Introduction

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Thiazolidinedione insulin sensitizers are a new class of antihyperglycemic agent that after chronic administration to animal models of non-insulin-dependent diabetes, improve glycemic control by enhancing insulin action in target tissues rather than by increasing insulin secretion. Numerous studies using a number of thiazolidinediones indicate that these agents enhance insulin-mediated suppression of hepatic glucose production and promote insulin-stimulated glucose transport into skeletal muscle and adipose tissue (Hulin et al., 1996). Increased glucose disposal, in adipose tissue at least, results from a combination of increased expression of GLUT-4 and increased translocation of GLUT-4 to the adipocyte cell surface in response to insulin (Young et al., 1995). In addition to improving insulin action in diabetic animal models, thiazolidinediones can influence adipocyte function in vitro. They increase glucose transport in differentiated 3T3-L1 adipocytes in culture, an action mediated by an insulin-independent increase in the expression of glucose transporters (Gibbs et al., 1989), and they can stimulate preadipocyte differentiation and expression of adipose-specific genes (Ibrahimi et al., 1994; Kletzien et al., 1992). The relevance of actions in adipose cells is underscored by the finding that the rank order of potency of thiazolidinediones as antidiabetic agents in vivo is highly correlated with their potencies as adipogenic stimulants in vitro (Lenhard et al., 1996).

Rosiglitazone (BRL-49653), a potent thiazolidinedione insulin sensitizer, has recently been identified as a high-affinity ligand for PPAR, a nuclear hormone receptor that is abundantly expressed in adipocytes and plays a central role as a regulator of terminal adipocyte differentiation (Lehmann et al., 1995; Tontonez et al., 1994). Two splice variants of PPAR have been identified. Human and murine PPAR₁ and PPAR₂ bind rosiglitazone with comparable affinities, and both  subtypes are activated equally by the compound in transactivation assays (Elbrecht et al., 1996). The activation of another PPAR isoform, PPAR (also known as fatty acid-activated receptor, or NUC-1), by high concentrations of rosiglitazone has been reported by some (Ibrahimi et al., 1994) but not all (Lehmann et al., 1995) researchers, and the relative contributions of activation of PPAR and PPAR to the adipogenic response to thiazolidinediones remain controversial.

The implication that one or more PPARs play a key role in the antidiabetic mechanism of thiazolidinediones has arisen entirely from correlations of compound potencies in animal models with potencies/affinities determined in binding and functional studies using recombinant PPAR isoforms (Willson et al., 1996). Direct evidence for the existence of specific binding sites for insulin sensitizers in normal rodent- and human-derived cells is lacking. In this report, to identify specific binding sites in intact rodent and human adipocytes, we describe the use of a high-specific-activity radioiodinated ligand, [¹²⁵I]SB-236636, that binds with high affinity and specificity to the LBD of recombinant hPPAR₁. The properties of these binding sites, assessed with a variety of PPAR subtype-selective ligands, are comparable in rat and human adipocytes and highly correlated with the pharmacology of binding to recombinant PPAR₁.

