From the Department of Biochemistry, Molecular
Biology & Biophysics, University of Minnesota, Minneapolis, Minnesota
55455 and the § Department of Nutritional Sciences,
University of Connecticut, Storrs, Connecticut 06269
Received for publication, February 20, 2001
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ABSTRACT |
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The rat acetyl-CoA carboxylase (ACC) Feeding a high carbohydrate, low fat diet to rodents increases the
expression of genes encoding enzymes involved in lipogenesis, the
conversion of carbohydrate to triglycerides, in the liver and adipose
tissue (for review, see Refs. 1 and 2). These include the central
enzymes of fatty acid biosynthesis: ATP-citrate lyase, acetyl-CoA
carboxylase (ACC),1 and fatty
acid synthase (FAS). In addition, enzymes of glycolysis, such as
pyruvate kinase (L-PK); of NADPH generation, such as malic enzyme; of fatty acid maturation, such as stearoyl-CoA desaturase and
of triglyceride formation, such as glycerol-3-phosphate acyltransferase are induced in response to dietary carbohydrate. For most of the genes
examined, transcription is the major site of control, although changes
in stability play a role in controlling mRNA induction for certain
gene products.
Both insulin and glucose play a role in the process by which high
carbohydrate diet induces lipogenic gene expression. The actions of
insulin are mediated at least in part by SREBP-1c (for review, see
Refs. 3-5). This basic/helix-loop-helix/leucine zipper transcription factor is induced at the level of transcription in
response to insulin (6-8). In turn, SREBP-1c binds to the promoters of
a number of the lipogenic enzyme genes to enhance their expression
(e.g. Refs. 9-11). Consequently, mice bearing a homozygous
deletion of the SREBP-1 gene show an impaired ability to induce
lipogenic enzymes in response to a high carbohydrate diet compared with
normal littermates (12). Similarly, transgenic mice overexpressing the
nuclear form of SREBP-1c in their liver have elevated rates of
lipogenesis and increased mRNA levels for many of the lipogenic
enzyme genes (13, 14).
In addition to insulin, glucose also provides an important signal for
induction of the lipogenic enzyme genes (15). Primary hepatocytes
cultured in the presence of constant insulin levels display a marked
induction of most lipogenic enzyme genes when glucose concentrations in
the media are elevated above fasting levels. This response requires
increased glucose metabolism; however, the intracellular pathway
responsible for mediating the actions of glucose remains unresolved.
Sequences important for the transcriptional induction by glucose have
been identified in three genes to date: FAS2,
L-PK, and S14 (16-19). S14 encodes
a 17-kDa gene product postulated to play a regulatory role in the
lipogenic process (20). In all three cases, the critical regulatory
element, designated the ChoRE, consists of two E box half-sites related
to the sequence 5'-CACG (19). These half-sites are found oriented in
either direct or inverted fashion, but the spacing between them is
critical for the ability to respond to glucose. Recently, we have
detected by EMSA a novel nuclear protein that recognizes the ChoREs of these three genes and designated this factor as the carbohydrate responsive factor or ChoRF (19). The identity of ChoRF is currently unknown. Although ChoRF has been predicted to be a member of the basic/helix-loop-helix family based on its binding site, it is distinct
from SREBP-1c by a number of criteria (21).
ACC catalyzes the first step in fatty acid synthesis and is under
complex control (for review, see Ref. 22). Two genes for ACC,
designated Studies on the control of the ACC Despite its apparent physiological significance, little is known about
the regulation of the ACC Primary Hepatocyte Culture and Transfections--
Primary
hepatocytes were isolated from male Harlan Sprague-Dawley rats
(180-260 g) maintained on a 12-h light/dark cycle with free access to
normal rat chow as described previously (32). After a 3-h attachment
period, cells were transfected with F1 reagent (Targeting Systems, San
Diego, CA) in modified Williams' E media containing 23 mM
HEPES, 0.01 µM dexamethasone, 0.1 unit/ml insulin, 50 unit/ml penicillin, 50 µg/ml streptomycin, and 5.5 mM
glucose for 12-14 h. Cells were then cultured in the same media containing either 5.5 or 27.5 mM glucose with or without
insulin with an overlay of 0.33 mg/ml Matrigel (Collaborative
Biomedical Products, Bedford, MA). In some experiments, 0.5 µM 3,5,3'-triiodo-L-thyronine was added as
indicated in figure legends. After 27 h, cells were harvested for
assay of luciferase activity. Luciferase activities are expressed as
relative light units measured per microgram of protein. All experiments
were repeated three times with duplicate samples for each point and
showed identical response patterns. Variation within duplicates is
generally less than 20%.
