From the Departments of § Nutrition and
¶ Biochemistry, Case Western Reserve University School of
Medicine, Cleveland, Ohio 44106-4935, the Department of
Biochemistry and Molecular Biology, University of Salamanca School of
Medicine, Salamanca E-37007, Spain, and the ** Department of
Biochemistry, University of Dundee, Dundee, Scotland, United
Kingdom
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ABSTRACT |
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CCAAT/enhancer-binding protein
(C/EBP) The CCAAT/enhancer-binding protein
(C/EBP)1 family includes
nuclear transcription factors C/EBP c/ebp In normal mice and cells, C/EBP Friedman et al. (43) showed that glucocorticoids are
essential for increased PEPCK gene transcription during diabetes.
Indeed, removal of the pituitary gland (44) or adrenalectomy (45) lessens or reverses many of the metabolic abnormalities of diabetic animals, including reducing blood glucose and PEPCK gene expression. At
the molecular level, a deletion of the glucocorticoid response modular
unit (GRU) of the PEPCK promoter prevents the increase in reporter gene
transcription in STZ-diabetic transgenic mice (43). The diabetic
response is also blocked in animals with the glucocorticoid receptor
antagonist RU-486 (46). Glucocorticoid-stimulated PEPCK gene
transcription is suggested to involve a cooperation of the GR with
factors binding to accessory factor sites AF-1, AF-2, and AF-3, and
through interaction of factors binding to the CRE (36). C/EBP isoforms
bind to three major sites on the PEPCK gene promoter, the CRE, the P3I
site, and the AF-2 element (26-27, 36-37, 47-50, Fig. 3). Because
C/EBP Experimental Animals--
Mice used in this study were obtained
by cross-breeding female mice heterozygous for a null mutation of the
c/ebp Analytical Procedures--
Blood was taken from the tails
in the morning, centrifuged, and plasma separated and frozen. Plasma
concentrations of non-esterified fatty acids and glucose were measured
with diagnostic reagent kits from Wako and Sigma, respectively. Insulin
and corticosterone levels in plasma were determined using
radioimmunoassay kits from Linco Research (St. Charles, MO) and ICN
Pharmaceuticals (Costa Mesa, CA). Glycogen was extracted from frozen
livers by homogenization in 6% perchloric acid, precipitated in
ethanol, hydrolyzed by boiling in 1 N HCl, and glucose
measured as for plasmas (51). Statistical comparisons between groups
were made using Student's t test.
Measurement of Gluconeogenesis Rate--
The rate of
gluconeogenesis was estimated in vivo in mice deprived of
food 4 h (noon) prior to receiving intraperitoneal injection of
2H2O (0.4% of body weight), and drinking water
was supplemented to 0.4% in 2H2O. Four hours
later (8 p.m.), mice were anesthetized by an intraperitoneal injection
of a solution containing ketamine HCl (65 mg/kg), acepromazine maleate
(2 mg/kg), and xylazine HCl (13 mg/kg) (Henry Schein, Port Washington,
NY) and blood collected from the abdominal aorta. Blood samples were
centrifuged at 13,000 rpm at 4 °C for 30 min, the plasma separated,
snap-frozen in liquid nitrogen, and stored at Hepatic Glucose Production Analysis--
Mice were fasted
overnight before injecting 5 µCi of
D-[3-3H]glucose (NEN Life Science Products)
in 100 µl of saline via tail vein. Blood samples (25 µl) for
glucose and radioactivity determinations were obtained at 5, 15, and 30 min from the tip of the tail. Serum was obtained after centrifugation
at 5,000 × g for 5 min, and glucose levels were
determined using the glucose oxidase method (Sigma). For radioactivity
determinations, 10 µl of blood was deproteinized with 200 µl of
20% trichloroacetic acid. Samples were centrifuged at 5,000 × g for 5 min, and the supernatants were evaporated to dryness
overnight at 65 °C under a hood. The residue was reconstituted in
200 µl of water, 5 ml of scintillation fluid was added, and the
samples were counted in a Liver Nuclear Protein--
Liver nuclear extracts were prepared
using the combined livers of 2-4 mice by a slight modification of the
method of Gorski et al. (27, 56). Freshly excised livers
were minced, homogenized, nuclei pelleted and lysed, and ammonium
sulfate-precipitated nuclear proteins recovered by centrifugation.
After dialysis, nuclear proteins were quantitated by Bradford (27),
aliquoted, and snap-frozen in liquid nitrogen. All buffers used
contained a mixture of proteases and phosphatase inhibitors with the
following final concentrations: 1 mg/ml antipain, chymostatin,
pepstatin, and leupeptin; 10 mg/ml aprotinin; 2.5 mM
benzamidine; 20 mg/ml trypsin inhibitor; 0.1 mM PMSF; 10 mM sodium fluoride, sodium orthophosphate, and sodium vanadate; and 5 nM Microcystin LR.
