From the Departments of Biochemistry and Nutrition, Case Western
Reserve University School of Medicine, Cleveland, Ohio 44106-4935, the § Department of Biochemistry, Meharry Medical College,
Nashville, Tennessee 37208, and the Department of Molecular
Biology, University of Dundee,
Dundee, DD1 4HN Scotland, United Kingdom
Received for publication, August 18, 2000, and in revised form, October 4, 2000
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ABSTRACT |
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Fifty percent of the mice homozygous
for a deletion in the gene for CCAAT/enhancer-binding protein The CCAAT/enhancer-binding protein
(C/EBP)1 family of
transcription factors has been implicated in the coordinated expression of genes involved in glucose homeostasis (1-5). For example the genes
for phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phophatase, glycogen synthase, and albumin are not expressed at birth in the livers
of mice that are homozygous for a deletion in the gene for C/EBP Another member of the C/EBP family of transcription factors, C/EBP In this study we further characterize the metabolism of C/EBP Materials--
Theophylline, dexamethasone, streptozotocin,
guanidine thiocyanate, and protease inhibitors for mammalian cell and
tissue extracts were purchased from Sigma. Anti-chicken IgG peroxidase was from Upstate Biotechnology (Lake Placid, NY). The following kits
were used in these studies: RETROscript® first strand
synthesis kit and the Stip-EZ® DNA probe synthesis kit
from Ambion Inc. (Austin, TX), the QuickPrep® Total RNA extraction kit
and the BIOTRAK® cAMP enzyme immunoassay system from Amersham
Pharmacia Biotech, and the RNeasy® Mini kit from Qiagen Inc.
(Valencia, CA). The insulin radioimmunoassay kit was from Linco
Research (St. Charles, MO); the cAMP radioimmunoassay kit was from
Amersham Life Sciences. Kits for the determination of blood ammonia and
[ Experimental Animals--
C/EBP Perinatal Studies--
Mice were delivered at day 19 of
gestation by cesarean section, and where indicated, they were injected
with 125 mg of Bt2cAMP/kg of body weight (12). All pups
were maintained in a humidicrib at 37 °C from birth to the
completion of the experiment (up to 4 h). When the pups of
phenotype B became lethargic due to a low blood glucose concentration,
they were killed together with their littermates. Blood was collected
by decapitation, blood glucose was measured using a Glucometer (Ames
Products, Indianapolis, IN), and insulin was measured by
radioimmunoassay. The liver was freeze-clamped, and total RNA was
isolated as described below, glycogen was measured (13), and cAMP was
determined using an enzyme immunoassay procedure. Glycogen was
extracted from frozen livers by homogenization in 6% KOH, precipitated
in ethanol, and hydrolyzed by boiling in 1 N HCl, and
glucose was measured using a Beckman glucose analyzer. Statistical
comparison between groups were performed using Student's t test.
Metabolic Measurements--
Adult C/EBP Systemic Glucose Production--
Mice were fasted overnight
before injecting into the tail vein 100 µl of 5 µCi of
D-[3-3H]glucose in 0.9% NaCl. Blood samples
(25 µl) were obtained via the tail vein at 5, 15, and 30 min, and
serum was used for the determination of glucose (glucose oxidase
method). For the determination of radioactivity, 10 µl of blood was
deproteinized with 200 µl of 20% trichloroacetic acid. Samples were
centrifuged, and the supernatants were evaporated to dryness overnight
at 65 °C. The residues were reconstituted in 200 µl of water, 5 ml
of scintillation fluid was added, and the samples were counted in a
RNA Extraction and Northern Blot Analysis--
Total RNA was
extracted from the liver and kidney of mice using a Quick Prep total
RNA kit (Amersham Pharmacia Biotech) by a modified acid-phenol
guanidine thiocyanate procedure that has been described in detail
previously (16). Northern blot analysis was carried out as described
previously (17) using 20 µg of total RNA. After electrophoresis of
the RNA, RNA in the gels was transferred to Gene Screen Plus® membrane
and hybridized with a probe. The probe for PEPCK was a 1.1-kilobase
PstI fragment from the 3'-end of the PEPCK cDNA that was
isolated as described previously (16). The probe for
glucose-6-phosphatase (Glc-6-P) mRNA was a fragment
(XbaI-PstI) from Glc-6-P cDNA (18). The C/EBP DNA Analysis--
DNA was isolated from the tail of mice by
lysis overnight at 55 °C in a buffer containing 50 mM
KCl, 10 mM Tris-HCl, pH 8.3, 2.5 mM
MgCl2, 0.1% gelatin, 0.45% Nonidet P-40, 0.45% Tween 20, and 24 µg of proteinase K. The DNA was digested with
EcoRI, and the resulting fragments were separated by
electrophoresis on 1% agarose gel, transferred to Gene Screen Plus®,
and hybridized to a cDNA probe for C/EBP Glucagon-stimulated cAMP Production in Liver in Vivo--
Fed
C/EBP Quantitative Reverse Transcription-PCR--
Quantitative
competitive reverse transcription-PCR was used to measure the relative
concentrations of phosphodiesterase mRNA in the livers of
C/EBP
After the PCR reaction, 8 µl of the reaction solution was subjected
to electrophoresis in a 1.6% agarose gel. The DNA bands were recorded
using an IS-500 digital imaging system (Alpha Innotech Corp. San
Leandro, CA), and the bands were quantitated with ImageQuant® software
(Molecular Dynamics, Sunnyvale, CA). The concentration of mRNA was
determined as the point where the internal control band is equal in
intensity to that of the test sample. To find that point, the ratios of
the intensities of test cDNA and internal control bands were
calculated and fitted to a regression curve using GraphPad Prism®
software (GraphPad Software, San Diego, CA).