	Materials and Methods

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All cell culture reagents were purchased from GIBCO (Paisley, Scotland, UK). 2-Deoxy-D-[2,6-³H]glucose was purchased from Amersham International (Berkshire, UK). SB-217092 [(±)-3-[4-[2-[N-(2-benzoxazolyl)-N-methylamino]ethoxy]3-iodophenyl]2-ethoxypropanoic acid], [¹²⁵I]SB-236636 [(S)-()-3-[4-[2-[N-(2-benzoxazoyl)-N-methylamino]ethoxy]3-[¹²⁵]iodophenyl]2-ethoxypropanoic acid; 2500 Ci/mmol], rosiglitazone (BRL-49653) [(±)-5-[[4-[2-[N-methyl-N-(2-pyridyl)amino]ethoxy]phenyl]methyl]2,4-thiazolidinedione], SB-219993 [(R)-(+)-3-[4-[2-[N-(2-benzoxazolyl)-N-methylamino]ethoxy]phenyl]2-(2,2,2-trifluoroethoxy)propanoic acid], SB-219994 [(S)-()-3-[4-[2-[N-(2-benzoxazolyl)-N-methylamino]ethoxy]phenyl]2-(2,2,2-trifluoroethoxy) propanoic acid], pioglitazone and troglitazone were synthesized in-house (fig. 1). Collagenase (136-169 U/mg CLS1) was obtained from Lorne Laboratories (Reading, UK), and bovine serum albumin (fraction V) was purchased from Boehringer-Mannheim (Sussex, UK). AEBSF was purchased from Calbiochem (Nottingham, UK). LY 171883, Wy 14643 and ETYA were purchased from BIOMOL Research Laboratories (Plymouth Meeting, PA). 2-Bromopalmitic acid was purchased from Aldrich (Gillingham, Dorset, UK), and 15-deoxy-^12,14-prostaglandin J₂ was obtained from Cayman Chemical (Ann Arbor, MI). HAWP 02500 (0.45-µm) filters were obtained from Millipore (Watford, UK). All other chemicals were purchased from Sigma (Poole, Dorset, UK). Kits for the estimation of glucose were obtained from Ciba-Corning, Halstead, Essex, UK. The 3T3-L1 fibroblast cell line was obtained from American Type Culture Collection (Rockville, MD).

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Structures of insulin sensitizers. Full chemical names of novel structures are given in the text.

Animals

Male Sprague-Dawley rats (weight, 200-250 g) used for adipocyte studies were from Charles River (Kent, UK), and female C57Bl/6 ob/ob obese mice (age, 9-10 weeks) were from Harlan Olac (Bicester, UK) and Jackson Laboratories (Bar Harbor, ME). Animals were maintained under a 12-hr light/dark cycle. Rats were fed RM1 diet (SDS; Special Diet Services, Witham, Essex, UK) and water ad libitum and maintained at 21 ± 2°C, whereas obese mice were maintained at 26 ± 2°C and fed powdered RM3 diet (SDS) and water ad libitum.

Test Systems

Baculovirus expression of full-length hPPAR₁. The PPAR₁ insert was excised from a clone containing the coding sequence of hPPAR₁ and ligated into pBacPAK8 [Clontech (Cambridge Bioscience), Cambridge, UK] for sense orientation (pBacPAK/PPAR₁) and into pBacPAK9 (Clontech) for antisense orientation (pBacPAK/PPAR₁-rev). The baculovirus transfer vectors were amplified in Escherichia coli JM 109 (Promega UK, Southampton, UK). The presence of insert was confirmed by PCR using pBacPAK-specific primers (Clontech), and the plasmids were purified using a commercial DNA purification system (Wizard; Promega).

E. coli expression of GST-hPPAR₁ LBD-fusion protein. A cDNA encoding amino acids 174 to 475 of hPPAR₁ was inserted into bacterial expression vector pGEX-5X-1. The chimera was expressed in XL-1 blue E. coli. Extracts were prepared by lysing the cells in 50 mM HEPES ( pH 7.9), 100 mM KCl, 1 mM dithiothreitol and 1% Triton X-100, followed by centrifugation for 30 min at 100,000 × g.

Culture of 3T3-L1 adipocytes. 3T3-L1 cells were grown to confluence and differentiated according to Frost and Lane (1985). Mature adipocytes were used between days 8 and 10 after differentiation. Differentiated cells were treated with compounds, dissolved in DMSO for 48 hr. Fresh compound was added each day, and the concentration of DMSO did not exceed 0.2% (v/v).

Preparation of adipocytes from rat and human adipose tissue. Rat adipocytes were prepared from epididymal adipose tissue according to Rodbell (1964) with the following modifications. Collagenase was used at a concentration of 2 mg/ml in a HEPES-buffered (30 mM, pH 7.4) Krebs' solution supplemented with 5.6 mM glucose, 200 nM adenosine and 4% (w/v) BSA.

Experimental Procedures

Determination of antihyperglycemic activity in the obese mouse. Insulin-sensitizer compounds were administered by dietary admixture to glucose-intolerant C57Bl/6 ob/ob obese mice. After 8 days of administration, the antihyperglycemic activity of each compound was determined from the measurement of changes in tolerance to an oral glucose load (Cantello et al., 1994). A 25% reduction in the area under the blood glucose vs. time curve (ED₂₅), compared with controls, was considered to be a half-maximal effective dose of compound (Cantello et al., 1994).

