A Role for C/EBP
in Regulating Peroxisome
Proliferator-activated Receptor
Activity during Adipogenesis in
3T3-L1 Preadipocytes*
Jonathan K.
Hamm,
Bae Hang
Park, and
Stephen R.
Farmer
From the Department of Biochemistry, Boston University School of
Medicine, Boston, Massachusetts 2118
Received for publication, January 29, 2001
 |
ABSTRACT |
The differentiation of 3T3-L1 preadipocytes is
regulated in part by a cascade of transcriptional events involving
activation of the CCAAT/enhancer-binding proteins (C/EBPs) and
peroxisome proliferator-activated receptor
(PPAR
) by
dexamethasone (DEX), 3-isobutyl-1-methylxanthine (MIX), and insulin. In
this study, we demonstrate that exposure of 3T3-L1 preadipocytes to DEX
and insulin fails to induce adipogenesis as indicated by a lack of C/EBP
, PPAR
2, and adipose protein 2/fatty acid-binding protein expression; however, PPAR
1 is expressed. Treatment of these
MIX-deficient cells with a PPAR
ligand, troglitazone, induces
C/EBP
expression and rescues the block in adipogenesis. In this
regard, we also show that induction of C/EBP
gene expression by
troglitazone in C3H10T1/2 cells ectopically expressing PPAR
occurs
in the absence of ongoing protein synthesis, suggesting a direct
transactivation of the C/EBP
gene by PPAR
. Furthermore, ectopic
expression of a dominant negative isoform of C/EBP
(liver-enriched
transcriptional inhibitory protein (LIP)) inhibits the induction of
C/EBP
, PPAR
2, and adipose protein 2/fatty acid-binding protein by
DEX, MIX, and insulin in 3T3-L1 cells without affecting the induction
of PPAR
1 by DEX. Exposure of LIP-expressing preadipocytes to
troglitazone along with DEX, MIX, and insulin induces differentiation
into adipocytes. Additionally, we show that sustained expression of C/EBP
in these LIP-expressing adipocytes requires constant exposure to troglitazone. Taken together, these observations suggest that inhibition of C/EBP
activity not only blocks C/EBP
and PPAR
2 expression, but it also renders the preadipocytes dependent on an
exogenous PPAR
ligand for their differentiation into adipocytes. We
propose, therefore, an additional role for C/EBP
in regulating PPAR
activity during adipogenesis, and we suggest an alternative means of inducing preadipocyte differentiation that relies on the
dexamethasone-associated induction of PPAR
1 expression.
 |
INTRODUCTION |
The differentiation of preadipocytes into mature fat cells is
regulated by a cascade of transcription factors that interact in a
complex fashion to control expression of several hundred adipogenic
genes (1, 2). Many different nuclear factors have been shown to
influence the adipogenic process, but two families of factors in
particular have received the most attention as follows: the CCAAT
enhancer-binding proteins
(C/EBPs)1 and the peroxisome
proliferator-activated receptors (PPAR) family of nuclear hormone
receptors. Three members of the C/EBP family,
,
, and
, have
been shown to play important roles in regulating adipose tissue
development in mice and preadipocyte differentiation in
vitro (3). In contrast, only the
form of the PPAR family is
considered to regulate adipogenesis in vitro and in
vivo (4). The temporal pattern of expression of these important
adipogenic factors, and control of their activity during adipogenesis,
is dependent on a variety of biological effectors and other
transcription factors (5-7).
The C/EBPs belong to a larger family of basic leucine zipper (bZIP)
transcription factors, which have a C-terminal leucine zipper domain
for dimerization and a basic domain for binding to DNA. There are at
least six members of this family,
,
,
,
,
, and
,
that can both homodimerize and heterodimerize with each other and bind
to the same C/EBP regulatory element in the promoters/enhancers of many
different genes. In addition, each family member can give rise to
several isoforms by a process of selective use of translational start
sites within each mRNA or by proteolysis of a larger precursor
protein (8). The C/EBP
mRNA, for instance, gives rise to at
least four isoforms corresponding to the following peptides, 38, 34, 30, and 20 kDa. The 34-kDa protein is often referred to as LAP
(liver-enriched transcriptional activator protein) since it has been
shown to be a potent transactivator of liver gene expression (9). The
20-kDa polypeptide, however, can inhibit hepatic gene expression and
is, therefore, referred to as LIP (liver-enriched transcriptional
inhibitory protein) (9). This LIP isoform of C/EBP
corresponds to
the C-terminal portion of the LAP protein that lacks the
transactivation domain but contains the basic leucine zipper region.
Consequently, LIP can act as a potent dominant negative repressor of
C/EBP
activity. In fact, ectopic expression of LIP, resulting in a
LAP/LIP ratio of ~1, blocks adipogenesis in preadipocytes in culture
(10).
PPAR
exists as two protein isoforms,
1 and
2, that are
generated by alternative splicing of at least three different
mRNAs, which are transcribed from the same gene (11, 12). PPAR
1 and
2 share almost all the same exon sequences, except
2 contains an additional 30 amino acids at the N terminus. Both isoforms of
PPAR
form an obligate heterodimer with the retinoid X receptor to
bind to regulatory elements within the promoters/enhancers of many
genes associated with lipid metabolism. Activation of PPAR
·RXR complexes requires association with a series of
ligands that include RXR ligands such as 9-cis-retinoic acid
as well as ligands for PPAR
(13). The latter includes
polyunsaturated fatty acids and their derivatives as well as the
thiazolidinedione family of insulin sensitizers such as troglitazone
(14-16). Transcription from the PPAR
gene has been detected in many
tissues in which the
1 isoform is the predominant transcript (17).
