From Research and Development, Dallas Veterans Affairs Medical Center and Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75216
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ABSTRACT |
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Transcription of the liver type pyruvate kinase
and lipogenesis enzyme genes is induced by high carbohydrate in liver.
We have found a novel protein factor in rat liver nuclei that binds to
the glucose response element (CACGTG motifs) of the pyruvate kinase
gene (Liu, Z., Thompson, K. S., and Towle, H. C. (1993) J. Biol. Chem. 268,12787-12795) and the "insulin
response element" of fatty acid synthase gene. The amounts of this
DNA-binding protein, termed "glucose response element binding
protein" (GRBP) in the nuclear extract, were increased in liver by a
high carbohydrate diet and decreased by starvation, high fat, and high
protein diet. GRBP also occurs in cytosols of liver and is dependent on
carbohydrate. Both the nuclear and the cytosolic GRBP showed similar
properties, except the former was more resistant to thermal
inactivation than the latter. Kinetics of glucose activation of the
cytosolic GRBP in a primary culture of hepatocytes indicated that a
half-maximum activation was achieved after 6 h, and glucose
concentration required for the maximum activation of the GRBP was
approximately 12 mM. Dibutyryl-cAMP, okadaic acid, and
forskolin inhibited glucose activation of both GRBP and liver pyruvate
kinase transcription. These results suggested that GRBP may be a factor
that recognizes the glucose response motif site and may be involved in
mediating carbohydrate response of the pyruvate kinase gene.
The liver, the principal site of lipogenesis, is responsible for
conversion of excess dietary carbohydrate to triglycerides. A high
carbohydrate diet induces the synthesis of several key enzymes involved
in glycolysis and lipogenesis, including pyruvate kinase, ATP citrate
lyase (ACL),1 ACC, and FAS
(reviewed in Refs. 1 and 2). This increased enzyme synthesis is
correlated to increased mRNA, resulting from glucose-induced
expression of the corresponding genes.
Glucose-stimulated liver type pyruvate kinase (LPK) gene expression in
liver is mediated through the glucose response element (GRE) that is
located within the region
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
183 to
96 base pairs upstream from the
cap site of the LPK gene (Fig. 1) (3).
Vaulont et al. (4) found three protein-binding sites within
this segment which they designated MLTF-like, HNF4, or LF-A1 and NF-1
as binding sites for the transcription factors. The MLTF-like site
contains a palindromic sequence, CACGGG (underlined in Fig.
1), separated by 5 bases that corresponds to the consensus binding site
(CACGTG) for MLTF/USF, c-Myc, and its related family members, and TFE3, SREBP/ADD1 (5-11). Among these factors, upstream stimulatory factor (USF) is the predominant factor of hepatic nuclear extracts.
Electrophoretic mobility shift assays demonstrated that USF does bind
to the carbohydrate or GRE of LPK which is "supershifted" by
antibodies against USF (12-14). However, several lines of evidence
rule out USF being the GRE-binding protein as follows: (a)
the USF-binding site from adenovirus major late promoter was unable to
replace the carbohydrate-responsive element of LPK (15); (b)
when dominant negative forms of USF, which are able to form a
heterodimer with endogenous USF, but are unable to bind to DNA, are
expressed in hepatocytes, they do not block LPK induction by glucose
(16); (c) mutant GRE constructs are able to retain glucose
responsiveness of chloramphenicol acetyltransferase transcription yet
lose ability to bind USF (16). Thus, these results rule out USF as the
specific carbohydrate-responsive factor that activates LPK gene
expression, and thus far such a factor has not been found.
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Fig. 1.
Sequence of the glucose response element of
the LPK gene ( 183 to
97).
Little is known about the nature of the signal transduction pathway for
the glucose stimulation of transcription of any of these genes. The
possible role of various glucose metabolites, including glucose-6-P
(17), 3-P-glycerate (18), and P-enolpyruvate (18) etc., has been
suggested but not proved. More recently, Doiron et al. (19)
showed that incubation of hepatocyte-derived transformed cells (At3F)
with xylitol results in induction of LPK, and they suggest that xylitol
is converted to xylulose-5-P (Xu-5-P) inside the cells, which in turn
activates LPK transcription. This idea for xylitol feeding is based on
the earlier demonstration that Xu-5-P activates specific protein
phosphatase (PP2A), which in turn activates the synthesis of hepatic
Fru-2,6-P2, the most potent activator of
phosphofructokinase, in response to increased glucose (20, 21).