Gel Supershifting--
Double-stranded oligodeoxynucleotides
containing sequences of the PEPCK promoter: CRE ( RNA Extraction and Northern Blot Analysis--
Total RNA was
extracted from mouse liver using the guanidine thiocyanate procedure as
described previously (46). Solutions were made in diethyl
pyrocarbonate-treated water and materials were rinsed in RNase-off
solution (CPG Inc.). RNA was purified through gradient centrifugation
in cesium chloride, resuspended, and the concentration determined with
reference to absorbance at 260 nm (A260/280 for
purity). 20 µg of total RNA were placed in 37% deionized formamide,
0.66 M formaldehyde gel loading solution and
size-fractionated by electrophoresis through a 1.4% agarose, 0.66 M formaldehyde gel in 1× Mops buffer. RNA was transferred overnight to a GeneScreen Plus membrane (NEN Life Science Products) and
cross-linked by vacuum-baking at 80 °C. Prehybridization was done at
65 °C, for 3 h, in Church buffer. Probes used were a
nick-translated 32P-labeled cDNA (106
dpm × µg Western Immunoblot Analysis--
Purified nuclei from 2-3 mouse
livers were resuspended in a lysis buffer, sonicated, and protein
quantitated by Bradford (27). Nuclear proteins were precipitated with
10% trichloroacetic acid, resuspended by sonication in 2× SDS-Laemmli
sample buffer, and electrophoresed in a 12% polyacrylamide (35:1
acrylamide:bisacrylamide) SDS gel along with molecular weight standards
(Life Technologies, Inc.). Liquid electroblotting transfer to a
polyvinylidene difluoride membrane (Millipore) was accomplished after
2.5 h at 200 V according to manufacturer's instructions
(Bio-Rad). Transcription factors were detected by primary antisera
(1:1,000) (anti-C/EBP Metabolic Profile of Adult c/ebp
The lower fasting glucose in c/ebp Gluconeogenesis and PEPCK Gene Expression Are Lower in
c/ebp
The contribution of in vivo gluconeogenesis to hyperglycemia
was estimated by administering 2H2O to control
and diabetic mice and measuring incorporation of the stable isotope
into plasma 2H-C-6-glucose, as described under "Materials
and Methods." Plasma glucose derived via gluconeogenesis (fractional
percent of endogenous glucose production) in
c/ebp
Failure to increase gluconeogenesis in the
c/ebp C/EBP
We tested whether nuclear protein binding to the PEPCK promoter sites
was modified in the liver by diabetes. Electrophoretic mobility shift
assays were performed as detailed under "Materials and Methods."
Quantification of bands from different gels of samples in duplicates
from three independent experiments was performed and presented in Fig.
7, and representative gel autorads are presented in Figs.
4-6.
Liver nuclear proteins bound to the CRE, P3I, and AF-2 oligonucleotides
with similar protein to DNA concentration ratios, and the banding
profiles obtained agreed with previously reported by others and us for
control mice (Figs. 4 and 5) (26, 27, 49, 50). Multiple bands shown
correspond to homo- and heterodimers of the C/EBP isoforms (40 and 32 kDa for
The absence of C/EBP
In summary, C/EBP Our current understanding of the transcriptional regulation
of the PEPCK gene promoter has derived from mutational analysis in vitro and in vivo using transgenic mice. Such
procedures give clues as to the potential regulatory proteins involved
in the control of gene expression. However, such results may be far
from the native chromosomal and whole body physiological contexts. The
liver-specific hormonal control of the PEPCK gene relies on a network
of transcription factors responsive to various extracellular signals
and integrated by coactivators. The necessity of each transcription
factor in an intact physiological setting can only be discriminated by
knocking out each family member. The knockout of the gene encoding
C/EBP An increase in plasma glucagon concentration is the primary trigger of
glycogenolysis and gluconeogenesis during overnight fasting (30).
Recent results from our laboratory have demonstrated that, under
glucagon or epinephrine stimulation, second messenger cAMP is
diminished in the liver and adipose tissue from
c/ebp Following STZ-diabetes, hyperglycemia results from a decrease in
insulin combined with a rise in glucocorticoids and glucagon. These
hormones synergistically stimulate increased PEPCK gene transcription
and gluconeogenesis while reducing insulin-dependent glucose utilization. C/EBP The main gene regulatory mechanisms for response to diabetes are
governed by glucocorticoids (43, 46). In vitro, C/EBP The phenomena that c/ebp and C/EBP
are members of the c/ebp gene
family and are highly expressed in mammalian liver and adipose tissue.
C/EBP
is essential for adipogenesis and neonatal gluconeogenesis, as
shown by the C/EBP
knockout mouse. C/EBP
binds to several
sequences of the phosphoenolpyruvate carboxykinase (PEPCK) gene
promoter with high affinity, and C/EBP
protein is increased 200% in
the livers of streptozotocin-diabetic mice, concurrent with increased
PEPCK mRNA. To elucidate the role of C/EBP
in the control of
gluconeogenesis during diabetes, we studied the levels of plasma
metabolites and hormones related to energy metabolism during diabetes
in adult mice heterozygous and homozygous for a null mutation of the
gene for C/EBP
. We also examined the expression of PEPCK and glucose
6-phosphatase mRNAs and regulation of blood glucose, including the
contribution of gluconeogenesis to blood glucose in
c/ebp
/
mice. C/EBP
was not essential
to basal PEPCK mRNA levels. However, C/EBP
deletion affected
streptozotocin-diabetic response by: (a) delaying
hyperglycemia, (b) preventing the increase of plasma free
fatty acids, (c) limiting the full induction of PEPCK and glucose 6-phosphatase genes, and (d) preventing the
increase in gluconeogenesis rate. Gel supershifts of transcription
factor C/EBP
, bound to CRE, P3I, and AF-2 sites of the PEPCK
promoter, was not increased in diabetic
c/ebp
/
mouse liver nuclei, suggesting
that C/EBP
does not substitute for C/EBP
in the diabetic response
of liver gene transcription. These results link C/EBP
to the
metabolic and gene regulatory responses to diabetes and implicate
C/EBP
as an essential factor underlying
glucocorticoid-dependent activation of PEPCK gene
transcription in the intact animal.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-,
-,
-,
-, and
D-binding protein, encoded by intronless genes located on different
chromosomes (1). The C/EBPs consist of homologous C-terminal basic DNA binding and leucine zipper dimerization domains, and less homologous N-terminal activation and attenuation domains (1). The enrichment of
C/EBPs in liver and adipose tissue suggested their physiological role
could be in the control of expression of genes for energy metabolism
(2-4). In adipose cells, C/EBP
and C/EBP
participate in the
differentiation of pre-adipocytes, including the transcription of
fat-specific genes (5-7). They also bind and transactivate a variety
of genes encoding key metabolic enzymes in the liver, including (but
not limited to) phosphoenolpyruvate carboxykinase (PEPCK) and tyrosine
aminotransferase (8-11), fatty acid synthesis enzyme acetyl-CoA
carboxylase (12), and the albumin gene (13). Additionally, C/EBP
participates in the induction of cytokines (14-16) and liver acute
phase response genes (17).
