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(Received for publication, August 19,
1994; and in revised form, October 17, 1994) From the
The effect of insulin on expression of CCAAT/enhancer binding
protein (C/EBP) A large body of evidence has shown that differentiation of 3T3
preadipocytes into adipocytes in cell culture serves as a faithful
model of the differentiation process in vivo (reviewed in (1) ). Two cell lines, i.e. the 3T3-L1 and 3T3-F442A,
have been most extensively characterized and are now widely used for
the study of preadipocyte
differentiation(2, 3, 4, 5, 6, 7) .
When subjected to an appropriate differentiation protocol, 3T3
preadipocytes lose their fibroblastic features, round-up, and acquire
the morphological and biochemical phenotype of adipocytes. Concomitant
with the accumulation of cytoplasmic triacylglycerol is the coordinate
expression of virtually every enzyme of the pathways of de novo fatty acid and triacylglycerol biosynthesis. In addition,
differentiating preadipocytes acquire the complement of proteins for
lipolysis of triacylglycerol, uptake, and intracellular translocation
of fatty acids, as well as responsiveness to lipogenic and lipolytic
hormones (1) . It has been established that these coordinate
changes in the cellular levels of proteins that give rise to the
adipocyte phenotype are almost entirely due to changes in the
transcription rates of the corresponding genes(8, 9) . Although the sequence of events which prompt preadipocyte
differentiation is not fully understood, compelling evidence indicates
that C/EBP C/EBP The C/EBP family of transcription factors share amino acid sequence
similarity within their C-terminal basic region/leucine zipper domain,
which confers the capacity to dimerize and bind DNA (reviewed by
McKnight(21) ). Members of the C/EBP family can form homo- and
heterodimers, all of which can bind to the same cis-regulatory
elements within the promoters/enhancers of genes regulated by the
C/EBP's. The temporal expression of C/EBP While members of the C/EBP family
have been implicated in the differentiation of 3T3-L1 preadipocytes,
the role(s) of these transcription factors in the mature,
fully-differentiated adipocyte has not been extensively investigated.
Recently, we reported that glucocorticoids exert rapid reciprocal
effects on the expression of C/EBP
The DNA fragment used as a probe
for C/EBP
Immune serum against a synthetic peptide
corresponding to an internal amino acid sequence of C/EBP
Figure 1:
Effect of
insulin on the expression of C/EBP. A, 3T3-L1 adipocytes in
monolayer culture were treated with 167 nM insulin (INS) for the indicated times. Whole cell lysates containing
equal cell equivalents (
Axel Kahn and colleagues (38) have found that
insulin can alter hepatic gene expression by glucose-dependent or
glucose-independent mechanisms. To ascertain whether glucose is
required for the regulation of C/EBP Inspection of Fig. 1A reveals that p30
Figure 2:
Rapid effect of insulin on C/EBP
Figure 3:
Gel shift analysis of C/EBP isoforms from
untreated 3T3-L1 adipocytes (ADIP) and those treated with insulin.
Nuclei were isolated and nuclear extracts were prepared from untreated
control adipocytes 11 days after initiating differentiation or
adipocytes treated with 167 nM insulin for 6 h (INS).
Gel shift analysis was performed using 3.0
Figure 4:
Effect of insulin or IGF-1 concentration
on expression of C/EBP
Figure 5:
Comparison of the effect of insulin on the
kinetics of expression of C/EBP
Figure 6:
A) Effect of insulin on nuclear run-on
transcription of the C/EBP
To ascertain whether the
mobility differences were due to phosphorylation of the C/EBP
Figure 7:
Effect of insulin and okadaic acid on the
post-translational modification of C/EBP
Since inhibition of a phosphatase
(by okadaic acid) might be expected to have the opposite effect of
insulin, it follows that insulin may function to activate a type 1 or
2A protein phosphatase. Activation of protein phosphatase type 1 by
insulin is well documented in several cell
types(42, 43) , including 3T3-L1
adipocytes(44) . Moreover, protein phosphatase type 1 has been
shown to be regulated during the cell cycle(45) , and this is
correlated with the predominance of the higher mobility band in
elutriation experiments. ( Ex
vivo labeling of 3T3-L1 adipocytes with
[
Figure 8:
Kinetic analysis of the effects of insulin
on expression of C/EBP
To ascertain whether
long-term exposure of 3T3-L1 adipocytes to insulin affects expression
of the C/EBP isoforms and the adipocyte-marker GLUT4, preadipocytes
were subjected to the standard differentiation protocol, which includes
insulin, after which the differentiated adipocytes were maintained for
4 days in medium with or without insulin. Northern analysis revealed
that insulin treatment beyond day 4 of adipocyte differentiation
markedly suppressed the expression of C/EBP