However, this Xu-5-P activation of PP2A by glucose happens rapidly,
within a few minutes, whereas glucose activation of gene transcription
is considerably slower, taking hours. Thus, it is questionable whether
the same mechanism of the glucose signaling applies to both short and
long term regulation. More recently Mourrieras et al. (22)
demonstrated that the effect of xylitol (on FAS and S14
genes) could be explained by increased glucose-6-P by conversion of
Xu-5-P to glucose-6-P. Attempts to identify a signaling metabolite in
whole cells where numerous metabolic intermediates are formed from a
major substrate such as glucose are futile without knowing the
individual steps involved in glucose activation of LPK gene expression.
A possibility that Xu-5-P-activated PP2A might be involved in
glucose-dependent activation of the LPK transcription
prompted us to search for a transcription factor responsible for the
glucose signaling. In the present study we report that we found a
factor binding to the GRE of LPK in rat liver which is induced by high
carbohydrate diet. We examined its DNA binding characteristics and
regulation of its activity using cultured hepatocytes.
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EXPERIMENTAL PROCEDURES |
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Materials--
[-32P]ATP (3,000 Ci/mmol) was
purchased from Amersham Pharmacia Biotech and poly(dI-dC) from
Pharmacia Biotech (Uppsala, Sweden). Okadaic acid was purchased from
Calbiochem, and dibutyryl-cAMP and forskolin were from Sigma.
Restriction enzymes and T4 polynucleotide kinase were
purchased from New England Biolabs (Beverly, MA). The Muta-Gene M13
in vitro mutagenesis kit was from Bio-Rad. Antibody against
USF1, USF2, Sp1, c-Myc, and HNF4 were from Santa Cruz Biotechnology
(Santa Cruz, CA). All other chemicals were reagent grade and were
obtained from commercial sources.
Plasmid Constructions and Oligonucleotides--
The promoter
region between positions 206 and
7 of the LPK gene (3) was obtained
by polymerase chain reaction amplification using rat kidney genomic DNA
as a template with outside primers containing engineered
XhoI and HindIII sites. The primers used were
CGCTCGAGCGGCTCTGCAGACAGGCCAAAG and CCAAGCTTGGGTCTGTGGGTCTGCTTTATAC. The
plasmid p(
206/
7)LPK was constructed by digesting pGEM-T vector
(Promega/Madison, WI) and was digested with XhoI and
HindIII. The digested fragment was ligated into the
XhoI and HindIII sites of the luciferase
expression plasmid, pGL3-Basic vector (Promega). Oligonucleotide-directed in vitro mutagenesis was performed
as described by Kunkel (23) using the Muta-Gene M13 in vitro
mutagenesis kit. Oligonucleotides used for the mutagenesis as well as
DNA binding experiments are listed in Tables I and II.
Animals-- Animals were starved for 48 h and then fed 24 h with NIH lab chow, high sucrose (20% casein, 60.2% sucrose, 15% cellulose, 2.25% minerals, and 2.25% vitamins) (24), high fat without starch (31% casein, 30.5% cellulose, 74% minerals, 1.5% vitamins, 1.5% corn oil, 1.5% peanut oil, and 27% lard) (25), or high protein diet (90% casein and 4% lard).
