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

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

In mammals, the peroxisome proliferator-activated receptor (PPAR) 1 family of nuclear hormone receptors consists of three subtypes encoded by separate genes: PPAR␣, 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).
PPAR␣ 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).
PPAR␥ 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 NH 2 -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).
Structurally distinct, selective RXR agonists have been identified that can activate PPAR␥/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.
Here, we report the identification and characterization of novel, non-TZD PPAR␥ 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. Plasmids-The chimeric receptor expression constructs, pcDNA3-hPPAR␥/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). pGEXhC-BP 1-453 , was constructed with a 1.5-kilobase pair NcoI-HindIII fragment encoding the NH 2 -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).

Materials-Cell
Binding Assay-GST-hPPAR␥ 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 A 600 ϭ 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 ϫ 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).

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␥2 and pSG5-hPPAR␦ plasmids were used to synthesize 35 Sradiolabeled 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-l volumes, and 2.5 l of phosphatebuffered saline Ϯ compound were added. These mixtures were incubated for 20 min at 25°C, separated into 4.5-l aliquots, and 0.5 l of distilled H 2 O or distilled H 2 O-solubilized trypsin were added. The protease digestions were allowed to proceed for 10 min at 25°C, then terminated by the addition of 95 l 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 EN 3 HANCE 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-hCBP 1-453 , GST-hPPAR␥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-hCBP 1-453 protein (1-2 g) bound to glutathione-Sepharose (10 l) was incubated with 0.2 g of purified hPPAR␥LBD or hPPAR␦LBD in 100 l 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 Ϯ the indicated compound (1 M). Samples were pelleted by centrifugation at 11,000 ϫ 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.
Cell Culture and Transactivation Assay-COS-1 cells were seeded at 12 ϫ 10 3 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% CO 2 . 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 l of LipofectAMINE, 0.00075 g of pcDNA3-PPAR/GAL4 expression vector, 0.045 g of pUAS(5X)-tk-luc reporter vector, and 0.0002 g 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% CO 2. The cells were then incubated for ϳ48 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 Ϯ increasing concentrations of test compound. Since the compounds were solubilized in Me 2 SO, control cells were incubated with equivalent concentrations of Me 2 SO; final Me 2 SO concentrations were Յ0.1%, 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).
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 g/ml streptomycin) at 37°C in 5% CO 2 as described previously (25). Confluent cells were incubated in medium A containing 0.150 M insulin and 1 M dexamethasone Ϯ PPAR ligand for 4 days at 37°C in 5% CO 2 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 g) 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 g/ml denatured salmon sperm DNA. Hybridization was carried out at the same temperature for 20 h in the same solution with a 32 P-labeled aP2 cDNA probe (2 ϫ 10 6 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 ϩ/ϩLepr db , 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) Ϯ 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 PPAR␥ 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␥ (K i ϭ 2, 15, and 14 nM, respectively). As expected, the TZDs AD-5075, BRL 49653, and troglitazone displaced the radiolabeled ligand differentially with K i values of 1, 24, and 250 nM, respectively. In contrast, a fourth non-TZD, L-165041 (a phenoxyacetic acid derivative), was far less potent (K i ϳ 730 nM) and WY-14643 failed to displace labeled AD-5075 from PPAR␥ at concentrations up to 30 M (data not shown).
One of the compounds, L-783483, was subsequently radiolabeled and shown to bind saturably and with high affinity to recombinant hPPAR␦. Scatchard analysis demonstrated a K D of Ϸ 1 nM for this binding interaction. 3 As shown in Fig. 2B, the K i for displacement of [ 3 H] 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 (K i ϭ 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.
PPAR␥ 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 ϳ25 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.
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␥, L-783483 and L-165461, induce the in vitro association of the hPPAR␥ LBD with the co-activator CBP (Fig. 4A). At higher concentrations (Ͼ5 M), 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-ac-tivator 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␣.
Novel Ligands Produce Transcriptional Activation of PPAR␥ 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).
Brown et al. (34) have recently reported the synthesis of a potent ligand for PPAR␦, 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 M) 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 PPAR␣ 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␥ 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.
Having identified a selective PPAR␦ 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 M L-165041 is presumably due to activation of PPAR␥.
Our results definitively indicate that while PPAR␥ activation was sufficient to induce adipocyte differentiation in 4 B. Zhang, unpublished data. 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.
In Vivo Consequences of PPAR␥ 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). We used the ligands described above to evaluate the relative in vivo effects of activating PPAR␥, 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 mice 5 ; 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).
In additional experiments, we used WY-14643 in the db/db mouse model to assess the metabolic effects of PPAR␣ 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).
Taken together, these results indicate that activation of the PPAR␥: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 ED 50 for glucose lowering in db/db mice correlates with the PPAR␥ binding affinity (K i ) of both TZD and non-TZD agonists (Fig. 8). Additional studies will be required to further define the physiological roles of PPAR␦.