Am J Physiol Endocrinol Metab 282: E222–E230, 2002;
10.1152/ajpendo.00189.2001.

Acetyl-coenzyme A synthetase is a lipogenic enzyme
controlled by SREBP-1 and energy status
HIROHITO SONE,1 HITOSHI SHIMANO,1 YUKI SAKAKURA,1 NORIYUKI INOUE,1MICHIYO AMEMIYA-KUDO,3 NAOYA YAHAGI,3 MITSUJIRO OSAWA,2 HIROAKI SUZUKI,1
TOMOTAKA YOKOO,1 AKIMITSU TAKAHASHI,1 KAORUKO IIDA,1
HIDEO TOYOSHIMA,1 ATSUSHI IWAMA,2 AND NOBUHIRO YAMADA1
1
Department of Internal Medicine, Institute of Clinical Medicine, and 2Department of Immunology,
Institute of Basic Medicine, University of Tsukuba, Tsukuba 305-8575; and 3Departmentof Metabolic Disease, School of Medicine, University of Tokyo, Tokyo 113-8655, Japan
Received 2 May 2001; accepted in final form 11 September 2001

Sone, Hirohito, Hitoshi Shimano, Yuki Sakakura,
Noriyuki Inoue, Michiyo Amemiya-Kudo, Naoya Yahagi,
Mitsujiro Osawa, Hiroaki Suzuki, Tomotaka Yokoo,
Akimitsu Takahashi, Kaoruko Iida, Hideo Toyoshima,
Atsushi Iwama, and Nobuhiro Yamada. Acetyl-coenzyme
A synthetase is a lipogenic enzyme controlled by SREBP-1
and energy status. Am J Physiol Endocrinol Metab 282:
E222–E230, 2002; 10.1152/ajpendo.00189.2001.—DNA microarray analysis on upregulated genes in the livers fromtransgenic mice overexpressing nuclear sterol regulatory element-binding protein (SREBP)-1a, identified an espressedsequence tag (EST) encoding a part of murine cytosolic acetylcoenzyme A synthetase (ACAS). Northern blot analysis of thelivers from transgenic mice demonstrated that this gene was
highly induced by SREBP-1a, SREBP-1c, and SREBP-2.
DNA sequencing of the 5⬘ flanking region of the murine
ACAS gene identified a sterol regulatory element with anadjacent Sp1 site. This region was shown to be responsible
for SREBP binding and activation of the ACAS gene by gel
shift and luciferase reporter gene assays. Hepatic and adipose tissue ACAS mRNA levels in normal mice were suppressed at fasting and markedly induced by refeeding, andthis dietary regulation was nearly abolished in SREBP-1
knockout mice, suggesting that the nutritional regulation of
the ACAS gene is controlled by SREBP-1. The ACAS gene
was downregulated in streptozotocin-induced diabetic mice
and was restored after insulin replacement, suggesting that
diabetic status and insulin also regulate this gene. Whenacetate was administered, hepatic ACAS mRNA was negatively regulated. These data on dietary regulation and
SREBP-1 control of ACAS gene expression demonstrate that
ACAS is a novel hepatic lipogenic enzyme, providing further
evidence that SREBP-1 and insulin control the supply of acetylCoA directly from cellular acetate for lipogenesis. However, its
high conservation among different species and the wide range ofits tissue distribution suggest that this enzyme might also play
an important role in basic cellular energy metabolism.