2-Deoxyglucose transport in 3T3-L1 adipocytes. For determination of glucose transport rates, differentiated 3T3-L1 cells were washed three times in DPBS and incubated in serum-free DMEM for 2 hr. After removal of the DMEM and three washes with DPBS, Krebs-Ringer phosphate buffer was added, and the cells were incubated for 15 min. 2-Deoxyglucose uptake was initiated by the addition of 2-deoxy-D-[2,6-³H]glucose (25 µM; 1 µCi/well). After 10 min, the medium was aspirated, and the cells were washed three times with ice-cold DPBS. The cell monolayers were allowed to air-dry and then dissolved in 1 M NaOH. Aliquots were removed for scintillation counting and protein estimation.

Western blot analysis of PPAR. 3T3-L1 fibroblasts and 3T3-L1 adipocytes were homogenized in lysis buffer (50 mM Tris·HCl, pH 7.5, 150 mM NaCl, 0.2 µM AEBSF, 1 mM EDTA, 10 µM leupeptin, 1 µM pepstatin and 1 µg/ml aprotinin). Then, 25 µg of protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes (Millipore, Watford, UK) (Young et al., 1995) and blocked overnight in blocking buffer (10 mM Tris·HCl, 150 mM NaCl, 5% nonfat dry milk, 0.05% Tween-20). Blots were sequentially incubated with rabbit polyclonal anti-PPAR serum and a horseradish peroxidase-coupled anti-rabbit secondary antibody for 1 hr, followed by three washes with Tris-buffered saline containing 0.05% Tween-20. Polyclonal antibodies to PPAR were raised in the rabbit, using the keyhole limpet hemacyanin-conjugated peptide SEKTQLYNRPHEEPSN (amino acids 87-102 of mouse PPAR₁ and amino acids 117-132 of mouse PPAR₂) as antigen. Blots were visualized with the supersignal CL-HRP substrate system according to the manufacturer's instructions (Pierce Chemicals, Chester, UK).

PPAR isoform mRNA expression. Total RNA was extracted from 3T3-L1 cells or adipocytes using a single-step extraction solution according to the manufacturer's instructions (TRIzol; Life Technologies, Renfrewshire, Scotland, UK). For PCR, 1 µg of total RNA was treated with RNase-free DNase I (Life Technologies) to remove contaminating genomic DNA before cDNA synthesis using Superscript II (Life Technologies) and oligo(dT) priming. PCR primers to each PPAR isoform were designed using a primer design package (Oligo v. 5.0; NBI, Plymouth, MN) and are shown in table 1. PCR was performed in a total volume of 50 µl with 0.5 µM concentration of primers, 25 ng of cDNA and 0.25 U of Taq polymerase (Life Technologies). After an initial denaturing step for 4 min, a two-step cycling protocol was used (68/94°C for 1 min each) for 30 cycles. Adipocyte content of specific PPAR isoforms was also determined by slot-blot analysis in which 15 µg of total RNA was hybridized with digoxygenin-labeled oligonucleotide probes for PPAR and PPAR (table 1). PPAR was hybridized with a digoxygenin-labeled cDNA probe synthesized by PCR from human adipose cDNA using the primers shown in table 1. A detailed description of this methodology is given elsewhere (Clapham et al., 1997).

Radioligand Binding Studies

Human PPAR₁ LBD-GST fusion protein. Binding of [¹²⁵I]SB-236636 to crude lysates of E. coli expressing GST-hPPAR₁ LBD fusion protein was carried out in 96-well plates at 4°C for 16 hr. The assay consisted of 1.4 µg of crude protein and 150 pM [¹²⁵I]SB-236636 in a total volume of 50 µl of lysis buffer. Competing compounds were dissolved in DMSO (final concentration in the incubation, <0.02%) and present at the concentrations indicated in the legends to figures. Bound ligand was separated from free on mixed cellulose acetate filters (Inoue et al., 1983). Nonspecific binding was assessed with 100 µM rosiglitazone.

