Fatty Acids, Eicosanoids, and Hypolipidemic Agents Identified as Ligands of Peroxisome Proliferator-Activated Receptors by Coactivator-Dependent Receptor Ligand Assay

Grigorios Krey, Olivier Braissant, Fabienne L’Horset, Eric Kalkhoven, Mai Perroud, Malcolm G. Parker and Walter Wahli

Institut de Biologie Animale (G.K., O.B., M.P., W.W.), Bâtiment de Biologie, Université de Lausanne, CH-1015 Lausanne, Switzerland,
Molecular Endocrinology Laboratory (F.L., E.K., M.G.P.), Imperial Cancer Research Fund, 44 Lincoln’s Inn Fields, London WC2A 3PX, U.K.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors controlling the expression of genes involved in lipid homeostasis. PPARs activate gene transcription in response to a variety of compounds including hypolipidemic drugs as well as natural fatty acids. From the plethora of PPAR activators, Scatchard analysis of receptor-ligand interactions has thus far identified only four ligands. These are the chemotactic agent leukotriene B4 and the hypolipidemic drug Wy 14,643 for the {alpha}-subtype and a prostaglandin J2 metabolite and synthetic antidiabetic thiazolidinediones for the {gamma}-subtype. Based on the hypothesis that ligand binding to PPAR would induce interactions of the receptor with transcriptional coactivators, we have developed a novel ligand sensor assay, termed coactivator-dependent receptor ligand assay (CARLA). With CARLA we have screened several natural and synthetic candidate ligands and have identified naturally occurring fatty acids and metabolites as well as hypolipidemic drugs as bona fide ligands of the three PPAR subtypes from Xenopus laevis. Our results suggest that PPARs, by their ability to interact with a number of structurally diverse compounds, have acquired unique ligand-binding properties among the superfamily of nuclear receptors that are compatible with their biological activity.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Fatty acids and carbohydrates, as major dietary constituents, participate together with several hormones in the regulation of gene expression in response to food intake and qualitative nutritional changes. This regulation operates on several metabolic pathways and involves mechanisms that control fuel utilization according to the availability of lipid and glucose and that govern the interconversion, transport, storage, mobilization, and use of these nutrients and their metabolites. Together these mechanisms ensure a healthy energy homeostasis, but alteration of this balance can lead to pathological states such as obesity, hyperlipidemia, diabetes, and the resulting cardiovascular diseases. Recently, peroxisome proliferator activated receptors (PPARs) have emerged as one of the central regulators of the nutrient/gene interactions (1, 2). They are lipid-activable transcription factors that belong to the nuclear hormone receptor superfamily and regulate genes controlling lipid and glucose metabolism and adipogenesis. They occur in three subtypes {alpha}, ß ({delta}, FAAR or NUC1), and {gamma}, in different species (Ref. 3 for review). In addition to various fatty acids and some arachidonic acid metabolites, a diverse group of substances called peroxisome proliferators, which includes the fibrate class of hypolipidemic drugs and certain plasticizers and herbicides, activate these transcription factors (4, 5, 6, 7). The diversity and relatively high concentrations of agents required for receptor activation has led to the hypothesis that PPAR activators exert their effects indirectly by either being metabolized in the cell to an active form or by inducing the release or synthesis of endogenous PPAR ligands (4, 6, 8). Therefore, determining whether peroxisome proliferators, fatty acids, and their metabolites are PPAR ligands is a key issue for a better understanding of the complex metabolic reactions they appear to modulate.

Recent work has shown that antidiabetic compounds of the thiazolidinedione class and a prostaglandin J2 metabolite are ligands of PPAR{gamma} (9, 10, 11), whereas the experimental hypolipidemic drug Wy 14,643 and the natural inflammation mediator leukotriene B4 (LTB4) are PPAR{alpha} ligands (12). These findings raise the intriguing possibility that a broad range of natural and synthetic molecules can interact directly with PPARs to regulate target gene expression. To test this hypothesis, we had to overcome one of the limitations of the classic binding assay, which is the availability of radiolabeled candidate ligands. To that end, we developed a novel in vitro ligand detector assay that we have termed Coactivator-dependent receptor ligand assay (CARLA). CARLA is based on the recently reported ligand-induced binding of transcriptional mediators, such as RIP 140 (13), TIF1 (14, 15), TIF2 (16), SRC-1 (17), SUG1 (15), and CBP (18), to classic nuclear receptors. Whereas SRC-1 and TIF2 function as transcriptional coactivators, the role of the other proteins remains unknown. Herein, we have used SRC-1, which promotes the transcriptional activity of the progesterone, glucocorticoid, estrogen, thyroid hormone, and retinoic-X receptors, through direct interaction with the ligand-activated form of the receptors (17). Using CARLA, we demonstrate that various naturally occurring fatty acids and metabolites, as well as synthetic hypolipidemic compounds, by virtue of their ability to induce PPAR/SRC-1 interactions, are bona fide PPAR ligands. The identification of fatty acids and eicosanoids as ligands of nuclear receptors implies that these compounds can regulate gene expression through the same mechanism of action used by steroid hormones.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
CARLA Assay: PPAR Ligands Induce PPAR/SRC-1 Interaction in Vitro
Application of Scatchard analysis in the identification of ligands for the three PPAR subtypes is technically and economically inefficient because a multitude of compounds are known to activate these receptors. One possibility for developing an alternative ligand assay for the rapid screening of a large number of compounds arose by the recently reported ligand-induced binding of the steroid receptor coactivator-1 (SRC-1) to nuclear hormone receptors (17). This approach is based on the hypothesis that exposing the ligand-binding domain (LBD) of the three PPAR subtypes to potential ligands would induce PPAR/SRC-1 interactions only in the cases where there is direct and specific binding of compounds to the LBDs of these receptors.

