From the Departments of Molecular Endocrinology and
Medicinal Chemistry, Merck Research Laboratories, Rahway, New
Jersey 07065
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ABSTRACT |
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The peroxisome proliferator-activated
receptors (PPARs) include three receptor subtypes encoded by separate
genes: PPAR In mammals, the peroxisome proliferator-activated receptor
(PPAR)1 family of nuclear
hormone receptors consists of three subtypes encoded by separate genes:
PPAR PPAR PPAR Structurally distinct, selective RXR agonists have been identified that
can activate PPAR Here, we report the identification and characterization of novel,
non-TZD PPAR Materials--
Cell culture reagents were obtained from Life
Technologies, Inc. [35S]Methionine and
EN3HANCE were purchased from NEN Life Science Products.
WY-14643 was obtained from Biomol (Plymouth Meeting, PA). All other
reagent-grade chemicals were from Sigma. The thiazolidinedione, AD-5075
(5-[4-[2-(5-methyl-2-phenyl-4-oxazoly)-2-hydroxyethoxy]benzyl]-2,4-thiazolidinedione), was kindly provided by Gerard Kieczykowski, Philip Eskola, Joseph F. Leone, and Peter A. Cicala (Merck Research Laboratories, Rahway, NJ).
[3H]2AD-5075 and
[3H]2L-783483 were prepared by Drs. David G. Melillo, Yui Sing Tang, and Allen N. Jones (Merck Research
Laboratories, Rahway, NJ).
Plasmids--
The chimeric receptor expression constructs,
pcDNA3hPPAR Binding Assay--
GST-hPPAR
For each assay, an aliquot of receptor, GST-hPPAR Assessment of Receptor Conformation by Partial Protease
Digestion--
The protease digestion assays were performed by the
method of Allan et al. (23) with previously described
modifications (2). The pSG5-hPPAR PPAR-CBP Pull-down Assay--
The
GST-hCBP1-453, GST-hPPAR Cell Culture and Transactivation Assay--
COS-1 cells were
seeded at 12 × 103 cells/well in 96-well cell culture
plates in high glucose Dulbecco's modified Eagle's medium containing
10% charcoal stripped fetal calf serum (Gemini Bio-Products, Calabasas, CA), nonessential amino acids, 100 units/ml penicillin G,
and 100 mg/ml streptomycin sulfate at 37 °C in a humidified atmosphere of 10% CO2. After 24 h, transfections were
performed with LipofectAMINE (Life Technologies, Inc.) according to the instructions of the manufacturer. Briefly, transfection mixes for each
well contained 0.48 µl of LipofectAMINE, 0.00075 µg of pcDNA3-PPAR/GAL4 expression vector, 0.045 µg of pUAS(5X)-tk-luc reporter vector, and 0.0002 µg of pCMV-lacZ as an internal control for transactivation efficiency. Cells were incubated in the
transfection mixture for 5 h at 37 °C in an atmosphere of 10%
CO2. The cells were then incubated for ~48 h in fresh
high glucose Dulbecco's modified Eagle's medium containing 5%
charcoal-stripped fetal calf serum, nonessential amino acids, 100 units/ml penicillin G, and 100 mg/ml streptomycin sulfate ± increasing concentrations of test compound. Since the compounds were
solubilized in Me2SO, control cells were incubated with
equivalent concentrations of Me2SO; final Me2SO
concentrations were Measurement of 3T3-L1 Preadipocyte Differentiation--
3T3-L1
cells (ATCC, Rockville, MD; passages 3-9) were grown to confluence in
medium A (Dulbecco's modified Eagle's medium with 10% fetal calf
serum, 100 units/ml penicillin, and 100 µg/ml streptomycin) at
37 °C in 5% CO2 as described previously (25). Confluent
cells were incubated in medium A containing 0.150 µM insulin and 1 µM dexamethasone ± PPAR ligand for 4 days at 37 °C in 5% CO2 with one medium change. Total
RNA was prepared from cells using the Ultraspec RNA isolation kit
(Biotecx, Houston, TX) and RNA concentration was estimated from
absorbance at 260 nm. RNA (20 µg) was denatured in
formamide/formaldehyde and slot blotted onto Hybond-N membrane
(Amersham Pharmacia Biotech). Prehybridization was performed at
42 °C for 1-3 h in 50% formamide and Thomas solution A containing
25 mM sodium phosphate, pH 7.4, 0.9 M sodium
chloride, 50 mM sodium citrate, 0.1% each of gelatin,
Ficoll, and polyvinylpyrollidone, 0.5% SDS, and 100 µg/ml denatured
salmon sperm DNA. Hybridization was carried out at the same temperature
for 20 h in the same solution with a 32P-labeled aP2
cDNA probe (2 × 106 cpm/ml). After washing the
membranes under appropriately stringent conditions, the hybridization
signals were analyzed with a PhosphorImager (Molecular Dynamics,
Sunnyvale, CA). The probe for mouse adipose fatty acid-binding protein
(aP2) was obtained from Dr. David Bernlohr (University of Minnesota,
Minneapolis, MN).
