Peroxisome Proliferator Activated Receptor ?, CCAAT/
Enhancer-binding Protein ?, and Cell Cycle Status Regulate
the Commitment to Adipocyte Differentiation*
(Received for publication, May 9, 1997, and in revised form, June 20, 1997)
Dalei Shao and Mitchell A. Lazar‡
From the Division of Endocrinology, Diabetes, and Metabolism, Departments of Medicine and Genetics,
University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
Terminal differentiation of stem cells is characterized
by cessation of cell proliferation as well as changes in
cell morphology associated with the differentiated state.
For adipocyte differentiation, independent lines of evi-
dence show that the transcription factors peroxisome
proliferator activated receptor ? (PPAR?) and CCAAT/
enhancer-binding protein ? (C/EBP?) as well as the tu-
mor suppressor retinoblastoma (Rb) protein are essen-
tial. How these proteins promote adipocyte conversion
and how they function cooperatively during the differ-
entiation process remain unclear. We have used retinoic
acid (RA) inhibition of adipogenesis to investigate these
issues. RA blocked adipogenesis of 3T3-L1 cells induced
to differentiate by ectopic expression of PPAR? and
C/EBP? independently or together. However, under
these circumstances RA was only effective at preventing
adipogenesis when added prior to confluence, suggest-
ing that factors involved in regulation of the cell cycle
might play a role in establishing the commitment state
of adipogenesis that is insensitive to RA. During differ-
entiation of wild type 3T3 L1 preadipocytes, we found
that Rb protein is hyperphosphorylated early in adipo-
genesis, corresponding to previously quiescent cells re-
entering the cell cycle, and later becomes hypophospho-
rylated. The data suggest that, together with the
coexpression of PPAR? and C/EBP?, permanent exit
from the cell cycle establishes the irreversible commit-
ment to adipocyte differentiation.
The molecular mechanisms relating to cell proliferation and
cell differentiation are inadequately understood. Adipocyte
conversion provides an excellent model system to study termi-
nal differentiation. In the case of 3T3-L1 cells, differentiation is
induced upon exposure of cells to a mixture of hormonal stim-
ulants including dexamethasone, isobutylmethylxanthine, in-
sulin, and fetal calf serum (1, 2). These pharmacological stim-
uli, or alternatives such as thiazolidinediones or other
activators of peroxisome proliferator activated receptors
(PPARs)1(3–6), are necessary for adipocyte differentiation of
3T3-L1 cells. During adipocyte conversion, a variety of tran-
scription factors are induced, including C/EBP?, PPAR?, and
C/EBP? (reviewed in Ref. 7). Enforced expression of PPAR? (8),
C/EBP? (9–11), or C/EBP? (10, 12, 13) stimulates adipogenesis
in NIH 3T3 fibroblasts, suggesting the essential roles of these
transcription factors in regulating adipogenesis. Furthermore,
combined expression of PPAR? and C/EBP? has synergistic
effects on promoting fat cell conversion in myoblasts (14).
Therefore, it is likely that PPAR? and C/EBP? function coop-
eratively to establish terminal adipocyte differentiation.
In addition to the expression of differentiated marker genes,
terminal differentiation is characterized by permanent with-
drawal of cells from the cell cycle. One protein that is involved
in cell cycle progression is the retinoblastoma susceptibility
gene product, Rb (15). Hypophosphorylation of Rb inhibits cell
cycle progression, and this inhibitory effect of Rb is lost upon
phosphorylation of the protein (16, 17). The involvement of Rb
in adipocyte differentiation is suggested by the observation
that ectopic expression of protein kinase C? in quiescent NIH
3T3 cells induces hypophosphorylation of Rb and promotes
adipogenesis (18), whereas Rb binding by SV40 large T antigen
interferes with adipogenic differentiation (19). Moreover,
Rb?/?mouse embryonic lung fibroblasts failed to undergo adi-
pocyte differentiation under appropriate conditions, and ec-
topic expression of Rb restored the adipogenic phenotype of the
Rb?/?cells (20). Together, these observations suggest an es-
sential role of Rb in adipocyte differentiation.
To explore early events in adipogenesis, we have used reti-
noic acid (RA), which normally inhibits adipocyte differentia-
tion of 3T3-L1 cells (21–23). Liganded RAR blocks C/EBP-
adipogenesis due to ectopic expression of C/EBP? or -? (13).
