From the Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455
Received for publication, November 3, 2000, and in revised form, December 5, 2000
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ABSTRACT |
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Transcription of a number of genes involved in
lipogenesis is stimulated by dietary carbohydrate in the mammalian
liver. Both insulin and increased glucose metabolism have been proposed
to be initiating signals for this process, but the pathways by which these effectors act to alter transcription have not been resolved. We have previously defined by electrophoretic mobility shift
assay a factor in nuclear extracts from rat liver, designated the
carbohydrate-responsive factor (Cho- RF), that binds to liver-type
pyruvate kinase and S14 promoters at sites critical
for regulation by carbohydrate. The sterol regulatory element binding
protein-1c (SREBP-1c) has also emerged as a major transcription factor
involved in this nutritional response. In this study, we examined the
relationship between SREBP-1c and ChoRF in lipogenic gene induction.
The two factors were found to possess distinct DNA binding
specificities both in vitro and in hepatocytes. Reporter
constructs containing binding sites for ChoRF were responsive to
glucose but not directly to insulin. On the other hand, reporter
constructs with an SREBP-1c site responded directly to insulin. The
S14 gene possesses binding sites for both ChoRF and SREBP,
and both sites were found to be functionally important for the response
of this promoter to glucose and insulin in hepatocytes. Consequently,
we propose that SREBP-1c and ChoRF are independent transcription
factors that mediate signals generated by insulin and glucose,
respectively. For many lipogenic enzyme genes, these two factors may
provide an integrated signaling system to support the overall
nutritional response to dietary carbohydrate.
In mammals, the ingestion of carbohydrate in excess of that
required to meet immediate energy needs triggers lipogenesis, the
conversion of simple carbohydrates into triglycerides. Lipogenesis occurs predominantly in the liver and adipose tissue and its activation by carbohydrate diet is accompanied by the induction of many of the key
enzymes involved in this metabolic conversion (for review see Refs.
1-3). Among these are enzymes of glycolysis, such as pyruvate kinase;
of fatty acid synthesis, such as acetyl CoA carboxylase and fatty acid
synthase (FAS)1; and of fatty
acid maturation and packaging, such as stearoyl CoA desaturase. The
increased production of these "lipogenic" enzymes results from
induction of their specific mRNAs and in most cases correlates with
increased transcription of the corresponding genes.
Two potential signaling pathways elicited in response to dietary
carbohydrate could play a role in lipogenic enzyme induction. Increased
insulin secretion by the The intracellular signaling pathway responsible for the induction of
lipogenic gene expression in response to glucose metabolism remains
unresolved (for reviews see Refs. 2 and 3). Two genes, the liver-type
pyruvate kinase (L-PK) and S14 genes, have been extensively
studied. The S14 gene product, a 17-kDa nuclear protein, is
hypothesized to play a regulatory role in lipogenesis (for review see
Ref. 7). Transfection analyses of the S14 and L-PK
promoters in primary hepatocytes led to the identification of a
conserved carbohydrate response element (ChoRE) that is necessary and
sufficient for control by glucose (8-11). This sequence consists of
two "half E box" motifs related to the sequence CACG (12). Definition of the ChoRE allowed the detection of a novel protein complex by EMSA that formed with active ChoREs but not with mutant ChoREs that failed to respond to glucose (12). The strong correlation between binding and function suggested that this complex, designated ChoRF, might be responsible for signaling through the ChoRE element. The identity of the protein(s) forming this complex is unknown.
The presence of E box-like sequences in the ChoRE suggests that a
member of the basic/helix-loop-helix/leucine zipper family binds this
site to mediate transcriptional responses to glucose. Among this large
family of factors, SREBP-1c has emerged as a candidate for ChoRF. SREBP
was first identified as a transcription factor that regulates genes
encoding enzymes involved in cholesterol uptake and biosynthesis (for
reviews see Refs. 13 and 14). SREBP has three isoforms: SREBP-1a, -1c,
and -2. SREBP-2 appears to be primarily involved in regulating genes of
cholesterol metabolism (13). SREBP-1c is predominant in tissues active
in lipogenesis such as liver and adipose (15, 16). The expression of
SREBP-1c itself was found to be rapidly increased by dietary
carbohydrate in the liver and adipose tissue, suggesting a role for
this factor in lipogenesis (17-19). The effects of dietary
carbohydrate were attributed to a direct action of insulin in
hepatocytes (19). Overexpression of SREBP-1c induced lipogenic gene
expression (e.g. FAS and acetyl CoA carboxylase) in mouse
liver and in cultured cells (17, 20, 21). Furthermore,
carbohydrate-induced lipogenic gene expression was severely impaired in
either SREBP-1 knockout mice or in hepatocytes overexpressing a
dominant negative form of SREBP-1 (22, 23). Together these data provide
a strong case for the role of SREBP-1c in lipogenic enzyme induction;
however, they do not directly demonstrate that SREBP-1c is the factor
that also mediates the glucose signaling pathway. In fact,
overexpression of a constitutively active form of SREBP-1c alone could
not maximally induce lipogenic gene expression in hepatocytes in low
glucose conditions (19). In addition, overexpression of constitutively active SREBP-1c was unable to activate constructs containing the L-PK
ChoRE either in COS cells or in mhAT3F cells (24). Thus, although
SREBP-1c might be implicated in mediating insulin signaling, its direct
involvement in the regulation of ChoREs remains in question.
In this study, we have investigated the relationship between ChoRF and
SREBP-1c with respect to lipogenic gene induction. We present evidence
indicating that ChoRF is distinct from SREBP-1c and acts through
different response elements. These data lead to a model in which both
ChoRF activation by glucose and SREBP activation by insulin play
integral roles in carbohydrate stimulation of gene expression.
Primary Hepatocyte Culture and Transfections--
Primary
hepatocytes were isolated from male Harlan Sprague-Dawley rats
(180-260 g) using the collagenase perfusion method as described
previously (25). Following an attachment for 3-6 h, cells were
transiently transfected using F1 reagent (Targeting Systems, San Diego,
CA) in modified Williams' E medium with 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 medium containing either 5.5 or 27.5 mM glucose for 30 h and harvested for luciferase
assay. For experiments in Figs. 6 and 8, cells were transfected with
medium containing 1.5 mM glucose and no insulin and
subsequently cultured with medium in varying glucose concentrations in
the absence or in the presence of 0.1 unit/ml insulin. In these
experiments, 0.33 mg/ml of Matrigel (Collaborative Biomedical Products,
Bedford, MA) was added to the medium after transfection. This treatment has been shown to enhance the maintenance of the differentiated phenotype in cultured hepatocytes (26). Results of luciferase assays
are expressed as relative light units measured per µg protein.
