The retinoid X receptor (RXR) agonist bexarotene can cause clinically significant hypothyroidism in cutaneous T cell lymphoma patients. The mechanism by which the RXR agonist produces this effect is unclear. We have studied the impact of a selective RXR agonist (rexinoid), LG100268, on rat thyroid axis hormones and show that the acute phase of hypothyroidism is associated with reduced pituitary TSH secretion. A single oral administration of LG100268 to naive Sprague Dawley rats causes a rapid and statistically significant decline in TSH levels (apparent in 0.5–1 h). Total T4 and T3 levels decline more gradually, reaching statistical significance 24 h after treatment. Increasing doses of LG100268 produce greater suppression of thyroid axis hormones. To investigate the mechanism(s) mediating this suppression, we determined pituitary TSHβ mRNA, TSH protein levels, and TRH-stimulated TSH secretion. Two hours after treatment, neither TSHβ mRNA nor TSH protein levels were altered by LG100268. However, LG100268 treatment reduced the area under the curve for TRH-stimulated TSH secretion by 54%. We have identified an unexpected mechanism by which rexinoids induce hypothyroidism by acutely reducing TSH secretion from the anterior pituitary. This mechanism is independent of the rexinoid's previously demonstrated inhibition of TSHβ gene transcription.
Previous data have shown that RXR-selective agonists (e.g., 3 and 4) are insulin sensitizers in rodent models of non-insulin-dependent diabetes mellitus (NIDDM). Unfortunately, they also produce dramatic increases in triglycerides and profound suppression of the thyroid hormone axis. Here we describe the design and synthesis of new RXR modulators that retain the insulin-sensitizing activity of RXR agonists but produce substantially reduced side effects. These molecules bind selectively and with high affinity to RXR and, unlike RXR agonists, do not activate RXR homodimers. To further evaluate the antidiabetic activity of these RXR modulators, we have designed a concise and systematic structure-activity relationship around the 2E,4E,6Z-7-aryl-3-methylocta-2,4,6-trienoic acid scaffold. Selected compounds have been evaluated using insulin-resistant rodents (db/db mice) to characterize effects on glucose homeostasis. Our studies demonstrate the effectiveness of RXR modulators in lowering plasma glucose in the db/db mouse model.
A common feature of many metabolic pathways is their control by retinoid X receptor (RXR) heterodimers. Dysregulation of such metabolic pathways can lead to the development of atherosclerosis, a disease influenced by both systemic and local factors. Here we analyzed the effects of activation of RXR and some of its heterodimers in apolipoprotein E −/− mice, a well established animal model of atherosclerosis. An RXR agonist drastically reduced the development of atherosclerosis. In addition, a ligand for the peroxisome proliferator-activated receptor (PPAR)γ and a dual agonist of both PPARα and PPARγ had moderate inhibitory effects. Both RXR and liver X receptor (LXR) agonists induced ATP-binding cassette protein 1 (ABC-1) expression and stimulated ABC-1-mediated cholesterol efflux from macrophages from wild-type, but not from LXRα and β double −/−, mice. Hence, activation of ABC-1-mediated cholesterol efflux by the RXR/LXR heterodimer might contribute to the beneficial effects of rexinoids on atherosclerosis and warrant further evaluation of RXR/LXR agonists in prevention and treatment of atherosclerosis.
The retinoid X receptor (RXR) contributes to the regulation of diverse biological pathways via its role as a heterodimeric partner of several nuclear receptors. However, RXR has no established role in the regulation of hematopoietic stem cell (HSC) fate. In this study, we sought to determine whether direct modulation of RXR signaling could impact human HSC self-renewal or differentiation. Treatment of human CD34(+)CD38(-)lin(-) cells with LG1506, a selective RXR modulator, inhibited the differentiation of HSCs in culture and maintained long-term repopulating HSCs in culture that were otherwise lost in response to cytokine treatment. Further studies revealed that LG1506 had a distinct mechanism of action in that it facilitated the recruitment of corepressors to the retinoic acid receptor (RAR)/RXR complex at target gene promoters, suggesting that this molecule was functioning as an inverse agonist in the context of this heterodimer. Interestingly, using combinatorial peptide phage display, we identified unique surfaces presented on RXR when occupied by LG1506 and demonstrated that other modulators that exhibited these properties functioned similarly at both a mechanistic and biological level. These data indicate that the RAR/RXR heterodimer is a critical regulator of human HSC differentiation, and pharmacological modulation of RXR signaling prevents the loss of human HSCs that otherwise occurs in short-term culture.
