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J Biol Chem, Vol. 273, Issue 49, 32679-32684, December 4, 1998


A Potent Antidiabetic Thiazolidinedione with Unique Peroxisome Proliferator-activated Receptor gamma -activating Properties*

Mauricio J. ReginatoDagger §, Shannon T. BaileyDagger , Samuel L. KrakowDagger , Chieko Minami, Shinichi Ishii, Hideho Tanaka, and Mitchell A. LazarDagger parallel

From the Dagger  Division of Endocrinology, Diabetes, and Metabolism, Departments of Medicine and Genetics and Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 and  Yokohama Research Center, Mitsubishi Chemical Corporation, Yokohama 227, Japan

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Thiazolidinediones (TZDs) constitute an exciting new class of antidiabetic compounds, which function as activating ligands for peroxisome proliferator-activated receptor gamma  (PPARgamma ). Until now, there has been an excellent correlation between in vivo hypoglycemic potency and in vitro binding and activation of PPARgamma by TZDs. We have characterized MCC-555, a novel thiazolidinedione ligand for PPARgamma with unique functional properties. The antidiabetic potency of this compound is greater than that of other TZDs, including BRL49653, yet its binding affinity for PPARgamma is less than <FR><NU>1</NU><DE>10</DE></FR> that of BRL49653. The effect of MCC-555 binding on PPARgamma transcriptional activity is highly context-specific such that it can function as a full agonist, partial agonist, or antagonist depending on the cell type or DNA binding site. These transcriptional properties are partly explained by unique partial agonism of coactivator recruitment to PPARgamma . The properties of MCC-555 are mechanistically distinct from those of the estrogen receptor partial agonist and antagonist tamoxifen because the N terminus of PPARgamma is not required for activation by MCC-555, and MCC-555 does not stimulate corepressor recruitment to PPARgamma . The context selectivity of MCC-555 may contribute to its enhanced hypoglycemic potency in vivo despite reduced affinity for PPARgamma relative to other TZDs.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Nuclear hormone receptors (NHRs)1 constitute a class of transcription factors with activity that is regulated by natural or synthetic lipophilic ligands (1). A number of NHRs are involved in developmental and/or metabolic processes, and modulation of NHR activity is an effective strategy in the treatment of a variety of cancers, such as breast cancer (2), prostate cancer (3), and acute promyleocytic leukemia (4), as well as metabolic diseases including thyroid disease (5) and diabetes. Non-insulin-dependent diabetes mellitus is a major cause of morbidity and mortality in industrialized nations and is characterized by a post-insulin receptor defect that has been difficult to target therapeutically until the recent discovery that thiazolidinediones (TZDs) enhance the actions of insulin at a level distal to the insulin receptor (6).

The mechanism of TZD action is not completely understood, but a number of lines of evidence point to their function as ligands for a member of the NHR superfamily called peroxisome proliferator-activated receptor gamma  (PPARgamma ), the natural ligand of which may be derived from or related to prostaglandin J2 (7-9). One of the most potent TZDs, BRL49653, binds to PPARgamma with an affinity in the nanomolar range (10), and the rank order of TZD potency for in vivo plasma glucose lowering correlates well with their rank order potency for PPARgamma activation (11, 12). Nevertheless, a number of questions remain with regard to the mechanism of TZD potentiation of insulin action. The main problem is that PPARgamma is primarily expressed in adipose tissue (13, 14), whereas muscle is ordinarily the main site of insulin-dependent glucose disposal in mammals. This apparent paradox has not yet been satisfactorily resolved.