Primary Hepatocyte Culture and Transfection-- Primary hepatocytes were prepared from male Sprague-Dawley rats (200-300 g) using collagenase (Life Technologies Inc.) perfusion method (26) and plated in collagen-coated six-well tissue culture plates (Primaria Falcon/Franklin Lakes, NJ) at a density of 1.2 × 106 cells/well in glucose-free Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10 nM dexamethasone, 0.1 unit/ml insulin, 100 units/ml penicillin, 100 µg/ml streptomycin, 10% dialyzed fetal bovine serum (Life Technologies, Inc.; glucose 1.4 mg/dl), and 5.5 mM glucose. After a 6-h attachment period, transfection was performed using synthetic liposome (Lipofectin from Life Technologies, Inc.) as described in Opti-MEM I reduced serum medium (27). After 12-14 h the media containing the liposome-DNA complex was removed and replaced with glucose-free Dulbecco's modified Eagle's medium supplemented as described above and containing either 5.5 or 27.5 mM glucose. Cells were then cultured further for 48 h and harvested for luciferase assay (luciferase assay system from Promega/Madison, WI).
Preparation of Extracts of Hepatocytes-- For preparation of cytosolic extract of hepatocytes, the cell pellets were washed twice with ice-cold phosphate-buffered saline (PBS) (sodium phosphate, 12 mM, pH 7.4, and 15 mM NaCl) and suspended in 5 volumes of ice-cold buffer A (20 mM Hepes/KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM sodium EDTA, 1 mM sodium EGTA, and 1 mM dithiothreitol) (DTT), supplemented with protease inhibitors (1 mM phenylmethylsulfonyl fluoride (PMSF), 1 µg/ml pepstatin A, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM benzamidine). After sitting on ice for 15 min, the cells were broken by passing 15 times through a 23-gauge needle. After centrifugation (27,000 × g) for 15 min at 4 °C, the supernatant solution was immediately assayed.
Preparation of Nuclear and Cytosolic Extracts of Rat Liver-- Liver nuclear extracts were prepared according to the method of Hattori et al. (28) with minor modifications. Livers (~10 g) were homogenized with three strokes in a Potter homogenizer in 15 ml of homogenization buffer (10 mM Hepes/KOH, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.74 mM spermidine, 1 mM DTT, and 0.5 mM PMSF) containing 0.3 M sucrose. The homogenate was mixed with 2 volumes of cushion buffer (2.2 M sucrose, 10 mM Hepes/KOH, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.74 M spermidine, 1 mM DTT, and 0.5 mM PMSF), was layered on top of a cushion buffer, and was centrifuged at 75,000 × g for 30 min at 4 °C. The precipitated nuclei were suspended in 5 volumes of nuclear lysis buffer (10% glycerol, 10 mM Hepes, pH 7.9, 420 mM NaCl, 0.1 mM EDTA, 3 mM MgCl2, 5 mM DTT, 0.5 mM PMSF, 0.5 µg/ml pepstatin A, 1 mM benzamidine, and 0.2 µg/ml leupeptin) and homogenized with 10 strokes of a hand-held Dounce homogenizer. After standing on ice for 30 min and centrifuging at 27,000 × g for 5 min at 4 °C, polyethylene glycol (PEG; Mr 8,000) was added to the supernatant solution to bring PEG to 25% to precipitate proteins in the extract. In some experiments it was necessary to remove USF in the extracts, and for this purpose USF was precipitated with 10% PEG and removed by centrifugation. The remaining protein in the supernatant solution was precipitated with the addition of PEG (25%). The pellet was dissolved in a buffer mixture (20% glycerol, 20 mM Hepes/KOH, pH 7.9, 100 mM KCl, 0.2 mM EDTA, 5 mM DTT, and 0.5 mM PMSF) and was centrifuged at 27,000 × g for 5 min at 4 °C to remove the insoluble materials. The protein concentration was determined using the Bradford method (29).
DNA Binding Assay Method--
Gel mobility shift assay was
performed as described by Liu et al. (13). Double-stranded
oligonucleotides were prepared by mixing equal amounts of the
complementary single-stranded DNAs in 50 mM NaCl, heating
to 70 °C for 15 min, and then cooling to room temperature. The
annealed oligonucleotides were labeled with 32P in the
presence of [-32P]ATP and polynucleotide kinase.