In contrast, transcription from the PPAR
2 promoter is highly adipose
tissue-selective giving rise to abundant production of the PPAR
2
polypeptide in addition to the more ubiquitous PPAR
1 isoform (18).
Ectopic expression of PPAR
2 or -
1 in non-adipogenic
fibroblasts under appropriate hormonal conditions results in potent
induction of adipocyte differentiation (19).
Various mouse cell lines have been used to delineate the many different
processes involved in regulating adipogenesis. Most notable are 3T3-L1
preadipocytes, which can be induced to differentiate into mature fat
cells following exposure to a mixture of hormonal inducers including
dexamethasone (DEX), isobutylmethylxanthine (MIX), insulin, and FBS.
MIX and DEX induce expression of C/EBP
and C/EBP
, respectively,
which in turn activate C/EBP
and PPAR
expression (10, 20, 21).
PPAR
and C/EBP
are then capable of cross-activating each other as
well as governing expression of the mature adipocytic phenotype (22,
23). The normal differentiation of preadipocytes in culture does not
require addition of an exogenous PPAR
ligand. In contrast,
non-adipogenic fibroblasts that ectopically express a C/EBP or PPAR
require exposure to a potent PPAR
ligand to undergo conversion into
adipocytes (24, 25). Preadipocytes have likely acquired the ability to
produce an appropriate ligand of PPAR
. The molecular mechanisms that
regulate production of such molecules are not known. Earlier studies by
others (26) have suggested a role for the sterol regulatory
element-binding proteins (SREBPs).
We demonstrate that attenuation of C/EBP
activity by omitting MIX
from the culture medium or ectopically expressing a dominant negative
form of C/EBP
(LIP) renders 3T3-L1 preadipocytes dependent on an
exogenous PPAR
ligand for their differentiation into adipocytes. These studies have also uncovered an alternative pathway of
adipogenesis, which involves a glucocorticoid-associated induction of
PPAR
1 in the absence of C/EBP
activity. Furthermore, activation
of PPAR
1 in the LIP-expressing cells with troglitazone directly activates C/EBP
gene expression.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Dexamethasone, 3-isobutyl-1-methylxanthine,
insulin, puromycin, aprotinin, leupeptin, and digitonin were purchased
from Sigma. Dulbecco's modified Eagle's medium (DMEM) and fetal
bovine serum were supplied by Life Technologies, Inc.
[
32P]dCTP and [
-32P[ATP were
purchased from PerkinElmer Life Sciences. Klenow fragment of DNA
polymerase I was obtained from Promega (Madison, WI). Troglitazone was
from Parke-Davis/Warner Lambert.
Antibodies--
We used a monoclonal anti-PPAR
antibody and
polyclonal antibodies against the C/EBPs,
,
, and
(Santa Cruz
Biotechnology, Santa Cruz, CA), and polyclonal anti-aP2 serum (kindly
provided by Dr. David Bernlohr, University of Minnesota, MN).
Plasmids and Stable Cell Lines--
The BOSC 23 packaging cells
(27), pBabe-Puro and pBabe-PPAR
-Puro retroviral expression vectors
(19), were kind gifts of Dr. Bruce Spiegelman (Dana Farber Cancer
Institute, Harvard Medical School). The pBabe vector expressing either
the LAP or LIP isoforms of C/EBP
were constructed by subcloning
corresponding PCR products of the C/EBP
cDNA (20) into the
BamHI and EcoRI sites of the pBabe-puro vector.
The LAP PCR fragments were generated using the following primers:
c
1 (5'-CGCGGATCCCCACCATGGAAGTGGCCAACTT) and c
-3
(5'-CCGGAATTCGCATCAAGTCCCGAAACCCGGT), and the LIP PCR fragments were
generated using c
-2 (5'-CGCGGATCCCCACCATGGCGGCCGGCTT) and c
-3
primers. Transfection of BOSC 23 packaging cells and subsequent
infection of target cells were performed as described by others (19,
27). Infected target cells were selected for 6-10 days in medium
containing 2.0 µg/ml puromycin.
Cell Culture--
Murine 3T3-L1 preadipocytes were cultured,
maintained, and differentiated as described previously (28, 29).
Briefly, cells were plated and grown for 2 days post-confluence in DMEM
supplemented with 10% calf serum. Differentiation was then induced
(Day 0) by changing the medium to DMEM containing 10% FBS, 0.5 mM 3-isobutyl-1-methylxanthine, 1 µM
dexamethasone, and 1.67 µM insulin. After 48 h,
cells were maintained in DMEM containing 10% FBS. 3T3-L1 cells
expressing either C/EBP
LIP or control vector and 10T1/2 cells
expressing PPAR
were differentiated by the same protocol for 3T3-L1
cells, except growth medium was DMEM containing 10% FBS and 2.0 µg
of puromycin, and the cells were differentiated and maintained in the
presence or absence of 10 µM troglitazone, except as noted.
Oil Red O Staining--
Oil Red O staining was performed
following the procedure described previously (29). The cells were then
photographed using phase contrast microscopy.
