From the Department of Biochemistry, Case Western Reserve
University School of Medicine, Cleveland, Ohio 44106-4935 and the
Edison Animal Biotechnology Center, Ohio University,
Athens, Ohio 45107
Received for publication, March 19, 2001, and in revised form, April 16, 2001
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ABSTRACT |
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The regulation of transcription of the
gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP)
(PEPCK-C) (4.1.1.32) during diabetes is a complex process that involves
a number of regulatory elements in the PEPCK-C gene promoter. The
accessory factor 2 (AF2)-binding region that is contained within the
glucocorticoid regulatory unit of the PEPCK-C gene promoter ( The cytosolic form of phosphoenolpyruvate carboxykinase
(PEPCK-C)1 (GTP) (4.1.1.32)
is a critical enzyme in hepatic gluconeogenesis and glyceroneogenesis
(1) and is induced during diabetes, where it is involved in the
increased rate of hepatic glucose output and triglyceride synthesis
characteristic of this disease. Transcription of the gene for PEPCK-C
is induced by cAMP (2-4) and glucocorticoids (5) and inhibited by
insulin (6-8) and high concentrations of glucose (4, 9). There are
three major factors involved in the induction of PEPCK-C during
diabetes. First, the concentration of insulin in the blood is
dramatically reduced resulting in the removal of the dominant negative
regulator of PEPCK-C gene transcription. Second, there is an
increase in the levels of glucagon and a subsequent elevation in the
concentration of hepatic cAMP; this stimulates PEPCK-C gene
transcription (10). Third, glucocorticoids are required to ensure the
elevated expression of the PEPCK-C gene and the resultant physiological
alterations, including increased gluconeogenesis (11) (12).
The mechanism(s) responsible for the induction of PEPCK-C gene
transcription during diabetes has been studied in some detail (13). The
effect of cAMP is exerted through two regions in the PEPCK-C gene
promoter; the cAMP-response element, which maps from The present study focuses on use of transgenic animals carrying a
mutated AF2 element (in the PEPCK-C gene promoter Materials--
Theophylline, Bt2cAMP,
streptozotocin, and dexamethasone were purchased from Sigma.
Strip-EZ DNA probe synthesis kit was purchased from Ambion (Austin,
TX). QuickPrep total RNA extraction kit was purchased from Amersham
Pharmacia Biotech. GeneScreen Plus and [ Molecular Probes--
The PEPCK-C probe used was a
1.1-kilobase PstI fragment from the 3'-end of the
PEPCK-C cDNA, as described previously (20). RNA was normalized with
a 752-nt SacI fragment from the mouse 18 S rRNA cDNA as
described by Oberbaumer (21). The 700-base pair SmaI
cDNA hGx fragment (22) was used to screen the hGx transgenic mice
by Southern blotting. All probes were synthesized using
[ Construction of Chimeric PEPCK-hGx Gene Containing a Mutation in
the AF2 Element in the PEPCK-C Gene Promoter--
A mutation was
introduced into the specific protein-binding domains of the PEPCK-C
gene promoter ( Generation of Transgenic Mice--
Recombinant DNA used for
microinjection was separated from plasmid sequences by digestion with
XbaI for WT-2000/hGx and AF2-2000/hGx or
XbaI-BglII for WT-490/hGx and AF2-490/hGx,
followed by electrophoresis. DNA was electroeluted from the agarose gel
and then used for microinjection into fertilized mouse eggs. The
procedure for the generation of transgenic mice has been described
previously (26). Briefly, fertilized eggs were flushed from the
oviducts of super-ovulated C57BL/6× SJL mice. 2 µl of DNA
solution (2 ng/µl) was injected into the male pronuclei of the
fertilized mouse eggs. Viable embryos were re-implanted into the
oviducts of pseudo-pregnant mice as described previously (26).
Transgenic mice were identified by genomic DNA analysis of tail samples
taken at 4 weeks of age. Briefly, small segments of mouse tail were
placed in a lysis buffer containing 50 mM KCl, 10 mM Tris-HCL, pH 8.3, 2.5 mM MgCl2,
0.01% gelatin, 0.45% Nonidet P-40, 0.45% Tween 20, and 24 µg of
proteinase K overnight at 55 °C. 1 µl of this solution, containing
the lysed tail cells, was used in a polymerase chain reaction to
amplify the hGx gene. The primers used were hGH1,
5'-TTGCAGCTAGGTGAGCT-3'; and hGH2, 5'-CTTTGGCCGTGGGGAGTG-3'.