For the validation of the interaction assay we determined whether the known PPAR ligands can induce interactions of the receptor with SRC-1. From the four PPAR ligands identified by classic receptor-ligand binding assays, BRL 49653 and 15d-{Delta}12,14-PGJ2 were identified using the LBD of the mouse (m) PPAR{gamma} (9, 10, 11) whereas Wy 14,643 and LTB4 were shown to bind to the Xenopus PPAR{alpha} LBD (12). Neither our group nor others (S. Kliewer, personal communication) was successful in obtaining soluble GST-PPAR LBD fusion protein when the LBDs were of mammalian origin, i.e. mouse or rat. In contrast, the LBDs of the three Xenopus PPAR subtypes, xPPAR{alpha}, ß and {gamma}, could be purified as glutathione-S-transferase (GST)-fusion proteins from Escherichia coli extracts. Because the dissociation constants (Kds) of both PPAR{gamma} ligands, BRL 49653 and 15d-{Delta}12,14-PGJ2, have not been determined with the Xenopus receptor and because we have observed species-specific differences in the activation of PPARs by their activators/ligands (see below and H. Keller and W. Wahli, unpublished results), relatively high concentrations (100 µM) of the PPAR ligands were used in the initial experiments. As shown in Fig. 1Go, the four known PPAR ligands induced binding of in vitro translated and 35S-radiolabeled SRC-1 to purified GST-xPPAR{alpha} LBD and xPPAR{gamma} LBD fusion proteins immobilized on glutathione Sepharose beads. These interactions were specific both to the PPAR LBDs and to SRC-1. Thus, the PPAR{alpha} ligands induced specific SRC-1/PPAR{alpha}-LBD interactions as was also the case for the PPAR{gamma} ligands that induced only PPAR{gamma}-LBD/SRC-1 interactions (see Fig. 2Go). In addition, there was no induced binding of SRC-1 by the PPAR LBDs when ligands such as 17ß-estradiol and 9-cis-retinoic acid were used. Similarly, PPAR ligands did not induce interactions between the PPAR LBDs and in vitro translated estrogen receptor (not shown), implying that these compounds do not enhance nonspecific protein-protein interactions, or chemically cross-link proteins. Finally, GST alone did not retain significant amounts of SRC-1 in the presence of ligands (not shown). In preliminary experiments, deletion of the activation function 2 core of PPAR{gamma} suppressed induced binding of SRC-1 (G. Krey and W. Wahli, unpublished). Taken together, the above results indicate that the ligand-dependent PPAR/SRC-1 interaction assay, termed coactivator-dependent receptor ligand assay or CARLA, provides a new method for the identification of PPAR ligands. In the following sections, we describe the use of CARLA for the identification of bona fide PPAR ligands, among the known activators of the three PPAR subtypes from Xenopus.



View larger version (52K):
[in this window]
[in a new window]
 
Figure 1. xPPAR{alpha} and -{gamma} Ligands Induce Specific Interactions of SRC-1 with the LBD of Their Corresponding Receptor Subtype

Autoradiogram showing that in vitro translated, 35S-labeled SRC-1 is retained on GST-fusion proteins of the xPPAR{alpha} and -{gamma} LBDs in the presence of known PPAR ligands. The ligands are Wy 14,643 (W) and LTB4 (L) for xPPAR{alpha} and BRL 49653 (B) and 15d-{Delta}12,14-PGJ2 (P) for xPPAR{gamma}. All ligands are at a concentration of 100 µM. The control lanes demonstrate the absence of specific PPAR-LBD/SRC-1 interactions either in the presence of carrier solvent (-), or in the presence of 100 µM of 17ß-estradiol (E), or 9-cis-retinoic acid (R). I represents 10% of the SRC-1 used in each reaction, and M is the protein molecular weight marker.

 


View larger version (46K):
[in this window]
[in a new window]
 
Figure 2. Identification of Ligands for the Three PPAR Subtypes {alpha}, ß, and {gamma} from Xenopus laevis

Autoradiogram (selected compounds) and quantification of the effect of natural and synthetic compounds on the retention of SRC-1 by the GST-xPPAR LBDs. The concentration of all compounds tested was 100 µM. Relative CARLA value of 100 for xPPAR{alpha} and -ß corresponds to the amount of SRC-1 retained by the corresponding LBDs in the presence of 100 µM ETYA. For xPPAR{gamma} LBD this value was set at 10. The average with SD of three independent experiments, not triplicates, is shown, most likely explaining some relatively high SD values. In the autoradiogram, input represents 10% of the SRC-1 used in each reaction, M is the protein molecular weight marker.

 
Fatty Acids Are Ligands of All Three PPAR Subtypes
The recent identification of the dihydroxy-fatty acid leukotriene B4 as PPAR ligand (12) strongly suggests that natural PPAR activators, which comprise a variety of unsaturated fatty acids, including the essential fatty acids linoleic, linolenic, and arachidonic acids (6, 7, 19), might also be able to interact directly with the receptors. Thus, we tested the ability of fatty acids to function as PPAR ligands by CARLA. The fatty acids analyzed included the {omega}3 polyunsaturated docosahexaenoic (C22:6), eicosapentaenoic (C20:5), and linolenic (C18:3) acids, the {omega}6 polyunsaturated linoleic (C18:2) and arachidonic (C20:4) acids, the {omega}12 monounsaturated petroselinic acid (C18:1), the {omega}9 monounsaturated oleic (C18:1), elaidic (C18:1 trans), erucic (22:1), and nervonic (C24:1) acids, as well as the dicarboxylic fatty acid 1,12-dodecanedioic acid (C12). In addition, we tested all three xPPAR subtypes with the above fatty acids to establish whether the receptor-ligand interactions are subtype specific. Many of the above compounds are required at a concentration of 50 µM for transcriptional activation of the receptor in transient transfection experiments. Thus, to avoid the possibility that weak ligands would go unoticed, we performed CARLA at the concentration of 100 µM (Fig. 2Go) before establishing dose-response curves for selected compounds (see Fig. 3Go). The results of Fig. 2Go show that each of the three receptors is promiscuous in its interactions with natural fatty acids, which correlates with their transcriptional activation of the three xPPAR subtypes (7, 19). Thus, there is an overlap in ligand recognition among the PPAR subtypes. This applies particularly to polyunsaturated fatty acids (PUFAs), which bind best to PPAR{alpha}, followed by xPPAR{gamma} and xPPARß. CARLA ED50s of PUFAs for xPPAR{alpha} are below 10 µM (e.g. linolenic acid, Fig. 3AGo) and between 10 and 100 µM for xPPARß (e.g. linoleic acid, Fig. 3BGo) and xPPAR{gamma} (e.g. eicosapentaenoic acid, Fig. 3CGo). Similarly, monounsaturated fatty acids (MUFAs) have a preference for xPPAR{alpha} in general. Among them, pretroselinic (C18:1/{omega}12), oleic (C18:1/{omega}9), and elaidic (C18:1/{omega}9-trans) acids are almost as efficient in stimulating SRC-1 binding to PPAR{alpha} as PUFAs. As an example, the CARLA ED50 of xPPAR{alpha} to the most potent of the PUFAs tested, i.e. linoleic acid (C18:2), is 7 µM (Fig. 3AGo), whereas that of petroselinic acid (C18:1) is 10 µM (not shown). It is interesting to note that even at the concentration of 100 µM, fatty acids (e.g. MUFAs) interact specifically with receptor subtypes (Fig. 2Go). Specificity of interactions at high ligand concentration is also observed with other natural or synthetic compounds as well (see below), suggesting that CARLA is functional at a wide range of ligand concentrations.