knockout mice die shortly after birth of profound
neonatal hypoglycemia (18-21). The knockout of c/ebp
results in a lethal phenotype following birth in a subset of the
homozygous offspring (15, 21-22). Surviving adult
c/ebp
/
mice display impaired macrophage
activation and reduced induction of hepatic genes encoding acute-phase
response proteins (15-16). c/ebp
/
females are infertile (15, 23). The epididymal fat pads and mammary
glands demonstrated impaired differentiation (22, 24), but no overt
disruption of glucose homeostasis was reported (15, 22). However, a
more detailed analysis revealed that
c/ebp
/
adult mice fail to regulate blood
glucose during fasting and in response to glucagon stimulation,
apparently due to lower cAMP levels (25).
expression, like gluconeogenesis, is
stimulated by cAMP (26, 27) and glucocorticoids (28), and
down-regulated by insulin (29). During streptozotocin (STZ)-diabetes,
liver C/EBP
mRNA is increased 3-fold while C/EBP
is decreased
(29). This change is reverted by insulin treatment in diabetic animals,
suggesting that glucagon, glucocorticoids, and possibly insulin action
may be expressed in part through C/EBP
. The hyperglycemia of
diabetes results from impaired insulin-dependent glucose
utilization and increased hepatic glucose output, via glycogenolysis,
and increased gluconeogenesis (30). The rate-limiting enzyme for
gluconeogenesis, PEPCK (EC 4.1.1.32), is controlled exclusively at the
transcription level in response to hormones and metabolites (31-32).
Glucagon and catecholamines (via cAMP) and glucocorticoids are positive
regulators, while insulin, in the presence of glucocorticoids, is a
negative signal (for review, see Ref. 31). PEPCK gene transcription is
regulated by several composite modular units in the promoter/enhancer
region, comprising two or more cis-acting DNA elements
(33-36). Mutational analysis of the sites in vitro and
reporter gene expression driven from mutated promoter constructs in
transgenic mice have identified the sites for tissue-specific and
hormonally regulated transcriptional response (for review, see Ref.
37). The transacting factors that bind to these sequences include CREB,
AP1, C/EBPs, HNFs, TR, RAR/RXR, and glucocorticoid receptor (GR) (see
Fig. 3 and Refs. 36 and 37). Many of the genes encoding these
transcription factors have been knocked out. HNF-4
and HNF-3
knockouts are embryonic lethal (38-40), and GR knockout mice die soon
after birth (41), while HNF-1
knockout mice die within 1 month from
birth (42).
has been found related to the main signals controlling PEPCK
gene expression, we decided to test whether C/EBP
inactivation
in vivo would provide integrative data for understanding
glucose homeostasis during diabetes. Our analysis suggests that
homozygous c/ebp
/
mice display decreased
blood lipids and impaired gluconeogenesis in response to diabetes. Our
results indicate there is a selective increase in C/EBP
protein in
liver nuclei, which binds with greater affinity to DNA sequences within
the PEPCK GRU during STZ-diabetes. In mice with a c/ebp
deletion, however, the normal induction of PEPCK and Glc-6-Pase during
STZ-diabetes is reduced, gluconeogenesis is decreased, and blood
glucose is significantly lower. The impairment in gluconeogenesis in
c/ebp
/
mice during diabetes suggests this
transcription factor is an essential mediator of glucocorticoid
signaling in the physiological context of the intact mouse.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
gene with homozygous male
c/ebp
/
mice. The generation and mixed
genetic background as well as the methods used for genotyping have been
described previously by Screpanti et al. (15). Adult male
and female c/ebp
/
,
c/ebp
+/
, and
c/ebp
+/+ mice were studied at 10-14 weeks of
age. Mice were housed in microisolater cages and were maintained on a
fixed 12-h light/dark cycle at Case Western Reserve University animal
facility. Animals had free access to water and were fed regular animal
chow (Harlan Teklad, Madison, WI) ad libitum. For
experimental diabetes, food was removed overnight, mice received a
single streptozotocin intraperitoneal injection (0.2 mg/g STZ in 0.05 M citrate, pH 5.0), and food was returned 4 h later.
Development of hyperglycemia (> 250 mg%, One Touch II blood glucose
meter; Lifescan Inc.) was considered as diabetic state. Animals were
sacrificed 3-4 days after STZ treatment. Excised livers were used
immediately for nuclear protein extraction or frozen in liquid nitrogen
and kept at
80 °C.
80 °C until
analyzed. Enrichment of 2H at carbon 6 of glucose was
assayed by mass spectrometry as hexamethylenetetramine (HMT) formed
from formaldehyde by periodate oxidation of the plasma glucose C-6
(52). Briefly, 200 µl of plasma was deproteinized, the supernatant
passed through columns AG1-X8 in the formate form and AG 50W-X8 in the
H+ form, and a neutral effluent evaporated to dryness.
Glucose content of the effluent was quantified in an automatic analyzer
(53). Formaldehyde obtained after periodate oxidation of glucose C-6 was treated with NH4OH to form HMT. The residue after
evaporation was taken up in methylene chloride. HMT formed was injected
into the gas chromatograph-mass spectrometer (HP5985; Hewlett Packard, Palo Alto, CA) without further derivatization. The samples were run
with standards of HMT of known deuterium enrichments (0.125-2.0 range). The deuterium enrichment of the samples was calculated in mole
percent excess from a linear regression equation of the standard curve.