In Vivo Studies--
Male db/db mice (10-11-week-old
C57BLKS/J-m +/+Leprdb, The Jackson Laboratory)
were housed five per cage and allowed ad lib. access to
ground rodent chow (Purina 5001) and water. The animal room was
maintained on a 12-h light/dark cycle (dark between 7 p.m. and
7 a.m.). The animals, and their food, were weighed every 2 days
and were dosed daily by gavage with vehicle (0.5%
carboxymethylcellulose) ± PPAR agonists at the indicated doses. Drug
suspensions were prepared every 1-7 days. Plasma glucose and
triglyceride concentrations were determined from blood obtained by tail
bleeds at 3-5-day intervals during the study. Glucose and triglyceride
determinations were performed on either an Alpkem RFA/2 320 Micro-Continuous Flow Analyzer (Astoria-Pacific International,
Clackamas, OR) or a Boehringer Mannheim Hitachi 911 automatic analyzer
(Boehringer Mannheim) using heparinized plasma diluted 1:6 (v/v) with
normal saline or utilizing glucose oxidase (Sigma) and glycerol kinase (Boehringer Mannheim), respectively. Lean animals were age-matched heterozygous mice maintained in the same manner. All in vivo
experiments were approved by the Institutional Animal Care and Use Committee.
Identification of Novel Synthetic Ligands for PPAR
One of the compounds, L-783483, was subsequently
radiolabeled and shown to bind saturably and with high affinity to
recombinant hPPAR PPAR
Binding of agonist to nuclear receptors is known to induce their
interaction with one or more members of a diverse group of nuclear
co-activator proteins, including SRC-1/NcoA-1, TIF2/GRIP-1/NcoA-2, and
CBP/p300 (28-30). These co-activators function by forming a bridge
with the basal transcriptional machinery and conferring a local
increase in histone acetyltransferase activity (31, 32). Using a GST
pull-down assay, we demonstrated that both the TZD AD-5075 and the new
non-TZD ligands with high affinity for PPAR Novel Ligands Produce Transcriptional Activation of PPAR
Brown et al. (34) have recently reported the synthesis of a
potent ligand for PPAR
Since the subsequent in vivo characterization of these
ligands employed murine systems (3T3-L1 cells and db/db
mice), a chimeric PPAR PPAR
Having identified a selective PPAR
Our results definitively indicate that while PPAR In Vivo Consequences of PPAR
We used the ligands described above to evaluate the relative in
vivo effects of activating PPAR
In additional experiments, we used WY-14643 in the db/db
mouse model to assess the metabolic effects of PPAR
Taken together, these results indicate that activation of the
PPAR, PPAR
, and PPAR
. PPAR
has been implicated as a
mediator of adipocyte differentiation and the mechanism by which
thiazolidinedione drugs exert in vivo insulin
sensitization. Here we characterized novel, non-thiazolidinedione
agonists for PPAR
and PPAR
that were identified by radioligand
binding assays. In transient transactivation assays these ligands
were agonists of the receptors to which they bind. Protease protection
studies showed that ligand binding produced specific alterations in
receptor conformation. Both PPAR
and PPAR
directly interacted
with a nuclear receptor co-activator (CREB-binding protein) in an
agonist-dependent manner. Only the PPAR
agonists were
able to promote differentiation of 3T3-L1 preadipocytes. In diabetic
db/db mice all PPAR
agonists were orally active
insulin-sensitizing agents producing reductions of elevated plasma
glucose and triglyceride concentrations. In contrast, selective
in vivo activation of PPAR
did not significantly affect
these parameters. In vivo PPAR
activation with WY-14653 resulted in reductions in elevated triglyceride levels with minimal effect on hyperglycemia. We conclude that: 1) synthetic
non-thiazolidinediones can serve as ligands of PPAR
and PPAR
; 2)
ligand-dependent activation of PPAR
involves an
apparent conformational change and association of the receptor ligand
binding domain with CREB-binding protein; 3) PPAR
activation (but
not PPAR
or PPAR
activation) is sufficient to potentiate
preadipocyte differentiation; 4) non-thiazolidinedione PPAR
agonists
improve hyperglycemia and hypertriglyceridemia in vivo; 5)
although PPAR
activation is sufficient to affect triglyceride
metabolism, PPAR
activation does not appear to modulate glucose or
triglyceride levels.
INTRODUCTION
Top
Abstract
Introduction
References
, PPAR
(also referred to as hNUC1, PPAR
, or FAAR), and
PPAR
(1). PPARs regulate gene transcription by binding to specific
direct repeat-1 response elements (peroxisome proliferator response
elements) in enhancer sites of regulated genes. Each receptor binds to
it's peroxisome proliferator response element as a heterodimer with a
retinoid X receptor (RXR). Like other nuclear receptors, the ligand
binding domain (LBD) of either PPAR
(2) or PPAR
(3) undergoes
conformational changes upon binding of known agonists. Such changes in
nuclear receptor conformation are thought to create a binding surface
(dependent upon the COOH-terminal AF-2 domain) that results in the
recruitment of one or more co-activator molecules and subsequent
transcriptional activation. Both PPAR
and PPAR
have been shown to
interact with a known nuclear receptor co-regulator (steroid receptor
co-activator 1; SRC-1) (4-6).
is expressed at high levels in liver and regulates the
expression of genes involved in the
oxidation of fatty acids as
well as other aspects of lipid metabolism (7, 8). Synthetic compounds
that induce peroxisome proliferation in rodents, including WY-14643,
and hypolipidemic agents such as clofibrate have been shown to
specifically bind to and activate PPAR
(5, 9). PPAR
is
ubiquitously expressed in a broad range of mammalian tissues (10).
