Acetyl-coenzyme A synthetase is a lipogenic enzyme controlled
by SREBP-1 and energy status
Hirohito
Sone1,
Hitoshi
Shimano1,
Yuki
Sakakura1,
Noriyuki
Inoue1,
Michiyo
Amemiya-Kudo3,
Naoya
Yahagi3,
Mitsujiro
Osawa2,
Hiroaki
Suzuki1,
Tomotaka
Yokoo1,
Akimitsu
Takahashi1,
Kaoruko
Iida1,
Hideo
Toyoshima1,
Atsushi
Iwama2, and
Nobuhiro
Yamada1
1 Department of Internal Medicine, Institute of Clinical
Medicine, and 2 Department of Immunology, Institute of Basic
Medicine, University of Tsukuba, Tsukuba 305-8575; and
3 Department of Metabolic Disease, School of Medicine,
University of Tokyo, Tokyo 113-8655, Japan
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ABSTRACT |
DNA microarray
analysis on upregulated genes in the livers from transgenic mice
overexpressing nuclear sterol regulatory element-binding protein
(SREBP)-1a, identified an espressed sequence tag (EST) encoding
a part of murine cytosolic acetyl-coenzyme A synthetase (ACAS).
Northern blot analysis of the livers 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 an adjacent 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, and this
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. When acetate 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 acetyl-CoA directly from cellular
acetate for lipogenesis. However, its high conservation among different
species and the wide range of its tissue distribution suggest that this
enzyme might also play an important role in basic cellular energy metabolism.
lipogenic enzyme; acetate; diabetes; insulin; transcription
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INTRODUCTION |
INTRACELLULAR
CHOLESTEROL and fatty acid synthesis are 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 and
activate 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 the transcription of genes involved in fatty acid synthesis,
whereas SREBP-2 is actively involved in the transcription of
cholesterogenic enzymes (13, 24). SREBP-1a is a stronger activator than SREBP-1c because of a longer transactivation domain, and
it has a wider range of target genes involved in both cholesterol and
fatty acid synthesis (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 a crucial 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 high carbohydrate diets, polyunsaturated fatty acid-enriched diets,
and fasting-refeeding regimens (12, 15, 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 intracellular enzyme 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 microbiological point of view (8,
27). ACAS activity has also been well known 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 and other 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 well understood. Because ACAS produces
acetyl-CoA, which is a key branching molecule for different metabolic
pathways, it could play an important role in energy metabolism in
mammals. In a search for new targets of SREBP-1, we cloned the murine
ACAS cDNA and analyzed its gene promoter. Investigation of the
tissue-specific expression profile and nutritional regulation
demonstrated a new aspect of this gene as a lipogenic enzyme.
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MATERIALS AND METHODS |
Materials.
Mice (C57BL/6NCrj) were obtained from Charles River Japan
(Yokohama, Japan). All animal experiments had been approved by a review
of the institutional board 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 to label
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 (free software distributed by the National
Institutes of Health, Bethesda, MD) was used to quantify intensity of
the detected bands.
cDNA cloning of the murine ACAS gene and its promoter region.
Poly(A)+ RNA samples were isolated from livers of male
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,
and homologs and other searches were performed in the National Center
for Biotechnology Information (NCBI) database to collect 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.
A liver cDNA library of SREBP-1a transgenic mice was prepared in the
pCMV7 vector. The library was screened for full-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
were analyzed by ABI Prism software version 3.0 (Perkin-Elmer).
Luciferase promoter assay of ACAS gene.
We obtained a clone containing the 5' flanking region of the ACAS gene
by screening 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 the pGEM3Zf 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 from 676, 463, and 331 bp 5' upstream of the ACAS
coding region) of the ACAS gene promoter were obtained by PCR with
forward primers pS676, ACTAGCTAGC GGAAGGTTCA TATTGGGGAT 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 as a 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 µg) or an empty vehicle vector (pCMV7) (0.2 µg) as a control, was
cotransfected with the indicated luciferase construct and pRL-SV40 (0.2 µg) into HepG2 cells by using SuperFect reagent (Qiagen, Hilden,
Germany). The cells were incubated in DMEM with 10% FCS and
cholesterol (10 µg/ml) and 25-hydroxycholesterol (1 µg/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 of the 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 · HCl, pH 7.6, 50 mM KCl, 0.05 mM EDTA, 2.5 mM
MgCl2, 8.5% glycerol, 1 mM dithiothreitol, 0.5 µg/ml
poly(dI-dC), 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 normalized by eight
different housekeeping genes (RNA Master Blots and 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).
