Molecular Characterization of New Selective Peroxisome ProliferatoreCActivated Receptor Modulators With Angiotensin Receptor Blocking Activ
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《糖尿病学杂志》
1 Center for Cardiovascular Research, Institute of Pharmacology and Toxicology, Charitee Campus Mitte, Charitee-Universittsmedizin Berlin, Berlin, Germany
2 GENFIT, Parc Eurasante, Loos, France
3 Max-Planck Institute for Molecular Genetics, Berlin, Germany
4 Franz Volhard Clinic, HELIOS Hospital Berlin, Charitee Campus Buch, Charitee-Universittsmedizin Berlin, Berlin, Germany
5 Department of Atherosclerose, Faculty of Pharmacy, Institute Pasteur Lille, University Lille II, UR 545 Institut National de la Santee et de la Recherche Meedicale, Lille, France
ARB, angiotensin type 1 receptor blocker; AT1R, angiotensin type 1 receptor; FRET, fluorescence resonance energy transfer; GST, glutathione S-transferase; ITT, insulin tolerance test; LBD, ligand binding domain; NCoR, nuclear receptor corepressor; PPAR, peroxisome proliferatoreCactivated receptor; PPRE, PPAR response element; RPE, R-phycoerythrin; SPPARM, selective PPAR modulators; TIF-2, transcriptional intermediary factor-2; TZD, thiazolidinedione
ABSTRACT
Selective peroxisome proliferatoreCactivated receptor (PPAR) modulation is a new pharmacological approach that, based on selective receptor-cofactor interactions and target gene regulation, should result in potent insulin sensitization in the absence of PPAR-mediated adverse effects. Here, we characterize two angiotensin receptor blockers (ARBs), telmisartan and irbesartan, as new selective PPAR modulators (SPPARMs). Analysis of PPAR protein conformation using protease protection showed that telmisartan directly interacts with the receptor, producing a distinct conformational change compared with a glitazone. Glutathione S-transferase pull-down and fluorescence resonance energy transfer assays revealed selective cofactor binding by the ARBs compared with glitazones with an attenuated release of the nuclear receptor corepressor and absence of transcriptional intermediary factor 2 recruitment by ARBs. Consistently, selective cofactor binding resulted in differential gene expression profiles in adipocytes (ARB versus glitazone treated) assessed by oligo microarray analysis. Finally, telmisartan improved insulin sensitivity in diet-induced obese mice in the absence of weight gain. The present study identifies two ARBs as new SPPARMs. SPPARM activity by ARBs could retain the metabolic efficacy of PPAR activation with reduction in adverse effects exerting in parallel AT1 receptor blockade. This may provide a new therapeutic option for better cardiovascular risk management in metabolic diseases and may initiate the development of new classes of drugs combining potent antihypertensive and antidiabetic actions.
The peroxisome proliferatoreCactivated receptors (PPARs) are members of the nuclear receptor superfamily of ligand-activated transcription factors (1). A series of coactivators/corepressors are involved in ligand-induced transcription of genes allowing a tissue- and ligand-specific activation of these target genes by PPARs (2). The most abundant isoform in adipose tissue, PPAR, plays an important role in the regulation of insulin sensitivity (3). Glitazones or thiazolidinediones (TZDs) are high-affinity ligands for this receptor and are currently used in the treatment of type 2 diabetes (4). Together with an improvement of insulin sensitivity and glucose tolerance, they improve lipid profiles (5). Given the side effects of glitazones, like weight gain, edema, and fluid retention, the characterization of new PPAR ligands that retain metabolic efficacy without exerting adverse actions plays a central role in the development of new therapeutic strategies for insulin resistance and type 2 diabetes (6).