Plasmid Constructs--
A rat genomic P1 library was screened by
Genome Systems (St. Louis, MO) using primers recognizing ACC Measurement of ACC PI and PII Transcripts by
RT-PCR--
Hepatocytes were incubated in 5.5 mM glucose
in the absence of insulin for 18 h with an overlay of Matrigel.
Cells were then washed and treated with media containing 5.5 or 27.5 mM glucose in the absence or presence of 0.1 unit/ml
insulin for 27 h. Total cell RNA was isolated using Trizol reagent
(Life Technologies, Gaithersburg, MD) according to the manufacturer's
protocol. RNA concentrations were determined spectrophotometrically.
RT-PCR reactions were run using the One-Step RT-PCR Kit (Qiagen,
Valencia, CA) using 0.1 µg of RNA. PI- and PII-specific
oligonucleotide primers were as described (28) except that the
forward PII-specific primer was changed to 5'-CACACCTCGGCGCAGGGGCTC.
For PI-specific primers, reactions were run for 29 cycles with an
annealing temperature of 58 °C. For PII-specific primers, reactions
were run for 24 cycles using an annealing temperature of 61 °C. As a
control, the following ribosomal protein L32-specific primers were
used: 5'-AAACTGGCGGAAACCCAGAG and 5'-GCAGCACTTCCAGCTCCTTG. Primers were annealed at a temperature of 58 °C and 24 cycles were used for the
PCR. Reaction products were subjected to electrophoresis in 2% low
melting point agarose gels and band intensities were compared by
imaging of ethidium bromide-stained gels.
To quantify PI transcripts, the LightCycler System (Roche Molecular
Biochemicals, Indianapolis, IN) was used. This system allows
amplification and detection by fluorescence of PCR products in the same
tube using a kinetic approach. RT-PCR reactions were run using SYBR
Green I master mix and the same primers as indicated above. Data was
acquired as a function of cycle number at a temperature of 82 °C,
1 °C below the melting point for the ACCI PCR product. LightCycler
software was used to compare amplification in the experimental samples
during the log-linear phase to a standard curve established from a
dilution series of control RNA.
Electrophoretic Mobility Shift Assay--
Nuclear proteins were
extracted from livers of rats using the procedure described by Koo and
Towle (19). The fraction that precipitated between 0 and 10%
polyethylene glycol 8000 (Hampton Research, Boston, MA) was used for
DNA binding experiments. EMSA was carried out under conditions
previously described (33). A typical reaction contained 100,000 cpm
(approximately 6 fmol) of 32P-labeled oligonucleotide with
15 µg of nuclear protein. Nonspecific competitors were 0.1 µg of
poly(pI-dC) and 1.9 µg of poly(dA-dT). Following incubation at room
temperature for 30 min, samples were subjected to electrophoresis on a
4.5% nondenaturing polyacrylamide gel and subjected to PhosphorImager
analysis. For competition EMSA, the indicated molar excess of unlabeled
oligonucleotide was added together with radiolabeled probe into the
sample prior to incubation.