View larger version (43K):
[in this window]
[in a new window]
 
Figure 3. Potency of Natural and Synthetic xPPAR Ligands as Determined by CARLA

A, xPPAR{alpha}; B, xPPARß; C, xPPAR{gamma}. The dose-response curves in each panel show CARLA values of each PPAR LBD with a variety of ligands. Each point represents the average of at least two independent experiments. The concentrations of the ligands at each point are indicated. The autoradiograms in each panel show representative CARLA experiments of the three xPPAR LBDs with different concentrations of their best respective ligands, as these were determined in Fig. 2Go. I and M are defined in Fig. 1Go.

 
In summary, the above data provide evidence that structurally diverse fatty acids have the possibility to directly act on gene expression by binding to and activating the three PPAR subtypes.

Arachidonic Acid Metabolites Are PPAR Ligands
The essential fatty acids, through arachidonate, are the precursors of the prostaglandin, thromboxane, and leukotriene families of compounds. These compounds, collectively known as eicosanoids, have important and diverse biological effects ranging from controlling platelet aggregation to chemotaxis. A number of eicosanoids have been shown to activate PPAR{alpha} in cellular transfection assays (10, 11, 12, 20, 21). Furthermore, two of these compounds, LTB4 and 15d-{Delta}12,14-PGJ2, are ligands for the PPAR{alpha} and {gamma}-subtypes, respectively (10, 11, 12). As discussed above, these results have also been confirmed by CARLA (Figs. 1Go and 2Go). Another arachidonic acid metabolite, which has been shown to be a strong activator of the human (h) PPAR{alpha}, is the lipoxygenation product 8(S) hydroxyeicosatetraenoic acid \[8(S)-HETE\]. Interestingly, 8(S)-HETE stereoselectively activates the receptor over its 8(R)-enantiomer (20). As shown in Fig. 2Go, when 8(S)-HETE is tested by CARLA it induces strong xPPAR{alpha}-SRC-1 interactions. Similarly, 8(R)-HETE is also a xPPAR{alpha} ligand, albeit of much lower affinity than its 8(S)-enantiomer. Indeed, dose-response curves reveal CARLA ED50s of 1 and 50 µM for 8(S)-HETE and 8(R)-HETE, respectively (Fig. 3AGo), indicating strong stereoselectivity in ligand binding. 8(S)-HETE is also a ligand of xPPARß and {gamma} with CARLA ED50 values of 20 and >50 µM for the two receptors, respectively (Figs. 2Go and 3BGo and results not shown), whereas no interaction was detected between 8(R)-HETE and the ß- and {gamma}-receptors (Fig. 2Go). In accordance with results of cellular transfection assays in which the chemotactic agent 5(S)-HETE fails to activate PPARs (20), this compound does not induce interactions of SRC-1 with either of the xPPAR subtypes (Fig. 2Go) and serves as an additional control for the validity of CARLA.

In contrast to 8(S)-HETE, which can induce xPPAR{alpha}-SRC-1 interactions at low concentrations, LTB4, an xPPAR{alpha} ligand with a Kd of 0.1 µM as determined by Scatchard analysis (12), is required at 100 µM concentration to induce maximum xPPAR{alpha}-SRC-1 interactions. In addition, even at this concentration, the amount of SRC-1 retained by the PPAR{alpha} LBD is significantly lower compared with that retained due to 8(S)-HETE, or the fatty acids ({approx}30%, Fig. 2Go). However, as illustrated in Fig. 3AGo, a reproducible biphasic CARLA behavior is observed with LTB4. Thus, the CARLA value due to 0.1 µM of this compound is {approx}40% of the value seen with 100 µM LTB4. This value then remains constant up to concentrations >= 10 µM, at which point the amount of retained SRC-1 is increased. The reason and significance of the unique CARLA behavior of this PPAR ligand remain to be elucidated.

In conclusion, the above results demonstrate that 8(S)-HETE with a ED50 of 1 µM for xPPAR{alpha} (Fig. 3AGo) is a potent natural ligand of this subtype. Note that xPPAR{alpha} has a 50-fold higher affinity for 8(S)-HETE than for its precursor, arachidonic acid (CARLA ED50{approx}50 µM, not shown). 8(S)-HETE, linolenic, and linoleic acids with ED50s of {approx}20 µM (Fig. 3BGo) are presently the best natural ligands for xPPARß. For xPPAR{gamma}, 15d-{Delta}12,14-PGJ2 with a CARLA ED50 value of 20 µM (Fig. 3CGo) is the most potent natural ligand identified so far for this subtype, as is also true for mPPAR{gamma} (10, 11).

Hypolipidemic Drugs Are Ligands of xPPAR{alpha}, -ß, and -{gamma}
PPAR activators also include synthetic hypolipidemic agents of experimental interest or that are already being used for the treatment of hyperlipidemia in humans. However, the molecular mechanism(s) of action of these drugs remain unclear. As a first step in analyzing these mechanisms with respect to PPAR biology, we determined whether hypolipidemic drugs directly bind to PPARs and whether they do so in a receptor subtype-specific manner. In addition to Wy 14,643 and BRL 49653, used above to validate the CARLA assay (Fig. 1BGo), we tested 5,8,11,14-eicosatetraynoic acid (ETYA), a synthetic arachidonic acid analog and potent xPPAR{alpha} activator (7), the medically important bezafibrate, clofibrate, ciprofibrate, fenofibrate, and nafenopin (also a fibrate), as well as the nonfibrate hypolipidemic drugs, benfluorex, gemfibrozil, and probucol, for their ability to induce interaction of SRC-1 with xPPAR{alpha}, -ß, and -{gamma}. As shown in Fig. 2Go, each of the PPARs interacted with two or more of the above mentioned compounds, i.e. PPAR{alpha} with ETYA and Wy 14,643, PPARß with ETYA and bezafibrate, and PPAR{gamma} with BRL 49653 and ciprofibrate. In addition, partial overlap in ligand recognition between the receptor subtypes was observed. This is most evident for ETYA with ED50s of 1 and 50 µM for PPAR{alpha} and -ß, respectively (Fig. 3Go, A and B), and with only low affinity for xPPAR{gamma} (Fig. 2Go). In contrast, some compounds exhibit a more strict subtype specificity, such as BRL 49653 for xPPAR{gamma} with an ED50 of 8 µM (Fig. 2Go and 3CGo) and Wy 14,643, which acts preferentially on xPPAR{alpha} (Fig. 2Go). Among the fibrate hypolipidemic agents, the most potent, with respect to PPAR subtypes, are Wy 14,643, with an ED50 of 30 µM, bezafibrate, with an ED50 of 5 µM, and ciprofibrate, with an ED50 of 30 µM for xPPAR{alpha}, -ß and -{gamma}, respectively (Fig. 3Go). The nonfibrate drugs, benfluorex, gemfibrozil, and probucol, do not interact with either PPAR subtype at the concentration tested (100 µM). These data provide evidence that the fibrate hypolipidemic agents are PPAR ligands to which they interact with subtype-specific preference. Consequently, the biological action of the hypolipidemic PPAR ligands could be mediated by one or all of the receptor subtypes, depending upon which ligand is being used. It is also interesting to note that the ED50 values of 1 and 30 µM for ETYA and Wy 14,643, respectively, in this interaction assay (Fig. 3AGo) correlate well with the ED50s of 0.8 and 20–30 µM for these compounds in a cellular transcriptional activation assay (7). Furthermore, the relatively poor xPPAR{alpha} activator clofibrate, with an ED50 of 200 µM in transfection assays (H. Keller and W. Wahli, unpublished), is required at concentrations of 100 to 1000 µM to induce interaction between PPAR{alpha} and SRC-1 (not shown).