Germline Transmission--
Six lines were generated for AF2
2000, four lines were generated for each of WT 390 and AF2 490 transgenes, and one line was generated for the WT 2000. All lines of
transgenic mice demonstrated the tissue-specific pattern of expression
characteristic of the endogenous PEPCK-C gene, with highest expression
levels in liver and expression in the kidney ~20% that of hepatic
expression. The transgenic mice were normal in body weight and showed
no evidence of hepatomegaly or other disorders.
Hormonal and Dietary Treatments--
Male mice heterozygous for
the transgene were back-crossed to C57BL/6× SJL WT females. Only mice
heterozygous for the transgene were used for analysis. There were no
gender differences noted in the level of expression of hGx mRNA, so
both male and female mice heterozygous for the transgene were used.
Adult animals were 2-4 months of age at the time of study; they were
given free access to water and were fed standard laboratory chow,
unless otherwise specified. Adult animals were killed between 9:00 and
11:00 a.m. Mice were injected intraperitoneally with 35 mg of
Bt2cAMP per kg of body weight and 30 mg of
theophylline per kg of body weight in 150 µl of saline or not
injected (controls). The animals were killed 2 h later, and their
livers and kidneys were isolated, frozen in liquid nitrogen, and stored
for analysis at
Animals were made diabetic by a single intraperitoneal injection of
streptozotocin (200 mg/kg body weight). Mice were fasted overnight
prior to injection and fed 2 h later. Diabetes was
confirmed by measuring the concentration of blood glucose (tail vein)
using glucose dipsticks and an ENCORE® glucometer
(VWR Scientific). Animals treated with insulin received 1 unit
per 30 grams of body weight. All diabetic animals had blood glucose
concentrations of more than 300 mg/dl. To determine the effect of
glucocorticoids on the PEPCK-C gene promoter in the transgenic mice,
the animals were injected with 1 µM dexamethasone, and
their livers and kidneys were removed 5 h later. The tissues were
frozen in liquid nitrogen and stored at RNA Analysis--
Total RNA was isolated from the liver using a
QuickPrep total RNA extraction kit from Amersham Pharmacia Biotech.
Total RNA was isolated from the kidneys using the modified acid-phenol
guanidine thiocyanate procedure described previously (28). Northern
blot analysis was performed as described previously (29) using 20 µg
of total RNA. Gels were blotted to GeneScreen Plus and hybridized with
PEPCK-C cDNA or hGx cDNA probes at 42 °C in hybridization buffer containing 50% formamide (deionized), 1% SDS, 1 M
sodium chloride, 110 µg/ml denatured salmon sperm, and 10% dextran
sulfate. The filters were washed at 55 °C in 2× SSC and 0.1% SDS
for 20 min and 1× SSC, 0.1% SDS for 20 min. The relative
radioactivity was determined using a PhosphorImager (Molecular
Dynamics, Sunnyvale, CA). The concentration of PEPCK-C mRNA and hGx
mRNA were expressed relative to the concentration of 18 S ribosomal
RNA to control for differences in loading of the RNA.
Developmental Expression of the PEPCK-C-hGx Transgene--
Male
mice heterozygous for the transgene were back-crossed to C57BL/6× SJL
non-transgenic females. On day 19 of gestation, the livers and kidneys
were removed from fetal mice, and the RNA was extracted and analyzed by
Northern analysis using an hGx cDNA probe. The concentration of hGx
mRNA was expressed relative to 18 S ribosomal RNA.
Effect of AF2 on the Developmental Expression of the PEPCK-hGx
Transgene--
The gene for PEPCK-C is not expressed in the liver
before birth (30), partly because of repression by insulin. Because the AF2 site has been implicated in the insulin regulation of PEPCK-C gene
transcription (18), we analyzed the role of the AF2 element in the
developmental expression of the PEPCK-C gene in the liver and kidney.
