The Rexinoid LG100754 Is a Novel RXR:PPAR{gamma} Agonist and Decreases Glucose Levels in Vivo

Rosemary M. Cesario, Kay Klausing, Haleh Razzaghi, Diane Crombie, Deepa Rungta, Richard A. Heyman1 and Deepak S. Lala2

Department of Nuclear Receptor Research (R.M.C., K.K., H.R., D.C., R.A.H., D.S.L.) and New Leads (D.R.), Ligand Pharmaceuticals, Inc., San Diego, California 92121

Address all correspondence and request for reprints to: Dr. Deepak S. Lala, Department of Biochemistry and Molecular Biology, Pharmacia Corporation, 700 Chesterfield Parkway North, St. Louis, Missouri 63198.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The RXR serves as a heterodimer partner for the PPAR{gamma} and the dimer is a molecular target for insulin sensitizers such as the thiazolidinediones. Ligands for either receptor can activate PPAR-dependent pathways via PPAR response elements. Unlike PPAR{gamma} agonists, however, RXR agonists like LG100268 are promiscuous and activate multiple RXR heterodimers. Here, we demonstrate that LG100754, a RXR:RXR antagonist and RXR:PPAR{alpha} agonist, also functions as a RXR:PPAR{gamma} agonist. It does not activate other LG100268 responsive heterodimers like RXR:liver X receptor{alpha}, RXR:liver X receptorß, RXR:bile acid receptor/farnesoid X receptor and RXR:nerve growth factor induced gene B. This unique RXR ligand triggers cellular RXR:PPAR{gamma}-dependent pathways including adipocyte differentiation and inhibition of TNF{alpha}-mediated hypophosphorylation of the insulin receptor, but does not activate key farnesoid X receptor and liver X receptor target genes. Also, LG100754 treatment of db/db animals leads to an improvement in insulin resistance in vivo. Interestingly, activation of RXR:PPAR{gamma} by LG100268 and LG100754 occurs through different mechanisms. Therefore, LG100754 represents a novel class of insulin sensitizers that functions through RXR but exhibits greater heterodimer selectivity compared with LG100268. These results establish an approach to the design of novel RXR-based insulin sensitizers with greater specificity.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE NUCLEAR HORMONE receptors are ligand-activated transcription factors regulating critical cellular pathways essential for mammalian physiology and development (1, 2). The RXRs play a central role in many of these functions through their ability to act as obligatory heterodimer partners for many members of the nuclear receptor family including RARs, TRs, PPARs, liver X receptors (LXRs), and bile acid receptor/farnesoid X receptor (BAR/FXR) (3). Studies using synthetic RXR-specific ligands (rexinoids) have identified at least two types of RXR-heterodimer complexes: nonpermissive heterodimers that can only be activated by the partners ligand and permissive heterodimers that can be activated either by a RXR or partner-specific ligand (4). Nonpermissive heterodimers include RXR:RAR, RXR:TR, and RXR:VDR. While RXR is completely silent in TR and VDR heterodimers, RXR:RAR can be activated by RXR ligands in the presence of a RAR ligand. Structurally distinct rexinoids have also been identified that activate RXR:RAR, even in the absence of a RAR ligand (5, 6). Thus, RXR:RAR is conditionally nonpermissive. Permissive heterodimers include RXR:PPARs, RXR:LXRs, RXR:BAR/FXR, and RXR:NGFI-B (nerve growth factor-induced gene B). Characterization of RXR:PPAR{gamma}, a molecular target for insulin sensitization, has provided biological evidence that the endogenous heterodimer is indeed permissive for RXR ligands. Supporting this are data showing that agonists for both receptors can participate in the control of common biological pathways such as adipocyte differentiation and carcinogenesis (7, 8). Moreover, both types of compounds act to improve insulin resistance in various animal models of diabetes (9). However, while PPAR{gamma} agonists such as thiazolidinediones are selective for the PPAR{gamma}:RXR heterodimer, the prototypic rexinoid LG100268 (10, 11) activates all other permissive heterodimers and RXR homodimers (9) and is likely to regulate multiple RXR-dependent pathways in vivo. Thus, in developing rexinoids as therapeutic agents, achieving a greater degree of dimer selectivity would be highly desirable.