We have studied a novel TZD called MCC-555 that is more potent than BRL49653 in vivo yet has lower affinity for PPARgamma . Moreover, MCC-555 binding activates transcription by the PPARgamma ligand-binding domain (LBD) differently in different contexts, in part because of novel effects on coactivator recruitment. This raises the possibility that only a subset of the functions of activated PPARgamma contributes to insulin sensitivity, and that therapeutic strategies for non-insulin-dependent diabetes mellitus that target these functions may lead to compounds with increased potency and decreased toxicity.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Hypoglycemic Potencies of TZDs-- Male KK-Ay mice obtained from CLEA Japan (Tokyo, Japan) were housed in individual cages in an animal room and given laboratory chow (MF, Oriental Yeast Co. Ltd.) and sterile water ad libitum. They were used for the experiment at the age of 11 weeks. MCC-555, pioglitazone, and BRL49653 were synthesized in crystalline forms, and purity was confirmed using standard criteria. Each test drug was suspended in 0.5% sodium carboxymethyl cellulose solution and administered orally for 4 days at a dose volume of 10 ml/kg of body weight. The control mice were given the sodium carboxymethyl cellulose solution in the same manner. Blood was collected in heparinized hematocrit capillary tubes under fed state on the day after the last administration. The capillary tubes were centrifuged, and plasma samples were separated. Plasma glucose levels were determined by a glucose oxidase method with a commercial kit (Glu Neo Shino-Test, Shino-Test Co.). The significance of differences between the control and each treatment were assessed by analysis of variance followed by Dunnett's two-tailed test. The dose that produces a 25% decrease in plasma glucose level from the control (ED25) was calculated by linear regression from the concentration-related efficacy curve. The relative hypoglycemic activities were estimated by the parallel line assay.

Northern Analysis-- 8-week old, male KK-Ay mice were used. For RNA determinations, MCC-555 and BRL49653 (30 mg/kg, suspended in 0.5% sodium carboxymethyl cellulose solution) were administered orally daily for 4 days. On day 5, mice were killed, and epididymal fat pads were removed and quickly frozen in liquid nitrogen. mRNA was prepared as described previously by homogenizing epididymal fat from KK-Ay mice with a Polytron homogenizer in 7 ml of 4 M guanidine thiocyanate, 20 mM sodium citrate (pH 7.0), 17 mM N-lauroyl sarcosine, and 100 mM beta -mercaptoethenol. After centrifugation (10,000 rpm for 10 min at 12 °C) the supernatant was sheared through a 20-gauge syringe three times. Total RNA was isolated from homogenates, and Northern blots were performed as described previously (15). The cDNA probes for aP2, PPARgamma , and leptin were labeled with 32P by using random hexamers.

Plasmids-- The expression vector tet-PPARgamma (LBD) and tet-luciferase reporter have been previously described (7). Full length mPPARgamma 2 in pCMX and the reporter construct acyl-coenzyme A X3-TK-luciferase are as described (16). PPARgamma Delta N (lacking amino acids 1-118) and GAL4-PPARgamma (LBD) expression plasmids were constructed using PCR. The (Gal4 UAS x 5)-SV40-luciferase reporter contained five copies of the Gal4 17-mer binding site and has been previously described (17). The LBD of mPPARgamma was also used in pGEX-5x-1 for expression of glutathione S-transferase (GST) fusion proteins. Steroid receptor coactivator-1 (SRC-1; kindly provided by B. O'Malley) was cloned into the Pst1 site of pCMX. All constructs were directly sequenced.

Coactivator Recruitment Assay-- The coactivator-dependent receptor ligand assay was performed as initially described by Krey et al. (18) with modifications described below. Briefly, GST fusion proteins were purified with glutathione-Sepharose beads (Amersham Pharmacia Biotech). GST fusion proteins bound to beads (100 µl of a 50% slurry) in GST binding buffer (50 mM KCl, 20 mM HEPES, pH 7.9, 2 mM EDTA, 0.1% Nonidet P-40, 10% glycerol, 0.5% nonfat dry milk, 5 mM dithiothreitol) was mixed with 5 µl of radiolabeled in vitro-translated SRC-1 and indicated ligand concentration. Ligand or vehicle control made up <2% of the total volume in all experiments. Fusion proteins, SRC-1, and ligand were incubated at 4 °C for 1 h with gentle rocking. The beads were then vigorously washed with 1 ml of GST binding buffer. Bound proteins were eluted by boiling in 20 µl of 2 × SDS loading buffer and analyzed by SDS-polyacrylamide gel electrophoresis. The GST fusion proteins were stained with Coomassie Blue for every experiment to ensure equal loading, and bound proteins were visualized by autoradiography and quantitated using a phosphorimager. Corepressor interaction experiments were performed under identical conditions using GST and GST-silencing mediator of retinoid and thyroid receptor (SMRT) receptor interaction domain (amino acids 982-1485) as described previously (33), except that 10 nM thyroid hormone or 50 µM BRL49653 and MCC-555 were added as indicated.