Neither the function, nor the array of genes regulated by this orphan
receptor, are presently known. However, some evidence suggests that
certain long-chain fatty acids may function as ligands of, and agonists
for PPAR
(9, 10).
has been shown to be expressed at high levels in mammalian
adipose tissue (11, 12). Two closely related isoforms (PPAR
1 and
PPAR
2), which differ by the addition of 30 NH2-terminal amino acids in PPAR
2, occur as a result of alternative promoter usage and mRNA splicing (11, 13). At the present time, no physiologically relevant differences in the function of these two
isoforms have been determined (14). It has become apparent that PPAR
plays an important regulatory role in adipocyte differentiation and
metabolism. The transcriptional activity of the aP2 (11), lipoprotein
lipase (15), and phosphoenolpyruvate carboxykinase (16) gene promoters
are up-regulated in adipocytes by PPAR
activation. Moreover,
ectopic overexpression of PPAR
in NIH/3T3 fibroblasts or in
myoblasts was shown to induce adipocyte differentiation (17, 18),
indicating that PPAR
is sufficient to function as an adipocyte
determination/differentiation factor. We and others have recently
demonstrated that the thiazolidinedione (TZD) insulin-sensitizing agents are specific PPAR
agonists (2, 18, 19). The in vivo antidiabetic activities of these compounds correlate with their ability to bind to, and activate, PPAR
in vitro (2, 20).
/RXR heterodimers; they have also been shown to
promote in vitro adipogenesis and in vivo insulin sensitization in rodents (21). These findings provide further support
for the role of PPAR
in regulation of adipocyte differentiation and
modulation of insulin action. However, the relative ability of PPAR
or PPAR
to exert similar physiologic effects has not been well characterized.
and PPAR
agonists. The novel compounds
differentially bound to and activated human PPAR
and PPAR
.
Binding of these ligands altered receptor conformations and induced the
association between the receptors and the coactivator CREB-binding
protein (CBP). Only PPAR
agonists were able to potentiate
adipogenesis of 3T3-L1 preadipocytes. In diabetic db/db
mice, the novel PPAR
agonists served as orally active
insulin-sensitizing agents that lowered both plasma glucose and
triglyceride concentrations. In contrast, in vivo
exposure to a PPAR
-selective compound was not sufficient to affect
glucose or triglyceride concentrations. Activation of PPAR
produced
a diminution in plasma triglycerides with minimal effects on glucose
levels in db/db mice and failed to promote the
differentiation of 3T3-L1 preadipocytes. These data strongly support
the role of PPAR
as the predominant mediator of insulin sensitization by compounds that are agonists of this receptor.
EXPERIMENTAL PROCEDURES
/GAL4, pcDNA3-hPPAR
/GAL4,
pcDNA3-mPPAR
/GAL4, pcDNA3-mPPAR
/GAL4, and
pcDNA3-mPPAR
/GAL4, were prepared by inserting the yeast GAL4 transcription factor DBD adjacent to the LBDs of hPPAR
, hPPAR
, mPPAR
, mPPAR
, and mPPAR
, respectively. The reporter construct, pUAS(5X)-tk-luc was generated by inserting five copies of the GAL4
response element upstream of the herpesvirus minimal thymidine kinase
promoter and the luciferase reporter gene (kindly provided by John
Menke, Merck Research Laboratories, Rahway, NJ). pCMV-lacZ contains the
galactosidase Z gene under the regulation of the cytomegalovirus
promoter. pSG5-hPPAR
2 and pSG5-hPPAR
were constructed by
subcloning the full-length cDNA for hPPAR
2 or hPPAR
(kindly provided by Dr. Azriel Schmidt, Merck Research Laboratories, West Point, PA), respectively, into the pSG5 mammalian expression vector (Stratagene, La Jolla, CA). pGEXKG-PPAR
LBD and pGEXKG-PPAR
LBD plasmids containing GST fused with the LBDs of hPPAR
(amino acids 176-477 of PPAR
1) or hPPAR
(amino acids 167-441) were
constructed by subcloning the LBD fragments into pGEXKG (22) digested
with XhoI and HindIII (HindIII
site was blunt-ended with T4 DNA polymerase). pGEXhCBP1-453, was constructed with a 1.5-kilobase pair NcoI-HindIII fragment encoding the
NH2-terminal 1-453 amino acids of human CBP ligated into
pGEXKG. pGEX-hPPAR
2 and pGEX-hPPAR
plasmids containing GST fused
to the full-length hPPAR
2 and hPPAR
, respectively, were
generated by subcloning the cDNAs encoding the entire receptors
into the SmaI site of pGEX-4T-2 (Amersham Pharmacia Biotech).