However, during normal adipogenesis RA exerts its inhibitory
function only when added in the first 24–48 h after exposure to
differentiating stimuli that are applied postconfluence. The
inhibitory function of RA is mediated by RA receptors (RARs),
which are down-regulated early in adipocyte differentiation
(23). However, ectopic expression of RAR in 3T3 L1 cells only
extends the period during which RA is effective in preventing
adipogenesis by an additional 24–48 h. Interestingly, at this
time, although PPAR? is already induced, the level of PPAR?
protein is diminished upon RA treatment. This could be ex-
plained by the fact that RA/RAR inhibits C/EBP function,
which may be responsible for maintaining the level of PPAR
during differentiation (23).
We first examined the effect of RA on adipocyte differentia-
tion due to incubation of wild type 3T3-L1 cells with the PPAR?
ligand BRL49653, as well as in 3T3-L1 cells that ectopically
express PPAR?. RA inhibited PPAR? activator-mediated fat
cell conversion, suggesting that RA blocks adipogenesis due to
endogenous PPAR?. The inhibitory effects of RA were also
* This work was supported by National Institutes of Health Grant
DK49780. The costs of publication of this article were defrayed in part
by the payment of page charges. This 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: University of Penn-
sylvania School of Medicine, 611 CRB, 415 Curie Blvd., Philadelphia,
PA 19104-6149; Tel.: 215-898-0198; Fax: 215-898-5408; E-mail:
1The abbreviations used are: PPAR, peroxisome proliferator acti-
vated receptor; C/EBP, CCAAT/enhancer-binding protein; RA, retinoic
acid; GM, growth medium; differentiation medium; Rb, retinoblastoma;
THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 1997 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 272, No. 34, Issue of August 22, pp. 21473–21478, 1997
Printed in U.S.A.
This paper is available on line at http://www.jbc.org
by guest on November 4, 2015
dominant over ectopic co-expression of both PPAR? and
C/EBP? when the cells were maintained in RA at the time of
gene transduction and thereafter. However, if added after gene
transduction but rather at the time of confluency, RA was no
longer effective at blocking differentiation of 3T3-L1 cells that
ectopically expressed these adipogenic transcription factors.
Thus, co-expression of PPAR? and C/EBP? in cells was not
sufficient to commit cells to undergo differentiation in the
presence of RA until the cells became postconfluent. Since
confluency is associated with withdrawal from the cell cycle, we
hypothesized that the stage of irreversible commitment to adi-
pocyte differentiation required exit from the cell cycle as well as
the co-expression of both PPAR? and C/EBP?. Indeed, during
adipocyte differentiation of wild type 3T3-L1 cells, confluent
cells undergo clonal expansion followed by permanent with-
drawal from the cell cycle that occurs at about the time RA
loses effectiveness in preventing differentiation. During this
process Rb protein shifts from a highly phosphorylated state to
its hypophosphorylated form. We conclude that the state of
RA-resistant commitment to adipocyte differentiation involves
not only expression of PPAR? and C/EBP? but also hypophos-
phorylation of Rb and withdrawal from the cell cycle.
MATERIALS AND METHODS
Cell Culture and Differentiation—3T3-L1 cells were obtained from
the American Type Culture Collection (ATCC, Rockville, MD). Cells
were cultured in growth medium (GM) containing 10% iron-enriched
fetal bovine serum in Dulbecco’s modified Eagle’s medium. For stand-
ard adipocyte differentiation, 2 days after cells reached confluency
(referred as day 0), cells were exposed to differentiation medium (DM)
containing 10% fetal bovine serum, 10 ?g/ml of insulin, 1 ?M dexa-
methasone, and 0.5 ?M isobutylmethylxanthine, for 48 h. Cells then
were maintained in postdifferentiation medium containing 10% fetal
bovine serum, and 10 ?g/ml of insulin. RA was dissolved in ethanol and
used at a concentration of 10 ?M. For BRL49653-induced adipogenesis,
cells were maintained in GM. 1 ?M BRL49653 was added to cells on day
0. Cells were exposed to BRL49653 constantly for 7–10 days until fat
cells were seen. For experiments involving RA, retrovirally infected
cells were studied in the following two protocols: 1) cells were main-
tained in the constant presence of 10 ?M RA from the time of infection
and then exposed to the presence or absence of 1 ?M BRL49653 at day
0, or 2) cells were grown in the absence of RA until day 0 and then
exposed to RA and various conditions. The first of these two conditions
corresponds to that described previously (13).