Binding reactions were carried out in a reaction mixture in a final
volume of 20 µl containing 20 mM Hepes/KOH, pH 7.9, 50 mM KCl, 5 mM DTT, 0.2 mM EDTA, 0.5 mM PMSF, 10% glycerol, and 2 µg of poly(dI-dC). A
typical reaction mixture contained 40,000 cpm (0.5-1 nM)
of the labeled DNA and 5-10 µg of nuclear or cytosolic extracts. The
reaction mixture was incubated at room temperature for 30 min and
loaded onto a 4.5% nondenaturing polyacrylamide gel. The
electrophoresis was run at 200 V at 4 °C in a Tris glycine buffer
system for 1-2 h. The gel was dried and exposed to Hyperfilm-MP
(Amersham Pharmacia Biotech) at
80 °C. To quantitate the DNA
binding the exposed film was scanned using an Alpha-Imager
scanner/densitometer (San Leandro, CA). "Supershift" assays were
carried out by preincubating the nuclear and cytosolic extracts (7.5 µg) with 2 µg each of anti-USF1, anti-USF2, anti-Sp1, anti-c-Myc
antibody, or anti-HNF4 (Santa Cruz Biotechnology) for 30 min at room
temperature before the binding probes and the gel mobility shift assay.
All experiments were performed at least three times.
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RESULTS |
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Changes in GRE-binding Protein with Diet-- Following 48 h starvation, rats were re-fed NIH lab chow, high sucrose, high fat, or high protein diet for 12 and 24 h. The livers were quickly removed, nuclear and cytosolic extracts prepared, and GRE-binding proteins in the extracts analyzed by gel shift assay. The results showed that a specific binding factor ("band 1") was present in the nuclear extract in low concentration and was difficult to detect, especially since an intense band later identified as USF was present in these extracts which migrated more slowly than band 1 protein. After precipitation of USF with PEG and subsequent concentration of the remaining extract enabled band 1 was detected more clearly. The results demonstrated that the nuclear extract contained two GRE-binding proteins (bands 1 and 2; Fig. 2, Liver), and only the band 1 protein changed with diet, i.e. the protein was extremely low upon starvation, induced by high sucrose diet in 12 and 24 h, and decreased with high fat or high protein diet. On the other hand, band 2 protein was unchanged with starvation or high sucrose diet and appeared to decrease with high protein diet (lane 6).
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To confirm that the identified GRE-binding protein (band 1) was contained in hepatocytes and not unique to nonparenchymal cells, isolated hepatocytes were examined. Nuclear extract of hepatocytes isolated from livers of rats fed high carbohydrate diet contained a highly intense band corresponding to band 1 and a trace amount of USF (Fig. 2, Hepatocytes). Extracts of hepatocytes isolated from livers of rats fed other diets or starved contained reduced amounts of band 1. These results indicated that band 1 is a hepatocyte-specific nuclear factor responsive to high carbohydrate, but the majority of the band 2 and USF observed in whole liver extracts appeared to be derived from nonparenchymal cells. band 2 protein was, however, absent or too low to be detected in these extracts.
GRE-binding Protein Was Also Present in Cytosol-- A similar DNA-binding protein was present in the cytosol of rat liver (Fig. 2, Liver) and hepatocytes (Fig. 2, Hepatocytes) and showed the same electrophoretic mobility as that of band 1. The cytosolic factor was also induced by high carbohydrate and decreased by starvation, high fat, or protein diet.
Specificity for Palindromic GRE Sequence-- The functional relevance of band 1 protein for the GRE site of LPK gene was examined by testing the effects of mutating the GRE region on both DNA binding and transcriptional activities. We prepared two mutant oligonucleotides in which one of the E boxes in the palindromic sequences CACGGG (E box 1) or CCCGTG (E box 2) was completely altered (Table I). The mutation of the E box 1 and 2 individually reduced the DNA binding activities of the nuclear GRBP by 53 and 72%, respectively, and those of the cytosolic factor by 62 and 52%, respectively (data not shown). These results indicated that both palindromic sequences are required for the maximum binding. We also prepared mutant oligonucleotides in which the palindromic sequences were systematically altered one base at a time (Table I), and the spacing between these sequence motifs also was altered by one base. Constructs of mutant oligonucleotides were the same as those used previously by Kaytor et al. (16) to examine the GRE-binding sites in the expression of S14 and LPK. The results (Fig. 3A) showed that band 1 binding to most of the mutants was decreased, compared with the wild type DNA, except for M5, M3/5, and Ml. Competitive binding assays also were performed in which unlabeled oligonucleotide DNAs were incubated with the labeled, wild type oligonucleotide in order to test the ability of mutant oligonucleotides to compete for the DNA-binding site. The results were similar to those of the binding experiments in that M5, M3/5, and Ml were the most effective in competing for the binding site (data not shown).