Figure 9:
Effect of chronic exposure to insulin on
the expression of C/EBP
This study shows that insulin reciprocally regulates the gene
encoding C/EBP In addition to regulating the
post-translational modification and transcription of C/EBP Reciprocal regulation of the C/EBP transcription factors is a
recurring theme. For example, during the acute-phase response of liver
or hepatocytes, cytokines suppress the expression of C/EBP The C/EBP transcription factors are also reciprocally
regulated in fully-differentiated adipocytes. For example, treatment of
3T3-L1 adipocytes with monocyte-conditioned medium (containing tumor
necrosis factor
Volume 270,
Number 2,
Issue of January 13, 1995 pp. 647-654
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
,
, and
Genes in Fully-differentiated 3T3-L1 Adipocytes (*)
, 
, and was investigated in
fully-differentiated 3T3-L1 adipocytes. Treatment of adipocytes with
insulin stimulated rapid dephosphorylation of C/EBP
, and repressed
the expression of C/EBP within 2-4 h, with >90%
suppression occurring at 24 h. While insulin induced expression of
C/EBP and C/EBP within 1 h and caused a >20-fold increase
by 4 h, expression returned to nearly pretreatment levels by 24 h. The
insulin concentration dependence of these effects was consistent with
involvement of the insulin receptor. Gel shift analysis revealed that 6
h of insulin treatment decreased the binding of nuclear C/EBP
while increasing binding of nuclear C/EBP and C/EBP. The
reciprocal effects of insulin on the steady-state levels of C/EBP
transcription factors can be accounted for kinetically and
quantitatively by changes in their mRNA levels, which can be accounted
for by effects on gene transcription. The effects of insulin on
adipocyte gene transcription (e.g. GLUT4) may be mediated, at
least in part, by down-regulation of C/EBP
and/or its
dephosphorylation.
(
)plays an essential role in this
process(10, 11, 12, 13, 14, 15) .
C/EBP appears to function both by inhibiting the clonal expansion
that precedes terminal differentiation (16) and by activating
the coordinate expression of a group of adipocyte genes whose promoters
possess C/EBP-binding
sites(10, 11, 17, 18) . Unequivocal
proof that C/EBP is essential for differentiation was obtained
using the antisense RNA approach(13, 19) . Expression
of a truncated C/EBP antisense RNA in 3T3-L1 preadipocytes blocked
expression of C/EBP, transcription of several adipocyte genes (i.e. 422/aP2, SCD1, and GLUT4), and accumulation of
cytoplasmic triacylglycerol(13) . More recently it was shown
that expression of C/EBP is not only necessary, but is sufficient,
to induce preadipocyte differentiation(14, 15) . Thus,
isopropyl-1-thio--D-galactopyranoside-induced expression
of C/EBP by 3T3-L1 preadipocytes harboring a LacSwitch C/EBP
expression vector system caused expression of adipocyte markers and
acquisition of the adipocyte phenotype(14) . In addition,
ectopic expression of C/EBP using a retroviral expression vector
was shown to induce adipogenesis in a variety of cell
lines(15) . mRNA has been shown to give rise to
two major alternative translation products, p42 and p30
(18, 20) , both of
which are expressed by 3T3-L1 adipocytes, liver, and white adipose
tissue. The two C/EBP isoforms possess some similar and some dissimilar
functional properties. While both isoforms transactivate the promoters
of certain adipocyte genes (18) , only p42
is antimitotic and capable of terminating clonal expansion.
Moreover, the relative levels of expression of the two isoforms differ
during hepatic development and during the differentiation of 3T3-L1
preadipocytes raising the possibility that they play different roles
during differentiation of these cell types. Although expression of
p30
precedes expression of p42
during development and differentiation, both isoforms are
expressed by terminally differentiated adipocytes and hepatocytes.
and C/EBP
during differentiation of 3T3-L1 preadipocytes, and the presence of a
C/EBP-binding site within the C/EBP
gene promoter, has led to the
hypothesis that C/EBP and/or C/EBP may be responsible for the
activation of expression of the C/EBP
gene(12) . Further
work will be required to clarify the roles of C/EBP and C/EBP
in preadipocyte differentiation.
, and , largely by altering
transcription of the corresponding genes(22) . In view of the
established roles of insulin and glucocorticoids on carbohydrate and
lipid metabolism in the adipocyte (23, 24, 25, 26) and the fact that a
number of genes which function in these processes are regulated by
C/EBP
, we examined the effect of insulin on the expression of
C/EBP, and in terminally-differentiated 3T3-L1
adipocytes. Our results suggest that insulin regulates C/EBP
through at least three mechanisms: post-translational modification,
transcription, and through induction of a dominant negative
transcription factor (LIP).