In conclusion, the fatty acid analog ETYA, bezafibrate, and the thiazolidinedione antidiabetic drug BRL 49653 are the most potent synthetic ligands identified so far for the xPPAR{alpha}, -ß, and -{gamma} subtypes, respectively. Our results also indicate that PPAR ligand recognition has been preserved through evolution as exemplified by the binding of BRL 49653 to PPAR{gamma} of lower vertebrates (this study) and of mammals (9, 11).

PPAR-Ligand Interaction and Transcriptional Activation
The CARLA profiles (dose-response curves) obtained with the fatty acids, as well as with Wy 14,643 and ETYA (Figs. 1Go and 3Go), correlate very well with the transcriptional activation profiles of the Xenopus PPAR receptors by these compounds (7, 19). However, several of the ligands studied herein by CARLA, i.e. 8(S)- and 8(R)-HETEs, bezafibrate, BRL 49653, and 15d-{Delta}12,14-PGJ2, have not been previously tested for their ability to transcriptionally activate the PPARs of Xenopus. Thus, we assessed whether the dose-dependent PPAR-SRC-1 interactions observed in vitro with these compounds parallel their effect on transcription in transfected cells. Because PPAR ligand binding can exhibit strong species-specific differences (H. Keller and W. Wahli, unpublished results), we examined in parallel the transcriptional activation profiles of the mouse and human PPAR{alpha} in response to 8(S)- and 8(R)-HETE and of the mouse and human PPAR{gamma} in response to BRL 49653 and 15d-{Delta}12,14-PGJ2. The Xenopus receptors present an excellent correlation between the ED50s obtained in CARLA and in transfection experiments (Fig. 4Go). For xPPAR{alpha} and its ligand 8(S)-HETE for example, the transcriptional activation ED50 is 2 µM compared with 1 µM in CARLA. Also consistent with the CARLA results, 8(S)-HETE is a much more efficient activator of PPAR-dependent transcription than its 8(R) stereoisomer. Furthermore, 8(S)-HETE, which did not induce xPPARß/SRC-1 interactions at low concentrations, also failed to significantly activate transcription through the ß-receptor (compare corresponding panels in Figs. 3Go and 4Go). Finally, species comparison indicates that the mPPAR{alpha} has a dose response to 8(S)- and 8(R)-HETEs similar to that of its Xenopus homolog, while the hPPAR{alpha} responds slightly better to both of these compounds (Fig. 4Go).



View larger version (36K):
[in this window]
[in a new window]
 
Figure 4. Ligand Dose-Responses of the Three PPAR Subtypes from Different Species

A, HeLa cells were transfected with the Cyp2XPAL reporter plasmid and expression vectors for the indicated PPARs. After transfection, cells were treated with the indicated concentration of ligand. Each point represents the average of at least two independent experiments. Relative CAT activity of 100 corresponds to the normalized CAT value obtained at the highest concentration of the most potent ligand used (10 µM for 8(S) HETE and BRL 49653 and 100 µM for bezafibrate). B, Autoradiogram of a CAT assay from HeLa cells transfected as in panel A. Triangles indicate the increasing concentration of ligand used. The concentrations of ligands were 0, 0.1, 1, and 10 µM for all compounds except for bezafibrate where CAT activity in the presence of 100 µM of this compound is also shown.

 
A good correlation between CARLA and transcriptional activation ED50 values is also observed for the other two Xenopus PPAR subtypes because the values for transcriptional activation are approximately 1 µM for bezafibrate (xPPARß), BRL 49653, and 15d-{Delta}12,14-PGJ2 (xPPAR{gamma}) (Fig. 4Go). The PPAR{gamma} from the three species exhibits a very similar activation by 15d-{Delta}12,14-PGJ2, in contrast to activation by BRL 49653, which is much more efficient for the mouse and human PPAR{gamma} than for the Xenopus receptor (ED50s at nanomolar vs. micromolar concentrations). However, the species-specific difference of PPAR{gamma} transcriptional activation by BRL 49653 correlates very well with the affinity of the respective receptors for this ligand. Indeed, Kds of 43 nM and 320 nM have been reported for the mPPAR{gamma} (9, 11), whereas in this study the CARLA ED50 value for the Xenopus receptor is of 10 µM.

Together, the above results demonstrate that the Xenopus PPAR ligands identified herein by CARLA are also activators, i.e. agonists, of PPAR-dependent transcription and that the ligand-receptor and transcriptional activation dose-response curves are in agreement. In addition, they show that the corresponding PPAR subtypes from different species respond similarly to ligands in terms of specificity and stimulation of transcription.

Compounds Negative in CARLA and Positive in Transactivation
All compounds, so far without exception, that induce PPAR-SRC-1 interaction are active in mediating transcriptional stimulation. Conversely, one could expect that compounds that fail to induce PPAR-SRC-1 interactions in CARLA are also unable to activate PPAR-dependent transcription. Indeed, this is the case with the nonfibrate hypolipidemic drugs, benfluorex, gemfibrozil, and probucol, as well as with the nervonic and 1,12-dodecanedioic acids, or 5(S) HETE that are negative both in CARLA (herein) and in transactivation (Ref. 7 and results not shown). In contrast to the above compounds, fibrates such as bezafibrate, ciprofibrate, and nafenopin significantly activate all three PPAR subtypes in HeLa cells, albeit to different levels (Fig. 5Go). At the same concentration (100 µM), fibrates either fail to induce strong PPAR-SRC-1 interactions in vitro or are subtype-specific ligands (compare Figs. 2Go and 5Go). Of these compounds, nafenopin appears to stimulate transcription 8- to 15-fold through all three xPPAR subtypes, which confirms previous observations in similar experiments (5). Also of interest is the observation that BRL 49653 confers some activation to xPPAR{alpha} and, to a lesser extent, to xPPARß, when present at micromolar concentrations, although this compound is not defined as xPPAR{alpha} or -ß ligand by CARLA (Fig. 2Go). Similarly, it also activates mPPAR{alpha} and hPPAR{alpha} (Fig. 5BGo) although it was defined as a mPPAR{gamma}-specific ligand (9, 11). Activation of PPARs by compounds that are not identified as ligands in two different assays, i.e. CARLA and classic ligand-binding assay, suggests that such compounds can indirectly influence PPAR-dependent transcription possibly as metabolites or by releasing endogenous ligands. Alternatively, it is possible that these compounds are indeed ligands but that they confer a distinct structural conformation to the receptor that excludes PPAR/SRC-1 interactions without, however, excluding interactions with other coactivators.



