Identification, Characterization, and Tissue Distribution of Human Peroxisome Proliferator-activated Receptor (PPAR) Isoforms PPARgamma 2 versus PPARgamma 1 and Activation with Retinoid X Receptor Agonists and Antagonists*

(Received for publication, October 1, 1996, and in revised form, November 26, 1996)

Ranjan Mukherjee §, Lily Jow , Glenn E. Croston and James R. Paterniti Jr.

From the Departments of  Cardiovascular Research and  New Leads Discovery, Ligand Pharmaceuticals, Inc., San Diego, California 92121

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

We describe the cloning, characterization, and tissue distribution of the two human peroxisome proliferator activated receptor isoforms hPPARgamma 2 and hPPARgamma 1. In cotransfection assays the two isoforms were activated to approximately the same extent by known PPARgamma activators. Human PPARgamma binds to DNA as a heterodimer with the retinoid X receptor (RXR). This heterodimer was activated by both RXR agonists and antagonists and the addition of PPARgamma ligands with retinoids resulted in greater than additive activation. Such heterodimer-selective modulators may have a role in the treatment of PPARgamma /RXR-modulated diseases like diabetes. Northern blot analysis indicated the presence of PPARgamma in skeletal muscle, and a sensitive RNase protection assay confirmed the presence of only PPARgamma 1 in muscle that was not solely due to fat contamination. However, both PPARgamma 1 and PPARgamma 2 RNA were detected in fat, and the ratio of PPARgamma 1 to PPARgamma 2 RNA varied in different individuals. The presence of tissue-specific distribution of isoforms and the variable ratio of PPARgamma 1 to PPARgamma 2 raised the possibility that isoform expression may be modulated in disease states like non-insulin-dependent diabetes mellitus. Interestingly, a third protected band was detected with fat RNA indicating the possible existence of a third human PPARgamma isoform.


INTRODUCTION

Peroxisome proliferator-activated receptors (PPARs)1 are members of the intracellular receptor superfamily. They play a role in lipid metabolism and metabolic diseases. There are three PPAR subtypes with distinct tissue distribution in Xenopus, mice, and humans: PPARalpha , PPARbeta (also called NUC1 or PPARdelta ), and PPARgamma (1-10). PPARgamma expression is observed in adipose tissue in rodents. Its expression is induced early in differentiation of 3T3-L1 preadipocytes into adipocytes, and its overexpression in fibroblasts induces them to differentiate into adipocytes (11). Two isoforms of mPPARgamma resulting from different promoters and alternate splicing have been identified (7, 12, 13). A human isoform, hPPARgamma 1, has been cloned from a human hematopoietic cell line and placenta (14, 15), and another from human fat (16) has been reported. Although a preliminary report on the distribution of PPARgamma in human tissues has been published (16), the distribution of PPARgamma 1 versus PPARgamma 2 has not been reported.

Thiazolidinediones are high affinity ligands and potent activators for PPARgamma . They decrease insulin resistance in insulin-responsive tissues including skeletal muscle (the primary site of insulin-stimulated glucose uptake) in patients with non-insulin-dependent diabetes mellitus (17). It is assumed that PPARgamma is the therapeutic target for these compounds; yet the presence of PPARgamma has not been conclusively demonstrated in human muscle. The identification of human PPARgamma isoforms and their tissue distribution will help in understanding their role in metabolic diseases like non-insulin-dependent diabetes mellitus and obesity.

We undertook to clone and characterize the tissue distribution of human PPARgamma 1 and PPARgamma 2 and compare it with that of human PPARalpha and PPARbeta . We compared the ability of PPARgamma agonists to activate the two isoforms. A PPARgamma antagonist would be a useful tool to dissect PPARgamma action and may also block adipocyte differentiation. Such a ligand that competitively antagonizes PPARgamma activity has not been reported. An alternative approach would be to block PPARgamma /RXR activation with an antagonist of RXR. Surprisingly, an RXR antagonist activated the PPARgamma /RXR heterodimer as did an RXR agonist. Greater than additive activation was seen with PPARgamma and RXR ligands.


EXPERIMENTAL PROCEDURES

5,8,11,14-Eicosatetraenoic acid and 2-bromopalmitate were purchased from Sigma, and 15-deoxy-Delta 12,14-prostaglandin J2 was obtained from Cayman Chemicals. BRL 49653, LG100268, and LG100754 were synthesized at Ligand Pharmaceuticals Inc.

