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Ligand Pharmaceuticals (M.D.L., R.J.A., M.F.B., D.L.C., J.D., N.I.H., R.A.H., D.J., K.K., S.L., D.E.M., C.M.M., K.B.M., P.-Y.M., K.M.O., B.P., D.R., J.S.T., M.S.U., M.W.), San Diego, California 92121; and Lilly Research Laboratories (C.L.B., M.A.C., G.J.E., M.M.F., T.A.G., H.H., C.M.-R., N.Y., A.R.-M.), Indianapolis, Indiana 46285
Address all correspondence and requests for reprints to: Dr. Mark D. Leibowitz, Ligand Pharmaceuticals, Inc., 10275 Science Center Drive, San Diego, California 92121. E-mail: mleibowitz{at}ligand.com.
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Abstract |
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agonists, such as rosiglitazone, produce insulin sensitization in rodent models of insulin resistance and type 2 diabetes. In sharp contrast to the TZDs that produce significant increases in body weight gain, RXR agonists reduce body weight gain and food consumption. Unfortunately, RXR agonists also suppress the thyroid hormone axis and generally produce hypertriglyceridemia. Heterodimer-selective RXR modulators have been identified that, in rodents, retain the metabolic benefits of RXR agonists with reduced side effects. These modulators bind specifically to RXR with high affinity and are RXR homodimer partial agonists. Although RXR agonists activate many heterodimer partners, these modulators selectively activate RXR:PPAR
and RXR:PPAR, but not RXR:RAR, RXR:LXR, RXR:LXRß, or RXR:FXR. We report the in vivo characterization of one RXR modulator, LG101506 (LG1506). In Zucker fatty (fa/fa) rats, LG1506 is a potent insulin sensitizer that also enhances the insulin-sensitizing activities of rosiglitazone. Administration of LG1506 reduces both body weight gain and food consumption and blocks the TZD-induced weight gain when coadministered with rosiglitazone. LG1506 does not significantly suppress the thyroid hormone axis in rats, nor does it elevate triglycerides in Sprague Dawley rats. However, LG1506 produces a unique pattern of triglycerides elevation in Zucker rats. LG1506 elevates high-density lipoprotein cholesterol in humanized apolipoprotein A-1-transgenic mice. Therefore, selective RXR modulators are a promising approach for developing improved therapies for type 2 diabetes, although additional studies are needed to understand the strain-specific effects on triglycerides. |
Introduction |
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Synthetic ligands for both RXR and PPAR produce insulin sensitization in animal models of type 2 diabetes (2, 3, 4). Although the well-characterized PPAR agonist rosiglitazone is specific for the RXR:PPAR heterodimer (5), the prototypic RXR-selective agonist LG100268 (LG268) (6) activates many RXR heterodimers and RXR homodimers (2, 7). Thus, rexinoids, when compared with a PPAR agonist, can signal through multiple RXR-dependent pathways (involving different partner receptors) with the potential to elicit not only broad therapeutic benefits, but also unwanted side effects. Tissue-selective modulators of other nuclear receptors have been identified and exploited clinically. The classic successful examples of this approach are the selective estrogen receptor modulators, such as raloxifene and tamoxifen.
The antidiabetic profile of LG268 in rodent models of insulin resistance is superior to that of thiazolidinedione (TZD) PPAR agonists that increase both fat mass and body weight. Rexinoid agonists reduce plasma glucose and insulin, body weight gain, and food consumption while preserving lean body mass (2, 8, 9). Unfortunately, they also suppress the thyroid hormone axis (10) and can produce rapid and dramatic increases in triglycerides (11). Thus, synthetic ligands for RXR that lack these side-effects have the potential to significantly improve upon the currently used thiazolidinedione insulin sensitizers for the treatment of type 2 diabetes. We chose to explore the identification of heterodimer-selective rexinoids to dissect the desirable effects from the undesirable side-effects.
