From the
Thiazolidinedione derivatives are antidiabetic agents that
increase the insulin sensitivity of target tissues in animal models of
non-insulin-dependent diabetes mellitus. In vitro,
thiazolidinediones promote adipocyte differentiation of preadipocyte
and mesenchymal stem cell lines; however, the molecular basis for this
adipogenic effect has remained unclear. Here, we report that
thiazolidinediones are potent and selective activators of peroxisome
proliferator-activated receptor
Adipocytes are highly specialized cells that play a critical
role in energy homeostasis. Their primary role is to store
triglycerides in times of caloric excess and to mobilize this reserve
during periods of nutritional deprivation. Adipocytes are derived from
a multipotent stem cell of mesodermal origin that also gives rise to
the muscle and cartilage lineages. Studies of the adipocyte
differentiation program have been facilitated by the availability of
established mesenchymal and preadipocyte cell lines that can be induced
to differentiate upon treatment with mixtures of hormonal stimulants
(reviewed in Ref. 1).
Adipocyte differentiation is characterized by
a coordinate increase in adipocyte-specific gene expression. In most
cases, these increases can be accounted for by activation of gene
transcription. Thus, considerable effort has been focused on the
identification of transcription factors that regulate
adipocyte-specific genes. Recently, an orphan member of the nuclear
receptor superfamily of ligand-activated transcription factors,
designated peroxisome proliferator-activated receptor
PPAR
To
further examine the role of PPAR
Thiazolidinediones are Selective Activators of PPAR
The effects of the thiazolidinediones on
wild-type PPAR
In summary, our demonstration that an adipogenic thiazolidinedione
is a high affinity ligand for PPAR
We thank Beverly Oliver, Deborah Noel, Bruce Wisely,
and Kelli Beck for expert technical assistance; Gyan Chandra, Stephen
Haneline, and Sandy Stinnett for sequence data; S. Prakash for
radiolabeled compound; Brian Champion and Alan Payne for assistance in
adipocyte differentiation assays; and Ken Batchelor, Dave Morris, Bob
Dougherty, Steve Blanchard, Ching Song, and Mike Luther for support and
discussion throughout the course of this work.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(PPAR
), a member of the
nuclear receptor superfamily recently shown to function in
adipogenesis. The most potent of these agents, BRL49653, binds to
PPAR
with a K
of approximately 40
nM. Treatment of pluripotent C3H10T1/2 stem cells with
BRL49653 results in efficient differentiation to adipocytes. These data
are the first demonstration of a high affinity PPAR ligand and provide
strong evidence that PPAR
is a molecular target for the adipogenic
effects of thiazolidinediones. Furthermore, these data raise the
intriguing possibility that PPAR
is a target for the therapeutic
actions of this class of compounds.
(PPAR
),
(
)
was shown to be expressed in an
adipose-specific manner and its expression induced early during the
course of differentiation of several preadipocyte cell
lines
(2, 3) . Subsequent studies revealed that forced
expression of PPAR
in fibroblasts resulted in adipocyte
differentiation; this differentiation was significantly enhanced in the
presence of weak PPAR
activators (4). These data demonstrated that
PPAR
plays a pivotal role in the adipogenic signaling cascade.
is one of a subfamily of PPARs encoded by independent
genes (5). To date, three mammalian PPARs, designated PPAR
,
PPAR
, and NUC-1, have been
identified
(6, 7, 8, 9, 10) .
PPARs regulate gene expression by binding to DNA sequence elements
termed PPAR response elements. PPAR response elements have been
identified in the regulatory regions of a number of genes encoding
enzymes that modulate lipid metabolism, indicating a physiological role
for the PPAR family in the regulation of lipid homeostasis (reviewed in
Ref. 11). PPARs have been shown to be activated to various degrees by
micromolar concentrations of long-chain fatty acids and a structurally
diverse group of compounds termed peroxisome proliferators that
includes the fibrate class of hypolipidemic
drugs
(5, 6, 7, 8, 9, 12) .
