism that activates the expression of D5D and D6D
in a fasted state. Our results from administration of fenofibrate, a PPARa ligand, indicate that PPARa is responsible
for the fasting induction of these desaturases. Furthermore, we and others previously reported that starvation
increases the expression of PPARa, which should enhancefasting-induced activation of D5D and D6D in addition to
the ligand activation of PPARa (27, 35). In a fasted state,lipolysis from adipose tissue is enhanced and circulating
free fatty acids enter the liver for b-oxidation that is controlled by PPARa. Many of these free fatty acids are essential fatty acids, and can be both ligands for both PPARa
and substrates for D5D and D6D. Activation of D5D and
D6D by PPARa in the liver might be important to produce
required amounts of PUFA.
Taken together, the expression of D5D and D6D could
be regulated by two distinct mechanisms, one through
SREBP-1c as is shown by its induction during refeeding,and the other through PPARa that is inducible by fasting.
Dual regulation of D5D and D6D by the two factors
SREBP-1 and PPARa, which possess opposing activation
properties depending on nutritional status, caused no remarkable change in the overall expression of these enzymes between fasting and refeeding as schematized inFig. 8. This dual regulation of both desaturases contributes to a stable production of PUFA that are essential forcellular functions regardless of energy state.
Further analysis of the promoter regions of the D5D
and D6D genes will be required. Interestingly, these genes

Fig. 8. Dual regulation of D5D and D6D expression by SREBP-1
and PPARa. AA, Arachidonic acid; EPA, eicosapentaenoic acid;
DHA, docosahexaenoic acid.

are located close to each other in an inverted orientation,
sharing an 11-kb upstream region (9). It would be intriguing if D5D and D6D shared in this region enhancers thatare important for nutritional regulation, such as an SRE
responsible for SREBP activation and/or a peroxisome
proliferator response element, which could explain PPAR
ligand activation. The current study suggests that PUFA
play an essential role in energy metabolism by regulating
or modifying these lipid transcription factors and that
D5D and D6D could be involved in this system by producing PUFA.
This study was supported by the Organization for Pharmaceutical Safety and Research (OPSR, Promotion of Fundamental
Studies in Health Science), and by Health Sciences Research
grants (Research on Human Genome and Gene Therapy)
from the Ministry of Health and Welfare, Japan.
Manuscript received 1 June 2001 and in revised form 8 October 2001.

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