In this communication, we report the biological characterization of the heterodimer-selective RXR modulator LG101506 [LG1506; (2E,4E,6Z)-7-(2-(2,2-difluoroethoxy)-3,5-di-tert-butylbenzene)-3-methylocta-2,4,6-trienoic acid] (12). This compound binds to the RXR with high affinity and induces a receptor conformation that results in selective activation of RXR:PPAR, RXR:PPAR, and RXR:PPAR
, but not RXR:RAR, RXR:LXR, or RXR:FXR heterodimers. In rodent models of insulin resistance, LG1506 administration produces antidiabetic activities similar to those seen after rosiglitazone administration. When administered in combination with rosiglitazone, LG1506 enhances the insulin-sensitizing activity of the TZD and blocks TZD-induced body weight gain. In addition, administration of LG1506 to humanized apolipoprotein A1-transgenic (hApoA1tg) mice leads to an elevation in high-density lipoprotein cholesterol (HDL-C) and enhances the activity of fenofibrate. LG1506 retains all the antidiabetic benefits of LG268 without suppressing the thyroid hormone axis. Interestingly, a unique pattern of triglycerides elevation is produced after the administration of LG1506 to Zucker rats.
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Materials and Methods |
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, -ß, or - and RAR, -ß, or -) were produced using a baculovirus expression system. Biotinylated oligonucleotides containing RXR or RAR response elements were used to couple the corresponding receptor dimers to yttrium silicate streptavidin-coated scintillation proximity assay beads. Receptor binding assays for RARs and RXRs were performed in a similar manner, as described by Boehm et al. (14) using [3H]-9-cis-retinoic acid as the radioligand for RXRs, and [3H]all-trans-retinoic acid (NEN Life Science Products-DuPont, Boston, MA) for the RARs. Ki values were determined by application of the Cheng-Prusoff equation (15). Test compounds were evaluated using an 11-point dose-response curve with concentrations ranging from 0.169 nM to 10 µM.
Cotransfection (CTF) assays
PPAR, PPAR, PPAR, LXR, LXRß, FXR, RAR, or RXR were constitutively expressed using plasmids containing the CMV promoter. The luciferase reporter constructs contained three to five copies of the cellular retinol-binding protein II response element for RXR (16), a thyroid receptor response element for RAR (17, 18), an LXR response element for LXR (19), an ecdysone receptor response element for FXR (20), an acyl coenzyme A oxidase-PPAR response element for PPAR and PPAR (21), or a cytochrome P450 4A1 PPAR response element for PPAR (22). Synergy assays for PPAR, PPAR, and RAR were performed as the standard CTF assays with the addition of EC20 concentrations of the appropriate specific ligand {rosiglitazone, GW501516, and (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthylenyl)-1-propenyl] benzoic acid (TTNPB), respectively}. All assays were performed in CV-1 cells, and compounds were tested in full log dilution, from 0.1 nM to 10 µM in duplicate. Efficacy was determined relative to reference molecules. The median effective concentration (EC50) values were determined by computer fit to a concentration-response curve. An EC50 value was not calculated if the maximum observed efficacy for the compound was less than 20%.
In vivo studies
All in vivo procedures were approved by the Ligand or Eli Lilly Institutional animal care and use committees, and principles of laboratory animal care (National Institutes of Health publication 85-23, revised 1985) were followed. Animals were housed in temperature-controlled rooms (70–74 F) with lights on from 0600–1800 h, with free access to water and food, except where noted below.