Binding Assay-- Ligand binding assay was performed essentially as described (8). Briefly, GST-PPARgamma fusion protein isolated from Escherichia coli DH5alpha cells was pelleted and lysed by sonication in Tris-EDTA-NaCl buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 100 mM NaCl). For competition binding assays, bacterial extracts (~300 µg of protein) were incubated at 4 °C for 2 h with 200 nM [3H]BRL49653 (specific activity, 18 Ci/mmol) in the absence or presence of unlabeled TZD in buffer containing 10 mM Tris (pH 8.0), 50 mM KCl, and 10 mM dithiothreitol. Bound was separated from free radioactivity by elution through 1-ml Sephadex G-25 desalting columns (Amersham Pharmacia Biotech). Bound radioactivity eluted from the column void volume was quantitated using a liquid scintillation counter.

Transient Transfection Transcription Assay-- 293T cells and JEG-3 cells were maintained in high glucose Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and then switched to high glucose Dulbecco's modified Eagle's medium with 10% charcoal-stripped fetal calf serum just before transfection. All transfections were normalized for total DNA and promoter concentration. All transfection experiments except the PPARgamma Delta N experiment (see Fig. 7A) were performed in 60-mm-diameter dishes using the calcium phosphate precipitation method with 1 µg of receptor expression vectors, 1 µg of luciferase reporter, and 0.5 µg of beta -galactosidase (beta -gal) expression vector. The calcium phosphate transfection experiments shown in Fig. 7A were performed using 24-well plates, with 20 ng of receptor expression vector, 50 ng of luciferase reporter, and 50 ng of beta -gal vector. BRL49653, MCC-555, or vehicle control (dimethyl sulfoxide) were added 16 h after transfections at concentrations described in legends. The following day, cells transfected were lysed in Triton X-100, and cell lysates were subjected to luciferase and beta -gal assays. Results were normalized to beta -gal activity, and fold activation was calculated.

3T3-L1 Adipocyte Differentiation-- 3T3-L1 cells were cultured and analyzed as described previously (19).

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

MCC-555 Is a Potent Antidiabetic Thiazolidinedione-- The structure of MCC-555 is shown and compared with that of BRL49653 and other TZDs in Fig. 1A. Fig. 1B shows that MCC-555 was more effective than BRL49653 or other TZDs in lowering plasma glucose in the mouse KK-Ay model of obesity and non-insulin-dependent diabetes mellitus. As indicated in the Fig. 1, the ED25 for MCC-555 was nearly 3-fold lower than that of BRL49653, the most potent of other TZDs tested (20).


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  		Fig. 1.   Increased antihyperglycemic potency of MCC-555 relative to BRL49653 and pioglitazone. A, chemical structure of MCC-555 compared with other TZDs. B, antihyperglycemic effects of MCC-555. KK-Ay mice were orally administered the indicated TZDs for 4 days. Shown are the mean ± S.E. of plasma glucose. **, p = 0.01 compared with placebo control (n = 4). ED25 values were calculated as described under "Materials and Methods."

MCC-555 Is an Activating Ligand for PPARgamma -- Antidiabetic TZDs have been shown to function as activating ligands for PPARgamma , and the abilities of TZDs to activate PPARgamma and to reduce plasma glucose are well correlated (11, 12). Using the well characterized PPAR binding site from the acyl-coenzyme A oxidase gene upstream of a luciferase reporter, MCC-555 activated PPARgamma -dependent transcription in a transient transfection assay, with maximal activation approximately equal to that of BRL49653 (Fig. 2). However, despite its greater antihyperglycemic potency, MCC-555 was less potent than BRL49653 in this activity, with an EC50 nearly 10-fold greater (Fig. 2). Using a direct binding displacement assay, we confirmed that MCC-555 binds directly to PPARgamma (Fig. 3). However, the ability of MCC-555 to bind to PPARgamma was more than a log order less than that of BRL49653; the EC50 of MCC-555 was 8 µM, whereas that of BRL49653 was 200 nM, similar to that reported by others (9, 10). This agreed well with the order of potency in PPARgamma activation assays but was reversed relative to the in vivo antidiabetic potencies of the TZDs.


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  		Fig. 2.   MCC-555 activates PPARgamma but with lower potency than BRL49653. A, MCC-555 is equally effective but less potent than BRL49653. 293T cells were transfected with 1 µg of reporter plasmid (acyl-coenzyme A PPRE x 3)-TK-luciferase, 0.5 µg of cytomegalovirus-beta -gal, and 1 µg of pCMX-PPARgamma . Normalized luciferase activity was plotted as fold activation relative to untreated cells.