Na channels of frog muscle fibers treated with 100 microM veratridine became transiently modified after a train of repetitive depolarizations. They open and close reversibly with a gating process whose midpoint lies 93 mV more negative than the midpoint of normal activation gating and whose time course shows no appreciable delay in the opening or closing kinetics but still requires more than two kinetic states. Like normal activation, the voltage dependence of the modified gating can be shifted by changing the bathing Ca2+ concentration. The instantaneous current-voltage relation of veratridine-modified channels is curved at potentials negative to -90 mV, as if external Ca ions produced a voltage-dependent block but also permeated. Modified channels probably carry less current than normal ones. When the concentration of veratridine is varied between 5 and 100 microM, the initial rate of modification during a pulse train is directly proportional to the concentration, while the rate of recovery from modification after the train is unaffected. These are the properties expected if drug binding and modification of channels can be equated. Hyperpolarizations that close modified channels slow unbinding. Allethrin and DDT also modify channels. They bind and unbind far faster than veratridine does, and their binding requires open channels.
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. 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) 1The 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, glutathioneS-transferase; CBP, CREB–binding protein; PAGE, polyacrylamide gel electrophoresis; CHAPS, 3-[(cholamidopropyl)dimethylammonio[-1-propanesulfonic acid]; UAS, upstream activating sequence. family of nuclear hormone receptors consists of three subtypes encoded by separate genes: PPARα, PPARδ (also referred to as hNUC1, PPARβ, or FAAR), and PPARγ (1Schoonjans K. Marin G. Staels B. Auwerx J. Curr. Opin. Lipidol. 1997; 8: 159-165Crossref PubMed Scopus (470) Google Scholar). 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 PPARγ (2Berger J. Bailey P. Biswas C. Cullinan C.A. Doebber T.W. Hayes N.S. Saperstein R. Smith R.G. Leibowitz M.D. Endocrinology. 1996; 137: 4189-4195Crossref PubMed Scopus (346) Google Scholar) or PPARα (3Dowell P. Peterson V.J. Zabriskie M. Leid M. J. Biol. Chem. 1997; 272: 2013-2020Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar) 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 PPARγ and PPARα have been shown to interact with a known nuclear receptor co-regulator (steroid receptor co-activator 1; SRC-1) (4DiRenzo J. Soderstrin M. Kurokawa R. Oliastro M.H. Ricote M. Ingrey S. Horlein A. Rosenfeld M.G. Glass C.K. Mol. Cell. Biol. 1997; 17: 2166-2176Crossref PubMed Scopus (255) Google Scholar, 5Krey G. Braissant O. L'Horset F. Kalkhoven E. Perroud M. Parker M.G. Wahli W. Mol. Endocrinol. 1997; 11: 779-791Crossref PubMed Scopus (908) Google Scholar, 6Zhu Y. Qi C. Calandra C. Rao M.S. Reddy J. Gene Expr. 1996; 6: 185-195PubMed Google Scholar). PPARα is expressed at high levels in liver and regulates the expression of genes involved in the β oxidation of fatty acids as well as other aspects of lipid metabolism (7Schoonjans K. Staels B. Auwerx J. Biochim. Biophys. Acta. 1996; 1302: 93-109Crossref PubMed Scopus (931) Google Scholar, 8Wahli W. Braissant O. Desvergne B. Chem. Biol. (Lond.). 1995; 2: 261-266Abstract Full Text PDF PubMed Scopus (265) Google Scholar). 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 PPARα (5Krey G. Braissant O. L'Horset F. Kalkhoven E. Perroud M. Parker M.G. Wahli W. Mol. Endocrinol. 