Animals and dietary manipulation.
Mice were housed in colony 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 gene allele 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 peritoneal injection of
8-wk-old C57BL/6 mice. Some of the diabetic mice received subcutaneous
insulin administration (100-400 U · kg
1 · day
1,
Novolet N, Novo Nordisk, Bagsvaerd, Denmark) for 7 days to
correct their blood glucose levels. The effect of acetate on the
hepatic ACAS gene was estimated by giving water containing
indicated concentrations of acetic acid for 24 h.
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RESULTS |
Cloning of mouse ACAS gene.
Results of the microarray analysis identified an EST clone, expression
of which was highly (10-fold) increased in the livers of transgenic
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 was
confirmed by Northern blot analysis of total RNA from livers 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 this clone was a part of the murine ACAS gene. We cloned a whole 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 is 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
the amino acid level. This cDNA 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). 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.

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Fig. 1.
Expression of the acetyl-CoA synthetase (ACAS) gene in
livers 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/low-carbohydrate 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.
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Fig. 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; amino acid 655; L instead of P) and
another reported sequence (19).
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Promoter analysis of the ACAS gene.
We obtained a clone 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 and is not conserved among SREs in
different SREBP target genes. Downstream of this SRE, a binding site of
Sp1 (CCCCGCCCC), an essential cofactor required for activation by
SREBPs, was also found in an inverted orientation, which made this
region a highly probable binding site for SREBPs. Upstream of the SRE,
the computer-assisted search found two Nkx6.1 sites and two C/EBP
sites, suggesting that this gene could be expressed and participate in
energy metabolism in pancreatic
-cells.

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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 is
shown and numbered in relation to the putative 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.
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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 the promoter 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-fold relative
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 with human embryonic kidney 293 cells (27-, 13-, and 6.6-fold, 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 activation (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 were
conducted (Fig. 6). The labeled SRE probe
was shifted by incubation 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.

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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 the context 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 the
empty 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 replaced by
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 the empty control
vector.
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Fig. 6.
Gel mobility shift assay demonstrating a direct binding
of SREBP to the SRE in the mouse ACAS gene promoter. A double-stranded
DNA fragment containing the SRE in the mouse ACAS gene promoter was
labeled with [ -32P]dCTP and incubated in the reaction
mixture with (lanes 2-4) or without (lane 1)
recombinant nuclear SREBP-1c protein. The shifted band of the DNA probe
and protein complex is indicated by the arrow (lane 2). In a
competition assay, a 1,000-fold molar excess of an unlabeled SRE probe
was added (lane 3). Specificity of SREBP-1 binding to the
SRE probe was confirmed 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 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.

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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.
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Effects of fasting and refeeding on ACAS expression in mice.
Recently, SREBP-1 has been reported to play a 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 and markedly upregulated by refeeding
in both liver and adipose tissue. This nutritional change is similar to
changes 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 by
refeeding is controlled mainly by SREBP-1. At the same 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.

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Fig. 8.
Nutritional regulation of ACAS gene expression in livers
and adipose 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 a high-carbohydrate diet.
Total RNA was extracted from livers and adipose tissue of each group
and subjected to Northern blot analysis using a radio-labeled mouse
ACAS cDNA probe.
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Effect of insulin-depleted diabetes on ACAS expression.
Because insulin has also been known to be important for lipogenic
enzyme expression, we estimated the effects 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 by insulin
administration.

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Fig. 9.
Insulin dependency of ACAS gene expression in livers from
treated 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.
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We also investigated the consequence of ACAS expression after
overloading the substrate of the enzyme in drinking water. Acetic acid
loading resulted in a significant 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.

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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 the indicated concentration of acetic acid for 24 h.
Total RNA was extracted from livers of each group and subjected to
Northern blot analysis by use of a radiolabeled mouse ACAS cDNA probe.
(Note that "fed" and "fasted" experiments were done separately
so that basal signal levels of these separate studies cannot be
compared directly.)
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DISCUSSION |
ACAS is a new member of the family of lipogenic enzymes.