or GST-hPPAR
fusion proteins
were generated in Escherichia coli (BL21 strain, Stratagene,
La Jolla, CA). Cells were cultured in LB medium (Life Technologies,
Inc.) to a density of A600 = 0.7-1.0 and
induced for overexpression by addition of isopropyl-1-thio-
-D-galactopyranoside to a final
concentration of 0.2 mM. The
isopropyl-1-thio-
-D-galactopyranoside-induced cultures
were grown at room temperature for an additional 2-5 h, before cells
were harvested by centrifugation for 10 min at 5000 × g. The GST-PPAR fusion proteins were purified from the cell
pellet using glutathione-Sepharose beads, following the procedure recommended by the manufacturer (Amersham Pharmacia Biotech).
, or
GST-hPPAR
, diluted 1:1000-1:3000, was incubated in TEGM (10 mM Tris, pH 7.2, 1 mM EDTA, 10% glycerol, 7 µl/100 ml of
-mercaptoethanol, 10 mM sodium molybdate,
1 mM dithiothreitol, 5 µg/ml aprotinin, 2 µg/ml
leupeptin, 2 µg/ml benzamide, and 0.5 mM
phenylmethylsulfonyl fluoride) containing 5-10% COS-1 cell
cytoplasmic lysate and 10 nM
[3H]2AD-5075 (21 Ci/mmol) or 2.5 nM [3H]2L-783483 (17 Ci/Mmol), ± test compound. Assays were incubated for ~16
h at 4 °C in a final volume of 300 µl. Unbound ligand was removed
by incubation with 200 µl of dextran/gelatin-coated charcoal, on ice,
for ~10 min. After centrifugation at 3000 rpm for 10 min at 4 °C,
200 µl of the supernatant fraction was counted in a liquid
scintillation counter. In these assays, the KD for either AD-5075 or L-783483 is
1 nM.
2 and pSG5-hPPAR
plasmids were used to synthesize 35S-radiolabeled PPAR
2
or PPAR
, respectively, in a coupled transcription/translation system
according to the protocol of the manufacturer (Promega, Madison, WI).
The transcription/translation reactions were subsequently aliquoted
into 22.5-µl volumes, and 2.5 µl of phosphate-buffered saline ± compound were added. These mixtures were incubated for 20 min at
25 °C, separated into 4.5-µl aliquots, and 0.5 µl of distilled
H2O or distilled H2O-solubilized trypsin were
added. The protease digestions were allowed to proceed for 10 min at 25 °C, then terminated by the addition of 95 µl of denaturing gel
loading buffer and boiling for 5 min. The products of the digestion
were separated by electrophoresis through a 1.5-mm 4-20% sodium
dodecyl sulfate-polyacrylamide gel (SDS-PAGE). After electrophoresis, the gels were fixed in 10% acetic acid (v/v):40% methanol (v/v) for
30 min, treated in EN3HANCE for a further 30 min, and dried
under vacuum for 2 h at 80 °C. Autoradiography was then
performed to visualize the radiolabeled digestion products.
LBD, and GST-hPPAR
LBD fusion
proteins were generated in E. coli strain DH5
(Life
Technologies, Inc.) as described above for the GST-hPPAR
and
GST-hPPAR
fusion proteins. The hPPAR
LBD and hPPAR
LBD were
generated by thrombin cleavage of glutathione-Sepharose-bound
GST-hPPAR
LBD and GST-hPPAR
LBD, respectively. The cleavage
products were shown to be pure by SDS-PAGE followed by Coomassie Blue
staining. GST-hCBP1-453 protein (1-2 µg) bound to
glutathione-Sepharose (10 µl) was incubated with 0.2 µg of purified
hPPAR
LBD or hPPAR
LBD in 100 µl of binding buffer (8 mM Tris, pH 7.4, 120 mM KCl, 8% glycerol,
0.5% CHAPS (w/v), 1 mg/ml bovine serum albumin) for 12-16 h at
4 °C ± the indicated compound (1 µM). Samples
were pelleted by centrifugation at 11,000 × g for
20 s and washed four times with cold binding buffer. The samples
were then suspended in denaturing gel loading buffer, incubated for 5 min at 100 °C, and electrophoretically separated by SDS-PAGE.
Proteins were then electroblotted onto polyvinylidene difluoride
membranes that were subsequently incubated with anti-human PPAR
LBD
or anti-human PPAR
LBD antibodies that had been raised against
purified recombinant hPPAR
LBD or hPPAR
LBD. After washing, the
filter was incubated with donkey anti-rabbit IgG conjugated to
horseradish peroxidase and the signals visualized using the Amersham
ECL system and Kodak X-Omat film.
0.1%, a concentration which was shown not to
effect transactivation activity. Cell lysates were produced using
Reporter Lysis Buffer (Promega, Madison, WI) according to the
manufacturer's instructions. Luciferase activity in cell extracts was
determined using Luciferase Assay Buffer (Promega, Madison, WI) in an
ML3000 luminometer (Dynatech Laboratories, Chantilly, VA).
-Galactosidase activity was determined using
-D-galactopyranoside (Calbiochem) as described
previously (24).