A human heart 5'-stretch cDNA library (Clontech) was screened with a mouse PPARgamma (7) probe at low stringency (35% formamide, 5 × SSC, 0.1% SDS, 100 µg/ml fish sperm DNA at 37 °C). Several positive clones were isolated and sequenced. Comparison with the mPPARgamma sequence indicated that one clone encoded the N terminus and another the C terminus of hPPARgamma , and their sequences overlapped by 485 base pairs. The complete hPPARgamma -coding region was reconstructed by a triple ligation using pBKCMV (Stratagene) digested with EcoRI and KpnI and utilizing the unique ScaI site in the coding region. This plasmid was then digested with NcoI, blunt-ended with Klenow enzyme, and redigested with KpnI. The liberated fragment was subcloned into pBKCMV at the XbaI site (blunted with Klenow enzyme) and the KpnI site to give pCMVhPPARgamma 1.

A third positive clone was isolated and sequenced. This sequence overlapped that of hPPARgamma 1 by 1268 base pairs but had a unique N terminus. The technique of crossover polymerase chain reaction was utilized to create pCMVhPPARgamma 2. The sequences of the two external primers were 5'-TGAGTCAGCTCGAGATATCAGTGTGAATTACAGC-3' and 5'-GATCCTAGGCGGCCGCTCAGAATAGTGCAACTGG-3'. The internal primers were 5' CATTACGGAGAGAGATCCAC-3' and 5'-ATGGTTGACACAGAGATG-3'. The polymerase chain reaction product was cloned into the SmaI site of pBKCMV, and the orientation was determined by restriction mapping.

Northern Blots

Human multiple tissue Northern blots were purchased from Clontech. Hybridization was done according to the manufacturer's protocol. The probe for hPPARalpha has been described (6). pCMVhPPARbeta (pCMVhNUC1) (18) was digested with EcoRI and the 500-base pair fragment was isolated. pCMVhPPARgamma 1 was digested with ScaI and KpnI and the 1-kilobase pair fragment isolated. This probe will recognize hPPARgamma 1 and hPPARgamma 2 RNA. All probes were labeled by random priming.

RNase Protection Assays

Four human white fat and two skeletal muscle samples were obtained from the National Disease Research Interchange (NDRI, Philadelphia) or the University of California (San Diego) tissue bank. Total RNA was isolated using standard techniques. A sample of human skeletal muscle RNA was purchased from Clontech.

A partial cDNA containing nucleotides 1-252 of hPPARgamma 2 (Fig. 1) was subcloned into the pCRII vector (Invitrogen). This was linearized with CelII and labeled antisense riboprobe made with the T7 RNA polymerase and Maxiscript in vitro transcription kit (Ambion). The adipocyte protein 2 (aP2, a kind gift from Dr. Bruce Spiegelman) cDNA was liberated with BamHI and subcloned into pGEM 3Zf(-) (Promega). The DNA was linearized with BclI and riboprobe made using SP6 RNA polymerase. RNase protection assay was done with an Ambion direct protect lysate RNase protection assay kit. Band intensities were quantitated by a PhosphorImager (Molecular Dynamics).


Fig. 1. Nucleotide and predicted amino acid sequence of hPPARgamma 2. The first three methionine codons are underlined. The translation stop codon is indicated by a dot and the stop codon upstream and in-frame with the first predicted methionine is indicated by an asterisk. The positions of the nucleotides and the predicted amino acids in the sequence are indicated on the side. The 30 amino acids at the N terminus unique to PPARgamma 2 are shown in bold type.
[View Larger Version of this Image (65K GIF file)]


Gel Mobility Shift Assays

Human hPPARgamma 2 was translated in vitro from pCMVhPPARgamma 2 using the TNT Coupled Reticulocyte Lysate System (Promega). The baculovirus/Sf21 cell system was used to express hRXRalpha (19). Gel retardations were performed as described (6). The sequence of the oligonucleotides containing PPREs from three genes are 5'-CTAGCGATATCATGACCTTTGTCCTAGGCCTC-3' (acyl-coenzyme A oxidase) (20), 5'-GATCCCCTTTGACCTATTGAACTATTACCTACATTA-3' (bifunctional enzyme) (21), and 5'-GATCCCCACTGAACCCTTGACCCCTGCCCTGCAGCA-3' (human apoA-1 "A" site) (22). The sequence of the upper strand is shown in all cases.

Co-transfection Assays

Transfections in CV-1 cells were performed as described (6, 23). The reporter plasmid pPPREA3-tk-Luc containing three copies of the PPRE identified in the acyl CoA oxidase (AOX) gene has been described (24). The beta -galactosidase expression plasmid pCH110 was used to normalize difference in transfection efficiencies. The normalized response is the luciferase activity of the extract divided by the beta -galactosidase activity of the same. Compounds were dissolved in Me2SO (vehicle). Each data point is the mean of triplicate transfections, and the error bars represent the standard error of the mean. Each experiment was repeated at least two times. A representative experiment is shown in each case.