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In order to test the biological activities of the mutants, the
5'-flanking region containing the LPK promoter (197 to +12; Fig. 1)
was prepared by polymerase chain reaction using rat genomic DNA as a
template with oligonucleotide primers based on the rat LPK gene
sequence (30, 31). A HindIII site at
7 and XhoI site at
206 were introduced by site-directed mutagenesis using a
synthetic oligonucleotide. The LPK promoter was then subcloned into the
polylinker of a luciferase reporter vector, pGL3-Basic vector
(Promega). LPK promoters containing the wild type and various mutant
GREs were constructed using oligonucleotide-directed in vitro mutagenesis, confirmed by DNA sequencing, and the activity of each was determined in primary cultured hepatocytes. The results, as
depicted in Fig. 3B, demonstrated that all the mutant
promoters except M5 and M3/5 showed significant loss of ability to
activate LPK transcription by high concentration of glucose. M5,
containing the perfect consensus CACGTG sequence, showed the highest
activity. These results were generally in agreement with the previously published results of Kaytor et al. (16) for LPK
transcription using chloramphenicol acetyltransferase transcription
system in which only the MLTF site sequence (from
170 to
145) of
the LPK promoter DNA was linked to
96 of the rest of the promoter
DNA, thus lacking the HNF4 and NF1 sites (Fig. 1). One significant difference was the result of M6 in which they (16) showed that this
mutant responds to glucose, whereas our result indicated no response.
This discrepancy could be due to the lack of the accessory sites such
as HNF4 and/or NF1 sites in the promoter they employed, which may
influence the glucose response, since these accessory proteins may
regulate the transcriptional activity. Thus, the above results
demonstrated that bases 1-4 and 6, as well as the spacing between the
consensus GRE sequences, were critical for the glucose response, but
base 5 may not be. These transcriptional activities were, in general,
similar to those of the DNA binding characteristics of band 1 protein,
although the effect on the latter was not as striking as the former
activities. Important differences were exhibited by M6 and Ml in which
the glucose-activated transcription was completely inhibited by the mutation, but the DNA binding activity was unaffected. This discrepancy suggested that an additional factor(s) may be required to confer a
higher degree of specificity of LPK transcription induced by glucose.
We have termed the band 1 protein as glucose response element binding
protein (GRBP) throughout the paper.
Specificity of GRBP for GRE Elements of Other Genes-- In order to further examine the specificity of GRBP, DNA binding activities of GRBP for the carbohydrate response elements of S14, FAS, ACC, and ACL genes were determined. All these enzyme promoters contain the consensus CACGTG motifs (Table II). Both the nuclear and cytosolic GRBP showed similar binding characteristics (Fig. 4); GRBP bound FAS/IRS nearly as well as the LPK GRE elements but S14 less strongly. It did not bind to the GREs of ACC, ACL, or FAS. The binding differences were not due only to the consensus sequence (CACGTG), since the DNAs of S14, FAS, and ACC contain the perfect sequence, whereas FAS/IRS contains a sequence CATGTG in which the important third base (for the transcription) is different, yet the DNA was bound. Moreover, none of these GRE elements of the genes are palindromic, unlike the LPK promoter.