Adenylyl Cyclase Activity--
Partially purified liver plasma
membranes were prepared from the livers of C/EBP Protein Isolation for Western Blotting--
Proteins were
isolated from the livers of C/EBP Electrophoresis and Western Blotting--
The N fraction as well
as the cytosolic and particulate fractions were sonicated for 20 s, and 20 µg of protein was diluted in 2× loading buffer containing
100 mmol/liter Tris-HCl, pH 6.8, 20% Cyclic AMP-dependent Protein Kinase Assay--
The
activity of protein kinase A (PKA) was measured using the Kemptide
assay (21) using cytosol and particulate fractions from the livers of
fasted C/EBP cAMP Degradation in Vitro--
Phosphodiesterase activity in
homogenates of livers from C/EBP Characteristics of Newborn C/EBP
The high levels of glycogen in the livers of animals of the B phenotype
suggested a defect in the ability of these mice to mobilize their
hepatic glycogen. C/EBP Metabolic Characteristics of C/EBP
The adult C/EBP Systemic Glucose Production--
Because C/EBP cAMP Metabolism in the C/EBP
The degradation of cAMP was tested by first administering the
phosphodiesterase (PDE) inhibitors theophylline (a non-selective inhibitor of PDE) or RO 20-1724 (a specific inhibitor of PDE 4) to fed
C/EBP
Because these data suggest an accelerated rate of degradation of cAMP
in the livers of C/EBP
Since cAMP levels can affect the activity of PKA, we next determined
the levels of both the regulatory (RI
A change in the ratio of regulatory to catalytic subunits of PKA has a
profound effect on the total activity of the enzyme at a given
concentration of cAMP. In Table II the
levels of RI The deletion of the gene for C/EBP One possible downstream target is PKA. We show that in C/EBP The observed alterations on the relative location of the isoforms of
the R subunit of PKA in the livers of C/EBP Alterations of the concentration of the regulatory subunits in
various fractions of the liver have profound implications in the
response of PKA to cAMP. For example, O'Brien et al. (30) demonstrate that dietary protein restriction or reduction of the caloric content of the diet resulted in a loss of RI Although the C/EBP The concentration of cAMP in tissues is regulated not only by adenylyl
cyclase but also by the activity of the various isoforms of PDE
isozymes (33). The PDE families of enzymes are comprised of multiple
isoforms within each family generated from alternative splicing of
their precursor RNA. For example, the PDE3 family consists of PDE3A and
PDE3B (34). PDE3A has been identified in smooth muscle, platelets, and
cardiac tissue, whereas PDE3B is most abundant in adipocytes and liver.
PDE4 is the largest member of the PDE families and is derived from at
least four different gene PDE4 products (35). However, there is little
information available concerning the tissue specificity of the members
in this family (36). Our data show a 25% increase in both mRNA levels for PDE3A and PDE3B and a 25% increase in PDE activity in the
livers of fasted C/EBP Abnormalities in the C/EBP The results of the present paper clearly demonstrate the far-reaching
metabolic consequences caused by the absence C/EBP (C/EBP
/
mice; B phenotype) die within 1 to 2 h after birth
of hypoglycemia. They do not mobilize their hepatic glycogen or induce
the cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK).
Administration of cAMP resulted in mobilization of glycogen, induction
of PEPCK mRNA, and a normal blood glucose; these mice survived beyond
2 h postpartum. Adult C/EBP
/
mice (A phenotype) also had
difficulty in maintaining blood glucose levels during starvation.
Fasting these mice for 16 or 30 h resulted in lower levels of
hepatic PEPCK mRNA, blood glucose,
-hydroxybutyrate, blood urea
nitrogen, and gluconeogenesis when compared with control mice. The
concentration of hepatic cAMP in these mice was 50% of controls, but
injection of theophylline, together with glucagon, resulted in a normal
cAMP levels. Agonists (glucagon, epinephrine, and isoproterenol) and
other effectors of activation of adenylyl cyclase were the same in
liver membranes isolated from C/EBP
/
mice and littermates. The
hepatic activity of cAMP-dependent protein kinase was 80%
of wild type mice. There was a 79% increase in the concentration of
RI
and 27% increase in RII
in the particulate fraction of the
livers of C/EBP
/
mice relative to wild type mice, with no change
in the catalytic subunit (C
). Thus, a 45% increase in hepatic cAMP
(relative to the wild type) would be required in C/EBP
/
mice to
activate protein kinase A by 50%. In addition, the total activity of
phosphodiesterase in the livers of C/EBP
/
mice, as well as the
concentration of mRNA for phosphodiesterase 3A (PDE3A) and PDE3B
was approximately 25% higher than in control animals, suggesting
accelerated degradation of cAMP. C/EBP
influences the regulation of
carbohydrate metabolism by altering the level of hepatic cAMP and the
activity of protein kinase A.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(C/EBP
/
mice) (1). This results in an absence of hepatic
glycogen and a delay in the initiation of gluconeogenesis. These
animals also have lower levels of plasma glucose than their wild type
counterparts and die within 2 h after birth (1-3).
,
also plays an important role in the regulation of glucose homeostasis
(2, 6-8). In mice with a deletion in the gene for this transcription
factor (C/EBP
/
mice), two distinct phenotypes have been observed
(phenotypes A and B) (2). Mice with phenotype A live to be adults but
have hypoglycemia as well as a lower than normal concentration of
plasma free fatty acids, triglycerides, and ketone bodies in the blood
after an overnight fast (4). These mice also have a compromised immune
system and an impaired ability to activate macrophages (9, 10). Newborn
C/EBP
/
mice with the phenotype B are unable to maintain normal
blood glucose levels and die within 2 h after birth (2).
/
mice during the perinatal period. Our goal is to identify the metabolic
defect(s) responsible for the death of C/EBP
/
mice (B phenotype)
and to determine the metabolic alterations that contribute to the
hypoglycemia noted in mice of the A phenotype that survive until adulthood.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-hydroxybutyrate were purchased from Sigma and for protein kinase A
were from Upstate Biotechnology (Lake Placid, NY). The SuperSignal®
chemiluminescent substrate kit was from Pierce. Antibodies against
RI
were from Biomol (Plymouth Meeting, PA), against RII
were from
Santa Cruz Biotechnology (Santa Cruz, CA), and against C
were from
Upstate Biotechnology (Lake Placid, NY). Imobolin-P® polyvinylidene
difluoride membranes were purchased from Millipore Corp (Bedford, MA).
-32P]dATP (3000 Ci/mmol),
[14C]NaHCO3 (1 Ci/mmol),
[3-3H]glucose (10-20 Ci/mmol), [3H]cAMP,
[
-32P]ATP and GeneScreen Plus® were purchased from
NEN. RNase-free DNase I and SuperTaq DNA polymerase were
purchased from Ambion Inc. (Austin, TX). NADH, Bt2cAMP,
cesium chloride, and anti-rabbit IgG peroxidase were purchased from
Roche Molecular Biochemicals, and RO 20-1724 was from Biomol.
/
mice were obtained for
this study by breeding female heterozygous animals with a targeted
deletion in the gene for C/EBP
with heterozygous male mice. The
generation of the C/EBP
/
mice and their genetic background have
been described by Screpanti et al. (9). Briefly, ES cell
clones from the CCE cell line (derived from the 129/Sv/Ev strain)
carrying the mutation were injected into C57Bl6 blastocysts and were
transplanted into the uteri of F1 (CBA×C57Bl6) foster mothers. Male
chimeras were mated to MF1 females, and offspring heterozygous for the
mutant allele were intercrossed to obtain homozygous mice. Adult male
and female mice were 8-12 weeks old at the time of their use.
Screening for C/EBP
/
mice was carried out by Southern analysis
as described previously (9). The animals were given free access to
water and standard chow (Tekland F6 8664 containing 24% protein, 6% fat, and 4.5% crude fiber and the remainder carbohydrate). The composition of the high carbohydrate diet used in this study was described previously (11). The mice were killed between 9 and 11 a.m., and where indicated, they were injected with Bt2cAMP (35 mg/kg of body weight) and theophylline (30 mg/kg of body weight).