Plasmid Constructs--
Sequences of ChoREs from the rat L-PK
and S14 genes, synthetic ChoREs derived from these
sequences (mut 3/5 and m3-6), and the consensus SRE-1 are shown in
Table I. Oligonucleotides containing these sequences (except mut3/5) were synthesized with BamHI
and BglII sites at the 5' and 3' ends, respectively. Each
oligonucleotide was ligated and treated with BamHI and
BglII to isolate a DNA fragment with two copies in a
head-to-tail orientation. Fragments containing two copies were then
inserted into the BamHI site of a PK(
The SREBP site mutation of the rat S14 gene was generated
by inverse PCR as described previously (12). Briefly, each
oligonucleotide creating a unique NsiI site was synthesized
and used to amplify the rat S14(
The cDNA encoding SREBP-1c (ADD-1) amino acids 1-403 (28) was
inserted into the CMV4 vector (29) for expression in primary hepatocytes. The plasmid pFACMV/SREBP-1c contained the pFA-CMV expression vector encoding the GAL4 DNA-binding domain (Stratagene, La
Jolla, CA) fused with human SREBP-1c transactivation domain (amino
acids 1-81). A fragment containing five copies of the GAL4-binding site was excised from pFR-Luc (Stratagene, La Jolla, CA) with KpnI and NheI and inserted into PK( Preparation of Nuclear Extracts--
Liver nuclear extracts were
prepared from male Harlan Sprague-Dawley rats that had been fed a high
carbohydrate diet for 16 h as described previously (10). The
polyethylene glycol 8000 (Hampton Research, Boston, MA) fraction that
precipitated between 0 and 6.7% was used for EMSA.
Crude nuclear extracts from COS-1 cells containing nuclear SREBP-1c
were prepared as described by Schreiber et al. (31). For
this purpose, COS-1 cells were transfected with CMV4/SREBP-1c (1)
for 16 h using LipofectAMINE (Life Technologies, Inc.) and then
cultured for an additional 30 h prior to harvest.
Electrophoretic Mobility Shift Assay--
EMSA was performed as
described previously (12). A typical reaction contained 100,000 cpm
(10-20 fmol) of 32P-labeled oligonucleotide either with 15 µg of the polyethylene glycol fraction from liver nuclear extracts or
7.5 µg of COS-1 cell nuclear extracts. Nonspecific competitors used
were 0.1 µg of poly(dI·dC) and 1.9 µg of poly(dA·dT) for EMSA
with liver nuclear extracts and 2 µg of poly(dI·dC) with COS-1 cell
nuclear extracts. Following incubation at room temperature for 30 min,
samples were subject to electrophoresis on a 4.5% nondenaturing
polyacrylamide gel and subjected to PhosphorImager analysis.
Antibody to SREBP-1(H-160) was purchased from Santa Cruz Biotechnology
(Santa Cruz, CA) and was added to nuclear extract for 20 min at 4 °C
prior to the addition of probe. For competition EMSA, a 10-, 25-, or
50-fold molar excess of unlabeled oligonucleotide was added together
with radiolabeled probe into the sample prior to the incubation.
Glucose Does Not Influence the Transactivating Potential of
SREBP-1c--
As noted above, several lines of experimental evidence
implicate SREBP-1c as the transcription factor responsible for
mediating the nutritional response of lipogenic genes to carbohydrate
feeding. SREBP-1c expression has been shown to respond rapidly to
insulin treatment, providing a direct link between insulin signaling
pathways and SREBP (17, 19). However, induction of most lipogenic
enzyme genes requires both insulin and elevated glucose metabolism in the hepatocyte, and no effects of glucose on SREBP expression have been
found in hepatocytes (23, 32). To test the possibility that SREBP
transactivation potential might be regulated by glucose, as suggested
by Foretz et al. (23), the following transfection experiment
was performed. A reporter construct containing two copies of a
consensus SREBP-binding site, SRE-1, linked to a basal TATA box
promoter from the pyruvate kinase gene was prepared. This construct was
cotransfected into primary hepatocytes with a eucaryotic expression
vector for the nuclear form of rat SREBP-1c (amino acids 1-403). Cells
were then incubated with either low (5.5 mM) or high (27.5 mM) glucose for 30 h. These conditions are identical
to those used for measuring glucose induction of L-PK or
S14 promoters in hepatocytes. Cotransfection of SREBP-1c expression vector resulted in robust induction of luciferase activity from the SRE-1-containing reporter construct both in low and high glucose, as expected (Fig.
1A). However, no significant
differences in the extent of induction were observed between samples
from low and high glucose conditions, suggesting that the
transactivation potential of SREBP-1c is not affected in the presence
of high glucose.
Because of the possibility that endogenous SREBPs might interfere with
the binding of the transfected SREBP-1c, an additional experiment was
performed using a recombinant SREBP-1c in which its DNA-binding domain
was replaced with the GAL4 DNA-binding domain. An expression vector for
this recombinant protein was cotransfected into hepatocytes with a
basal TATA box promoter construct containing five copies of the
GAL4-binding site. Again, expression of the SREBP fusion protein
activated promoter activity, but no significant differences were found
between low and high glucose conditions (Fig. 1B). These
data argue against the possibility that SREBP-1c activity is enhanced
by signals generated by glucose metabolism in primary hepatocytes.
ChoRF and SREBP Possess Distinct DNA Binding Specificities--
We
recently showed by EMSA that a series of glucose-responsive ChoREs, but
not glucose-unresponsive oligonucleotides, can form a novel protein
complex, designated ChoRF, when incubated with partially purified rat
liver nuclear proteins (12). Consequently, ChoRF was suggested to be
responsible for mediating effects of glucose on genes containing the
ChoRE site, such as S14 and L-PK. Among the
glucose-unresponsive oligonucleotides used in that study was a
consensus SRE-1, a strong binding site for SREBP. These data suggested
that ChoRF is not identical with the well characterized SREBP dimer. To
further verify this observation, competition EMSA was performed. In
this experiment, liver nuclear proteins were incubated with a
radiolabeled ChoRE probe from the L-PK promoter, and the abilities of
various oligonucleotides to compete for the binding of the ChoRF were
tested. As expected, all natural and modified oligonucleotides with
functional ChoRE activity were able to compete for ChoRF binding with
L-PK ChoRE with the degree of competition increasing as a greater
excess of oligonucleotide was added (Fig.
2A). On the other hand,
an oligonucleotide containing the consensus SRE-1 was unable to
interfere with ChoRF binding when tested over the same range of
concentrations. These data indicate that the ChoRF complex is not
identical to the SREBP-1c homodimer.
Because it was still possible that SREBP-1c might be a part of a larger
ChoRF complex together with other components, the ability of an
antibody to SREBP-1 to disrupt the formation of ChoRF was tested. As a
control, the binding of SREBP-1c to an SRE-1 oligonucleotide was
compared. Attempts to detect SREBP-1c binding using nuclear extracts
were unsuccessful because of its low abundance, as noted by others
(33). To circumvent this problem, extracts were prepared from COS-1
cells that were transfected with a vector overexpressing the nuclear
form of SREBP-1c. Using the resultant nuclear extracts, formation of a
specific band was observed with the consensus SRE-1 (Fig.