However, as no binding of these compounds to the PPARs has been
reported, PPARs have remained ``orphan'' receptors.
in adipocyte differentiation, we
sought to identify activators of PPAR
. We report here that a class
of compounds, termed thiazolidinediones
(13) , are
PPAR
-selective ligands. Thiazolidinediones are known to have
marked adipogenic effects on preadipocyte and mesenchymal stem cells
in vitro(14, 15, 16, 17) and
dramatic antidiabetic effects in animal models of
NIDDM
(13, 18) . Our data provide strong evidence that
PPAR
is the molecular target for the adipogenic effects of
thiazolidinediones and, furthermore, suggest that PPAR
may be the
target for the antidiabetic actions of this class of compounds.
Plasmids
GAL4-PPAR chimera expression constructs
contain the translation initiation sequence and amino acids 1-76
of the glucocorticoid receptor fused to amino acids 1-147 of the
yeast transcription factor GAL4, including the DNA binding domain, in
the pSG5 expression vector (Stratagene). cDNAs encoding amino acids
167-468, 138-440, and 174-475 of murine PPAR,
NUC-1, and PPAR
1 (ref. 8) were amplified by polymerase chain
reaction and inserted C-terminal to GAL4 in the pSG5 expression vector
(Stratagene) to generate plasmids pSG5-GAL4-PPAR
, pSG5-GAL4-NUC-1,
and pSG5-GAL4-PPAR
, respectively. The regions of the PPARs
included in the chimeras should contain the ligand binding domains
based on their homology to ligand binding domains of characterized
nuclear receptors (19, 20). The chimeras initially contained the
translation start site and N-terminal 262 amino acids of the
glucocorticoid receptor, including the
transcriptional transactivation domain (21). However, as these
chimeras had high basal activity in CV-1 cells, a 0.6-kilobase
BglII fragment containing the
domain was
removed, leaving the translation start site and amino acids 1-76
of the glucocorticoid receptor. Wild-type receptor expression vectors
were generated by insertion of cDNAs encoding murine PPAR
, NUC-1,
PPAR
1, and PPAR
2 into the expression vector pSG5
(Stratagene). Reporter plasmid (UAS)
-tk-CAT was generated
by insertion of five copies of a GAL4 DNA binding element into the
BamHI site of pBLCAT2 (Ref. 22). The reporter aP2-tk-CAT was
generated by insertion of the 518-bp EcoRI/XbaI
fragment containing the enhancer of the aP2 gene
(23) into the
BamHI site of pBLCAT2.
Cotransfection Assay
CV-1 cells were plated in
24-well plates in DME medium supplemented with 10% delipidated fetal
calf serum. In general, transfection mixes contained 10 ng of receptor
expression vector, 100 ng of the reporter plasmid, 200 ng of
-galactosidase expression vector (pCH110, Pharmacia) as internal
control, and 200 ng of carrier plasmid. Transfections were done with
Lipofectamine (Life Technologies, Inc.) according to the
manufacturer's instructions. Cell extracts were prepared and
assayed for chloramphenicol acetyltransferase (CAT) and
-galactosidase activities as described previously
(24) .
Ligand Binding Assay
cDNA encoding amino acids
174-475 of PPAR1 was amplified via polymerase chain reaction
and inserted into bacterial expression vector pGEX-2T (Pharmacia).
GST-PPAR
LBD was expressed in BL21(DE3)plysS cells and extracts
prepared as described previously
(25) . For saturation binding
analysis, bacterial extracts (100 µg of protein) were incubated at
4 °C for 3 h in buffer containing 10 mM Tris (pH 8.0), 50
mM KCl, 10 mM dithiothreitol with
[
H]-BRL49653 (specific activity, 40 Ci/mmol)
(18) in the presence or absence of unlabeled BRL49653. Bound was
separated from free radioactivity by elution through 1-ml Sephadex G-25
desalting columns (Boehringer Mannheim). Bound radioactivity eluted in
the column void volume and was quantitated by liquid scintillation
counting.