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A possibility that GRBP may bind to the neighboring HNF4 site of LPK
gene was examined using a probe LII (32) (147 to
124; Fig. 1),
which contains the HNF4 site (
145 to
122; Fig. 1) and an additional
four bases (GATC) extending into the MLTF site. The results (data not
shown) demonstrated that GRBP bound weakly to LII but not at all to the
HNF4 site, indicating that GRBP bound only to the MLTF site (
146 at
the 3' end) and not the 5' region of the HNF4. In addition,
immunoreactivity of GRBP toward HNF4 antibodies was determined by gel
shift assay. The results (Fig. 5) showed
that a heavy band of HNF4 was present in the nuclear extract (Fig. 5,
lane 3) which was supershifted after addition of the HNF4
antibodies (lane 4). The remaining band in the
antibody-treated sample (lane 4) corresponded to GRBP
(compare lanes 1 and 2). On the other hand, GRBP
did not react with the HNF4 antibodies. There was a band binding weakly
to the HNF4 oligonucleotide (lanes 5 and 6) which
was supershifted after incubation with HNF4 antibody. Both partially
purified nuclear (lanes 7 and 8) and cytosolic (lanes 9 and 10) GRBP failed to react with the
antibodies. These results clearly demonstrated that GRBP is not the
same as HNF4.
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Identity of GRBP-- The transcription factors thus far suspected to bind to the glucose-responsive promoter sites of LPK and other genes include USF, Sp1, and c-Myc families of proteins. In order to see if GRBP is related to any of these factors, the nuclear and the cytosolic extracts were reacted with specific antibodies and subjected to gel mobility shift assay. The results showed that USF in the liver nuclear extract supershifted by the USF antibody treatment (Fig. 6), but none of the antibodies against USF1, Sp1, and c-Myc affected the electrophoretic mobility of GRBP, suggesting that GRBP does not belong to any of these families of factors.
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Heat Lability of GRBP--
Previously Liu et al. (13)
described a hepatic nuclear factor that binds to the GRE site
(MLTF-like site) of LPK and is resistant to heating (7 min at
70 °C). In contrast, both nuclear and cytosolic GRBP were completely
inactivated by the heat treatment as discussed below. The results also
ruled out GRBP as a member of NF1 family which binds to the 149- to
126-base pair region (Fig. 1) of the LPK promoter, since it has been
shown to be resistant to heating at 90 °C for 5 min (32). Moreover,
the heat inactivation studies revealed that the cytosolic GRBP was more
sensitive to heating, since a half-maximum inactivation occurred at
40 °C, whereas the nuclear GRBP was more resistant, with a
half-maximum at 56 °C (data not shown), suggesting there was a
structural difference between the GRBPs in the cytosols and the nuclei.
Kinetics of GRBP Activation by Glucose in Hepatocytes-- To gain insight into the glucose signaling mechanism, kinetics of glucose activation of GRBP in cytoplasm of hepatocytes were followed using the DNA binding assay. The primary hepatocytes, isolated from rats starved for 24 h, were incubated in a medium containing 10 mM lactate for 4 h. At 0 time the medium was switched to glucose (27.5 mM), and at the indicated time intervals the cells were harvested, and changes in the DNA binding activity of cytosolic GRBP were determined. The activity of GRBP increased linearly with time up to 12 h and reached a half-maximal value in 6 h and the maximum in 12 h (Fig. 7A). The activity in the hepatocytes incubated in lactate remained constant during the same period.
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Effect of Varying Glucose Concentration on GRBP Activity-- Hepatocytes isolated from rats starved for 48 h were incubated with varying concentrations of glucose for 12 h, and cytosolic GRBP activity was determined. The cells incubated in 10 mM lactate served as a control. The results showed that half-maximum activation of GRBP was achieved at approximately 12 mM and maximum activation at approximately 30 mM glucose (Fig. 7B), which is comparable to the range of glucose concentration required for LPK mRNA synthesis (34).
Specificity for Sugar-- None of the sugars examined, fructose, ribose, and xylitol, at 20 mM were as effective as glucose in GRBP activation (Table III). 2-Deoxyglucose, which is known to be phosphorylated in vivo but not metabolized further (35), also activated GRBP, suggesting that 2-deoxyglucose-6-P could serve as an activator. 3-O-Methylglucose, which is not phosphorylated, failed to activate GRBP, suggesting that a phosphorylated sugar is an activator. These results of the glucose analogs were similar to the effect on LPK (34), ACC, and FAS gene transcription (35). Unfortunately, we were unable to obtain nuclei from these hepatocytes to examine the effect of these sugars and sugar analogs on the nuclear GRBP.