Preparation of Whole Cell Extracts--
At the indicated times,
cultured cells grown in 10-cm dishes were rinsed with
phosphate-buffered saline (140 mM NaCl, 2.7 mM
KCl, 1.5 mM KH2PO4, 8.1 mM Na2HPO4, pH 7.4) and then
harvested in 1 ml of ice-cold buffer containing 50 mM Tris
(pH 7.4), 100 mM NaCl, 1% sodium deoxycholate, 4% Nonidet
P-40, 0.4% SDS, 5 µM aprotinin, and 50 µM
leupeptin. Lysates were vortexed for 1 min and centrifuged for 15 min
at full speed (13,000 rpm) in a microcentrifuge. Pellets were discarded
and supernatants stored at
80 °C. Protein content of supernatants
was determined using the BCA kit (Amersham Pharmacia Biotech).
Gel Electrophoresis and Immunoblotting--
Proteins were
separated in SDS-polyacrylamide (acrylamide from American
BioAnalytical) gels as described previously (23) and transferred to
polyvinylidene difluoride membrane (Bio-Rad) in 25 mM Tris,
192 mM glycine. After transfer, the membrane was blocked
with 4% nonfat dry milk in PBST for 1 h at room temperature. After incubation with the primary antibodies specified above, horseradish peroxidase-conjugated secondary antibodies (Sigma) and an
enhanced chemiluminescent (ECL) substrate kit (PerkinElmer Life
Sciences) were used for detection.
RNA Analysis--
Total RNA was harvested according to the
procedure of Chomczynski and Sacchi (30). Cells were lysed in
buffer containing 4 M guanidinium isothiocyanate. Lysates
were extracted with acid phenol/chloroform, and RNA was precipitated in
50% isopropyl alcohol overnight at
20 °C. Northern blot
analysis was performed on 20 µg of each sample RNA as described.
cDNA probes for C/EBP
and PPAR
were labeled using Klenow
fragment of DNA polymerase I and [
-32P[dCTP by random priming.
Preparation of Nuclear Protein Extracts--
Nuclear protein
extracts were prepared essentially as described. Cells were washed
twice with ice-cold phosphate-buffered saline and then lysed in nuclear
lysis buffer (10 mM Tris (pH 7.6), 10 mM NaCl,
3 mM MgCl2, 0.5% Nonidet P-40). Samples were spun at low speed in a clinical centrifuge. Supernatants were discarded, and nuclei were lysed in nuclear extraction buffer (20 mM HEPES (pH 7.9), 350 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA (pH 8.0),
25% glycerol). Nuclear extracts were incubated on ice for 15 min and
centrifuged at full speed (13,000 rpm) at 4 °C. The resulting
supernatants were stored at
80 °C. Protein concentrations were
determined using the BCA protein assay kit (Amersham Pharmacia Biotech).
Electrophoretic Mobility Shift Assay--
DNA binding assays
were performed as described previously (31). Ten micrograms of nuclear
extract was incubated with 3 µg of poly(dI-dC), 2 µl of carrier mix
(50 mM MgCl2, 340 mM KCl), and
delta buffer (0.1 mM EDTA, 40 mM KCl, 25 mM HEPES (pH 7.6), 8% Ficoll, 1 mM
dithiothreitol) at 4 °C for 15 min. Double-stranded oligonucleotides
corresponding to a C/EBP-binding site (5'-gatccGCGTTGCGCCACGATG-3' and
5'-CATCGTGGCGCAACGCggatc-3') were end-labeled with
[
-32P[ATP using T4 polynucleotide kinase according to
the manufacturer (New England Biolabs). For supershift experiments,
antibody was added, and samples were incubated at room temperature for
1.5 h. Reactions were mixed with labeled probes on ice for 30 min and resolved on nondenaturing 6% polyacrylamide (39.5:0.5
acrylamide/bisacrylamide) gels at 200 V for 2-2.5 h at 4 °C in TBE
buffer (80 mM Tris borate, 2 mM EDTA (pH 8.0)).
Gels were vacuum-dried for 1 h before exposure to Biomax MR-1
autoradiography film (Eastman Kodak Co.). Antibodies used for C/EBP
supershifts were the same as described above. Goat anti-rabbit IgG
antibodies (Sigma) were used in negative control experiments.
 |
RESULTS |
To understand the roles of various inducers and the C/EBPs in
regulating adipogenesis, we generated 3T3-L1 cell lines expressing vector DNA alone (designated L1-V cells) or the dominant negative C/EBP
, LIP (designated L1-LIP cells). To determine the effect of the
different inducers on expression of PPAR
and C/EBP
, we stimulated
L1-V cells to differentiate by exposure to different combinations of
insulin, dexamethasone (DEX), and isobutylmethylxanthine (MIX) in the
presence or absence of 10 µM troglitazone. Cells were
maintained according to the procedure described under "Experimental Procedures," and total protein was harvested 4 days after induction. Fig. 1, lane 7, shows the
abundant expression of PPAR
and C/EBP
by 4 days of
differentiation following exposure to DEX, MIX, insulin, and FBS. The
presence of troglitazone appears to attenuate expression of these
transcription factors (lane 8). Omission of DEX and/or MIX
from the culture medium significantly attenuates differentiation of
these preadipocytes into mature fat cells as indicated by the barely
detectable expression of C/EBP
and aP2 in each case (lanes 1, 3, and 5). Interestingly, PPAR
1 is abundantly
expressed in cells exposed to either DEX or MIX alone but not to the
same extent as that when the two inducers are used together (compare
lane 7 with lanes 3 and 5). PPAR
2,
however, appears to be more highly expressed in cells exposed to DEX
compared with those exposed to MIX (compare lane 5 with
lane 3). Activation of PPAR
1 in these differentiation-compromised cells (cells deprived of DEX or MIX) by
exposure to troglitazone resulted in an extensive induction of C/EBP
and aP2 (lanes 4 and 6) and a corresponding
conversion of the preadipocytes into morphologically distinct
adipocytes (data not shown).