Full-length hPPAR₁. Cell pellets from Sf9 cells transfected with PPAR₁ were solubilized with 30 mM HEPES (pH 7.9) containing 300 mM KCl, 1 mM dithiothreitol, 1 mM AEBSF and 1% Triton X-100. Binding experiments were performed as detailed for GST-hPPAR₁ LBD fusion protein.

3T3-L1 adipocytes. 3T3-L1 cells were grown to confluence and differentiated in 35-mm-diameter six-well plates. Cell monolayers were washed three times in phosphate-buffered saline and incubated for 1 hr at 37°C in DMEM containing 30 pM [¹²⁵I]SB-236636 and varying concentrations of competing compounds. The cell monolayers were then washed three times with phosphate-buffered saline and dissolved in 1 ml of 1 M NaOH. Aliquots of the cell lysate were counted for cell-associated [¹²⁵I]SB-236636. Nonspecific binding of [¹²⁵I]SB-236636 (obtained in the presence of 10 µM rosiglitazone) was 30% of total binding.

Rat adipocytes. After preparation, adipocytes were rinsed in DMEM/Ham's F-12 nutrient mix medium containing 15 mM HEPES (pH 7.4) supplemented with 200 nM adenosine. After three washes to remove collagenase and BSA, 0.5 ml of cells was aliquoted (triplicate incubations) into tubes containing [¹²⁵I]SB-236636 to yield a final concentration of the radioligand of 30 pM. Each adipocyte preparation was diluted to an adipocrit of 10% (v/v) before aliquoting. Binding was carried out at 37°C for 1 hr in a shaking water bath. Cell-associated radioactivity was assessed after separation of cells from the medium by centrifugation of cells (200 µl of incubation medium, in duplicate) through silicone oil (Dow Corning 200/200 cs) for 20 sec at 10,000 × g using a Beckman Instruments (Palo Alto, CA) microfuge (Green, 1983). Cell pellets were then obtained by cutting the microfuge tube, and cell-associated radioactivity was measured using a Wallac (Gaithersburg, MD) Wizard  Counter. Nonspecific binding was assessed in the presence of 10 µM rosiglitazone and was 50% of total binding.

Human adipocytes. The methodology was essentially the same as that described for rat adipocyte binding except incubations were carried out in quintuplicate and the DMEM/F-12 medium was supplemented with 25 mM HEPES, pH 7.4, containing 200 nM adenosine and 100 nM p-aminoclonidine to suppress basal lipolysis (Berlan and Lafontan, 1982; Larrouy et al., 1991). Specific binding was 30% of total binding. Nonspecific binding was determined in the presence of 10 µM rosiglitazone.

	Results

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Evaluation of SB-236636 as a Radioligand

To assess the potential of [¹²⁵I]SB-236636 as a radioiodinated ligand to detect molecular targets for rosiglitazone in insulin-responsive cells, the insulin-sensitizing properties of the nonradioactive racemic compound, SB-217092, were determined in the obese mouse and differentiated 3T3-L1 cells. SB-217092 was equipotent with rosiglitazone as an antihyperglycemic agent in the obese mouse (table 2). In addition, SB-217092 was a potent stimulant of glucose transport in differentiated 3T3-L1 adipocytes. The EC₅₀ value was 9 nM, which is comparable with that of rosiglitazone (EC₅₀ = 11 nM) (table 2).

Binding of [¹²⁵I]SB-236636 to Human PPAR₁ LBD and Recombinant Full-Length Human PPAR₁