The percent contribution of gluconeogenesis was calculated by comparing
the 2H enrichment of glucose with that of body water, as
measured in mouse urine by isotopic exchange with
[U-13C3]acetone (54).
-scintillation counter. The rate of
hepatic glucose production (HGP) was calculated using steady-state
equations (55). The gluconeogenesis rate during overnight fasting was
obtained by multiplying the fraction of plasma glucose derived via
gluconeogenesis times HGP. Statistical comparisons between groups were
made using Student's t test.
94/
77),
5'-CCCCTTACGTCAGAGGCTCTAG-3' (underlined
consensus CRE); P3I (
249/-232),
5'-CTAGACGTTGTGTAAGGACTCA-3' (underlined C/EBP
site homologous sequence); and AF-2 (
420/-406), 5'-GCGGCTGTGGTGTTTTGAAAC-3'(underlined consensus
IRS) were synthesized using an Applied Biosystems 380A DNA synthesizer, and gel-purified before use (CWRU Core Laboratory). The annealed double-stranded oligomers were labeled by filling-in the overhanging ends with the Klenow fragment of DNA polymerase and
[
-32P]dCTP, gel-purified, and used at 20,000 cpm/ml in
the reaction mixture for nuclear protein binding assay. Binding
reactions were carried out as described (27). Briefly, 20 µl of
mixture consisted of 3-14 fmol of labeled probe (60,000 cpm), 10 mM Tris (pH 7.9), 5 mM MgCl2, 1 mM dithiothreitol, 1 mM EDTA, 50 mM
NaCl, 10% glycerol, 50 µg of bovine serum albumin, and 1 µg of
poly(dI-dC) as non-specific competitor. Following 15 min at room
temperature, either antisera against C/EBP
, C/EBP
, CREB, p-CREB,
or preimmune serum were added. Binding reactions continued for another
10 min before electrophoresis. Protein-DNA complexes and free probe
were resolved on a 20 × 20-cm2 4% acrylamide (55:1
acrylamide:bisacrylamide) gel in 0.5× Tris borate/EDTA at 100 V for
2 h. The gel was dried and exposed to Kodak X-ARO5 film. The
specific band intensities were quantitated by optical densitometry
using a Digiscan scanner (U.S. Biochemical Corp.) and the
autoradiographic signals integrated (27). The means from these
experiments, including total binding and relative amount of C/EBP
and C/EBP
binding to each separate oligonucleotide, on the same
autoradiography were calculated as relative percentage of the control
wild-type or C/EBP
/
signals integrated in arbitrary
units. The values are means of samples in duplicates of three
experiments. S.E. were 10-15% of the average values.
1) for PEPCK (1.1-kilobase pair fragment;
kind gift of Dr. R. W. Hanson) and glucose 6-phosphatase
(1.25-kilobase pair fragment; kindly provided by Dr. D. Massillon).
After hybridization overnight at 65 °C, the filter was washed
extensively in 2× sodium chloride/sodium citrate (SSC)/0.1% SDS at
room temperature, and exposed to Kodak BioMax autoradiographic film at
80 °C. For re-probing, the blots were stripped at 80 °C in
0.1× SSC, 0.1% SDS during 15 min or until no counts. The specific
band intensities were quantitated by optical densitometry using a
Digiscan scanner (U. S. Biochemical Corp.) and the autoradiographic
signals integrated (27). The relative levels of mRNA were expressed
as a percentage of mRNA hybridization in liver from wild-type
control mice detected on the same Northern blot after correction for
ribosomal RNA (28 S) to account for loading differences.
and anti-C/EBP
, Santa Cruz Biotechnology,
Santa Cruz, CA; anti-CREB and anti-p-CREB, from Dr. D. Ginty, Harvard
Medical School), followed by goat anti-rabbit horseradish peroxidase
conjugate (1:5,000) (Amersham Pharmacia Biotech). Detection was done
with ECL detection system (Amersham Pharmacia Biotech) as per
manufacturer's instructions and membranes exposed to Kodak X-ARO5
film. The specific band intensities were quantitated by optical
densitometry using a Digiscan scanner (U.S. Biochemical Corp.) and the
autoradiographic signals integrated (27). The relative levels of
transcription factors were the means of three or four experiments as a
percentage of the arbitrary densitometric units of wild-type or mutant
control mice liver detected on the same blot.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
Mice--
The
involvement of C/EBP
in liver gene expression has been suggested by
previous studies in adult liver and hepatoma cell lines. In order to
determine the potential role of C/EBP
on glucose homeostasis
in vivo, we studied an animal model lacking the
transcription factor. Morning plasma glucose from mice fed overnight
ad libitum was not significantly different in the
c/ebp
/
mice compared with the other
groups, indicating lack of a role of C/EBP
on euglycemia in the fed
state (Table I). However, when food was
withheld overnight (approximately 12 h), glucose levels decreased
in mutant homozygous mice by 31% compared with wild-type controls
(p < 0.05) (Table I). Corticosterone values were not
different among the three groups of mice (Table I). Plasma insulin in
the fed state was not significantly different between normal (0.89 ng/ml) and c/ebp
+/
mice (0.87 ng/ml), but
it was 47% lower (p < 0.05) in
c/ebp
/
mice. Plasma insulin was similar
in c/ebp
/
mice and wild-type mice during
fasting (0.32 ng/ml; Ref. 25). The corticosterone and insulin
concentrations agree with values reported for mice at the same time of
the day (57).