RESULTS

A human heart cDNA library was screened with a probe corresponding to the mouse PPARgamma (7). Three overlapping clones were identified, purified, and sequenced. The nucleotide sequence is shown in Fig. 1. The longest open reading frame starting from the nucleotide at position 91 coded for a polypeptide of 505 amino acids. There was an in-frame stop codon upstream of this methionine suggesting the translation initiation occurred from this codon. The second and third methionine codons were at positions 29 and 31 in the amino acid sequence. The first and third methionine codons in hPPARgamma 2 were in a context appropriate for translation initiation, i.e. the Kozak sequence (25), and were conserved between mice and man. The second methionine codon was unique to human PPARgamma 2, and the corresponding amino acid in mice was isoleucine.

Amino acid sequence comparison indicated 97% identity overall between hPPARgamma 2 and mPPARgamma 2. The DNA binding domains were 83% conserved between hPPARgamma 2 and hPPARalpha or hPPARbeta . Further, three amino acids were present between the two cysteines in the D-box (amino acids 177-179), a characteristic feature of all PPARs known to date. Based on these observations we believe this human isoform is hPPARgamma 2.

To determine if there are multiple translation start sites for hPPARgamma 2, as in mPPARgamma 2, coupled in vitro transcription/translation reactions were performed in the presence of [35S]methionine and pCMVhPPARgamma 2 as template. Two bands were observed by PAGE (Fig. 2). The upper band (57 kDa) corresponded to translation initiation from the methionine at position 1. The lower band (53 kDa) probably corresponded to translation initiation from the methionine at position 31. We cannot rigorously discount translation initiation from the methionine at position 29. However, since this methionine was not within a good Kozak sequence and was also absent in mPPARgamma 2, we think it is unlikely. Indeed, in vitro transcription/translation of pCMXmPPARgamma 1 (10) and pCMVhPPARgamma 1 also gives rise to bands that comigrate with the lower band observed with pCMVPPARgamma 2. Hence, in analogy with mPPARgamma 1, we called the smaller polypeptide hPPARgamma 1.


Fig. 2. In vitro translation of hPPARgamma 2 cDNA indicates two translation initiation sites. Coupled transcription/translation of pCMVhPPARgamma 2, pCMVhPPARgamma 1, and pCMXmPPARgamma 1 was performed with a TNT kit (Promega) and [35S]methionine. The products were resolved on a 12% SDS-polyacrylamide gel and visualized by fluorography. The positions of standard molecular markers are shown on the left side.
[View Larger Version of this Image (38K GIF file)]


PPARs bind to PPREs as heterodimers with RXRs (24). Mobility shift assays using in vitro-translated hPPARgamma 2 (Fig. 3) and recombinant baculovirus-expressed RXRalpha were performed. PPARgamma 2 alone did not form a complex with oligonucleotides containing PPRE sequences identified in the promoters from the enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (bifunctional enzyme), acyl-CoA oxidase, and the A site of the apoA-1 gene (lanes 1-3). Similarly, no complex was observed with RXRalpha and the PPRE-containing oligonucleotides (lanes 7-9). However, with PPARgamma 2 and RXRalpha , retarded complexes are clearly observed (lanes 4-6). Similar results were obtained with hPPARgamma 1 (data not shown). We concluded that hPPARgamma 1 and hPPARgamma 2 bound to PPREs as heterodimers with RXR. Complexes were also observed between hPPARgamma 1 or hPPARgamma 2 and RXRbeta and RXRgamma (data not shown).


Fig. 3. hPPARgamma 2 binds to PPREs as a heterodimer with RXR. Gel retardation reactions were performed with 1 µl of in vitro-translated hPPARgamma 2 and 1 µg of recombinant baculovirus-expressed hRXRalpha . Oligonucleotides containing PPREs from the bifunctional enzyme (lanes 1, 4, and 7), acyl-coenzyme A oxidase (lanes 2, 5, and 8) and apoA-1 (lanes 3, 6, and 9) genes were used as probes.
[View Larger Version of this Image (64K GIF file)]


The transcriptional response of hPPARgamma 2 to PPARgamma activators was determined in a cotransfection assay (Fig. 4A) and compared with hPPARgamma 1 (Fig. 4B). PPARgamma 1 and PPARgamma 2 are activated by BRL 49653 with an EC50 of approximately 100 nM and by 15-deoxy-Delta 12,14-prostaglandin J2 (an endogenous PPARgamma ligand) (26, 27) with an EC50 around 3 µM. They are also activated by 5,8,11,14-eicosatetraenoic acid and 2-bromopalmitate. The response of hPPARgamma 2 to these four activators is very similar to that of hPPARgamma 1. We conclude that both hPPARgamma 1 and hPPARgamma 2 are similarly activated by known PPARgamma activators.