View larger version (35K):
[in this window]
[in a new window]
 
Figure 5. Fibrates and BRL 49653 Activate the Three PPAR Subtypes

A, Activation of xPPAR{alpha}, -ß and -{gamma} by the indicated compounds. HeLa cells were transfected as described in Fig. 4AGo and were treated with the indicated compounds. Normalized CAT activity was determined and plotted as fold induction relative to CAT activity from cells treated with no ligand. Ligand concentration was 100 µM for all compounds except for 8(S) HETE and BRL 49653 (10 µM). B, Activation of Xenopus, mouse, and human PPAR{alpha} by 10 µM BRL 49653. Fold induction values are as in panel A.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The CARLA Screen
Herein, we have described CARLA as a novel and reliable assay for the identification of PPAR ligands and have provided evidence that many of the known PPAR activators are also ligands for the receptors. Thus, we have identified natural and synthetic ligands for all three PPAR subtypes from Xenopus. It is noteworthy that all the identified ligands are activators of PPAR-dependent transcription and, thus, are PPAR agonists, whereas antagonists, as suggested from previous observations with the progesterone receptor (17), would probably not be detected by this assay.

Compared with the classic ligand-binding assay, CARLA presents major technical advantages: it does not require radioactive labeling of candidate ligands, it is sensitive and, in addition, identifies only agonist ligands. The technical simplicity of the assay makes possible the screening of a large number of compounds with simultaneous economic advantages in terms of material and time. In addition, the fact that this assay is based on a physical-functional interaction between two proteins in response to ligand, and not merely on the association of a lipophilic molecule with a globular protein domain, suggests that it should be less prone to artifacts. However, the assay depends on soluble GST-receptor fusion protein produced in bacteria, which can not always be taken for granted (see Results). In addition, some compounds, which might induce conformational changes that may result in interactions with coactivators different from SRC-1, would escape identification as ligands when SRC-1 is used in the assay system. Finally, but not least, it should be noted that the CARLA screen should be applicable to the identification of ligands for other orphan members of the nuclear hormone receptor superfamily.

Our results also indicate that there is an excellent correlation between the CARLA ED50 values and transactivation ED50s observed in transient transfection experiments. However, further work, beyond the scope of the present study, is necessary to establish how and if the CARLA ED50 values relate to affinity constants of the receptors for their ligands.

PPARs: Promiscuous Receptors with Overlapping Ligand Recognition
We have demonstrated that the three PPAR subtypes from Xenopus are able to bind a wide spectrum of compounds ranging from fatty acids and arachidonate metabolites and analogs to synthetic hypolipidemic drugs. The amazing ability of the PPAR LBDs to accommodate such a variety of molecular structures implies that these receptors, in contrast to the steroid/thyroid hormone receptors with their stringent ligand specificity, have evolved under different selection pressures. In addition, there is an important overlap in ligand recognition between the three PPAR subtypes, especially for polyunsaturated fatty acids. Overlap in ligand recognition does not necessarily imply functional similarities between PPAR{alpha}, -ß, and -{gamma}, since differences in the affinities of each subtype for the ligands, as well as the tissue distribution of the subtypes, may be important determinants of subtype- and tissue-specific recognition and regulation of PPAR target genes.

Our data also indicate that, in general, ligand recognition by PPAR subtypes has been preserved through evolution. However, together with results from others (9, 11), they also show that species-specific differences exist in the affinity of a receptor subtype for a given ligand, as is suggested by the fact that hPPAR{alpha} is more efficiently activated by 8(S) HETE than either the mouse or Xenopus receptors, whereas the mouse and human PPAR{gamma} are more efficiently activated by BRL 49653 than their Xenopus homolog. It remains to be investigated whether species-specific differences in affinity for natural ligands reflect species-specific physiological peculiarities, such as peroxisomal proliferation.

Fatty Acids as Hormones
In mammals, synthesis of the {omega}6 and {omega}3 series of fatty acids depends on the nutritional intake of the essential fatty acids, linoleic, linolenic, and arachidonic acids. Production of many biologically important molecules, such as prostaglandins, thromboxanes, and leukotrienes, depends on the intracellular availability and levels of the essential fatty acids and their derivatives. Our identification of unsaturated fatty acids as PPAR ligands provides the most conclusive evidence to date that part or all of PPAR-dependent transcription (reviewed in 3 results from the direct activation of the receptor by these molecules and further implicates PPARs as key control factors in the maintenance of energy homeostasis. Consequently, fatty acids that share properties of classic hormones, i.e. the ability to directly interact with nuclear receptors and to regulate transcription, can no longer be considered as simple biological substrates. Even though the actual concentration of fatty acids in the cell nucleus, where PPARs are localized, has not been extensively documented, the levels of free (nonesterified) fatty acids in human blood plasma (22) are sufficiently high for PPAR activation. This implies that high-affinity binding of fatty acids to PPARs would not be an advantage as these receptors have evolved to sense ligands whose concentrations are naturally high and even very high in situations of lipid overload.

Although the PPAR subtypes overlap in ligand recognition, there is a clear preference of the {alpha}-subtype for PUFAs. It remains still to be demonstrated whether this preference is linked to the adipolytic function of PPAR{alpha} (review in 3 . Similarly, the strong preference of the monounsaturated fatty acids for PPAR{alpha} suggests that this receptor may play a specific role in situations of nutritional lipid overload. In this context, it will be of interest to examine possible connections between ligand preference, differential tissue distribution of the PPAR subtypes, and the fatty acid content of each tissue. It should be noted that a reverse relationship appears to exist between affinity for ligand and overlap in ligand recognition by PPARs. Thus, in contrast to fatty acids, the eicosanoids 8(S)-HETE and 15d-{Delta}12,14-PGJ2, which naturally occur at lower concentrations, are high- affinity PPAR ligands exhibiting a more strict subtype specificity than their polyunsaturated fatty acid precursors.

8(S)-HETE Is a High-Affinity PPAR{alpha} Ligand
As mentioned above, we have identified the hydroxyeicosatetraenoic acid 8(S)-HETE as a high-affinity ligand of the PPAR{alpha} and have confirmed previous observations that it is a strong activator of hPPAR{alpha} (20). 8(S)-HETE is a naturally occurring compound that is thought to result from the NADPH-dependent metabolism of arachidonic acid by monooxygenases (cytochrome P 450) (23). Although a considerable amount of information is available on the biological action of the 5(S)-, 12(S)-, and 15(S)-HETE regioisomers (review in 24 , little is known about the 8(S)-isomer. It is noteworthy, however, that 8(S)-HETE has been found in a variety of tissues (e.g. liver, brain, epidermis) and cell types (e.g. neutrophils, smooth muscle, oocytes) (Ref. 24 and references therein). It is tempting to speculate that binding of 8(S)-HETE to PPAR{alpha} results in the stimulation of fatty acid oxidative pathways that would lead to its own neutralization and degradation, in a situation similar to that described recently for another chemotatic eicosanoid, LTB4 (12).