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Fig. 1.
Effect of different effectors on
PPAR and C/EBP
expression. 3T3-L1 cells transfected with vector DNA alone
were induced to differentiate in media containing insulin and FBS in
the presence or absence of dexamethasone (D),
isobutylmethylxanthine (M), or 10 µM
troglitazone (Trog). Four days later, whole cell extracts
were prepared, and 100 µg of each sample was subjected to Western
blot analysis for PPAR , C/EBP , and aP2 expression. I,
1.67 µM insulin; D, 1 µM
dexamethasone; M, 0.5 mM isobutylmethylxanthine;
CRM, cross-reacting material.
|
|
Earlier studies by us and others (10, 21, 29) have shown that one of
the mechanisms by which DEX and MIX induce adipogenesis is to enhance
expression of C/EBP
and C/EBP
during the initial few hours of
adipogenesis in 3T3-L1 cells, which in turn activate expression of
PPAR
and C/EBP
. Fig. 1 suggests that these effectors may in fact
be regulating two separate pathways in which DEX alone can induce
PPAR
1 expression, but MIX is required along with DEX for C/EBP
expression. Consequently, since MIX has been shown to regulate C/EBP
(20), we questioned whether the induction of C/EBP
by troglitazone
in cells deprived of MIX was due to a corresponding
troglitazone-associated induction of C/EBP
. In the experiment
presented in Fig. 2, 3T3-L1 cells were
exposed to insulin and DEX in the presence or absence of MIX or
troglitazone, and total protein extracts harvested at the indicated
times were subjected to Western blot analysis. At 4 h
post-induction, C/EBP
is expressed in cells treated with the
complete set of inducers (lanes 3 and 4), whereas
very little C/EBP
is produced in the absence of MIX (Fig. 2,
lanes 1 and 2). Addition of troglitazone has no
significant effect on C/EBP
expression in the presence or absence of
MIX (compare lane 1 with lane 2, and lane
3 with lane 4). This figure also shows that expression
of PPAR
1 occurs much earlier (at 24 h) in cells exposed to
mixture lacking MIX than cells cultured in the complete mixture (Fig.
1, compare lanes 5 and 7). By 72 h after
treatment, PPAR
1 is abundantly expressed in both populations of
cells (minus or plus MIX); however, PPAR
2 and C/EBP
are expressed
to any significant extent only in cells exposed to DEX and MIX (compare
lanes 9 and 11). The presence of troglitazone
resulted in an extensive induction of C/EBP
and PPAR
2 in
MIX-deprived cells but also enhanced expression of C/EBP
in cells
exposed to MIX.

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Fig. 2.
Time course of protein expression during
adipogenesis with and without MIX and troglitazone. At the
indicated times following induction of differentiation of L1-vector
cells in the presence or absence of isobutylmethylxanthine
(ID versus DIM) and troglitazone
(Trog), total protein extracts were collected and analyzed
by Western blot analysis for expression of PPAR , C/EBP , and
C/EBP .
|
|
The induction of C/EBP
by troglitazone suggests that PPAR
may be
capable of directly transactivating the C/EBP
gene. To test this
idea, we ectopically expressed PPAR
2 in C3H10T1/2 mesenchymal stem
cells to create a cell line (10T-P
) whose differentiation into
adipocytes was dependent on an exogenous PPAR
ligand. The Northern
blot in Fig. 3 shows that exposure of
these cells to 10 µM troglitazone results in the
induction of C/EBP
mRNA expression that also occurs in the
absence of ongoing protein synthesis (+ cycloheximide). These data are
consistent with the notion that PPAR
is interacting directly with
the C/EBP
gene to enhance C/EBP
mRNA production.

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Fig. 3.
PPAR induces
C/EBP mRNA expression in the absence of
ongoing protein synthesis. 10T1/2 cells ectopically expressing
PPAR 2 were exposed to DMEM containing 1 µM DEX, 0.5 mM MIX, 1.67 µM insulin, and 10% FBS for
48 h. Cells were maintained in 10% FBS for an additional 24 h and then treated with or without cycloheximide (CHX, 5 µg/ml), in the presence or absence of 5 µM troglitazone
(Trog). Total RNA was harvested at the indicated times
post-treatment and analyzed by Northern blot for C/EBP and PPAR
mRNA expression.
|
|
To determine if the selective effect of omitting MIX on C/EBP
compared with PPAR
1 expression was primarily due to its role in
regulating C/EBP
, we generated a 3T3-L1 cell line ectopically expressing a dominant negative isoform of C/EBP
(designated L1-LIP cells). Fig. 4A shows abundant
expression of LIP (3rd lane) in proliferating
3T3-L1 preadipocytes compared with undetectable levels of this
polypeptide in the vector cells (1st lane). Also shown is the ectopic expression of the LAP (34 kDa) isoform of C/EBP
in a corresponding L1-LAP cell line (2nd lane).
