Novel Peroxisome Proliferator-activated Receptor (PPAR) gamma  and PPARdelta Ligands Produce Distinct Biological Effects*

Joel BergerDagger §, Mark D. LeibowitzDagger , Thomas W. Doebber, Alex Elbrecht, Bei Zhang, Gaochou Zhou, Chhabi Biswas, Catherine A. Cullinan, Nancy S. Hayes, Ying Li, Michael Tanen, John Ventre, Margaret S. Wu, Gregory D. Bergerparallel **, Ralph Mosleyparallel , Robert Marquisparallel Dagger Dagger , Conrad Santiniparallel , Soumya P. Sahooparallel , Richard L. Tolmanparallel §§, Roy G. Smith¶¶, and David E. Moller

From the Departments of Molecular Endocrinology and parallel  Medicinal Chemistry, Merck Research Laboratories, Rahway, New Jersey 07065

    ABSTRACT
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Abstract
Introduction
References

The peroxisome proliferator-activated receptors (PPARs) include three receptor subtypes encoded by separate genes: PPARalpha , PPARdelta , and PPARgamma . PPARgamma 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 PPARgamma and PPARdelta 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 PPARgamma and PPARdelta directly interacted with a nuclear receptor co-activator (CREB-binding protein) in an agonist-dependent manner. Only the PPARgamma agonists were able to promote differentiation of 3T3-L1 preadipocytes. In diabetic db/db mice all PPARgamma agonists were orally active insulin-sensitizing agents producing reductions of elevated plasma glucose and triglyceride concentrations. In contrast, selective in vivo activation of PPARdelta did not significantly affect these parameters. In vivo PPARalpha 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 PPARgamma and PPARdelta ; 2) ligand-dependent activation of PPARdelta involves an apparent conformational change and association of the receptor ligand binding domain with CREB-binding protein; 3) PPARgamma activation (but not PPARdelta or PPARalpha activation) is sufficient to potentiate preadipocyte differentiation; 4) non-thiazolidinedione PPARgamma agonists improve hyperglycemia and hypertriglyceridemia in vivo; 5) although PPARalpha activation is sufficient to affect triglyceride metabolism, PPARdelta activation does not appear to modulate glucose or triglyceride levels.

    INTRODUCTION
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Abstract
Introduction
References

In mammals, the peroxisome proliferator-activated receptor (PPAR)1 family of nuclear hormone receptors consists of three subtypes encoded by separate genes: PPARalpha , PPARdelta (also referred to as hNUC1, PPARbeta , or FAAR), and PPARgamma (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 PPARgamma (2) or PPARalpha (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 PPARgamma and PPARalpha have been shown to interact with a known nuclear receptor co-regulator (steroid receptor co-activator 1; SRC-1) (4-6).

PPARalpha is expressed at high levels in liver and regulates the expression of genes involved in the beta  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 PPARalpha (5, 9). PPARdelta 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 PPARdelta (9, 10).

PPARgamma has been shown to be expressed at high levels in mammalian adipose tissue (11, 12). Two closely related isoforms (PPARgamma 1 and PPARgamma 2), which differ by the addition of 30 NH2-terminal amino acids in PPARgamma 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 PPARgamma 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 PPARgamma activation. Moreover, ectopic overexpression of PPARgamma in NIH/3T3 fibroblasts or in myoblasts was shown to induce adipocyte differentiation (17, 18), indicating that PPARgamma 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 PPARgamma agonists (2, 18, 19). The in vivo antidiabetic activities of these compounds correlate with their ability to bind to, and activate, PPARgamma in vitro (2, 20).

Structurally distinct, selective RXR agonists have been identified that can activate PPARgamma /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 PPARgamma in regulation of adipocyte differentiation and modulation of insulin action. However, the relative ability of PPARalpha or PPARdelta to exert similar physiologic effects has not been well characterized.