Angiotensin type 1 receptor (AT1R) blockers (ARBs) prevent the binding of angiotensin II to the AT1R and are widely used for the treatment of hypertension and related cardiovascular and organ damage (7). Recent clinical trials revealed a substantial lower risk in developing type 2 diabetes for patients treated with ARBs in comparison with other hypertensive treatment (8). In addition, the ARB telmisartan has been shown to improve metabolic parameters in rodents and type 2 diabetic patients (9,10,11). Recently, a subgroup of ARBs has been characterized as PPAR ligands independent of their AT1R blocking actions (11,12,13). Although designed to bind to the AT1R, these ARBs have been shown to activate the PPAR ligand binding domain (LBD) in the e蘭ol/l range (11,13).
The underlying molecular mechanisms of PPAR activation by ARBs are unknown. Here, we characterize the molecular interactions between PPAR and two PPAR-activating ARBs, telmisartan and irbesartan. Glutathione S-transferase (GST) pull-down and fluorescence resonance energy transfer (FRET) assays revealed selective cofactor recruitment by ARBs. Oligo microarray analysis of RNA from 3T3-L1 adipocytes treated with the ARBs or the full agonist pioglitazone demonstrated qualitative differences in gene expression profiles. Finally, telmisartan improved insulin sensitivity in diet-induced obese mice in the absence of weight gain. Taken together, we have identified two ARBs, telmisartan and irbesartan, that are already widely in clinical use as new selective PPAR modulators (SPPARMs).
RESEARCH DESIGN AND METHODS
3T3-L1 preadipocytes and COS-7 cells were purchased from the American Type Culture Collection. Cell culture reagents and lipofectamine 2000 were from Invitrogen. Trypsin and DMSO were purchased from Sigma. 35S-labeled methionine was purchased from Amersham. Irbesartan, telmisartan, eprosartan, pioglitazone, and rosiglitazone were kindly provided by the manufacturers. The plasmids DR1eC6 x PPAR response element (PPRE)-TkpGl3basic, hPPAR2-pSG5, and murine transcriptional intermediary factor-2 (TIF-2)-pSG5 were described elsewhere (14). Antibodies were purchased from Santa Cruz (PPAR [E-8] sc7273) and Abcam (glyceraldehyde-3-phosphate dehydrogenase, ab9485).
Cell culture and differentiation.
Mouse 3T3-L1 preadipocytes were cultured as described elsewhere (13). Adipocytes were serum deprived for 16 h and then treated with vehicle or various effectors as outlined. The isolation and differentiation of human preadipocytes is described elsewhere (15).
Quantitative real-time PCR.
RNA isolation, reverse transcription, and quantitation of gene expression were performed as previously described using an ABI 7000 sequence detection system for real-time PCR (13). Human glyceraldehyde-3-phosphate dehydrogenase and mouse mitochondrial subunit 18s RNA were chosen for real-time PCR as endogenous controls (housekeeping genes). Sequences of primers and probes are shown in the supplemental data.
Protein analysis.
Protein isolation was performed as previously described (16). Proteins were separated in 10% SDS polyacrylamide gels and transferred to polyvinylidene difluoride membrane (Amersham). After incubation with the primary antibody for PPAR at a 1:200 dilution, a secondary horseradish-conjugated antibody (Amersham) was added, and an enhanced chemiluminescent substrate kit (Amersham) was used for detection.
Assessment of receptor conformation by partial protease digestion.
Two microliters of [35S]-methionineeClabeled, full-length, human PPAR2 synthesized using the rabbit reticulocyte lysate and T7 RNA polymerase system (Promega) were preincubated with DMSO or the ligands for 60 min at 25°C in 40 e蘬 digestion buffer (20 mmol/l KCl, 10 mmol/l Tris-HCl, pH 8.0, and 10 mmol/l dithiothreitol). Samples were removed before and 10, 30, and 60 min after adding trypsin to 75 e蘥/ml final concentration. The protease digestion was immediately terminated by the addition of denaturing gel loading buffer and boiling for 10 min. The products of digestion were separated by electrophoresis through a 10% SDS polyacrylamide gel. After drying, the gel was analyzed by autoradiography.
GST pull-down.