Effects of Glucose and Insulin on ACC Transcript Levels from PI and
PII Promoters--
Previous work in one of our laboratories (H. C. F.) used an RT-PCR assay to monitor transcripts from PI and PII
promoters in a variety of tissues. These studies demonstrated
PI-generated transcripts were elevated in response to feeding a high
carbohydrate, low fat diet in the liver, while transcripts from the PII
promoter did not respond (28). We wished to verify that this pattern of
control can be recapitulated in primary hepatocytes and to examine the
contribution of insulin and glucose to the process. Consequently,
primary hepatocytes were isolated from rats and cultured under
conditions of low glucose (5.5 mM) minus insulin, low
glucose plus insulin, high glucose (27.5 mM) minus insulin or high glucose plus insulin for 27 h. RNA was isolated from these hepatocytes and used in an RT-PCR assay with primers that would specifically detect PI or PII transcripts, as described previously (28). As shown in Fig. 1, transcripts
from the PI promoter are barely detectable in hepatocytes maintained in
low glucose media in the absence of insulin. Treatment with insulin in
the presence of low glucose caused a significant increase above the
basal levels. Likewise, increasing glucose from 5.5 to 27.5 mM in the absence of insulin caused a modest increase in
the level of PI-generated transcripts. However, when cells were treated
with both high glucose and insulin, a marked induction in PI-generated
transcripts was observed. Using a real time RT-PCR system to quantify
PI transcripts, increases of 9-, 6.6-, and 48-fold were found in cells
treated with insulin alone, high glucose alone, or the combined
treatment, respectively, compared with the low glucose minus insulin
state. By contrast, PII transcripts were only marginally increased by addition of insulin in low glucose conditions, but no effect of glucose
was observed. A control using ribosomal protein L32-specific primers
indicated comparable amounts of RNA in each sample. In this experiment,
it is not possible to directly compare levels of PI and PII transcripts
to each other. However, the results demonstrate that primary
hepatocytes are capable of responding to signals of glucose and insulin
in a manner similar to the whole animal fed high carbohydrate diet by
stimulating production of transcripts from the PI promoter.
Glucose Stimulates Activity of the ACC PI Promoter--
To
determine whether the regulation of PI-generated ACC mRNA is
mediated at the transcriptional level, transfection experiments were
performed. A segment of the ACC PI gene from Identification of a Possible ChoRE in the ACC PI Promoter--
The
ACC PI promoter from
To test the possible significance of this element from the ACC PI
promoter, we asked whether ChoRF recognized an oligonucleotide containing this sequence. ChoRF was detected as a gel shift band that
formed between rat liver nuclear proteins and various ChoREs (19). In
Fig. 4A, an oligonucleotide
containing the putative ChoRE of the ACC PI promoter ( Functional Role for the ACC PI Promoter ChoRE--
To test the
functional significance of the putative ChoRE in the ACC PI promoter,
we examined the effects of mutating this site. For this experiment, an
ACC PI promoter fragment from
To test also whether this element can function in an independent manner
to confer a glucose response, an oligonucleotide containing the
putative ChoRE of the ACC PI promoter was linked in two copies to a
minimal L-PK promoter containing sequences from
Given the published data suggesting that SREBP-1c is a major player in
response of animals to a high carbohydrate diet, it was of interest to
examine whether SREBP-1c was capable of stimulating through the ACC
ChoRE site. The same constructs tested above were examined. For this
experiment, a vector that drove expression of the nuclear form of
SREBP-1c was co-transfected with the various reporter constructs. As
can be seen in Fig. 6B, nuclear SREBP-1c expression resulted
in increased reporter activity from a construct containing a consensus
SREBP-binding site. However, no effect was observed on the activity of
the ACC PI ChoRE-containing construct or on an L-PK
ChoRE construct. A modest induction of rat S14
ChoRE-containing construct has been observed previously and attributed
to the presence of a CACGTG motif in this element (21), a generic
binding site for basic/helix-loop-helix factors. These data suggest
that SREBP-1c is not the factor responsible for mediating effects of
glucose on the ACC PI promoter ChoRE.
ACC is generally recognized as the rate-limiting step for fatty
acid biosynthesis. Consequently, the enzymatic activity of ACC is
tightly controlled by a variety of mechanisms including covalent
modification, allosteric control, and alterations in its polymeric
state (22, 34). In addition, diet and hormonal factors influence the
level of ACC production (22). In this regard, ACC is a member of a
family of lipogenic enzymes that are induced under conditions favoring
energy storage and repressed under conditions of energy demand.
The ACC The control of ACC The induction of lipogenic enzyme genes in response to feeding of a
high carbohydrate diet can be attributed to the actions of insulin and
glucose. Stimulation of most lipogenic enzyme genes requires the
presence of both insulin and elevated glucose in primary hepatocytes.