2B). Addition of anti-SREBP-1 antibody completely disrupted
the band formed between SREBP-1c and the SRE-1 oligonucleotide and led
to the appearance of a slower migrating supershifted band. However, the
intensity of the ChoRF complex on the L-PK ChoRE was undiminished in
the presence of anti-SREBP-1 antibody. Thus, SREBP-1c is not likely to
be a component of the ChoRF complex.
To complement this experiment, we were interested in testing whether
oligonucleotides with ChoRE activity can compete with SREBP binding to
its DNA recognition site. To test the binding affinity of various
ChoREs to SREBP-1c, a competition EMSA was again performed. Although
the SRE-1 oligonucleotide itself could compete for the binding of
SREBP-1c to radiolabeled SRE-1 probe, several ChoREs were unable to
disrupt the SREBP-1c band with SRE-1 over the same concentration range
(Fig. 3). It is noteworthy that the wild
type rat S14 ChoRE competes to some degree with SREBP-1c for binding. The rat S14 ChoRE contains one perfect CACGTG
element, a common binding site for basic/helix-loop-helix/leucine
zipper factors including SREBP (28). To verify that the presence of the
CACGTG sequence could lead to competition for SREBP binding, the
adenovirus major late promoter upstream stimulatory
factor-binding site, which also contains a perfect CACGTG
sequence but is glucose-unresponsive, was used for competition EMSA. As
predicted, the adenovirus upstream stimulatory factor-binding site
could compete for the SREBP-1c binding with SRE-1 to a similar degree
to rat S14 ChoRE (data not shown). Consequently, the
ability to compete for SREBP-1c binding does not correlate with the
ability of various oligonucleotides containing ChoRE activity to
support a glucose response.
To further exclude a role for SREBP-1c in the glucose regulation of
ChoREs, it was important to confirm the conclusions of the in
vitro binding experiments in the context of the hepatocyte. For
this purpose, the ability of SREBP-1c to activate ChoRE-containing constructs was tested by cotransfection assays. As a control, the
reporter construct containing two copies of the SRE-1 oligonucleotide was used. Transfection of this construct into hepatocytes gave a
minimal level of luciferase activity only slightly higher than that
obtained with the construct lacking SRE-1 sites. Cotransfection of a
construct overexpressing a nuclear form of SREBP-1c resulted in a
robust increase in reporter activity. An approximately 25-fold induction of luciferase activity by SREBP-1c was achieved by adding only 2.5 ng of expression vector, and a maximal induction of 35-fold was observed with 10 ng of vector (Fig.
4). No further increase in luciferase
activity was observed by adding 40 ng of expression vector. In contrast
to the results seen with SRE-1, only a modest increase of reporter
activity was observed when SREBP-1c expression vector was cotransfected
with constructs containing two copies of various ChoREs. Unlike the
SRE-1 construct, further induction of ChoREs was observed by adding up
to 40 ng of SREBP-1c expression vectors. These data suggest that
SREBP-1c has a much lower affinity for the ChoREs than for the SRE-1
sequence. It is noteworthy that the luciferase activity driven by the
L-PK ChoRE was not induced by addition of SREBP-1c expression in
primary hepatocytes, which is consistent with data observed in COS
cells or in mhAT3F hepatoma cells (24). In fact, the relative induction
of various ChoREs by SREBP-1c correlates with SREBP-1c binding affinity
examined in EMSA, in which SREBP-1c showed the highest affinity for the rat S14 ChoRE and the lowest affinity for the rat L-PK
ChoRE (Fig. 3).
The same ChoRE-containing constructs that were tested for their
activation by SREBP-1c were evaluated for their ability to respond to
glucose in primary hepatocytes. As shown in Fig.
5A, all
ChoRE-containing constructs were induced at least 7-fold by high
glucose. The SRE-1 construct, however, showed no significant induction
by high glucose. The effects of high glucose are directly compared with
the effects of SREBP-1c overexpression in Fig. 5B. There was
no correlation between the relative induction by glucose with that by
constitutively active SREBP-1c. Hence, it is unlikely that SREBP-1c is
the factor that specifically recognizes ChoREs and mediates glucose
signaling in hepatocytes.
Glucose and Insulin Differentially Activate through ChoRF and
SREBP-binding Sites--
The above data on the distinct nature of
ChoRF and SREBP led us to speculate that both factors may both be
involved in mediating the effects of carbohydrate feeding on lipogenic
gene induction. We hypothesize that SREBP is primarily involved in
mediating insulin effects and ChoRF in mediating the effects of
glucose. To test this possibility, we compared reporter constructs that
contained binding sites for only ChoRF or SREBP for their responses to
glucose or insulin. One test construct contained two copies of the rat S14 ChoRE linked to a basal TATA box promoter. The other
construct contained a region of the rat FAS promoter between
The promoter activity of the ChoRE-containing construct in high glucose
without insulin was less than that observed in the presence of both
high glucose and insulin. Because glucokinase expression is known to be
regulated by insulin and critical for effective glucose metabolism, the
low level of glucokinase expression might in part limit the response to
glucose when insulin is absent. Indeed, when a glucokinase expression
vector was introduced together with the reporter gene, the level of
reporter gene activity at 27.5 mM glucose in the absence of
insulin was substantially increased. Interestingly, expression of
glucokinase also increased reporter gene activity at 5.5 mM
glucose. Under low glucose conditions, glucokinase is sequestered in
the nucleus bound in an inactive form by the glucokinase regulatory
protein (34). The overexpression of glucokinase likely oversaturated
the amount of glucokinase regulatory protein, and the excess
glucokinase was then able to use the available glucose for stimulating
glycolysis and promoter activity from the ChoRE construct. This effect
was not seen at 1.5 mM glucose, where substrate is
limiting. These results thus support the conclusions that the ChoRE is
primarily a glucose-responsive element.
The results with the SREBP-binding site shown in Fig. 6B are
sharply different. In this case, insulin simulates promoter activity at
each glucose concentration tested, even at 1.5 mM glucose
where substrate is limiting. Changing glucose concentrations has little or no effect in the absence of insulin and only a modest effect in its
presence. Moreover, overexpression of glucokinase in cells without
insulin had no effect on the ability of the cells to respond to
glucose. Hence, the SREBP-binding site present in the FAS promoter would appear to be predominantly an insulin-responsive element. These
data suggest that glucose and insulin may function through distinct
transcription factors to mediate their effects following carbohydrate ingestion.
S14 Gene Expression Is Regulated by Glucose and Insulin
through ChoRF and SREBP-binding Elements--
We previously reported
that mutations in the ChoRE sequence located between
To test the roles of ChoRE and SREBP-binding sites in the induction of
S14 gene expression, we prepared a construct that contained the ChoRE-containing region of the S14 gene ( The ChoRE was defined as a regulatory sequence within the L-PK and
S14 promoter regions that conferred a response to changes in glucose metabolism in primary hepatocytes (8-11). By EMSA, we
subsequently found a novel complex, ChoRF, that formed between this
site and liver nuclear proteins (12). The ChoRF complex was detected
with a variety of wild type and variant ChoREs that supported glucose
responsiveness in transfection assays but not with mutants of these
oligonucleotides that were unresponsive. Consequently, we proposed that
ChoRF was responsible for the induction of L-PK and S14
gene expression observed when animals are fed a high carbohydrate diet
and that it might also mediate the induction of many other lipogenic
enzyme genes following the nutritional stimulus. In this model, the
role of insulin was suggested to be largely permissive in allowing
enhanced glycolysis under conditions of elevated glucose levels. In
particular, glucokinase expression is dependent on insulin levels, and
its expression is critical for the ability of the hepatocyte to respond
to elevated glucose levels (5, 37).