Like 8(S)-HETE, the arachidonic acid analog ETYA, a compound with very diverse biological effects (Ref. 7 and references therein), has also been shown to be a potent xPPAR{alpha} ligand. The identification of these two high-affinity PPAR{alpha} ligands could eventually lead to the development of medically important subtype-specific ligands and to an increased understanding of the biological functions of PPAR{alpha}.

Bezafibrate Is a High-Affinity Ligand of xPPARß
The biological function of PPARß is the least understood of the three PPAR subtypes despite the fact that it is the most widely expressed one (5, 25). Here we have identified bezafibrate as the first specific and high-affinity ligand for this receptor. Bezafibrate is actually the only fibrate from those tested that exhibits sensus stricto classic hormonal ligand characteristics, i.e. subtype specificity and low ED50 both in CARLA and transcriptional activation. It will be of interest to examine whether PPARß from mouse and human (26, 27, 28) are also activated by this compound as it is still an open question whether xPPARß is actually the homolog of the mammalian PPARß (or {delta}, or NUC I) or whether it represents a different PPAR subtype. Furthermore, the availability of a PPARß-specific ligand provides a precious tool in investigating the biological function of this receptor.

In contrast to bezafibrate and ciprofibrate, which have been identified as PPAR ligands by CARLA, nafenopin, a nonligand by CARLA definition, activates all three PPARs (Refs. 4 and 5 and present study). Furthermore, we have observed that BRL 49653, a PPAR{gamma}-specific ligand, activates PPAR{alpha} in the HeLa cells used in this study. Thus, it is possible that nafenopin or other fibrates and BRL 49653 activate some PPAR subtypes indirectly, either by inducing production of endogenous PPAR ligands or by posttranslational modification of the receptor. Alternatively, these compounds could be modified and converted to ligands of different PPAR subtypes in the cell. It should also be noted that BRL 49653 has been reported not to have an effect on mPPAR{alpha} transcriptional activity in CV-1 cells (9, 11). However, in these studies chimeric receptors with a heterologous DNA-binding domain were used. Thus, activation of PPAR{alpha} by these two compounds may be cell type and/or receptor N-terminal domain-dependent. Nevertheless, as previously mentioned, we cannot exclude the possibility that compounds negative in CARLA are true PPAR ligands. According to this possibility, binding of nafenopin or BRL 49653 to the receptor could cause distinct conformational changes that do not favor PPAR/SRC-1 interactions. Thus, the effect of these compounds in PPAR-dependent transcription could be mediated by other coactivator(s). However, considering the ability of fibrates (e.g. bezafibrate, ciprofibrate) to induce favorable LBD conformation for receptor/co-activator interactions, we think that this is an unlikely possibility. Also arguing against this possibility is the fact that classic Scatchard analysis has failed to identify BRL 49653 as PPAR{alpha} ligand (9).

Activation of PPAR{alpha} by BRL 49653 could be consistent with the observation that this antidiabetic compound exhibits hypolipidemic properties as well (29). Accumulating evidence suggests that PPAR{alpha} is the receptor subtype regulating fatty acid catabolism (reviewed in 3 , although it may also have a direct role in adipogenesis (20, 30). Thus, it is currently believed that the activity of hypolipidemic drugs is mediated through PPAR{alpha}. Similarly, the major function attributed to PPAR{gamma} is that of key regulator of adipogenesis (reviewed in 31 . We have demonstrated herein that hypolipidemic agents, such as the fibrates, are also PPARß and {gamma} ligands. Thus, the hypolipidemic effects of these compounds may also be a result of lipid clearance via PPAR{gamma} (storage of circulating triglycerides in adipose tissue) and/or activation of lipid catabolism through PPARß. Future investigation, aided by the identification of subtype-specific ligands, will clarify whether fatty acid catabolism and adipogenesis depend on a particular PPAR subtype or on the combined action of the three receptors.

Conclusions
The development of the CARLA screen and the identification of synthetic and natural PPAR ligands opens many experimental routes for the analysis of the PPAR function. The fact that certain fatty acids and eicosanoids can act as ligands of nuclear receptors reveals a novel role for these molecules in the overall lipid metabolism and cellular energy balance and implicates the function of PPARs in processes such as transport, uptake, activation, oxidation, or storage of fatty acids, as well as clearance of biologically very active eicosanoids. In addition, the hypoglycemic effects of PPAR{gamma} agonists imply that these receptors may have key functions in the interface between lipid and carbohydrate metabolism. It should be possible now, through comparative analysis of PPAR ligand and LBD structures, to define the structural requirements for ligand binding to this subfamily of nuclear receptors. The subsequent development of subtype-specific agonists and antagonists, in addition to their potential utility in treating lipid disorders, should also provide powerful tools for deciphering the precise role of PPARs in the metabolic pathways they regulate.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Reagents
ETYA, probucol, bezafibrate, fenofibrate, benfluorex, gemfibrozil, 17ß-estradiol, docosahexaenoic acid, eicosapentaenoic acid, linolenic acid, linoleic acid, arachidonic acid, petroselinic acid, oleic acid, elaidic acid, erucic acid, nervonic acid, and 1,12-dodecanedioic acid were from Sigma Chemical Co. (St. Louis, MO). Leukotriene B4 (LTB4), 5(S)-HETE, 8(S)-HETE, and 8(R)-HETE were from Cascade Biochem. Ltd. (Berkshire, UK) 15d-{Delta}12,14-PGJ2 was from Cayman Chemical Co. (Ann Arbor, MI). Wy 14,643 was from Chemsyn Science Laboratories (Campro Scientific, Veenendaal, The Netherlands); ciprofibrate was a kind gift of P. Bernard; nafenopin was a kind gift of W. Bencze (Ciba-Geigy, Summit, NJ); BRL 49653 was a kind gift of S. Kliewer (Glaxo-Wellcome, Research Triangle Park, NC); and 9-cis-retinoic acid was a gift of M. Klaus (Hoffmann-La Roche, Basel, Switzerland). All compounds were dissolved in ethanol, with the exception of 15d-{Delta}12,14-PGJ2 (dimethyl acetate), LTB4, 5(S)-HETE, and 9-cis-retinoic acid (all in dimethylsulfoxide).