We have observed a similar pattern of regulation for the ACC
PI-generated transcripts in hepatocytes. Although either elevated
glucose or insulin can cause a modest increase, the two signals
synergize to greatly elevate ACC mRNA levels when simultaneously
present. A similar situation has been reported for induction of ACC
mRNA in primary adipocytes (37). We have recently presented
evidence that two transcription factors are involved in the induction
of lipogenic enzyme genes by insulin and glucose (21). One of these
factors is SREBP-1c. The expression of SREBP-1c is elevated in
hepatocytes in response to insulin (8, 38). In turn, SREBP-1c binds to
the promoters of several lipogenic enzyme genes, including fatty acid
synthase (6, 9), stearoyl-CoA desaturase (11), and glycerol-3-phosphate
acyltransferase (10). In each of these cases, co-transfection of an
SREBP-1c expression vector together with promoter/reporter plasmids
containing the SREBP-binding site led to induction of reporter gene
activity. Based on this evidence, SREBP-1c is responsible, at least in
part, for mediating effects of insulin on lipogenic enzyme induction.
In addition to SREBP-1c, an independent transcription factor, ChoRF,
appears to be critical for regulation of lipogenic enzyme genes by
glucose. ChoRF was identified by its ability to bind to the ChoRE of
L-PK and S14 promoters (19). This element is responsible for conferring a response of these genes to glucose in
hepatocytes and is not recognized by SREBP-1c. We further postulated that many of the lipogenic enzyme genes would require both SREBP-1c and
ChoRF during induction by high carbohydrate diet. This has been shown
to be the case for two genes to date: the S14 gene (21) and
the FAS gene.2 In both of these cases, SREBP-1c- and
ChoRF-binding sites are present and can function independently to
mediate modest effects of insulin and glucose, respectively. However,
together the two sites give a strong synergy to the combined treatment.
It is noteworthy that the ChoRF- and SREBP-1c-binding sites of the
S14 and FAS genes are located at considerable distances
from each other (>1000 bp). Hence, the role of ChoRF in synergizing
with SREBP appears distinct from the synergy reported earlier for SREBP
and other factors such as Sp1 or NF-Y (39-42). In these cases,
auxiliary factors binding to sites adjacent to the SREBP-1c-binding
site functionally cooperate to give effective SREBP action.
For ACC, the data of this study strongly implicate ChoRF as a
transcription factor regulating ACC PI promoter activity in response to
glucose. ACC thus adds a fourth gene to the list of previously
characterized ChoRE-containing genes. Interestingly, two of these, ACC
and FAS, are the central enzymes of fatty acid biosynthesis. In these
two genes, the ChoREs are identical in their core motifs and the
arrangement of these motifs. However, the location of these sites is
markedly different; in the FAS gene the element is located several
thousand base pairs upstream from the transcription start site, whereas
the ACC element is located in the proximal promoter region.
A role for SREBP-1c in the control of ACC gene expression has been
suggested by several observations. First, in studies on fasted-refed
mice, the amount of nuclear SREBP-1c was found to increase in parallel
with the amounts of mRNA encoding lipogenic genes, including ACC
(7). Second, transgenic mice that express the nuclear form of SREBP-1c
in their liver show increased levels (2.1-2.4-fold) of ACC mRNA
and elevated (4-fold) levels of hepatic triglycerides (13, 14).
Furthermore, in two mouse models of diabetes, the ob/ob mouse and the
transgenic aP2-SREBP-1c mouse (which overexpress SREBP-1c only in
adipose), nuclear levels of SREBP-1c were elevated in liver. In both
models, increased ACC mRNA levels (2.5- and 4.7-fold, respectively)
were found and correlated with increased rates of fatty acid synthesis
and triglyceride accumulation in the liver. It should be noted that
only total ACC mRNA was measured in these studies. Thus, extreme
changes in SREBP-1c may have provoked alterations in PII promoter
activity, which responds to this transcription factor in adipocytes.
Finally, mice bearing a homozygous deletion of the SREBP-1 gene show a blunted response to feeding of a high carbohydrate diet (12). The
normal 20-fold induction of ACC seen in mice upon a fast-refeed is
reduced to ~6-fold in the mutant mice. We predict that this remaining
response observed in the SREBP-1 knockout mice is likely to be mediated
by the actions of ChoRF binding to the ACC PI promoter.