For this purpose, chimeric genes were constructed that contained either
the wild type promoter (WT) or a gene with a deletion for AF2 element
(AF2) in the The Role of AF2 in the Regulation of PEPCK-hGx Gene Transcription
during Diabetes--
To dissect the role of AF2 in the response of the
PEPCK-C gene to diabetes and to the subsequent administration of
insulin, the animals were made diabetic by injection of
streptozotocin (the blood glucose concentration was in excess of 300 mg/dl in these mice). In all animals, the endogenous gene for PEPCK-C
responded to diabetes as expected, i.e. the
mRNA increased in diabetes and decreased when insulin was injected
(Fig. 3B). The WT-2000/hGx transgene was induced 1.7-fold by diabetes as shown by an increased hepatic hGx mRNA; insulin reduced its expression by half when administered for 8 h. The difference in the time course of
response to insulin may be attributable to differences in half-life
between PEPCK-C and hGx mRNAs. Transcription of the AF2-2000/hGx
transgene in the liver was not induced by diabetes but was inhibited
50% by insulin treatment (Fig. 3A) (**, p < 0.05). This finding strongly suggests that AF2 is necessary for the
induction of PEPCK-C by diabetes but that it is not involved in the
response of the PEPCK-C gene promoter to insulin.
The AF2 regulatory element in the PEPCK-C gene promoter contains a
glucocorticoid-binding site, as well as an insulin regulatory element. Because glucocorticoids are required for the increase in
PEPCK-C gene transcription that occurs during diabetes (12), we next
determined the response of the AF2-2000/hGx transgene to
glucocorticoids. Dexamethasone (1 µM) was administered to
both WT-2000/hGx and AF2-2000/hGx transgenic mice; the animals were killed 5 h later. The administration of dexamethasone had no
effect on the concentration of mRNA for hGx in the livers of
WT-2000/hGx and AF2-2000/hGx transgenic mice (Fig.
4A). The lack of response of
the transgenes to dexamethasone was expected, because it is well
established that glucocorticoids cause an increase in the secretion of
insulin (11); this insulin secretion would, in turn, prevent the
induction of transcription from the PEPCK-C gene promoter (12). For
this reason, we determined the response to glucocorticoids of both
transgenes in the kidney. There was a 3-fold induction of hGx mRNA
in the kidneys of AF2-2000/hGx transgenic mice, accompanied by a
5.5-fold induction of hGx mRNA in the kidneys of WT-2000/hGx mice
(Fig. 4B) (*, p < 0.05). Thus the loss of
one accessory factor 2-binding site resulted in a 60% reduction in the
glucocorticoid response of the transgene. These data are consistent
with the in vitro studies of Scott et al. (15,
32) showing a similar reduction in PEPCK-C gene transcription when one
accessory factor 2 element of the GRU was deleted.
We have shown that the AF2-binding site in the PEPCK-C gene promoter is
critical for the regulation of PEPCK-C mRNA by glucocorticoids. The
AF2-binding site has an overlapping GRE and a putative IRS that can
potentially bind C/EBP Effect of the AF2 Regulatory Element on the Response of the PEPCK-C
Gene Promoter to Alterations in the Carbohydrate Content of the
Diet--
Feeding mice a diet high in carbohydrate (or a high
fat/carbohydrate-free diet) markedly decreases (or increases) the
transcription of the gene for PEPCK-C in the liver (20). This effect is
presumably because of the secretion of insulin by the pancreas that in
turn represses hepatic PEPCK-C gene transcription. To determine whether the AF2 regulatory element in the PEPCK-C gene promoter is involved in
the response of the gene to dietary carbohydrate, WT-2000/hGx and
AF2-2000/hGx transgenic mice were fed a high carbohydrate diet for 1 week. This diet did not significantly alter the blood glucose
concentration of the animals. Control mice fed a normal lab chow diet
had a blood glucose level of 124 mg/dl ± 6.4, whereas mice fed a
high carbohydrate diet had a blood glucose level of 114 mg/dl ± 12.9. The high carbohydrate diet dramatically decreased the level of
hGx mRNA in the livers of both the WT-2000/hGx and AF2-2000/hGx
transgenic mice. This demonstrates that the AF2 site in the PEPCK-C
gene promoter is not required for the transcriptional response of the
promoter to dietary carbohydrate and presumably to insulin (Fig.
5). Mice that were fed the high
carbohydrate diet for 1 week were then injected with
Bt2cAMP and theophylline to stimulate transcription from
the PEPCK-C gene promoter. There was no difference in the response to
Bt2cAMP between the WT-2000/hGx and AF2-2000/hGx
transgenes, indicating that the AF2 site is not required for the effect
of cAMP on PEPCK-C gene transcription in the liver. Finally, the mice
were fed a diet in which the protein content was not changed, but the
carbohydrate was replaced with fat (high fat/carbohydrate-free diet).