Construction of Plasmids and Retroviral Infection—pLXSN-PPAR?2
was generated by insertion of a 1.7-kilobase pair SalI fragment of
mouse PPAR?2 cDNA into the XhoI site of pLXSN (neor) (24). pTS13-
C/EBP? was generated by insertion of a 1.26 kilobase pair EcoRI-
BamHI fragment of C/EBP? into the BamHI site of the TS13 vector
(HgmBr). Standard calcium phosphate-DNA transfections were per-
formed. To generate retrovirus-producing packaging cells, 293T cells
were transfected with 7.5 ?g of plasmid DNA and viral gag and pol
plasmids (25). 48 h post-transfection, filtered viral supernatants from
the ecotropic packaging cell line were used to infect 3T3 L1 cells. Two
days after infection, cells were selected in G418 (400 ?g/ml; Life Tech-
nologies, Inc.) or hygromycin B (200 ?g/ml) for 10–14 days. For double
infection, pLXSN-PPAR? cells were infected with virus containing
TS13-C/EBP? for 48 h. Cells then were selected in G418 plus hygromy-
cin growth medium.
Oil Red O Staining—Dishes were washed three times with phos-
phate-buffered saline, fixed by 10% formalin in phosphate buffer for 1 h
at room temperature. After fixation, cells were washed once with phos-
phate-buffered saline and stained with a filtered oil red O stock solution
(0.5 g of oil red O (Sigma) in 100 ml of isopropyl alcohol) for 15 min at
room temperature. Cells then were washed twice with water for 15 min
each and visualized.
Western (Immunoblot) Analysis—3T3-L1 cells were lysed in cell ly-
sate buffer (500 ?l for the 10-cm dish and 150 ?l for the 60-mm dish),
and cells were incubated on ice for 30 min, followed by centrifugation at
17,000 rpm at 4 °C for 30 min. Supernatant was collected, and protein
concentration was determined by Bio-Rad protein assay. 60–100 ?g of
protein was subjected to 10% polyacrylamide gel electrophoresis. Pro-
teins were then transferred to nitrocellulose membrane, and Ponceau-S
(Sigma) staining was performed to verify equal loading/transfer. The
membrane was incubated first with primary antibody (anti-PPAR?,
1:1500 (23); anti-C/EBP?, 1:300 (Santa Cruz Biotechnology, Inc.)) for
2 h, followed by secondary antibody (horseradish peroxidase-conju-
gated) for 1 h. Blots were developed using ECL chemiluminescence
detection reagent (Amersham Life Sciences, Inc.) and visualized by
exposure to autoradiography film.
BrdUrd Labeling Assay—Cells were incubated with BrdUrd (Amer-
sham Corp.) for 24 h and then trypsinized, washed twice in phosphate-
buffered saline, and fixed with 70% ethanol on ice for 30 min or longer.
Fixed cells were incubated with anti-BrdUrd and then fluorescein iso-
thiocyanate-conjugated secondary antibody. BrdUrd-positive cells were
sorted by flow cytometry.
[3H]Thymidine Incorporation Assay—Cells were incubated with 1
?Ci/ml of [3H]thymidine for 24 h and then harvested, washed, and
resuspended in 0.5 ml of 0.3 N NaOH and incubated on ice for 30 min
prior to the addition of 0.5 ml of 20% trichloroacetic acid. Precipitated
DNA was filtered through a Whatman GFC filter, washed, dried, and
counted in a liquid scintillation counter.
RA Inhibits PPAR? Ligand-mediated Adipogenesis in 3T3 L1
Cells—During DM-induced 3T3-L1 differentiation, RA is only
effective in preventing adipogenesis when added during the
first 24–48 h (day 0 and day 1), when PPAR? has not yet been
induced (7). Indeed, under circumstances when PPAR? is al-
ready expressed, RA can lead to the loss of expression of
PPAR?, consistent with its ability to block the transcriptional
activity of C/EBP?, which induces PPAR? during adipogenesis
(12, 23). To further the use of RA as a tool for understanding
the stages of adipocyte differentiation, we first tested the abil-
ity of RA to affect the adipogenesis of 3T3-L1 cells that is
induced by the PPAR?2 activator, BRL49653. BRL49653 in-
duces adipogenesis of post-confluent 3T3-L1 cells, typically
causing 20–70% of cells to differentiate into adipocytes within
7–10 days (26). This is presumably caused by the activation of
an endogenous low level of PPAR? in the preadipocytes (23)
and subsequently the activation of the differentiation program
and fat cell conversion. These results were confirmed in Fig. 1
(column 1, row 2), where fat cell differentiation is indicated by
FIG. 1. Adipocyte differentiation of 3T3-L1 cells. Retroviral in-
fected cells that expressing vector alone (control, column 1), PPAR?