We previously described a novel RXR homodimer antagonist, LG100754, that functioned as a RXR:RAR and RXR:PPAR{alpha} activator in cotransfection assays (5). In this study we have examined the activity of this compound on permissive RXR heterodimers. We demonstrate that, in addition to RXR:PPAR{alpha}, LG100754 is also active on RXR:PPAR{gamma} heterodimers. Surprisingly, it is inactive on other permissive heterodimers such as RXR:LXR{alpha}, RXR:LXRß, RXR:NGFI-B, and RXR:BAR/FXR, indicating greater selectivity than LG100268. LG100268 and LG100754 show differential recruitment of coactivators to the RXR:PPAR{gamma} heterodimer, suggesting distinct mechanisms of activation by the two classes of rexinoids. We further demonstrate that LG100754 activates endogenous RXR:PPAR{gamma} heterodimer-mediated pathways through its ability to induce adipocyte differentiation of 3T3-L1 cells. Also, like LG100268, LG100754 is able to block TNF{alpha}-mediated inhibition of insulin receptor (IR) phosphorylation in mature adipocytes. However, unlike LG100268, LG100754 does not activate key FXR and LXR target genes. Finally, we show that LG100754 treatment of db/db animals prevents the rise in blood glucose levels, suggestive of an improvement in insulin resistance.

Our data indicate that the RXR homodimer antagonist LG100754 is a novel RXR:PPAR{gamma} agonist that represents a new class of rexinoids that are more dimer selective. Such compounds may be useful in improving insulin resistance and offering benefit for the treatment of human metabolic disease.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
RXR:RXR and RXR:PPAR Activation by LG100754 and LG100268
Using a transient cotransfection assay in CV-1 cells we compared the ability of the two rexinoids, LG100268 and LG100754, to activate RXR homodimers and RXR:PPAR heterodimers. As previously described, LG100268 the prototypic RXR agonist, is a potent and efficacious activator of RXR homodimers; in contrast LG100754 is completely inactive (Fig. 1AGo) and is a RXR homodimer antagonist (5). Interestingly, both LG100754 and LG100268 are active on RXR:PPAR{alpha} (Fig. 1BGo and Ref. 5). In addition, as shown in Fig. 1CGo, both rexinoids are active on the RXR:PPAR{gamma} heterodimer with comparable efficacy. Both are also active on RXR:PPAR{delta}, although not as much as on RXR:PPAR{gamma} (Fig. 1DGo).



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Figure 1. Dimer Selective Activation by LG100754

A, LG100754 does not activate RXR homodimers whereas LG100268 is a potent and efficacious RXR:RXR agonist. B, Both LG100754 and LG100268 activate RXR:PPAR{alpha} heterodimers. C, Both rexinoids activate RXR:PPAR{gamma} heterodimers. D, Activation of RXR:PPAR{delta} by rexinoids.

 
LG100268 is a Promiscuous Activator of Multiple RXR Heterodimers Whereas LG100754 Exhibits Greater Selectivity
To further characterize the activity of LG100754, we compared its ability to activate other RXR heterodimers in similar experiments. Specifically, we tested the permissive heterodimers, all known to respond to LG100268 to determine whether LG100754 is more selective in activating these complexes, including RXR:LXR{alpha}, RXR:LXRß, RXR:BAR/FXR, and RXR:NGFI-B. In each case, the response of a specific dimer to the rexinoids was determined on its cognate hormone response element (HRE). Surprisingly, while LG100268 acts as a strong and potent agonist of RXR:LXR{alpha}, RXR:LXRß, RXR:FXR, and RXR:NGFI-B heterodimers, LG100754 is inactive on all four heterodimers (Fig. 2Go A–D). Further, while combination of LG100268 with either FXR or LXR ligands had an additive effect, LG100754 did not (Fig. 2Go, E and F). In our studies, in addition to RXR:PPAR, the only other heterodimer LG100754 is capable of activating RXR:RAR (5). Thus, while LG100268 acts as a highly promiscuous agonist of RXR heterodimers, LG100754 exhibits greater selectivity toward RXR:PPARs. Other RXR antagonists structurally related to LG100754 also exhibit a similar selectivity for RXR:PPARs (data not shown). These data suggest that, in general, this class of RXR homodimer antagonists may be more dimer selective than agonists such as LG100268.