Cell Culture
3T3-L1 preadipocytes were
maintained and induced to differentiate into adipocytes as described
previously(27) , except that insulin was withdrawn from the
medium on day 4. Using this protocol, >95% of the cells begin to
acquire the adipocyte phenotype 3-4 days after initiating
differentiation. Fully-differentiated 3T3-L1 adipocytes, 11-14
days after induction of differentiation, were switched to fresh media
(Dulbecco's modified Eagle's media, 10% fetal bovine serum)
16-24 h before subjecting cells to insulin or IGF-1 treatment.
Insulin was dissolved in 0.1 M HCl and IGF-1 was dissolved in
10 mM acetic acid. Okadaic acid (Boehringer Mannheim) was
dissolved in dimethyl sulfoxide.Analysis of RNA
Cellular RNA was isolated from
3T3-L1 cells using guanidine thiocyanate followed by
ultracentrifugation through CsCl(28) . The amount of C/EBP
and C/EBP mRNA was assessed by Northern blot
analysis(28) . Total RNA (20 µg) was separated by
electrophoresis in horizontal 1.2% agarose gels containing 6.5%
formaldehyde. RNA was transferred to nylon membranes (Hybond-N;
Amersham Corp.) overnight and covalently bonded to the membrane by
exposure to ultraviolet light (UV Stratalinker 1800; Stratagene). Blots
were prehybridized for at least 6 h (42 °C) in a solution
containing 50% formamide, 4
SSC, 5 Denhardt's
solution, 50 mM phosphate buffer, pH 7.0, 100 µg/ml yeast
tRNA, 0.5 mg/ml sodium pyrophosphate, and 1% sodium dodecyl sulfate
(SDS). Hybridization was carried out for at least 16 h at 42 °C in
an identical solution containing 4 10
dpm of
labeled probe per ml. In general, hybridized blots were washed three
times in a solution containing 0.5 SSC, 0.1% SDS at 60 °C.
Autoradiography was at -80 °C with Kodak X-Omat AR film
(Eastman Kodak Co.) and an intensifying screen for the times indicated
in the figure legends. Results were quantified using a laser
densitometer (LKB Ultroscan XL). mRNA was an 900-base pair SacI/HindIII fragment complementary to the 3` end of
the C/EBP
coding region, as well as part of the 3`-untranslated
region (+1175 to +2078 nucleotides relative to
transcriptional start site). The cDNA fragment for C/EBP is full
length and was cloned from a 3T3-L1 adipocyte library as reported
previously(29) . The cDNA fragment used as a probe for
C/EBP mRNA was as described(12) . Isolated C/EBP
,
C/EBP, or C/EBP DNA probes were labeled to high specific
activity (
1
10
dpm/µg) by random hexamer
priming(30) .Cell Lysates and Immunoblotting
3T3-L1 adipocyte
monolayers (10 cm) were washed once with 10 ml of phosphate-buffered
saline and scraped in 1 ml of a lysis buffer containing 1% SDS, 60
mM Tris-Cl, pH 6.8. Lysates were boiled for 3 min, vortexed,
then boiled for an additional 7 min prior to storing at -35
°C. Western analysis was performed as described
previously(13) . Results were quantified using laser
densitometry. When presented graphically for C/EBP, the results
represent the sum of p30 and
p42
, and when presented for C/EBP
, the
results represent the sum of LAP and LIP.Preparation of Antibodies
Peptides corresponding
to amino acids 278-295 (LRNLFKQLPEPLLASAGH) of C/EBP and
115-130 (ARGPLKREPDWGDGDA) of C/EBP were synthesized
(Protein Peptide Facility; Department of Biological Chemistry, Johns
Hopkins University School of Medicine) with an additional N-terminal
cysteine residue and then cross-linked through the N-terminal-SH to
keyhole limpet hemocyanin. Immunization of rabbits and collection of
preimmune and immune sera were performed by HRP (Denver, PA). Antiserum
used for Western blots or supershifting was specific for either
C/EBP
or C/EBP and interactions with other C/EBP isoforms
were not observed.
(present
in both p42 and p30
) was
prepared as described previously(18) . In some experiments,
immune sera to C/EBP
and C/EBP were generously provided by
Dr. Steve McKnight(12) .
Preparation of Nuclear Extracts
Nuclei were
purified from 3T3-L1 cells by a modification (31) of the
procedure of Dignam et al.(32) . Nuclear extracts were
prepared as described by Lavery and Schibler (33) using a 1
NUN solution (0.3 M NaCl, 1 M urea, 1%
Nonidet P-40, 25 mM HEPES, pH 7.9, and 1 mM dithiothreitol). Protein concentration was determined using the
BCA Protein Assay Reagent (Pierce Chemical Co.) and ranged from 8.5 to
10 mg/ml.Gel Shift Analysis
A double stranded
oligonucleotide corresponding to the C/EBP-binding site in the 422(aP2)
promoter (10) was labeled using
[-P]ATP and DNA polymerase (Klenow
fragment; New England Biolabs). The gel shift mixture (40 µl)
contained 0.33 M urea, 0.1 M NaCl, 0.33% Nonidet
P-40, 25 mM HEPES, pH 7.9, 10 mM dithiothreitol, 10%
glycerol, 5 µg of acetylated bovine serum albumin, 3 µg of
poly[d(I-C)], and 3.0
10
dpm of P-labeled oligonucleotide. This mixture (including 6
µl of antiserum and/or preimmune sera as indicated) was incubated
on ice for 30 min, then at room temperature for 90 min prior to
electrophoresis on 6% polyacrylamide gels.