Saturation binding of [¹²⁵I]SB-236636 to a crude lysate of E. coli expressing GST-hPPAR₁ LBD fusion protein yielded a K_D value of 70 nM (fig. 2). Competition of [¹²⁵I]SB-236636 binding by a number of thiazolidinedione antihyperglycemic agents with different in vivo potencies is shown in table 3. The rank order of binding affinities (IC₅₀ values) was rosiglitazone (41 nM) > pioglitazone (4830 nM) > troglitazone (7970 nM). In addition, binding was stereoselective, because SB-219994 [(S)-enantiomer; fig. 1) possessed an IC₅₀ value of 2.1 nM, whereas the corresponding (R)-enantiomer, SB-219993, had an IC₅₀ value of 2770 nM (table 3). The PPAR₁ LBD may indeed have absolute specificity for the (S)-configuration because the batch of SB-219993 used in these studies contained a small percentage (0.26%) of SB-219994.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Saturation binding of [125I]SB-236636 to GST-hPPAR1 LBD. E. coli lysate (1.4 µg of protein) was incubated with increasing concentrations of [125I]SB-236636 in the absence () or presence () of rosiglitazone (100 µM). Specific binding of [125I]SB-236636 to GST-PPAR LBD is indicated (). Inset, Scatchard analysis. Linear regression yielded a KD value for [125I]SB-236636 of 70 nM. Results are mean ± S.D. of three separate experiments.

View this table: [in this window] [in a new window]   TABLE 3 Competition of [125I]SB-236636 binding in E. coli-expressed GST-hPPAR1 LBD and baculovirus-expressed full-length hPPAR1 Competition binding (IC50 values) for a series of insulin sensitizers.

Specific binding of [¹²⁵I]SB-236636 was detected in crude extracts of Sf9 cells transfected with hPPAR₁. No specific binding was detectable in extracts of nontransfected cells or cells transfected with PPAR or the PPAR₁ insert in the reverse (antisense) orientation. Competition experiments showed that rosiglitazone was a potent competitor of binding (IC₅₀ = 62 nM; table 3). Again, competition of [¹²⁵I]SB-236636 binding showed stereoselectivity; the IC₅₀ for SB-219994 [(S)-enantiomer] was 1.8 nM compared with 1210 nM for SB-219993 [(R)-enantiomer]. Thus, ligand binding affinities measured using only the LBD of PPAR₁ (GST-PPAR₁ LBD) are comparable with those determined using the full-length receptor.

Binding Studies in Intact Cells

3T3-L1 cells. In nonconfluent, nondifferentiated 3T3-L1 fibroblasts, no specific binding of [¹²⁵I]SB-236636 was detected. However, differentiation of these cells to lipid-filled adipocytes lead to the appearance of specific binding of [¹²⁵I]SB-236636 (2000 dpm/mg of protein at a free ligand concentration of 30 pM). The appearance of [¹²⁵I]SB-236636 binding sites during differentiation coincided with the expression of PPAR. PPAR₁ and ₂ mRNA, determined by PCR and slot-blot analysis, and PPAR₁ and ₂ protein, as assessed by Western blotting, were present only in differentiated 3T3 cells displaying adipocyte-like morphology (table 4, fig. 3). Competition of [¹²⁵I]SB-236636 binding in differentiated 3T3-L1 cells by insulin sensitizers showed the same rank order of potency as that determined at recombinant PPAR in cell-free extracts. IC₅₀ values were SB-219994 (0.6 nM) > rosiglitazone (4 nM) > SB-219993 (700 nM) > troglitazone (2000 nM).

View larger version (42K): [in this window] [in a new window]   Fig. 3.   A, Reverse transcription-PCR analysis of PPARs from 3T3-L1 fibroblasts, 3T3-L1 adipocytes, rat adipocytes and human adipocytes. RNA was extracted from 70% to 80% confluent 3T3-L1 fibroblasts, day 10 postdifferentiation 3T3-L1 adipocytes, rat adipocytes and human subcutaneous adipocytes. PCR was performed on 200 ng of 3T3-L1 and rat adipose cDNA and 25 ng of human adipose cDNA. +, cDNA; , non-reverse transcription control; m, low-molecular-weight density markers. B, Western blot analysis of PPAR isoforms from 3T3-L1 fibroblasts (a) and 3T3-L1 adipocytes (b). Total cell homogenates were prepared from 70% to 80% confluent 3T3-L1 fibroblasts and day 10 postdifferentiation 3T3-L1 adipocytes as described in Materials and Methods.