/
mice (A
phenotype) were given 5 mg of glucose/kg of body weight orally by
gavage and then fasted for 16 and 30 h. Blood was taken from the
tails of mice at 30 min, and after 16 and 30 h of fasting,
and plasma was separated and frozen. Blood glucose was measured using
glucose oxidase method (Glucose Trender Kit, Sigma), whereas the
concentration of
-hydroxybutyrate was determined using an
enzymatic kit (Sigma). The blood urea nitrogen (BUN)
levels were measured on a Beckman automated analyzer at MetroHealth, Schwartz Nutrition Center, Cleveland, Ohio. Ammonia levels
were measured on blood plasma after an overnight fast by an enzymatic
kit (Sigma) based on reductive amination using L-glutamate dehydrogenase (14). Insulin was determined using a radioimmunoassay.
-scintillation spectrometer. The rate of systemic glucose production
was calculated using steady-state equations. (15) Statistical
comparison between groups was made using Student's t test.
cDNA probe was a 0.7-kilobase BamHI fragment of
the mouse cDNA (9). The concentration of 18 S rRNA was determined
by Northern blotting using a 752-nucleotide SacI fragment of
a cDNA made from mouse 18 S rRNA. The signal from this
hybridization was used to standardize the concentration of RNA on the
Northern blots. All probes were labeled by using
[
32P]dATP and the Strip-EZ RNA & DNA probe synthesis
and removal kit (Ambion Inc., Austin, TX).
.
/
mice and control littermates (WT) (8-12 weeks of age)
were anesthetized with avertin, the liver was clamped, and a biopsy was
taken for the measurement of basal cAMP. The mice were then injected
with glucagon (50 µg/kg of body weight) via the portal vein, and the
liver was biopsied 1 min later for the assay of glucagon-induced cAMP.
C/EBP
/
and wild type mice were given an intraperitoneal
injection of either theophylline (30 mg/kg of body weight) or RO
20-1724 (15 mg/kg of body weight). Thirty min later, the mice were
anesthetized with avertin, the liver was clamped to prevent bleeding,
and a liver biopsy was obtained. This was used for the basal
concentration of cAMP. Glucagon (50 µg/kg of body weight) was
injected into the portal vein, and one min later, another piece of
liver was removed for the quantitation of cAMP. The liver samples were
quickly frozen and assayed for cAMP using an enzyme immunoassay
(Amersham Pharmacia Biotech).
/
and control littermates. This procedure involves four
steps: first, total RNA was isolated from the liver; second, reverse
transcription was performed to create cDNAs; third, competitive PCR
was preformed; and fourth, the DNA bands were quantitated. The RNA was
isolated from the livers of mice using the Amersham Pharmacia Biotech
QuickPrep® total RNA extraction kit. Trace amount of genomic DNA was
removed from the sample by treatment with 10 units of RNase-free DNase
I, and the RNA was further purified by using a Qiagen RNeasy Mini
Kit®. Reverse transcription was performed at 42 °C for 2 h
using an Ambion RETROscript kit®. Briefly, 2 µg of total RNA was
reverse-transcribed in a reaction mixture containing 0.5 mM
dNTP, 5 µM random primer, 1× buffer (50 mM Tris-HCl, pH 8.3, 75 mM KCl, 30 mM MgCl2, and 50 mM
dithiothreitol), 10 units of RNase inhibitor, and 100 units of reverse
transcriptase. The competitive PCR reaction was carried out in a volume
of 25 µl that contained 2 µl of the solution from the
reverse-transcribed reaction with varied concentrations of an internal
DNA control fragment as well as 0.2 mM dNTP mix, 1× PCR
buffer (20 mM Tris-HCl, pH 8.4, and 50 mM KCl),
3 mM MgCl2, 0.8 µM mixed primer,
and 0.5 units of SuperTaq DNA polymerase. The competitive PCR reaction was performed at 94 °C for 1 min, at 55 °C for 1 min, and at
72 °C for 1 min for 30 cycles. For each RNA sample, one reverse
transcriptase reaction and eight PCR reactions were performed. The
internal DNA control fragments were constructed as follows. The
internal control for PDE3A was obtained from a 482-bp segment of the
mouse PDE3A by deleting a 178-bp StyI fragment. The internal
control for PDE3B was generated by deleting a 124-bp SacII
segment of the 682-bp XhoI-StuI cDNA
fragment, and for the PDE4B internal control, the fragment was obtained
by deleting a 152-bp NsiI fragment from the 1968-bp
EcoRI cDNA.
and control mice as
described previously (19), with slight modifications. Briefly, the
livers were homogenized (Dounce homogenizer) in 5 ml of Buffer A (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 100 µM phenylmethylsulfonyl fluoride, 3 mM
benzamidine, and 1 µM leupeptin) containing 0.25 M sucrose. The homogenates were centrifuged at 17,000 × g for 10 min, and the pellet was suspended in 4 ml of the
homogenization buffer. This suspension was layered onto 42.3% (w/w)
sucrose in Buffer A and centrifuged at 30,000 rpm for 90 min in a SW40
rotor. The interfacial material was collected and washed twice with
Buffer A by centrifuging at 86,000 × g for 45 min. The
final pellet was re-suspended in Buffer A and stored at
80 °C
until used. The yield of membrane protein, as determined by the method
of Lowry (20), was about 15 mg of protein/g of liver; there was no
difference in the yield of liver membranes of C/EBP
and control
mice. Adenylyl cyclase activity in the membrane preparations was
assayed for 10 min at 30 °C using 300 µM
[
-32P]ATP as a substrate, as described previously
(19).
/
and wild type mice that were
fasted overnight. A piece of frozen liver was homogenized in 15 ml/g of
tissue of ice-cold homogenizing buffer consisting of 20 mM
Tris, pH 7.6, 0.1 mM EDTA, 0.5 mM EGTA, 0.1%
Triton-X, 250 mM sucrose, and 50 µl/5 ml protease
inhibitor mixture. Homogenates were centrifuged at 19,500 × g for 30 min at 4 °C to separate cellular debris,
mitochondria, nuclei, and plasma membranes. This pellet was
re-suspended in 4 ml of homogenizing buffer (N fraction). The
supernatant was then centrifuged at 100,000 × g for 30 min at 4 °C. The resulting supernatant (the cytosolic fraction) was
removed, and the remaining pellet (particulate fraction) was
re-suspended in 4 ml of homogenizing buffer. The particulate fraction
contained membranes of the endoplasmic reticulum and the Golgi complex.
The concentration of protein was measured with the Bio-Rad protein
assay using bovine serum albumin as a standard.
-mercaptomethanol, 4% SDS,
0.2% bromphenol blue, 20% glycerol and separated by 10% SDS-PAGE.