Nuclear Run-on Transcription Analysis
After the
indicated treatments of 3T3-L1 adipocytes (day 12), nuclei were
isolated from transcriptional run-on assays as described by Cornelius et al.(34) . Equal numbers of nuclei were incubated
for 25 min at 28 °C in the presence of
[-P]UTP (6000 Ci/mmol; DuPont). Reactions
were terminated by addition of RNase-free DNase, and then
P-labeled RNA was isolated by guanidine thiocyanate
extraction and CsCl density gradient centrifugation. After partial
hydrolysis in 0.2 M NaOH,
P-labeled RNA was
extracted once with chloropane (35) and recovered by ethanol
precipitation. Labeled transcripts (
20
10 dpm/ml) were hybridized for 72 h with cDNAs covalently linked to
nylon membrane (Hybond-N; Amersham). Blots were washed to high
stringency, and hybridized RNA was visualized by autoradiography.
Effects of Insulin on the Cellular Levels of
C/EBP
In
view of the well documented effects of insulin on adipose tissue
metabolism and of the activating effect of C/EBP, C/EBP, and C/EBP on adipocyte gene
transcription during preadipocyte differentiation, it was of interest
to assess the effect of insulin on the expression of C/EBP family
members in fully-differentiated 3T3-L1 adipocytes. As shown in Fig. 1, A and B, treatment of adipocytes with
insulin suppressed expression of both the 42- and 30-kDa C/EBP
isoforms. A decrease was first observed between 2 and 4 h after the
addition of insulin, with repression of C/EBP continuing for at
least 24 h. In contrast, insulin caused an increase in C/EBP (LAP
and LIP; (36) and (37) ) that was observed at 2 h and
was maximal at 4 h. Expression of C/EBP was also induced by
insulin within 2 h and was sustained for 10 h. Induction of both
C/EBP
and C/EBP by insulin was transient, with expression
decreasing toward pretreatment levels by 24 h. These results show that
insulin reciprocally regulates expression of at least 3 members of the
C/EBP family of transcription factors in fully differentiated 3T3-L1
adipocytes.
200 µg of protein) were subjected to
SDS-PAGE, and immunoblotted using antisera against C/EBP
,
C/EBP, and C/EBP. These results are representative of at
least six independent time course experiments.
42 and
30 refer to p42 and
p30
isoforms, respectively. LAP and LIP refer to the liver activator protein and liver inhibitory
protein of C/EBP
, respectively; and refers to C/EBP
. B, results in A were quantified by laser densitometry
and the results are shown graphically relative to the maximal level of
expression of each C/EBP.
by insulin, cells were
incubated overnight in glucose-free media (with or without pyruvate)
prior to insulin treatment. Western blot analysis showed that while
overall expression of C/EBP was repressed by incubation in
glucose-free medium, regulation of C/EBP by insulin was identical
to that shown in Fig. 1. This includes the rapid effect of
insulin on post-translational modification and the subsequent
suppression of C/EBP protein. Therefore, insulin regulates
C/EBP through glucose-independent mechanisms. consists
of two bands, and that the top band is absent after 2 h of insulin
treatment. A more extensive time course of insulin effects on
expression of C/EBP
in 3T3-L1 adipocytes reveals that the top band
of p30 is about 50% depleted by 15 min, and is
completely absent at 30 min (Fig. 2). The corresponding increase
in the bottom band suggests that these mobilities might reflect
structural differences within p30
, perhaps due to
post-translational modification. A similar, although less obvious,
shift in mobility of p42
occurs with similar
kinetics (Fig. 2). Evidence described in a later section
suggests that insulin treatment alters the phosphorylation of the two
C/EBP
isoforms.
.
3T3-L1 adipocytes in monolayer culture were treated with insulin (167
nM) for the indicated times. Whole cell lysates containing
equal cell equivalents 200 µg of protein) were subjected to
SDS-PAGE, and immunoblotted using antisera against
C/EBP
.