Metabolic characteristics of c/ebp+/+,
c/ebp
+/
, and c/ebp
/
mice
/
mice
suggested that either the rate of glucose uptake from the circulation
is greater or that a decrease in glucose production exists. HGP was
tested under steady-state conditions and calculated as mg/min × kg (as described under "Materials and Methods"). In agreement with
published levels, HGP in wild-type mice was 36.9 ± 4.98 mg/min × kg (58), while c/ebp
/
and
c/ebp
+/
mice showed a significantly lower
HGP of 47% and 21%, respectively (p < 0.01) (Table
I). Hepatic glycogen concentration in the fed state was 43% lower in
the c/ebp
/
mice compared with the wild
type (p < 0.05), suggesting a possible deficiency in
glycogen stores. However, the rate of gluconeogenesis (fraction of
endogenous glucose production) was 53% lower (p < 0.01) in c/ebp
/
mice (Table I),
indicating that decreased HGP was associated with a deficiency in gluconeogenesis.
/
Mice during Diabetes--
During
streptozotocin-induced diabetes, a lack of insulin and an increase in
glucagon and glucocorticoids cause a potent induction in liver
gluconeogenic enzymes and hyperglycemia. To test the contribution of
C/EBP
to liver gluconeogenesis during diabetes, mice were
treated with STZ and blood was taken from the tails everyday
thereafter. The third day following injection, both STZ-treated wild-type and c/ebp
+/
mice had reached
hyperglycemic values of 24.7 and 19.5 mM, respectively, which represents 2.7-fold in normal mice (8.47 mM basal)
and 2-fold in mutant c/ebp
+/
(9.41 mM) (p < 0.01). Glucose levels in
c/ebp
/
mice were also increased from 8.47 to 21.2 mM, which represented a 2.5-fold increase from
mutant mice glucose basal levels (p < 0.01) (Fig.
1). The difference between
STZ-c/ebp
/
and STZ-wild type mice blood
glucose was close to 15% (p < 0.05).
View larger version (14K):
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Fig. 1.
Metabolic response to STZ-diabetes in
c/ebp -deficient
mice. A and B, plasma glucose and free fatty
acids were measured in plasmas of mice mutated in none, one, or two
alleles of the c/ebp
gene. Mice received 0.2 mg/g STZ for
diabetes and were sacrificed 3 days later. C, plasma glucose
derived via gluconeogenesis (fractional percentage of endogenous
glucose production) in +/+, +/
, and
/
mice was measured with
2H2O. Enrichment of 2H in C-6 of
glucose was assayed in plasmas by mass spectrometry as described under
"Materials and Methods." Results are means ± S.E. from 6-12
animals per group. *, significantly reduced compared with +/+ controls,
p < 0.05. **, significantly reduced compared with +/+
controls, p < 0.01.
+/+, c/ebpB+/
,
and c/ebpB
/
mice shows that gluconeogenesis
was 10% lower in c/ebp
/
compared with
wild-type mice (NS) (Fig. 1). During diabetes, gluconeogenesis did not
change significantly in c/ebp
/
mice
(62.9% control versus 62.6% diabetes) but it did increase significantly by 20% in wild-type and mutant heterozygous mice (p < 0.05). These results support that the
contribution of gluconeogenesis (via pyruvate) is over 80% of the
total plasma glucose in wild-type mice, but remains constant at
approximately 60% in the c/ebp
/
mice.
/
mice during diabetes could result
from a failure to fully induce enzymatic machinery and/or from a
decrease in availability of substrates and energy. Lipolysis from the
adipose tissue is impaired in c/ebp
/
mice
(25), and transcription factor C/EBP
transactivates genes involved
in lipid metabolism. When non-esterified plasma fatty acids were
measured during diabetes, a 113% decrease in
c/ebp
/
mice was found compared with the
wild-type mice (Fig. 1). The expression of the key gluconeogenic genes
PEPCK and glucose 6-phosphatase in diabetic mice was studied by
Northern blot analysis. RNA was extracted from livers of control and
STZ-treated c/ebp
/
,
c/ebp
+/
, and wild-type mice after 3-4 days
of STZ injection. RNA was electrophoresed, blotted and probed with
cDNAs encoding the enzymes as detailed under "Materials and
Methods." A representative blot is shown in Fig.
2. Quantification of signals in bands
from different blots of samples in duplicates from three or more
independent experiments are shown as diagram in Fig. 2. PEPCK mRNA
signal intensity was similar in wild-type and
c/ebp
/
mice before STZ. However,
following STZ, PEPCK mRNA was increased in normal mice livers,
while the increase was significantly less by 35% in mutant mice
(p < 0.01; Fig. 2). Changes in glucose 6-phosphatase mRNA levels paralleled those of PEPCK (p < 0.01;
Fig. 2).
View larger version (41K):
[in a new window]
Fig. 2.
Autoradiograph of Northern blot reflecting
changes in levels of hepatic PEPCK and Glc-6-Pase mRNAs in
normoglycemic and diabetic c/ebp +/+ and
c/ebp
/
mice. Total cellular RNA was
isolated from livers of mice under fasting or diabetic conditions and
PEPCK, Glc-6-Pase, and ribosomal RNA detected as described under
"Materials and Methods." A representative hybridization from a
Northern blot of six different mice is shown. The relative levels of
mRNA were determined by densitometry and expressed as a percentage
of mRNA hybridization in liver from +/+ control mice detected on
the same Northern blot after correction for ribosomal RNA (28 S) to
account for loading differences. Results are means ± S.E. of 4-6
animals per group. *, significantly reduced compared with +/+ control
p < 0.05.
Contributes to Nuclear Protein Binding to DNA Elements
That Regulate the PEPCK Promoter Activity during Diabetes--
The
lower PEPCK mRNA in the STZ-diabetic mutant mice led us to
investigate the effect of diabetes and lack of C/EBP
on the PEPCK
promoter activity. The sequences up to
500 base pairs of the PEPCK
gene are sufficient to carry out the control of its expression by
glucocorticoids, cAMP, and insulin. In vitro, C/EBP
and
C/EBP
have been shown to interact with CRE, P3I, and IRS sites of
the proximal PEPCK promoter (Fig. 3).