Adipocyte Differentiation Assay
C3H10T1/2 cells
were grown in a 24-well plate in DME medium (Life Technologies, Inc.)
supplemented with 10% fetal calf serum. Medium and compound were
exchanged every 3 days. Cells were stained at day 7 with Oil Red O and
photographed.
Northern Analysis
C3H10T1/2 cells were grown in
225-mm flasks in DME medium (Life Technologies, Inc.) supplemented with
10% fetal calf serum. Medium was exchanged every 3rd day and fresh
compound added. Cells were harvested on day 7 and poly(A) RNA prepared using the PolyATract® system 1000 (Promega).
-A
transient cotransfection assay was used to screen for PPAR
activators. As mammalian cell lines contain endogenous nuclear
receptors that can complicate interpretation of the results, we used an
established chimera system
(26) . Chimeras were constructed that
fused the putative ligand binding domains of the three murine PPAR
subtypes (
,
, NUC-1) to the DNA binding domain of the yeast
transcription factor GAL4. The structural organization of the GAL4-PPAR
chimeras is shown schematically in Fig. 1A.
Figure 1:
Thiazolidinediones are selective
activators of PPAR. A, schematic representation of the
chimeras used in this study. Chimeras include the translation
initiation site and amino acids 1-76 of the glucocorticoid
receptor (GR), amino acids 1-147, including the DNA
binding domain, of GAL4, and the putative ligand binding domains of
PPAR
, NUC-1, and PPAR
. See ``Materials and
Methods'' for additional details. MCS, multiple cloning
site. B, CV-1 cells were cotransfected with chimeric receptor
expression plasmids pSG5-GAL4-PPAR
, pSG5-GAL4-NUC-1, or
pSG5-GAL4-PPAR
and the reporter plasmid (UAS)
-tk-CAT.
Cells were treated with either vehicle alone (0.1% Me
SO), 1
10
M or 1
10
M Wy14,643, or 1
10
M thiazolidinediones and cell extracts subsequently
assayed for CAT activity. Similar results were obtained in two
independent experiments performed in triplicate. C, chemical
structures of BRL49653, pioglitazone, ciglitazone, and
englitazone.
Expression plasmids for the GAL4-PPAR chimeras were cotransfected
into CV-1 cells with a reporter construct containing five copies of the
GAL4 DNA binding site upstream of the thymidine kinase (tk) promoter
driving chloramphenicol acetyltransferase (CAT) gene expression. As
previously reported, all three PPAR subtypes were activated by high
concentrations of the peroxisome proliferator Wy14,643
(Fig. 1B)
(5, 6, 7, 8, 12) .
Whereas the PPAR-GAL4 chimera was activated in the presence of 1
10
M Wy14,643, activation of the
PPAR
and NUC-1 chimeras required 1
10
M Wy14,643 (Fig. 1B). Using this assay,
we identified four compounds that were efficacious activators of the
GAL4-PPAR
chimera at a concentration of 1
10
M, yet had little or no activity on either the
GAL4-PPAR
or GAL4-NUC-1 chimeras (Fig. 1B), even
when concentrations as high as 1
10
M were used (data not shown). These chemicals failed to
activate a control chimera that lacked a ligand binding domain (data
not shown). Interestingly, these four compounds, termed BRL49653,
pioglitazone, ciglitazone, and englitazone, fall into a class of
structurally related antidiabetic agents designated thiazolidinediones
(Fig. 1C).
were examined next. Two isoforms of PPAR
,
termed PPAR
1 and PPAR
2, have been identified and shown to
differ in their amino
termini
(2, 8, 9, 10) . Expression
vectors for the two PPAR
isoforms were cotransfected into CV-1
cells with a reporter plasmid containing the enhancer of the
adipocyte-specific aP2 gene driving expression of the tk-CAT construct.
The aP2 enhancer has previously been shown to contain two PPAR
response elements and to confer responsiveness to
pioglitazone
(2, 27) . Both PPAR
1 and PPAR
2
were activated by BRL49653 and pioglitazone in a dose-dependent and
saturable manner (Fig. 2). Interestingly, although the
half-maximal concentration of activation with pioglitazone was 4
10
M for both PPAR
isoforms,
the EC
with BRL49653 was 3
10
M and 1
10
M for the
PPAR
1 and PPAR
2 isoforms, respectively (Fig. 2). Thus,
the two PPAR
isoforms have distinct activation profiles.