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Figure 2. LG100268 Is a Promiscuous Activator of Multiple RXR Heterodimers Whereas LG100754 Is Selective

Shown are the dose-response curves of various heterodimers to LG100268 and LG100754. While LG100268 activates RXR:LXR{alpha}, RXR:LXRß, RXR:FXR, and RXR: NGFI-B, LG100754 is inactive or only weakly active. A, Activation of RXR:LXR{alpha} by LG100268 and LG100754. B, Activation of RXR:LXRß by LG100268 and LG100754. C, Activation of RXR:FXR by LG100268 and LG100754. D, Activation of RXR:NGFI-B by LG100268 and LG100754. E, LG100268(1 µM) and 22(R)OHC(10 µM) activate RXR: LXR{alpha}; LG100754(1 µM) does not. Combination of LG100268 (1 µM) + 22(R)OHC (10 µM) shows additive induction; LG100754 (1 µM) + 22(R)OHC (10 µM) does not. F, LG100268(1 µM) and CDCA (50 µM) activate RXR:FXR; LG100754 (1 µM) does not. Combination of LG100268 (1 µM) + CDCA (50 µM) shows additive induction; LG100754 (1 µM) + CDCA (50 µM) does not.

 
LG100754 and LG100268 Lead to Distinct Recruitment of Coactivators to RXR:PPAR{gamma} Heterodimers
Transcriptional activation of nuclear receptors by their ligands occurs after binding to the ligand binding domain (LBD) and induction of a conformational change that facilitates coactivator recruitment. Recent biochemical and crystallographic studies have demonstrated that the structure of the receptor-LBD is ligand specific (12, 13, 14, 15). We have also shown that LG100268 and LG100754 binding to RXR elicit different interactions between RXR and components of the basal transcriptional machinery (5). Based on these data, we speculated that the two different rexinoids would lead to differential recruitment of cofactors to the RXR:PPAR{gamma} heterodimer. To test this, we employed a modified mammalian two-hybrid system that was set up to detect coactivator interactions with the RXR:PPAR{gamma} heterodimer in the presence of rexinoids. This system consists of a coactivator receptor interacting domain (RID) fused to the Gal4-DNA binding domain, a GAL4 response element driving the luciferase cDNA, and the PPAR LBD fused to the VP16 activation domain. Neither component by itself or in combination can respond to rexinoids. Introduction of the RXR LBD minus its DNA-binding domain allows heterodimer formation; however, no RXR homodimer activity is measured since the RXR-LBD cannot bind DNA (data not shown). Similar assays have been used to demonstrate that LG100268 can recruit the coactivator, steroid receptor coactivator-1 (SRC-1), to the RXR:PPAR{gamma} heterodimer (16). We therefore used our assay to first compare the ability of LG100268 and LG100754 to recruit SRC-1 to RXR:PPAR{gamma}.

As shown in Fig. 3AGo, LG100268 induces an interaction of RXR:PPAR{gamma} with the RID of SRC-1; surprisingly, LG100754 does not. Similar results were obtained using SRC-2 or SRC-3 RIDs (data not shown). These data indicate that, in this assay, LG100268, but not LG100754, can promote interactions with the SRC family of coactivators. To determine whether other cofactors might be recruited by LG100754, we compared the ability of both rexinoids to recruit the coactivator cAMP response element-binding protein-binding protein (CBP). LG100754 was a weak recruiter of CBP in this assay (2-fold); LG100268 also recruited CBP much more weakly than SRC-1 (Fig. 3BGo). Since LG100754 is clearly active on RXR:PPAR{gamma} heterodimers, these data suggest that either the weak recruitment of CBP in response to LG100754 is sufficient for activation, or other classes of coactivators are required for its activity. To test the latter, we examined the ability of both compounds to recruit TR-associated protein 220 (TRAP220), a coactivator identified as part of a larger complex recruited to the activation function (AF-2) domain of the TR (17). In contrast to SRC-1, LG100754 was able to recruit TRAP220 5-fold to RXR:PPAR{gamma} (Fig. 3CGo). Interestingly, the promiscuous rexinoid LG100268 recruited TRAP220 as well (Fig. 3DGo), suggesting it places the heterodimer in a conformation that is more flexible for coactivator binding compared with LG100754. Therefore, different rexinoids may induce unique conformational changes within RXR:PPAR{gamma} and activate the heterodimer using different coactivators. Our data suggest that although both LG100268 and LG100754 are active on the RXR:PPAR{gamma} heterodimer, they activate it via different mechanisms.



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Figure 3. Coactivator Recruitment to the RXR: PPAR{gamma} Heterodimer by LG100268 and LG100754 Using a Modified Mammalian Two-Hybrid Assay

A, LG100268 recruits SRC-1 to the heterodimer whereas LG100754 does not. B, Both LG100268 and LG100754 are weak recruiters of the coactivator CBP. Shown below in each case is a schematic indicating the various components of the assay. C and D, LG100754 selectively recruits TRAP220 to the RXR:PPAR heterodimer while LG100268 lacks selectivity. The same assay was used except the coactivator RID used was derived from TRAP220. Ligand concentrations used were 1 µM for both.