There was a robust 3.4-fold stimulation in the level of hGx mRNA in
the livers of the WT-2000/hGx mice as compared with hGx mRNA in the
livers of the same lines of mice fed a control diet (Fig. 5). In
contrast, the AF2-2000/hGx transgenic mice had a 50% increase in
response to the high fat/carbohydrate-free diet.
If a high carbohydrate diet inhibits transcription from the PEPCK-C
gene promoter via the action of insulin then the AF2 site is clearly
not required for the insulin effect. It is possible that insulin
inhibits PEPCK-C gene transcription via another site on the promoter.
In this regard, we have recently found that sterol regulatory
element-binding protein 1c (SREBP-1c) is an intermediate in the action
of insulin on transcription of the PEPCK-C gene in
hepatocytes.3 Hasty et
al. (35) have reported that the addition of 5.5 to 25 mM glucose to hepatocytes in culture increases the
concentration of mature SREBP-1c in the nucleus, most likely by
inducing the level of mRNA for the transcription factor. In
addition, reporter gene analysis demonstrated a glucose-induced
increase in transcription from the SREBP-1c gene promoter (35). It is
thus possible that dietary glucose, which induces insulin secretion in
the mice, increased the level of SREBP-1c in the liver leading to a
repression of PEPCK-C gene transcription. If so, this must occur at a
site on the PEPCK-C gene promoter distinct from the AF2-binding site.
In the absence of the AF2 site, there is a blunted transcriptional
response of the PEPCK-C gene promoter to a high fat/carbohydrate-free diet. This suggests that this site is needed to achieve the full stimulation of transcription when the concentration of insulin is
decreased by feeding the mice a high fat/carbohydrate-free diet. This
is very similar to the response of the AF2-2000/hGx transgene to
diabetes where the stimulatory response is blunted (see Fig. 3). We
assume that the increase in transcription of the hepatic AF2-2000/hGx
transgene caused by feeding a high fat/carbohydrate-free diet is partly
because of the stimulatory effect of glucocorticoids on the PEPCK-C
gene promoter. Alternatively, the AF2 site is adjacent to a
PPAR-binding domain that maps at 451 to
353) has been implicated in the action of both insulin and
glucocorticoids on PEPCK-C gene transcription. To determine the role of
AF2 in these processes, we have generated a mouse model bearing a
transgene that contains the PEPCK-C gene promoter with a mutation in
the AF2-binding region. This promoter is linked to the structural gene
for human growth hormone that is biologically inactive (AF2-2000/hGx). In the absence of the AF2 regulatory element, the transcription of the
transgene in the liver is not induced by diabetes but is inhibited by
the administration of insulin. There is also a marked reduction in the
response of the AF2-2000/hGx gene in the kidney to the administration
of glucocorticoids. The AF2-2000/hGx gene in the liver responds
normally to a high carbohydrate diet with a marked decrease in gene
transcription. This suggests that insulin is not exerting its usual
negative effect on the PEPCK-C gene promoter through the AF2 site. In
contrast, the response of this transgene to a high
fat/carbohydrate-free diet is severely blunted. Our results support a
role for the AF2 site in the PEPCK-C gene promoter in the effect of
glucocorticoids, but not insulin, on PEPCK-C gene transcription in the liver.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
90 to
83, and an up-stream regulatory element termed P3(I), which
spans bases
250 to
234 in the promoter (14). The regulation of
PEPCK-C gene expression by glucocorticoids is accomplished through a
glucocorticoid regulatory unit (GRU) in the promoter located between
451 and
353 (15). The GRU includes a linear array of three
accessory factor-binding sites, AF1 (
451 to
439), AF2 (
416 to
407), and AF3 (
326 to
321), and two glucocorticoid receptor-binding sites, GR1 and GR2 (
386 to
353) (15). The GRU can
bind a variety of transcription factors. The AF1 site binds the chicken
ovalbumin up-stream promoter transcription factor and hepatic nuclear
factor 4 (HNF-4). Both the CCAAT enhancer-binding protein (C/EBP)
family members C/EBP
and C/EBP
and HNF-3 bind to the AF2
site, but it has been shown that HNF-3 mediates PEPCK-C gene
transcription (16). The AF2-binding site also contains an overlapping
glucocorticoid response element (GRE) and an insulin regulatory
sequence (IRS), which regulate the glucocorticoid and insulin
responses. A third accessory site has been characterized by Scott
et al. (15) and shown to bind chicken ovalbumin upstream promoter transcription factor and up-stream stimulatory factor. A
single mutation in any one of these three AF elements results in a 60%
decrease of the glucocorticoid response in hepatocytes, whereas a
mutation in any two AF elements completely eliminates the
glucocorticoid response of the PEPCK-C gene promoter mediated by the
GRU (15). As suggested by the overlapping GRE and IRS within the AF2
element, the AF1, AF2, and AF3 are also required for other hormone
responses. Both the AF1 (17) and AF3 contain GRE and retinoic acid
response elements (15) and are necessary for the response of the
PEPCK-C gene promoter to retinoic acid. The AF2 element contains an IRS
(18) that has been proposed to mediate part of the negative response of
the gene to insulin (19).