(L1-PPAR?2, column 2), C/EBP? (L1-C/EBP?, column 3), or both (col-
umn 4) were cultured under various conditions. Cells were fixed and
stained with Oil Red O on culture dishes as described under “Materials
and Methods.” Row 1, after selection for 10 days in G418 media, cells
were split and grown in GM. Row 2, same as row 1, but 1 ?M BRL49653
(BRL) was added to cells at time of confluence. Row 3, same as row 1,
but 10 ?M RA was present from the time of retroviral infection. Row 4,
same as row 2, but RA was present from the time of retroviral infection.
Row 5, same as row 1, but RA was added at the time of confluence.
Commitment to Adipocyte Differentiation
by guest on November 4, 2015
oil red O staining. RA was able to inhibit BRL49653-induced
differentiation (Fig. 1, column 1, row 3). Fig. 2 shows that
BRL49653 treatment induced both PPAR? and C/EBP? and
that, consistent with the oil red O staining, this induction was
blocked by RA. Note that adipocytes express two forms of
C/EBP?, referred to in this paper as C/EBP?-LAP and C/EBP?-
LIP using nomenclature derived from similar alternative
translation products for C/EBP? (27).
blocked PPAR? induction by BRL49653, we next examined
whether expression of PPAR? would be sufficient to establish
insensitivity to RA by testing the efficacy of RA on cells that are
ectopically expressing PPAR?2 (designated as L1-PPAR?2).
Fig. 3A shows that these cells constitutively express PPAR?2
protein. In a typical experiment, approximately 20–50% of
L1-PPAR?2 cells spontaneously differentiated into fat cells in
the absence of any adipogenic stimuli in 7–10 days postconflu-
ency, due either to a low level of endogenous ligand or consti-
tutive activity of the ectopic PPAR? (see Ref. 28 and Fig. 1,
column 2, row 1). The addition of the PPAR? ligand BRL49563
accelerated and enhanced the differentiation process, with
nearly 100% of cells differentiating into fat cells. This is shown
by oil red O staining in Fig. 1 (column 2, row 2). In addition,
Fig. 3 shows that the adipocyte markers C/EBP?-LAP and
C/EBP?-LIP were markedly induced (lane 5). However, when
maintained in the presence of RA, cells that were grown in GM
with or without the PPAR? ligand BRL49653 did not convert
into fat cells, documented both by oil red O staining (Fig. 1,
column 2, row 4) and C/EBP? expression (Fig. 3, lanes 6 and 7).
The presence of RA had no effect on the ectopic expression of
PPAR? (Fig. 3, compare lanes 6 and 7 to lanes 4 and 5). These
results suggest that RA is able to block adipogenesis induced by
ectopic PPAR? protein expression, and expression of PPAR? is
not able to override the inhibitory effect of RA on adipogenesis.
RA Is Ineffective When Added to Postconfluent Cells That
Ectopically Express PPAR? or C/EBP?—Above we showed
that RA blocks the function of PPAR? to induce fat cell conver-
sion, suggesting that additional factor(s) may be necessary to
be insensitive to RA. Previously we have shown that adipogen-
esis in cells that ectopically express either C/EBP? or C/EBP?
is also blocked when cells are maintained in the presence of RA
from the onset of ectopic gene expression (Fig. 1 and Ref. 13).
This was confirmed in Fig. 1 (column 3, row 3, where the
ectopic C/EBP?-expressing cells are referred to as L1-C/EBP?
cells). Interestingly, when RA was added not at the onset of
ectopic expression of either PPAR? or C/EBP?, but rather at a
later time when cells were confluent, it was no longer effective
at blocking differentiation (Fig. 1, row 5). Constitutive expres-
sion of C/EBP? in the L1-C/EBP? cells is demonstrated in Fig.