regulated at the transcriptional level, mainly by sterol

regulatory element-binding proteins (SREBPs), transcription factors belonging to the basic-helix-loop-helix
leucin zipper family (2–4, 28). SREBPs are synthesized
in a membrane-bound form. Upon sterol deprivation,
nuclear SREBPs are cleaved to enter the nucleus andactivate the transcription of genes involved in cholesterol and fatty acid synthesis by binding to sterol regulatory elements (SREs) or to palindromic sequences
called E boxes within their promoter regions (3, 5, 26,
28). SREBPs consist of three isoforms (SREBP-1a,
SREBP-1c, and SREBP-2), where SREBP-1a and -1c
are generated from a single gene through alternative
splicing (14, 31).
Cumulative lines of evidence, including normal,transgenic, and knockout mice on diet studies, established that SREBP-1 plays a role in regulating thetranscription of genes involved in fatty acid synthesis,
whereas SREBP-2 is actively involved in the transcription of cholesterogenic enzymes (13, 24). SREBP-1a is astronger activator than SREBP-1c because of a longer
transactivation domain, and it has a wider range oftarget genes involved in both cholesterol and fatty acidsynthesis (22, 23). Transgenic mice overexpressing nuclear SREBP-1a in the liver demonstrated a marked
induction of cholesterogenesis and lipogenesis resulting in engorged fatty livers (22).
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 (9, 11). Recent
in vivo studies demonstrated that SREBP-1c plays acrucial role in the dietary regulation of most hepatic
lipogenic genes. These include studies of the effects of
the absence or overexpression of SREBP-1 on hepatic
lipogenic gene expression (22–24), as well as physiological changes of SREBP-1c protein in normal mice
after dietary manipulations, such as placement on highcarbohydrate diets, polyunsaturated fatty acid-enriched diets, and fasting-refeeding regimens (12, 15,

Address for reprint requests and other correspondence: H. Shimano, Dept. of Internal Medicine, Institute of Clinical Medicine,Univ. of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan (E-mail: hshimano@md.tsukuba.ac.jp).

The costs of publication of this article were defrayed in part by thepayment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.

lipogenic enzyme; acetate; diabetes; insulin; transcription

INTRACELLULAR CHOLESTEROL and fatty acid synthesis are

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ACAS AS A LIPOGENIC ENZYME

25, 29, 30). Recent studies suggest that insulin or
insulin-facilitated glucose uptake mediates lipogenesis
through SREBP-1c induction (7, 10, 18).
Acetyl-CoA synthetase (ACAS) is an intracellularenzyme that catalyzes the formation of acetyl-coenzyme A (acetyl-CoA) from coenzyme A and acetate (17).
ACAS is known to be involved in ethanol and acetate
metabolism of bacteria, and its molecular characterization has been well described from a microbiologicalpoint of view (8, 27). ACAS activity has also been wellknown among researchers in ruminology to play a
crucial role in energy production of ruminants, because
volatile fatty acids (also known as short-chain fatty
acids), produced through fermentation of cellulose andother fibers in rumen, are their main source of energy.
Even in other mammals, including rodents and humans, acetate, a major component of volatile fatty
acids, can contribute considerably as an energy source
as a result of fermentation of dietary fibers (20).
Beyond this limited information, the molecular characterization of ACAS in mammals has not been wellunderstood. Because ACAS produces acetyl-CoA,
which is a key branching molecule for different metabolic pathways, it could play an important role inenergy metabolism in mammals. In a search for new
targets of SREBP-1, we cloned the murine ACAS cDNAand analyzed its gene promoter. Investigation of thetissue-specific expression profile and nutritional regulation demonstrated a new aspect of this gene as alipogenic enzyme.
MATERIALS AND METHODS