Fig. 4. hPPARgamma 1 and hPPARgamma 2 are activated by PPARgamma activators. CV-1 cells were cotransfected with the expression plasmids pCMVhPPARgamma 2 (A) or pCMVhPPARgamma 1 (B) and the pPPREA3-tk-Luc reporter (24). PPARgamma activators were added at increasing concentrations as shown. Luciferase and beta -galactosidase activity was measured as indicated under "Experimental Procedures."
[View Larger Version of this Image (37K GIF file)]


Since PPARgamma 2 binds to PPREs as a heterodimer with RXR, we next determined the transcriptional response of the PPARgamma 2/RXR heterodimer to an RXR ligand. LG100268 (28) is a highly selective RXR ligand (Kd ~3 nM). Both BRL 49653 and LG100268 transcriptionally activated the PPARgamma 2/RXR heterodimer (Fig. 5A), and the transcriptional response observed with both ligands was greater than that observed individually. RXR agonists activated a reporter containing the hydratase (bifunctional enzyme) PPRE. They also induced expression of the hydratase gene in vivo, and increased induction is seen with a combination of RXR and PPAR agonists.2


Fig. 5. hPPARgamma 2 and RXR cooperatively activate transcription. A, CV-1 cells were transfected with the expression plasmids pCMVhPPARgamma 2, pRSmRXRbeta (38), and pPPREA3-tk-Luc as reporter. BRL 49653 was added to a final concentration of 100 nM, and LG100268 (LG 268) or LG100754 (LG 754) to 1 µM, respectively. B, CV-1 cells were transfected with pRShRXRalpha (39) and pPPREA3-tk-LUC. LG100268 was added to a final concentration of 100 nM where indicated, and the concentration of LG100754 is shown. vehicle, Me2SO.
[View Larger Version of this Image (19K GIF file)]


Recently LG100754, another high affinity RXR ligand (Kd ~12 nM), has been described as an RXR/RXR homodimer antagonist on a CRBPII-tk-Luc reporter (29). To determine if the response of PPARgamma 2/RXR to BRL 49653 will be antagonized by LG100754 binding to RXR, a cotransfection assay with PPARgamma 2/RXR was performed (Fig. 5A). Surprisingly, LG100754, like LG100268, is an agonist of hPPARgamma 2/RXR, and activation by the combination of BRL 49653 and LG100754 is greater than the individual compounds. It is also an agonist of hPPARgamma 1/RXR (data not shown) and hPPARalpha /RXR (30).

Since LG100754 is a high affinity RXR ligand and also activates the hPPARgamma /RXR heterodimer, we determined whether LG100754 also activates RXR homodimers using the same reporter used for the PPAR assays (pPPREA3-tk-Luc). Since the consensus PPRE and RXR response element are of the DR-1 type (24), it would be interesting to compare the effect of RXR modulators on the two response elements. LG100268 strongly activated the RXR/RXR homodimer on PPREA3-tk-Luc (Fig. 5B). LG100754 was a very weak activator of the RXR homodimer. Interestingly, it antagonized the activation of RXR/RXR by LG100268 (Fig. 5B). Hence, LG100754 acted as a PPARgamma /RXR heterodimer agonist but as an RXR homodimer antagonist on the same response element, a PPRE. This was the first demonstration of an RXR ligand having such dimer-selective effects on the same reporter. This dimer-selective activity is probably not due to LG100754 binding with high affinity to PPARgamma since LG100754 displaces labeled BRL 49653 from PPARgamma only at very high concentrations in a DNA-dependent ligand binding assay using PPARgamma /RXR heterodimers (40) (data not shown).

We next determined the tissue distribution of human PPARgamma RNA (using a probe common to PPARgamma 1 and PPARgamma 2) and compared it with that of hPPARalpha and hPPARbeta by Northern blotting (Fig. 6A). Human PPARalpha was found predominantly in skeletal muscle, liver, heart, and kidney, a distribution similar to that reported for mPPARalpha . PPARbeta RNA was more ubiquitously expressed with maximal expression in placenta and skeletal muscle. One band approximately 2 kilobases in length was observed with the PPARgamma probe. Human PPARgamma is expressed in the insulin-responsive tissues (skeletal muscle, heart, and liver) and is consistent with the distribution in mice (31).