4 (lanes 5 and 7). The vector used to ectopically express C/EBP?
does not express the smaller translation product of C/EBP?,
called C/EBP?-LIP (23). Thus, C/EBP?-LIP expression serves
as a marker of adipogenesis in these experiments (29). Note
that C/EBP?-LIP was undetectable in the L1-C/EBP? cells at
day 0 (Fig. 4, lane 5), consistent with their lack of adipocyte
phenotype. In contrast, continued incubation of the L1-C/EBP?
cells for 9 days postconfluency did induce adipogenesis (Fig. 1
(column 3, row 1) and Fig. 4 (note C/EBP-LIP expression in
lane 6)). Consistent with the cell morphology shown in Fig. 1,
this adipose conversion of L1-C/EBP? cells was blocked when
cells were grown in media containing RA (Fig. 1, column 3, row
3; Fig. 4, lane 8).
PPAR? and C/EBP? Are Mutually Regulated—Note that
while allowing the cells to reach confluency prior to the addi-
tion of RA appeared to prevent the effects of RA on the ectopic
C/EBP?- and PPAR?-expressing cells, RA normally prevents
adipocyte differentiation of wild type 3T3-L1 cells. Those cells
however, do not express PPAR? or C/EBP? until they have
begun to differentiate. Thus, the state of refractoriness to RA
seems to require both confluency and expression of PPAR? and
C/EBP?. Indeed, during normal 3T3-L1 differentiation, RA
loses effectiveness at times when PPAR? and C/EBP? are
simultaneously expressed in the cells (23). Since C/EBP? (13)
and C/EBP? induce PPAR? (12, 13), we considered the possi-
bility that PPAR? and C/EBP? may induce each other, leading
after some time to a state that is refractory to RA inhibition of
adipogenesis. To determine whether PPAR? is able to activate
C/EBP? gene expression, L1-PPAR? cells were collected prior
to differentiation and subjected to Western analysis. Fig. 3
shows that a low but detectable level of C/EBP? protein was
present in day 0 L1-PPAR? preadipocytes (lane 3). This is
notably different from wild type 3T3-L1 cells, which do not
express C/EBP? on day 0 (e.g. lane 1). Thus, ectopic PPAR?
expression induced a low level of expression of C/EBP?. The
C/EBP? expression was even greater on day 7, consistent with
the adipocyte phenotype of these cells. Interestingly, RA
blocked expression of C/EBP? despite continued expression of
the ectopic PPAR?, suggesting that RA inhibits PPAR? induc-
tion of C/EBP?. This would be consistent with the results of
others indicating that liganded RAR can interfere with PPAR-
FIG. 2. RA inhibits BRL-mediated adipogenesis of 3T3 L1 cells.
Day 0 postconfluent 3T3-L1 preadipocytes maintained in the growth
media were exposed to 1 ?M of BRL49653, in the absence or presence of
10 ?M RA. Cell extracts were prepared on the indicated days and
subjected to Western immunoblot to detect the expression of PPAR?
and C/EBP?. The arrows indicate the two PPAR? isoforms (?1 and ?2)
as well as the two translation products of C/EBP? (LAP and LIP).
FIG. 3. Ectopic expression of PPAR? in 3T3 L1 cells. A Western
blot of PPAR? and C/EBP? protein expression in L1-PPAR?2 cells is
shown. After selection for 10 days in G418 media, cells were split and
grown in the absence (lanes 4 and 5) or presence (lanes 6 and 7) of 10 ?M
RA. cells were maintained in the absence (lanes 4 and 6) or presence
(lanes 5 and 7) of 1 ?M BRL49653 for 7 days. Cell extract at day 0 is also
shown (lane 3). Wild type 3T3-L1 preadipocytes on day 0 (PreAd, lane 1)
and adipocytes on day 7 (Ad, lane 2) after adipogenic stimulation with
DM are shown for comparison.
Commitment to Adipocyte Differentiation
by guest on November 4, 2015
mediated gene transcription (31).
In a reciprocal experiment we examined the expression of
endogenous PPAR? in L1-C/EBP? cells. Fig. 4 shows that en-
dogenous PPAR? was expressed in the day 0 L1-C/EBP? prea-
dipocytes (lane 7), whereas expression of PPAR? was undetect-
able in day 0 control preadipocytes (lane 1). Expression of
PPAR? in the L1-C/EBP? cells was abolished by treatment
with RA from the time of C/EBP? expression (lanes 7 and 8),
consistent with the ability of RA to inhibit transcriptional
activation by C/EBP? (13).
RA Inhibits Adipogenesis of Preadipocytes Coexpressing Ec-
topic PPAR? and C/EBP?—The above results demonstrate
that PPAR? and C/EBP? can activate each other’s expression.