Concomitant with this work, SREBP-1c emerged as a major factor
regulating production of lipogenic enzymes. SREBP-1c gene expression was found to increase rapidly in response to insulin in hepatocytes (23, 32). In addition, SREBP-binding sites were identified within the
promoter regions of a number of lipogenic genes, including FAS,
glycerol-3-phosphate acyltransferase, and stearoyl-CoA desaturase (38-40). Transgenic mice overexpressing SREBP-1c in liver exhibited elevated lipogenesis and induced the program of lipogenic enzyme expression observed with carbohydrate feeding (21). Furthermore, mice
bearing a homozygous disruption of the SREBP-1 gene were impaired in
their nutritional response to high carbohydrate diet (22). Together,
these results indicated that SREBP is a major transcription factor
regulating lipogenic genes in response to carbohydrate feeding and
raised the possibility that ChoRF might be identical to SREBP-1c.
Consequently, we undertook the present study to analyze this question.
The data from the current study support a model in which ChoRF and
SREBP-1c are discrete transcription factors that both play a role in
the induction of lipogenic enzyme genes and are primarily responsible
for mediating signals generated by glucose and insulin, respectively.
Although both recognize sequences related to the E box consensus
CACGTG, the binding patterns of the two factors are distinct. ChoRF
binding was not competed by a consensus SRE-1 site, and SREBP binding
was not competed by most ChoRE sites. ChoRF binding requires two E box
half-sites related to the sequence CACG (12). These two half-sites are
found in either an inverted orientation with 9-base pair spacing or a
direct orientation with 7-base pair spacing, and the spacing has been
shown to be critical to ChoRF binding and function (11). ChoRF does not
bind directly to oligonucleotides with a single CACGTG motif in which
the half sites are situated in an inverted orientation without spacing between them. SREBP can bind to the CACGTG motif in vitro
(28), but in naturally occurring genes characterized to date, all
SREBP-binding sites consist of E box half sites related to the sequence
(Py)CAC. In the consensus SRE-1 site, these half sites are found as
direct repeats with a 1-base pair spacing; however, much variation is found in the sequence and arrangement of naturally occurring
SREBP-binding sites. The similarity in the nature of the binding sites
suggests that ChoRF and SREBP may be related family members of the
basic/helix-loop-helix/leucine zipper transcription factor family.
In addition to differences in binding specificities, a number of other
lines of evidence suggest that ChoRF and SREBP are distinct factors.
First, ChoRF migrates on EMSA more slowly than the SREBP-1c dimer
formed on the consensus SRE-1 site. Second, ChoRF binding is strongly
inhibited by poly(dI·dC) in EMSA, whereas SREBP is routinely assayed
in the presence of this competitor. Third, SREBP was shown to bind
tightly to a strong cation exchanger, S-Sepharose (33), whereas ChoRF
did not bind to this resin at 0.1 M
NaCl.2 Fourth, an antibody to
SREBP did not interfere with ChoRF binding. Although it is possible
that the epitope is precluded in the ChoRF complex, this same antibody
efficiently competed for SREBP binding. Finally, SREBP expression is
induced by feeding a carbohydrate diet (18), whereas ChoRF binding
activity is unaffected (12). Together these data demonstrate that ChoRF
is likely a distinct molecular entity from SREBP.
Based on this information, we propose the following model for the
induction of lipogenic genes by high carbohydrate diet (Fig. 9). Elevated blood glucose levels
following a meal promote secretion of insulin from the pancreatic
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cell in response to elevated blood glucose
could act as the primary signal. Alternatively, increased glucose
metabolism itself might lead to alterations in gene expression.
Attempts to sort out the respective roles of these pathways have been
most effectively carried out in cultured primary hepatocytes. Treatment
of hepatocytes with insulin and high glucose levels mimics the
lipogenic response seen in the animal following dietary carbohydrate
(4). However, neither signal alone is able to recapitulate the
response. Treatment of hepatocytes with insulin alone (in low glucose
conditions) does stimulate the expression of the glucokinase gene (5).
Enhancement of pyruvate kinase gene expression, on the other hand, is
mainly dependent on increases in glucose concentration. When
glucokinase is expressed constitutively, induction of pyruvate kinase
mRNA occurs in the absence of insulin (6). For most of the
remaining lipogenic enzyme genes, however, both insulin and glucose are required for the induction process.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
40) Luc construct.
The PK(
40) Luc construct is based on the pGL3 basic vector (Promega,
Madison, WI) with a modified polylinker region. The L-PK gene sequence
from
40 to +12 that by itself exhibits basal promoter activity was
inserted between PstI and XhoI sites. Two copies
of mut3/5 sequences were excised from the 2Xmut3/5 PK(
96) CAT
construct (25) with BamHI and PstI and inserted
into PK(
40) Luc construct. The FAS (
150/
43) sequence was excised
from the FAS promoter construct described previously (27) and inserted
into KpnI and MluI sites of PK(
40) Luc
construct.
The sequence of oligonucleotides used for EMSA or generation of
reporter constructs
1601/+18)/pBluescript SK
II (+) plasmid. PCR products were digested at the introduced
restriction enzyme site, purified by gel electrophoresis, ligated, and
transformed into Escherichia coli. This procedure resulted
in an 8-base pair mutation at positions
139 to
131 of the
S14 promoter. Mutant constructs were isolated, and rat
S14 sequences were excised with PstI and
XhoI to clone into the modified pGL3 vector. The rat
S14 sequences from
1467 to
1395 were PCR amplified
either from the wild type rat S14 (
4316/+18) CAT or the
Mut1 construct that contained a mutation in the ChoRE (25) and inserted
into the HindIII site of either the wild type or SREBP site
mutated rat S14(
290/+18) Luc construct.
40) Luc
construct. The cDNA encoding the human pancreatic glucokinase (30)
was inserted into XbaI site of CMV4 vector for expression in
primary hepatocytes. All plasmid constructs were confirmed by DNA sequencing.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Glucose does not modify the transactivating
ability of SREBP-1c in primary hepatocytes. A, the
2XSRE-1 PK(-40) Luc construct (1.9 µg) was cotransfected with 2.5 ng
of CMV4/SREBP-1c(1-403) expression vector. Cells were cultured in 5.5 or 27.5 mM glucose for 30 h. Luciferase activity is
shown as the percentage of relative light units measured with the value
at 27.5 mM glucose as 100%. Values represent the means (± S.E.) of three independent experiments, each with duplicate
transfections. B, the 5XGAL4 PK( 40) Luc construct (1.9 µg) was cotransfected with 5 ng of pFACMV/SREBP1c expression vector.