 
LG100754 Is a Selective Activator of Endogenous RXR Heterodimers
To extend our transfection studies, we examined the ability of LG100754 to act as a dimer-selective activator of endogenous RXR:PPAR{gamma}/FXR/LXR pathways in relevant cells. Adipocyte differentiation is a well established PPAR{gamma}-dependent pathway, and both LG100268 and PPAR{gamma}-ligands induce differentiation in 3T3-L1 preadipocytes (14, 18, 20, 21). We therefore tested the ability of LG100754 to promote adipogenesis in this cell line. Consistent with its ability to activate the RXR:PPAR{gamma} heterodimer in transfections, LG100754, like LG100268, induces differentiation in a dose-dependent manner at concentrations comparable to those at which it activates RXR:PPAR{gamma} in vitro, and 100 nM of either rexinoid was equally effective (Fig. 4Go, A and B). Thus, LG100754 can activate endogenous RXR:PPAR{gamma} heterodimers and drive cellular PPAR{gamma}-dependent pathways. Next, we looked at the ability of both rexinoids, at the same concentration, to induce intestinal bile-acid binding protein (IBABP) gene expression, a FXR target gene in differentiated Caco-2 cells (22, 23). These cells have low IBABP, which is strongly induced by FXR ligands (22). Using a semiquantitative RT-PCR method, we show strong induction of IBABP with chenodeoxycholic acid (CDCA), a FXR ligand, consistent with previous results (Ref. 22 and Fig. 4CGo). LG100268 also induces IBABP very significantly; in contrast, LG100754 is unable to induce IBABP expression either alone or in the presence of CDCA (Fig. 4CGo). ABCA1 was recently identified as a direct LXR target gene in macrophages and is induced by LG100268 and LXR ligands in vivo and in vitro (24). Interestingly, it can also be up-regulated indirectly but less effectively by BRL (rosiglitazone, a PPAR{gamma} ligand) through RXR:PPAR{gamma} (25). We therefore speculated that LG100754 would behave like BRL and induce ABCA1 less effectively than LG100268 or 22(R)hydroxycholesterol [22(R)OHC], a LXR ligand (24). To test this, we treated RAW264.7 cells, a mouse macrophage cell line, with compounds and used a highly quantitative real-time PCR method to look for differences. As predicted, while LG100268 and 22(R)OHC, RXR:LXR agonists, induce ABCA1 significantly (6- to 13-fold), both LG100754 and BRL, selective RXR:PPAR{gamma} agonists, are weak activators (~2.5 fold, Fig. 4DGo). No further increase was observed by combination of 22(R)OHC with LG100754. Similar results were obtained with ApoE, another LXR target gene (data not shown). Thus, concentrations of LG100754 that are as effective as LG100268 in inducing adipogenesis, a PPAR{gamma}-dependent pathway, are completely ineffective on IBABP, a FXR target gene, and less effective on ABCA1, a LXR target gene. Moreover, the weak induction of ABCA1 by LG100754 is most likely via RXR:PPAR{gamma} since it is also induced by BRL, and these cells express low levels of PPAR{gamma} (data not shown). These data confirm that LG100754, unlike LG100268, is a dimer-selective agonist of cellular PPAR{gamma} but not of FXR or LXR.



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Figure 4. Selective Activation of Endogenous RXR-Heterodimers

A, Quantitative measurement of adipocyte differentiation. 3T3-L1 cells were treated with increasing amounts of either LG100268 or LG100754 in the presence of insulin as described in Materials and Methods. Triglyceride accumulation was determined by measuring the release of glycerol using a kit from Sigma (see Materials and Methods). B, Oil red O staining of 3T3-L1 cells treated with either insulin, insulin + LG100268 (100 nM), or insulin + LG100754 (100 nM). C, Semiquantitative RT-PCR assay using Caco-2 cells. LG100268 and CDCA (RXR:FXR activators) induce IBABP expression but not LG100754 (PPAR-selective). No further increase was observed with a combination of LG100754 and CDCA. GAPDH was used as a control for equal loading; both mRNAs were within the linear range of the reaction. D, Quantitative real-time PCR for ABC1 in RAW264.7 cells. LG100268 and 22(R)OHC (RXR:LXR activators) induce ABC1 expression significantly. LG100754 and BRL (rosiglitazone), PPAR{gamma}-selective activators, are weak activators. Combination of 22(R)OHC with LG100754 does not show further increase.