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  		Fig. 3.   MCC-555 binds PPARgamma but with 50-fold lower affinity than BRL49653. Competition binding assays were performed using bacterial extracts containing GST-PPARgamma LBD and 200 nM [3H]BRL49653 in the presence of increasing concentrations of cold BRL49653 or MCC-555. Data are presented as mean of triplicate points and were normalized to reactions performed with vehicle alone (0.5% dimethyl sulfoxide).

MCC-555 Is a Partial Agonist and/or Antagonist of Chimeric Forms of PPARgamma -- One of the cardinal features of members of the NHR superfamily is the ability of LBD to function independently when fused to a heterologous polypeptide (21). This feature has been exploited for many NHRs, including PPARgamma . Indeed, the binding of ligand to PPARgamma observed by others and in the present work involves the fusion of the LBD to glutathione S-transferase (8-10). Moreover, much of the work on ligand-dependent activation of PPARgamma is derived from studies fusing the PPARgamma LBD to a heterologous DNA-binding domain (DBD), most frequently that of Gal4. We therefore decided to compare the properties of MCC-555 and BRL49653 in the context of the Gal4-PPARgamma fusion protein. Fig. 4A shows that both MCC-555 and BRL49653 activated transcription by Gal4-PPARgamma , and the EC50 value for the compounds again were consistent with the reduced binding affinity of MCC-555. However, unlike the activation of wild-type PPARgamma by MCC-555, the maximal activation achieved by MCC-555 was consistently ~2 fold less than that caused by saturating concentrations of BRL49653. Thus, in this context, MCC-555 functioned as a partial agonist of PPARgamma activation. This predicted that at high concentration MCC-555 would displace BRL49653 and function as an inhibitor of BRL49653-stimulated activation of Gal4-PPARgamma . Indeed, Fig. 4B shows that MCC-555 antagonized BRL49653-dependent activation when present at a very high relative concentration. This effect was not attributable to destabilization of the Gal4-PPARgamma chimera, because Western analysis with Gal4 antibody confirmed equal concentrations of the protein in the presence and absence of MCC-555 (data not shown).


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  		Fig. 4.   MCC-555 is a partial agonist of Gal4-PPARgamma . A, dose response of activation of Gal4-PPARgamma by MCC-555 and BRL-49653. 293T cells were transfected with 1 µg of reporter plasmid (Gal4 binding site x 5)-SV40-luciferase and 1 µg of Gal4-PPARgamma LBD and treated with indicated TZD. B, MCC-555 acts as partial agonist of Gal4-PPARgamma . Effects of increasing doses of MCC-555 on Gal4-PPARgamma activation by BRL49653 are shown.

The behavior of MCC-555 as a partial agonist of Gal4-PPARgamma contrasted with its full activation of wild-type PPARgamma . To determine whether this was in some way an artifact of the chimeric receptor, we fused the PPARgamma LBD to an alternate DBD from the tetracycline operon. This chimeric receptor was previously used to analyze prostaglandin activators of PPARgamma (7). Fig. 5A shows that tet-PPARgamma -dependent transcription was markedly stimulated by BRL49653. In contrast, however, MCC-555 was not at all effective until near millimolar concentrations were provided (Fig. 5B). Moreover, Fig. 5C shows that in the presence of activating concentrations of BRL49653, MCC-555 now functioned essentially as a pure antagonist. This effect of MCC-555 was attributable to competitive binding to PPARgamma rather than interference with expression of the chimeric PPARgamma , because the tet-PPARgamma remained able to interact with retinoid X receptor as demonstrated by mammalian two-hybrid assay (Fig. 5D).


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  		Fig. 5.   MCC-555 acts as a pure antagonist of tet-PPARgamma . A, 15 µM MCC-555 does not activate tet-PPARgamma LBD. B, dose response of activation of tet-PPARgamma LBD by MCC-555 and BRL49653. C, effects of increasing doses of MCC-555 on tet-PPARgamma activation by BRL49653. D, MCC-555 does not prevent expression of tet-PPARgamma . A mammalian two-hybrid experiment was performed with tet-luciferase reporter, using cotransfected retinoid X receptor-VP16 as described previously (45). 293T cells were transfected with 1 µg of reporter plasmid tet-TK-luciferase and pSG5-tet-PPARgamma LBD and then treated with indicated TZD or appropriate vehicle control (dimethyl sulfoxide).