Rat adipocytes. Initial experiments showed that specific binding of tracer amounts of [¹²⁵I]SB-236636 to intact rat white adipocytes reached equilibrium within 40 min at 37°C (table 5). Scatchard analysis of saturation binding of [¹²⁵I]SB-236636, assessed in three separate adipocyte preparations, yielded a K_D value of 2.4 nM and a B_max of 170 fmol/mg of protein (fig. 4).

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Saturation binding of [125I]SB-236636 to rat adipocytes. Cells were incubated with increasing concentrations of [125I]SB-236636 in the presence or absence of rosiglitazone (10 µM). Inset, Scatchard analysis indicating specific binding. Linear regression yielded a KD value for [125I]SB-236636 of 2.4 nM. Results are mean ± S.D. from three separate experiments.

Human adipocytes. To determine whether human adipocytes possess a population of high-affinity binding sites similar to those present in rat adipocytes, a parallel series of experiments was conducted using adipocytes freshly prepared from human mammary adipose tissue. In human adipocytes, as in rat adipocytes, cell-specific binding of [¹²⁵I]SB-236636 reached equilibrium within 40 min at 37°C (table 5). Scatchard analysis of a saturation binding study performed with a typical adipocyte preparation yielded a K_D value of 0.8 nM with a B_max of 49 fmol/mg of protein (fig. 5). Thus, the affinity and number of insulin-sensitizer binding sites in rat and human adipocytes are comparable.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Saturation binding of [125I]SB-236636 to human adipocytes. Cells were incubated with increasing concentrations of [125I]SB-236636 in the presence or absence of rosiglitazone (10 µM). Inset, Scatchard analysis indicating specific binding. Linear regression yielded a KD value for [125I]SB-236636 of 0.5 nM.

	Discussion

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It is well established that thiazolidinediones improve glycemic control in animal models of non-insulin-dependent diabetes mellitus by increasing insulin sensitivity of the liver, muscle and adipose tissues (Hulin et al., 1996). The potency with which these agents bind to, and activate, PPAR, a ligand-activated nuclear receptor that is a key regulator of adipogenesis, is highly correlated with antidiabetic activity in vivo and suggests that this receptor might be pivotal in the insulin-sensitizing mechanism (Lehmann et al., 1995). This link has, however, been derived exclusively from studies using recombinant PPAR in cell-free or transfected cell systems, and evidence for the existence of specific binding sites for insulin sensitizers in intact rodent- or human-derived cells in a more physiological setting is lacking.

To identify and characterize specific binding sites for insulin sensitizers in cells and tissues, a high-specific-activity radioiodinated ligand, [¹²⁵I]SB-236636, was developed. The choice of ligand was predicated on the following: (1) it must contain iodine to allow radiolabeling to a high specific activity, thereby enabling detection of low abundance receptors. (2) It must be cell penetrant and have a high affinity for the molecular target or targets, as demonstrated by potent insulin-sensitizing activity both in vivo and in vitro. SB-217092 [derived from the -ethoxyacid SB-213068, which is a highly potent antihyperglycemic agent and high-affinity PPAR ligand (Buckle et al., 1996)], was chosen as the iodine-containing insulin sensitizer because it had comparable potency to rosiglitazone as an antidiabetic agent in the obese mouse and as a stimulant of glucose transport in 3T3-L1 adipocytes. The observation that the (S)-enantiomer of -substituted -phenylpropanoic acid antihyperglycemic agents is considerably more potent than the (R)-enantiomer prompted us to radioiodinate only the (S)-enantiomer of SB-217092 to produce [¹²⁵I]SB-236636 (D. Haigh, personal communication).