As observed previously by McKnight and co-workers (10), ectopic
expression of LIP completely blocks the ability of DEX, MIX, insulin,
and FBS to induce the differentiation of 3T3-L1 preadipocytes to
differentiate into fat cells. The data shown in Fig. 4B
confirm this result, but they also show that troglitazone can reverse
this inhibitory action of LIP as judged by the accumulation of Oil Red
O-positive lipid droplets following exposure of the L1-LIP cells to
troglitazone.

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Fig. 4.
Ectopic expression of a dominant negative
isoform of C/EBP (LIP) inhibits adipogenesis
in 3T3-L1 preadipocytes. A, total proteins harvested
from proliferating preadipocytes, which ectopically express either
vector (V), LAP, or LIP were subjected to Western blot
analysis using an anti-C/EBP antibody. CRM,
cross-reacting material. B, confluent L1-vector and L1-LIP
cells were induced to differentiate in the presence or absence of
troglitazone (Trog). At day 7, cells were fixed and stained
for neutral lipids with Oil Red O.
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|
To gain insight into the effect of LIP and troglitazone on the
differentiation of preadipocytes, L1-LIP cells were induced to
differentiate as in Fig. 1, where MIX and/or DEX were omitted from the
culture medium. Extracts of total protein were then harvested at day 4 and subjected to Western blot analysis. Fig.
5 shows that expression of LIP greatly
attenuates adipogenesis under all hormonal conditions as indicated by a
lack of C/EBP
and aP2 expression (lanes 1, 3, 5, and
7). DEX is capable of enhancing PPAR
1 expression above
the low basal levels produced in the presence of insulin and FBS, but
it has no positive effect on PPAR
2 expression (compare lane
1 and 3). Addition of troglitazone along with DEX
induces adipogenesis as indicated by the expression of C/EBP
,
PPAR
2, and aP2. It is worth noting that DEX has a similar effect on
gene expression in the presence or absence of LIP (compare Fig. 5, lanes 3 and 4 with Fig. 1, lanes 3 and
4). In contrast, exposure of LIP cells to MIX and insulin
only slightly enhances PPAR
1 with no PPAR
2 expression, and when
troglitazone is added under these conditions it does not induce
adipogenesis (i.e. minimal aP2 and C/EBP
expression).
This pattern of gene expression differs significantly from that
observed in the absence of LIP. Specifically, MIX induces both PPAR
1
and -2 in the L1-vector cells, and consequently, exposure of these
cells to troglitazone promotes adipogenesis (compare Fig. 1,
lanes 5 and 6, with Fig. 4, lanes 5 and 6). Taken together, these data are consistent with a
model in which DEX is capable of priming the preadipocytes to be
responsive to troglitazone even in the absence of C/EBP
; this likely
involves induction of PPAR
1 expression. MIX, however, is only
capable of a similar priming process if C/EBP
is actively expressed
in the absence of LIP. These data also strongly suggest that inhibiting
C/EBP
activity blocks production of an endogenous activator of
PPAR
, which renders the 3T3-L1 preadipocytes dependent on an
exogenous PPAR
ligand for their differentiation into adipocytes.

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Fig. 5.
Troglitazone induces adipogenic gene
expression in L1-LIP cells exposed to dexamethasone. 3T3-L1
preadipocytes expressing LIP were induced to differentiate by exposure
to the indicated combinations of the adipogenic inducers, DEX
(D), MIX (M), and insulin (I) in the
presence or absence of 10 µM troglitazone
(Trog). At 4 days post-induction, whole cell proteins were
extracted and analyzed by Western blot for expression of PPAR ,
C/EBP , and aP2.
|
|
To gain more insight into the ligand dependence of these LIP-expressing
preadipocytes, we analyzed the temporal pattern of gene expression
following exposure to troglitazone as well as determining the optimum
dose of troglitazone required to induce adipogenesis. In the experiment
shown in Fig. 6, confluent L1-LIP cells
were exposed to DEX, MIX, and insulin in the presence or absence of
troglitazone, and total cellular proteins were subjected to Western
blot analysis. The combination of DEX, MIX, and insulin is capable of
initiating the early phase of adipogenesis in these LIP-expressing
cells as indicated by induction of C/EBP
as well as PPAR
1
(compare lane 2 and 4 with lane 1).
Exposure of these cells to troglitazone appears to have no significant
effect on this pattern of gene expression during the first 2 days.
After this time, however, troglitazone is essential for the induction of C/EBP
and aP2 expression. Taken together, the studies shown above
demonstrate that culturing LIP cells in troglitazone for 6 days, along
with an initial priming with DEX, MIX, and insulin, results in their
conversion into adipocytes based on accumulation of lipid droplets in
>95% of the cells (Fig. 4B) and the abundant expression of
PPAR
2, C/EBP
, and aP2 (Fig. 6). To establish the troglitazone
dose dependence of LIP cells, both L1-LIP and L1-V cells were exposed
to differentiation medium containing DEX, MIX, insulin, and increasing
concentrations of troglitazone. Total protein samples were harvested 6 days later and subjected to Western blot analysis of the indicated
proteins. Fig. 7 demonstrates that expression of both C/EBP
and PPAR
2 increased substantially with increasing doses of troglitazone. Expression of aP2 also seemed proportionate to troglitazone concentration, correlative to the number
of cells accumulating lipid droplets (data not shown). LIP expression
was unaffected by the PPAR
ligand.