MCC-555 Functions as a Partial Agonist in Recruitment of Coactivators to PPARgamma -- The paradigm of ligand-dependent activation by NHRs suggests that one or more coactivators are recruited to the LBD in the presence of ligand (22-24). Fig. 6A shows that a saturating concentration of BRL49653 indeed caused recruitment of two coactivators, cAMP response element-binding protein (CBP) (25) and SRC-1 (26), to the PPARgamma LBD. The results with a saturating concentration of MCC-555 are shown for comparison. Note, however, that the magnitude of coactivator binding, especially SRC-1, in Fig. 6A is less in the presence of saturating concentration of MCC-555 than of BRL49653. This is seen better in the dose-response study for ligand-induced recruitment of SRC-1 to GST-PPARgamma shown in Fig. 6B. As predicted from the reduced affinity of MCC-555 for the PPARgamma , the EC50 for maximal SRC-1 binding was approximately one log greater for MCC-555 than for BRL49653. However, even at saturating concentrations of MCC-555, the maximum amount of SRC-1 recruited to the same amount of GST-PPARgamma is remarkably less than that recruited by saturating concentrations of BRL49653. Phosphorimager quantitation of three similar experiments indicates that maximal binding of SRC-1 to GST-PPARgamma was ~5-fold greater in the presence of BRL49653 than MCC-555 (Fig. 6C). We propose this to be the novel mechanism whereby MCC-555 functions as a variable partial agonist and antagonist of PPARgamma LBD function.


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  		Fig. 6.   MCC-555 recruits coactivator SRC-1 with 10-50-fold less potency than BRL49653. Coactivator-dependent receptor ligand assay was performed using purified GST-PPARgamma LBD incubated with 5 µl of radiolabeled in vitro-translated SRC-1 or CBP in the presence of the indicated ligand at 4 °C for 1 h. After vigorous washing bound proteins were eluted and analyzed by SDS-polyacrylamide gel electrophoresis. A, both MCC-555 and BRL49653 recruit CBP and SRC-1 to PPARgamma . B, TZD dose response of SRC-1 recruitment to PPARgamma . C, phosphorimager quantitation of the mean of the results of the experiment shown in B plus two additional repetitions of the same experiment.

The ability of MCC-555 binding to PPARgamma to recruit both CBP and SRC-1 contrasts with the mechanism of action of a widely studied estrogen receptor (ER) partial agonist, tamoxifen. Tamoxifen is unable to induce the conformational change required for the ER LBD to bind coactivator (23, 27). Indeed, tamoxifen binds competitively with estrogen to the ER LBD but fails to activate through the activation function 2 (AF2) activation domains. Transcriptional activation by tamoxifen requires the N-terminal A/B domain of the ER that is not present in Gal4 fusions (28). In contrast, MCC-555 activation of full length PPARgamma on PPREs does not require the N-terminal A/B domain of PPARgamma . Fig. 7A shows that although the ED50 of MCC-555 was higher than for BRL49653, consistent with its reduced affinity for PPARgamma , saturating concentrations of the two ligands were equally effective in activating PPARgamma Delta N (lacking amino acids 1-118).


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  		Fig. 7.   The A/B domain of PPARgamma is not required for activation by MCC-555, and MCC-555 does not enhance corepressor interaction with PPARgamma . A, effect of PPARgamma A/B domain on MCC-555 activity. 293T cells were transfected with 1 µg of reporter plasmid (acyl-coenzyme A PPRE x 3)-TK-luciferase, 0.5 µg of cytomegalovirus-beta -gal, and 1 µg of pCMX-PPARgamma Delta N, lacking amino acids 1-118 of PPARgamma 2. Shown is the mean ± S.E. (n = 4) fold activation by MCC-555 and BRL49653 at the indicated concentrations of ligand. B, effect of MCC-555 on SMRT binding to PPARgamma . Reticulocyte lysate-translated PPARgamma 2 and thyroid hormone receptor were incubated with GST alone or GST-SMRT (982-1485) (33), and bound protein was analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. Input lane shows 50% of input protein. C, phosphorimager quantification of experiment shown in B.