Zucker fatty (fa/fa) rat studies
Female Zucker obese (fa/fa) or lean rats were obtained from Harlan (Indianapolis, IN) at 6 wk of age. After a 2-wk acclimation period, rats for efficacy studies were prebled and assigned to experimental groups to minimize the variance between groups based on the measured plasma glucose levels and body weight (six animals per group). Compounds were administered by oral gavage once daily between 0730 and 0830 h for 14 d: LG268 (30 mg/kg), rosiglitazone (10 mg/kg), LG1506 (3, 10, or 30 mg/kg), or LG1506 (3 or 30 mg/kg) plus rosiglitazone (1 mg/kg). The dosing vehicle was 0.085% povidone (ISP Technologies, Inc., New Milford, CT), 1.5% lactose (Quest International, New York, NY), 0.026% Tween 80 (Sigma-Aldrich Corp., St. Louis, MO), and 0.2% (vol/vol) Antifoam (Dow Corning, Midland, MI) with control animals receiving dosing vehicle only. Insulin, TSH, and triglycerides levels were determined from blood samples collected from the tail vein of unfasted conscious animals 3 h after treatment on d 2, 7, and 14. Body weight gain was determined by subtracting the starting body weight of each animal on d –1 from its weight on d 14. The rats were fasted overnight after the last dose and were subjected to an oral glucose tolerance test (OGTT) the following morning. Glucose and insulin levels were measured at 0 min (immediately before the glucose challenge) and 15, 30, 60, and 120 min after challenge (2 g glucose/kg body weight). The insulin resistance index was calculated by multiplying the insulin area under the curve (AUC) by the glucose AUC. For the study described in Fig. 5
, lean or obese Zucker rats were sorted based upon body weight and pretreatment triglycerides (six animals per group). Animals were treated for 7 d as described above with vehicle or LG1506 (1, 3, or 30 mg/kg).
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Lipoproteins were separated by fast protein liquid chromatography, and cholesterol was quantitated with an in-line detection system based on that described by Kieft et al. (24). Briefly, 35-µl serum samples (from 50-µl pooled samples) were applied to a Superose 6 HR 10/30 size exclusion column (Pharmacia Biotech, Piscataway, NJ) and eluted with PBS (pH 7.4; diluted 1:10), containing 5 mM EDTA, at 0.5 ml/min. Cholesterol reagent from Roche Diagnostics (Indianapolis, IN) at 0.16 ml/min was mixed with the column effluent through a T-connection; the mixture was then passed through a 15-m x 0.5-mm knitted tubing reactor (Aura Industries, New York, NY) immersed in a 37 C water bath. The colored product produced in the presence of cholesterol was monitored in the flow stream at 505 nm, and the analog voltage from the monitor was converted to a digital signal for collection and analysis. The change in voltage corresponding to the change in cholesterol concentration was plotted vs. time, and the AUC corresponding to the elution of HDL-C was calculated using Turbochrome software (version 4.12F12, PerkinElmer, Norwalk, CT).
Sprague Dawley rat studies
Sprague Dawley rats were purchased from Harlan (San Diego, CA) at 6 wk of age. After a 2-wk acclimation period, the rats were assigned (based on weight) to individual groups (seven animals per group). An acute study was performed in which TSH and triglycerides levels were measured 2 h after a single dose of vehicle, rosiglitazone (30 mg/kg), LG268 (3, 10, 30 mg), or LG1506 (30 mg/kg). An additional study focused on triglycerides was performed in which rats were administered vehicle, LG268 (30 mg/kg), or LG1506 (1, 3, 10, or 30 mg/kg) for 7 d, with triglycerides levels measured 2 h after treatment on d 1, 3, 5, and 7. An acute LG1506 dose-response study focused on TSH was conducted using eight animals per group. TSH was measured 2 h after a single dose of vehicle, LG268 (30 mg/kg), or LG1506 (1, 3, 10, or 30 mg/kg). The dosing vehicle was 1% (wt/vol) carboxymethylcellulose and 0.25% Tween-80.
Statistical analysis
All results are expressed as the mean ± SEM. Data were analyzed by either one- or two-way ANOVA, followed by Dunnett’s or Tukey’s test. In the two-way analysis, the time factor was considered a repeated measure. Data that did not meet the assumptions for parametric statistics (normal distribution and equality of variance) were transformed using Box-Cox transformations (25) before analysis. Any specific transformations used are described in the figure legends. All statistical analysis was performed using JMP 5.1 (SAS Institute, Inc., Cary, NC), with the level set at 0.05.