Materials. Mice (C57BL/6NCrj) were obtained fromCharles River Japan (Yokohama, Japan). All animal experiments had been approved by a review of the institutionalboard of animal welfare. Streptozotocin was purchased from
Sigma (St. Louis, MO). TRIzol reagent (GIBCO-BRL, Rockville, MD) was used to isolate total RNA. [␣-32P]dCTP and
Hybond-N⫹ membrane were purchased from Amersham
Pharmacia Biotech (Uppsala, Sweden). The BcaBEST Labeling Kit (TaKaRa Biomedicals, Kyoto, Japan) was used tolabel radioactive probes. The BAS 2000 system (Fuji Photo
Film, Tokyo, Japan) was used to detect the signals of Northern and dot blot analysis. NIH Image version 1.62 (freesoftware distributed by the National Institutes of Health,Bethesda, MD) was used to quantify intensity of the detectedbands.
cDNA cloning of the murine ACAS gene and its promoter
region. Poly(A)⫹ RNA samples were isolated from livers ofmale SREBP-1a transgenic and male normal control mice
(C57BL/6NCrj) of 8 wk of age and applied to Gene Expression
Microarray serviced by Incyte Genomics (St. Louis, MO). A
514-bp expressed sequence tag (EST) (AA537637), which
encoded a part of the murine ACAS cDNA, was located, andhomologs and other searches were performed in the National
Center for Biotechnology Information (NCBI) database tocollect the related ESTs. 5⬘-Rapid amplification of cDNA ends
(RACE) was performed by using the 5⬘ RACE system version
2.0 (GIBCO-BRL) to extend a known sequence to get further
upstream probes for more efficient screening of libraries. A
probe was obtained by PCR with a forward primer, S39,
GGACAAGGTGTTCGGAACTTG, and a reverse primer,
AS209, ACAGAACGCCGGTGCAGCTC.

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A liver cDNA library of SREBP-1a transgenic mice wasprepared in the pCMV7 vector. The library was screened forfull-length cDNA of ACAS using the probe just described.
Obtained clones were sequenced three times by dye-terminator cycle sequencing using the Dye Primer Cycle Sequencing
Kit (Perkin-Elmer, Wellesley, MA) and chemiluminescence
sequencers (model 377, ABI 100, Perkin-Elmer) and wereanalyzed by ABI Prism software version 3.0 (Perkin-Elmer).
Luciferase promoter assay of ACAS gene. We obtained aclone containing the 5⬘ flanking region of the ACAS gene byscreening the BAC library of mouse genomic DNA (Incyte
Genomics). An EcoRI fragment (a 7.8-kb fragment) containing the promoter and 5⬘UTR of ACAS was subcloned into thepGEM3Zf vector (Promega, Madison, WI), was designated as
pGEM3/ACAS/BAC/EcoRI 5⬘ flanking region of the ACAS
gene, and was sequenced. Three DNA fragments (sizes from676, 463, and 331 bp 5⬘ upstream of the ACAS coding region)of the ACAS gene promoter were obtained by PCR withforward primers pS676, ACTAGCTAGC GGAAGGTTCATATTGGGGAT CTGTGC; pS436, ACTAGCTAGC GTAACCCAAC CCTTGTCACT CCAAG; and pS331, ACTAGCTAGC
GCCTCCTCGC CTGTCACCTC TG, and a 3⬘ primer, pAS1,
TCCGCTCGAG CGCATCAAGT TCCGAACACC TTGTC.
They were ligated into XhoI-NheI sites of the pGL3-Basic
vector (Promega) and designated as pGL3-ACAS676,
ACAS463, and ACAS331, respectively. Transfection and luciferase assays were performed as previously described (1)except that pRL-SV40 (Tokyo Ink, Tokyo, Japan) was used asa reference plasmid instead of pSV␤gal. Either an expression
plasmid of SREBP-1a, -1c, or -2, under the regulation of the
CMV early promoter (pCMV-SREBP-1a, -1c, -2) (0.2 ␮g) or an
empty vehicle vector (pCMV7) (0.2 ␮g) as a control, wascotransfected with the indicated luciferase construct andpRL-SV40 (0.2 ␮g) into HepG2 cells by using SuperFect
reagent (Qiagen, Hilden, Germany). The cells were incubatedin DMEM with 10% FCS and cholesterol (10 ␮g/ml) and25-hydroxycholesterol (1 ␮g/ml) to suppress endogenous
SREBP activity. The Dual Luciferase System (Picagene Dual
Seapansy, Toyo Ink, Tokyo, Japan) was used to measure
firefly and seapansy luciferase activities with a luminometer,
Lumat LB9507 (Berthod, Berlin, Germany), according to the
manufacturer’s instruction.
Gel shift assay. The DNA probe was prepared by annealing
both strands of the SRE (see Fig. 3) containing sequence ofthe mouse ACAS gene promoter, GGGCTACACCCCATCACTCCACGGGCC, and was labeled with [␣-32P]dCTP by
the Klenow enzyme, followed by purification on G50 Sephadex columns. The labeled DNA was incubated with a recombinant SREBP-1 protein (100 ng) in a mixture containing 10
mM Tris 䡠 HCl, pH 7.6, 50 mM KCl, 0.05 mM EDTA, 2.5 mM
MgCl2, 8.5% glycerol, 1 mM dithiothreitol, 0.5 ␮g/ml poly(dIdC), 0.1% Triton X-100, and 1 mg/ml nonfat milk for 30 min
on ice. The DNA-protein complexes were resolved on a 4.6%
polyacrylamide gel.
ACAS gene expression profile in various tissues. Tissue
survey of murine and human ACAS mRNA levels was performed using Poly(A)⫹ RNA dot blots that were normalizedby eight different housekeeping genes (RNA Master Blotsand Human Multiple Tissue Expression Array; Clontech,
Palo Alto, CA) according to the manufacturer’s instructions.
Probes used for murine and human blots were produced by
PCR with a forward primer S39 and a reverse primer AS209(for murine blots) and a forward primer, h3S, GACACTCTCGTGTGGGACAC, and a reverse primer, h2AS,
CCTTGTTGTCTGTCCTGTGAGC (for human blots), respectively, which were set on the basis of reported ESTs
(AW007194 and AW242634).