Therefore, it is likely that the inability of RA to prevent fat cell
conversion in L1-PPAR? or C/EBP? cells when added postcon-
fluency is due to the coexpression of PPAR? and C/EBP?. We
next tested whether the requirement for confluency could be
overcome by forcing cells to express both PPAR? and C/EBP?
at higher levels. For these experiments, we doubly infected 3T3
L1 cells with LXSN-PPAR?2 (neor) and TS13-C/EBP? (hygro-
mycinr). These cells grew slowly, perhaps related to growth
suppressive properties of C/EBP? in other cell lines (30); cells
that express only ectopic C/EBP? grew somewhat slowly but
closer to normal than those ectopically expressing both PPAR?
and C/EBP?, suggesting that PPAR? provided a second growth-
inhibitory signal in 3T3-L1 cells. In any case, the cells co-
expressing C/EBP? and PPAR? spontaneously differentiated
into adipocytes during 10 days of selection, even prior to con-
fluency. However, when cells were selected and maintained in
the presence of RA, they were able to grow to confluence at
normal rates, and no fat cells were observed in the absence or
presence of BRL49653 (Fig. 1, column 4, row 1). The ability of
RA to prevent differentiation of cells that ectopically express
both PPAR? and C/EBP? suggested that concomitant expression
of these two proteins was not sufficient to establish a commit-
ment state to adipogenesis that is no longer responsive to RA.
PPAR?, C/EBP?, and Withdrawal from the Cell Cycle Are
Necessary for Commitment to Adipogenesis—We demonstrated
that RA is able to inhibit 3T3-L1 adipogenesis due to C/EBP?
and PPAR?, alone or in combination, provided that RA is added
at the time of gene transduction. In contrast, RA did not have
an appreciable effect when added to cells postconfluency. Since
confluency is associated with withdrawal of cells from the cell
cycle and since cell proliferation and differentiation are often
mutually exclusive events, we hypothesized that both cell cycle
arrest and the expression of adipogenic genes are necessary for
the commitment to fat cell differentiation. It is known that
standard differentiation medium induces mitosis of quiescent
3T3-L1 cells prior to cell cycle withdrawal and completion of
differentiation along adipogenic lineage. Although RA blocks
differentiation, it does not prevent the mitosis and resultant
increase in cell number due to the adipogenic stimulation (22).
Fig. 5A shows the time course of the mitotic response to adipo-
genic stimulation in the absence and in the presence of RA,
using BrdUrd incorporation as a measure of DNA synthesis. In
both cases, a major increase in BrdUrd incorporation occurs on
days 1 and 2 following adipogenic stimulation. Thus, the cells
become postmitotic on day 3 and beyond. These data indicate
that, although RA prevented adipocyte differentiation, it did
not block the clonal expansion that occurs following adipogenic
stimulation, although it appears to be quantitatively reduced.
Note that in the presence or absence of RA, the adipogenically
stimulated cells return to a quiescent state characterized by
few mitotic events. However, these states are fundamentally
different as shown in Fig. 5B. The RA-treated cells are quies-
cent due to contact inhibition, because reseeding them at low
density allows them to re-enter the cell cycle. In contrast, Fig.
5B shows that adipocytes differentiated by the standard pro-
tocol in the absence of RA are permanently postmitotic and do
not divide after reseeding at low density. These results suggest
FIG. 4. Ectopic C/EBP? induces expression of PPAR?. L1-C/
EBP? cells were grown in the absence (lanes 5 and 6) or presence of RA
(lanes 7 and 8) in GM. Day 0 and day 7 cells were harvested, and whole
cell extract was prepared and subjected to Western blot. C/EBP? ex-
pression in wild type preadipocytes (day 0) and adipocytes (day 7) are
shown for comparison in lanes 1 and 2, respectively. Cells infected with
LXSN control are shown in lanes 3 and 4.
FIG. 5. Role of clonal expansion and cell cycle exit in adipocyte
differentiation. A, clonal expansion and subsequent quiescence of
quiescent 3T3-L1 cells after exposure to adipogenic stimulation in the
presence and absence of RA. Cells were labeled by BrdUrd for 24 h at
various time points during differentiation. BrdUrd-positive cells were
labeled by fluorescence and sorted by FACScan. Shown is the mean and
range of two separate experiments. B, cells expanded during differen-
tiation are permanently postmitotic, whereas cells induced to divide by
differentiation medium in the presence of RA remain capable of re-
entering the cell cycle. Cells at various time points during the differen-
tiation process were reseeded at lower density in GM with 1 ?M/ml
[3H]thymidine. Cells were harvested and assayed for3H uptake 24 h
later. Each time point was performed in triplicate, and each experiment
was performed twice with similar results.