2000 to +73) linked
to the human growth hormone structural gene (hGx), which lacks a
receptor-binding site and thus is biologically inactive. We have
analyzed the transcriptional response of the transgene to diabetes,
insulin replacement, administration of glucocorticoids, and alterations
in dietary protein and carbohydrate. The results suggest that the AF2
element of the GRU is necessary for induction of PEPCK-C mRNA by
glucocorticoids during diabetes and that the effect of insulin on this
site is modest at best.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-32P]dATP (3000 Ci/mmol) were purchased from
PerkinElmer Life Sciences. Restriction enzymes and proteinase K were
purchased from Roche Molecular Biochemicals. The synthetic diets (20)
used in these studies were purchased from Nutritional Biochemical Corp.
(Cleveland, OH). Human recombinant insulin was obtained from Calbiochem.
32P]dATP and a Strip-EZ DNA random-primed StripAble
DNA probe synthesis and removal kit from Ambion (Austin, Texas).
490 to +72) or (
2000 to +72) using a variation of
the method of Kunkel (23) as described by Liu et al. (24).
The AF2 mutation replaced the entire sequence of the AF2 element (
414
to
405) from the wild type sequence of 5'-ACCGATATCA-3' to a
nonspecific sequence of 5'-TGGTGTTTTG-3'. The mutated AF2 or the WT
promoter fragment was used to construct the transgene for AF2 or WT
control lines, respectively. Briefly, either the
2000-base pair
SacI-BglII or the
490
XbaI-BglII region of the cytosolic form of the
PEPCK-C gene promoter from the rat was ligated to a 2.2-kilobase
BamHI-EcoRV fragment containing the human growth
hormone (hGx) reporter gene. The hGx reporter gene has a frameshift
mutation in exon 5 that affects the 55 carboxyl-terminal amino acids
and thus does not produce active growth hormone (25). Fig. 1 presents a
diagram of the genes used in this study and the location of the mutated
AF2 element.
70 °C.
70 °C. For dietary studies, the mice were fed either a high carbohydrate diet that contained 81.5% glucose, 12.2% casein, 0.3%
DL-methionine, 4% cottonseed oil, 2% brewers' yeast, and
1% mineral mix with vitamins for 1 week or a high
fat/carbohydrate-free diet that contained 64% casein, 22% nutritive
fiber, 11% vegetable oil, 2% brewers' yeast, and 1% mineral mix
plus vitamins. (27). As controls, mice were fed normal laboratory chow
ad libitum. The animals were anesthetized, and the liver and
kidneys were removed, frozen in liquid nitrogen, and stored at
70 °C for subsequent analysis.
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
2000 or
490 region of the PEPCK-C gene promoter that
was ligated to the hGx structural gene (Fig.