Glucocorticoids signal through the composite element GRU containing two
different glucocorticoid receptor binding elements together with sites
for auxiliary factors, termed AF-1, AF-2, and AF-3 (Fig. 3). All of the
sites in the GRU are necessary for maximal response to glucocorticoids (8, 35-36, 50) and to mediate synergism between factors binding at the
CRE and GRU (8, 36, 59).
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Fig. 3.
Schematic representation of the PEPCK GRU
located between 455 and
349 base pairs upstream from the
transcription start site. The PEPCK GRU consists of three
accessory factor binding sites (AF-1, AF-2, and AF-3) necessary for the
full response to glucocorticoids (8, 36, 49, 50), and two adjacent
glucocorticoid receptor binding sites, GR1 and GR2. The AF-2 site
contains an IRS (
416 to
407), which mediates a negative effect of
insulin on PEPCK gene transcription (49). The CRE site is more than 200 base pairs from the GRU and can cooperate with the GRU for
glucocorticoid responsiveness of the PEPCK gene (59). The sequences
that bind C/EBPs are indicated by
in the AF-2, P3I, and CRE
elements (26, 27, 49, 50).
, and 38, 34, and 20 kDa for
), together with other
possible factors cross-dimerizing with C/EBPs or independently binding
to the sites. As shown in Fig. 7,
diabetes increased total binding of wild-type mice liver nuclear
extracts by 150%, 170% and 150% to CRE, P3I, and AF-2 sites,
respectively, which could be partially supershifted by antibodies
specific to C/EBP
but not to C/EBP
. Indeed, binding activity of
C/EBP
increased by diabetes 125%, 181%, and 171% over control to
CRE, P3, and AF-2, respectively (Fig. 7B). Since C/EBP
mRNA had been reportedly induced in rat liver by diabetes and
repressed by insulin (29), the increase in the C/EBP
binding to DNA
is likely result of translation of a message increased by diabetes
rather than a posttranslational event. Western blot analysis was
performed with three different liver nuclear extracts obtained from the
pooled livers of 2-3 mice of each condition and phenotype, and probed
for C/EBP
and C/EBP
. The results in Fig. 4 demonstrated that
indeed, the increase in DNA binding with diabetes in wild-type mice was
paralleled by a 187% enrichment in C/EBP
protein. Scanning of
silver-stained liver nuclear proteins, separated in SDS-PAGE, did not
show differences in protein banding profile, or relative area of peaks,
between control and diabetic state (data not shown), suggesting that
the enrichment of transcription factor C/EBP
in liver nuclei with
diabetes was specific. Western blot analysis in Fig. 5 shows that
C/EBP
nuclear protein is 25% decreased, in agreement with others
results showing a down-regulation of the protein with diabetes (60).
Accordingly, binding activity of C/EBP
decreased 17% to CRE and P3
and 40% to AF-2 site with diabetes (Fig. 7C). Neither
binding activity of transcription factor CREB nor its Ser-133
phosphorylated form was detected in nuclear extracts from wild-type
mice, independent of the diabetic or normal state. These results agree
with previous results reported by Granner and colleagues (49). However,
Western blots showed that CREB and p-CREB were present in the liver
nuclei, although no changes in either form appeared with diabetes
(results not shown). Hence, C/EBP
was the major transcription factor
responsible for the increase in DNA binding to the PEPCK promoter and,
presumably, in transactivation of the PEPCK gene during diabetes in the
liver in vivo.
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Fig. 4.
Increased C/EBP
binding in nuclear proteins from wild-type diabetic mice to sites
from the PEPCK promoter. Mice received 0.2 mg/g streptozotocin for
diabetes and were sacrificed 3 days later. Liver nuclear extracts were
prepared from control and diabetic mice as outlined under "Materials
and Methods." Mobility shift assays were performed using
-32P-labeled oligonucleotides containing sequences CRE
(
94 to
77) and P3 I (
249 to
232) from the PEPCK promoter. The
complexes were supershifted using anti-C/EBP
antiserum.
Lower panel, 10-40 µg of liver nuclear protein
from diabetic and control mice were electrophoresed, blotted and probed
with anti-C/EBP
antiserum. The major band corresponded to 38-kDa
isoform as compared with molecular weight standards.
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Fig. 5.
No effect of diabetes on
C/EBP binding in nuclear proteins from
wild-type diabetic mice to sites from the PEPCK promoter. Liver
nuclear proteins were prepared from wild-type control and diabetic mice
as outlined in the legend to Fig. 4. Mobility shift assays were
performed with
-32P-labeled oligonucleotides containing
sequences CRE (
94 to
77) and P3 I (
249 to
232) from the PEPCK
promoter and supershifted using specific anti-C/EBP
antiserum.
Lower panel, 20-40 µg of liver nuclear protein
from diabetic and control mice were electrophoresed, blotted, and
probed with anti-C/EBP
antiserum. The major band corresponded to
42-kDa isoform as compared with molecular weight standards.
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Fig. 6.
Binding of liver nuclear proteins from
diabetic c/ebp /
mutant
mice to sites from the PEPCK promoter. Mice were rendered diabetic
and sacrificed 3 days later and nuclear extracts prepared as described
under "Materials and Methods." Binding to
32P-labeled oligonucleotides of sequences CRE (
94 to
77) and AF-2 (
420 to
406) from the PEPCK promoter were
supershifted by specific binding to anti-C/EBP
, anti-C/EBP
,
anti-CREB, and anti-p-CREB specific antibodies. Lower
panel, 20-40 µg of liver nuclear protein from diabetic
and control mice were electrophoresed, blotted, and probed with
anti-C/EBP
antiserum. The major band corresponded to 42-kDa isoform
as compared with molecular weight standards.
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Fig. 7.