 
Inhibition of TNF{alpha}-Mediated Hypophosphorylation of the IR by LG100754 and LG100268
IR tyrosine kinase activity is an absolute requirement for the biological activities of insulin. TNF{alpha} has been shown to affect this proximal step of insulin signaling by inhibiting tyrosine phosphorylation of the IR (26). This TNF{alpha}-mediated insulin resistance has previously been shown to be completely blocked by thiazolidinediones in mature adipocytes treated with TNF{alpha} (27). That this effect appears to be mediated through the RXR:PPAR{gamma} heterodimer was shown by the observation that a similar inhibition was achieved with other heterodimer agonists such as 15-deoxy-{Delta}12, 14 PGJ2, a PPAR{gamma} ligand, and LG100268 (27). We therefore examined the ability of the RXR:PPAR{gamma}-selective rexinoid LG100754 to overcome TNF{alpha} inhibition of IR phosphorylation. For this, we first differentiated 3T3-L1 preadipocytes using a standard mixture of isobutylmethylxanthine (IBMX), dexamethasone, and insulin as described in Materials and Methods. Differentiated adipocytes were then treated with TNF{alpha} (5ng/ml) for a period of 1 or 2 d. At day 2 we observed a strong inhibition of insulin-induced IR tyrosine phosphorylation. Importantly, at the same time, we did not observe any significant decrease in IR levels in the same experiment (Fig. 5AGo). Next, we tested the ability of the RXR:PPAR{gamma}-selective rexinoid LG100754 to block the inhibitory effects of TNF{alpha} on day 2 and compared its activity with LG100268. Consistent with its ability to activate RXR:PPAR{gamma}, LG100754 completely blocks the ability of TNF{alpha} to inhibit insulin-dependent IR phosphorylation (Fig. 5BGo). Incubation of differentiated 3T3-L1 cells with LG100268 also leads to a complete block of the inhibitory effects of TNF{alpha} (Fig. 5BGo). Rosiglitazone, the synthetic PPAR{gamma} ligand, also blocked the effects of TNF{alpha} (Ref. 24 and data not shown). Our data indicate that, like LG100268 and rosiglitazone, LG100754 can also improve TNF{alpha}-mediated insulin resistance in mature adipocytes. These results establish LG100754 as a bona fide activator of endogenous RXR:PPAR{gamma} heterodimers and regulator of insulin-dependent signaling pathways.



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Figure 5. LG100268 and LG100754 Inhibit TNF{alpha}-Mediated Hypophosphorylation of IR in Differentiated 3T3-L1 Cells

A, 3T3-L1 cells were differentiated using a standard mixture of IBMX, insulin, and dexamethasone as described in Materials and Methods. Treatment of cells for 2 d with 5 ng/ml TNF{alpha} leads to a decreased response to insulin as determined by a decrease in insulin-dependent IR phosphorylation. No significant change in IR levels is observed under the same conditions. B, Incubation of differentiated 3T3-L1 cells with either LG100268 or LG100754 in combination with TNF{alpha} completely blocks the cytokine-mediated inhibition of insulin-dependent IR phosphorylation.

 
Treatment of db/db Mice with LG100754 Stabilizes Glucose Levels and Improves Insulin Resistance in Vivo
RXR:PPAR{gamma} is a target for insulin-sensitizing drugs, and two PPAR{gamma} ligands, rosiglitazone (Avandia) and pioglitazone (Actos) are used clinically for the treatment of diabetes. It has been shown previously that treatment of db/db mice with rosiglitazone or LG100268 leads to a decrease in glucose levels, indicating an improvement in insulin resistance (9). This may be due, at least in part, to their ability to block the inhibitory effects of TNF{alpha} in these animals. Collectively, our results suggested that LG100754 would also act as an insulin sensitizer in vivo. To test this, db/db animals were treated with the RXR:PPAR selective compound, and its ability to decrease glucose levels was compared with LG100268. As shown in Fig. 6Go, treatment with 100 mg/kg LG100754 completely blocks the increase in glucose levels observed in untreated animals, suggesting that LG100754 can improve insulin resistance in vivo. Our results indicate that LG100754 is a novel rexinoid that serves as a RXR:PPAR{gamma} agonist in vivo, but exhibits a much more restricted pattern of RXR-heterodimer activation in vitro compared with LG100268 (Fig. 7Go).



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Figure 6. LG100268 is an Insulin Sensitizer in Vivo

Treatment of db/db mice with LG100754 (100 mg/kg) blocks an increase in glucose, suggesting an improvement in insulin resistance in these hyperglycemic animals. LG100268 (30 mg/kg) treatment of db/db animals also leads to an improvement in insulin resistance.