Fig. 6. Tissue distribution of human PPARgamma . A, Northern blots (Clontech) with 2 µg of poly(A)+ RNA from various human tissues were hybridized with hPPARalpha -, hPPARbeta -, or hPPARgamma -specific probes. A blot was stripped and hybridized to beta -actin as a control. RNase protection assays were performed with RNA from fat or skeletal muscle from different individuals. A PPARgamma probe was used in B and an aP2 probe in C. Lane 1, undigested probe; lane 2, probe digested with RNase in a mock hybridization. In the other lanes, 10 µg of RNA was used except in lane 6 (B) and lane 4 (C) where 2 µg were used. The 170-nucleotide-long protected fragment is denoted by an asterisk (see "Results"). The position of the expected protected band is denoted by an arrow (C).
[View Larger Version of this Image (68K GIF file)]


Since the probe used in the Northern blot experiments could not distinguish between PPARgamma 1 and PPARgamma 2 RNA, we developed an RNase protection assay to distinguish the two isoforms. The majority of insulin-stimulated glucose uptake occurs in skeletal muscle, therefore, we determined the expression of PPARgamma 2 versus PPARgamma 1 in muscle. Since mPPARgamma 2 expression is restricted to fat (32), and the commercial blot used in the Northern analysis did not have a sample of fat RNA, we included human fat RNA in the study (Fig. 6B). Two bands (78 and 252 nucleotides long) were observed in all adipose tissue samples arising from protection of the probe by PPARgamma 1 and PPARgamma 2 RNA, respectively, as shown. In contrast to the findings in mice (31), PPARgamma 1 was expressed at higher levels in all human fat samples studied. Quantitation of the band intensities indicated that the ratio of PPARgamma 1 to -gamma 2 varied in the human samples (from 2 (lane 5) to 10 (lane 7)).

With RNA from human skeletal muscle, we observed the protected fragment due to hPPARgamma 1 but not from PPARgamma 2 in all three samples. This was not observed with yeast RNA, which was used as a negative control.

To test whether PPARgamma 1 RNA observed in muscle was solely due to fat contaminating the muscle samples, we performed RNase protection assays with the muscle RNA and a mouse aP2 probe and compared that with a sample of fat RNA. aP2 (adipocyte protein 2) gene expression is fat-specific (33). Very little specific protection of the probe was seen with 10 µg of muscle RNA (Fig. 6C, lanes 5-7) while an intense band is seen with only 2 µg of fat RNA (lane 4). The autoradiogram was deliberately overexposed (see lane 3) to reveal any protected bands in lanes 5-7. The smear observed in lanes 5-7 was probably due to nonspecific hybridization between human RNA and the mouse probe and is also seen with yeast RNA (lane 8). We concluded that PPARgamma was expressed in human skeletal muscle and PPARgamma 1 was the predominant isoform in this tissue. In contrast, both PPARgamma 1 and PPARgamma 2 were expressed in human fat and at much higher levels compared with muscle.

Interestingly, a third protected fragment (170 nucleotides long) was also observed (denoted by an asterisk) in all four fat samples but not in the muscle samples (Fig. 6B). This could be simply due to RNase digestion in regions of imperfect hybridization. However, the ratio of the intensity of this fragment compared with PPARgamma 2 varied in the different fat samples, hence, it is unlikely that this was due to breakdown of the larger protected fragment. We therefore hypothesized a third isoform of PPARgamma in humans that may arise due to alternate splicing and promoter usage.


DISCUSSION

We have cloned the cDNA for a second isoform of the human PPARgamma , hPPARgamma 2. Sequence comparison with mPPARgamma 2 revealed 97% amino acid identity. Human PPARgamma 2 bound to PPREs as a heterodimer with RXR and was activated by the PPARgamma ligands BRL 49653 and 15-deoxy-Delta 12,14-prostaglandin J2. Based on these observations we believe that hPPARgamma 2 was a genuine member of the PPAR subfamily. The amino acid sequence shown in Fig. 1 was identical to the hPPARgamma 2 amino acid sequence predicted from the cDNA isolated from a human adipose library (16). However, our clone contained an additional 90 nucleotides of 5'-untranslated sequence including the upstream in-frame translation stop codon.

PPARgamma 2, like PPARgamma 1, bound to PPREs as a heterodimer with RXRs as do all the PPARs known to date. Human PPARgamma 1 and hPPARgamma 2 have similar activation profiles in reponse to BRL 49653 and 15-deoxy-Delta 12,14-prostaglandin J2. Interestingly, there was only 63% identity in the N-terminal 30 amino acids between human and mouse PPARgamma 2, far less than in the rest of the polypeptide (98%). This suggests that the N terminus coded by a different exon (13) has diverged more rapidly than the rest of the protein during evolution. The function of these amino acids is unclear.