Another property that contributes to the ability of tamoxifen to function as an antagonist for the ER is stabilization of corepressor binding to the ER LBD (29-32). We have previously shown that PPARgamma interacts in solution with nuclear receptor corepressor and SMRT (33), and SMRT has been suggested to be involved in PPARgamma function (30). Fig. 7, B and C, shows that PPARgamma interacted with the receptor interaction domain of SMRT to a considerably lesser extent than thyroid hormone receptor and that, unlike triiodothyronine binding to thyroid hormone receptor, BRL49653 binding did not lead to significant displacement of SMRT from PPARgamma . Moreover, MCC-555 did not enhance the binding of SMRT to PPARgamma . Similar results were obtained with nuclear receptor co-repressor (N-CoR) (data not shown). Thus there are three major differences between the properties of MCC-555 and tamoxifen on PPARgamma and ER, respectively: (i) MCC-555 recruits coactivators to PPARgamma , albeit to a lesser degree than BRL49653; (ii) transcriptional activation by MCC-555 does not require the A/B domain of PPARgamma ; and (iii) MCC-555 does not enhance PPARgamma binding to corepressors.

Potency of MCC-555 in Adipocyte Differentiation-- We considered the possibility that reporter assays might not reflect the true potency of MCC-555. Therefore we assessed the ability of MCC-555 to induce adipocyte differentiation of 3T3-L1 cells, a well established property of TZDs (34) that is mediated by PPARgamma (19, 35). MCC-555 induced adipocyte differentiation as measured by induction of the adipocyte-specific, PPARgamma -responsive aP2 gene (Fig. 8) as well as by morphologic criteria (data not shown). The maximal induction of aP2 was similar for MCC-555 and BRL49653. However, the EC50 for MCC-555 was ~10-fold greater for than for BRL49653, again consistent with the relative abilities of these compounds to bind and activate PPARgamma but inconsistent with their abilities to reduce blood glucose levels in vivo.


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  		Fig. 8.   MCC-555 induces adipogenesis but with lower potency than BRL49653. 3T3-L1 cells were exposed to different concentrations of TZDs as indicated. Northern analysis of 10 µg of total RNA probed with aP2 cDNA is shown. Equal RNA loading was confirmed by rRNA staining (not shown).

Effects of MCC-555 on PPARgamma -regulated Gene Expression In Vivo-- Although MCC-555 was less potent in adipocyte differentiation in vitro, we considered that its increased in vivo potency might still involve PPARgamma but relate to a favorable pharmacokinetic profile relative to that of the multiple other TZDs for which in vitro and in vivo effects are better correlated. We therefore examined epididymal adipose tissue of KK-Ay mice for the expression of PPARgamma -responsive genes. We chose aP2 as a gene that is up-regulated by PPARgamma ligands (34, 35) and leptin as a gene that is down-regulated by PPARgamma ligands (36-39). We also studied the expression of PPARgamma itself, which is only minimally responsive to TZDs in adipocytes where it is already expressed at high levels (36). Fig. 9 shows that aP2 was induced from its already high levels by treatment with BRL49653 as well as by MCC-555. Interestingly, the effect of BRL49653 was greater after 1 day of treatment, whereas after 4 days aP2 expression was greatest in mice treated with MCC-555. Similar conclusions regarding the in vivo potency of the TZDs pertained to the down-regulation of leptin gene expression, although this was more modest. PPARgamma expression was not detectably different in adipose from TZD-treated mice in this experiment. These results suggest that MCC-555 is at least as potent as BRL49653 at regulating PPARgamma -responsive genes in vivo.


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  		Fig. 9.   Regulation of PPAR-responsive genes from adipose tissue of TZD treated KK-Ay mice. Northern analysis of epididymal adipose tissue of KK-Ay mice treated with BRL49653 or MCC-555 (30 mg/kg) for either 1 or 4 days as indicated. 3T3-L1 day 0 (D0, preadipocyte) and day 8 (D8, adipocyte) RNA was used as control. 10 µg of total RNA was probed with cDNAs for aP2, leptin, and PPARgamma . 28 S rRNA ethidium bromide staining is also shown as evidence of equal loading.