The [35S]-methionineeClabeled cofactors TIF-2 (AA 412-813), the coactivators vitamin D interacting protein (DRIP205, AA 529-790), and NCoR (AA 1990-2310) were synthesized by using a Quick T7 TnT kit (Promega). After bacterial expression of GST-PPAR LBD (AA 209-505) fusion protein and subsequent lysis, supernatants were incubated with glutathione-Sepharose 4B beads (Amersham), and proteins were quantified by sodium SDS gel electrophoresis and coomassie blue staining. A total of 15 e蘥 GST-PPAR fusion protein were incubated with 4 e蘬 [35S]-methionineeClabeled nuclear receptor interacting domain cofactors in the presence of increasing amounts of compound or vehicle for 2 h at room temperature in GST binding buffer. Bound proteins were washed four times and boiled and separated by SDS-PAGE. Bands were visualized by autoradiography and quantified by a FLA-3000 PhosphorImager (Fujifilm).
FRET.
The cofactor peptides were synthesized and bound to biotin. GST-PPAR and cofactor peptides were labeled with allophycocyanin coupled to anti-GST antibody and R-phycoerythrin (RPE) coupled to streptavidine, respectively. Increasing amounts of compounds were incubated in the assay buffer with 35 nmol/l GST-PPAR, 26.3 nmol/l allophycocyanin-labeled anti-GST antibody, 1.25 nmol/l RPE strepavidine, and 5eC30 nmol/l of biotinylated cofactor peptide at 4°C for 4 h in 384-well plates. RPE was excited at 495 nm, and emission was measured at 635 nm (RPE emission) and at 670 nm (allophycocyanin emission). Fluorescence intensities were measured with a Genesis Freedom 200 (Tecan). Plots of fluorescence intensity (intensity at 670 nm/intensity at 635 nm) versus ligand concentration were calculated. Dose-response curves were done in triplicate using each cofactor.
Transfection and luciferase reporter assays.
Transient transfection and luciferase assays were performed as previously described (16). COS-7 cells were plated in 24-well dishes and transfected using lipofectamine 2000 with 150 ng DR1eC6 x PPRE-TkpGl3 basic; 20 ng hPPAR2-pSG5; 0, 50, 100, or 200 ng mTIF-2-pSG5; or the corresponding empty control vector up to 200 ng and 3 ng pRL-CMV, a renilla luciferase reporter vector. After 3 h, the medium was replaced by 10% fetal bovine serum Dulbecco’s modified Eagle’s medium plus the indicated ARBs, pioglitazone or vehicle (DMSO), and luciferase activities were measured after 36 h using the Dual-Luciferase Reporter Assay System (Promega). Transfection experiments were performed in triplicate and repeated at least three times.
Gene expression analysis.
After starvation of 3T3-L1 adipocytes for 16 h (Dulbecco’s modified Eagle’s medium with 0.5% fetal bovine serum), cells were incubated with the different ligands for 24 h. Isolated total RNA of three independent experiments was pooled. cRNA targets were amplified and fluorescently labeled with cyanine 3eCor cyanine 5eCCTP (Perkin Elmer/NEN Life Science) starting from 1 e蘥 of total RNA in each reaction using a Low RNA Input Fluorescent Linear Amplification Kit (Agilent). Yields of cRNA were determined by ultraviolet spectrometry (NanoDrop ND1000). Aliquots (1.2 e蘥) of labeled cRNA were combined and hybridized to Agilent mouse oligo microarrays (G4121A; Agilent) following the manufacturer’s protocol. Four independent measurements were carried out for each treatment using exchanged dye-labeled RNA probes (dye swap). After washing, the microarray slides were scanned using an Agilent microarray scanner (G2565BA), and image analysis was performed with Feature Extraction software 7.1.1 (Agilent). The array data are available as the NCBI Gene Expression Omnibus series "GSE2486."
Glucose uptake.