The hPPARgamma 2/RXR and hPPARgamma 1/RXR heterodimers were activated by the RXR modulators LG100268 and LG100754. They increased the transcriptional response seen with the PPARgamma agonist BRL 49653. This is consistent with our previous studies showing that RXR modulators increase the responsiveness of the PPARalpha /RXR heterodimer (6, 24). LG100754 was interesting because it is an agonist of PPARgamma /RXR but an RXR/RXR antagonist. Binding of LG100754 to RXR may lead to distinct conformational changes of the receptor dimer such that PPARgamma /RXR is read as an activator by the transcriptional machinery, but the RXR/RXR homodimer is transcriptionally silent. Such compounds like LG100268, LG100754, and BRL 49653 may therefore modulate distinct but overlapping sets of target genes and might have a role in the treatment of PPAR/RXR-modulated diseases like diabetes.

The tissue distribution of hPPARgamma is important for the therapeutic activity of drugs targeting the PPARgamma /RXR heterodimer. Thiazolidinediones act as insulin sensitizers in skeletal muscle and are high affinity PPARgamma ligands (34). Structure activity relationship indicates a good correlation between in vivo potency and in vitro activity of thiazolidinediones (35), implicating PPARgamma as the therapeutic target for these compounds. However, earlier data indicated PPARgamma is expressed at high levels, specifically in adipose tissue in rodents (11, 32), and is essentially undetectable in muscle where approximately 80% of insulin-stimulated glucose uptake occurs (36). It was not clear how PPARgamma expressed almost exclusively in adipose tissue could have the effect of insulin sensitization in skeletal muscle and raised the possibility that insulin sensitization by thiazolidinediones in skeletal muscle was not mediated by PPARgamma -dependent mechanisms.

Our data demonstrated that PPARgamma was expressed in human skeletal muscle, fat, and heart, tissues where the majority of insulin-stimulated glucose uptake occurs. Further, while both PPARgamma 1 and PPARgamma 2 are expressed in human fat, the dominant isoform in human muscle is PPARgamma 1. These findings are consistent with recently published data in mice (31) and suggest that PPARgamma 1 might be the relevant target for thiazolidinediones in human skeletal muscle. The close conservation of sequence, subtype, and tissue distribution of PPARgamma between mice and humans is consistent with the observation that thiazolidinediones act as insulin sensitizers in both species (17, 37). However, we do not yet know the distribution of PPARgamma 1 versus PPARgamma 2 protein in these tissues.

The ratio of the intensities of the PPARgamma 1 and -gamma 2 isoforms varied in the four individual fat samples, hinting that isoform expression may be modulated. Further, only PPARgamma 1 was detected in muscle, not PPARgamma 2, pointing to differential expression of PPARgamma isoforms in tissues. Therefore, an analysis of PPARgamma isoform distribution in skeletal muscle and fat in normal, obese, and diabetic individuals might yield valuable information and is currently underway.

We used a commercially available Northern blot to determine PPARgamma distribution. Although we did not observe hybridization to placenta and lung RNA on this blot we note that PPARgamma expression was observed in these tissues (15). We cannot explain this other than as a variation between individuals. For an accurate determination it was important to assay expression levels in several individuals as was done in our RNase-protection assays.

Thiazolidinediones appear to act as insulin sensitizers in vivo through activation of PPARgamma /RXR. Our data indicate the presence of PPARgamma in insulin-responsive tissues in humans. One may speculate that thiazolidinediones bind to and activate PPARgamma altering the expression of key genes in target tissues rendering them more responsive to insulin, increasing glucose uptake, lowering hepatic glucose output, and lowering hyperglycemia. Since RXR modulators are also able to activate the PPARgamma /RXR heterodimer, they could activate a set of thiazolidinedione-responsive genes and may therefore either alone or in combination with thiazolidinediones have utility in the treatment of non-insulin-dependent diabetes mellitus.


FOOTNOTES

*   The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U79012[GenBank].


§   To whom correspondence should be addressed: Dept. of Cardiovascular Research, Ligand Pharmaceuticals, Inc., 9393 Towne Centre Dr., San Diego, CA 92121. Tel.: 619-535-3900; Fax: 619-535-3906; E-mail: rmukherjee{at}ligand.com.
1   The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; hPPAR, human PPAR; mPPAR, mouse PPAR; tk, thymidine kinase; RXR, retinoid X receptor; PPRE, peroxisome proliferator response element; LG100268, 6-[1-(3,5,5,8-8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)-cyclopropyl]-nicotinic acid; LG100754, (2E,4E, 6Z)-7-(3-N-propoxy-5,6,7,8-tetrahydro-5,5,8,8-tetramethylnaphthalen-2-yl)-3-methylocta-2,4,6-trienoic acid.
2   R. Mukherjee, L. Jow, G. E. Croston, and J. R. Paterniti, Jr., manuscript in preparation.