1997; 11: 779-791Crossref PubMed Scopus (908) Google Scholar, 9Forman B.M. Chen J. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4312-4317Crossref PubMed Scopus (1889) Google Scholar). PPARδ is ubiquitously expressed in a broad range of mammalian tissues (10Schmidt A. Endo N. Rutledge S.J. Vogel R. Shinar D. Rodan G.A. Mol. Endocrinol. 1992; : 1634-1641PubMed Google Scholar). 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 PPARδ (9Forman B.M. Chen J. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4312-4317Crossref PubMed Scopus (1889) Google Scholar, 10Schmidt A. Endo N. Rutledge S.J. Vogel R. Shinar D. Rodan G.A. Mol. Endocrinol. 1992; : 1634-1641PubMed Google Scholar). PPARγ has been shown to be expressed at high levels in mammalian adipose tissue (11Tontonoz P. Erding H. Graves R.A. Budavari A.I. Spiegelman B.M. Genes Dev. 1994; 8: 1224-1234Crossref PubMed Scopus (2021) Google Scholar, 12Vidal-Puig A. Jimenez-Linan M. Hamann A. Lowell B.B. Hu E. Spiegelman B.M. Flier J.S. Moller D.E. J. Clin. Invest. 1996; 97: 2553-2561Crossref PubMed Scopus (597) Google Scholar). Two closely related isoforms (PPARγ1 and PPARγ2), which differ by the addition of 30 NH2-terminal amino acids in PPARγ2, occur as a result of alternative promoter usage and mRNA splicing (11Tontonoz P. Erding H. Graves R.A. Budavari A.I. Spiegelman B.M. Genes Dev. 1994; 8: 1224-1234Crossref PubMed Scopus (2021) Google Scholar, 13Zhu Y. Qi C. Korenberg J.R. Chen X.N. Noya D. Sambasiva Rao M. Reddy J.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7921-7925Crossref PubMed Scopus (609) Google Scholar). At the present time, no physiologically relevant differences in the function of these two isoforms have been determined (14Spiegelman B.M. Diabetes. 1998; 47: 507-514Crossref PubMed Scopus (1655) Google Scholar). It has become apparent that PPARγ plays an important regulatory role in adipocyte differentiation and metabolism. The transcriptional activity of the aP2 (11Tontonoz P. Erding H. Graves R.A. Budavari A.I. Spiegelman B.M. Genes Dev. 1994; 8: 1224-1234Crossref PubMed Scopus (2021) Google Scholar), lipoprotein lipase (15Schoonjans K. Peinado-Onsurbe J. Lefebvre A.M. Heyman R.A. Briggs M. Deeb S. Staels B. Auwerx J. EMBO J. 1996; 15: 5336-5348Crossref PubMed Scopus (1037) Google Scholar), and phosphoenolpyruvate carboxykinase (16Tontonoz P. Hu E. Devine J. Geale E.G. Spiegelman B.M. Mol. Cell. Biol. 1995; 15: 351-357Crossref PubMed Google Scholar) gene promoters are up-regulated in adipocytes by PPARγ activation. Moreover, ectopic overexpression of PPARγ in NIH/3T3 fibroblasts or in myoblasts was shown to induce adipocyte differentiation (17Hu E. Tontonoz P. Spiegelman B.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9856-9860Crossref PubMed Scopus (587) Google Scholar, 18Tontonoz P. Hu E. Spiegelman B.M. Cell. 1994; 79: 1147-1156Abstract Full Text PDF PubMed Scopus (3165) Google Scholar), indicating that PPARγ 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 PPARγ agonists (2Berger J. Bailey P. Biswas C. Cullinan C.A. Doebber T.W. Hayes N.S. Saperstein R. Smith R.G. Leibowitz M.D. Endocrinology. 1996; 137: 4189-4195Crossref PubMed Scopus (346) Google Scholar, 18Tontonoz P. Hu E. Spiegelman B.M. Cell. 1994; 79: 1147-1156Abstract Full Text PDF PubMed Scopus (3165) Google Scholar, 19Lehmann J.M. Moore L.B. Smith-Oliver T.A. Wilkison W.O. Willson T.M. Kliewer S.A. J. Biol. Chem. 1995; 270: 12953-12956Abstract Full Text Full Text PDF PubMed Scopus (3496) Google Scholar). The in vivo antidiabetic activities of these compounds correlate with their ability to bind to, and activate, PPARγ in vitro (2Berger J. Bailey P. Biswas C. Cullinan C.A. Doebber T.W. Hayes N.S. Saperstein R. Smith R.G. Leibowitz M.D. Endocrinology. 