Cells were cultured as above. Luciferase activity is shown as the
percentage of relative light units measured with the value at 27.5 mM glucose as 100%. Values represent the means (± S.E.)
of two experiments, each with duplicate or triplicate
transfections.
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Fig. 2.
The ChoRF complex does not contain
SREBP. A, formation of the ChoRF complex is not
disrupted by the presence of a consensus SRE-1 oligonucleotide. EMSA
was performed with the 0-6.7% polyethylene glycol fraction of rat
liver nuclear protein (15 µg) and the rat L-PK ChoRE oligonucleotide
as described under "Experimental Procedures." Various ChoREs or the
consensus SRE-1 oligonucleotide (Table I) were added in increasing
amounts as indicated to each binding reaction. B, formation
of the ChoRF complex is not disrupted by the presence of the
anti-SREBP-1 antibody. EMSA was performed either with rat liver nuclear
protein (15 µg) and the rat L-PK ChoRE oligonucleotide (lanes
2-4) or with nuclear extracts from COS-1 cells that overexpressed
nuclear form of SREBP-1c (7.5 µg) and the consensus SRE-1
oligonucleotide (lanes 5-7). Anti-SREBP-1 antibody was
added at 0.1 or 0.2 µg/µl to each binding reaction.
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Fig. 3.
Formation of the SREBP-1c complex is not
disrupted by the presence of ChoRE oligonucleotides. EMSA was
performed with nuclear extracts of COS-1 cells that overexpressed the
nuclear form of SREBP-1c (7.5 µg) and the consensus SRE-1
oligonucleotide. Various ChoREs or the consensus SRE-1 oligonucleotide
were added in increasing amounts to each binding reaction, as
indicated. Ab, antibody.
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Fig. 4.
SREBP-1c is not a potent activator of
ChoRE-containing constructs in primary hepatocytes. Primary
hepatocytes were cotransfected with luciferase reporter plasmids that
contain two copies of various ChoREs or a SRE-1 oligonucleotide linked
to the PK( 40) basal promoter (1.9 µg) and with varying amounts of a
SREBP-1c expression vector. Cells were cultured in 5.5 mM
glucose for 30 h. Luciferase activity is shown as fold induction
of luciferase activity in cell extracts transfected with CMV4/SREBP-1c
expression vector compared with the value obtained with the cells
transfected with control expression vector. Values represent the means
(± S.E.) of two independent experiments, each with duplicate
transfections.
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Fig. 5.
Comparison of the sequence requirements for
the glucose response and SREBP-1c-dependent activation in
primary hepatocytes. Each construct shown in Fig. 4 was compared
for its response to glucose and to exogenous SREBP-1c expression in
primary hepatocytes. A, cells were cultured in 5.5 or 27.5 mM glucose for 30 h. Luciferase activity is shown as
fold induction of the luciferase activity in cell extracts treated with
high glucose compared with the value obtained for cells treated with
low glucose. Values represent the means (± S.E.) of two independent
experiments, each with duplicate transfections. B,
the data shown in Fig. 4 with 2.5 ng of CMV4/SREBP-1c expression vector
is presented for the comparison with A.
150 and
43 linked to the same basal promoter. This region of the FAS gene has
been shown to bind to SREBP and together with two adjacent accessory
factor-binding sites to mediate responses to the combined actions of
insulin and glucose (27). Each construct was transfected into primary
hepatocytes, and cells were incubated in varying glucose concentrations
with or without insulin. As shown in Fig. 6A, the ChoRE-containing
construct responded to a change in glucose concentrations between 5.5 and 27.5 mM even in the absence of insulin in the medium.
Note that in these conditions sufficient glucokinase should remain in
the cell following isolation of hepatocytes from normal animals to
support this partial glucose response. However, this construct was
unresponsive to insulin when glucose levels were at 1.5 or 5.5 mM. These data suggest that insulin is not required as a
direct signal for mediating the carbohydrate response through the
ChoRE.
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Fig. 6.
The ChoRE- and SREBP-binding sites regulate
hepatic responses to glucose and insulin, respectively. Primary
hepatocytes were transfected with luciferase reporter plasmids that
contain either two copies of the rat S14 ChoRE or a rat FAS
( 150/
43) sequence linked to the PK(
40) basal promoter and with
either 10 ng of an empty CMV4 vector or 10 ng of a glucokinase
expression vector. Cells were cultured in 1.5, 5.5, or 27.5 mM glucose in the absence or presence of 0.1 unit/ml
insulin for 30 h. Luciferase activity is shown as the percentage
of relative light units measured with the value for the 2X rat
S14 ChoRE PK(
40) Luc construct at 27.5 mM
glucose with insulin set at 100%. Values represent the means (± S.E.)
of two experiments, each with duplicate transfections.
1448 and
1422
of the rat S14 gene promoter diminished the ability of this
promoter to support a response to glucose in cells cultured in the
presence of insulin (25). Recently, Mater et al. (35)
reported on the presence of an SREBP-binding site in the proximal
S14 promoter between
139 and
131 and demonstrated that
this site was responsible for the suppression of S14
promoter activity by polyunsaturated fatty acids. These observations
raised the possibility that both of these binding sites might be
important for the regulation of S14 gene expression by
carbohydrate diet. Previously, S14 mRNA had been shown
to respond to increased glucose levels in primary hepatocyte cultures
(36), but effects of insulin on S14 gene expression in
cultured cells have not been reported. We therefore tested whether
S14 mRNA expression is regulated by insulin in cultured
hepatocytes. Cells were first incubated in the absence of insulin and
low glucose for 18 h. Then cells were switched to medium with or
without insulin under both low and high glucose conditions. When
S14 mRNA levels were monitored in cells treated for
8 h with insulin, a striking increase was observed even in low
glucose conditions (Fig. 7). This
response occurred rapidly with changes being detectable as early as
1 h after insulin treatment (data not shown). Addition of glucose
in the absence of insulin also led to a significant increase in
S14 mRNA levels, although the action of glucose was
clearly less dramatic than insulin. Again, we presume that this
reflects at least in part the important role of insulin in stimulating
glucose metabolism. When cells were treated with both insulin and high
glucose, a further increase in S14 mRNA was detected
that was more than additive of the independent actions of the two
effectors. These data suggest that expression of the S14
gene is regulated by two different pathways, one stimulated by insulin
directly (as observed in low glucose conditions) and one by glucose
metabolism.
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Fig. 7.
Insulin and glucose induce
S14 mRNA levels in primary hepatocytes.
After attachment, primary hepatocytes were washed several times in
medium with 5.5 mM glucose and no insulin and then cultured
in this medium for 18 h with an overlay of Matrigel. Cells were
then washed and treated with medium containing either 5.5 or 27.5 mM glucose in the absence or presence of 0.1 unit/ml
insulin for 8 h. Total RNA was extracted, and S14
mRNA was determined using a competitive reverse transcriptase-PCR
assay as described by Kaytor et al. (48). Values represent
the means (± S.E.) of four samples. *, p < 0.01 versus low glucose-insulin group.