Acknowledgments

We thank Rich Heyman and Patricia Hoener for critical comments on the manuscript, Lawrence Hamann for synthesizing BRL 49653, Dr. Jerrold Olefsky for providing a human skeletal muscle biopsy sample, Regis Saladin for isolating human fat RNA, Johan Auwerx for useful suggestions, Kay Klausing for the DNA-dependent ligand displacement assay, and Sharon Dana, Thuan Le, Hung Lam, and members of the Departments of Cardiovascular Research, Retinoid Research, and New Leads Discovery for help.


REFERENCES

  1. Issemann, I., and Green, S. (1990) Nature 347, 645-650 [CrossRef][Medline] [Order article via Infotrieve]
  2. Dreyer, C., Krey, G., Keller, H., Givel, F., Helftenbein, G., and Wahli, W. (1992) Cell 68, 879-887 [Medline] [Order article via Infotrieve]
  3. Gottlicher, M., Widmar, E., Li, Q., and Gustafsson, J. A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 4653-4657 [Abstract]
  4. Sher, T., Yi, H. F., McBride, W. O., and Gonzales, F. J. (1993) Biochemistry 32, 5598-5604 [Medline] [Order article via Infotrieve]
  5. Schmidt, A., Endo, N., Rutledge, S. J., Vogel, R., Shinar, D., and Rodan, G. A. (1992) Mol. Endocrinol. 6, 1634-1641 [Abstract]
  6. Mukherjee, R., Jow, L., Noonan, D., and McDonnell, D. P. (1994) J. Steroid Biochem. Mol. Biol. 51, 157-166 [CrossRef][Medline] [Order article via Infotrieve]
  7. Chen, F., Law, S. W., and O'Malley, B. W. (1993) Biochem. Biophys. Res. Commun. 196, 671-677 [CrossRef][Medline] [Order article via Infotrieve]
  8. Zhu, Y., Alvares, K., Huang, Q., Rao, M. S., and Reddy, J. K. (1993) J. Biol. Chem. 268, 26817-26820 [Abstract/Free Full Text]
  9. Aperlo, C., Pognonec, P., Saladin, R., Auwerx, J., and Boulukos, K. E. (1995) Gene (Amst.) 162, 297-302 [CrossRef][Medline] [Order article via Infotrieve]
  10. Kliewer, S. A., Forman, B. M., Blumberg, B., Ong, E. S., Borgmeyer, U., Mangelsdorf, D. J., Umesono, K., and Evans, R. M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7355-7359 [Abstract]
  11. Tontonoz, P., Hu, E., and Spiegelman, B. M. (1994) Cell 79, 1147-1156 [Medline] [Order article via Infotrieve]
  12. Tontonoz, P., Hu, E., Graves, R. A., Budavari, A. I., and Spiegelman, B. M. (1994) Genes Dev. 8, 1224-1234 [Abstract]
  13. Zhu, Y., Qi, C., Korenberg, J. R., Chen, X. N., Noya, D., Rao, M. S., and Reddy, J. K. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7921-7925 [Abstract]
  14. Greene, M. E., Blumberg, B., McBride, O. W., Yi, H. F., Kronquist, K., Kwan, K., Hsieh, L., Greene, G., and Nimer, S. D. (1995) Gene Expr. 4, 281-299 [Medline] [Order article via Infotrieve]
  15. Lambe, K. G., and Tugwood, J. D. (1996) Eur. J. Biochem. 239, 1-7 [Abstract]
  16. Elbrecht, A., Chen, Y., Cullinan, C. A., Hayes, N., Leibowitz, M. D., Moller, D. E., and Berger, J. (1996) Biochem. Biophys. Res. Commun. 224, 431-437 [CrossRef][Medline] [Order article via Infotrieve]
  17. Suter, S. I., Nolan, J. J., Wallace, P., Gumbiner, B., and Olefsky, J. M. (1992) Diabetes Care 15, 193-203 [Abstract]
  18. Jow, L., and Mukherjee, R. (1995) J. Biol. Chem. 270, 3836-3840 [Abstract/Free Full Text]
  19. Allegretto, E. A., McClurg, M. R., Lazarchik, S. B., Clemm, D. L., Kerner, S. A., Elgort, M. G., Boehm, M. F., White, S., Pike, J. W., and Heyman, R. A. (1993) J. Biol. Chem. 268, 26625-26633 [Abstract/Free Full Text]
  20. Osumi, T., Wen, J., and Hashimoto, T. (1991) Biochem. Biophys. Res. Commun. 175, 866-871 [Medline] [Order article via Infotrieve]