RESULTS AND DISCUSSION
and
PPAR
--
Known PPAR
ligands include the prostaglandin
metabolite 15-deoxy-
12,14-PGJ2 (26) and the synthetic
thiazolidinedione antidiabetic agents, which bind with high affinity
and specificity to this receptor (2, 19). Using a combination of
molecular modeling and directed chemical
synthesis,2 we synthesized a
series of structurally distinct non-TZD compounds, which are PPAR
and/or PPAR
agonists (Fig. 1). As
depicted in Fig. 2A, a binding
assay employing the radiolabeled TZD AD-5075 and recombinant PPAR
was used to demonstrate that three of these compounds,
L-796449, L-165461, and L-783483
(all phenylacetic acid derivatives), were potent ligands for PPAR
(Ki = 2, 15, and 14 nM, respectively).
As expected, the TZDs AD-5075, BRL 49653, and troglitazone displaced
the radiolabeled ligand differentially with Ki
values of 1, 24, and 250 nM, respectively. In contrast, a
fourth non-TZD, L-165041 (a phenoxyacetic acid derivative),
was far less potent (Ki ~ 730 nM) and
WY-14643 failed to displace labeled AD-5075 from PPAR
at
concentrations up to 30 µM (data not shown).
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Fig. 1.
Structures of PPAR
ligands.
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Fig. 2.
Novel ligands bind to PPAR
and/or PPAR
. Competition curves
generated by incubation of 10 nM
[3H]2AD-5075 with GST-hPPAR
(A)
or 2.5 nM [3H]2L-783483 with
GST-hPPAR
(B). The displacement of radioligand
after incubation in the presence of the indicated concentration of each
unlabeled compound for ~16 h is plotted. Similar results were
obtained in at least two independent experiments performed in
duplicate.
. Scatchard analysis demonstrated a
KD of
1 nM for this binding
interaction.3 As shown in
Fig. 2B, the Ki for displacement of
[3H]2L-783483 by cold compound
was 1 nm. Titration of other compounds in this PPAR
binding
assay revealed that L-796449, L-165461, and
L-165041 also bound to PPAR
with high affinity (Ki = 2, 3, and 6 nM, respectively). The
TZDs AD-5075, BRL 49653, and troglitazone (Fig. 2B) and
WY-14643 (data not shown) were unable to displace labeled
L-783483 from the receptor.
and PPAR
Ligands Alter Receptor Conformation and Mediate
Co-activator Association--
We reported previously that saturating
concentrations of TZDs can induce an alteration in the conformation of
PPAR
, as assessed by generation of a major protease-resistant band
following partial protease digestion of recombinant receptor protein
(2). In addition, Dowell et al. (3) reported that
selected PPAR
activators, including clofibrate and WY-14643, induce
similar conformational changes upon incubation with recombinant
PPAR
. Both of these effects are analogous to changes in estrogen
receptor (ER
) conformation that have been observed following the
binding of known agonists (e.g. estradiol)(27). In contrast,
ER
antagonists induce different, and more limited, changes in the
pattern of fragments produced following limited protease digestion
(27). When incubated with PPAR
, the TZD AD-5075 protects a fragment
of ~25 kDa from trypsin digestion (Fig.
3A, upper panel).
On the other hand, no protection is evident when PPAR
is treated
with AD-5075 (Fig. 3A, lower panel). As shown for
PPAR
in the top panel of Fig. 3B, the novel PPAR
/
ligand, L-165461 produced a protease protection
pattern that was indistinguishable from that observed using the known TZD agonist AD-5075. L-165461, however, also protected a
fragment of PPAR
from digestion (Fig. 3B, lower
panel). In contrast, treatment with L-165041 alters
the conformation of PPAR
, but not PPAR
(Fig. 3C), as
expected based upon it's affinity for the respective receptors. These
results demonstrate that the newly identified PPAR
and PPAR
ligands produce altered, and, presumably, active conformations of the
receptors to which they bind.
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Fig. 3.
PPAR ligands alter the conformation of
receptors to which they bind. 35S-PPAR (upper
panels) or 35S-PPAR
(lower panels) were
synthesized in vitro in a coupled transcription/translation
system. Labeled receptor was subsequently incubated with 0.1% dimethyl
sulfoxide (Control) or 10 µM AD-5075
(A), 10 µM L-165461
(B), or 10 µM L-165041
(C), followed by incubation with distilled H2O
or increasing concentrations of trypsin. Digestion products were
analyzed by SDS-PAGE followed by autoradiography. An
arrowhead indicates the 25-kDa protease-resistant fragment
of PPAR
(A, AD-5075 and B,
L-165461) or PPAR
(B, L-165461,
and C, L-165041).
, L-783483
and L-165461, induce the in vitro association of
the hPPAR
LBD with the co-activator CBP (Fig.