1996; 137: 4189-4195Crossref PubMed Scopus (346) Google Scholar,20Willson T.M. Cobb J.E. Cowan D.J. Wiethe R.W. Correa I.D. Prakash S.R. Beck K.D. Moore L.B. Kliewer S.A. Lehmann J.M. J. Med. Chem. 1996; 39: 665-668Crossref PubMed Scopus (658) Google Scholar). Structurally distinct, selective RXR agonists have been identified that can activate PPARγ/RXR heterodimers; they have also been shown to promote in vitro adipogenesis and in vivo insulin sensitization in rodents (21Mukherjee R. Davies P.J.A. Crombie D.L. Bischoff E.D. Cesario R.M. Jow L. Hamann L.G. Boehm M.F. Mondon C.E. Nadzan A.M. Paterniti J.R. Heyman R.A. Nature. 1997; 386: 407-410Crossref PubMed Scopus (578) Google Scholar). These findings provide further support for the role of PPARγ in regulation of adipocyte differentiation and modulation of insulin action. However, the relative ability of PPARα or PPARδ to exert similar physiologic effects has not been well characterized. Here, we report the identification and characterization of novel, non-TZD PPARγ and PPARδ agonists. The novel compounds differentially bound to and activated human PPARγ and PPARδ. Binding of these ligands altered receptor conformations and induced the association between the receptors and the coactivator CREB-binding protein (CBP). Only PPARγ agonists were able to potentiate adipogenesis of 3T3-L1 preadipocytes. In diabetic db/dbmice, the novel PPARγ agonists served as orally active insulin-sensitizing agents that lowered both plasma glucose and triglyceride concentrations. In contrast, in vivoexposure to a PPARδ-selective compound was not sufficient to affect glucose or triglyceride concentrations. Activation of PPARα 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 PPARγ as the predominant mediator of insulin sensitization by compounds that are agonists of this receptor. 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). The chimeric receptor expression constructs, pcDNA3hPPARγ/GAL4, pcDNA3-hPPARδ/GAL4, pcDNA3-mPPARγ/GAL4, pcDNA3-mPPARδ/GAL4, and pcDNA3-mPPARα/GAL4, were prepared by inserting the yeast GAL4 transcription factor DBD adjacent to the LBDs of hPPARγ, hPPARδ, mPPARγ, mPPARδ, and mPPARα, 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-hPPARγ2 and pSG5-hPPARδ were constructed by subcloning the full-length cDNA for hPPARγ2 or hPPARδ (kindly provided by Dr. Azriel Schmidt, Merck Research Laboratories, West Point, PA), respectively, into the pSG5 mammalian expression vector (Stratagene, La Jolla, CA). pGEXKG-PPARγLBD and pGEXKG-PPARδLBD plasmids containing GST fused with the LBDs of hPPARγ (amino acids 176–477 of PPARγ1) or hPPARδ (amino acids 167–441) were constructed by subcloning the LBD fragments into pGEXKG (22Guan K.L. Dixon L.E. Anal. Biochem. 1991; 192: 262-267Crossref PubMed Scopus (1663) Google Scholar) digested with XhoI and HindIII (HindIII site was blunt-ended with T4 DNA polymerase). pGEXhCBP1–453, was constructed with a 1.5-kilobase pairNcoI-HindIII fragment encoding the NH2-terminal 1–453 amino acids of human CBP ligated into pGEXKG. pGEX-hPPARγ2 and pGEX-hPPARδ plasmids containing GST fused to the full-length hPPARγ2 and hPPARδ, respectively, were generated by subcloning the cDNAs encoding the entire receptors into the SmaI site of pGEX-4T-2 (Amersham Pharmacia Biotech). GST-hPPARγ or GST-hPPARδ 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 = 