1). These chimeric genes were used to
generate transgenic animals. Heterozygous, transgenic males were then
mated to C57BL/6× SJL non-transgenic females. Each litter consisted of
non-transgenic controls and heterozygous transgenic littermates. The
livers and kidneys of 19-day-old fetuses were collected from each line,
and the level of hepatic and kidney hGx mRNA transcribed from the
2000 and
490 segments of the PEPCK-C gene promoter, with and without the AF2 regulatory element, were measured by Northern hybridization (Fig. 2). Chimeric genes
containing the WT-2000, AF2-2000, and WT-490 promoters expressed very
little hGx mRNA in the liver and kidney before birth. The
AF2-490/hGx gene, however, had an unexpected high level of expression
before birth in both the kidney and liver compared with WT (*,
p < 0.05). This result demonstrates the importance of
the AF2 element in the PEPCK-C gene promoter for the correct
developmental expression of this gene before birth. The gene for
PEPCK-C is not expressed in the liver before birth (30). The high
levels of glucose from the maternal circulation repress expression of
the gene in the fetal liver and the subsequent production of insulin by
the fetal pancreas. At birth, there is a decrease in the concentration
of glucose in the blood, accompanied by a decrease in secretion of
insulin and an increase in hepatic cAMP (31); this initiates
transcription of the gene for PEPCK-C in the liver. In the absence of
the AF2 site, we noted a premature appearance of hGx mRNA in the
livers of 19-day-old fetal mice. This occurred only when the AF2
mutation was in the PEPCK-C gene promoter from
490 to +73, suggesting that a region of the promoter between
490 and
2000 contains an
element required to suppress the premature expression of PEPCK-C in the
fetal liver. More work will be required to identify this region of the
PEPCK-C gene promoter.
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Fig. 1.
The chimeric genes used for the generation of
the transgenic mice. A, the WT-2000/hGx chimeric gene
was made by ligating the 2000-base pair
SacI-BglII fragment of the PEPCK-C gene promoter
to the inactive human growth hormone (hGx) and the 5' end of the bovine
growth hormone gene containing a splice acceptor site and the poly(A)
tract (bGH). The AF2-2000/hGx chimeric gene contains a
block mutation introduced into the AF2 region of the PEPCK-C gene
promoter between 414 and
405, as described under "Experimental
Procedures." B, the WT-490/hGx chimeric gene was made by
ligating the 490 XBAI-BglII fragment of the PEPCK-C gene
promoter to the inactive human growth hormone (hGx) structural gene
described for A. The AF2-490 gene promoter contains a block
mutation from
414 to
405. The entire GRU is shown in the
bracket; the relative positions of AF1, AF2, and the
GR-binding sites GR1 and GR2 are presented.
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Fig. 2.
The effect of a mutation in the AF2 region of
the PEPCK-C gene promoter on transcription from the promoter in the
fetal liver. Male mice heterozygous for the transgene were
mated with non-transgenic C457BL/6× SJL females. On day 19 of
gestation, the mice were delivered, and their livers and kidneys were
removed for mRNA and analysis by Northern blotting. A,
hepatic expression of WT-490/hGx, AF2-490/hGx, WT-2000/hGx, and
AF2-2000/hGx is presented as the relative ratios of hGx mRNA
normalized to 18 S rRNA. B, renal expression of WT-490/hGx,
AF2-490/hGx, WT-2000/hGx, and AF2-2000/hGx is presented as the ratio
of hGx mRNA to 18 S rRNA. The values are expressed as the mean ± the S.E. for 5 to 7 mice (*p < 0.05 relative to WT
controls).
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Fig. 3.
The effect of a mutation in the AF2 region of
the PEPCK-C gene promoter on transcription from the promoter during
diabetes and after the administration of insulin. WT-2000/hGx or
AF2-2000/hGx transgenic mice (8-12 weeks of age) were untreated
(controls), made diabetic with streptozotocin, or made diabetic
(D) and given insulin for 2 or 8 h. Livers were
collected, and mRNA was extracted and analyzed by Northern
blotting. A, the level of hGx mRNA was measured and
normalized to 18 S rRNA for the WT-2000/hGx and AF2-2000/hGx mice.
B, the concentration of endogenous PEPCK-C mRNA from
livers of the same animals was measured by Northern blotting and
normalized to 18 S rRNA. The values are expressed as the mean ± S.E. for 5 to 7 mice. A statistical comparison of the results is shown;
*, p < 0.05 difference in the control values for hGx
or PEPCK-C mRNAs as compared with the diabetic values; **,
p < 0.05 difference in the diabetic values for hGx or
PEPCK-C mRNAs as compared with values noted 8 h after insulin
administration.
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Fig. 4.