The current study clearly demonstrates that expression of the mouse
acetyl-CoA synthetase gene is nutritionally regulated in the same
fashion as all other known 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 was restored by insulin supplement, also a well known response of
lipogenic enzymes in a diabetic state. Therefore, nutritional
regulation of ACAS followed a lipogenic pattern. This is not
surprising, as ACAS is one of the enzymes responsible for the
production of acetyl-CoA, an initial substrate for lipogenesis. In a
nutritional state favorable for lipogenesis, acetyl-CoA is produced
from glycolysis and transported from mitochondria to the cytosol
through a sequence of steps. However, lipogenic induction of cytosolic
ACAS suggests that direct production of acetyl-CoA from free acetate 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 this gene 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 dominant factor for the expression of most lipogenic
genes in the liver (24). Absence of hepatic/adipocytic induction of the ACAS gene in refed SREBP-1 KO mice in the current study supports the notion that ACAS is another target of SREBP-1 as a
lipogenic enzyme. Upregulation of the ACAS gene by SREBPs has already
been 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 assays showed that the ACAS promoter was activated by
SREBP-1a, -1c, or -2, consistent with the observation that the ACAS
mRNA was increased in livers from transgenic mice overexpressing any of
the SREBPs. The relative activity of each SREBP isoform for the ACAS
SRE as estimated by Northern blot analysis of transgenic livers (Fig.
1) was similar to that of classic SRE: SREBP-1a > SREBP-2 > SREBP-1c (21). This is presumably due to a high similarity
between the SRE in the ACAS promoter and the classic SRE originally
found in the low-density lipoprotein receptor promoter.
The current studies with STZ-induced diabetic mice demonstrated that
insulin regulates ACAS gene expression. This is consistent with the
previous report on changes 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 a good 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 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 predicted from
the control by SREBPs (19). These observations suggest
that ACAS might have some physiological roles 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 of
acetate between free acetate and acetyl-CoA though cytoplasmic and mitochondrial pathways. One hypothesis of why such a pathway exists is
to provide a means by 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
physiological roles and regulation of both enzymes in cellular energy metabolism.
In the current studies, we cloned and identified the murine ACAS gene
as a target of SREBPs and a new member 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 fuel source than glucose, especially for individuals
with impaired glucose tolerance and diabetes.
 |
ACKNOWLEDGEMENTS |
We thank Hiroshi Kajikawa, PhD, Chief Researcher, Digestive
Microbiology Laboratory of National Institute of Animal Industry (Tsukuba, Japan); Kenji Tayama, Ph.D., Manager, Central Research Institute, 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.
 |
FOOTNOTES |
This work is supported by the Promotion of Fundamental Studies in
Health Science of the Organization for Pharmaceutical Safety and
Research (OPSR), Health Sciences Research Grants (Research on Human
Genome and Gene Therapy) from Ministry of Health and Welfare, and a
research project grant of the University of Tsukuba. H. Sone is
the recipient of a Grant-in-Aid for Scientific Research from the Japan
Society for the Promotion of Science (no. 12770632).
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{at}md.tsukuba.ac.jp).
The costs of publication of this
article were defrayed in part by the
payment 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.
Received 2 May 2001; accepted in final form 11 September 2001.
 |
REFERENCES |
1.
Amemiya-Kudo, M,
Shimano H,
Yoshikawa T,
Yahagi N,
Hasty AH,
Okazaki H,
Tamura Y,
Shionoiri F,
Iizuka Y,
Ohashi K,
Osuga Ji Harada K,
Gotoda T,
Sato R,
Kimura S,
Ishibashi S,
and
Yamada N.
Promoter analysis of the mouse sterol regulatory element-binding protein (SREBP)-1c gene.
J Biol Chem
275:
31078-31085,
2000[Abstract/Free Full Text].
2.
Briggs, MR,
Yokoyama C,
Wang X,
Brown MS,
and
Goldstein JL.
Nuclear protein that binds sterol regulatory element of low density lipoprotein receptor promoter. I. Identification of the protein and delineation of its target nucleotide sequence.
J Biol Chem
268:
14490-14496,
1993[Abstract/Free Full Text].
3.
Brown, MS,
and
Goldstein JL.
The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor.
Cell
89:
331-340,
1997[ISI][Medline].
4.
Brown, MS,
and
Goldstein JL.
A proteolytic pathway that controls the cholesterol content of membranes, cells, and blood.