In the present experiments, insulin treatment causes a modest elevation
of ACC PI-generated transcript levels when added in low glucose
conditions and synergized with glucose to increase levels dramatically
in the combined treatment. However, we did not observe an effect of
insulin on ACC PI promoter activity for the In summary, we have shown that the activity of ACC gene is
transcribed from two promoters, denoted PI and PII, that direct
regulated expression in a tissue-specific manner. Induction of ACC, the
rate-controlling enzyme of fatty acid biosynthesis, occurs in the liver
in response to feeding of a high carbohydrate, low fat diet, conditions
that favor enhanced lipogenesis. This induction is mainly due to
increases in PI promoter activity. We have used primary cultured
hepatocytes from the rat to investigate glucose regulation of ACC
expression. Glucose and insulin synergistically activated expression of
ACC mRNAs transcribed from the PI promoter with little or no effect on PII mRNAs. Glucose treatment stimulated PI promoter activity in
transfection assays and a glucose-regulated element was identified (
126/
102), homologous to those previously described in other responsive genes, including L-type pyruvate kinase,
S14 and fatty acid synthase. Mutation of this element
eliminated the response to glucose. This region of the ACC PI promoter
was able to bind a liver nuclear factor designated ChoRF that interacts
with other conserved glucose-regulated elements. This ACC PI element is
also capable of conferring a strong response to glucose when linked to
a heterologous promoter. We conclude that induction of ACC gene
expression under lipogenic conditions in hepatocytes is mediated in
part by the activation of a glucose-regulated transcription factor,
ChoRF, which stimulates transcription from the PI promoter. Similar
mechanisms operate on related genes permitting the coordinate induction
of the lipogenic pathway.
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and
, exist. The
form is expressed predominantly in the heart and skeletal muscle and may play a role in regulating fatty acid oxidation (23). The
gene is expressed ubiquitously and
its expression is induced by nutritional and hormonal signals that
promote increased lipogenesis (24-26). In chickens, diet-induced changes in ACC
abundance have been shown to be mediated by
alterations in the rate of transcription (25). The ACC
gene is
transcribed from two promoters, designated PI and PII (27). Studies on
the physiological regulation of these two promoters indicate that the
PII promoter is fairly uniformly expressed in all tissues and does not
respond to dietary changes (28). The PI promoter is expressed
predominantly in adipose and liver and its utilization is markedly
elevated in response to diets inducing lipogenesis (24, 28). Hence, the
20-fold increase in ACC mRNA in livers of fasted rats that have
been shifted to a high carbohydrate diet (26) is largely a consequence
of PI promoter induction.
promoter function have largely
focused on the PII promoter to date. Although it is not strongly
influenced by dietary status in animals, the PII promoter shows a
modest ability (~3-fold) to respond to glucose/insulin in the 30A5
adipocyte cell line (29). This regulation has been mapped to a region
of the PII promoter between
340 and
249. Within this region,
binding sites for both SREBP-1 and Sp1 have been identified as being
important (29, 30). It has been proposed that Sp1 binding to the ACC
PII promoter is controlled by glucose through a mechanism involving
dephosphorylation by protein phosphatase 1 (31).
PI promoter. Although an intrinsic
repressor element has been described (22), the role of this element in
regulation of the PI promoter is unknown. In this study, we examined
the control of PI promoter function in primary rat hepatocytes and
identified glucose as a major signal controlling its expression. We
also identify a glucose responsive sequence in the PI promoter with
similarity to the previously characterized ChoRE and demonstrate that
this element can bind to ChoRF. Thus, the PI promoter of the ACC
gene is regulated in a manner similar to other lipogenic enzyme genes
in response to glucose.
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exon 1. A single positive 85-kilobase clone was obtained. Digestion with
BamHI yielded a 12-kilobase subclone including exon 1 and
the PI promoter region. PCR was used to generate products of this
subclone corresponding to
1049 to +21 or
220 to +21 containing
additional BamHI and XhoI restriction sites on
their 5'- and 3'-ends, respectively. These fragments were cleaved with
these restriction enzymes and inserted into the corresponding sites of
a modified pGL3 luciferase reporter vector described previously (21).
The ChoRE mutant of ACC PI promoter (
220/+21) was obtained by inverse
PCR as described previously (19). This mutation introduced a 6-bp
sequence that changed nucleotides
109 to
104 from CGTGGG to ATGCAT.
Oligonucleotides containing the ACC PI promoter sequence from
126 to
102 were synthesized with additional BamHI and
BglII at their 5'- and 3'-ends, respectively.
Oligonucleotides were ligated and a fragment with two copies oriented
in a head-to-tail fashion was inserted in the modified pGL3 vector. All
other plasmids constructs have been reported previously (21).
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Fig. 1.
Effect of glucose and insulin on ACC mRNA
transcripts from PI and PII promoters in primary hepatocytes.