Plasmid Constructions
The mammalian expression vectors (pSG5) for the Xenopus PPAR{alpha}, -ß, and -{gamma} have been previously described (5). The mPPAR{alpha} (4) was a kind gift of S. Green; the mPPAR{gamma}1 (27) was a kind gift of J. M. Lehmann; the hPPAR{alpha} (32) was a kind gift of F. Gonzalez; and the hPPAR{gamma} (33) was a kind gift of A. Elbrecht and M. D. Leibowitz. All PPAR cDNAs were cloned in the pSG5 vector. The SRC-1 cDNA (17), PCR amplified from a human B cell cDNA library, was cloned in the BamHI site of pSG5. The reporter plasmid Cyp2XPAL contains two copies of the CYP4A6 PPRE cloned in palindromic orientation upstream of the minimal herpes simplex virus thymidine kinase promoter in the pBLCAT8+ plasmid (12). The GST-PPAR LBD fusions were constructed as follows: the xPPAR{alpha} and -ß cDNA sequences corresponding to the LBD (codons 167–470 and 93–397, for {alpha} and ß, respectively) were amplified by PCR and cloned into the BamHI site of pGEX1 vector (Pharmacia, Piscataway, NJ); the xPPAR{gamma} LBD (codons 214–477) was excised as a BamHI fragment from the ER-PPAR chimera (5) and cloned into the corresponding site of pGEX1.

CARLA Pulldowns
One milliliter of overnight cultures of Escherichia coli BL2 strain, transformed with the GST-PPAR LBD fusion plasmids, was used to inoculate 50 ml of LB medium. The bacteria were incubated at 37 C until the culture reached A600 of 0.6. At that point, IPTG was added at final concentration of 0.1 mM, and the culture was incubated for a further 3 h at 28 C. Bacterial cells were harvested by centrifugation and suspended in one tenth of the culture volume in NETN buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P40) supplemented with 1 mM dithiothreitol, 0.5% dry milk, and protease inhibitors (Complete, Boehringer Mannheim, Indianapolis, IN). Bacteria cells were then lysed by mild sonication. After centrifugation to remove the cell debris, glutathione Sepharose 4B beads (Pharmacia) equilibrated in NETN were added to the sonication extracts (250 µl of beads suspension per 50 ml of culture volume), and the GST-PPAR LBD fusions were allowed to bind to the beads for 1 h at 4 C with constant rotation. The glutathione-bound fusion proteins were washed several times with NETN and were resuspended in the appropriate volume of the same buffer. CARLA was performed in a total volume of 1 ml per reaction containing 2–3 µg of fusion protein. In each reaction, 20 µl of a 1:10 dilution in NETN of 35S-labeled, in vitro translated (TNT, Promega, Madison, WI) SRC-1 were added in ice (0.2% final concentration of translation reaction mix). The compounds tested were added at the appropriate concentrations. Probucol and nervonic acid initially precipitated in ice but were completely dissolved at 4 C. After incubation of the reaction mixture for 4 h at 4 C with constant rotation, the immobilized fusion proteins were washed three times with 0.5 ml NETN, without milk. The glutathione-Sepharose-bound proteins were dried under vacuum before being resuspended in SDS sample buffer and subjected to SDS-PAGE. Before being dried and exposed to autoradiography, the polyacrylamide gels were lightly stained with Coomasie brilliant blue to control that equal quantities of fusion proteins were used in each reaction. The amounts of retained SRC-1 were determined by image analysis on a GS 250 molecular Imager (Bio-Rad Laboratories, Richmond, CA) instrument.

Cell Culture and Transfections
Cell culture conditions and transfections of HeLa cells have been previously described (7). For each transfected plate (3 cm diameter), 2.5 x 105 cells and 0.1 µg PPAR expression plasmid were used along with 2 µg of the reporter and 0.5 µg of the CMV-ß-gal (34) internal control plasmid. Twelve hours posttransfection, cells were incubated for a further 24 h with culture medium containing the ligands.


    ACKNOWLEDGMENTS
 
We would like to acknowledge P. Devchand, J. M. Lehmann, S. Green, F. Gonzalez, A. Elbrecht, and M. Leibowitz for plasmids. We are grateful to E. Beale, S. Basu-Modak, and T. Lemberger for critical reading of the manuscript. Special thanks go to T. Lemberger for stimulating discussions and for generously sharing his ideas.


    FOOTNOTES
 
Address requests for reprints to: Walter Wahli, Institut de Biologie Animale, Bâtiment de Biologie, Université de Lausanne, CH-1015 Lausanne, Switzerland.

This work was supported by the Swiss National Science Foundation and the Etat de Vaud. G. K. was supported by Ciba-Geigy. E. K. is a Netherlands Organization for Scientific Research (N.W.O.) fellow.