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Results |
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, -ß, or -, with minimal affinity for RAR, -ß, or - (Table 1), thus defining both compounds as selective RXR ligands (rexinoids). We have not identified compounds that are selective for RXR, -ß, or -. As this is the case, we have used the RXR isotype for all further molecular profiling. In standard CTF assays, LG268 functioned as a PPAR, PPAR, LXR, and FXR agonist, whereas LG1506 was active only in the PPAR assay (Table 2). In the LXR assay, LG1506 was a weak antagonist (data not shown). To more clearly define the transcriptional potential of LG1506, CTF synergy assays were developed. In the synergy assays, the EC20 concentration of a known ligand for the RXR heterodimeric partner was added to the standard CTF assay. Using this approach, LG268 was active in the PPAR, PPAR, and RAR synergy assays. In contrast, the modulator LG1506 did not activate the RXR:RAR heterodimer in the synergy assay and thus demonstrated heterodimer selectivity (Table 2). These data differentiated LG1506 from rexinoid agonists (exemplified here by LG268) and prompted us to investigate whether LG1506 retained the superior antidiabetic properties of an RXR agonist, but had reduced negative side effects.
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agonist rosiglitazone (10 mg/kg). The combination arm included LG1506 plus a submaximal dose of rosiglitazone (3 or 30 mg/kg LG1506 plus 1 mg/kg rosiglitazone). The study also included fa/fa and lean control groups administered the dosing vehicle only. After 14 d of compound administration, the rats were fasted overnight and subjected to an OGTT. As shown in Fig. 1A, results from the monotherapy arm of the study demonstrated that LG1506, LG268, and rosiglitazone all reduced the hyperinsulinemia associated with this rodent model of insulin resistance. In contrast, insulin levels remained elevated in the control fa/fa rats, whereas the lean control rats maintained normal insulin levels throughout the study period. LG1506 treatment resulted in a dose-dependent reduction in both glucose and insulin excursions after the oral glucose challenge, whereas both LG268 and rosiglitazone reduced the excursions to levels observed in the lean control rats (Fig. 1, B and C).
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). In the combination arm of the study, both the 3 and 30 mg/kg doses of LG1506 enhanced the activity of a submaximal dose of rosiglitazone (Fig. 1E). In fact, the 1 mg/kg dose of rosiglitazone plus the 30 mg/kg dose of LG1506 were as efficacious as the maximally efficacious dose of rosiglitazone (10 mg/kg), both of which normalized the insulin resistance index to that of the lean control animals.
Administration of a high dose of rosiglitazone (10 mg/kg) for 14 d to Zucker fa/fa rats increased body weight gain significantly (Table 3). In contrast, a fully efficacious dose of LG268 significantly reduced weight gain, as we previously reported (8). Although combined administration of rosiglitazone and LG268 did not further enhance insulin-lowering efficacy, body weight gain was restrained to the same extent observed in animals dosed with LG268 alone (Table 3).
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, the 1 mg/kg dose of rosiglitazone was a submaximally efficacious dose, although it produced an increase in weight gain (Fig. 2). In comparison, neither the 3 nor the 30 mg/kg dose of LG1506, alone or in combination with rosiglitazone, increased weight gain compared with the obese control animals. In fact, the 30 mg/kg dose of LG1506 in combination with rosiglitazone produced significantly less weight gain than that in the fa/fa controls. The combination study revealed that administration of 3 mg/kg LG1506 plus a submaximal dose of rosiglitazone led to significant insulin sensitization, with no drug-induced weight gain, and a higher dose of LG1506 in combination with rosiglitazone normalized the insulin resistance index while reducing weight gain. All animals in this study remained healthy and had weight gain at least comparable with that of the control lean animals.