After attachment, hepatocytes were washed in media with 5.5 mM glucose and no insulin and then cultured in this media
for 18 h with an overlay of Matrigel. Cells were then washed and
treated with either 5.5 mM glucose or 27.5 mM
glucose in the absence or presence of 0.1 unit/ml insulin for 27 h. All cultures contained 0.5 µM
3,5,3'-triiodo-L-thyronine. Total RNA was extracted and
PI-generated (A) or PII-generated (B) transcripts
were assayed using RT-PCR. As a control for RNA levels, mRNA for
ribosomal protein L32 was monitored (C). Numbers
on the left-hand side represent molecular weight markers and
numbers on the right-hand side the estimated sizes of RT-PCR
products. The PI products correspond to mRNAs including exons 1, 4, and 5 (285 bp) and 1 and 5 (238 bp) (28). The PII products are derived
from mRNAs including exons 2, 3, 4, and 5 (213 bp), exons 2, 4, and
5 (152 bp), and 2 and 5 (105 bp).
1049 to +21 was
initially tested. This segment was fused to a luciferase reporter gene
and the resulting construct was introduced into primary hepatocytes. Cells were subsequently maintained in varying glucose and insulin conditions as described above for 27 h, prior to measurement of luciferase activities. The level of reporter gene activity was minimal
in cells maintained in low glucose, regardless of whether insulin was
present or not (Fig. 2). In contrast, a
strong elevation in promoter activity was observed in cells treated
with high glucose. Insulin did not increase this level and may, in
fact, have caused a modest decrease. By comparison, a fragment from the
L-PK promoter from
183 to +12, which was previously shown
to be glucose responsive (17), supported a 5-fold increase in the
presence of high glucose, whereas a basal L-PK promoter
from
96 to +12 lacking the ChoRE was unresponsive. These experiments
indicated that the ACC PI promoter was indeed responsive to glucose in
primary hepatocytes and that at least in part the induction of
PI-generated mRNA is due to increased transcription.
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Fig. 2.
ACC PI promoter activity is induced by
glucose. Primary hepatocytes were transfected with luciferase
reporter vectors containing 5'-flanking sequences of the ACC PI
promoter ( 1049/+21) or the L-PK promoter (
183/+12 or
96/+12). Cells were cultured in either 5.5 or 27.5 mM
glucose in the absence or presence of 0.1 unit/ml insulin, as
indicated, for 27 h. All cultures contained 0.5 µM
3,5,3'-triiodo-L-thyronine. Luciferase values were measured
and represent the average of duplicate samples (±range of values).
This experiment was performed three times with comparable
results.
1049 to +21 was examined for sequences that
might function as a ChoRE. One site was found that matched the
previously identified ChoREs from the S14,
L-PK, and FAS genes. This sequence contained a direct
repeat of the E box half-site 5'-CACG separated by 7 bp (Fig.
3). Identical sequence and spacing has
recently been shown to support a glucose response of the FAS promoter.2
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Fig. 3.
Sequence alignment identifies a potential
ChoRE in ACC PI promoter. Sequences shown for previously
characterized ChoREs from the rat S14 gene (18), the mouse
S14 gene (19), the rat L-PK gene (16, 17), and
the FAS gene (Footnote 2) are aligned to a region of the rat ACC PI
promoter. Arrows indicate the positions and orientation of
5'-CACG motifs shown to be important for supporting the glucose
response. Leftward arrows below the sequence indicate
that the motif is found on the bottom strand of the DNA. Note that
direct repeats of this motif are separated by 7 bp, whereas inverted
repeats are separated by 9 bp.
126/
102) was
found to form two major bands on EMSA with rat liver nuclear proteins.
The slower migrating and predominant band comigrated with the
previously identified ChoRF band observed with L-PK and
S14 ChoREs. To confirm that this band is due to binding of
the same complex, a competition experiment was performed. An
oligonucleotide containing the ChoRE of the L-PK gene was
radiolabeled and used as a probe in EMSA. The ability of the putative
ACC PI ChoRE to compete for ChoRF binding was assessed. As shown in
Fig. 4B, the ACC oligonucleotide was a strong competitor for
ChoRF binding and appeared to compete at least as effectively as the
L-PK oligonucleotide itself. ChoRE-containing oligonucleotides from the rat S14 and FAS genes also
competed for ChoRF binding, whereas oligonucleotides containing binding sites for SREBP or HNF-4 were ineffective. Hence, the putative ChoRE of
the ACC PI promoter is indeed an effective binding site for the
ChoRF.