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Fig. 6.
Time course of adipogenic gene expression
following exposure of L1-LIP cells to troglitazone. L1-LIP cells
were induced to differentiate with DEX, MIX, insulin, and FBS in the
presence or absence of 10 µM troglitazone
(Trog). Total protein extracts were harvested on the
indicated days after induction. Western blot analysis was performed as
described using antibodies for the indicated proteins.
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Fig. 7.
Troglitazone dose-dependent
rescue of adipogenic gene expression in L1-LIP cells. L1-LIP cells
and L1-V cells (L1) were induced to differentiate as in Fig.
6 in the presence of varying concentrations of troglitazone
(Trog). Six days after induction, whole cell proteins were
harvested and subjected to Western blot analysis for expression of the
indicated proteins. L1-V controls (0 and 10 µM
troglitazone) are included.
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|
The LIP polypeptide retains the C-terminal basic leucine zipper region
of the full-length C/EBP
protein, and therefore, it can dimerize
with other C/EBP
isoforms and bind to DNA. It was important,
therefore, to determine what effect LIP and/or troglitazone may have on
the DNA binding activity of the different C/EBPs during adipogenesis.
Consequently, L1-V and L1-LIP cells were treated to differentiate in
the presence or absence of troglitazone, and nuclear proteins were
harvested at day 1 and day 6. The electrophoretic mobility shift assay
presented in Fig. 8 shows binding of
nuclear protein complexes to an oligonucleotide corresponding to the
C/EBP regulatory element within the promoter of the C/EBP
gene (31). The profile and intensity of binding observed at day 1 is very similar
in the LIP and vector cells, with one exception. There is a faster
migrating species in the LIP cells that likely corresponds to LIP-LIP
homodimers (Fig. 8A). Troglitazone has no significant effect
on the overall binding activity in either cell line. Fig. 8A
also shows that the complexes present in L1-V samples at day 6 migrate
with a slightly larger mass than the day 1 complexes, which is probably
due to the presence of C/EBP
. This same shift in migration is
observed in the LIP samples following exposure to troglitazone for 6 days. To examine the composition of these DNA-protein complexes, a
series of supershift assays were performed using antibodies
corresponding to C/EBP
, C/EBP
, and C/EBP
. Fig. 8B, lane
4, demonstrates that a proportion of the complexes expressed at
day 6 in L1-V cells consist of C/EBP
homodimers. As expected, there
is a significant increase in C/EBP
binding activity in LIP cells
following exposure to troglitazone for 6 days (Fig. 8B,
compare lane 12 with lane 8). In fact, the
C/EBP
binding activity is slightly higher in the LIP cells plus
troglitazone compared with that expressed in the vector cells.
Furthermore, ectopic expression of LIP has not affected the ability of
C/EBP
to bind to the C/EBP regulatory element. This figure also
shows the existence of LIP-LIP homodimers binding to the C/EBP
oligonucleotide since the faster migrating complexes present in the LIP
cells can be supershifted selectively with an anti-C/EBP
antibody
(lanes 6 and 10). C/EBP
is minimally expressed
at 6 days under all conditions since there is no detectable supershift
with an anti-C/EBP
antibody.

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Fig. 8.
Changes in C/EBP DNA binding activity during
adipogenesis in L1-V and L1-LIP cells. L1-V and L1-LIP cells were
treated to differentiate in the presence or absence of 10 µM troglitazone (Trog). A, at 1 and
6 days after induction (d1 and d6, respectively),
nuclear proteins were harvested and subjected to electrophoretic
mobility shift assay, as described under "Experimental Procedures."
B, supershift analysis of C/EBP DNA binding activity in L1-V
and L1-LIP cells induced in the presence or absence of troglitazone.
Day 6 nuclear protein samples from L1-V and L1-LIP cells induced to
differentiate in the standard inducers, with or without troglitazone,
were analyzed by supershift analysis using antibodies to C/EBP
(C ), C/EBP (C ), C/EBP
(C ), or IgG ( ).
|
|
Our observation that LIP cells require an exogenous PPAR
ligand such
as troglitazone for their complete conversion into adipocytes suggests
that they are not capable of producing the endogenous ligand(s) or
activators. Alternatively, it is possible that they express PPAR
1 at
levels below a threshold required for activation by the endogenous
activator(s). To determine the reason for the ligand dependence of the
LIP cells, we induced PPAR
and C/EBP
to fully differentiated
levels by exposing LIP cells to DEX, MIX, insulin, and 10 µM troglitazone for 6 days. We then questioned whether
these LIP adipocytes still require the exogenous PPAR
ligand to
maintain normal adipocyte gene expression. This was achieved by
withdrawing troglitazone from half the cultures at day 6 and measuring
expression of PPAR
, C/EBP
, and aP2 in these and a control set of
cultures that were maintained in troglitazone for the entire
experiment. The Western blot in Fig. 9
shows abundant levels of PPAR
, C/EBP
, and aP2 following 7 days of
exposure of LIP cells to troglitazone. Withdrawal of the exogenous
ligand at day 6, however, results in an extensive dedifferentiation as indicated by a drop in expression of C/EBP
and aP2 to virtually undetectable levels by day 10 (4 days of withdrawal). Interestingly, the abundance of both PPAR
1 and -
2 remains constant throughout this period even in the absence of troglitazone. Notably, expression of
LIP does not increase when troglitazone is withdrawn; on the contrary,
it appears to decrease (Fig. 9).