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CTF activity of LG1506, a study was conducted in hApoA1tg mice to investigate the lipid-altering properties of the compound. Administration of LG1506 to hApoA1tg mice increased HDL-C to the level seen after treatment with 100 mg/kg fenofibrate. Combined treatment with LG1506 and fenofibrate produced greater increases in HDL-C at all doses compared with the monotherapy arm of the study (Table 4). These results demonstrate that the RXR modulator LG1506 can have significant impact on the RXR:PPAR heterodimer in vivo, and in vivo activation of both sides of the heterodimer produces increased activity.
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). To evaluate this effect, we conducted an LG1506 dose-response study using 1, 3, 10, or 30 mg/kg LG1506 and 30 mg/kg LG268 as the positive control (Fig. 3B). Although the absolute values are different in these two studies, LG268 reduced TSH by the same percentage in both experiments (49% and 50%, respectively). Animals treated with all doses of LG1506 in excess of 1 mg/kg showed a trend for reduced TSH levels, although none of the decreases was statistically significant.
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). To explore these results in more detail, Sprague Dawley rats were treated for 7 d with LG1506 (1, 3, 10, or 30 mg/kg) or a maximally efficacious dose of LG268 (30 mg/kg). Triglycerides levels were determined from plasma collected 2 h after treatment on d 1, 3, 5, and 7. LG1506 did not alter triglycerides levels at any dose on any day of this study; however, LG268 administration led to a rapid and sustained increase in triglycerides (Fig. 3D).
During the fa/fa rat efficacy study described in Fig. 1 we determined both TSH and triglycerides levels. As we had seen in Sprague Dawley rats, administration of LG268 (30 mg/kg) to fa/fa rats for 14 d decreased TSH levels for the duration of the study (Fig. 4A). Although the mean TSH levels in animals treated with LG1506 (3, 10, or 30 mg/kg) are lower than those in the vehicle-treated animals, none of the reductions was statistically significant (Fig. 4A). The selective RXR modulator LG1506 does not significantly alter TSH levels in two different rat strains, suggesting that such a selective modulator might not cause this side effect in man. As expected based upon the previous 7-d Sprague Dawley rat study, LG268 administration led to a rapid and sustained increase in triglycerides (Fig. 4B). Interestingly, although fa/fa rats receiving LG1506 had control levels of triglycerides after three doses, triglycerides were higher after seven doses. Furthermore, this increase in triglycerides had an inverted dose response; the greatest increase in triglycerides was at the lowest dose of the modulator. Similar results were found in other studies and when other selective RXR modulators were studied (data not shown).
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). On d 3, triglycerides were elevated (but not statistically different from the control level) in lean rats after the administration of both the 1 and 3 mg/kg doses of LG1506. On d 7, lean animals receiving 3 mg/kg LG1506 had triglycerides levels statistically higher than those in control animals. In the obese fa/fa rats, both 1 and 3 mg/kg LG1506 doses produced significant increases in triglycerides on d 3, although only the 3 mg/kg dose was significant on d 7. These data indicate that at low doses, LG1506 retains the unwanted side effect of triglycerides elevation in the Zucker strain of rat (regardless of obesity phenotype). Data from these studies clearly indicate that selective RXR modulators are devoid of the hypertriglyceridemia associated with the administration of LG268 to Sprague Dawley rats. In addition, the kinetic pattern of the elevation in triglycerides seen after administration of the RXR modulator LG1506 to fa/fa rats is distinct from that seen after administration of the RXR agonist LG268, suggesting that different and/or multiple mechanisms underlie the effects.
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Discussion |
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agonist rosiglitazone.