Commitment to Adipocyte Differentiation
by guest on November 4, 2015
that after the clonal expansion that occurs in the early phase of
adipogenesis, cells permanently exit from the cell cycle.
The Phosphorylation State of Rb as a Marker of the Commit-
ment to Adipocyte Differentiation—Interestingly, the time after
adipogenic stimulation at which the cells are permanently
postmitotic corresponds to the time at which cells become re-
fractory to the effects of RA, i.e. between 48 and 72 h after
beginning the differentiation protocol (23). This further sug-
gested the role of cell cycle events in adipocyte differentiation.
The Rb protein plays a major regulatory role in the withdrawal
of cells from the cell cycle, and recently it was shown that Rb is
required for adipogenic differentiation in a less well character-
ized model involving embryonic lung fibroblasts (20). We there-
fore examined expression and phosphorylation of Rb protein in
3T3-L1 cells before and during differentiation in the presence
and absence of RA. The immunoblot in Fig. 6 shows that
confluent, quiescent 3T3-L1 cells (day 0) contained both hyper-
phosphorylated and hypophosphorylated forms of Rb protein.
The total amount of Rb protein was relatively constant during
differentiation. Adipogenic stimulation led to an increase in
hyperphosphorylation of Rb during the first 2 days, consistent
with the process of clonal expansion that would be aided by
hyperphosphorylation and, thereby, inactivation of Rb. As ad-
ipogenesis proceeded, as confirmed by expression of PPAR?
and C/EBP?, the Rb protein began to shift to its hypophospho-
rylated form (Fig. 6A, lane 3), corresponding to cells exiting the
cell cycle. By day 7, when nearly 100% adipocyte conversion
was achieved, Rb protein was almost entirely hypophosphory-
lated. The stage of RA insensitivity corresponds to the time of
withdrawal from the cell cycle in addition to the expression of
the adipogenic factors PPAR? and C/EBP? (day 3).
Fig. 6B shows that the presence of RA at the time of adipo-
genic stimulation altered the phosphorylation state of Rb. The
degree of hyperphosphorylation on day 1 was reduced, consist-
ent with the reduced percentage of cells that underwent clonal
expansion (Fig. 5A). Furthermore, in the presence of RA, Rb
was present in both hyper- and hypophosphorylated forms on
all days after exposure to adipogenic stimulation. Consistent
with the lack of adipogenesis, there was no induction of PPAR?
or C/EBP? in the presence of RA (Fig. 6B). These data sug-
gested a correlation between terminal differentiation and the
hypophosphorylation of Rb protein. Together with our earlier
demonstration of the role of confluency in determining RA
insensitivity of cells that ectopically express both PPAR? and
C/EBP?, we conclude that irreversible commitment to adipo-
genic differentiation requires both the expression of the adipo-
genic transcription factors and the cell cycle arrest associated
with hypophosphorylation of Rb.
We have used the ability of RA to inhibit adipogenesis as a
tool to explore the molecular events that occur during the
adipogenic differentiation process. In particular, we were in-
terested in pursuing the observation that at early times after
exposure of confluent 3T3-L1 cells to adipogenic stimulation,
the cells become committed to the differentiation pathway and
no longer respond to RA. RA was able to block BRL49653-
mediated differentiation in wild type 3T3-L1 cells, as well as
adipogenesis due to ectopic expression of C/EBP?, PPAR?, or
both, even in the presence of the PPAR? ligand BRL49653.
These results strongly suggest that the insensitivity to RA
inhibition that occurs during normal differentiation is not due
solely to the concomitant expression of PPAR? and C/EBP?.
However, we have noted that allowing C/EBP?- or PPAR?-
expressing cells to reach confluence reproduces the committed,
RA-irreversible state that occurs after about 48 h during wild
type 3T3-L1 cell differentiation. Consistent with this notion, we
found that during standard 3T3-L1 cell differentiation, at the
point at which cells normally become refractory to the effects of
RA, cells are not only expressing PPAR? and C/EBP? but are
exiting the cell cycle following clonal expansion.