Proc Natl Acad Sci USA
96:
11041-11048,
1999[Abstract/Free Full Text].
5.
Brown, MS,
Ye J,
Rawson RB,
and
Goldstein JL.
Regulated intramembrane proteolysis: a control mechanism conserved from bacteria to humans.
Cell
100:
391-398,
2000[ISI][Medline].
6.
Crabtree, B,
Gordon MJ,
and
Christie SL.
Measurement of the rates of acetyl-CoA hydrolysis and synthesis from acetate in rat hepatocytes and the role of these fluxes in substrate cycling.
Biochem J
270:
219-225,
1990[ISI][Medline].
7.
Foretz, M,
Pacot C,
Dugail I,
Lemarchand P,
Guichard C,
Le Liepvre X,
Berthelier Lubrano C,
Spiegelman B,
Kim JB,
Ferre P,
and
Foufelle F.
ADD1/SREBP-1c is required in the activation of hepatic lipogenic gene expression by glucose.
Mol Cell Biol
19:
3760-3768,
1999[Abstract/Free Full Text].
8.
Garre, V,
Murillo FJ,
and
Torres-Martinez S.
Isolation of the facA (acetyl-CoA synthetase) gene of Phycomyces blakesleeanus.
Mol Gen Genet
244:
278-286,
1994[ISI][Medline].
9.
Goodridge, AG.
Dietary regulation of gene expression: enzymes involved in carbohydrate and lipid metabolism.
Annu Rev Nutr
7:
157-185,
1987[ISI][Medline].
10.
Hasty, AH,
Shimano H,
Yahagi N,
Amemiya-Kudo M,
Perrey S,
Yoshikawa T,
Osuga Ji Okazaki H,
Tamura Y,
Iizuka Y,
Shionoiri F,
Ohashi K,
Harada K,
Gotoda T,
Nagai R,
Ishibashi S,
and
Yamada N.
Sterol regulatory element-binding-1 protein is regulated by glucose at the transcriptional level.
J Biol Chem
276:
37402-37408,
2000[Medline].
11.
Hillgartner, FB,
Salati LM,
and
Goodridge AG.
Physiological and molecular mechanisms involved in nutritional regulation of fatty acid synthesis.
Physiol Rev
75:
47-76,
1995[Free Full Text].
12.
Horton, JD,
Bashmakov Y,
Shimomura I,
and
Shimano H.
Regulation of sterol regulatory element binding proteins in livers of fasted and refed mice.
Proc Natl Acad Sci USA
95:
5987-5992,
1998[Abstract/Free Full Text].
13.
Horton, JD,
Shimomura I,
Brown MS,
Hammer RE,
Goldstein JL,
and
Shimano H.
Activation of cholesterol synthesis in preference to fatty acid synthesis in liver and adipose tissue of transgenic mice overproducing sterol regulatory element-binding protein-2.
J Clin Invest
101:
2331-2339,
1998[Abstract/Free Full Text].
14.
Hua, X,
Yokoyama C,
Wu J,
Briggs MR,
Brown MS,
Goldstein JL,
and
Wang X.
SREBP-2, a second basic-helix-loop-helix-leucine zipper protein that stimulates transcription by binding to a sterol regulatory element.
Proc Natl Acad Sci USA
90:
11603-11607,
1993[Abstract].
15.
Kim, HJ,
Takahashi M,
and
Ezaki O.
Fish oil feeding decreases mature sterol regulatory element-binding protein 1 (SREBP-1) by down-regulation of SREBP-1c mRNA in mouse liver. A possible mechanism for down-regulation of lipogenic enzyme mRNAs.
J Biol Chem
274:
25892-25898,
1999[Abstract/Free Full Text].
16.
Kim, JB,
Spotts GD,
Halvorsen YD,
Shih HM,
Ellenberger T,
Towle HC,
and
Spiegelman BM.
Dual DNA binding specificity of ADD1/SREBP1 controlled by a single amino acid in the basic helix-loop-helix domain.
Mol Cell Biol
15:
2582-2588,
1995[Abstract].
17.
Knowles, SE,
Jarrett IG,
Filsell OH,
and
Ballard FJ.
Production and utilization of acetate in mammals.
Biochem J
142:
401-411,
1974[ISI][Medline].
18.
Koo, SH,
and
Towle HC.
Glucose regulation of mouse S14 gene expression in hepatocytes. Involvement of a novel transcription factor complex.