Ciglitazone and englitazone, although less potent, also activated both
PPAR
isoforms (Fig. 2). No significant activation of
wild-type PPAR
or NUC-1 was seen in the presence of 1
10
M of the thiazolidinediones (data not
shown). Likewise, the thiazolidinediones failed to activate other
nuclear receptors including the human retinoic acid receptor
and
the human thyroid hormone receptor
(data not shown). Thus, the
thiazolidinediones are potent and selective activators of PPAR
.
Figure 2:
Thiazolidinediones activate wild-type
PPAR1 and PPAR
2. CV-1 cells were cotransfected with
expression plasmids for PPAR
1 (A) or PPAR
2
(B) and reporter plasmid aP2-tk-CAT. Cells were treated with
increasing concentrations of BRL49653 (open diamonds),
pioglitazone (open triangles), ciglitazone (closed
squares), and englitazone (stars), and cell extracts
subsequently assayed for CAT activity. Similar results were obtained in
two independent experiments performed in
triplicate.
Thiazolidinedione BRL49653 Is a High Affinity PPAR
Although it has been shown that members of the PPAR
subfamily of nuclear receptors are activated by micromolar
concentrations of fatty acids and hypolipidemic drugs such as clofibric
acid and Wy14,643, none of these compounds have been shown to interact
directly with the
PPARs
(5, 6, 7, 8, 9, 12) .
To test for binding of BRL49653 to PPAR
Ligand
, the region extending from
the end of the conserved DNA binding domain to the C terminus of
PPAR
, which includes the putative ligand binding domain
(LBD)
(19, 20) , was expressed in Escherichia coli as a fusion protein with glutathione S-transferase
(GST-PPAR
LBD). Radiolabeled BRL49653 bound specifically and
saturably to GST-PPAR
LBD with a K
of 43 nM (Fig. 3, A and B). No
binding was detected in control extracts from bacteria expressing
glutathione S-transferase (data not shown). Consistent with
the dose-response data for PPAR
in the transient transfection
assay, BRL49653 was the most effective competitor for binding of
tritiated BRL49653 to GST-PPAR
LBD, followed by pioglitazone
(Fig. 3C). Ciglitazone and englitazone also competed for
binding of tritiated BRL49653 to GST-PPAR
LBD, albeit less
efficiently than pioglitazone (Fig. 3C). Thus, all four
thiazolidinediones bound directly to PPAR
. In control experiments,
dexamethasone failed to compete with tritiated BRL49653 for binding to
PPAR
(Fig. 3C). These data represent the first
description of PPAR ligands and demonstrate that PPAR
is a
bona fide member of the steroid/thyroid hormone/retinoid
family of ligand-activated transcription factors.
Figure 3:
The thiazolidinedione BRL49653 binds
PPAR with high affinity. A, bacterial extracts containing
the glutathione S-transferase-PPAR
ligand binding domain
fusion protein (GST-PPAR
LBD) were incubated with increasing
concentrations of tritiated BRL49643 in the absence (total binding;
closed circles) or presence (nonspecific binding; closed
squares) of a 500-fold excess of nontritiated BRL49653. Specific
binding of tritiated BRL49653 to GST-PPAR
LBD is indicated
(open triangles). B, Scatchard analysis. Specific
binding of BRL49653 in A was transformed by Scatchard analysis
and plotted. Linear regression yielded a K of 43 nM.
Similar results were obtained in three independent experiments
performed in duplicate. C, competition binding analysis was
performed with GST-PPAR
LBD and 10 nM tritiated BRL49653
in the presence of a 100-, 500-, or 2500-fold excess of unlabeled
BRL49653 (closed squares), pioglitazone (open
squares), englitazone (closed circles), ciglitazone
(open circles), or dexamethasone (closed triangles).
Similar results were obtained in two independent experiments performed
in duplicate.