0.7–1.0 and induced for overexpression by addition of isopropyl-1-thio-β-d-galactopyranoside to a final concentration of 0.2 mm. The isopropyl-1-thio-β-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-hPPARγ, or GST-hPPARδ, 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 β-mercaptoethanol, 10 mm sodium molybdate, 1 mm dithiothreitol, 5 μg/ml aprotinin, 2 μg/ml leupeptin, 2 μg/ml benzamide, and 0.5 mmphenylmethylsulfonyl 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 K Dfor either AD-5075 or L-783483 is ≈1 nm. The protease digestion assays were performed by the method of Allan et al. (23Allan G.F. Long X. Tsai S.Y. Weigel N.L. Edwards D.P. Tsai M.-J. O'Malley B.W. J. Biol. Chem. 1992; 267: 19513-19520Abstract Full Text PDF PubMed Google Scholar) with previously described modifications (2Berger J. Bailey P. Biswas C. Cullinan C.A. Doebber T.W. Hayes N.S. Saperstein R. Smith R.G. Leibowitz M.D. Endocrinology. 1996; 137: 4189-4195Crossref PubMed Scopus (346) Google Scholar). The pSG5-hPPARγ2 and pSG5-hPPARδ plasmids were used to synthesize 35S-radiolabeled PPARγ2 or PPARδ, 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. The GST-hCBP1–453, GST-hPPARγLBD, and GST-hPPARδLBD fusion proteins were generated in E. coli strain DH5α (Life Technologies, Inc.) as described above for the GST-hPPARγ and GST-hPPARδ fusion proteins. The hPPARγLBD and hPPARδLBD were generated by thrombin cleavage of glutathione-Sepharose-bound GST-hPPARγLBD and GST-hPPARδLBD, 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 hPPARγLBD or hPPARδLBD 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 PPARγLBD or anti-human PPARδLBD antibodies that had been raised against purified recombinant hPPARγLBD or hPPARδLBD. 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. 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). β-Galactosidase activity was determined using β-d-galactopyranoside (Calbiochem) as described previously (24Hollons T. Yoshimura F.K. Anal. Biochem. 1989; 182: 411-418Crossref PubMed Scopus (76) Google Scholar). 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 (25Kohanski R.A. Frost S.C. Lane M.D. J. Biol. Chem. 1986; 261: 12272-12281Abstract Full Text PDF PubMed Google Scholar). Confluent cells were incubated in medium A containing 0.150 μminsulin 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). Male db/db mice (10–11-week-old C57BLKS/J-m +/+Lepr 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) ± 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 vivoexperiments were approved by the Institutional Animal Care and Use Committee. Known PPARγ ligands include the prostaglandin metabolite 15-deoxy-Δ12,14-PGJ2 (26Forman B.M. Tontonoz P. Chen J. Brun R.P. Spiegelman B.M. Evans R.M. Cell. 1995; 83: 803-812Abstract Full Text PDF PubMed Scopus (2754) Google Scholar) and the synthetic thiazolidinedione antidiabetic agents, which bind with high affinity and specificity to this receptor (2Berger J. Bailey P. Biswas C. Cullinan C.A. Doebber T.W. Hayes N.S. Saperstein R. Smith R.G. Leibowitz M.D. Endocrinology. 1996; 137: 4189-4195Crossref PubMed Scopus (346) Google Scholar, 19Lehmann J.M. Moore L.B. Smith-Oliver T.A. Wilkison W.O. Willson T.M. Kliewer S.A. J. Biol. Chem. 1995; 270: 12953-12956Abstract Full Text Full Text PDF PubMed Scopus (3496) Google Scholar). Using a combination of molecular modeling and directed chemical synthesis, 2R. 