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Fig. 4.
Putative ChoRE of ACC PI promoter binds to
ChoRF. A, EMSA was performed with the 0-10%
polyethylene glycol 8000-precipitated fraction of rat liver nuclear
proteins (15 µg) and various radiolabeled probes, as indicated. The
arrow indicates the position of the previously described
ChoRF complex. rS14(m3-5) is a variant form of the rat S14 ChoRE that
retains activity, but has reduced affinity for binding USF (44).
B, EMSA was performed as described in A using a
radiolabeled probe containing the L-PK ChoRE. Various
oligonucleotides were added in increasing amounts as indicated to each
binding reaction. Position of the ChoRF complex is indicated. The FAS
ChoRE oligonucleotides contained the sequence from 7218 to
7194 of
the rat FAS gene. L-PK and S14 ChoREs
represented sequences from
171 to
142 and
1448 to
1422 of these
genes, respectively. The HNF-4 oligonucleotide was derived from the
L-PK gene (
146/
124). The consensus SREBP-binding site,
SRE, was defined previously (21).
220 to +21 was used. As can be seen in
Fig. 5, this segment supported a robust
response of the ACC PI promoter to glucose, indicating that sequences
between
1049 and
220 are not critical for glucose regulation. It
should be noted that this deletion removes a DNA sequence reported by
others to possess intrinsic repressor activity (22). Introduction of a
6-bp mutation that disrupted the putative ChoRE site of the ACC PI
promoter resulted in a complete loss of the glucose response of this
promoter. Consequently, this site is necessary to support ACC PI
promoter activation by glucose.
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Fig. 5.
Functional role of ChoRE from the ACC PI
promoter. Primary hepatocytes were transfected with luciferase
reporter vectors containing 5'-flanking sequences of the ACC PI
promoter ( 220/+21) or the same sequence containing a 6-bp mutation
within the putative ChoRE sequence. Cells were cultured in 5.5 mM glucose or 27.5 mM glucose in the presence
of 0.1 unit/ml insulin. Values are average of duplicates (±range of
values) and are representative of three experiments with comparable
results.
40 to
+12. This construct was compared with a series of similarly organized plasmids containing oligonucleotides from other genes. As seen in Fig.
6A, a construct containing the
ACC oligonucleotide linked to the minimal L-PK promoter
supported a strong response to glucose. Two previously characterized
ChoREs from the rat S14 and L-PK genes also
supported a glucose response. However, this effect was not observed
when a consensus SREBP-binding site was fused to the L-PK
minimal promoter. These data indicate that the ChoRE-like sequence of
the ACC PI promoter can indeed function as a glucose-responsive sequence in primary hepatocytes and, in fact, in this transfection assay appears to be the strongest ChoRE that we have characterized to
date.
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Fig. 6.
ACC PI ChoRE can support a response to
glucose. A, all constructs contain two copies of the
indicated oligonucleotides fused in a head-to-tail fashion to the
L-PK( 40/+12) basal promoter in the reporter plasmid pGL3.
Constructs were transfected into primary hepatocytes and cells were
incubated in the 5.5 mM or 27.5 mM glucose in
the presence of 0.1 unit/ml insulin for 27 h. Luciferase values
represent averages of duplicates and are representative of three
experiments with comparable results. B, hepatocytes
were transfected with 2 µg of the luciferase reporter vectors used in
A and 10 ng of a SREBP-1c expression vector (21). Cells were
maintained in 5.5 mM glucose in the presence of 0.1 unit/ml
insulin for 27 h. Luciferase values represent averages of
duplicates (±range of values) and are representative of three
experiments with similar results. Note the broken axes used in this
figure.