Diabetes-induced increase in
C/EBP protein-DNA binding is reduced in
c/ebp
/
mice and is partially substituted
for by C/EBP
. A, total binding
to DNA sites was calculated relative to control wild-type or
c/ebpP
/
mice after integration of the
scanned autoradiographs (represented in Figs. 4-6). B, DNA
binding due to C/EBP
is increased by diabetes in wild-type mice
liver nuclear extracts. Expressed relative percentage of total binding
in A. C, DNA binding due to C/EBP
expressed
relative to total binding in A. The means from these
experiments include total binding and relative amount of C/EBP
and
C/EBP
binding in each separate oligonucleotide are presented. The
values are means of samples in duplicates of three experiments. S.E.
were 10-15% of the average values.
, however, did not totally prevent PEPCK
expression or hyperglycemia after STZ-diabetes in the mice (Figs. 1 and
2). Next we studied whether STZ-diabetes had changed the nuclear
protein binding to the PEPCK promoter sites in
c/ebp
/
mice. As shown in Fig. 6, liver
nuclear proteins from control and STZ-treated
c/ebp
/
mice bound to CRE and AF-2 sites
of the PEPCK promoter. The number of DNA-protein complexes decreased
from 6, in wild-type mice (Figs. 4 and 5), to 2 in
c/ebp
/
mice (Fig. 6), but it was not
changed by STZ-diabetes in any mice group. Whether the reduction of
bands was due to the absence of C/EBP
and/or of other proteins that
necessitate this factor for DNA binding required further analysis.
Total binding of diabetic nuclear extracts from mutant mice to the P3,
CRE, and AF-2 sites was significantly reduced, by 32%, 38%, and 20%,
respectively (Fig. 7A), as compared with extracts from
livers of wild-type mice (170%, 150%, and 150%; p < 0.01) (Fig. 7). The increase in total binding did not appear to change
the number of bands, but an increase in one of the two major complexes
was detected (Fig. 6). The increased binding was supershifted by
anti-C/EBP
antibody. In the mutant mice, binding of liver nuclear
proteins to C/EBP
increased with diabetes to 130% (P3) and 131%
(CRE), but it did not change for AF-2 (105%) (Fig. 7C).
Antibody specific to CREB supershifted a band only in mutant mice
extracts (Fig. 6). Together, antibodies to C/EBP
and CREB
supershifted almost all of the protein-DNA complexes, and in both
control and diabetic extracts from c/ebp
/
mice (Fig. 6). The anti-p-CREB antibody used failed to supershift any
bands on the sites tested (only AF-2 shown; Fig. 6), despite the
addition of inhibitors of protein phosphatases during the nuclear
protein preparation (see "Materials and Methods").
is increased with diabetes in the liver, it can
bind to three DNA sites that stimulate PEPCK gene transcription, and
can only be partially substituted for by C/EBP
, and probably also
CREB, to contribute to the induction of genes involved in the hepatic
diabetic response, such as PEPCK and glucose 6-phosphatase (Fig.
2).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
resulted in a lethal failure to synthesize glycogen and to
induce PEPCK and Glc-6-Pase mRNA at the time of birth (18). The
knockout of c/ebp
displayed a phenotype A, lethal for the
same cause as the c/ebp
knockout, and a phenotype B,
which survives by partial genetic complementation with no overt disruption of glycemia (21, 25). However, adult
c/ebp
/
mice (B phenotype) had a 50%
reduction in hepatic glucose production after an overnight fast, which
caused hypoglycemia, demonstrating that C/EBP
is essential for
glucose homeostasis (Table I).
/
mice (25). In addition to having
lower initial hepatic glycogen levels, liver glycogenolysis was
impaired, consistent with reduced hepatic cAMP concentration (25). We
have demonstrated here that C/EBP
is essential for increasing
gluconeogenesis during fasting and diabetes, despite the observation
that PEPCK and Glc-6-Pase mRNA levels are similar after an
overnight fast in wild-type and c/ebp
/
mice. Thus, both glycogenolysis, which is activated through
phosphorylation by cAMP-regulated protein kinase A, and
gluconeogenesis, controlled in part by cAMP at the level of gene
expression, appear to be compromised. C/EBP
has also been shown to
repress insulin gene expression in cultured cell lines (61). However,
c/ebp
/
mice present a compromised immune
response (15), but do not show increased insulin levels. In contrast,
c/ebp
/
mice demonstrated lower insulin
levels and hypoglycemia that is only evident after overnight fasting.
We also found that C/EBP
is required for the normal increase in
plasma non-esterified fatty acids (FFA) during diabetes and fasting.
Gluconeogenesis is stimulated in part by increased oxidation of FFA and
subsequent activation of pyruvate carboxylase by acetyl-CoA. The
decreased plasma FFA levels are likely caused by decreased rate of FFA
release from adipose tissue (25). Consequently, a significant decrease
in FFA and glycerol from adipose tissue in
c/ebp
/
mice may also contribute to lower
gluconeogenesis. The observation that C/EBP
deficiency impairs the
rise in plasma FFA and gluconeogenesis demonstrates that C/EBP
is
required for cAMP-mediated responses in both liver and adipose cells.
The putative target genes that respond to C/EBP
and increase cAMP
levels are currently under investigation in our laboratory.