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. we synthesized a series of structurally distinct non-TZD compounds, which are PPARγ and/or PPARδ agonists (Fig. 1). As depicted in Fig. 2 A, a binding assay employing the radiolabeled TZD AD-5075 and recombinant PPARγ was used to demonstrate that three of these compounds,L-796449, L-165461, and L-783483 (all phenylacetic acid derivatives), were potent ligands for PPARγ (K i = 2, 15, and 14 nm, respectively). As expected, the TZDs AD-5075, BRL 49653, and troglitazone displaced the radiolabeled ligand differentially with K ivalues 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 ∼ 730 nm) and WY-14643 failed to displace labeled AD-5075 from PPARγ at concentrations up to 30 μm (data not shown).Figure 2Novel ligands bind to PPARγ and/or PPARδ. Competition curves generated by incubation of 10 nm[3H]2AD-5075 with GST-hPPARγ (A) or 2.5 nm [3H]2L-783483 with GST-hPPARδ (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.View Large Image Figure ViewerDownload (PPT) One of the compounds, L-783483, was subsequently radiolabeled and shown to bind saturably and with high affinity to recombinant hPPARδ. Scatchard analysis demonstrated aK D of ≈ 1 nm for this binding interaction. 3M. Leibowitz, unpublished data. As shown in Fig. 2 B, the K i for displacement of [3H]2L-783483 by cold compound was 1 nm. Titration of other compounds in this PPARδ binding assay revealed that L-796449, L-165461, andL-165041 also bound to PPARδ with high affinity (K i = 2, 3, and 6 nm, respectively). The TZDs AD-5075, BRL 49653, and troglitazone (Fig. 2 B) and WY-14643 (data not shown) were unable to displace labeledL-783483 from the receptor. We reported previously that saturating concentrations of TZDs can induce an alteration in the conformation of PPARγ, as assessed by generation of a major protease-resistant band following partial protease digestion of recombinant receptor protein (2Berger J. Bailey P. Biswas C. Cullinan C.A. Doebber T.W. Hayes N.S. Saperstein R. Smith R.G. Leibowitz M.D. Endocrinology. 1996; 137: 4189-4195Crossref PubMed Scopus (346) Google Scholar). In addition, Dowell et al. (3Dowell P. Peterson V.J. Zabriskie M. Leid M. J. Biol. Chem. 1997; 272: 2013-2020Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar) reported that selected PPARα activators, including clofibrate and WY-14643, induce similar conformational changes upon incubation with recombinant PPARα. Both of these effects are analogous to changes in estrogen receptor (ERα) conformation that have been observed following the binding of known agonists (e.g. estradiol)(27). In contrast, ERα antagonists induce different, and more limited, changes in the pattern of fragments produced following limited protease digestion (27McDonnell D.P. Clemm D.L. Hermann T. Goldman M.E. Pike J.W. Mol. Endocrinol. 1995; 9: 659-669Crossref PubMed Google Scholar). When incubated with PPARγ, the TZD AD-5075 protects a fragment of ∼25 kDa from trypsin digestion (Fig.3 A, upper panel). On the other hand, no protection is evident when PPARδ is treated with AD-5075 (Fig. 3 A, lower panel). As shown for PPARγ in the top panel of Fig. 3 B, the novel PPARγ/δ 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 PPARδ from digestion (Fig. 3 B, lower panel). In contrast, treatment with L-165041 alters the conformation of PPARδ, but not PPARγ (Fig. 3 C), as expected based upon it's affinity for the respective receptors. These results demonstrate that the newly identified PPARγ and PPARδ 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 (28Kamei Y. Xu L. Heinzel T. Torchia J. Kurokawa R. Gloss B. Lin S.C. Heyman R.A. Rose D.W. Glass C.K. Rosenfeld M.G. Cell. 1996; 85: 403-414Abstract Full Text Full Text PDF PubMed Scopus (