Here, we report the identification and characterization of novel, non-TZD PPARgamma and PPARdelta agonists. The novel compounds differentially bound to and activated human PPARgamma and PPARdelta . Binding of these ligands altered receptor conformations and induced the association between the receptors and the coactivator CREB-binding protein (CBP). Only PPARgamma agonists were able to potentiate adipogenesis of 3T3-L1 preadipocytes. In diabetic db/db mice, the novel PPARgamma agonists served as orally active insulin-sensitizing agents that lowered both plasma glucose and triglyceride concentrations. In contrast, in vivo exposure to a PPARdelta -selective compound was not sufficient to affect glucose or triglyceride concentrations. Activation of PPARalpha 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 PPARgamma as the predominant mediator of insulin sensitization by compounds that are agonists of this receptor.

    EXPERIMENTAL PROCEDURES

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, pcDNA3hPPARgamma /GAL4, pcDNA3-hPPARdelta /GAL4, pcDNA3-mPPARgamma /GAL4, pcDNA3-mPPARdelta /GAL4, and pcDNA3-mPPARalpha /GAL4, were prepared by inserting the yeast GAL4 transcription factor DBD adjacent to the LBDs of hPPARgamma , hPPARdelta , mPPARgamma , mPPARdelta , and mPPARalpha , 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-hPPARgamma 2 and pSG5-hPPARdelta were constructed by subcloning the full-length cDNA for hPPARgamma 2 or hPPARdelta (kindly provided by Dr. Azriel Schmidt, Merck Research Laboratories, West Point, PA), respectively, into the pSG5 mammalian expression vector (Stratagene, La Jolla, CA). pGEXKG-PPARgamma LBD and pGEXKG-PPARdelta LBD plasmids containing GST fused with the LBDs of hPPARgamma (amino acids 176-477 of PPARgamma 1) or hPPARdelta (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-hPPARgamma 2 and pGEX-hPPARdelta plasmids containing GST fused to the full-length hPPARgamma 2 and hPPARdelta , respectively, were generated by subcloning the cDNAs encoding the entire receptors into the SmaI site of pGEX-4T-2 (Amersham Pharmacia Biotech).

Binding Assay-- GST-hPPARgamma or GST-hPPARdelta 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-beta -D-galactopyranoside to a final concentration of 0.2 mM. The isopropyl-1-thio-beta -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).

For each assay, an aliquot of receptor, GST-hPPARgamma , or GST-hPPARdelta , 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 beta -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 approx 1 nM.

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-hPPARgamma 2 and pSG5-hPPARdelta plasmids were used to synthesize 35S-radiolabeled PPARgamma 2 or PPARdelta , 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.

PPAR-CBP Pull-down Assay-- The GST-hCBP1-453, GST-hPPARgamma LBD, and GST-hPPARdelta LBD fusion proteins were generated in E. coli strain DH5alpha (Life Technologies, Inc.) as described above for the GST-hPPARgamma and GST-hPPARdelta fusion proteins. The hPPARgamma LBD and hPPARdelta LBD were generated by thrombin cleavage of glutathione-Sepharose-bound GST-hPPARgamma LBD and GST-hPPARdelta 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 hPPARgamma LBD or hPPARdelta 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 PPARgamma LBD or anti-human PPARdelta LBD antibodies that had been raised against purified recombinant hPPARgamma LBD or hPPARdelta 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.

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 <= 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). beta -Galactosidase activity was determined using beta -D-galactopyranoside (Calbiochem) as described previously (24).

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.

    RESULTS AND DISCUSSION

Identification of Novel Synthetic Ligands for PPARgamma and PPARdelta -- Known PPARgamma ligands include the prostaglandin metabolite 15-deoxy-Delta 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 PPARgamma and/or PPARdelta agonists (Fig. 1). As depicted in Fig. 2A, a binding assay employing the radiolabeled TZD AD-5075 and recombinant PPARgamma was used to demonstrate that three of these compounds, L-796449, L-165461, and L-783483 (all phenylacetic acid derivatives), were potent ligands for PPARgamma (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 PPARgamma 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 PPARgamma and/or PPARdelta . Competition curves generated by incubation of 10 nM [3H]2AD-5075 with GST-hPPARgamma (A) or 2.5 nM [3H]2L-783483 with GST-hPPARdelta (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.