The ability of RA to block PPAR?-induced adipocyte differ-
entiation suggests that liganded endogenous RAR can interfere
with the function of PPAR? in addition to its ability to block
transcriptional activity of C/EBPs (13). Indeed, the ability of
liganded RAR to block PPAR transactivation can be demon-
strated in transfected cells (Ref. 31 and data not shown). How-
ever, during normal adipogenesis, RA is effective at times prior
to expression of PPAR?, suggesting that its natural target is
C/EBP?-mediated transcription (13).
We have now shown that RA blocks adipocyte differentiation
of confluent 3T3-L1 cells when added prior to expression of
adipogenic transcription factors and also prevents adipogenesis
of cells that constitutively express C/EBP? and PPAR? when
added prior to confluency. Thus, excluding the situation where
RAR is limiting (23), preadipocytes enter a state of commit-
ment to differentiation that is not reversible by RA when
PPAR? and C/EBP? are expressed and growth cessation has
occurred. In the absence of RA, cells that ectopically express
PPAR? and C/EBP? undergo adipocyte differentiation before
cells reach confluency, indicating that cell-cell contact may not
be necessary for adipogenesis to occur. However, these cells
grow very slowly, suggesting that expression of adipogenic
genes may contribute to the cessation of cell growth that is also
required for cells to undergo adipogenic differentiation.
Both PPAR? and C/EBP? activate several adipocyte-specific
genes. Coexpression of PPAR? and C/EBP? has been shown to
have a synergistic effect and strongly promote fat cell differen-
FIG. 6. Role of clonal expansion and cell cycle exit, Rb phos-
phorylation, and expression of PPAR? and C/EBP? in the com-
mitment to adipocyte differentiation. Expression and phosphoryl-
ation of Rb protein in confluent 3T3-L1 cells exposed to adipogenic
stimulation in the absence (A) and presence (B) of 10 ?M RA. The time
course of PPAR? and C/EBP? induction in the same experiment is
shown for comparison.
Commitment to Adipocyte Differentiation
by guest on November 4, 2015
tiation of fibroblastic (32) and myogenic cell lines (14). C/EBP?
has known antimitotic effects, suggesting that its role in pro-
moting differentiation is at least partially related to suppres-
sion of cell growth (30). Expression of C/EBP? in 3T3-L1 cells
reduced their growth rate, and cells that ectopically expressed
both PPAR? and C/EBP? grew extremely slowly and under-
went adipocyte conversion even before they reached confluence.
However, we found that co-expression of these two proteins was
not sufficient to commit cells to undergo differentiation in a
manner that was irreversible by RA unless the cells were growth-
arrested. These observations suggest that growth arrest played a
positive role, along with the specific differentiation factors, in
determining whether cells proliferate or differentiate.
During the normal differentiation process, we found that Rb
protein is first hyperphosphorylated and then hypophosphory-
lated. This correlated well with the cell cycle status of the cells
both during and after clonal expansion. While this work was in
progress, another group also showed that Rb protein levels
were stable throughout adipogenesis but reported that the Rb
protein was hypophosphorylated throughout the differentia-
tion process (33). The discrepancy appears to be due to the use
of different Rb antibodies, and using the same antibody as we
used, those investigators have more recently found changes in
Rb phosphorylation similar to those reported here.2The role of
Rb in adipogenesis has been predicted by other studies. In
addition to being required for the adipogenesis of mouse lung
fibroblasts (20), Rb is necessary for myoblastic differentiation
(35). Indeed, there is evidence that RB interacts with adipo-
genic C/EBP proteins (34) as well as with the myogenic differ-
entiation factor Myo D (35). However, the precise role of Rb in
differentiation should be further pursued.
It is unlikely that cell cycle arrest is merely a downstream
effect of adipogenic differentiation factors because expression
of C/EBP and PPAR? complement but cannot replace the im-
portant effects of cell growth arrest on the ability of 3T3-L1
cells to irreversibly commit to adipocyte differentiation. Taken
together, our results suggest that cell cycle arrest is as important
as the expression of adipogenic genes in promoting cell differen-
tiation and the irreversible commitment of preadipocytes to this
Acknowledgment—We thank E. Huang for comments on the
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Commitment to Adipocyte Differentiation
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Dalei Shao and Mitchell A. Lazar
1997, 272:21473-21478.J. Biol. Chem.
the Commitment to Adipocyte
, and Cell Cycle Status Regulate
, CCAAT/ Enhancer-binding
Peroxisome Proliferator Activated
CELL BIOLOGY AND METABOLISM:
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