Proteins were electrophoretically transferred to Immobilon-P®
polyvinylidene difluoride membranes and stained with Coomassie stain
(45% methanol, 10% acetic acid, 2.5% Coomassie Blue R250) to ensure
even loading. For detection of the subunits of protein kinase A, RI
,
RII
, and C
, polyvinylidene difluoride membranes were incubated in
blocking buffer containing 5% nonfat dried milk in 10 mmol/liter Tris, pH 7.4, 150 mmol/liter NaCl, and Tween 20 (TBS-T) for
1 h at room temperature. Membranes were then incubated with
primary antibody diluted in blocking buffer for 1 h as follows:
RI
(1:250), RII
(1:1000), C
(1:250), washed 3 times for 5 min
each in TBS-T. The membranes were then incubated with secondary
antibody diluted in blocking buffer for 1 h as follows: rabbit
anti-chicken IgG peroxidase (1:1000) for RI
and anti-rabbit IgG
peroxidase (1:1000) for RII
and C
. Membranes were washed 3 times
for 5 min each in TBS-T. All incubations were at room temperature.
Immunoreactive proteins were detected using the SuperSignal
Chemiluminescent Substrate® kit, and the density of the immunoreactive
bands was measured by scanning densitometry. Western blots were first
reacted with RI
, washed as described by Pierce, and reacted again
with RII
and then with C
.
/
and wild type mice. Livers were homogenized in
ice-cold Kemptide homogenizing buffer (1 g of liver/9 ml of buffer).
The buffer contained 20 mM Tris, pH 7.6, 0.1 mM
EDTA, 0.5 mM EGTA, 0.1% Triton-X, and 250 mM
sucrose and 50 µl/5 ml protease inhibitor mixture. The homogenate was centrifuged at 19,500 × g for 30 min at 4 °C. The
cytosolic fraction was used for the measurement of PKA activity.
Samples were diluted 1:1 with the Kemptide homogenization buffer, and
the phosphorylation of the Kemptide substrate was measured using the
PKA assay kit with [
-32P]-ATP. Duplicate samples were
assayed at 30 °C at 30 s intervals for 5 min for three
C/EBP
/
and three wild type mice. The amount of labeled Kemptide
was determined using a Beckman LS scintillation spectrometer. Activity
in the presence of protein kinase A inhibitor peptide was subtracted
from total activity to account for nonspecific activity.
/
and control mice was
determined by the procedure of Shahid and Nicholson (22). About
0.2 g of liver was homogenized in 2 ml of Buffer A containing 0.25 M sucrose using a glass homogenizer fitted with a
motor-driven Teflon pestle. The homogenates were filtered though two
layers of cheese cloth and used immediately for the assay of
phosphodiesterase activity. Approximately 25 µg of protein equivalent
of the homogenate was incubated with 1 µM
[3H]cAMP for 5 min at 30 °C. The reaction was
terminated by placing the samples into boiling water; the unhydrolyzed
[3H]cAMP was separated by using alumina columns. In other
assays in which the rate of cAMP degradation was also measured in the homogenate, 50 µg of protein was incubated with 1 µM
[3H]cAMP at 30 °C, and the labeled cyclic nucleotide
that was not hydrolyzed was measured at various time intervals up to 6 min by separation on alumina columns as described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
Mice Exhibiting the A and
B Phenotypes--
Mice heterozygous for a deletion in the gene for
C/EBP
were mated, and the pups were delivered by cesarean section on
days 19 to 20 of gestation. The newborn mice were stimulated by touch after birth and maintained at 37 °C in a humidicrib.
C/EBP
/
mice identified as being of the B phenotype were
typically lethargic, had difficulty breathing, and remained cyanotic
until they died within 2 h after delivery. The C/EBP
/
pups
that were viable and breathing well were classified as the phenotype A. The plasma glucose levels of the B phenotype were about 50% that of
control pups (wild type) (p < 0.01) (Fig.
1). The C/EBP
/
mice (B phenotype) also had difficulty in mobilizing hepatic glycogen compared with wild
type littermates, as was evident from the higher level
(p < 0.03) of hepatic glycogen in these animals (70 mg/g of liver glycogen in C/EBP
/
versus 40 mg/g of
liver glycogen in the wild type mice). Because maintenance of normal
blood glucose in the neonate depends on the capacity to mobilize
hepatic glycogen as well as to initiate hepatic gluconeogenesis, we
measured the ability of these animals to initiate gluconeogenesis by
measuring the level of mRNA for PEPCK, the last of the
gluconeogenic enzymes to develop in newborn mammals (23). As shown in
Fig. 1, the level of PEPCK mRNA in C/EBP
/
mice (B phenotype)
was only 30% that of either the A phenotype or the wild type control
animals. PEPCK gene expression is down-regulated by insulin (24). We therefore measured the concentration of insulin in the plasma of
C/EBP
/
mice and control mice; the insulin levels were found to
be the same (data not shown).
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Fig. 1.
The characterization of
C/EBP /
mice of the A and B phenotypes
during the perinatal period. C/EBP
/
mice and control
littermates (WT) were delivered by cesarean section and maintained in a
humidicrib at 37 °C for 2 to 4 h. The mice were killed, blood
glucose was measured, and their genomic DNA was screened. The livers
were removed and quickly frozen for the determination of the
concentration of glycogen and PEPCK mRNA. The values for PEPCK
are presented as a ratio of the mRNAs for PEPCK relative to 18 S
rRNA and are expressed as the mean ± S.E. for three-five mice
(*, p < 0.0001;**, p < 0.03; ***,
p < 0.01).
/
mice at 19 to 20 days of fetal life
were delivered by cesarean section and given an intraperitoneal
injection of Bt2cAMP (125 mg/kg) immediately after delivery. These mice mobilized their hepatic glycogen to the same extent as control littermates (to a level of 35 mg/g of liver) (data
not shown). Surprisingly, mice with the B phenotype responded immediately to the Bt2cAMP by breathing normally and
becoming less lethargic. All of the C/EBP
/
mice injected with
Bt2cAMP survived for up to 4 h, the duration of the
experiment (Fig. 2). Since the
administration of Bt2cAMP rescued the C/EBP
/
mice (B
phenotype) from death within the first 2 h after birth, we considered it important to determine whether endogenous levels of cAMP
in the livers of B-phenotype mice were different from the normal
littermates. For this experiment, the animals were delivered at 19-20
days of fetal life. The newborn animals were treated as one group
representing a mixture of both A and B phenotypes. The C/EBP
/
mice had the same level of cAMP as control mice (data not shown),
suggesting that there is no defect upstream of cAMP production. The
biochemical basis for the decreased viability of C/EBP
/
mice of
the B phenotype remains enigmatic, but it seems likely that their
inability to respond to the normal concentration of cAMP may be due to
an alteration in downstream target(s) required to activate critical
metabolic processes during the perinatal period.
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Fig. 2.
The rate of survival of
C/EBP /
mice and control littermates during
the perinatal period in response to cAMP and measurement of cAMP.