Before attempting to identify binding sites in whole cells and subcellular fractions, the characteristics of [¹²⁵I]SB-236636 binding to recombinant PPAR isoforms were determined. Binding of [¹²⁵I]SB-236636 was PPAR specific. Although [¹²⁵I]SB-236636 bound with high affinity to both a fusion protein containing the LBD of hPPAR₁ and to full-length hPPAR₁, no specific binding was detectable in extracts of Sf9 cells transfected with a plasmid containing PPAR₁ in antisense orientation. No specific binding could be detected to either a hPPAR LBD GST fusion protein (data not shown) or full-length mouse PPAR. This could simply be a consequence of low intrinsic affinity of [¹²⁵I]SB-236636 for PPAR and PPAR or may result from incorrect folding of the recombinant proteins after cell lysis. The latter possibility is considered unlikely because we also were unable to detect specific radioligand binding in intact cell types known to express high levels of PPAR or PPAR (data not shown). Moreover, direct binding of [³H]leukotriene B₄ to E. coli-expressed PPAR LBD-GST fusion protein has recently been reported, suggesting that the LBD, of this PPAR isoform at least, folds correctly after cell lysis (Devchand et al., 1996).

The binding affinity of the thiazolidinedione rosiglitazone at GST-hPPAR₁ LBD (IC₅₀ = 41 nM), which was assessed using [¹²⁵I]SB-236636 as ligand, is comparable to that reported for rosiglitazone at mPPAR (K_D = 43 nM) using [³H]rosiglitazone as ligand (Lehmann et al., 1995); this is predicted from the high homology (98%) of the LBDs of the human and murine receptors. However, the binding affinity of rosiglitazone for full-length hPPAR₁ determined in our studies is 8-fold higher than the affinity of rosiglitazone for recombinant hPPAR₁ and ₂ reported recently (Elbrecht et al., 1996) using [³H]AD-5075 as a radioligand. The reason for this discrepancy is unclear. In the present study, we also now show that ligand binding to PPAR₁ is stereoselective, with the (S)-enantiomer of the -trifluoroethoxy propanoic acid insulin sensitizer, SB-219994, having an IC₅₀ value of 1000-fold lower than that of the (R)-enantiomer, SB-219993. Thus, ligand enantioselectivity of PPAR is the same as that displayed by PPAR, which is preferentially activated by 8-(S)- but not 8-(R)-HETE (Yu et al., 1995).

In a search for thiazolidinedione binding sites in insulin-sensitive target tissues, [¹²⁵I]SB-236636 binding studies were performed initially using cytosolic and membrane fractions and nuclear extracts prepared from rat liver, skeletal muscle and adipose tissue. No specific radioligand binding was detected in any subcellular fraction, possibly reflecting low abundance of the receptor or liberation of endogenous ligand on cell lysis, which masks specific binding. However, specific, saturable binding was observed in freshly prepared rat epididymal and human mammary tissue-derived adipocytes. The rank order of potency and enantioselective competition of [¹²⁵I]SB-236636 binding by insulin sensitizers in intact adipocytes was identical with the pharmacology shown by recombinant PPAR₁ and strongly supports the contention that the specific binding site in adipocytes is PPAR. In addition, specific binding was undetectable in undifferentiated 3T3-L1 cells, in which PPAR is not expressed (table 4, fig. 3). The appearance of specific binding sites coincided with terminal differentiation and expression of PPAR (Tontonez et al., 1994). Further support that the adipocyte binding site for [¹²⁵I]SB-236636 is PPAR is provided by the demonstration that putative PPAR-, and PPAR-, selective agonists, including the nonmetabolizable fatty acid 2-bromopalmitate, the hypolipidemic agent Wy 14643, the leukotriene D₄ antagonist LY 171883 and the synthetic arachidonic acid analog ETYA, were all poor competitors. All of these agents also are low-potency PPAR ligands, as assessed from radioligand binding or functional transactivation studies.

Scatchard analysis of saturation binding curves produced binding site densities of 170 and 49 fmol/mg of protein for rat and human adipocytes, respectively, values that are close to those of other nuclear hormone receptors such as thyroid (Inoue et al., 1983) and glucocorticoid (Sheppard and Funder, 1996) receptors. The affinity of the insulin-sensitizer binding site for [¹²⁵I]SB-236636 in intact adipocytes was 30-fold (rat) to 140-fold (human) higher than the K_D value measured with recombinant PPAR₁ LBD. In addition, thiazolidinedione and acyclic antihyperglycemic agents were more potent competitors (1.5-38-fold) of radioligand binding in intact adipocytes than of binding to either full-length hPPAR₁ or hPPAR₁ LBD in cell-free extracts, although the rank order of binding affinities was maintained in all the test systems. Moreover, in absolute terms, potencies of insulin sensitizers as stimulants of glucose transport in 3T3-L1 adipocytes were in closer agreement with IC₅₀ values determined in intact adipocytes than with those assessed using recombinant PPAR protein.