BRL49653 Promotes Differentiation of C3H10T1/2 Stem Cells
to Adipocytes
Induction of PPAR message is a very early
event during the course of differentiation of several preadipocyte cell
lines, preceding the induction of other adipocyte markers such as aP2
and C/EBP
(2, 3, 4) . Recently, it was shown
that ectopic expression of PPAR
in fibroblasts induces the entire
adipocyte differentiation program as measured by lipid accumulation and
induction of adipocyte-specific genes
(4) . These data
demonstrate a causal role for PPAR
in adipogenesis. To test for an
effect of a PPAR
-selective ligand on adipogenesis, we treated
pluripotent C3H10T1/2 stem cells with BRL49653. Untreated C3H10T1/2
cells express PPAR
as demonstrated by Northern analysis
(Fig. 4C). Treatment of C3H10T1/2 cells with BRL49653
resulted in efficient adipocyte differentiation as judged by Oil Red O
staining (Fig. 4, A and B) and Northern
analysis using the adipocyte-specific markers aP2 and adipsin
(Fig. 4C). PPAR
expression levels were increased
approximately 3-fold in response to treatment with BRL49653
(Fig. 4C). Significant adipocyte differentiation was
seen at concentrations of BRL49653 as low as 1
10
M (data not shown). These data provide evidence that
ligand-mediated activation of PPAR
is sufficient to initiate the
adipogenic signaling cascade in a mesenchymal stem cell line.
Furthermore, our results provide a mechanistic explanation for the
reported adipogenic effects of several thiazolidinediones on
preadipocyte cell
lines
(14, 15, 16, 17) .
Figure 4:
BRL49653 promotes differentiation of
C3H10T1/2 cells to adipocytes. C3H10T1/2 cells were treated for 7 days
with either vehicle alone (0.1% MeSO) (A) or 1
10
M BRL49653 (B) and
subsequently stained for lipid accumulation with Oil Red O.
Magnification is
40. C, Northern analysis was
performed with 2 µg of poly(A)
RNA prepared from
C3H10T1/2 cells that were either untreated (day 0) or treated for 7
days with 1
10
M BRL49653. Blots
were hybridized with
P-labeled aP2, adipsin, PPAR
,
and GAPDH cDNA probes. An equivalent amount of intact RNA was run in
each lane as indicated by the GAPDH cDNA probe. Exposure times were
1.5, 24, 48, and 7 h for aP2, adipsin, PPAR
, and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) blots,
respectively.
Thiazolidinediones are insulin sensitizers that significantly reduce
glucose, lipid, and insulin levels in animal models of NIDDM and
obesity
(13) . Our results suggest that PPAR may be a target
for the antidiabetic effects of these agents. How might activation of
an adipocyte-specific transcription factor account for these diverse
therapeutic effects? In recent years there has been a growing awareness
that NIDDM is not only a derangement of glucose homeostasis, but is
also characterized by elevated levels of circulating lipids. Although
the mechanism is poorly characterized, increases in lipid levels have
been shown to interfere with glucose disposal (reviewed in Ref. 28).
Thus, activation of PPAR
in adipose may affect glucose usage in
other tissues such as skeletal muscle, the primary site of glucose
disposal, through an indirect mechanism involving modulation of lipid
levels. Alternatively, activation of PPAR
may regulate signaling
molecules secreted by adipose such as tumor necrosis factor-
or
the ob gene product
(29, 30) . These secreted
products could, in turn, modulate glucose metabolism in other tissues.
provides compelling evidence
that this nuclear receptor plays a critical role in adipogenesis.
Clearly, an understanding of the PPAR
signaling cascade may lead
to insights into the molecular mechanisms regulating energy homeostasis
and the defects underlying obesity and NIDDM.
, peroxisome proliferator-activated
receptor
; NIDDM, non-insulin-dependent diabetes mellitus; DME
medium, Dulbecco's modified Eagle's medium; CAT,
chloramphenicol acetyltransferase; tk, thymidine kinase; GST,
glutathione S-transferase.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.