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Inhibition of GRBP by cAMP, Okadaic Acid, and
Forskolin--
Transcriptional and DNA binding activities of the
cytosolic GRBP were sensitive to inhibition by dibutyryl-cAMP
(Bt2cAMP), okadaic acid, a potent inhibitor of PP2As, and
forskolin, activator of adenylcyclase, in hepatocytes cultured in the
presence of lactate or glucose prior to glucose administration (data
not shown). A half-maximum inhibition of GRBP activity was elicited
with approximately 0.1 mM Bt2cAMP and 90%
inhibition with 1 mM, and the transcription activity showed
comparable inhibition with the same concentrations of
Bt2cAMP. Forskolin also inhibited both GRBP and the
transcriptional activities, suggesting that increased cAMP in the
hepatocytes is responsible for the inactivation of both activities.
Okadaic acid at 1 nM inhibited the GRBP and the
transcriptional activities 40 and 20%, respectively, and at 10 nM over 90 and 65%, respectively. Phorbol ester (1 mM) showed no inhibition of GRBP under these conditions
(data not shown), suggesting that protein kinase C was not responsible
for inactivation. Cycloheximide (10 µM) had no effect on
the glucose activation (data not shown), suggesting that protein
synthesis was not involved in increased GRBP in the hepatocytes. These
results further suggest that GRBP may be involved in glucose
stimulation of LPK transcription. Moreover, the observed inhibitory
effects of cAMP and okadaic acid together imply that phosphorylation/dephosphorylation may be involved in the regulation of
activity of GRBP, and a phosphorylation of the binding protein inactivates the activity.
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DISCUSSION |
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The presence of two CACGTG-type E box motifs within the GREs of LPK promoter suggests that a member of the basic/helix-loop-helix/leucine zipper family of transcription factors binds to the site (36, 37) and is involved in carbohydrate activation of transcription. Among the potential protein factors binding to the site, USF appears to be a predominant hepatic nuclear factor. USF in hepatic nuclear extracts does bind to the site (12, 13, 38) but has been excluded as the right factor of the LPK GRE in vivo based on several lines of evidence (12, 16), as discussed in the Introduction. We report herein a novel factor which bound to the "MLTF-like" sequence as demonstrated by electrophoretic mobility shift assay, and which depended on carbohydrate diet in vivo, and thus may play an important role in carbohydrate regulation of LPK expression. The evidence in support for this suggestion is as follows: (a) the factor occurred in the nuclei of rat livers and was induced by high carbohydrate diet, no other glucose responsive factor found; (b) the factor showed specificity for the MLTF-like sequence of the LPK promoter but not CACGTG-like E box of other genes of ACC, ACL, or FAS; (c) the factor showed specificity, although weak, toward palindromic CACGTG sequence; (d) the kinetics and the concentration of glucose required for the activation of GRBP were near physiological range; and (e) both the DNA binding and the transcription activities were inhibited to comparable degree by Bt2cAMP, forskolin, and okadaic acid consistent with the previous observation that glucose-induced expression of LPK gene is inhibited by cAMP (39) and also suggests that these activities are regulated by phosphorylation/dephosphorylation.
It was somewhat surprising to find the similar GRBP in the cytosolic extracts of rat livers. Other factors such as USF, HNF4, or the band 2 protein were absent in the cytosols. The cytosolic and nuclear GRBPs appear to be related since all the characteristics thus far uncovered were similar. The similar properties include electrophoretic mobility, DNA sequence binding specificity, immunoreactivity, solubility in salt and PEG, and chromatographic behavior on ion exchange columns. The only difference between the nuclear and cytosolic factors was that the former was considerably more heat-resistant than the latter, suggesting they have different structures.
We can exclude GRBP from a number of known families of transcription factors. GRBP was not HNF4 which is capable of binding to the adjacent site just 3' to the GRBP-binding site (Fig. 1) and appears to serve as an accessory factor in the glucose activation (4, 13, 40). GRBP was not USF, Sp1, or c-Myc since it failed to react with any of the antibodies against those transcription factors that are known to interact with MLTF-like sequence (41). These results suggested that GRBP may be a new and unique factor.