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neal injection of 8-wk-old C57BL/6 mice. Some of the diabetic
mice received subcutaneous insulin administration (100–400
U 䡠 kg⫺1 䡠 day⫺1, Novolet N, Novo Nordisk, Bagsvaerd, Denmark) for 7 days to correct their blood glucose levels. The effectof acetate on the hepatic ACAS gene was estimated by giving
water containing indicated concentrations of acetic acid for 24 h.
RESULTS
Fig. 1. Expression of the acetyl-CoA synthetase (ACAS) gene inlivers from wild-type (WT) and transgenic mice overexpressing sterol
regulatory element-binding protein (SREBP)-1a, -1c, and 2. Northern blot analysis of total RNA from livers of SREBP-1a, -1c, and -2
transgenic mice is shown. Mice were fed on a high-protein/lowcarbohydrate diet for 2 wk to induce the phosphoenolpyruvate carboxykinase (PEPCK) promoter used for transgene expression. This
diet suppresses the basal level of the ACAS mRNA.

Animals and dietary manipulation. Mice were housed incolony cages with a 12:12-h light-dark cycle and were fed a
regular chow diet until the dietary manipulations. C57BL/6
mice 8 wk of age were used for dietary and streptozotocin (STZ)
studies. Mice homozygous for the disrupted SREBP-1 geneallele B (SREBP-1⫺/⫺) were handled as previously described(24). Transgenic mice overexpressing human nuclear
SREBP-1a, -1c, and -2 in the liver have been previously
described (22). The fasting (24 h) and refeeding (12 h) protocol was as previously described (24). To prepare diabetic
mice, STZ (100 mg/kg) or saline was administered by perito-

Cloning of mouse ACAS gene. Results of the microarray analysis identified an EST clone, expression ofwhich was highly (10-fold) increased in the livers oftransgenic mice overexpressing nuclear SREBP-1a(22) compared with that of wild-type mice. This clone(AA537637) had a high similarity to bacterial ACAS.
The increase of this gene transcript by SREBP-1a wasconfirmed by Northern blot analysis of total RNA fromlivers of SREBP-1a transgenic mice (Fig. 1). The hepatic mRNA level of this gene was also elevated by
SREBP-1c and SREBP-2, demonstrating that any
member of the SREBP family can induce this gene. On
the basis of the high similarity to bacterial homologs
and the data we will present later, we assumed that thisclone was a part of the murine ACAS gene. We cloned awhole ACAS cDNA by screening a mouse liver cDNA
library using the 5⬘ RACE method. The murine cDNA
sequence of ACAS open reading frame consisted of 2103
bp coding for 701 amino acids (Fig. 2). The sequence isFig. 2. cDNA and amino acid sequences of the mouse ACAS gene. This
cDNA sequence was essentially identical to a clone that was registered by
P. S. Haghighi and co-workers in the
NCBI GenBank as a murine ACAS
cDNA (AF216873) with four mismatched amino acids (amino acids
498–500; LQS instead of PAI; aminoacid 655; L instead of P) and anotherreported sequence (19).