Effect of Insulin on the Binding of C/EBP Isoforms to
a C/EBP Binding-site Oligonucleotide
Gel shift
experiments were performed to verify that the insulin-induced changes
in C/EBP isoform levels described above correlate with changes in their
DNA binding capacity. Thus, nuclear extracts, prepared from 3T3-L1
adipocytes that had been treated or not with insulin for 6 h, were
subjected to gel shift analysis using an oligonucleotide probe
corresponding to the C/EBP-binding site in the 422(aP2)
promoter(10) . All three C/EBP isoforms (, , and
) bind at this site. Although the gel shift banding patterns were
complex (Fig. 3, lanes 2 and 3), use of
isoform-specific antibodies and changes in isoform level due to insulin
treatment made it possible to identify DNA-protein complexes containing
each of the C/EBP isoforms. While addition of preimmune sera stabilized
the binding of C/EBP isoforms to this probe, the relative intensities
of the bands were similar, and no ``new'' bands were observed (Fig. 3, lanes 4 and 5). Supershift
experiments with antisera against C/EBP
and C/EBP gave rise
to DNA-protein complexes with low mobility. This facilitated detection
of DNA-protein complexes containing C/EBP
and revealed that 6 h of
insulin treatment suppressed the relative amount of C/EBP
available for binding to the C/EBP-binding site (Fig. 3, compare lanes 6 and 7). In a similar fashion, supershifting
C/EBP and C/EBP revealed that insulin increased the relative
amount of C/EBP
-containing DNA-protein complexes (Fig. 3,
compare lanes 8 and 9), and supershifting C/EBP
and C/EBP revealed that insulin increased the relative amount of
C/EBP-containing DNA-protein complex (Fig. 3, compare lanes 10 and 11). Supershifting with antiserum
specific to the N-terminal region of C/EBP
(recognizes only LAP)
revealed that the band of highest mobility contains LIP/LIP homodimers, (
)and it is presumed that the intermediate
C/EBP-containing band is composed of LAP/LIP heterodimers.
Therefore, LIP appears to predominate over LAP (and C/EBP) in both
the basal and insulin-stimulated states (see ``Discussion'').
At 24 h, suppression of C/EBP
and induction of C/EBP could
still be observed, however, binding of C/EBP
was the same as in
control adipocyte extracts (data not shown). It should be noted that
all of the oligonucleotide-protein complexes detected by gel shift
analysis with nuclear extracts from control and insulin-treated
adipocytes were supershifted by a combination of antibodies to all
three C/EBP isoforms (Fig. 3, lanes 12 and 13). Identical gel shift patterns were observed with the
C/EBP-binding site from the C/EBP promoter (data not shown). Taken
together, these findings are consistent with the results of Western
blot analysis, and show that insulin induces changes in the relative
cellular levels of C/EBP transcription factors.
10 dpm
of a P-labeled oligonucleotide corresponding to the
C/EBP-binding site from the 422/aP2 gene promoter, and 8 µg of
nuclear protein. Oligonucleotide-protein complexes were separated on a
6% polyacrylamide gel at 9 V/cm for 4 h. Free-labeled oligonucleotide
was run off the gel. Autoradiography was performed at -80 °C
for 16 h. Supershifting was performed using the indicated combinations
of antiserum (2 µl each) with preimmune (PI) serum added
to bring the total sera volume to 6 µl.
refers to antiserum
to C/EBP; to antiserum to C/EBP; and to antiserum
to C/EBP
.
Concentration Dependence and Specificity of the Insulin
Response
To ascertain whether the insulin-induced changes in
cellular levels of C/EBP, C/EBP, and C/EBP are mediated
by the insulin receptor or by the insulin-like growth factor-1 (IGF-1)
receptor, the dependence of these changes on concentration of insulin
or IGF-1 was assessed. As illustrated in Fig. 4, the
concentration of insulin for half-maximal effects on the expression of
C/EBP
, C/EBP, and C/EBP occurred around 3-10
nM insulin. This is in good agreement with the reported K
for insulin with the insulin receptor of intact
3T3-L1 adipocytes(39) . While IGF-1 did not influence the
expression of C/EBP or C/EBP in these cells, IGF-1 induced
expression of C/EBP with a concentration dependence consistent
with an effect mediated by the IGF-1 receptor. These findings support
the view that insulin regulates expression of these C/EBP family
members through the insulin receptor, and that IGF-1 also induces
C/EBP
through the IGF-1 receptor.
(A), C/EBP (B), and C/EBP (C).
The indicated concentrations of insulin or IGF-1 were added to 3T3-L1
adipocytes for 24 h (C/EBP
) or 2 h (C/EBP and C/EBP) on
day 11 after initiating differentiation. After lysis and Western
analysis, results were quantified by laser densitometry and are
presented relative to the maximal level of expression for each C/EBP.
Results from the insulin concentration dependence are representative of
three independent experiments while the IGF-1 experiment was performed
once. The half-maximal insulin effect on the expression of C/EBP
,
C/EBP, and C/EBP was observed at
3-10 nM insulin. While IGF-1 induced expression of C/EBP
with a
half-maximal effect at 10 nM, IGF-1 did not influence the
expression of either C/EBP
or
C/EBP.