C/EBP
/
mice (both A and B phenotypes) and control littermates
(WT) were delivered by cesarean, injected with 125 µg/kg of body
weight of Bt2cAMP, and maintained in a humidicrib at
37 °C for 4 h. The mice were then killed, and their genomic DNA
was screened. The number of mice that survived out of the total number
treated for each group are represented in parenthesis below each group
(wild type controls with no cAMP, C/EBP
/
mice without cAMP, and
C/EBP
/
injected with cAMP).
/
(phenotype A) and Wild
Type Mice--
Based on the blunted response of the C/EBP
/
mice
(B phenotype) to the endogenous concentrations of hepatic cAMP and the resulting aberrations in carbohydrate metabolism, we extended this
study to adult animals (the A phenotype) to determine the differences
in metabolic response from adult wild type control mice. To
simulate the fed state, adult mice were given glucose orally (5 g/kg),
and blood was taken from the tail vein 30 min later. The mice were then
fasted for 30 h, blood was collected from the tail vein at 16 and
30 h, and the concentrations of glucose,
-OH butyrate, and BUN
were determined. The concentration of glucose in the blood of the
C/EBP
/
mice was 25% lower than that of wild type control
animals, whereas the level
-hydroxybutyrate was 50% lower than the
wild type animals (Fig. 3). The lowered concentration of
-hydroxybutyrate probably reflects a decreased oxidation of fatty acids in the C/EBP
/
mice because these
animals exhibit lower fasting free fatty acids levels than wild type
counterparts (4).
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Fig. 3.
The effect of starvation on the concentration
of glucose and -hydroxybutyrate in the blood
plasma of C/EBP
/
and wild type mice.
C/EBP
/
mice and control littermates (WT) (8-12 weeks of age)
were fed 5 g of glucose by oral gavage and then starved for 16 and
30 h, at which time the concentration of glucose and
-hydroxybutyrate were determined. The values are expressed as the
mean ± S.E. of six mice for the blood glucose and four-five mice
for
-hydroxybutyrate.(*, p < 0.05; **,
p < 0.002).
/
mice resemble "sparse fur mice" (25) that
have a defect in the urea cycle enzyme, ornithine transcarbamylase, and
exhibit lower than normal levels of BUN, elevated blood ammonia, and
premature hair loss. For this reason, we also investigated alterations
in ammonia metabolism in the C/EBP
/
mice. In 16-h fasted
animals, the concentration of BUN was 40% lower in C/EBP
/
mice
compared with control littermates, suggesting derangement in ammonia
metabolism or urea production; however, this value returned to control
levels after 30 h of fasting (Fig.
4). To confirm a lower rate of flux
through the urea cycle, the concentration of ammonia was determined in
the plasma of C/EBP
/
mice that had been fasted for 16 h
(Fig. 4, inset). C/EBP
/
mice had blood ammonia levels
of 425 µg/dl as compared with 200 µg/dl in wild type mice. This
suggests that ammonia metabolism in the C/EBP
/
mice is
compromised, an abnormality that may contribute to the premature death
of these animals, as noted earlier by Screpanti et al.
(9).
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Fig. 4.
The effect of starvation on the content of
blood urea nitrogen and ammonia. C/EBP /
mice and control
littermates (WT) (8-12 weeks of age) were fed 5 g of glucose by
oral gavage and then starved for 16 and 30 h, at which time the
concentration of BUN was determined. At 16 h of fasting, the
concentration of plasma ammonia (shown as boxes) was measured as
described under "Experimental Procedures." The values are 453.50 ± 50 and 206.83 ± 30.58 for CEBP
/
and wild type, respectively,
and are expressed as the mean ± S.E. for three-five mice for BUN
and 6 mice for ammonia. (*, p < 0.05, **,
p < 0.01).
/
mice have
difficulty maintaining normal levels of blood glucose during fasting,
systemic glucose production was determined in vivo. In these
experiments [3H]glucose was injected into the tail vein
of conscious C/EBP
/
mice that had been fasted overnight, and the
rate of dilution of the [3H]glucose was then measured. As
shown in Fig. 5, glucose production in
the C/EBP
/
mice was half that of control littermates, suggesting a defect in gluconeogenesis. The levels of insulin were the same in
both the wild type and C/EBP
/
mice (data not shown). The levels
of mRNA for two gluconeogenic enzymes, PEPCK and Glc-6-P, were then
measured in the livers of C/EBP
/
mice and control littermates
fed a high carbohydrate diet for 1 week and then injected with
Bt2cAMP (Fig. 6). In livers
of normal animals, both hepatic PEPCK and Glc-6-P mRNA levels were
repressed by a high carbohydrate diet were induced by the
administration of Bt2cAMP. The inhibitory response of the
PEPCK gene to a high carbohydrate diet and normal induction by
Bt2cAMP was blunted in the livers of C/EBP
/
mice in
comparison to control animals. In addition, a high carbohydrate diet,
rather than repressing Glc-6-P mRNA as in control animals, induced
its levels in the livers of the C/EBP
/
mice. One possible explanation for this finding is that C/EBP
is involved in the repression of Glc-6-P gene transcription by carbohydrate but not in the
repression of PEPCK gene transcription. Although PEPCK and Glc-6-P
share a common set of regulatory signals, they respond in a different
manner to high concentrations of glucose (18). Our findings suggest
that C/EBP
is involved in controlling the response of the gene for
Glc-6-P to dietary carbohydrate.
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Fig. 5.
Systemic glucose production by
C/EBP /
mice and wild type mice after an
overnight fast. C/EBP
/
mice and control littermates (WT)
(8-12 weeks of age) were fasted overnight, and the rate of hepatic
glucose production and concentration of plasma glucose in the plasma
were determined as outlined under "Experimental Procedures."
Details of the procedure for the determination of systemic glucose
output were presented by Liu et al. (4). The values are
expressed as the mean ± S.E. for six mice in each group. *,
p < 0.04; **, p < 0.05.
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Fig. 6.
Northern blot analysis of hepatic PEPCK and
Glc-6-Pase mRNA in wild type and
C/EBP /
mice fed a high carbohydrate diet
and treated with cAMP. C/EBP
/
mice and control littermates
(8-12 weeks of age) were fed a high carbohydrate (CHO) diet
for 1 week before the determination of hepatic PEPCK and Glc-6-P
mRNA. The mice were killed, the livers were freeze-clamped, and
total RNA was extracted. In some of the experiments shown in this
figure, Bt2cAMP (35 mg/kg of body weight) and theophylline
(30 mg/kg of body weight) were administered by intraperitoneal
injection, and the mice were killed 2 h later for the
determination of PEPCK and Glc-6-Pase mRNA. The level of mRNA
was determined by Northern blotting. The hepatic mRNA was
standardized against 18 S rRNA in the same tissue using a
PhosphoImager® and expressed as a ratio. The values are
expressed as the mean ± S.E. for five mice in each group. *,
p < 0.02.