Received for publication January 30, 1997. Accepted for publication March 17, 1997.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Desvergne B, Wahli W 1995 PPAR: a key nuclear factor in nutrient/gene interactions? In: Bauerle PA (ed) Inducible Gene Expression. Birkhäuser, Boston, vol 1:142–176
  2. Wahli W, Braissant O, Desvergne B 1995 Peroxisome proliferator activated receptors: transcriptional regulators of adipogenesis, lipid metabolism and more... Chem Biol 2:261–266[Medline]
  3. Lemberger T, Desvergne B, Wahli W 1996 PPARs: a nuclear receptor signaling pathway in lipid metabolism. Annu Rev Cell Dev Biol 12:335–363[CrossRef][Medline]
  4. Issemann I, Green S 1990 Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferator. Nature 347:645–650[CrossRef][Medline]
  5. Dreyer C, Krey G, Keller H, Givel F, Helftenbein G, Wahli W 1992 Control of the peroxisomal beta-oxidation pathway by a novel family of nuclear hormone receptors. Cell 68:879–887[Medline]
  6. Göttlicher M, Widmark E, Li Q, Gustafsson J-Å 1992 Fatty acids activate a chimera of the clofibric acid-activated receptor and the glucocorticoid receptor. Proc Natl Acad Sci USA 89:4653–4657[Abstract]
  7. Keller H, Dreyer C, Medin J, Mahfoudi A, Ozato K, Wahli W 1993 Fatty acids and retinoids control lipid metabolism through activation of peroxisome proliferator-activated receptor-retinoid x receptor heterodimers. Proc Natl Acad Sci USA 90:2160–2164[Abstract]
  8. Krey G, Keller H, Mahfoudi A, Wahli W 1995 PPARs: nuclear receptors controlling peroxisomal fatty acid ß-oxidation. In: Wanders RJA, Schutgens RBH, Tabak HF (eds) Function and Biogenesis of Peroxisomes in Relation to Human Disease. North-Holland Press, Amsterdam, pp 167–200
  9. Lehmann JM, Moore LB, Smith-Oliver TA, Wilkison WO, Willson TM, Kliewer SA 1995 An antidiabetic thiazolidinedione is a high affinity ligand for the nuclear receptor PPAR{gamma}. J Biol Chem 270:12953–12956[Abstract/Free Full Text]
  10. Kliewer SA, Lenhard JM, Willson TM, Patel I, Morris DC, Lehmann JM 1995 A prostaglandin J2 metabolite binds peroxisome proliferator-activated receptor gamma and promotes adipocyte differentiation. Cell 83:813–819[Medline]
  11. Forman BM, Tontonoz P, Chen J, Brun RP, Spiegelman BM, Evans RM 1995 15-deoxy-delta(12, 14)-prostaglandin J2 is a ligand for the adipocyte determination factor PPAR-gamma. Cell 83:803–812[Medline]
  12. Devchand PR, Keller H, Peters JM, Vazquez M, Gonzalez FJ, Wahli W 1996 The PPAR{alpha}-leukotriene B4 pathway to inflammation control. Nature 384:39–43[CrossRef][Medline]
  13. Cavaillès V, Dauvois S, L’Horset F, Lopez G, Hoare S, Kushner PJ, Parker MG 1995 Nuclear factor RIP140 modulates transcriptional activation by the estrogen receptor. EMBO J 14:3741–3751[Abstract]
  14. Le Douarin B, Zechel C, Garnier J-M, Lutz Y, Tora L, Pierrat B, Heery D, Gronemeyer H, Chambon P, Losson R 1995 The N-terminal part of TIF1, a putative mediator of the ligand-dependent activation function (AF-2) of nuclear receptors, is fused to B-raf in the oncogenic protein T18. EMBO J 14:2020–2033[Abstract]
  15. Vom Baur E, Zechel C, Heery D, Heine MJS, Garnier JM, Vivat V, Le Douarin B, Gronemeyer H, Chambon P, Losson R 1996 Differential ligand-dependent interactions between the AF-2 activating domain of nuclear receptors and the putative transcriptional intermediary factors mSUG1 and TIF1. EMBO J 15:110–124[Abstract]
  16. Voegel JJ, Heine MJS, Zechel C, Chambon P, Gronemeyer H 1996 TIF2, a 160 kDa transcriptional mediator for the ligand-dependent activation function AF-2 of nuclear receptors. EMBO J 15:3667–3675[Abstract]
  17. Oñate SA, Tsai SY, Tsai M-Y, O’Malley BW 1995 Sequence and characterization of a coactivator for the steroid hormone receptor superfamily. Science 270:1354–1357[Abstract]
  18. Kamei Y, Xu L, Heinzel T, Torchia J, Kurokawa R, Gloss B, Lin S-C, Heyman RA, Rose, DW, Glass CK, Rosenfeld MG 1996 A CBP integrator complex mediates transcriptional activation and AP-1 inhibition by nuclear receptors. Cell 85:403–414[Medline]
  19. Krey G, Keller H, Mahfoudi A, Medin J, Ozato K, Dreyer C, Wahli W 1993 Xenopus peroxisome proliferator activated receptors: genomic organization, response element recognition, heterodimer formation with retinoid X receptor and activation by fatty acids. J Steroid Biochem Mol Biol 47:65–73[CrossRef][Medline]
  20. Yu K, Bayona W, Kallen CB, Harding HP, Ravera CP, Mcmahon G, Brown M, Lazar MA 1995 Differential activation of peroxisome proliferator-activated receptors by eicosanoids. J Biol Chem 270:23975–23983[Abstract/Free Full Text]
  21. Hertz R, Berman I, Keppler D, Bar-Tana J 1996 Activation of gene transcription by prostacyclin analogues is mediated by the peroxisome-proliferators activated receptor (PPAR). Eur J Biochem 235:242–247[Abstract]
  22. Murray RK, Granner DK, Mayes PA, Rodwell VW 1988 Harper’s Biochemistry, ed 21. Appleton and Lange, Norwalk, CT, p 226
  23. Fitzpatrick FA, Murphy RC 1989 Cytochrome P-450 metabolism of arachidonic acid: formation and biological actions of "epoxygenase"-derived eicosanoids. Pharmacol Rev 40:229–241[Medline]
  24. Spector AA, Gordon JA, Moore SA 1988 Hydroxyeicosatetraenoic acids (HETEs). Prog Lipid Res 27:271–323[CrossRef][Medline]
  25. Braissant O, Foufelle F, Scotto C, Dauça M, Wahli W 1996 Differential expression of peroxisome proliferator-activated receptors (PPARS): tissue distribution of PPAR-{alpha}, -ß, and -{gamma} in the adult rat. Endocrinology 137:354–366[Abstract]
  26. Schmidt A, Endo N, Rutledge SJ, Vogel R, Shinar D, Rodan GA 1992 Identification of a new member of the steroid hormone receptor superfamily that is activated by a peroxisome proliferator and fatty acids. Mol Endocrinol 6:1634–1641[Abstract]
  27. Kliewer SA, Forman BM, Blumberg B, Ong ES, Borgmeyer U, Mangelsdorf DJ, Umesono K, Evans RM 1994 Differential expression and activation of a family of murine peroxisome proliferator-activated receptors. Proc Natl Acad Sci USA 91:7355–7359[Abstract]
  28. Amri E-Z, Bonino F, Ailhaud G, Abumrad NA, Grimaldi PA 1995 Cloning of a protein that mediates transcriptional effects of fatty acids in preadipocytes. Homology to peroxisome proliferator-activated receptors. J Biol Chem 270:2367–2371[Abstract/Free Full Text]
  29. Nolan J, Ludvik B, Beerdsen P, Joyce M, Olefsky J 1994 Improvement in glucose tolerance and insulin resistance in obese subjects treated with troglitazone. N Engl J Med 331:1188–1193[Abstract/Free Full Text]
  30. Brun RP, Tontonoz P, Forman BM, Ellis R, Chen J, Evans RM, Spiegelman BM 1996 Differential activation of adipogenesis by multiple PPAR isoforms. Genes Dev 10:974–984[Abstract]
  31. Spiegelman BM, Flier JS 1996 Adipogenesis and obesity: rounding out the big picture. Cell 87:377–389[Medline]
  32. Sher T, Yi HF, McBride OW, Gonzalez FJ 1993 cDNA cloning, chromosomal mapping, and functional characterization of the human peroxisome proliferator activated receptor. Biochemistry 32:5598–5604[Medline]
  33. Elbrecht A, Chen YL, Cullinan CA, Hayes N, Leibowitz MD, Moller DE Berger J 1996 Molecular cloning, expression and characterization of human peroxisome proliferator activated receptors gasmma-1 and gamma-2. Biochem Biophys Res Commun 224:431–437[CrossRef][Medline]
  34. MacGregor GR, Caskey CT 1989 Construction of plasmids that express E. coli beta-galactosidase in mammalian cells. Nucleic Acids Res 17:2365[Medline]