Effect of Insulin on mRNA Levels of C/EBP
To determine whether changes in the expression
of the C/EBP proteins were due to changes in steady-state levels of
mRNA, Northern analysis was performed on total RNA isolated from
fully-differentiated 3T3-L1 adipocytes treated with insulin for
different periods of time. Analysis of C/EBP,
, and , C/EBP, and
C/EBP mRNA levels revealed that the effects of insulin on these
C/EBP isoforms could be accounted for by changes in the cellular levels
of their respective mRNAs (Fig. 5, A and B).
Insulin caused a decrease in the expression of C/EBP
mRNA, the
greatest decrease occurring between 1 and 2 h of treatment, with low
levels of expression persisting until at least 24 h. In contrast,
insulin transiently induced the level of C/EBP and C/EBP
mRNAs, which reached a maximum at 2 and 1 h, respectively (Fig. 5B). It is evident, therefore, that insulin
regulates expression of C/EBP
, C/EBP, and C/EBP
primarily by regulating the steady-state levels of their respective
mRNAs.
, C/EBP, and C/EBP mRNAs. A, insulin (167 nM) was added to 3T3-L1 adipocytes on
day 12 after initiation of differentiation, and total RNA was prepared
from two independent cell monolayers after 0, 1, 2, 4, 6, 10, or 24 h.
Equal amounts of RNA (20 µg) were electrophoresed, and analyzed by
Northern blotting using DNA fragments complementary to C/EBP
,
C/EBP, or C/EBP mRNAs. Results are representative of three
independent experiments. Autoradiography was for 48 h at -80
°C for C/EBP
and C/EBP; 24 h for C/EBP
. B,
results in A were quantified by laser densitometry and the
mean result ± range are shown graphically relative to the
maximal level of expression.
Effect of Insulin on Transcription of the C/EBP
To ascertain
whether the rapid changes in the mRNA levels for C/EBP,
C/EBP, and C/EBP Genes
,
C/EBP, and C/EBP were due to changes in gene transcription,
the effect of insulin on nuclear run-on transcription was evaluated. As
shown in Fig. 6, insulin markedly (
65%) repressed
C/EBP
gene transcription within 1 h and this suppressed rate of
transcription was maintained for at least 4 h. In contrast, insulin
induced (3-fold) transcription of both the C/EBP
and
C/EBP genes by 1 h (Fig. 6). Other experiments showed
induction levels of up to 8-fold. These changes in transcription
precede and are comparable in magnitude to effects of insulin on the
cellular levels of C/EBP
, C/EBP, and C/EBP mRNAs (see Fig. 5). Therefore, insulin rapidly regulates the expression of
C/EBP
, C/EBP, and C/EBP mRNAs, in large part, by
altering rates of transcription.
, C/EBP, and C/EBP genes.
Nuclei were prepared from fully-differentiated 3T3-L1 adipocytes that
were untreated, or treated with 167 nM insulin for 1 or 4 h.
After incubation of nuclei with [
P]UTP and
isolation of RNA, 18
10 dpm of P-labeled RNA were used for hybridization of blots
containing 1 mg of DNA complementary for C/EBP
, C/EBP,
C/EBP, pBluescript, or genomic DNA. Filters were washed to high
stringency, and exposed to film for 7 days at -80 °C. Results
are representative of two independent experiments. B, results
in A were quantified by laser densitometry, normalized to the
genomic signal, and the results for each C/EBP homologue expressed in
arbitrary units.
Effect of Insulin on the Phosphorylation State of
C/EBP
As shown above (Fig. 2) insulin rapidly
(within 15 min) alters the mobility of p30 and
p42
by SDS-PAGE. Following exposure to insulin the
initial double-band pattern of each isoform reverts to a single,
higher-mobility band, i.e. corresponding to
41 and 29
kDa, reaching 50 and 100% completion within 15 and 30 min,
respectively. The rapidity of the response to insulin and the fact that
blocking protein synthesis with cycloheximide did not prevent the
response (results not shown) suggested that the changes in mobility of
the C/EBP
isoforms were the result of post-translational
modification, such as phosphorylation.
isoforms, the effect of okadaic acid, a potent inhibitor of
serine/threonine protein phosphatases 1 and
2A(40, 41) , was tested in the absence and presence of
insulin. Insulin treatment generated the higher mobility forms of the
two C/EBPs (most evident with p30), whereas
treatment with 1.5 µM okadaic acid for 45 min, either in
the absence or presence of insulin, gave rise to the lower mobility
forms (Fig. 7A).