/
A-phenotype and Wild Type
Mice--
We previously noted that the basal level of cAMP in the
livers of C/EBP
/
mice was about half that of littermates (4). The concentration of hepatic cAMP was 296.68 ± 32.98 pmol/g as compared with 581.63 ± 92.98 pmol/g of liver in control
littermates (Fig. 7A). After
the administration of glucagon into the portal vein, the concentration
of hepatic cAMP increased to 495.32 ± 84.17 pmol/g of liver in
C/EBP
/
mice and to 1162.96 ± 171.11 pmol/g of liver in the
wild type mice (Fig. 7A). Although the fold change in the
concentration of cAMP was about the same, the absolute level of the
cyclic nucleotide in the livers of C/EBP
/
mice was markedly
different from that noted in wild type animals. The relatively lower
concentration (50%) of hepatic cAMP in the C/EBP
/
mice might be
due to the inability of the liver to synthesize cAMP at the appropriate
rate or might have resulted from an increase in cAMP degradation. To
test these possibilities, basal and activated adenylyl cyclase
activities were measured in liver plasma membranes of C/EBP
/
and wild type mice (Table I). In response
to glucagon, cholera toxin, or forskolin, and isoproterenol, membranes
from the C/EBP
/
mice synthesized cAMP at the same rate as those from control littermates. This indicates that the capacity to produce
cAMP was intact in the livers of these mice and was not altered by a
deletion of the gene for C/EBP
. However, there appeared to be an
accelerated rate of degradation of cAMP in levels of these mice.
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Fig. 7.
Effect of glucagon, theophylline, and
RO-20-1724 on the concentration of cAMP in the livers of adult
C/EBP /
mice and wild type mice.
Panel A, fed C/EBP
/
mice and control littermates
(WT) (8-12 weeks of age) were anesthetized with avertin, the liver was
clamped, and a biopsy was taken for the measurement of basal cAMP.
Glucagon (50 µg/kg of body weight) was then injected via the portal
vein, and the liver was biopsied 1 min later for the assay of
glucagon-induced cAMP. The data in this panel were redrawn
from Liu et al. (4). *, p < 0.05. Panel B, fed C/EBP
/
mice and control littermates
(WT) (8-12 weeks of age) were administered theophylline (30 mg/kg of
body weight) by intraperitoneal injection. Thirty min later, the
animals were anesthetized with avertin, the liver was clamped, and a
biopsy was taken for the measurement of basal cAMP. Glucagon (50 µg/kg of body weight) was then injected via the portal vein, and the
liver was biopsied 1 min later for the assay of glucagon- induced cAMP.
Panel C, the protocol was the same as in B except
that the RO 20-1724 (15 mg/kg of body weight) was injected instead of
theophylline. The values are expressed as the mean ± the S.E. of
the mean for 6 mice in each group.
Adenylyl cyclase activity in the livers of C/EBP/
and wild
type mice
/
mice and control
littermates (wild type). The activity of adenylyl cyclase was
determined as outlined under "Experimental Procedures." Values are
the mean ± S.E. for six mice in each group.
/
and control mice. The concentration of cAMP was then
measured in liver biopsies taken before and after glucagon injection
into the portal vein (Fig. 7, panels B and C).
After the administration of theophylline or RO 20-1724, the basal
levels of cAMP in the livers of C/EBP
/
mice were the same as
control animals, and there was no significant difference in the
response of cAMP to glucagon injection.
/
mice, we determined the total activity
of PDE in liver homogenates of fed mice using 1 µM cAMP as substrate. The specific activity of PDE was 56.7 pmol/min/mg of
protein in C/EBP
/
mice as compared with 44.1 in the livers of
control animals (Fig. 8). The level of
mRNA for PDE 3A, 3B, and 4B was also measured using a liver biopsy
taken 1 min after glucagon injection. The results show that the
concentrations of PDE 3A and PDE 3B mRNA were 25% higher in
C/EBP
/
mice (p < 0.01 for PDE 3B); no
difference in the levels of PDE 4 was detectable. It is important to
note that PDE 3B is the major phosphodiesterase isoform in the liver,
and its mRNA was 100-fold higher that of PDE3A. In agreement with
these findings, the rate of cAMP degradation in vitro was
determined from parallel experiments in which diluted liver homogenates
were incubated with 1 µM [3H]cAMP. As
shown in Fig. 9, the disappearance
(degradation) of [3H]cAMP by liver extracts from C/EBP
/
mice was more rapid than noted for controls.
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Fig. 8.
The activity of phosphodiesterase and the
level of PDE 3A, PDE3B, and PDE4B in the livers of
C/EBP /
mice and wild type mice.
Livers from fed C/EBP
/
and control littermates (WT) (8 to 12 weeks of age) were analyzed for total phosphodiesterase activity. Fed
C/EBP
/
mice and control littermates were anesthetized with
avertin, glucagon (50 µg/kg of body weight) was then injected via the
portal vein, and the liver was biopsied 1 min later for the measurement
of mRNA for PDE3A, PDE3B, and PDE4B as described under
"Experimental Procedures." The concentration of PDE mRNA was
assessed using competitive reverse transcription-PCR, as described
under "Experimental Procedures." Values for PDE mRNA are
expressed as a fold-change from the level of PDE3A mRNA in the
livers of wild type mice. To quantify the relative abundance of the
levels of PDE mRNA, values for PDE3A (WT) were designated as equal
to 1. This gave a ratio of 1:129:2.9 for PDE3A, PDE3B, and PDE4B,
respectively. The values are expressed as the mean ± S.E. for
four animals for PDE3A, three animals for PDE3B, and four animals for
PDE 4B. The activity of phosphodiesterase was determined as described
under "Experimental Procedures." The activity of phosphodiesterase
is expressed as the mean ± S.E. for eight animals in each group
(*, p < 0.02).
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Fig. 9.
Degradation of cAMP by homogenates of livers
from C/EBP /
and wild type mice. Liver
homogenates from fed C/EBP
/
mice and control littermates (WT)
(8-12 weeks of age) were incubated with 1 µM
[3H]cAMP and 1 mM 5'AMP for 1-6 min. At
intervals during this period the reactions were terminated by boiling
in water for 3 min, and the level of cAMP was determined over 6 min;
the concentration of unhydrolyzed [3H]cAMP was determined
as outlined under "Experimental Procedures." The values are
expressed as the mean ± S.E. for four animals in each group and
represent the percent change in [3H]cAMP from 0 time.
and RII
) and the catalytic
(C
) subunits of PKA in the nuclear (N), cytosolic (C) and particulate (P) fractions of livers from
adult C/EBP
/
and wild type mice by Western blotting (Fig.