gene expression is positively regulated by glucocorticoids and by cAMP, suggesting increased C/EBP
could mediate the synergistic effects of cAMP and glucocorticoids on PEPCK
gene transcription. C/EBP
binds in vitro to the AF-2,
CRE, and P3I sites in the PEPCK promoter responsible for glucocorticoid and cAMP activation (Figs. 4-6). During STZ-diabetes, C/EBP
, and not C/EBP
, increases its binding to the composite glucocorticoid response unit of the PEPCK promoter (Figs. 4 and 5). In the
c/ebp
/
mice, C/EBP
only partially
substitutes for C/EBP
binding to the AF-2 or CRE sites in the PEPCK
promoter (Figs. 6 and 7), and does not mediate an increase in
gluconeogenesis (Fig. 1). The presence of C/EBP binding sites in the
C/EBP
promoter (62) suggests that induction of C/EBP
expression
may depend on C/EBP
. Furthermore, the increase in total binding to
AF-2, CRE, and P3 sites from the PEPCK promoter by liver nuclear
proteins during diabetes is significantly reduced in the absence of
C/EBP
. This suggests that no other transcription factors of the same
or different family can substitute for C/EBP
to fully stimulate
PEPCK transcription during diabetes. However, the HNF-3 family members
can also bind to AF-2 site in vitro (8, 50), and HNF-3
and HNF-3
are able to interact in vitro with the
glucocorticoid receptor, thus being potential accessory factor-2 (36)
for the full response of the PEPCK promoter to glucocorticoids. The
knockout of hepatic nuclear factors HNF-3
and HNF-4
are both
embryonic lethal (38-40). However, in HNF-3
knockout mice, a
50-70% reduction in basal mRNA levels for gluconeogenic enzymes
PEPCK and tyrosine aminotransferase occurred, despite an up-regulation
of the expression of HNF-3
and HNF-3
(63). Thus, HNF-3
could
be an accessory factor in the physiological context. The fact that, in
the absence of C/EBP
, PEPCK gene expression and gluconeogenesis are
impaired during diabetes is reasonable indirect evidence that HNF-3
does not substitute for C/EBP
in the glucocorticoid-mediated
diabetic response of the PEPCK promoter. Natural mutations affecting
genes encoding HNF-4
and HNF-1
have been found in humans with
maturity onset diabetes of youth type 1 and 2, respectively (64), which result in a phenotype mainly affecting insulin secretion. Whether the
rate of gluconeogenesis and/or transcription of gluconeogenic enzymes
are also affected in these patients remains to be explored.
gene expression is increased by glucocorticoids (28). C/EBP
has
displayed interaction, physically and functionally, with glucocorticoid receptor to synergistically mediate expression of genes involved in
liver glucocorticoid responsiveness (65, 66). C/EBP
, but not
C/EBP
or C/EBP
, has recently been shown to specifically interact
with the glucocorticoid receptor (67, 68). However, neither
dimerization nor DNA binding of the glucocorticoid receptor is required
for the glucocorticoid response of the PEPCK promoter (68, 69). A
putative coactivator protein, CBP/p300 binds functionally to the
N-terminal sequences of C/EBP
(and not C/EBP
or -
) (70), in a
manner that allows it to regulate transcription in response to
dexamethasone (66, 71). If the mechanisms for glucocorticoid mediated
PEPCK gene transcription involve interactions between CPB/p300 and
C/EBP
, this mechanism would be defective in C/EBP
knockout mice.
Indeed, we have found that induction of PEPCK mRNA in the
c/ebp
/
mice is less responsive to
administered
glucocorticoids.2 There is,
however, no defect in the cAMP induction of PEPCK gene transcription
when c/ebp
/
mice are treated with
Bt2-cAMP (25), indicating that C/EBP
, although critical
for the full response to hormones such as glucocorticoids, glucagon,
and epinephrine, is not required for the response to cAMP. Overall,
these results link C/EBP
to the metabolic and gene regulatory
responses to diabetes in liver and adipose tissue and indicate C/EBP
is required for glucocorticoid-dependent activation of PEPCK gene transcription.
knockout decreases
gluconeogenesis and reduces circulating lipid and glucose levels
reinforces the suggestion made by McKnight and colleagues in 1989 that
C/EBPs are at the center of integration of molecular control of
carbohydrate and lipid metabolism (4). Moreover, the fact that a single trans-acting factor can act globally to regulate both glucose and lipid
concentration in the whole animal suggests that C/EBP
could be a
novel therapeutic target for treating multiple metabolic disorders.
C/EBP
is essential for expression of genes involved in the normal
development of the ovaries and the mammary glands (23, 24), processes
that involve steroid hormone receptor-mediated gene expression. It is
temping to speculate that the unique activation domain of C/EBP
(1)
could provide interaction sites for multiple steroid hormone receptors.
Understanding the interaction between C/EBP
and glucocorticoid
receptors would potentially be of therapeutic interest in the design of
specific drugs based on abolition and/or selective increase of
interactions involved in metabolic processes such as inflammation,
diabetes, and obesity.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Satish Kalhan's laboratory for
performing the HMT analysis, and Dr. Henri Brunengraber and Dr.
Michelle Beylot for 2H2O analysis. We also
acknowledge Dr. W. Stanley's laboratory for plasma FFA determination.
We thank Sandra Ferguson for skillful care and genotyping of the
c/ebp/
mice.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grant DK-50272 (to J. E. F.), DK-25541 (to R. W. Hanson), and NATO Collaborative Research Grant 960189 (to J. E. F. and C. A.).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.
Trainee supported by Metabolism Training Program Grant
DK-07319 from the National Institutes of Health.
To whom correspondence should be addressed: Dept. of Nutrition,
Case Western Reserve University School of Medicine, 10900 Euclid Ave.,
Cleveland, OH 44106-4935. Tel.: 216-368-1616; Fax: 216-368-6644;
E-mail: jef8{at}po.cwru.edu.
2 C. Croniger, J. E. Friedman, and R.W. Hanson, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: C/EBP, CCAAT/enhancer-binding protein; PEPCK, phosphoenolpyruvate carboxykinase; STZ, streptozotocin; FFA, free fatty acid; Glc-6-Pase, glucose 6-phosphatase; GR, glucocorticoid receptor; HNF, hepatic nuclear factor; AP, activator protein; CREB, cAMP response element-binding protein; GRU, glucocorticoid response modular unit; Mops, 4-morpholinepropanesulfonic acid; IRS, insulin receptor sequence; CRE, cAMP response element; AF, accessory factor; HGP, hepatic glucose production; HMT, hexamethylenetetramine.
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REFERENCES |
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