 


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Figure 7. Diagram Illustrating the Selectivity of LG100754 Compared with LG100268 in Activating Permissive RXR Heterodimers

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In a previous publication, we identified LG100754, a unique rexinoid, that functioned as an antagonist of RXR homodimers but as a dimer-selective activator of RXR:RAR and RXR:PPAR{alpha} heterodimers (5). In this study, we have focused on its activity on other permissive RXR dimers. Like the promiscuous rexinoid LG100268, LG100754 activates RXR:PPAR{gamma} in transfection assays; however, unlike LG100268, it does not activate other permissive RXR heterodimers. Based on these data, we predicted that LG100754 would act as a selective agonist of endogenous PPAR{gamma}-dependent pathways. This was demonstrated through its ability to induce PPAR{gamma}-dependent adipocyte differentiation, but inability to activate IBABP and ABCA1, key FXR and LXR target genes, respectively. That LG100754 is a bona fide RXR:PPAR{gamma} agonist with insulin-sensitizing activity was shown via its effects on inhibition of TNF{alpha}-mediated hypophosphorylation of the IR. Consistent with its ability to modulate cellular PPAR-dependent pathways, LG100754 treatment of db/db animals led to a decrease in blood glucose levels, indicating an improvement in insulin resistance. Although in vivo LG100754 was less efficacious than LG100268, this may be due to its poor bioavailability, and circulating concentrations are low in db/db mice (data not shown). Nevertheless, as demonstrated by its ability to activate several endogenous PPAR{gamma}-dependent pathways, but its inability to induce key FXR and LXR target genes, LG100754 serves as a novel rexinoid whose activation profile is markedly distinct from that of LG100268. These data provide, for the first time, proof for the concept that rexinoid activity can be restricted to specific permissive RXR heterodimers.

Ligand binding to nuclear receptors has been shown to induce alterations within the LBD of receptors, including a drastic change in the AF-2 domain (helix 12) as well as changes within helices 3–5. These conformational changes together are believed to facilitate interactions of the receptor with coactivators. Subsequently, coactivators are able to enhance transcriptional activation through the receptor via mechanisms that include histone acetylation and contacts with the basal transcriptional machinery (28, 29). x-Ray crystallographic studies have demonstrated, that for the ER{alpha} and PPAR{gamma}, different ligands for the two receptors can induce different conformations within their LBDs (12, 13, 14). Further support for this comes from biochemical studies with ER{alpha} and the VDR, which showed different receptor ligands lead to distinct coactivator interactions (15, 30). Different PPAR{gamma} agonists can also have distinct effects on coactivator recruitment. For example, the TZD MCC-555, a partial agonist of PPAR{gamma}, is a weaker recruiter of SRC-1 compared with rosiglitazone, a full agonist (31). These data suggest that the structure of the ligand-bound LBD region that recruits cofactors is ligand specific. In this regard we showed that the ability to recruit coactivators to RXR:PPAR{gamma} by LG100268 and LG100754 is very different. LG100268 was a strong recruiter of SRC-1 to the heterodimer while LG100754 was completely inactive, while both compounds recruited CBP weakly. In contrast, LG100754 induced a significant interaction with TRAP220 (as did LG100268). LG100754 thus appears to have a different coactivator recruitment profile than LG100268. While SRC-1, CBP, and TRAP220 are all recruited to the AF-2 domains of receptors, TRAP220 belongs to a distinct class of coactivators. Both CBP and SRC-1 possess histone acetyltransferase activity (HAT) whereas TRAP220, which is part of a large TR-associated complex, does not (28). Although the precise mechanism by which SRC and TRAP complexes contribute to overall nuclear receptor function is unclear, our data suggest that LG100754 might recruit a different subset of coactivators to RXR:PPAR. Thus, the mechanism by which LG100754 activates RXR:PPAR{gamma} is likely to be different from that of LG100268. Activation of RXR:PPAR{gamma} by LG100268 or rosiglitazone also leads to differential cofactor recruitment with LG100268 recruiting SRC-1, but rosiglitazone recruiting CBP (16). Our data with LG100754 suggest a third mechanism for activating RXR:PPAR{gamma}.