10). We found significant changes in
the regulatory subunits of PKA in the livers of C/EBP
/
mice. The
concentration of RI
was 79% higher in the particulate fraction and
17% higher in the cytosolic fraction as compared with wild type mice,
whereas the concentration of RII
in the livers of C/EBP
/
mice
was increased by 27% in particulate fraction and 5% in the cytosolic
fraction as compared with wild type littermates. In contrast, the
concentration of the catalytic subunit of PKA in C/EBP
/
mice was
the same as wild type (Fig. 10). RII
antibody reacted with two
protein bands, one at 56 kDa and another at 52 kDa; the 56-kDa band is
characteristic of a phosphorylated form of RII
(28). The
concentration of the 52-kDa band (non-phosphorylated RII
) was higher
in the P and C fractions as compared with wild type, whereas the
phosphorylated band was similar to wild type mice in the P and C
fractions. The potential physiological significance of this result is
not clear.
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Fig. 10.
The activity of protein kinase A and the
concentration of its subunits in fractions from the livers of
C/EBP /
and wild type mice. The
concentration of the subunits of PKA in the various fractions from the
liver was determined by Western blotting using antibodies against
RI
, RII
, and C
. The figure shows as an insert the Western blot
for the various PKA subunits present in the liver fractions prepared as
described under "Experimental Procedures." N, nuclei,
mitochondria, and cell debris; P, Goli membranes;
C, cytosol fraction. The values are presented as a
percentage of the concentration of the various PKA subunits in the
livers of wild type mice. The activity of PKA was assayed over a 2-min
time period using the cytosolic fraction isolated from the livers of
C/EBP
/
and wild type mice. The values are expressed as the
mean ± S.E. of the mean for determinations for three animals in
each group (*, p < 0.05; **p, < 0.06).
, RII
, and C
were measured by scanning the Western
blots in Fig. 10. The values for the particulate and cytosolic
fractions are represented as percent of wild type. For RII
, the
phosphorylated (56 kDa band) and the unphosphorylated (52-kDa band)
forms of RII
were combined when scanned to give the total RII
.
Using the equations of Houge et al. (26), we calculated the
increase in cAMP required to give 50% activation of PKA in the
cytosolic and particulate fractions of the livers of C/EBP
/
mice
as compare with wild type animals. For example, the observed 79%
increase in the RI
subunit in the particulate fraction of the livers
of C/EBP
/
mice requires a 33% increase in cAMP for a 50%
activation of PKA. With the observed increase in the level of
regulatory subunits of PKA, the concentration of cAMP in the livers of
C/EBP
/
mice is critical in determining the total activity of the
enzyme. The result could be a failure to fully stimulate many of the
cAMP-dependent processes vital to the metabolic function of
the liver.
The effect of the relative concentration of the various isoforms of PKA
on the relative concentration of cAMP required for a 50% activation of
PKA in the livers of C/EBP/
mice
/
mice and are expressed as a percentage of wild type
control mice. The required increase in the concentration of cAMP in the
livers of C/EBP
/
mice required for 50% activation of PKA was
calculated from the following equations from Houge et al.
(26).
(1)
(2)
(3)
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
markedly alters the normal
initiation of glucose homeostasis in the immediate perinatal period.
Only 50% of the C/EBP
/
mice (animals with the A phenotype) survive the first hours after birth. C/EBP
/
mice (B phenotype) display profound hypoglycemia despite the fact that they have higher
than normal levels of hepatic glycogen (Fig. 1). Kawai and Arinze (46)
demonstrate that in newborn guinea pigs the response of hepatic
glycogenolysis to administered glucagon in the first 3-4 h after birth
is blunted when compared with the response beyond 4 h. A similar
age-dependent response was observed with epinephrine and
isoproterenol. In contrast, cAMP itself induced glycogenolysis
independent of age, suggesting that the retarded rate of hepatic
glycogen mobilization might be due to a delayed responsiveness of the
receptor-coupling system in the livers of newborn guinea pigs. The
results of the present studies suggest that C/EBP
/
mice (B
phenotype) do not respond appropriately to the normal stimuli that
occur at birth and that these can be by-passed by an injection of
Bt2cAMP immediately after birth.
/
mice (A phenotype) there is a 79% increase in the concentration of the
RI
regulatory subunit and a 27% increase in the concentration of
the RII
in the particulate fractions of the liver as compared with
wild type. There was no difference in the levels of the C
subunit of
PKA. This change in the R/C ratio could account for the 25% decrease
in PKA activity noted in the livers of C/EBP
/
mice (A
phenotype). In fact, a calculation of the required increase in the
concentration of cAMP needed to cause a 50% activation of PKA activity
increases by 45% for fractions RI
+ RII
combined in the
particulate fractions from livers of C/EBP
/
mice as compared
with wild type controls (Table II). A similar pattern of change in the
ratio of RI
and RII
relative to C
has been noted in the
regenerating liver (27). In the first 36 h of regeneration after
70% hepatectomy, the levels of both RI
and RII
increase 30 to
50%, whereas the concentration of C
in the liver remains constant.
This leads to a disproportion between the R and C subunits of PKA that
diminishes the concentration of C
during the cAMP burst that occurs
with liver regeneration (27). The increase in the R subunit during
liver regeneration has been interpreted as a response to the increase
in cAMP, since the elevation of the R subunit of PKA may be a method of
down-regulating PKA activity (hysteresis).
/
mice may also be of
significance. RI
is the predominant regulatory subunit of PKA in the
cytosol of hepatocytes, whereas RII
predominates in the
cytoskeleton, the Golgi apparatus, microtubules, and nucleus (28). We
have noted an increase in the concentration of both of the R subunits
of PKA in the particulate fraction of liver cell and a decrease in the
presence of these proteins in the cytosolic fraction (Fig. 10). In
contrast to our results, Ekanger et al. (27), in their
studies of regenerating liver, note that the change in the increased
concentrations of the RI
and RII
(relative to the C
)
was constant in the cytosol and particulate fractions of the liver. The
reason for the selective increase that we observe in the particulate
fraction of C/EBP
/
mice is not clear, but it could be related to
the concentration of PKA-anchoring protein in the liver. Anchoring
proteins bind specifically to RII and control the movement of PKA to
the particulate fraction of the liver, thus partitioning its activity
in the cell (29). The concentration of anchoring protein in the livers
of C/EBP
/
mice has not been determined.
in the cytosol of rat livers and an increase in the amount of the RII
subunit. This
was accompanied by a sharp reduction in the level of the catalytic
subunit of PKA in the particulate fraction of the liver cell. In
addition, the activation of glycogen phosphorylase and the
phosphorylation of the cAMP regulatory element-binding protein (CREB)
by glucagon was lower in hepatocytes isolated from rats fed a 0.5%
protein diet as compared with control animals that had been fed a
standard diet containing 15% protein (30). This dietary-induced shift
in the ratio of the RI
and RII
subunits of PKA in the liver would
explain the blunted response of these animals to glucagon, despite the
fact that the concentration of hepatic cAMP is the same as wild type
mice, since the two subunits of PKA have different affinities for cAMP
(31).