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ABSTRACT
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gene is expressed from two promoters and variably spliced to
include or exclude alternative exons within the 5'-untranslated region
(27). Transcripts from the PII promoter are expressed in all tissues
examined in the rat and do not respond to fasting or refeeding a high
carbohydrate diet (28). Consistent with these previous observations, we
did not detect any major changes in the levels of PII-generated
transcripts in response to alterations in glucose or insulin in the
cultured rat hepatocytes. Consequently, the PII promoter likely
provides enzyme to serve basic cellular needs, such as synthesis of
membrane phospholipids. In contrast, PI-generated transcripts are
expressed predominantly in adipose and liver, two major sites of
triglyceride formation in mammals, and are induced under conditions
favoring lipogenesis (24, 28). Thus, it is reasonable to speculate that
the PI promoter provides enzyme for meeting the enhanced needs of
tissues capable of lipogenesis under favorable conditions. Consistent
with this notion, we have found that transcripts from the PI promoter
are increased dramatically by glucose and insulin in the primary
hepatocyte. Given that an overall 20-fold increase is observed in total
ACC mRNA in the fast refeed response in rats (12, 26), the PI
promoter is likely the predominant one used under lipogenic conditions
in the liver.
promoter usage in chickens appears to be somewhat
different than that observed in rats. Yin et al. (35) recently demonstrated that transcripts from both PI and PII promoters are elevated in liver when 12-day-old chicks are refed a high carbohydrate, low-fat diet after a 24-h fast. PII-generated transcripts were induced 10.5-fold, whereas PI-generated transcripts were induced
6.4-fold. Similarly, thyroid hormone regulation of ACC promoters shows
a species-specific variation. In rats, changes in thyroid hormone were
found to affect predominantly PI-generated transcripts (36). However,
in chickens, the activity of the PII promoter, as well as the PI
promoter, was influenced (35). Whether these differences solely reflect
species-specific variation or might also be due to developmental
differences between 12-day-old chicks and adult rats remains to be determined.
1049/+21 construct.
Furthermore, co-transfection of a vector expressing nuclear SREBP-1c
did not enhance activity of the ACC(
1049/+21)luciferase reporter.3 Two explanations
may account for this discrepancy. The most likely is that an
SREBP-binding site on the ACC PI promoter exists outside of the region
that we have tested. This situation would be somewhat unusual for
SREBP, since all previously characterized functional sites in both
cholesterogenic and lipogenic genes lie within 300 bp upstream of the
transcriptional start site. The second explanation is that ACC may not
be directly acted upon by SREBP-1c. In this scenario, the effects of
insulin on ACC PI transcription would be indirectly mediated by
stimulation of another gene product or occur at the level of
mRNA stability. For example, glucokinase gene expression is
induced independently by insulin (43) and has been shown to be
dependent on SREBP-1c function (8). If glucokinase expression is
stimulated by insulin/SREBP-1c, it could contribute to the overall
effect of carbohydrate diet on ACC PI activity by affecting the rate of
glucose metabolism and the activation of ChoRF.
promoter PI is
regulated by glucose in primary rat hepatocytes. This regulation is
mediated by a conserved DNA element, the ChoRE, found in several other
glucose-regulated genes that binds to the hepatic factor ChoRF. Given
the central role of ACC in mediating the lipogenic response of the
liver, ChoRF, together with SREBP, are likely to be major regulators of
the hepatic responses to conditions affecting lipid metabolism.
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ACKNOWLEDGEMENTS |
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We thank Ross E. Newman for technical assistance, Angela Dutcher and Jenny Xanthos for help with the LightCycler system, and Yangha Moon for preparation of the initial ACC reporter construct.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant DK26919 (to H. C. T.) and United States Department of Agriculture Hatch Grant CONS00665 (to H. C. F.).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.
¶ To whom correspondence should be addressed: Dept. of Biochemistry, Molecular Biology & Biophysics, 6-155 Jackson Hall, 321 Church St. SE., Minneapolis, MN 55455. Tel.: 612-625-3662; Fax: 612-625-5476; E-mail: towle@mail.ahc.umn.edu.
Published, JBC Papers in Press, February 28, 2001, DOI 10.1074/jbc.M101557200
2 Rufo, C., Teran-Garcia, M., Nakamura, M., Koo, S.-H., Towle, H. C., and Clarke, S. D. (2001) J. Biol. Chem. 276, in press.
3 B. L. O'Callaghan and H. C. Towle, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: ACC, acetyl-CoA carboxylase; FAS, fatty acid synthase; L-PK, L-type pyruvate kinase; SREBP, sterol regulatory element-binding protein; ChoRE, carbohydrate response element; ChoRF carbohydrate responsive factor, EMSA, electrophoretic mobility shift assay; RT-PCR, reverse transcriptase-polymerase chain reaction; bp, base pair(s).
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