  21. Zhang, B., Marcus, L. L., Sajjadi, R. G., Alvares, K., Reddy, J. K., Subramani, S., Rachubinski, R. A., and Capone, J. P. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 7541-7545 [Abstract]
  22. Widom, R. L., Ladias, J. A. A., Kouidou, S., and Karathanasis, S. K. (1991) Mol. Cell. Biol. 11, 677-687 [Medline] [Order article via Infotrieve]
  23. Berger, T. S., Parandosh, Z., Perry, B., and Stein, R. B. (1992) J. Steroid Biochem. Mol. Biol. 41, 733-738 [CrossRef][Medline] [Order article via Infotrieve]
  24. Kliewer, S. A., Umesono, K., Noonan, D. J., Heyman, R. A., and Evans, R. M. (1992) Nature 358, 771-774 [CrossRef][Medline] [Order article via Infotrieve]
  25. Kozak, M. (1986) Cell 44, 283-292 [Medline] [Order article via Infotrieve]
  26. Kliewer, S. A., Lenhard, J. M., Wilson, T. M., Patel, I., Morris, D. C., and Lehmann, J. M. (1995) Cell 83, 813-819 [Medline] [Order article via Infotrieve]
  27. Forman, B. M., Tontonoz, P., Chen, J., Brun, R. P., Spiegelman, B. M., and Evans, R. E. (1995) Cell 83, 803-812 [Medline] [Order article via Infotrieve]
  28. Boehm, M. F., Zhang, L., Zhi, L., McClurg, M. R., Berger, E., Wagoner, M., Mais, D. E., Suto, C. M., Davies, P. J. A., Heyman, R. A., and Nadzan, A. M. (1995) J. Med. Chem. 38, 3146-3155 [Medline] [Order article via Infotrieve]
  29. Koch, S. C., Dardashti, L. J., Hebert, J. J., White, S. K., Croston, G. E., Flatten, K. S., Heyman, R. A., and Nadzan, A. M. (1996) J. Med. Chem. 39, 3229-3234 [CrossRef][Medline] [Order article via Infotrieve]
  30. Lala, D. S., Mukherjee, R., Schulman, I. G., Koch, S. C., Dardashti, L., Nadzan, A., Croston, G. E., Evans, R. M., and Heyman, R. A. (1996) Nature 383, 450-453 [CrossRef][Medline] [Order article via Infotrieve]
  31. Vidal-Puig, A., Jimenez-Linan, M., Lowell, B. B., Hamann, A., Hu, E., Spiegelman, B., Flier, J. S., and Moller, D. E. (1996) J. Clin. Invest. 97, 2553-2561 [Abstract/Free Full Text]
  32. Tontonoz, P., Graves, R. A., Budavari, A. I., Erdjument-Bromage, H., Lui, M., Hu, E., Tempst, P., and Spiegelman, B. M. (1994) Nucleic Acids Res. 22, 5628-5434 [Abstract]
  33. Graves, R. A., Tontonoz, P., and Spiegelman, B. M. (1992) Mol. Cell. Biol. 12, 1202-1208 [Abstract]
  34. Lehmann, J. M., Moore, L. B., Smith-Oliver, T. A., Wilkison, W. O., Willson, T. M., and Kliewer, S. A. (1995) J. Biol. Chem. 270, 12953-12956 [Abstract/Free Full Text]
  35. Willson, 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., and Lehmann, J. M. (1996) J. Med. Chem. 39, 665-668 [CrossRef][Medline] [Order article via Infotrieve]
  36. Olefsky, J. (1995) Curr. Opin. Endocrinol. Diabetes 2, 290-299
  37. Eldershaw, T. P. D., Rattigan, S., Cawthorne, M. A., Buckingham, R. E., Colquhoun, E. Q., and Clark, M. G. (1995) Horm. Metab. Res. 27, 169-172 [Medline] [Order article via Infotrieve]
  38. Mangelsdorf, D. J., Borgmeyer, U., Heyman, R. A., Zhou, J. Y., Ong, E. S., Oro, A. E., Kakizuka, A., and Evans, R. E. (1992) Genes Dev. 6, 329-344 [Abstract]
  39. Mangelsdorf, D. J., Ong, E. S., Dyck, J. A., and Evans, R. M. (1990) Nature 345, 224-229 [CrossRef][Medline] [Order article via Infotrieve]
  40. Kurokawa, R., DiRenzo, J., Boehm, M., Sugarman, J., Gloss, B., Rosenfeld, M. G., Heyman, R. A., and Glass, C. K. (1994) Nature 371, 528-531 [CrossRef][Medline] [Order article via Infotrieve]

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.