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Fig. 3. DNA sequence of 5⬘ flanking
region of the mouse ACAS gene. The
promoter and a part of the 5⬘UTR sequence of the mouse ACAS gene isshown and numbered in relation to theputative translation initiation site. A
sequence (ATCACTCCAC) highly similar to the sterol regulatory element(ATCACCCCAC) and inverted Sp1 site
(CCCCGCCCC), two Nkx6.1 sites, and
two C/EBP sites were found and underlined.

highly preserved among many species; i.e., 92% homologous with human, 74% with Drosophila, 63% with
Caenorhabditis elegans, 58% with Saccharomyces cerevisiae (yeast), and 64% with Escherichia coli at theamino acid level. This cDNA was essentially identicalto a clone that was registered by P. S. Haghighi andco-workers in the NCBI GenBank as a murine ACAS
cDNA (AF216873). Very recently, Luong et al. (19)reported the amino acid sequences of human and murine ACAS and their regulation by SREBP and, furthermore, demonstrated that they have an ACAS activity when overexpressed in the cultured cells.
Promoter analysis of the ACAS gene. We obtained aclone containing the mouse ACAS gene from a mouse
genomic DNA BAC library. DNA sequencing of the 5⬘
flanking region of the mouse ACAS gene is shown in
Fig. 3. We identified a sequence (ATCACTCCAC at⫺350 bp) that is highly similar to a classic SRE (ATCACCCCAC). The only mismatched base (6th T instead of C) was at the position of a residue that separates two direct repeats of PyCAC in the consensus andis not conserved among SREs in different SREBP target genes. Downstream of this SRE, a binding site ofSp1 (CCCCGCCCC), an essential cofactor required for
activation by SREBPs, was also found in an invertedorientation, which made this region a highly probablebinding site for SREBPs. Upstream of the SRE, thecomputer-assisted search found two Nkx6.1 sites andtwo C/EBP␣ sites, suggesting that this gene could be

expressed and participate in energy metabolism inpancreatic ␤-cells.
For the promoter analysis, the luciferase reporter
gene containing this region (668 bp) was constructed
and tested for SREBP activation in transfection studies in HepG2 cells, a hepatic cell line of human origin(Fig. 4). The marked increase in luciferase activity of
the ACAS promoter was observed by expression of
nuclear SREBP-1a by cotransfection. Deletion of thepromoter sequence upstream of the SRE did not essentially change the luciferase activity, whereas further
deletion of the SRE completely abolished the SREBP
activation. The data demonstrate that the SRE is absolutely required for SREBP activation of this gene.
Activation of the ACAS promoter was compared among
isoforms of the SREBP family. Expression of nuclear
SREBP-1a, -1c, and -2 resulted in 89-, 57-, and 45-foldrelative increases in luciferase activity, respectively.
The data demonstrate that every member of the
SREBP family can activate this ACAS promoter, although SREBP-1a has the highest transcriptional activity. Similar activation by nuclear SREBP-1a, -1c,and -2 was also observed in transfection studies withhuman embryonic kidney 293 cells (27-, 13-, and 6.6fold, respectively). Replacement of the tyrosine residue
for arginine in the basic region of SREBP (YR mutation) has been shown to result in loss of its binding
activity to an SRE (16). Transfection of the YR mutant
SREBP-1a and -1c did not cause any significant acti-

Fig. 4. Deletion studies with the SRE-containing promoter region of the ACAS gene. Luciferase reporter
genes containing the mouse ACAS promoter region(668 bp) and its sequential deleted fragments in thecontext of presence or absence of the SRE were constructed and transfected with a CMV promoter expression vector of nuclear SREBP-1a or an empty
control vector, and pRL-SV40 as a reference plasmid
into HepG2 cells. Luciferase activity was measured
and normalized to the sea urchin activity. Values are
represented as degree of change compared with theempty control vector.