Another aspect of this study is the elimination of RXR homodimer agonist activity as a requirement for activating PPAR response elements via RXR. In the context of the RXR:PPAR{gamma} pathway, the degree to which the LG100268 response is PPAR dependent vs. RXR homodimer dependent was a question that remained to be determined. Since LG100754 lacks the ability to activate RXR:RXR, our data indicate that RXR homodimer activity is not critical for activating endogenous RXR:PPAR pathways. An interesting question is why LG100754 exhibits selectivity toward RXR:PPAR{gamma} and does not activate other RXR-permissive dimers. The recent crystal structures of RXR:RAR and RXR:PPAR{gamma} revealed that both RAR and PPAR{gamma} use a similar mechanism for heterodimerization (32, 33). However, the PPAR{gamma} dimer with RXR reveals an asymmetrical interaction between PPAR{gamma} AF-2 helix and helix 7 of RXR, which is lacking in RXR:RAR. This interaction could stabilize the PPAR{gamma} AF-2 in a position that permits interactions with coactivators even in the absence of a PPAR{gamma} agonist and may provide a structural basis for the permissivity of this heterodimer. However, other factors, including binding of corepressors, the conformation of RXR, the specific ligand itself, and the precise nature of the HRE, are also likely to play a role in regulating permissiveness. Crystal structures and mutational studies of other permissive and nonpermissive RXR dimers should shed more light on other mechanisms that may be important in determining permissivity. It is possible that different permissive RXR partners mediate distinct allosteric interactions with RXR. Such differences could be exploited to design novel dimer-selective rexinoids. Thus, it may be possible to synthesize more specific and potent RXR:PPAR-selective rexinoids or RXR ligands that activate exclusively RXR:PPAR{gamma} (or, if desired, both RXR:PPAR{gamma} and RXR:LXR{alpha}, for example). Regulating multiple but very specific RXR-dimer combinations by a single compound would be a novel approach to modulate RXR-dependent pathways in vivo with greater selectivity. Conceptually, this should be easier to accomplish through the design of an appropriate ligand for RXR, the common partner, than to expect a ligand to bind to and activate two or more completely different partners.

In conclusion, our data indicate that LG100754, a RXR:RXR antagonist, represents a novel class of rexinoids that, unlike prototypic agonists such as LG100268, is more selective for RXR:PPAR heterodimers in vitro and can function as an insulin sensitizer in vivo. A number of RXR-containing heterodimers (e.g. the PPARs, the LXRs, and BAR/FXR) either are or likely will be important drug discovery targets for the treatment of diabetes, cardiovascular disease, obesity, and other lipid disorders. RXR homodimer antagonists that exhibit unique heterodimer selectivity may be beneficial for the treatment of human metabolic disease. Our results establish an approach to the design of novel RXR-based insulin sensitizers with greater dimer selectivity.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Transfections
Transfections were carried out as previously described (5). Briefly, CV-1 cells were cotransfected with either one of the following receptor expression plasmids: pCMXRXR{alpha}, pCMVSPORT-LXRß, pCMX-LXR{alpha}, pCMXPPAR{alpha}, pCMXPPAR{gamma} alone, or with pCMXNGFI-B, or pCMXBAR/FXR in combination with pCMXRXR{alpha}. The luciferase reporter constructs contained three to five copies of either a LXR response element (for LXR{alpha}/ß), an ecdysone receptor response element (for BAR/FXR), a NGFIB response element (for NGFI-B) or acyl coenzyme A oxidase-PPAR response elements (for PPAR{alpha}/{gamma}) (5, 9). Cofactor recruitment experiments were done by transfecting a Gal4Coactivator RID [SRC-1 (amino acids 380–851), CBP (1–356 amino acids), or TRAP220 (amino acids 442–693)], a VP16-PPAR{gamma}LBD fusion construct, a RXRLBD expression plasmid, and a Gal4 response element linked to a minimal promoter-luciferase construct. ß-Galactosidase was included as an internal control. Experiments were carried out at least three times with comparable results.

Cell Culture and Differentiation
3T3-L1 cells were obtained from ATCC (Manassas, VA) and maintained in DMEM supplemented with 10% calf serum and gentamicin sulfate. For differentiation studies, cells were plated at approximately 80% confluency and treated 2 d after cells were 100% confluent. Solvent or LG100268 or LG100754 was added to cells in the presence of insulin (10 µg/ml) for 7 d with a media and compound change on day 3. During the differentiation assay, cells were treated in DMEM with 10% FBS medium. Differentiation was monitored via oil red O staining and triglyceride accumulation.

Oil Red O Staining
Oil red O staining of adipocytes was performed by fixing the cells with 4% formalin in PBS, followed by a 5-min incubation with 0.5% oil red O stain in propylene glycol, and destaining for 2 min in 85% propylene glycol. Stained cells were covered with PBS and stored at 4 C.