. A, 3T3-L1
adipocytes were treated for 45 min with MeSO (vehicle for
okadaic acid; CONT), 167 nM insulin (INS),
1.5 µM okadaic acid (OA), insulin after a 5-min
pretreatment with okadaic acid (O+A), or okadaic acid
after a 5-min pretreatment with insulin (I+O). Whole cell
lysates containing equal cell equivalents (200 µg of protein)
were subjected to SDS-PAGE, and immunoblotted using antisera generated
against C/EBP
. B, 3T3-L1 adipocytes were incubated under
serum-free conditions overnight. After a wash in phosphate-free,
serum-free media, adipocytes were incubated with
[P]orthophosphate for 2 h, then insulin or not
for another hour. C/EBP
was immunoprecipitated with antiserum
against C/EBP using protein A-Sepharose, separated by SDS-PAGE,
and visualized with autoradiography at -80 °C. C,
nuclear extracts were prepared from adipocytes treated with 1.5
µM okadaic acid for 45 min. After precipitation with 12.5%
trichloroacetic acid, the pellet was rinsed with cold acetone, and
dissolved in 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl, 1 mM dithiothreitol, and 0.1% SDS at 37
°C for 8 h. After addition of Triton X-100 to 1%, 50 units of calf
intestinal phosphatase were added to half the sample prior to an
overnight incubation at 37 °C. C/EBP was analyzed following
SDS-PAGE by Western blot analysis. All results above are representative
of at least two independent experiments.
)Whether this phosphatase acts to
directly dephosphorylate C/EBP or acts through a second messenger
pathway which leads to dephosphorylation remains unclear.P]orthophosphate followed by
immunoprecipitation and SDS-PAGE revealed that both the 42- and 30-kDa
forms of C/EBP
are highly phosphorylated. Nevertheless, the gross
level of phosphorylation of both isoforms did not appear to be affected
by insulin treatment (Fig. 7B). It is possible that an
insulin (and okadaic acid-)-sensitive phosphatase targets only one of
multiple phosphorylation sites on C/EBP and would, therefore, only
lead to small fractional changes which would make the dephosphorylation
event difficult to detect. Phosphoamino acid analysis of
p30 from 293 cells transiently transfected with a
p30
expression vector showed that this isoform is
phosphorylated on serine and threonine (in an
1:1 ratio), but not
on tyrosine.
As expected, inhibition of tyrosine kinase
activity with genestein (Calbiochem) inhibited the rapid effects of
insulin on C/EBP post-translational modification (results not
shown). Further evidence that post-translational modification of
C/EBP is due to phosphorylation is provided in Fig. 7C. Nuclear extracts from 3T3-L1 adipocytes
treated with okadaic acid, and prepared in the presence of phosphatase
inhibitors (30 mM -glycerol phosphate and 1 mM orthovanadate) gave rise to the low mobility band of both
C/EBP isoforms. When the extracts were treated with calf
intestinal phosphatase, the low mobility band was lost and the high
mobility band was observed (Fig. 7C). These experiments
strongly suggest that insulin, in addition to suppressing the
expression of C/EBP within several hours, also acutely stimulates
dephosphorylation of both C/EBP isoforms.Temporal Relationship of Down-regulation of
C/EBP
In view of the
findings that both C/EBP and GLUT4 mRNA Caused by Insulin (Fig. 5) and GLUT4
mRNA(46) ; Fig. 8) are down-regulated by insulin in
terminally differentiated 3T3-L1 adipocytes, it was of interest to
ascertain whether the kinetics of these processes were compatible with
a causal relationship. It should be noted that during differentiation
of 3T3-L1 preadipocytes, C/EBP serves as a transactivator of the
GLUT4 gene whose promoter possesses a C/EBP-binding
site(13, 17) . As shown in Fig. 8, following
the insulin-induced drop in the C/EBP message, the decline of
C/EBP protein is virtually coincident with the decline of GLUT4
message in 3T3-L1 adipocytes. Although this result is consistent with
trans-activation of the GLUT4 gene by C/EBP in the adipocyte, the
possibility that dephosphorylation of C/EBP is involved cannot be
ruled out (see ``Discussion'').
mRNA and protein, and GLUT4 mRNAs. 3T3-L1
adipocytes were treated with 167 nM insulin for various times
prior to preparation of whole cell lysates or total RNA. The amount of
C/EBP protein and C/EBP or GLUT4 mRNAs were analyzed by
immunoblot and Northern analyses, respectively. Protein and mRNA levels
were quantified using laser densitometry and are represented
graphically relative to their maximal levels of
expression.
and GLUT4 mRNAs (Fig. 9A) without affecting the expression of
C/EBP or C/EBP (Fig. 9B). Some cells were
treated with or without insulin for an additional 4 days, i.e. to day 12, at which time cell monolayers were stained for
triacylglycerol with oil red-O. Cell monolayers chronically treated
with insulin had fewer adipocytes with large unilocular triacylglycerol
vacuoles, and had a higher proportion of adipocytes with a multilocular
adipocyte phenotype. These findings are consistent with a causal
relationship between insulin-induced suppression of C/EBP
, and the
reduced levels of GLUT4 mRNA and other mRNAs (e.g. SCD1 mRNA;
results not shown), which give rise to the adipocyte phenotype.