One of the compounds, L-783483, was subsequently radiolabeled and shown to bind saturably and with high affinity to recombinant hPPARdelta . Scatchard analysis demonstrated a KD of approx  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 PPARdelta binding assay revealed that L-796449, L-165461, and L-165041 also bound to PPARdelta 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.

PPARgamma and PPARdelta 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 PPARgamma , 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 PPARalpha activators, including clofibrate and WY-14643, induce similar conformational changes upon incubation with recombinant PPARalpha . Both of these effects are analogous to changes in estrogen receptor (ERalpha ) conformation that have been observed following the binding of known agonists (e.g. estradiol)(27). In contrast, ERalpha antagonists induce different, and more limited, changes in the pattern of fragments produced following limited protease digestion (27). When incubated with PPARgamma , 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 PPARdelta is treated with AD-5075 (Fig. 3A, lower panel). As shown for PPARgamma in the top panel of Fig. 3B, the novel PPARgamma /delta 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 PPARdelta from digestion (Fig. 3B, lower panel). In contrast, treatment with L-165041 alters the conformation of PPARdelta , but not PPARgamma (Fig. 3C), as expected based upon it's affinity for the respective receptors. These results demonstrate that the newly identified PPARgamma and PPARdelta 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-PPARgamma (upper panels) or 35S-PPARdelta (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 PPARgamma (A, AD-5075 and B, L-165461) or PPARdelta (B, L-165461, and C, L-165041).

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 PPARgamma , L-783483 and L-165461, induce the in vitro association of the hPPARgamma LBD with the co-activator CBP (Fig. 4A). At higher concentrations (>5 µM), the PPARdelta -selective compound, L-165041, was able to induce weak association between hPPARgamma LBD and CBP (not shown), as expected given its weak PPARgamma binding activity. In addition, the potent PPARdelta ligands (but not the TZD) were able to promote an association of hPPARdelta LBD with this co-activator (Fig. 4B). Both PPARgamma and PPARalpha reportedly undergo a ligand-induced association with SRC-1 (4-6). Our results show that hPPARgamma and hPPARdelta 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 PPARgamma or PPARdelta . It is worth noting that Dowell et al. (33) reported recently that p300 (a homologue of CBP) can function as a co-activator for PPARalpha .


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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 hPPARgamma -LBD (A) or hPPARdelta -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 hPPARgamma (A) or hPPARdelta (B) with CBP is indicated by the presence of a band.

Novel Ligands Produce Transcriptional Activation of PPARgamma or PPARdelta -- 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 PPARgamma ligands (L-796449, L-783483, and L-165461) produced robust transactivation of the UAS reporter gene, in cells co-transfected with GAL4-hPPARgamma (Fig. 5A). In contrast, weak transactivation by PPARgamma was observed with L-165041, and WY-14643 failed to activate GAL4-hPPARgamma . All four new compounds with high affinity for PPARdelta (L-783483, L-796449, L-165461, L-165041) demonstrated similar, and substantial, degrees of UAS reporter gene transactivation in cells expressing GAL4-hPPARdelta (Fig. 5B). As predicted, both WY-14643 and AD-5075 failed to activate GAL4-hPPARdelta . These experiments were repeated using GAL4 chimeric receptors containing murine PPARgamma or PPARdelta LBDs with similar results (not shown).


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Fig. 5.   Novel ligands are PPAR agonists. Transactivation by hPPARgamma (A), hPPARdelta (B), or mPPARalpha (C) in COS-1 cells transiently cotransfected with pSG5-hPPARgamma /GAL4, pSG5-hPPARdelta /GAL4, or pSG5-mPPARalpha /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 (triangle , AD-5075; , L-796449; black-square, L-165461; black-triangle, L-783483; black-diamond , L-165041; , WY14643).

Brown et al. (34) have recently reported the synthesis of a potent ligand for PPARdelta , GW 2433, which also exhibits PPARalpha 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 PPARdelta , 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 PPARdelta agonists, have extremely weak activity (30 µM) and lack receptor selectivity (9).