We reasoned that an appropriate heterodimer-selective RXR modulator would need to retain high-affinity binding to RXR to maintain receptor specificity. The molecule would have to retain the desirable antidiabetic/metabolic efficacy and reduce/eliminate the undesirable side effects, such as suppression of the thyroid hormone axis and elevation of triglycerides. We believed that an RXR partial agonist could sensitize the in vivo homeostatic network to the activity of endogenous ligands for partner receptors such as the PPARs (28). This hypothesis led us to use in vitro synergy assays to explore in a sensitive manner the activities of our compounds on various heterodimers. We used a PPAR synergy assay to screen for desirable antidiabetic activities and an RAR synergy assay as a marker to screen against undesirable side effects. The synergy assays are more sensitive at detecting agonist activity and/or reflect mechanistically relevant receptor conformations that are distinct from those identified in the standard CTF assays. As expected based upon this increased sensitivity, numerous compounds are active in the synergy assays, but negative in the corresponding standard CTF assays. The most promising selective RXR modulators would be those that activate only RXR:PPAR and RXR:PPAR, thus mimicking the effects of the thiazolidinediones and fibrates, respectively. The selective RXR modulator LG1506 described in this report does not activate RXR:RAR, RXR:LXR, or RXR:FXR heterodimers. The ability of modulators to be selectively devoid of agonist activity on RXR:RAR and RXR:LXR is important, because both RAR and LXR agonists have been shown to increase triglycerides (29, 30, 31, 32, 33). It is important to note, however, that the kinetics of the increase in triglycerides induced after the administration of an RXR agonist are not the same as those seen after the administration of an RAR agonist, suggesting that the RXR:RAR heterodimer cannot fully account for all the observed increases.
All our CTF assays were conducted using RXR. It is possible that RXRß or RXR would produce different results. However, we think that this is unlikely, because we have been unable to identify rexinoids that are selective for any of the RXR subtypes, and the binding of both LG268 and LG1506 is essentially the same to all three RXRs. Because the RXR isotypes are not identical in either sequence or tissue distribution, it is still possible that they could interact in heterodimer-specific ways, although we are not aware of any demonstration of such specificity.
The in vivo activity of LG1506 is compared with that of the RXR agonist LG268 that activates all four heterodimers. LG1506 demonstrates an efficacious antidiabetic profile when administered alone and has the ability to enhance the activity of the PPAR agonist rosiglitazone when administered in combination. Although LG1506 was positive in the PPAR CTF synergy assay, it was inactive in the standard CTF assay. It has been shown that full PPAR agonist activity, as measured in CTF and cofactor recruitment assays, is not needed to obtain significant insulin-sensitizing benefits in vivo (28, 34). Therefore, it is not surprising that administration of LG1506 resulted in significant insulin sensitization in the absence of in vitro PPAR agonist activity in the standard CTF assay. Alternatively, one could argue that the insulin-sensitizing benefit is due to the PPAR agonist activity of LG1506, because a number of reports have shown that PPAR agonists can function as insulin-sensitizing agents (35, 36, 37). With these alternatives offered, the most likely explanation is that both PPAR and PPAR agonist activities of LG1506 contribute to the insulin-sensitizing benefits of the selective RXR modulator.
The third member of the PPAR family, PPAR, could also play a role in the observed activity of LG1506 and/or LG268. We did not explore this possibility in detail. Although the initial demonstration that PPAR activation influenced cholesterol metabolism was made in db/db mice (38), the more dramatic metabolic effects that have been demonstrated recently have been in nonhuman primates (39) or have used molecular manipulations to dramatically increase receptor activation (40). Nevertheless, it is possible that some fraction of the observed metabolic activity of the rexinoids could be mediated through PPAR. In particular, it is possible that some of the advantageous activity with respect to body weight gain could result from activation of PPAR.