1467 to
1395) linked to S14 proximal promoter sequences from
290 to +18, including the SREBP-binding site. Mutations were
introduced into the ChoRE, the SREBP-binding site, or both sites in the
context of this construct. These various plasmids were then tested for
their ability to respond to glucose, insulin, or the combination of
these factors. With the wild type construct, a significant induction
was observed in either the transition from 5.5 to 27.5 mM
in the absence of insulin or by the addition of insulin in cells
maintained in 5.5 mM glucose, suggesting both effectors can
act on this construct (Fig. 8). When
cells were treated with both high glucose and insulin, promoter
activity was enhanced synergistically compared with treatment with
either component alone. Both the ChoRE and SREBP-binding sites
appear to be critical for this synergistic action of glucose and
insulin. A mutation that disrupted the ChoRE site resulted in a
significant reduction in the presence of high glucose either in the
absence or presence of insulin. On the other hand, the effects of
insulin on this construct were not diminished at 5.5 mM
glucose, suggesting that the insulin effect is mediated by sequences
other than the ChoRE. The actions of insulin on this construct are
clearly more pronounced in the presence of high glucose, suggesting
that the S14 proximal promoter (
290/+18) may contain
additional sequences that mediate glucose responsiveness. A mutation
that disrupted the SREBP-binding site had an even more dramatic effect,
completely disrupting the effect of insulin and leaving a construct
with a modest, but significant, response (~6-fold) to glucose.
Combining the ChoRE and SREBP-binding site mutations resulted in a
promoter that was no longer affected by either glucose or insulin.
These data support the role of both ChoRE and SREBP-binding sites in the overall response to glucose and insulin and suggest that the factors binding to these sites function in a synergistic fashion to regulate S14 promoter activity.
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Fig. 8.
Role of the S14 ChoRE and
SREBP-binding site in supporting responses to glucose and insulin in
hepatocytes. Primary hepatocytes were transfected with the rat
S14 ( 1467/
1395)(
290/+18) Luc construct that was
derived from either wild type S14 sequences (WT)
or sequences harboring mutations in the ChoRE (ChoRE mut) or
SREBP-binding site (SREBP mut). Cells were then cultured in
either 5.5 or 27.5 mM glucose in the absence or in the
presence of 0.1 unit/ml insulin for 30 h. Luciferase activity is
shown as the percentage of relative light units measured with the value
of wild type construct at 27.5 mM glucose with insulin set
at 100%. Values represent the means (± S.E.) of three experiments,
each with duplicate transfections.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cell. Insulin acts on the hepatocyte insulin receptor and leads to the
induction of SREBP-1c mRNA and protein. SREBP binds to the promoter
region of many lipogenic enzyme genes where it functions to stimulate
transcription. Simultaneously, increased glucose levels following a
carbohydrate meal lead to elevated glycolysis in the hepatocyte. This
is mediated in part by the presence of the high Km
glucokinase, which allows glycolysis in the hepatocyte to be increased
proportionately with postprandial glucose levels. Because glucokinase
expression is induced by insulin, perhaps through SREBP-1c (23), the
two pathways are partially coupled to each other. Increased glucose
metabolism in the hepatocyte results in the generation of an unknown
intracellular signal that activates the ChoRF. ChoRF binds to a
distinct site from SREBP-1c on the promoter regions of many lipogenic
enzyme genes to activate their expression. In many cases, ChoRF may
function synergistically with SREBP to activate transcription of
lipogenic enzyme genes and promote lipogenesis in the liver.
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Fig. 9.
Model for the regulation of lipogenic enzyme
genes by glucose and insulin. See text for explanation.
In support of this model, we have shown that the S14 gene
product is induced directly by insulin, in addition to its stimulation by glucose. This possibility was suggested by earlier studies in which
diabetic animals were shown to possess reduced levels of
S14 mRNA, whereas insulin treatment normalized its
expression (41). However, it is difficult to distinguish direct effects of insulin from those of glucose metabolism in such experiments. We
have shown in this report that S14 gene expression is
regulated independently and synergistically by glucose and insulin. Two regulatory sequences, the ChoRE at 1435 and an SREBP-binding site at
135, are required for the synergistic activation of S14 promoter activity. The critical roles of these two sites were demonstrated by mutations that disrupted either or both sites. Clearly,
the S14 promoter construct with both sites mutated is completely incapable of responding to glucose and/or insulin. Mutation
of the SREBP-binding site resulted in a complete loss in the ability of
the S14 promoter to respond to insulin, consistent with the
role of SREBP in mediating insulin action. Similarly, a construct that
contained an SREBP-binding site from the FAS promoter also responded
primarily to insulin. The SREBP-binding site mutant of the
S14 promoter construct retained the ability to respond to
glucose, but the overall activity of this construct was greatly reduced
compared with the wild type construct. This effect presumably reflects
the multiple roles of insulin in stimulating aspects of glucose
metabolism as well as its direct action on SREBP. Mutations of the
ChoRE site of the S14 gene, on the other hand, are reduced
in their ability to respond to glucose but retain most of the insulin
responsiveness. These data suggest that ChoRF is primarily mediating a
glucose signal. The ability of the two copy ChoRE construct to respond
to glucose in the absence of insulin also supports this model. However,
the ChoRE mutant construct still retains significant glucose
responsiveness. Several explanations could account for this
observation. First, the S14 proximal promoter (
290/+18)
might contain additional elements capable of contributing to a glucose
response. Second, ChoRF might have some ability to bind the SREBP site
and mediate the glucose response. Third, SREBP itself might be
stimulated by glucose. The latter explanation seems less likely because
the glucose effect was not observed with the FAS construct that
contains only SREBP sites (Fig. 6). Investigations are currently
underway to explore these possibilities.
Because the induction of many or most lipogenic enzyme genes in
cultured hepatocytes requires both glucose and insulin, we suggest that
dual regulation by ChoRF and SREBP-1c might be a common mechanism for
transcriptional control. We have found evidence for the fatty acid
synthase gene to support this contention. In the FAS promoter, a pair
of SREBP-binding sites is found overlapping an E box site at
approximately 65 (38). This site has been shown to be critical for
supporting a 3-6-fold induction of insulin in cultured cells or
hepatocytes (17, 27, 42). However, the induction of FAS that occurs in
the whole animal upon feeding carbohydrate diet is on the order of
25-30-fold, suggesting that additional regulatory sequences exist (43,
44). Recently, Rufo et al. (45) presented evidence for the
role of an upstream enhancer of the fatty acid synthase gene that
contributes to the overall nutritional response. We found that this
region contains a site for binding of ChoRF and that this site is
functional in supporting a response to
glucose.3 Thus, FAS may be
regulated in a similar manner to S14 via a synergistic activation by both ChoRF and SREBP. That this may also be true for
other lipogenic genes is suggested by the phenotype of SREBP-1 knockout
mice. Although these mice are impaired with respect to their ability to
respond to carbohydrate feeding, most of the lipogenic genes retain a
significant level of induction even in the absence of SREBP-1 (22). For
example, FAS mRNA is induced 10-fold in the knockout mice, whereas
S14 mRNA levels increase 6-fold. These data imply that
another factor in addition to SREBP-1 is capable of mediating
nutritional responses to carbohydrate diet.