4A). At higher concentrations
(>5 µM), the PPAR
-selective compound,
L-165041, was able to induce weak association between
hPPAR
LBD and CBP (not shown), as expected given its weak PPAR
binding activity. In addition, the potent PPAR
ligands (but not the
TZD) were able to promote an association of hPPAR
LBD with this
co-activator (Fig. 4B). Both PPAR
and PPAR
reportedly
undergo a ligand-induced association with SRC-1 (4-6). Our results
show that hPPAR
and hPPAR
can also be induced to associate with
CBP following ligand binding, suggesting an important role for this
co-activator in transcriptional activation mediated by these receptors.
Furthermore, this ligand-dependent co-activator association
suggests that the novel ligands are agonists for either PPAR
or
PPAR
. It is worth noting that Dowell et al. (33) reported
recently that p300 (a homologue of CBP) can function as a co-activator
for PPAR
.
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Fig. 4.
PPAR ligand binding induces PPAR-CBP
association. The GST-hCBP1-453 fusion protein bound
to glutathione-Sepharose beads was incubated with either hPPAR -LBD
(A) or hPPAR
-LBD (B) ± the indicated ligand.
Proteins associated with the beads were eluted in denaturing sample
buffer. They were subsequently separated by SDS-PAGE, and PPAR LBDs
were visualized by immunoblotting with anti-LBD antibodies.
Ligand-dependent association of hPPAR
(A) or
hPPAR
(B) with CBP is indicated by the presence of a
band.
or
PPAR
--
In order to examine their activity as agonists in a
cell-based context, the non-TZD ligands were incubated with COS-1 cells that had been co-transfected with chimeric receptors composed of the
GAL4 DBD and a PPAR LBD along with a GAL4-responsive reporter gene.
Both AD-5075 and the new high affinity PPAR
ligands
(L-796449, L-783483, and L-165461)
produced robust transactivation of the UAS reporter gene, in cells
co-transfected with GAL4-hPPAR
(Fig. 5A). In contrast, weak
transactivation by PPAR
was observed with L-165041, and
WY-14643 failed to activate GAL4-hPPAR
. All four new compounds with
high affinity for PPAR
(L-783483, L-796449, L-165461, L-165041) demonstrated similar, and
substantial, degrees of UAS reporter gene transactivation in cells
expressing GAL4-hPPAR
(Fig. 5B). As predicted, both
WY-14643 and AD-5075 failed to activate GAL4-hPPAR
. These
experiments were repeated using GAL4 chimeric receptors containing
murine PPAR
or PPAR
LBDs with similar results (not shown).
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Fig. 5.
Novel ligands are PPAR agonists.
Transactivation by hPPAR (A), hPPAR
(B), or mPPAR
(C) in COS-1 cells transiently
cotransfected with pSG5-hPPAR
/GAL4, pSG5-hPPAR
/GAL4, or
pSG5-mPPAR
/GAL4 and both pUAS(5X)-tk-luciferase and
pCMV-lacz then incubated with the indicated concentrations
of ligand for 48 h. The figure plots normalized luciferase
activity; similar results were obtained in two independent experiments
performed in triplicate (
, AD-5075;
, L-796449;
,
L-165461;
, L-783483;
,
L-165041;
, WY14643).
, GW 2433, which also exhibits PPAR
activity. However, no assessment of potential biological effects of
this ligand have been reported. Other than GW 2433 and the compounds
reported here, there are no other known ligands for PPAR
, which are
suitable for use as tools to explore the potential physiologic roles of
this receptor. By comparison, fatty acids such as linoleic acid, which
have been reported to function as PPAR
agonists, have extremely weak
activity (30 µM) and lack receptor selectivity (9).
receptor composed of a cDNA encoding the
murine PPAR
LBD and the GAL4 DBD was used to evaluate their PPAR
activity. WY-14643, a potent and specific PPAR
agonist (3, 9), was
used as the positive control. In COS-1 cells transfected with
GAL4-mPPAR
, neither the four Merck ligands nor AD-5075 stimulated
reporter gene transactivation, whereas WY-14643 evoked a robust
transcriptional response (Fig. 5C). Thus, PPAR agonists with
the following profiles were available for further biological
evaluation: potent and selective PPAR
activity (WY-14643); potent
and selective PPAR
activity (AD-5075); potent PPAR
and PPAR
activity (L-165461, L-783483, L-796449); and selective PPAR
activity
(L-165041).
but Not PPAR
or PPAR
Activation Is Sufficient to
Promote Preadipocyte Differentiation--
It is clear that TZD
compounds, which are potent activators and selective ligands for
PPAR
, can promote in vitro differentiation of 3T3-L1
preadipocytes (35, 36). A similar adipogenic effect of TZDs has been
observed using cultured bone marrow stromal cells (37).4 Moreover, forced
overexpression of PPAR
in fibroblasts (18) or cultured myoblasts
(17) is sufficient to drive adipocyte differentiation. The role of the
other PPAR isoforms in adipogenesis, however, is less clear. Brun
et al. (39) reported recently that ectopic overexpression of
PPAR
in NIH/3T3 cells followed by stimulation with WY-14643 was
sufficient to induce adipogenesis, while overexpression of PPAR
, in
the absence of ligand, was ineffective. Other investigators have also
reported that high concentrations of PPAR
activators, including
8(S)-HETE, WY-14643 (40), or bezafibrate (41), can promote
differentiation of 3T3-L1 cells. Amri et al. (42) have implicated a role for PPAR
in adipogenesis by showing that 3T3-C2 fibroblasts, which overexpress PPAR
(FAAR in their paper), could be
induced to express selected adipocyte genes after stimulation with
fatty acids.
ligand, L-165041, we
sought to use it, and the PPAR
agonist WY-14643, to definitively assess the role of activation of these receptors versus
PPAR
on adipocyte differentiation. To do this we measured aP2
mRNA expression in differentiating 3T3-L1 cells as a sensitive
measure of adipogenesis. The level of aP2 mRNA is well correlated
with both lipid accumulation and up-regulation of other adipocyte
genes, including GLUT4 (36). As depicted in Fig.