J Biol Chem
275:
5200-5207,
2000[Abstract/Free Full Text].
19.
Luong, A,
Hannah V,
Brown M,
and
Goldstein J.
Molecular characterization of human acetyl-CoA synthetase, an enzyme regulated by sterol regulatory element-binding proteins.
J Biol Chem
275:
458-466,
2000.
20.
McNeil, NI.
The contribution of the large intestine to energy supplies in man.
Am J Clin Nutr
39:
338-342,
1984[Abstract].
21.
Murthy, VK,
and
Steiner G.
Hepatic acetate levels in relation to altered lipid metabolism.
Metabolism
22:
81-84,
1973[ISI][Medline].
22.
Shimano, H,
Horton JD,
Hammer RE,
Shimomura I,
Brown MS,
and
Goldstein JL.
Overproduction of cholesterol and fatty acids causes massive liver enlargement in transgenic mice expressing truncated SREBP-1a.
J Clin Invest
98:
1575-1584,
1996[Abstract/Free Full Text].
23.
Shimano, H,
Horton JD,
Shimomura I,
Hammer RE,
Brown MS,
and
Goldstein JL.
Isoform 1c of sterol regulatory element binding protein is less active than isoform 1a in livers of transgenic mice and in cultured cells.
J Clin Invest
99:
846-854,
1997[Abstract/Free Full Text].
24.
Shimano, H,
Yahagi N,
Amemiya Kudo M,
Hasty AH,
Osuga J,
Tamura Y,
Shionoiri F,
Iizuka Y,
Ohashi K,
Harada K,
Gotoda T,
Ishibashi S,
and
Yamada N.
Sterol regulatory element-binding protein-1 as a key transcription factor for nutritional induction of lipogenic enzyme genes.
J Biol Chem
274:
35832-35839,
1999[Abstract/Free Full Text].
25.
Thewke, DP,
Panini SR,
and
Sinensky M.
Oleate potentiates oxysterol inhibition of transcription from sterol regulatory element-1-regulated promoters and maturation of sterol regulatory element-binding proteins.
J Biol Chem
273:
21402-21407,
1998[Abstract/Free Full Text].
26.
Tontonoz, P,
Kim JB,
Graves RA,
and
Spiegelman BM.
ADD1: a novel helix-loop-helix transcription factor associated with adipocyte determination and differentiation.
Mol Cell Biol
13:
4753-4759,
1993[Abstract].
27.
Van den Berg, MA,
de Jong Gubbels P,
Kortland CJ,
van Dijken JP,
Pronk JT,
and
Steensma HY.
The two acetyl-coenzyme A synthetases of Saccharomyces cerevisiae differ with respect to kinetic properties and transcriptional regulation.
J Biol Chem
271:
28953-28959,
1996[Abstract/Free Full Text].
28.
Wang, X,
Briggs MR,
Hua X,
Yokoyama C,
Goldstein JL,
and
Brown MS.
Nuclear protein that binds sterol regulatory element of low density lipoprotein receptor promoter. II. Purification and characterization.
J Biol Chem
268:
14497-14504,
1993[Abstract/Free Full Text].
29.
Worgall, TS,
Sturley SL,
Seo T,
Osborne TF,
and
Deckelbaum RJ.
Polyunsaturated fatty acids decrease expression of promoters with sterol regulatory elements by decreasing levels of mature sterol regulatory element-binding protein.
J Biol Chem
273:
25537-25540,
1998[Abstract/Free Full Text].
30.
Yahagi, N,
Shimano H,
Hasty A,
Amemiya-Kudo M,
Okazaki H,
Tamura Y,
Iizuka Y,
Shionoiri F,
Ohashi K,
Osuga J,
Harada K,
Gotoda T,
Nagai R,
Ishibashi S,
and
Yamada N.
A crucial role of sterol regulatory element-binding protein-1 in the regulation of lipogenic gene expression by polyunsaturated fatty acids.
J Biol Chem
274:
35840-35844,
1999[Abstract/Free Full Text].
31.
Yokoyama, C,
Wang X,
Briggs MR,
Admon A,
Wu J,
Hua X,
Goldstein JL,
and
Brown MS.
SREBP-1, a basic-helix-loop-helix-leucine zipper protein that controls transcription of the low density lipoprotein receptor gene.
Cell
75:
187-197,
1993[ISI][Medline].
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