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Fig. 9.
L1-LIP cells require exposure to troglitazone
throughout the differentiation process in order to maintain adipogenic
gene expression. L1-LIP cells were induced to differentiate and
maintained in the presence of 10 µM troglitazone
(Trog). Six days after induction, troglitazone was either
withdrawn (WD) from the media or maintained at a
concentration of 10 µM (+). Total protein extracts were
harvested at 1 (day 7, d7), 2 (day 8, d8), 3 (day
9, d9), and 4 (day 10, d10) days later. Samples
were subjected to Western blot analysis for PPAR , C/EBP , aP2, and
LIP expression.
|
|
 |
DISCUSSION |
The differentiation of 3T3-L1 cells into mature adipocytes
requires their exposure to a mixture of hormonal inducers including DEX, MIX, insulin, and FBS. These effectors have been shown to activate
a cascade of transcriptional events that culminate in expression of the
mature adipocytic phenotype. Most notably, they facilitate the
induction of C/EBP
and C/EBP
, which together activate expression
of PPAR
and C/EBP
(10, 20, 21, 29). The data presented in this
study suggest an additional role for C/EBP
, and the effectors that
control its expression, in regulating the production of PPAR
ligands. These studies further show that adipogenesis can be induced in
3T3-L1 preadipocytes in the absence of C/EBP
by exposing the cells
to an exogenous PPAR
ligand. This alternative mechanism appears to
depend on the ability of insulin and DEX to induce PPAR
1 expression,
which is then capable of inducing C/EBP
and PPAR
2 expression
following exposure to troglitazone. Induction of C/EBP
gene
expression in the absence of an exogenous PPAR
ligand depends on MIX
and/or C/EBP
expression. It appears, therefore, that expression of
C/EBP
during adipogenesis can be regulated by at least two
independent pathways. One pathway involves a MIX-associated induction
of C/EBP
, which transactivates a C/EBP regulatory element within the
promoter of the C/EBP
gene (32). The other mechanism can occur in
the absence of C/EBP
due to a DEX-associated induction of PPAR
1,
which is also capable of transactivating the C/EBP
gene in the
presence of troglitazone.
Previous studies have shown that an important role for MIX and DEX is
to induce expression of C/EBP
and C/EBP
, respectively, which in
turn activate C/EBP
and PPAR
2 expression through C/EBP regulatory
elements in the promoters of the corresponding genes (10, 31-34). Our
data are consistent with these observations since inhibition of
C/EBP
activity by either omitting MIX or expressing LIP blocks both
C/EBP
and PPAR
2 expression. Of interest is the observation that
DEX can induce PPAR
1 expression in the absence of C/EBP
activity,
which may be due to a DEX-associated induction of C/EBP
. Other
studies, however, have shown that ectopic expression of C/EBP
alone
in 3T3 fibroblasts does not induce C/EBP
, PPAR
1, or -
2
expression (10, 29). Despite the abundant expression of PPAR
1 in the
C/EBP
-deficient preadipocytes, they are incapable of expressing the
adipogenic program unless exposed to an exogenous PPAR
ligand
(i.e. troglitazone) along with the normal mixture of
hormonal inducers. This observation suggests that C/EBP
may play a
role in regulating processes that lead to production of PPAR
ligands/activators. Further support for this notion are the data in
Fig. 9 showing a continuing requirement of the LIP cells for
troglitazone to maintain adipogenic gene expression even after they
have completely converted into adipocytes by a 6 -day exposure to the
PPAR
ligand.
Mechanisms by Which C/EBP
May Regulate PPAR
Activity--
The most likely determinant of PPAR
activity is the
availability of ligands within the preadipocyte. Even though the
natural cellular ligand for PPAR
has not been identified, evidence
suggests that derivatives of polyunsaturated fatty acids are potent
activators of PPAR
both in in vitro assays as well as
in vivo (13, 16) Mechanisms that control the cellular
production of polyunsaturated fatty acids or their derivatives may play
an important role in regulating adipogenesis. In this regard, studies
have shown that ADD1/SREBP-1, a transcription factor that is linked to
processes controlling fatty acid production, appears to be involved in
the production of endogenous PPAR
ligands (26). In fact,
ADD1/SREBP-1 is induced early during adipogenesis, and its ectopic
expression in non-adipogenic cells can enhance fat cell formation by
directly activating the PPAR
2 gene as well as stimulating production
of PPAR
ligands (26, 35, 36). It is conceivable, therefore, that a
role for C/EBP
in regulating PPAR
activity may involve induction
and/or activation of ADD1/SREBP-1.