6, preadipocyte differentiation correlated with PPAR
binding affinity; AD-5075 and the Merck agonists, L-796449, L-783483, and
L-165461, produced robust preadipocyte differentiation. In
contrast, concentrations of WY-14643, which were within, or
substantially above, the range needed for transactivation of murine
PPAR
in COS-1 cells, failed to promote differentiation. Although
3T3-L1 cells express significant levels of PPAR
(43), L-165041 did not increase the expression of aP2 mRNA at
concentrations shown to selectively activate PPAR
in COS-1 cells.
The modest effect at 30 µM L-165041 is
presumably due to activation of PPAR
.
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Fig. 6.
PPAR agonists
potentiate adipogenesis. Confluent 3T3-L1 preadipocytes were
incubated ± the indicated concentrations of ligand for four days.
Total RNA was isolated, and 20 µg of denatured RNA was analyzed by
slot blot using a radiolabeled mouse aP2 cDNA probe. Normalized aP2
mRNA levels are plotted; similar results were obtained in at least
two independent experiments conducted in triplicate.
activation was
sufficient to induce adipocyte differentiation in 3T3-L1 cells,
activation of PPAR
or PPAR
had no significant effect on this
process. It should be noted that the adipogenic effects reported by
others with PPAR
and/or PPAR
activators were observed with
nonselective receptor agonists (fatty acids) and/or very high ligand
concentrations (e.g. 0.5 mM WY-14643 in data
reported by Yu et al. (40)) where modest activation of
PPAR
can be expected to occur.
Versus PPAR
or PPAR
Activation--
The in vivo insulin-sensitizing action of
TZD's has been attributed to their PPAR
activity since, in general,
beneficial effects on hyperglycemia and hypertriglyceridemia observed
with this class of agent correlates with in vitro potency in
PPAR
binding or transactivation assays (2, 20). However, the ability of PPAR
or PPAR
activation to affect insulin sensitivity is not
well characterized. In vivo metabolic effects similar to the TZD's have been reported with selective RXR ligands that activate RXR:PPAR
heterodimers in transfected CV-1 cells (21). However, such
compounds can also activate RXR:PPAR
(44) and are likely to activate
RXR:PPAR
heterodimers as well. Houseknecht et al. (45)
reported recently that in vivo administration of conjugated linoleic acid normalizes impaired glucose tolerance in young Zucker diabetic fatty rats; this finding suggests that activation of multiple
PPARs might exert insulin-sensitizing effects, since linoleic acid is
known to activate all three PPAR subtypes (9, 46). Furthermore, some
evidence suggests that weak PPAR
activators (including clofibrate or
bezafibrate) can exert insulin-sensitizing effects in rats (47) or man
(48).
, PPAR
, and PPAR
in obese, insulin-resistant db/db mice. As shown in Fig.
7A, in vivo
treatment of these mice with L-796449 (at 10 mg/kg/day), a
potent Merck PPAR
agonist, or AD-5075 (at 2 mg/kg/day), resulted in
robust reductions of both plasma glucose and triglycerides. Similar
effects have been observed with other Merck PPAR
agonists, including L-165461 and L-783483 (data not shown). In
contrast, in vivo treatment with L-165041, a
potent PPAR
-selective agonist, did not significantly affect either
glucose or triglycerides at 30 mg/kg/day (Fig. 7B). However,
L-165041, at the same in vivo exposure level
(and even at a 3-fold lower dose), did affect plasma cholesterol in
db/db mice5; we
have observed that this response is associated with PPAR
, but not
PPAR
, in vitro activity. As expected, given the weak activity of L-165041 on PPAR
, it did lower glucose in
db/db mice when administered at higher doses (Fig.
8).
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Fig. 7.
Effects of PPAR agonists in
db/db mice. Male db/db mice were dosed
daily by gavage with vehicle or the indicated dose of PPAR agonist.
Blood samples were obtained from the tail of each mouse every 3-5 days
for the determination of both plasma glucose and triglyceride
concentrations. Each data point represents the mean (±S.E.) of 7-10
individual mice. All results are representative of at least two
independent experiments. A, treatment with either 2 mg/kg/day of the TZD AD-5075 or 10 mg/kg/day of the Merck PPAR
agonist L-796449. B, treatment with either 30 mg/kg/day of the Merck PPAR
agonist L-165041 or 10 mg/kg/day of the Merck PPAR
agonist L-796449.