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Fig. 5. Activation of the ACAS promoter luciferase gene by SREBPs.
A firefly luciferase reporter gene containing the mouse ACAS promoter region (668 bp) was constructed and transfected with a CMV
promoter expression vector of nuclear SREBP-1a, -1c, 2 or an empty
control vector, and a reference plasmid into HepG2 cells. A mutant
version of SREBP-1a (1aM) and -1c (1cM) expression plasmids,in
which the tyrosine residue in the basic region of SREBP was replacedby arginine, was also tested. Firefly luciferase activity was measured
and normalized to the seapansy luciferase activity. Values are represented as degree of change compared with the value from theempty control vector.

vation (Fig. 5), suggesting that SREBP activation of
the ACAS promoter is mediated through its binding to
the SRE. To confirm the direct binding of SREBP to the
SRE in the ACAS promoter, gel mobility shift assays wereconducted (Fig. 6). The labeled SRE probe was shifted byincubation with SREBP-1 protein, and the specific binding
was confirmed by a supershift after addition of SREBP-1
antibody. These promoter studies confirmed that the SRE
in the ACAS gene promoter is responsible for SREBP
activation of ACAS gene expression.
ACAS gene expression profile in various tissues. The
tissue survey of human and mouse ACAS gene expression is shown in Fig. 7, A and B, respectively. In both
species, ACAS was ubiquitously expressed in almost
every tissue tested. Particularly in mice, it was highly
expressed in kidney, liver, submaxillary gland, epididymus, and testis.
Effects of fasting and refeeding on ACAS expression
in mice. Recently, SREBP-1 has been reported to playa crucial role in hepatic expression of lipogenic enzyme
genes, especially in nutritional regulation, as was observed in fasted and refed mice (24). As shown by
Northern blot analysis in Fig. 8, ACAS expression was
significantly downregulated in a fasted state andmarkedly upregulated by refeeding in both liver andadipose tissue. This nutritional change is similar tochanges of other lipogenic enzymes that are controlled
by SREBP-1 (24). This refeeding induction of the ACAS
gene was nearly abolished in the livers and adipose
tissue of SREBP-1 knockout (KO) mice (Fig. 8), indicating that lipogenic induction of the ACAS gene byrefeeding is controlled mainly by SREBP-1. At thesame time, a slight but significant increase in ACAS
RNA was seen in the SREBP-1 KO mice as well as
wild-type mice, suggesting that other factors contribute to expression to a lesser degree.
Effect of insulin-depleted diabetes on ACAS expression. Because insulin has also been known to be important for lipogenic enzyme expression, we estimated theeffects of insulin depletion and its supplementation on

the hepatic mRNA level of ACAS. As shown in Fig. 9,
STZ-induced diabetic mice showed markedly decreased
ACAS expression in the livers compared with normal
control mice, expression that was totally restored byinsulin administration.
We also investigated the consequence of ACAS expression after overloading the substrate of the enzyme
in drinking water. Acetic acid loading resulted in asignificant decrease of ACAS expression in both fasted
and fed mice (Fig. 10). A dose-dependent suppression
was observed for acetate concentrations between 0 and
10% in fasted animals. In fed animals, acetate overloading also suppressed ACAS expression. There was no
significant difference in either food or water consumption that might have affected SREBP-1c expression.
DISCUSSIONACAS is a new member of the family of lipogenic
enzymes. The current study clearly demonstrates that
expression of the mouse acetyl-CoA synthetase gene isFig. 6. Gel mobility shift assay demonstrating a direct binding of
SREBP to the SRE in the mouse ACAS gene promoter. A doublestranded DNA fragment containing the SRE in the mouse ACAS
gene promoter was labeled with [␣-32P]dCTP and incubated in thereaction mixture with (lanes 2–4) or without (lane 1) recombinantnuclear SREBP-1c protein. The shifted band of the DNA probe andprotein complex is indicated by the arrow (lane 2). In a competition
assay, a 1,000-fold molar excess of an unlabeled SRE probe wasadded (lane 3). Specificity of SREBP-1 binding to the SRE probe wasconfirmed by a supershift after the addition of anti-human SREBP-1
monoclonal antibody (2A4, ATCC, 25 ng/ml; lane 4).