PPAR
Ligand-dependent Induction of C/EBP
Expression--
Several investigations have demonstrated that
activation of PPAR
in a variety of different fibroblast lines
results in expression of many adipogenic genes including C/EBP
(19,
22-25, 37). Similarly, ectopic expression of C/EBP
in
non-adipogenic cells can induce PPAR
expression (22, 23). In fact,
Spiegelman and co-workers (22) have suggested that cross-regulation
between C/EBP
and PPAR
is important in maintaining the
differentiated state. The molecular mechanisms involved in such a
cross-regulatory process are not known. It is very likely that C/EBP
directly transactivates PPAR
2 gene expression through the C/EBP
regulatory elements within the PPAR
2 promoter (33, 34). The data
presented in Fig. 3 suggest that PPAR
may also be capable of a
similar direct transactivation of the C/EBP
gene based on the
observation that activation of PPAR
by troglitazone in 10T1/2 cells
induces C/EBP
mRNA expression in the absence of ongoing protein
synthesis. For most PPAR
target genes, PPAR
initiates
transcription by binding to cognate PPAR regulatory elements at DR-1
sites within the promoter/enhancer regions of the genes (18, 38). It
seems likely that similar DR-1 sites exist within the C/EBP
gene.
Analysis of sequences in the 5'-flanking region of the C/EBP
gene
have identified DR-1 elements that bind strongly to particular COUP-TF proteins, but very weakly to
PPAR
.2 We are presently
determining whether this or any other elements facilitate the
PPAR
-dependent induction of C/EBP
gene expression.
What Are the Roles of C/EBP
and/or C/EBP
in Regulating
Adipogenesis?--
Several studies performed in a variety of cultured
cell systems have led to a model for the transcriptional control of
adipogenesis, which involves the sequential activation of C/EBPs and
PPAR
. The function of C/EBP
and C/EBP
in this process is to
induce expression of both PPAR
2 and C/EBP
. Investigations using
mice lacking C/EBP
and/or C/EBP
suggest an alternative role for
these C/EBPs in regulating adipose tissue formation and function in the
animal (39). Specifically, C/EBP
(
/
)·C/EBP
(
/
) mice express defects in lipid accumulation despite normal expression of
C/EBP
and PPAR
. However, primary embryonic fibroblasts derived from these knock-out animals have lost the potential to undergo adipogenesis and, in so doing, do not express PPAR
or C/EBP
in
response to DEX, MIX, insulin, and FBS. Taken together, these observations suggest a role for C/EBP
and/or C/EBP
in regulating PPAR
and C/EBP
gene expression in cultured cells (cell lines or
mouse embryonic fibroblasts) exposed to a restricted set of inducers. In preadipocytes in adipose tissue, however, PPAR
and C/EBP
may be activated by an alternative mechanism due to the presence of effectors not present in the culture system. As mentioned above, there appears to be a role for C/EBP
and C/EBP
in
facilitating the formation and function of adipose tissue in
vivo that is independent of C/EBP
and PPAR
expression,
which may involve production of PPAR
ligands.
In summary, we propose an alternative model for the transcriptional
control of adipogenesis (Fig. 10) that
incorporates the conclusions drawn from these studies with those
already presented by others (10, 21, 22). In this model, C/EBP
and
C/EBP
regulate production of PPAR
ligands as well as PPAR
2 and
C/EBP
expression. Additionally, we suggest that physiological
effectors can induce expression of PPAR
1 in the absence of C/EBP
and C/EBP
as part of a default pathway. This event can then initiate
a cascade of transcription factor expression, commencing with C/EBP
,
which in turn induces expression of the entire adipogenic program
providing the preadipocyte is exposed to PPAR
ligands. Further
dissection of the transcriptional events that regulate production of
PPAR
ligands should provide a greater understanding of the processes controlling adipogenesis.

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|
Fig. 10.
Transcriptional control of
PPAR ligand production is a central component
of the signaling cascade that regulates adipogenesis. Initiation
of adipogenesis involves induction of C/EBP , C/EBP , and PPAR 1
in response to exposure of preadipocytes to a variety of physiological
effectors including insulin, glucocorticoids, and agonists that elevate
cAMP. C/EBP and C/EBP activate expression of C/EBP and
PPAR 2 as well as stimulate a pathway that leads to production of
PPAR ligands. Ligand-activated forms of PPAR 1 and PPAR 2 can
directly induce expression of C/EBP to establish a positive feedback
loop in which C/EBP maintains expression of the PPARs. The
synergistic activity of C/EBP and PPAR ensures expression of the
entire adipogenic gene program.
|
|
 |
ACKNOWLEDGEMENTS |
We thank Drs. Bruce Spiegelman, David
Bernlohr, and Steve McKnight for their generosity in providing
reagents. We also thank Dr. Marthe Moldes for critical reading of the
manuscript and valuable suggestions.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant DK51586.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biochemistry,
Boston University School of Medicine, 715 Albany St., Boston, MA 02118. Tel.: 617-638-4186; Fax: 617-638-5339; E-mail:
farmer@biochem.bumc.bu.edu.
Published, JBC Papers in Press, February 27, 2001, DOI 10.1074/jbc.M100797200
2
Y. Xie, J. Hamm, and S. Farmer, unpublished data.
 |
ABBREVIATIONS |
The abbreviations used are:
C/EBP, CCAAT/enhancer-binding protein;
DMEM, Dulbecco's modified Eagle's
medium;
DEX, dexamethasone;
MIX, 3-isobutyl-1-methylxanthine;
FBS, fetal bovine serum;
PPAR, peroxisome proliferator-activated receptor;
aP2, adipose protein 2/fatty acid-binding protein;
LIP, liver-enriched
transcriptional inhibitory protein;
LAP, liver-enriched transcriptional
activator protein;
PCR, polymerase chain reaction;
SREBP, sterol
regulatory element-binding protein.
 |
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