The GRE and the insulin response element of FAS gene have been
localized +283 to +303 nucleotides (12) and 71 to
50 nucleotides (42), respectively (Table II), and USF family of transcription factors
have been shown to bind to both sites (43, 44). We found that GRBP did
not bind to the GRE of the FAS gene even though it contains a perfect E
box sequence CACGTG. However, GRBP did bind to the "insulin response
element" of the FAS gene which contains an E box with a sequence
CATGTG as tightly as to the GRE site of the LPK gene (Fig. 7). Wang and
Sul (43) demonstrated that USF1 and USF2 as the major factors binding
to the FAS-IRS site and the same complex also bind the E box sequence
of S14 and LPK GREs. Thus, it is not surprising that GRBP was able to
bind to the FAS-IRS site, even though the GRBP is not USF, and the
E-box is not palindromic. The results suggested that GRBP bound not only to the consensus CAXXTG core but also the 3' extension
of the FAS gene, and the latter region may be equally important for strong interaction.
The observation that cAMP and forskolin inhibited the activation of cytosolic GRBP may suggest that phosphorylation of GRBP may be involved in the inactivation. This is further supported by the fact that okadaic acid, a potent inhibitor of PP2As, may inhibit dephosphorylation and activation of the inactive form of the phosphorylated GRBP. The protein kinase involved in the phosphorylation of GRBP could be cAMP-dependent protein kinase, but a few preliminary results indicated that it was not the case. Thus, the effect of cAMP may not be direct but probably more complex and may require additional enzyme(s). The activation of the cytosolic GRBP probably involves dephosphorylation of phosphorylated GRBP which is stimulated by glucose. Evidence in support of the suggestion was the observation that okadaic acid in nanomolar concentrations inactivated cytosolic GRBP, which can be explained by its inhibition of a PP2A, thus maintaining GRBP in a phospho form.
Doiron et al. (19) showed that the LPK transcription in hepatocytes and mhAT3F hepatoma cells was increased when these cells were incubated in xylitol, and they suggested that the same xylulose-5-P (Xu-5-P)-mediated mechanism may apply for the LPK transcription as that originally demonstrated for short term regulation of glucose signaling in Fru-2,6-P2 formation (20). Unfortunately, Doiron et al. (19) failed to demonstrate the formation and the concentration of Xu-5-P in their cells under the conditions of LPK transcription; consequently, the rates of Xu-5-P formation and LPK induction cannot be correlated. We found that the activation of the LPK transcription in hepatocytes took at least 3-6 h (Fig. 7), while the rate of Xu-5-P formation is less than 5 min (in perfused liver) (20). Thus, these two rates are vastly different and do not seem to support the Xu-5-P-mediated mechanism and Xu-5-P as the glucose signaling compound.
In summary, we have found a protein factor binding to the glucose
response elements of LPK and FAS/IRS genes in both nuclear and
cytosolic extracts of rat liver. The nuclear and cytosolic factors
exhibited similar activities and appeared to be related but not
identical. The DNA binding activity was inhibited by low glucose
in vivo and by cAMP in hepatocytes and was activated by high
glucose in vivo. Purification, characterization, and
determination of their roles in regulation of LPK are under way.
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ACKNOWLEDGEMENTS |
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We thank Dr. Sarah McIntire for critical review of this manuscript and Drs. Steven L. McKnight and Richard Gaynor for their advice.
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FOOTNOTES |
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* 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: Research (151B),
Dallas VA Medical Center, 4500 S. Lancaster Rd., Dallas, TX 75216. Tel.: 214-372-7028; Fax: 214-372-9534; E-mail:
kuyeda6400{at}aol.com.
The abbreviations used are: ACL, ATP citrate lyase; GRBP, glucose response element binding protein; LPK, liver pyruvate kinase; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; GRE, glucose response element; FAS, fatty-acid synthase; ACC, acetyl-CoA carboxylase; PP2A, protein phosphatase; USF, upstream stimulatory factor; PEG, polyethylene glycol; Xu-5-P, xylulose-5-P; IRS, insulin-responsive sequence.
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REFERENCES |
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