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ACAS AS A LIPOGENIC ENZYME

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Fig. 7. Tissue distribution of human and mouse ACAS
gene expression. Human (A) and mouse (B) poly(A)⫹RNA dot blots (RNA Master Blots and Human Multiple
Tissue Expression Array, Clontech) were hybridized
with a radiolabeled mouse ACAS cDNA probe as described in MATERIALS AND METHODS.

nutritionally regulated in the same fashion as all otherknown lipogenic enzymes. Hepatic/adipose ACAS expression was suppressed by fasting and highly induced
by refeeding, which is a typical feeding response of
lipogenic genes. It was also suppressed in a state of
insulin depletion by administration with STZ and wasrestored by insulin supplement, also a well known

response of lipogenic enzymes in a diabetic state.
Therefore, nutritional regulation of ACAS followed alipogenic pattern. This is not surprising, as ACAS is
one of the enzymes responsible for the production ofacetyl-CoA, an initial substrate for lipogenesis. In anutritional state favorable for lipogenesis, acetyl-CoA
is produced from glycolysis and transported from mi-

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Fig. 8. Nutritional regulation of ACAS gene expression in livers andadipose tissue from WT and SREBP-1-deficient mice after fasting
and refeeding treatment. Five male WT and SREBP-1-deficient
knockout (KO) mice were fasted (24 h) and refed (12 h) with ahigh-carbohydrate diet. Total RNA was extracted from livers andadipose tissue of each group and subjected to Northern blot analysis
using a radio-labeled mouse ACAS cDNA probe.

tochondria to the cytosol through a sequence of steps.
However, lipogenic induction of cytosolic ACAS suggests that direct production of acetyl-CoA from freeacetate in the cytosol might play a role in lipogenesis.
The relative contribution of this pathway to lipogenesis
remains unknown, and it awaits gene KO mice of thisgene to estimate this. This enzyme could be more
important in ruminants in which glycolytic activity is
low and acetate is a main source of energy.
The ACAS gene is a target of SREBPs. The expression of the EST clone from the ACAS gene was upregulated by SREBP-1a, which led us to clone this gene.
Recently, we reported that SREBP-1c is a dominantfactor for the expression of most lipogenic genes in theliver (24). Absence of hepatic/adipocytic induction of
the ACAS gene in refed SREBP-1 KO mice in thecurrent study supports the notion that ACAS is another target of SREBP-1 as a lipogenic enzyme. Upregulation of the ACAS gene by SREBPs has alreadybeen shown in the first report of this gene (19). The
SRE sequence was found in the promoter region of the
ACAS gene and was confirmed to be responsible for
SREBP activation by promoter analysis. Luciferase

Fig. 9. Insulin dependency of ACAS gene expression in livers fromtreated diabetic mice. Streptozotocin (STZ)-treated diabetic mice
(C57BL6) (D) and insulin-supplemented mice (I) were prepared as
described in MATERIALS AND METHODS. N, normal control mice. Total
RNA was extracted from livers of each group and subjected to
Northern blot analysis using a radio-labeled mouse ACAS cDNA
probe.