An alternative model proposed by Foretz et al. (19, 23) suggests that SREBP-1c is regulated by insulin at the level of transcription and by glucose post-transcriptionally to mediate carbohydrate induction of lipogenic genes. This hypothesis was based on the observation that an adenoviral expression vector for the nuclear form of SREBP-1c enhanced expression of L-PK or FAS more effectively in cells cultured in high glucose than in cells in low glucose. However, no alterations in SREBP-1c mRNA or protein levels were found in hepatocytes exposed to varying glucose levels (23, 32). As shown in Fig. 1, glucose does not affect the transactivation potential of SREBP on cotransfected reporter constructs, further arguing against this model. A couple of explanations might account for the apparent discrepancy between these experiments. First, in a study by Foretz et al. (23), measurements were made on mRNA levels produced from endogenous genes. If SREBP and ChoRF can function synergistically, as suggested by our data, then the enhanced mRNA levels could be accounted for by the dual regulation of these gene products by exogenous SREBP and endogenous ChoRF stimulated by the high glucose conditions. A second explanation arises from the observation that glucose acts not only to stimulate transcription but also to stabilize mRNA levels for many lipogenic enzyme genes (2). Hence, in measuring mRNA levels in their study, Foretz et al. (23) could have been observing the effects of SREBP stimulation of transcription and glucose stabilization of mRNA. In the cotransfection experiments, effects at the level of mRNA stability would not be observed.
Mice bearing a homozygous deletion of the SREBP-1 gene display an impaired response to a carbohydrate diet in the induction of any of 10 lipogenic enzyme gene products examined, suggesting a broad role of SREBP in the nutritional induction (22). Although these results clearly demonstrate an essential role for SREBP in the nutritional induction pathway, they must be interpreted with some caution. Expression of glucokinase is critical for the ability of the hepatocyte to increase its rate of glycolysis in response to glucose. If SREBP-1c is directly responsible for the induction of glucokinase by insulin, as suggested by recent results (19), then the phenotype of the SREBP-1 null hepatocyte could arise from a defect in expression of this critical gene and indirectly affect other lipogenic enzyme genes. Similarly, studies in which SREBP-1 is overexpressed have shown that many or most lipogenic enzyme genes are induced (21). These data certainly support a broad role for SREBP in the nutritional stimulation of lipogenic enzyme genes. However, again the possibility that overexpression of SREBP could induce glucokinase gene expression and turn on increased glucose metabolism must be considered. In fact, as shown in Fig. 6, overexpression of glucokinase in hepatocytes does result in elevated promoter activity from a construct containing a ChoRE site even at fasting glucose levels. Thus, the demonstration of a direct role of SREBP in regulation of any lipogenic enzyme gene by carbohydrate feeding requires the detection of a functional SREBP-binding site that when mutated reduces or eliminates the ability of the promoter to respond to insulin and/or glucose.
If most of the lipogenic enzyme genes are indeed regulated by both
ChoRF and SREBP similarly to S14 and FAS, what might be the
physiological basis for this dual regulation? One possibility is that
this control ensures that lipogenesis is not inappropriately turned on
in the liver unless both anabolic signals from insulin and glucose
metabolism are in agreement. Another possibility is that this system
serves to provide finer level regulation on the levels of individual
gene products and their control by multiple effectors of the lipogenic
process. In this regard, it is interesting to note that polyunsaturated
fatty acids, which function to repress lipogenesis, mediate their
actions by blocking formation of nuclear SREBP-1c (35, 46, 47). On the
other hand, glucagon inhibition of lipogenic enzyme gene production
occurs at least in part by inhibition of glycolysis and hence
inactivation of the ChoRF factor. Thus, these two factors are able to
integrate multiple physiological cues in determining appropriate levels
of production for the variety of lipogenic enzyme genes.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Timothy Osborne (University of California, Irvine) for the gift of pFACMV/SREBP-1c and Dr. Bruce Spiegelman (Dana-Farber Cancer Institute, Harvard Medical School) for the plasmid containing SREBP-1c sequences.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant DK26919.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: 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, December 8, 2000, DOI 10.1074/jbc.M010029200
2 S.-H. Koo and H. C. Towle, unpublished results.
3 C. Rufo, M. Teran-Garcia, S.-H. Koo, H. C. Towle, and S. D. Clarke, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are: FAS, fatty acid synthase; L-PK, liver-type pyruvate kinase; ChoRE, carbohydrate response element; ChoRF, carbohydrate-responsive factor; SREBP, sterol regulatory element-binding protein; SRE-1, sterol regulatory element; EMSA, electrophoretic mobility shift assay; PCR, polymerase chain reaction.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Hillgartner, F. B.,
Salati, L. M.,
and Goodridge, A. G.
(1995)
Physiol. Rev.
75,
47-76 |
2. | Towle, H. C., Kaytor, E. N., and Shih, H.-M. (1997) Annu. Rev. Nutr. 17, 405-33[CrossRef][Medline] [Order article via Infotrieve] |
3. | Girard, J., Ferre, P., and Foufelle, F. (1997) Annu. Rev. Nutr. 17, 325-52[CrossRef][Medline] [Order article via Infotrieve] |
4. |
Decaux, J.-F.,
Antoine, B.,
and Kahn, A.
(1989)
J. Biol. Chem.
264,
11584-11590 |
5. |
Iynedjian, P. B.,
Jotterand, D.,
Nouspikel, T.,
Asfari, M.,
and Pilot, P. R.
(1989)
J. Biol. Chem.
264,
21824-21829 |
6. |
Doiron, B.,
Cuif, M.-H.,
Kahn, A.,
and Diaz-Guerra, M.-J.
(1994)
J. Biol. Chem.
269,
10213-10216 |
7. | Cunningham, B. A., Moncur, J. T., Huntington, J. T., and Kinlaw, W. B. (1998) Thyroid 8, 815-825[Medline] [Order article via Infotrieve] |
8. | Bergot, M.-O., Diaz-Guerra, M.-J. M., Puzenat, N., Raymondjean, M., and Kahn, A. (1992) Nucleic Acids Res. 20, 1871-1878[Abstract] |
9. |
Liu, Z.,
Thompson, K. S.,
and Towle, H. C.
(1993)
J. Biol. Chem.
268,
12787-12795 |
10. |
Shih, H.-M.,
and Towle, H. C.
(1994)
J. Biol. Chem.
269,
9380-9387 |
11. |
Shih, H.-M.,
Liu, Z.,
and Towle, H. C.
(1995)
J. Biol. Chem.
270,
21991-21997 |
12. |
Koo, S.-H.,
and Towle, H. C.