Since the subsequent in vivo characterization of these ligands employed murine systems (3T3-L1 cells and db/db mice), a chimeric PPARalpha receptor composed of a cDNA encoding the murine PPARalpha LBD and the GAL4 DBD was used to evaluate their PPARalpha activity. WY-14643, a potent and specific PPARalpha agonist (3, 9), was used as the positive control. In COS-1 cells transfected with GAL4-mPPARalpha , 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 PPARalpha activity (WY-14643); potent and selective PPARgamma activity (AD-5075); potent PPARgamma and PPARdelta activity (L-165461, L-783483, L-796449); and selective PPARdelta activity (L-165041).

PPARgamma but Not PPARdelta or PPARalpha Activation Is Sufficient to Promote Preadipocyte Differentiation-- It is clear that TZD compounds, which are potent activators and selective ligands for PPARgamma , 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 PPARgamma 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 PPARalpha in NIH/3T3 cells followed by stimulation with WY-14643 was sufficient to induce adipogenesis, while overexpression of PPARdelta , in the absence of ligand, was ineffective. Other investigators have also reported that high concentrations of PPARalpha 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 PPARdelta in adipogenesis by showing that 3T3-C2 fibroblasts, which overexpress PPARdelta (FAAR in their paper), could be induced to express selected adipocyte genes after stimulation with fatty acids.

Having identified a selective PPARdelta ligand, L-165041, we sought to use it, and the PPARalpha agonist WY-14643, to definitively assess the role of activation of these receptors versus PPARgamma 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 PPARgamma 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 PPARalpha in COS-1 cells, failed to promote differentiation. Although 3T3-L1 cells express significant levels of PPARdelta (43), L-165041 did not increase the expression of aP2 mRNA at concentrations shown to selectively activate PPARdelta in COS-1 cells. The modest effect at 30 µM L-165041 is presumably due to activation of PPARgamma .


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Fig. 6.   PPARgamma 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.

Our results definitively indicate that while PPARgamma activation was sufficient to induce adipocyte differentiation in 3T3-L1 cells, activation of PPARalpha or PPARdelta had no significant effect on this process. It should be noted that the adipogenic effects reported by others with PPARalpha and/or PPARdelta 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 PPARgamma can be expected to occur.

In Vivo Consequences of PPARgamma Versus PPARdelta or PPARalpha Activation-- The in vivo insulin-sensitizing action of TZD's has been attributed to their PPARgamma activity since, in general, beneficial effects on hyperglycemia and hypertriglyceridemia observed with this class of agent correlates with in vitro potency in PPARgamma binding or transactivation assays (2, 20). However, the ability of PPARalpha or PPARdelta 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:PPARgamma heterodimers in transfected CV-1 cells (21). However, such compounds can also activate RXR:PPARalpha (44) and are likely to activate RXR:PPARdelta 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 PPARalpha activators (including clofibrate or bezafibrate) can exert insulin-sensitizing effects in rats (47) or man (48).

We used the ligands described above to evaluate the relative in vivo effects of activating PPARgamma , PPARdelta , and PPARalpha 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 PPARgamma 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 PPARgamma agonists, including L-165461 and L-783483 (data not shown). In contrast, in vivo treatment with L-165041, a potent PPARdelta -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 PPARdelta , but not PPARgamma , in vitro activity. As expected, given the weak activity of L-165041 on PPARgamma , 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 PPARgamma agonist L-796449. B, treatment with either 30 mg/kg/day of the Merck PPARdelta agonist L-165041 or 10 mg/kg/day of the Merck PPARgamma agonist L-796449. C, treatment with either 10 mg/kg/day of the PPARalpha agonist WY-14643 or 2 mg/kg/day of AD-5075.


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Fig. 8.   PPARgamma 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 PPARgamma binding.

In additional experiments, we used WY-14643 in the db/db mouse model to assess the metabolic effects of PPARalpha 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 PPARalpha 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).

Taken together, these results indicate that activation of the PPARgamma :RXR heterodimer through either PPARgamma , 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 PPARalpha nor PPARdelta 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 PPARgamma binding affinity (Ki) of both TZD and non-TZD agonists (Fig. 8). Additional studies will be required to further define the physiological roles of PPARdelta .

    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 PPARdelta 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.

    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.

Dagger 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.

Dagger Dagger 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.

    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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Abstract
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