Fig. 10. Acetic acid loading resulted in a significant decrease of
ACAS expression in both fasted and fed mice. Mice (C57BL6) were
fed ad libitum (A) or fasted (B) and were given water containing theindicated concentration of acetic acid for 24 h. Total RNA wasextracted from livers of each group and subjected to Northern blot
analysis by use of a radiolabeled mouse ACAS cDNA probe. (Notethat “fed” and “fasted” experiments were done separately so thatbasal signal levels of these separate studies cannot be compareddirectly.)

assays showed that the ACAS promoter was activatedby SREBP-1a, -1c, or -2, consistent with the observation that the ACAS mRNA was increased in livers fromtransgenic mice overexpressing any of the SREBPs.
The relative activity of each SREBP isoform for the
ACAS SRE as estimated by Northern blot analysis oftransgenic livers (Fig. 1) was similar to that of classic
SRE: SREBP-1a ⬎ SREBP-2 ⬎ SREBP-1c (21). This ispresumably due to a high similarity between the SRE
in the ACAS promoter and the classic SRE originallyfound in the low-density lipoprotein receptor promoter.
The current studies with STZ-induced diabetic micedemonstrated that insulin regulates ACAS gene expression. This is consistent with the previous report onchanges in hepatic ACAS enzyme activity in STZ-induced rats (21). Because insulin is important for
SREBP-1c expression, insulin-dependent ACAS expression can be explained at least partially by its
activation of SREBP-1c.
Physiological roles of ACAS gene in mammals. The
decreased ACAS expression in the mouse liver by oral
administration of acetate is implicative. The suppression of the enzyme expression by excess substrate is agood contrast to the regular mechanism of lipogenic
enzyme regulation, in which conversion of excess energy to lipids is free from a negative feedback control.
There may be a regulatory system for cytosolic production of acetyl-CoA by excess exogenous acetate. In
addition, this gene is highly expressed in many other

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tissues, as well as in lipogenic organs. We also observed a considerable expression of this gene in cultured cells such as 293 cells (data not shown). The
ACAS gene expression in the cultured cells is reported
to be partially under sterol regulation, as predictedfrom the control by SREBPs (19). These observations
suggest that ACAS might have some physiologicalroles other than in lipogenesis. From this standpoint, it
is important to identify and clone a mitochondrial
ACAS. This enzyme produces acetyl-CoA in mitochondria and would be involved in ketogenesis or ATP
production in the tricarboxylic acid cycle and should be
regulated in a different way from the cytosolic enzyme.
Crabtree et al. (6) have proposed futile cycling ofacetate between free acetate and acetyl-CoA thoughcytoplasmic and mitochondrial pathways. One hypothesis of why such a pathway exists is to provide a meansby which free acetate levels can be controlled (i.e.,
buffered). This hypothesis is attractive when one considers that ACAS is expressed in all tissues studied.
There could be other functions for ACAS as well. Further studies are needed to clarify the physiologicalroles and regulation of both enzymes in cellular energy
metabolism.
In the current studies, we cloned and identified themurine ACAS gene as a target of SREBPs and a newmember of the lipogenic enzyme family. Acetyl-CoA
plays a pivotal role in cellular fuel metabolism. Further
studies on ACAS might open up a new aspect of glucose
and fatty acid metabolism and have therapeutic implications, because acetate is known to be a better fuelsource than glucose, especially for individuals with
impaired glucose tolerance and diabetes.
We thank Hiroshi Kajikawa, PhD, Chief Researcher, Digestive
Microbiology Laboratory of National Institute of Animal Industry(Tsukuba, Japan); Kenji Tayama, Ph.D., Manager, Central ResearchInstitute, Mitsukan Group (Handa, Japan); and Hiromitsu Nakauchi, MD, PhD, Professor of Immunology, Institute of Basic Medicine,
University of Tsukuba (Tsukuba, Japan) for fruitful discussion. We
are also grateful to Alyssa H Hasty, Ph.D., Vanderbilt University
Medical Center (Nashville, TN), for careful reading of the manuscript.
This work is supported by the Promotion of Fundamental Studies
in Health Science of the Organization for Pharmaceutical Safety andResearch (OPSR), Health Sciences Research Grants (Research on
Human Genome and Gene Therapy) from Ministry of Health andWelfare, and a research project grant of the University of Tsukuba.
H. Sone is the recipient of a Grant-in-Aid for Scientific Research fromthe Japan Society for the Promotion of Science (no. 12770632).
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