, C/EBP, C/EBP, and GLUT4. 3T3-L1
preadipocytes were differentiated by the standard protocol until day 4.
At this time, half of the cells continued to receive insulin every 2
days with feeding. Total RNA was harvested on day 0, and then daily
starting at day 2. Northern analysis was used to evaluate the
expression of: A, C/EBP
and GLUT4 mRNA levels, as well
as, B, C/EBP and C/EBP. Data were quantified using
laser densitometry and are represented graphically in arbitrary
units.
, and those encoding C/EBP and C/EBP in
fully-differentiated 3T3-L1 adipocytes. While insulin represses the
expression of C/EBP
for at least 24 h, insulin rapidly and
transiently induces the expression of C/EBP and C/EBP (Fig. 1). These changes are due largely to changes in
steady-state levels of their respective mRNAs (Fig. 5), which
are correlated with changes in the rates of transcription of the
corresponding C/EBP genes (Fig. 6). In addition to regulating
C/EBP
by repressing its expression, insulin may also regulate the
activity of C/EBP itself by controlling its state of
phosphorylation. Indeed, it has been reported (47) that
C/EBP can be phosphorylated in vitro on serine 299 by
protein kinase C, and that phosphorylation attenuated its binding to
DNA. Evidence presented in this paper indicates that C/EBP exists
in a phosphorylated state in 3T3-L1 adipocytes (Fig. 7B), and that insulin promotes its apparent
dephosphorylation by an okadaic acid-sensitive phosphatase (Fig. 7A), presumably protein phosphatase 1 or 2A.
Although insulin-activated dephosphorylation of C/EBP has the
potential to regulate transcription of adipocyte genes (e.g. GLUT4), the findings presented in this paper do not specifically
address this issue. It should be noted, however, that the time frame
within which apparent dephosphorylation of C/EBP occurs is
consistent with the rate at which GLUT 4 transcription and mRNA
fall(46) . Thus, apparent dephosphorylation of C/EBP is
complete within 30 min (Fig. 2) and the rate of GLUT4 gene
transcription reaches a minimum in less than 2 h (46) . In this
connection, it has been reported that insulin transiently activates
type 1 protein phosphatase in several cell types including 3T3-L1
adipocytes(42, 43, 44) . Insulin has also
recently been shown to promote dephosphorylation and suppression of
CREB (cAMP response element-binding protein) activity by a type 1
protein phosphatase(45) . Further work will be necessary to
determine whether insulin-promoted dephosphorylation of C/EBP,
repressed transcription of the C/EBP gene, or other factors are
responsible for the physiological effects of insulin on the expression
of GLUT4 and other adipocyte genes.,
insulin transiently induces the expression of C/EBP, as well as
both forms of C/EBP
(LAP and LIP). Western and gel-shift analyses
suggest that the level of LIP predominates over that of LAP or
C/EBP in both the basal and insulin-stimulated states ( Fig. 1and Fig. 3). Since LIP can act as a dominant
negative inhibitor of C/EBP-regulated gene transcription by forming
inactive heterodimers (37) , the induction of LIP by insulin
would most likely offset (or at least dampen) the increases in LAP or
C/EBP
, and would further accentuate the loss of C/EBP
. while
markedly increasing the expression of C/EBP and
C/EBP(48) . This results in the induction of a number of
acute-phase response proteins such as serum amyloid
A(49, 50) ,
-acid glycoprotein (51, 52, 53) , and complement component
C3(54) , whose gene promoters contain critical C/EBP-binding
sites.) causes an induction of C/EBP while
suppressing the expression of
C/EBP(55, 56, 57) . A variation of this
reciprocity is observed with 3T3-L1 adipocytes treated with
glucocorticoids(22) . While expression of C/EBP is
transiently decreased and C/EBP is transiently increased, no
change is observed in the expression of C/EBP
. The current report
demonstrates that in mature adipocytes, insulin also reciprocally
regulates the C/EBPs. In this case, C/EBP is persistently
suppressed, and both C/EBP and C/EBP are transiently induced.
The fact that different hormones/cytokines give rise to similar (i.e. reciprocal) patterns of expression of the C/EBP isoforms
both during (12) and after terminal cell differentiation (see
above) suggests that the C/EBPs play a central role in controlling gene
transcription in a variety of metabolic situations. Presumably, the
unique, but overlapping, sets of genes that are transcriptionally
activated or repressed in each metabolic state would depend on the
specific combination of active trans-acting factors (e.g. the
glucocorticoid receptor), the complement of C/EBP homo- and
heterodimers, and the cis-elements in each of the gene
promoters affected. This concept extends the hypothesis (58) that C/EBP
serves as a central regulator of energy
metabolism.
)))
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