Triglyceride Accumulation
The triglyceride accumulation assay used a modified method from Sigma. 3T3-LI cells were plated at 3,500 cells per well in a 96-well plate. After 7 d of treatment, cells were rinsed two times with PBS and 50 µl of 0.1% NP-40/well was added to lyse cells. Triglyceride assay solution (100 µl/well, Sigma Diagnostics) was added and the plates were incubated for 1 h at 37 C and read at 540 nm. All treatments were done in triplicate.

Gene Induction and Semiquantitative and Quantitative PCR
Caco-2 cells were differentiated as described previously (22) and treated with compound at appropriate concentrations for 24 h; RNA was harvested using TRI Reagent (Sigma no. T9424) and processed with DNase. RT-PCR was done using the SuperScript One-step RT-PCR with Platinum Taq (Life Technologies, Inc., Gaithersburg, MD; no. 10928–034). IBABP primers were as described previously (22); GAPDH was used as a control. For ABCA1, RAW 264.7 cells were treated overnight with receptor ligands. RNA was prepared and subjected to real-time PCR analysis on an ABI Prism 7700 Sequence Detector (ABI Advanced Technologies, Inc., Columbia, MD). ABCA1 primer and probes were: 5' CAT CGA CAT GGT GAA GAA CCA (forward), 5'GAA GCG GTT CTC CCC AAA C (reverse), CAT GGC CGA TGC CCT GGA G (probe). Each data point was the average of 12 values; amplification efficiencies were normalized against cyclophilin.

TNF{alpha}-Induced Insulin Resistance
Two-day postconfluent 3T3-L1 cells were treated with IBMX (0.52 mM), dexamethasone (1 µM), and insulin (10 µg/ml) for 3 d. On day 3, media containing 10 µg/ml insulin was added and cells were incubated an additional 4 d. To induce insulin resistance, differentiated cells were treated with TNF{alpha} (5 ng/ml) for 1–2 d. Protein collection for tyrosine phosphorylation studies was done as described (23). Rexinoids were added at the same time TNF{alpha} treatment was started. Anti-IR antibody (Oncogene Science, Inc., Manhasset, NY) was used for overnight immunoprecipitation in RIPA buffer containing aprotinin (2 µg/ml), pepstatin (1 µM), leupeptin (10 µM), sodium orthovanadate (1 mM), and glycerol-2-phosphate (25 mM) at 4 C. Western blot detection of phosphotyrosine was performed using antiphosphotyrosine antibody clone 4G10 (Upstate Biotechnology, Inc., Lake Placid, NY) at a 1 µg/ml concentration.

db/db Glucose Studies
Female diabetic, 47-d-old, C57BLKS/J-m+/+db mice were dosed with vehicle, LG100268 (30 mg/kg) or LG100754 (100 mg/kg) once daily for 14 d. Blood was drawn after a 3-h fast from the tip of the tail on indicated days, and plasma glucose levels were measured by adoption of the glucose oxidase Trinder protocol (Sigma).

Reagents, RXR, and PPAR{gamma} Compounds
IBMX, dexamethasone, and insulin were purchased from Sigma. Recombinant human TNF{alpha} was purchased from R & D Systems (Minneapolis, MN). LG100268 and LG100754 were synthesized and solvated (90% ethanol and 10% DMSO) at Ligand Pharmaceuticals, Inc. (San Diego, CA).


    ACKNOWLEDGMENTS
 
We thank Dr. Ron Evans for helpful discussions and Drs. Martin Seidel and Chris Glass for critical reading of the manuscript. We also thank Neetu Shah and Charles Bolten for help with quantitative real time PCR.


    FOOTNOTES
 
1 Present address: X-Ceptor Therapeutics, 4757 Nexus Center Drive, Suite 2000, San Diego, California 92121. Back

2 Present address: Department of Biochemistry and Molecular Biology, Mail Zone AA4E, Pharmacia Corporation, 700 Chesterfield Parkway North, St. Louis, Missouri 63198. Back

Abbreviations: AF-2, Activation function 2; BAR, bile acid receptor; BRL, BRL49653; CBP, cAMP response element-binding protein; CDCA, chenodeoxycholic acid; FXR, farnesoid X receptor; HRE, hormone response element; IBABP, intestinal bile acid binding protein; IBMX, isobutylmethylxanthine; IR, insulin receptor; LBD, ligand binding domain; LXR, liver X receptor; NGFI-B, nerve growth factor-induced gene B; RID, receptor interacting domain; 22(R)OHC, 22(R)hydroxy cholesterol; SRC-1, steroid receptor coactivator 1; TRAP220, TR-associated protein 220.

Received for publication September 6, 2000. Accepted for publication April 23, 2001.


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