One of the potential advantages of the rexinoid approach is that multiple RXR partners can be activated simultaneously. This benefit is clearly demonstrated in the combination study with rosiglitazone and LG1506 in Zucker fa/fa rats. In the respective monotherapy studies, rosiglitazone normalized the insulin resistance index when administered at 10 mg/kg, and LG1506 caused a dose-dependent decrease at the doses administered (3, 10, and 30 mg/kg). However, combined administration of a submaximal dose of rosiglitazone (1 mg/kg) plus LG1506 (30 mg/kg) normalized the insulin resistance index. Even more interesting and exciting is the finding that LG1506 blocked the drug-induced weight gain associated with rosiglitazone treatment. A significant increase in body weight gain was seen after the administration of a submaximal dose of rosiglitazone (1 mg/kg), whereas this dose of rosiglitazone plus LG1506 (30 mg/kg) resulted in a significant decrease in body weight gain. Importantly, all animals in this study remained healthy and achieved at least the weight gain of the lean control animals. The lack of drug-induced weight gain after mono- and combination therapy with LG1506 can result from multiple pathways. Ogilvie et al. (8) demonstrated, using intracerebroventricular injections, that RXR agonists act directly in the central nervous system to regulate food intake and satiety. When administered intracerebroventricularly, LG1506 also has these effects (data not shown). In addition, numerous reports have shown that PPAR activity is associated with increased fatty acid ß-oxidation, and a lack of weight gain has been observed when a PPAR agonist is administered to insulin-resistant rodents and nonhuman primates (35, 36, 37).
Our data demonstrate that the dose of a PPAR agonist required to achieve maximal efficacy can be reduced when used in combination with a selective RXR modulator. Thus, combination therapy may reduce the incidence of additional side effects associated with PPAR agonist therapy, such as edema and the potential for congestive heart failure.
The selective RXR modulators identified and characterized to date demonstrate PPAR agonist activity in the standard CTF assay. These compounds demonstrate a lipid-altering effect in vivo, as shown in the hApoA1 transgenic mouse. Although lipid metabolism in rodents is not identical with that in humans (e.g. rodents do not express cholesterol ester transfer protein), rodent models have been and continue to be used extensively for both basic research and drug discovery. For example, the fibrate class of hypolipidemic drugs raise HDL-C and lower triglycerides levels in the hApoA1-transgenic mouse and in humans. When used alone, LG1506 produced a dose-dependent increase in HDL-C that was equivalent to the elevation produced by 100 mg/kg fenofibrate. Coadministration of LG1506 with 100 mg/kg fenofibrate produced greater increases in HDL-C at all doses of LG1506 tested.
Administration of the heterodimer-selective RXR modulator LG1506 did not significantly suppress the thyroid hormone axis, as seen after the administration of RXR agonists (10, 26). Because the LG268-mediated reduction in thyroid hormones has been shown to result from a decrease in TSH secretion from the anterior pituitary, which then leads to a reduction in T3 and T4 levels (10), we carefully monitored TSH levels in the Zucker fa/fa rat efficacy study and in the acute studies in Sprague Dawley rats. No significant changes in TSH levels were observed in any of the in vivo studies with LG1506. The mechanism by which rexinoids alter TSH levels is not understood. Although neither LG268 nor LG1506 binds to the RAR, LG268 clearly has agonist activity in the RXR:RAR CTF synergy assay, whereas LG1506 has absolutely no agonist activity in that assay. These data clearly demonstrate the importance of the sensitive RXR:RAR CTF synergy assay to differentiate between rexinoid agonists (such as LG268) that suppress the thyroid axis and heterodimer-selective modulators (such as LG1506) that do not.
Administration of RXR agonists, including LG268, to naive animals is known to cause a rapid elevation in triglycerides levels (11, 27). Therefore, triglycerides levels were carefully monitored after the administration of selective RXR modulators to Sprague Dawley rats under conditions where the RXR agonist LG268 raised triglycerides levels. Both acute and chronic studies were conducted, and no alteration in levels of triglycerides were seen after the administration of LG1506 to Sprague Dawley rats. These data definitively demonstrate in normal rats that the activities of LG1506, with respect to triglycerides, are distinctly differentiated from those of the RXR agonists.