    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The treatment of non-insulin-dependent diabetes mellitus has recently been improved by the identification of TZDs as novel agents with the unique ability to potentiate insulin action at a level of signaling that is distal to the insulin receptor. The discovery that TZDs are nanomolar PPARgamma ligands and that their abilities to activate PPARgamma correlate with their enhancement of insulin action suggests that these two actions of TZDs are related. Our results are very important in this context. There are a number of current theories of TZD action. PPARgamma is mainly expressed in adipose tissue, and thus one possibility is that PPARgamma activation stimulates insulin-mediated glucose uptake to such a large extent in adipose tissue that blood sugar is lowered despite the fact that muscle is ordinarily the main site of insulin-dependent glucose disposal in mammals. Another possibility is that TZD activation of PPARgamma induces adipocytes to send an insulin-enhancing signal to muscle; possibilities include reduced levels of adipocyte-derived tumor necrosis factor alpha  (40) or free fatty acids (41). It is also possible that the very low amounts of PPARgamma in muscle are sufficient for TZDs to directly affect insulin action in that tissue (42), or that TZDs have other direct effects on muscle besides activation of PPARgamma .

The ability of MCC-555 to function as a potent antidiabetic agent and to possess novel PPARgamma -activating properties has a number of potential implications. Certainly, if PPARgamma activation is sufficient for the antidiabetic effects of TZDs, the potency of MCC-555 indicates that partial or context-dependent agonism of PPARgamma is sufficient for these antidiabetic effects. Moreover, the antidiabetic potency of MCC-555 is increased relative to BRL49653, which has higher affinity for PPARgamma . The effects on adipose gene expression in vivo are likely to be related to more favorable pharmacokinetics of MCC-555, leading to stimulation of PPARgamma -responsive genes in adipose tissue. However, MCC-555 is also associated with reduced cardiac and hematopoietic side effects relative to other TZDs (20). Thus, it is possible either that 1) the differential ability of MCC-555 to activate the PPARgamma LBD depending on the context of the DBD to which it is fused is somehow favorable in terms of antidiabetic effects or 2) a subset of the full range of agonistic effects of other TZDs, such as BRL49653, is somehow detrimental to the therapeutic effects of these full agonists. Of course, it is also possible that MCC-555 has other targets in addition to PPARgamma . There may thus be additional or alternative targets of MCC-555 in fat or muscle that contribute to its unique pharmacological and therapeutic profile in diabetes.

The unique properties of MCC-555 are not completely understood. Unlike the ER partial agonist tamoxifen, the A/B domain of PPARgamma was not essential for full activation from a PPRE-containing reporter gene. While this work was under review, Blanco et al. (43) reported that p300/CBP-associated factor functions as a nuclear receptor coactivator by binding to the receptor DBD (43). This could explain the ability of MCC-555 to fully activate PPARgamma transcription from the PPRE, because that process requires the PPARgamma DBD, which is absent from the Gal4 and tet fusion proteins. By contrast, the ability of MCC-555 to function as a full agonist with full-length PPARgamma suggests that SRC-1, the recruitment of which by MCC-555 is submaximal, is not rate-limiting in this situation. This is consistent with the recent description of promoter-specific requirements for p300/CBP-associated factor, CBP, SRC-1, and other coactivators (44). We also cannot rule out the possibility of a specific coactivator that plays a critical role in insulin action and is differentially recruited by MCC-555.

    ACKNOWLEDGEMENTS

We thank Jinsong Zhang for the Gal4-PPARgamma construct and Shamina Rangwala and Myles Brown for helpful discussions. We also thank Hiroaki Ueno, Masahiko Morioka, Ichirou Suehiro, and Takayuki Ohe for the synthesis of TZDs. We also thank Dick Goodman and Bert O'Malley for providing CBP and SRC-1 cDNAs, respectively.

    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.

§ Present address: Dept. of Cell Biology, Harvard Medical School, Boston, MA 02115.

parallel To whom correcpondence should be addressed: University of Pennsylvania School of Medicine, 611 CRB, 415 Curie Blvd., Philadelphia, PA 19104-6149; Tel.: 215-898-0210; Fax: 215-898-5408; E-mail: lazar{at}mail.med.upenn.edu.

The abbreviations used are: NHR, nuclear hormone receptor; TZD, thiazolidinedione; PPARgamma , peroxisome proliferator-activated receptor gamma ; LBD, ligand-binding domain; GST, glutathione S-transferase; SMRT, silencing mediator of retinoid and thyroid receptor; beta -gal, beta -galactosidase; DBD, DNA-binding domain; CBP, cAMP response element-binding protein; SRC-1, steroid receptor coactivator-1; ER, estrogen receptor; aP2, adipocyte protein 2.
    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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