Murine adipocytes express two forms of PPAR, ₁ and ₂, which arise from differential promoter use and alternative splicing (Zhu et al., 1997). Recently, the molecular cloning of hPPAR₁ and ₂ homologs was reported (Elbrecht et al., 1996). The question arises of whether binding sites in intact adipocytes represent PPAR₁, PPAR₂, or both. Although relative expression of the two isoforms in adipocytes at the protein level has not been rigorously quantified, because isoform-specific antibodies are not available, murine and human recombinant ₁ and ₂ isoforms appear to have identical ligand-binding properties and show comparable transactivational responses to rosiglitazone and other thiazolidinediones (Elbrecht et al., 1996; Lehmann et al., 1995). It therefore is likely that [¹²⁵I]SB-236636 binding sites in adipocytes consist of ₁ and ₂ components.

The endogenous ligand or ligands for PPAR have not been unequivocally identified, although recently, Forman et al. (1995) and Kliewer et al. (1995) demonstrated that prostanoids can activate PPAR in transactivation assay systems. Of a range of arachidonic acid metabolites tested, 15-deoxy-^12,14-prostaglandin J₂ was the most potent activator of PPAR and inducer of adipogenesis. The prostanoid also displaced [³H]rosiglitazone binding to PPAR LBD, although with only low affinity (K_i  2.5 µM). In contrast, the present results show that 15-deoxy-^12,14-prostaglandin J₂ was a potent competitor of [¹²⁵I]SB-236636 binding in human adipocytes (IC₅₀ = 45 nM) and further supports the conclusion, drawn from the competition studies with rosiglitazone and a number of other insulin sensitizers, that the high-affinity binding site for [¹²⁵I]SB-236636 in human adipocytes is PPAR. The discrepancy in binding affinities of 15-deoxy-^12,14-prostaglandin J₂ determined in intact adipocytes and at recombinant PPAR in cell lysates is entirely consistent with the data obtained for thiazolidinediones and acyclic insulin sensitizers. The apparent increase in ligand affinity and potency in the intact cell assay is perhaps not surprising because the receptor would be in the optimal conformation and also have access to partner proteins, such as retinoid × receptor, with which PPAR heterodimerizes. Although 15-deoxy-^12,14-prostaglandin J₂ can undoubtedly promote adipogenesis in vitro, the physiological relevance is unclear because its synthesis by adipose tissue has not been reported. Furthermore, the role of prostanoids in the regulation of PPAR function in fully differentiated lipid-filled adipocytes, as opposed to preadipocytes, is unknown.

Examples from two other chemical classes of orally active antidiabetic agent were tested for their ability to bind to PPAR₁ in cell-free extracts and intact adipocytes. The biguanide metformin was inactive, but surprisingly, the sulfonylurea insulin secretagogue glibenclamide had an IC₅₀ value of 1000 nM in rat adipocytes and an IC₅₀ value at recombinant PPAR₁ LBD of 5000 nM. Direct insulin-sensitizing properties of relatively high millimolar concentrations of sulfonylureas in rat adipocytes in culture have been reported, and activation of PPAR may be associated with this response (Altan et al., 1985; Zuber et al., 1985).

In summary, this is the first report detailing the use of a radioiodinated ligand that binds with high affinity and specificity to both rodent and hPPAR to identify specific binding sites in intact rat and human adipocytes. We have shown that for a range of thiazolidinedione and acyclic insulin sensitizers and other PPAR ligands, there is a high correlation between binding affinities determined in rat and human adipocytes with those measured in binding assays using recombinant PPAR. It therefore is very likely that PPAR is the molecular species to which [¹²⁵I]SB-236636 binds in intact adipocytes. This ligand will prove useful in identifying and characterizing binding sites in cell types other than adipocytes, in which insulin action is enhanced by antihyperglycemic compounds such as rosiglitazone.