RXR agonists, such as LG268, produce the same pattern of triglycerides elevation in both Sprague Dawley and Zucker fa/fa rats. It was therefore totally unexpected that LG1506 would produce a different pattern in Zucker compared with Sprague Dawley rats. The fact that triglycerides levels in Zucker rats are increased more at lower doses of LG1506 suggests that the compound activates multiple pathways that impact triglycerides. Furthermore, the compound appears to activate a pathway(s) that increases levels of triglycerides at low doses and to activate another pathway(s) that opposes the increase at higher doses.
These observations are not specific to LG1506, because analogous delayed increases in triglycerides have been observed after the administration of other heterodimer-selective RXR modulators to Zucker rats (data not shown). Additional studies will be required to understand the mechanism of this unexpected difference between strains. The insulin resistance phenotype of the fa/fa rat may contribute to the unusual lipid findings. We presume that the increase in triglycerides observed in Zucker rats after treatment with LG1506 is not mediated through activation of LXR. Although agonist activity at either partner of the RXR:LXR heterodimer is associated with hypertriglyceridemia in rodents (11, 33), LG1506 is a weak LXR antagonist in CTF assays.
In conclusion, selective RXR modulators offer an interesting and potentially beneficial approach to the identification and development of novel therapies for type 2 diabetes. Although we do not fully understand the precise molecular mechanisms involved, heterodimer-selective RXR modulators demonstrate a number of advantages when used alone and in combination with PPAR and/or PPAR agonists. At the same time, one must be cautious, because all our work relied upon rodent models and, as such, may not precisely reflect the activity of the compounds in man. Although we have identified RXR modulators that do not significantly suppress the thyroid hormone axis, the unexpected pattern of triglycerides elevation that has been unmasked remains an issue that requires additional study to obtain a solution.
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Acknowledgments |
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Footnotes |
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Current address of M.M.F.: Amgen, Thousand Oaks, California 91320.
Current address of R.A.H.: Exelixis, San Diego, California 92121.
Current address of D.J.: Neurocrine Biosciences, San Diego, California 92130.
Current address of C.M.M.: Johnson & Johnson Pharmaceutical Research and Development, San Diego, California 92121.
Current address of P.-Y.M.: Genomics Institute of the Novartis Research Foundation, San Diego, California 92121.
Current address of K.M.O. and B.P.: Pfizer Global Research and Development, La Jolla, California 92121.
First Published Online November 3, 2005
Abbreviations: Apo, Apolipoprotein; AUC, area under the curve; CTF, cotransfection; FXR, farnesoid X receptor; h, humanized; HDL-C, high-density lipoprotein cholesterol; LG268, LG100268; LXR, liver X receptor; OGTT, oral glucose tolerance test; PPAR, peroxisome proliferator-activated receptor; RAR, retinoic acid receptor; RXR, retinoid X receptor; tg, transgenic; TZD, thiazolidinedione.
Received June 9, 2005.
Accepted for publication October 21, 2005.
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References |
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(PPAR). J Biol Chem 270:12953–12956) ligand. Medicinal Chem Res 7:325–334 ligands. J Biol Chem 277:12503–12506/ agonist with beneficial effects on insulin resistance and carbohydrate and lipid metabolism in ob/ob mice and obese Zucker rats. J Lipid Res 43:1855–1863 activators improve insulin sensitivity and reduce adiposity. J Biol Chem 275:16638–16642 activator. Am J Physiol 283:H949–H9457 alters lipid metabolism in db/db mice. FEBS Lett 473:333–336[CrossRef][Medline] agonist promotes reverse cholesterol transport. Proc Natl Acad Sci USA 98:5306–5311. PLOS Biol 2:e294This article has been cited by other articles:
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  P. Germain, P. Chambon, G. Eichele, R. M. Evans, M. A. Lazar, M. Leid, A. R. De Lera, R. Lotan, D. J. Mangelsdorf, and H. Gronemeyer International Union of Pharmacology. LXIII. Retinoid X Receptors Pharmacol. Rev., December 1, 2006; 58(4): 760 - 772. [Abstract] [Full Text] [PDF]
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