(2000)
J. Biol. Chem.
275,
5200-5207 |
13. | Brown, M. S., and Goldstein, J. L. (1997) Cell 89, 331-340[Medline] [Order article via Infotrieve] |
14. | Osborne, T. F. (2000) J. Biol. Chem. 276, 32379-32382[CrossRef] |
15. | Tontonoz, P., Kim, J. M., Graves, R. A., and Spiegelman, B. M. (1993) Mol. Cell. Biol. 13, 4753-4759[Abstract] |
16. |
Shimomura, I.,
Shimano, H.,
Horton, J. D.,
Goldstein, J. L.,
and Brown, M. S.
(1997)
J. Clin. Invest.
99,
838-845 |
17. |
Kim, J. B.,
Sarraf, P.,
Wright, M.,
Yao, K. M.,
Mueller, E.,
Solanes, G.,
Lowell, B. B.,
and Spiegelman, B. M.
(1998)
J. Clin. Invest.
101,
1-9 |
18. |
Horton, J. D.,
Bashmakov, Y.,
Shimomura, I.,
and Shimano, H.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
5987-5992 |
19. |
Foretz, M.,
Pacot, C.,
Dugail, I.,
Lemarchand, P.,
Guichard, C.,
Liepvre, X. L.,
Berthelier-Lubrano, C.,
Spiegelman, B.,
Kim, J. B.,
Ferre, P.,
and Foufelle, F.
(1999)
Mol. Cell. Biol.
19,
3760-3768 |
20. |
Shimano, H.,
Horton, J. D.,
Shimomura, I.,
Hammer, R. E.,
Brown, M. S.,
and Goldstein, J. L.
(1997)
J. Clin. Invest.
99,
846-854 |
21. |
Shimomura, I.,
Shimano, H.,
Korn, B. S.,
Bashmakov, Y.,
and Horton, J. D.
(1998)
J. Biol. Chem.
273,
35299-35306 |
22. |
Shimano, H.,
Yahagi, N.,
Amemiya-Kudo, M.,
Hasty, A. H.,
Osuga, J.-I.,
Tamura, Y.,
Shionoiri, F.,
Iizuka, Y.,
Ohashi, K.,
Harada, K.,
Gotoda, T.,
Ishihashi, S.,
and Yamada, N.
(1999)
J. Biol. Chem.
274,
35832-35839 |
23. |
Foretz, M.,
Guichard, C.,
Ferre, P.,
and Foufelle, F.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
12737-12742 |
24. | Moriizumi, S., Gourdon, L., Lefrancois-Martinez, A.-M., Kahn, A., and Raymondjean, M. (1998) Gene Expr. 7, 103-113[Medline] [Order article via Infotrieve] |
25. |
Kaytor, E. N.,
Shih, H.-M.,
and Towle, H. C.
(1997)
J. Biol. Chem.
272,
7525-7531 |
26. | Shih, H.-M., and Towle, H. C. (1995) BioTechniques 18, 813-816[Medline] [Order article via Infotrieve] |
27. |
Magana, M. M.,
Koo, S.-H.,
Towle, H. C.,
and Osborne, T. F.
(2000)
J. Biol. Chem.
275,
4726-4733 |
28. | Kim, J. B., Spotts, G. D., Halvorsen, Y.-D., Shih, H.-M., Ellenberger, T., Towle, H. C., and Spiegelman, B. M. (1995) Mol. Cell. Biol. 15, 2582-2588[Abstract] |
29. |
Andersson, S.,
Davis, D. L.,
Dahlback, H.,
Jornvall, H.,
and Russell, D. W.
(1989)
J. Biol. Chem.
264,
8222-8229 |
30. | Miller, S. P., Anand, G. R., Karschnia, E. J., Bell, G. I., LaPorte, D. C., and Lange, A. J. (1999) Diabetes 48, 1645-1651[Abstract] |
31. | Schreiber, E., Matthias, P., Muller, M. M., and Schaffner, W. (1989) Nucleic Acids Res. 17, 6419[Medline] [Order article via Infotrieve] |
32. | Azzout-Marniche, D., Becard, D., Guichard, C., Foretz, M., Ferre, P., and Foufelle, F. (2000) Biochem. J. 350, 389-393[CrossRef][Medline] [Order article via Infotrieve] |
33. |
Briggs, M. R.,
Yokoyama, C.,
Wang, X.,
Brown, M. S.,
and Goldstein, J. L.
(1993)
J. Biol. Chem.
268,
14490-14496 |
34. | Agius, L., and Peak, M. (1997) Biochem. Soc. Trans. 25, 145-150[Medline] [Order article via Infotrieve] |
35. |
Mater, M. K.,
Thelen, A. P.,
Pan, D. A.,
and Jump, D. B.
(1999)
J. Biol. Chem.
274,
32725-32732 |
36. |
Mariash, C. N.,
Seelig, S.,
Schwartz, H. L.,
and Oppenheimer, J. H.
(1986)
J. Biol. Chem.
261,
9583-9586 |
37. |
Vaulont, S.,
and Kahn, A.
(1994)
FASEB. J.
8,
28-35 |
38. |
Magana, M. M.,
and Osborne, T. F.
(1996)
J. Biol. Chem.
271,
32689-32694 |
39. |
Ericsson, J.,
Jackson, S. M.,
Kim, J. B.,
Spiegelman, B. M.,
and Edwards, P. A.
(1997)
J. Biol. Chem.
272,
7298-7305 |
40. |
Tabor, D. E.,
Kim, J. B.,
Spiegelman, B. M.,
and Edwards, P. A.
(1998)
J. Biol. Chem.
273,
22052-22058 |
41. | Jump, D. B., Bell, A., Lepar, G., and Hu, D. (1990) Mol. Endocrinol. 4, 1655-1660[Abstract] |
42. |
Moustaid, N.,
Beyer, R. S.,
and Sul, H. S.
(1994)
J. Biol. Chem.
269,
5629-5634 |
43. |
Paulauskis, J. D.,
and Sul, H. S.
(1989)
J. Biol. Chem.
264,
574-577 |
44. | Blake, W. L., and Clarke, S. D. (1990) J. Nutr. 120, 1727-1729[Medline] [Order article via Infotrieve] |
45. | Rufo, C., Gasperikova, D., Clarke, S. D., Teran-Garcia, M., and Nakamura, M. T. (1999) Biochem. Biophys. Res. Commun. 261, 400-405[CrossRef][Medline] [Order article via Infotrieve] |
46. |
Xu, J.,
Nakamura, M. T.,
Cho, H. P.,
and Clarke, S. D.
(1999)
J. Biol. Chem.
274,
23577-23583 |
47. |
Yahagi, N.,
Shimano, H.,
Hasty, A. H.,
Amemiya-Kudo, M.,
Okazaki, H.,
Tamura, Y.,
Iizuka, Y.,
Shionoiri, F.,
Ohashi, K.,
Osuga, J.-I.,
Harada, K.,
Gotada, T.,
Nagai, R.,
Ishibashi, S.,
and Yamada, N.
(1999)
J. Biol. Chem.
274,
35840-35844 |
48. | Kaytor, E. N., Qian, J., Towle, H. C., and Olson, L. K. (2000) Mol. Cell. Biochem. 210, 13-21[CrossRef][Medline] [Order article via Infotrieve] |