(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) ,
, 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.
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 (
)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) .
C/EBP 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.
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 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.
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, 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).
The DNA fragment used as a probe
for C/EBP 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) .
Immune serum against a synthetic peptide
corresponding to an internal amino acid sequence of C/EBP (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) .
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 (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.
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 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.
Inspection of Fig. 1A reveals that p30 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.
Figure 2:
Rapid effect of insulin on C/EBP.
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
.
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 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
.
Figure 4:
Effect of insulin or IGF-1 concentration
on expression of C/EBP (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
.
Figure 5:
Comparison of the effect of insulin on the
kinetics of expression of C/EBP, 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.
Figure 6:
A) Effect of insulin on nuclear run-on
transcription of the C/EBP, 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.
To ascertain whether the
mobility differences were due to phosphorylation of the C/EBP
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/EBP
s (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).
Figure 7:
Effect of insulin and okadaic acid on the
post-translational modification of C/EBP. A, 3T3-L1
adipocytes were treated for 45 min with Me
SO (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.
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. ()Whether this phosphatase acts to
directly dephosphorylate C/EBP
or acts through a second messenger
pathway which leads to dephosphorylation remains unclear.
Ex
vivo labeling of 3T3-L1 adipocytes with
[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.
Figure 8:
Kinetic analysis of the effects of insulin
on expression of C/EBP 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.
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 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.
Figure 9:
Effect of chronic exposure to insulin on
the expression of C/EBP, 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.
This study shows that insulin reciprocally regulates the gene
encoding C/EBP, 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.
In addition to regulating the
post-translational modification and transcription of C/EBP,
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
.
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 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.
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 ) 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.