The effect of a mutation in the AF2 region of
the PEPCK-C gene promoter on the glucocorticoid stimulation of
transcription from the promoter. WT-2000/hGx or AF2-2000/hGx
transgenic mice (8-12 weeks of age) were either untreated (controls)
or given an injection of 1 µM dexamethasone
(DEX). 5 h later the livers were collected, and
mRNA was extracted and analyzed by Northern blotting. A,
the level of hepatic hGx mRNA was normalized to 18 S rRNA for the
WT-2000/hGx and AF2-2000/hGx mice. B, the level of hGx
mRNA in the kidneys was normalized to renal 18 S rRNA in the
WT-2000/hGx and AF2-2000/hGx mice. The values are presented relative
to wild type control mice and expressed as the mean ± S.E. for 3 to 5 mice (*, p < 0.05 relative to WT controls).
and HNF-4; both can then act as accessory
factors for the glucocorticoid response. In animals with a deletion in
the gene for C/EBP
, the glucocorticoid response is totally
blocked.2 C/EBP
binds to the PEPCK-C gene promoter through the cAMP-response element-1
and P3(I) regions, both of which are downstream from the AF2-binding
site and GRU. Yamada et al. (33) have determined that
C/EBP
is an accessory factor for the glucocorticoid response by
acting via the cAMP-response element region in the PEPCK-C gene
promoter. This suggests a complex mechanism for the regulation of
PEPCK-C by glucocorticoids. Recently Leahy et al. (34) have stressed the role of CREB-binding protein in coordinating the regulation of the PEPCK-C promoter. CREB-binding protein can bind both
steroids and C/EBP
, which interact with the promoter. It is likely
that the AF2 region interacts with the GR or with a protein that binds
the GR. Here we note that the lack of an AF2 site results in a 60%
reduction in the increase in hGx mRNA when stimulated with
glucocorticoids. Thus, the absence of a binding site for AF2 may
disrupt the ability to form the needed complex with CREB-binding
protein and C/EBP
.
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Fig. 5.
The effect of a mutation in the AF2 region of
the PEPCK-C gene promoter on its transcriptional response to
alterations in the carbohydrate content of the diet. WT-2000/hGx
or AF2-2000/hGx transgenic mice (8-12 weeks) were fed for 1 week a
normal laboratory chow (control), a high carbohydrate diet (High
CHO), high fat/carbohydrate-free diet (CHO free), or
high carbohydrate diet and given an intraperitoneal injection of
dibutyryl cAMP and theophylline (high CHO + cAMP). Their
livers were excised, and mRNA was extracted and analyzed by
Northern blotting. The hGx mRNA was normalized to 18 S rRNA, and
the values were expressed as the mean ± S.E. for 3-5 mice. A
statistical difference was observed at the 0.05 probability level. *, control mice as compared with mice fed a
high carbohydrate diet; **, control mice as compared with mice fed a
high fat/carbohydrate-free diet; ***, mice fed a high carbohydrate diet
as compared with mice fed the high carbohydrate diet and injected with
cAMP.
451 to
439 (36). A diet high in
fat increases the concentration of PPAR
in the liver (37).
Therefore, in the absence of the AF2 site, PPAR
might be less
effective in inducing PEPCK-C gene transcription. Clearly, further work
is needed to establish the exact effect of a high fat/carbohydrate-free
diet on the control of PEPCK-C gene transcription.
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ACKNOWLEDGEMENTS |
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We are indebted to Dr. Ifeanyi J. Arinze for critically reading the manuscript and to Jianqi Yang for help in preparing the figures.
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FOOTNOTES |
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* This work was supported in part by Grant DK-22451 from the National Institutes of Health, and P. S. L. was supported in part by Metabolism Training Program DK-07319 from the National Institutes of Health.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. Tel.: 216-368-3634; Fax: 216-368-4544; E-mail: rwh@po.cwru.edu.
Published, JBC Papers in Press, April 17, 2001, DOI 10.1074/jbc.M102422200
2 C. M. Croniger and R. W. Hanson, unpublished results.
3 K. Chakravarty, P. Ferre, F. Foufelle, and R. W. Hanson, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: PEPCK-C, cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32); GRU, glucocorticoid regulatory unit; AF, accessory factor; GR, glucocorticoid receptor; HNF, hepatic nuclear factor; C/EBP, CCAAT enhancer-binding protein; GRE, glucocorticoid response element; IRS, insulin regulatory sequence; WT, wild type; CREB, cAMP-response element-binding protein; SREBP-1c, sterol regulatory element-binding protein 1c.
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