/
mice (A phenotype) survive to adulthood,
they display critical metabolic abnormalities. They have pronounced hypoglycemia associated with fasting and an impaired hepatic glucose production (4). There was a blunted rate of hepatic glucose production
caused by glucagon injection into 18-h fasted mice that were infused
with somatostatin to clamp the insulin and glucagon output from the
pancreas (4). The level of cAMP in the livers of C/EBP
/
mice (A
phenotype) was about half that noted in control littermates, and the
response to glucagon was also less robust. In addition, these mice have
a lower rate of epinephrine-induced release of free fatty acids from
epididymal adipose tissue in vitro (4). This may explain the
lower concentration of blood ketone bodies noted in the blood of
C/EBP
/
mice after fasting (Fig. 3). In addition to alterations
in the rate of hepatic glucose output, insulin sensitivity in the
C/EBP
/
mice was greater, resulting in a rate of whole body
glucose disposal that was 77% higher than noted in control littermates
(32). This is in part due to an increased response of muscle from the
C/EBP
/
mice to insulin stimulation; the insulin-stimulated
phosphorylation of the insulin receptor and phosphatidylinositol
3-kinase activities as well as insulin receptor kinase substrate-1 and
Akt-Ser473 were all about 2-fold greater in the skeletal
muscle of the C/EBP
/
mice as compared with littermates (32).
This suggests that the marked drop in the concentration of blood
glucose in the C/EBP
/
mice (A phenotype) during fasting is due
in part to an accelerated rate of removal of glucose by muscle as well
as a diminished rate of gluconeogenesis. This may also contribute to
the profound hypoglycemia noted in C/EBP
/
mice (B phenotype) in
the immediate perinatal period.
/
mice. It is known that interleukin-3 and
-4 activate PDE3 in FDCP2 myeloid cells (37), and the concentration of
interleukin-6 increased in fasted C/EBP
/
mice (9). It is
intriguing to speculate that the increased PDE activity observed in the
C/EBP
mice is accomplished through the increase of interleukin-6, which could in turn cause a cascade effect through the insulin receptor
substrate 2 (IRS-2), phosphatidylinositol 3-kinase, protein kinase B pathway and ultimately affect PDE3B activity. Another possibility would be a direct effect of C/EBP
on the promoter for
the PDE3A and PDE3B genes. Little is known about the transcriptional regulation of PDE genes. It is known that cAMP down-regulates the
expression of the gene for PDE3 (38). It is thus possible that in the
absence of C/EBP
there is decreased inhibition of gene
transcription, leading to an accumulation of PDE3 mRNA. This would
require that C/EBP
be involved as a negative regulator of PDE gene
transcription, for which there is no direct information to date.
However, a cAMP regulatory binding protein (CREB)-binding site is
present in the PDE3B gene
promoter2; this might serve
as a binding site for C/EBP
, as occurs with the PEPCK gene promoter
(39). A third possibility is that C/EBP
may directly regulate one of
the G proteins in the adenylyl cyclase pathway. This is unlikely since
we found no changes in the relative levels of
Gs
1, Gs
2,
Gi
2, Gq
, G
2,
and G
1 in membranes of livers of C/EBP
/
mice and control littermates, as determined by Western blotting (data
not shown). This result agrees with the data on adenylyl cyclase
activation in the same liver membranes (Table I).
/
mice are not limited to carbohydrate
and lipid metabolism; they also extend to amino acid metabolism. The
concentration of BUN in C/EBP
/
mice after 16 h of fasting is half that of control littermates, a value that is reflected in the
2-fold increase in blood ammonia in the C/EBP
/
mice. After
30 h of fasting, the concentration of BUN decreases to near normal
levels, indicating a sparing of amino nitrogen characteristic of
prolonged starvation. The levels of BUN have been shown to increase in
humans during the first few days of fasting and to return to normal
levels by the end of 2 weeks (40). Rats also spare body protein; this
is reflected in a decreased concentration of BUN at 24 h of
fasting (41-43). We have determined the concentration of 20 amino
acids in the blood of C/EBP
/
mice and control littermates that
were fasted for 30 h. The most notable difference was a 3-fold
increase in the concentration of taurine relative to control
littermates (1035 µmol/liter versus 333 µmol/liter), a 2-fold increase in ornithine (127 µmol/liter
versus 64 µmol/liter), and a 30% increase in glutamine
(647 µmol/liter versus 452 µmol/liter). The higher
levels of ammonia, ornithine, and glutamine in the blood of the
C/EBP
/
mice may reflect a decreased rate of urea cycle activity
in the livers of these mice. C/EBP
is required for the
glucocorticoid induction of transcription of the gene for arginase, a
critical urea cycle enzyme (44). It has been proposed that C/EBP
is
involved in the regulation of basal expression of the gene for arginase
and that C/EBP
controls the high level of expression of the gene in
the adult mouse in response to dietary protein (45). C/EBP
/
mice
have the appearance of sparse fur, a phenotype associated with a defect
in the gene for ornithine transcarbamylase caused in part by elevated
levels of blood ammonia.
. A series of
relatively small changes in the rate of hepatic cAMP degradation and a
shift in the pattern of expression of the genes for the isoforms of the
regulatory and catalytic subunits of PKA result in a lower
concentration of cAMP in the liver. When combined with the increased
requirement for cAMP to attain activation of PKA in the livers of
C/EBP
/
mice, the result is a failure to respond to glucagon in a
normal fashion. Thus, a 25% increase in the activity of PDE3 in the
liver ensures a rapid enough removal of hepatic cAMP to cause the
animals to have lowered rate of hepatic glucose output in response to
fasting and to glucagon administration.
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ACKNOWLEDGEMENT |
---|
We thank Dr. Satish Kalhan for assistance in the analysis of amino acids and other metabolites.
![]() |
FOOTNOTES |
---|
* This research was supported in part by National Institutes of Health Grants DK-25541 (to R. W. H.) and DK-50272 (to J. E. F.) and National Science Foundation Grant MCB 9905070 (to I. J. 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.
¶ This author was on sabbatical leave from the Division of Vascular Biology, National Cardiovascular Center, Research Institute, 5-7-1 Fujishiro-dai, Suita, Osaka 565-8565, Japan.
Supported by National Institutes of Health Metabolism Training
Program, DK-07319. To whom correspondence should be addressed: Dept. of
Biochemistry, Case Western Reserve University School of Medicine,
Cleveland, Ohio 44106-4935. Tel.: 216-368-5302; Fax: 216-368-4544;
E-mail: cmc6@po.cwru.edu.
Published, JBC Papers in Press, October 6, 2000, DOI 10.1074/jbc.M007576200
2 V. Manganiello personal communication.
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ABBREVIATIONS |
---|
The abbreviations used are: C/EBP, CCAAT/enhancer-binding protein; Bt2cAMP, dibutyryl cyclic AMP; PEPCK, phosphoenolpyruvate carboxykinase; BUN, blood urea nitrogen; Glc-6-P, glucose-6-phosphatase; WT, wild type; PCR, polymerase chain reaction; bp, base pair; PKA, cAMP-dependent protein kinase A, PDE, phosphodiesterase.
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REFERENCES |
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