C, treatment with either 10 mg/kg/day of the PPAR
agonist
WY-14643 or 2 mg/kg/day of AD-5075.
View larger version (20K):
[in a new window]
Fig. 8.
PPAR binding
affinity correlates with the ED50 for reduced plasma
glucose in db/db mice. To evaluate the
correlation between PPAR binding and in vivo antidiabetic
activity, the approximate ED50 values for glucose lowering
in db/db mice were plotted versus the
Ki values determined for PPAR
binding.
on glucose and triglyceride levels. As shown in Fig. 7C, a dose of 10 mg/kg/day of WY-14643 was sufficient to normalize elevated triglyceride levels in db/db mice. The effect of WY-14643 on glucose
levels was minimal relative to the effects of either AD-5075 or
L-796449 (Fig. 7A), both of which normalized
glucose and triglyceride levels. Based on these results, we conclude
that in vivo activation of PPAR
preferentially modulates
triglyceride metabolism without substantially affecting insulin
sensitivity. This is consistent with clinical findings where
therapeutic doses of fibrates reliably lower elevated triglycerides but
produce variable, and/or subtle, effects on glucose metabolism (38, 48,
49).
:RXR heterodimer through either PPAR
, with a TZD or non-TZD, or RXR (21) is sufficient to promote preadipocyte differentiation and
in vivo insulin sensitization. In contrast, activation of neither PPAR
nor PPAR
results in a comparable effects on
adipogenesis or glucose homeostasis. With respect to in vivo
insulin sensitization, it is important to note that the
ED50 for glucose lowering in db/db mice
correlates with the PPAR
binding affinity (Ki) of
both TZD and non-TZD agonists (Fig. 8). Additional studies will be required to further define the physiological roles of PPAR
.
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ACKNOWLEDGEMENTS |
---|
We are indebted to Gerard Kieczykowski,
Philip Eskola, Joseph F. Leone, and Peter A. Cicala (Merck Research
Laboratories, Rahway, NJ) for the preparation of AD-5075 and Dominick
F. Gratale for synthesis of L-783483. We thank David G. Melillo, Yui Sing Tang, and Allen N. Jones (Merck Research
Laboratories, Rahway, NJ) for the preparation of
[3H]2AD-5075 and
[3H]2L-783483. Dr. David Bernlohr (University
of Minnesota, Minneapolis, MN) generously provided the probe for mouse
adipose fatty acid binding protein. Dr. Azriel Schmidt (Merck Research
Laboratories, West Point, PA) provided the human PPAR cDNA and
has been a source of information and intellectual support throughout
this work. John Menke (Merck Research Laboratories, Rahway, NJ)
prepared and kindly provided pUAS(5X)-tk-luc. The assistance of Roger
Meurer, Michael Forrest, Charlotte Trainor, Michele Mariano, and
Beverly A. Shelton in conducting the in vivo
experiments is greatly appreciated. We would also like to thank Dr.
Johan Auwerx (Institut Pasteur, Lille, France) for ongoing intellectual consultation.
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FOOTNOTES |
---|
* 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.
These authors contributed equally to this work.
§ To whom correspondences should be addressed at: Merck Research Laboratories RY80N-C31, 126 E. Lincoln Ave., Rahway, NJ 07065. Tel.: 732-594-4738; Fax: 732-594-3925; E-mail: joel_berger{at}merck.com.
¶ Present address: Ligand Pharmaceutical, Inc., 9393 Towne Center Dr., San Diego, CA 92121.
** Present address: Pfizer Central Research, Eastern Point Rd., Groton, CT 06340.
Present address: SmithKline Beecham Pharmaceuticals, 709 Swedeland Rd., King of Prussia, PA 19406.
§§ Present address: Geron Corp., 230 Constitution Dr., Menlo Park, CA 94025.
¶¶ Present address: Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030.
2 R. L. Tolman, S. P. Sahoo, C. Santini, C. Liang, G. D. Berger, R. W. Marquis, W. Han, D. Gratale, D. Von Langen, R. Mosley, J. Berger, M. D. Leibowitz, T. W. Doebber, K. MacNaul, B. Zhang, R. G. Smith, and D. E. Moller, manuscript in preparation.
3 M. Leibowitz, unpublished data.
4 B. Zhang, unpublished data.
5 M. D. Leibowitz, C. Fiévet, N. Hennuyer, J. Peinado-Onsurbe, H. Duez, J. Berger, C. A. Cullinan, C. P. Sparrow, J. Baffic, G. D. Berger, C. Santini, R. W. Marquis, R. Tolman, J.-C. Fruchart, R. G. Smith, D. E. Moller, and J. Auwerx, submitted for publication.
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ABBREVIATIONS |
---|
The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; RXR, retinoid X receptor; LBD, ligand binding domain; SRC-1, steroid receptor co-activator 1; TZD, thiazolidinedione; DBD, DNA binding domain; GST, glutathione S-transferase; CBP, CREB-binding protein; PAGE, polyacrylamide gel electrophoresis; CHAPS, 3-[(cholamidopropyl)dimethylammonio[-1-propanesulfonic acid]; UAS, upstream activating sequence.
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REFERENCES |
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