Day 8 adipocytes were incubated for 72 h with the indicated ligands. After washing, cells were incubated for 30 min ± 100 nmol/l insulin followed by addition of deoxy-D-glucose (Sigma) and 0.5 e藽i/ml deoxy-D-[2,6;3H] glucose (Amersham) in a final concentration of 0.1 mmol/l for the last 6 min. The uptake was stopped by adding ice-cold PBS containing 20 e蘥/ml cytochalasin B (Sigma). After cell lysis, radioactivity was measured. The glucose transporter independent uptake was assessed by performing the same procedure, having 10 e蘥/ml cytochalasin B present as a background value.
Animals.
Male C57BL/6J mice, aged 4eC5 weeks, were purchased from Harlan Winkelmann (Borchen, Germany). All mice were housed in a temperature-controlled (25°C) facility with a 12-h light/dark cycle. Mice were fed with a high-fat diet (60% kcal from fat) (17) for 16 weeks followed by randomization to either a vehicle-treated (0.5% Tween80/H2O), a pioglitazone-treated (10 mg · kgeC1 · dayeC1), or a telmisartan-treated (3 mg · kgeC1 · dayeC1) group. Animals were treated by oral gavage for 10 weeks. Body weight and food intake were determined throughout the experiment (twice a week). At the start and end of treatment, body composition was determined by nuclear magnetic resonance (Bruker’s Minispec MQ10). After 10 weeks’ treatment, blood samples were collected from overnight-fasted animals by retroorbital venous puncture under isofluran anesthesia for analysis of serum insulin (mouse-insulin ELISA; Linco Research, St. Charles, MO) and glucose (colorimetric glucose test; Cypress Diagnostics, Langdorp, Belgium). An oral glucose tolerance test was performed using a dose of 2 g/kg body wt glucose. After 1 week, an insulin tolerance test (ITT) was performed by intraperitoneally injecting 0.5 units/kg body wt insulin (Actrapid; Novo Nordisk). Tail vein blood was used for glucose quantification with a Glucometer (Precision Xtra; Abbott) during the oral glucose tolerance test and ITT. One week after the ITT, blood pressure was invasively measured in the abdominal aorta using a solid-state pressure transducer catheter (Micro-Tip 3F; Millar Instruments) under isoflurane anesthesia. Afterward, animals were killed and organs were dissected. All animal procedures were in accordance with institutional guidelines and were approved.
Statistical analysis.
Results from real-time PCR of cell lines, transfections, glucose uptakes, and animal experiments were analyzed by ANOVA followed by multiple comparison testing or with paired/unpaired t tests, as appropriate. Data are expressed as means ± SD or as indicated. Human data are presented as box and whisker plots and analyzed by an H test (Kruskal-Wallis) or U test (Mann-Whitney) as appropiate. Results were considered to be statistically significant at P < 0.05. For statistical analysis of microarray data, we used the significance analysis of microarrays (18).
RESULTS
PPAR-activating ARBs negatively autoregulate PPAR expression in murine and human adipocytes.
A specific property of agonists for certain nuclear receptors is the downregulation of the receptor on mRNA and/or protein level upon ligand activation (19,20,21). We therefore tested the ability of ARBs to downregulate PPAR in 3T3-L1 and human adipocytes. Telmisartan and irbesartan downregulated PPAR1 and 2 mRNA levels in 3T3-L1 adipocytes (Figs. 1A and B). Western blotting experiments revealed modestly reduced PPAR protein levels after telmisartan and irbesartan treatment (Fig. 1C). Eprosartan did not affect PPAR mRNA or protein expression (Figs. 1A and C). Consistently, human adipocytes showed reduced levels of PPAR mRNA levels after incubation with pioglitazone, telmisartan, and irbesartan (Fig. 1D). The noneCPPAR-activating ARB eprosartan had no effect (Fig. 1D). Together, these data demonstrate that PPAR-activating ARBs moderately downregulate PPAR expression, a typical characteristic of PPAR receptor ligands.
Telmisartan induces different PPAR conformation changes compared with a TZD-full PPAR agonist.
Conformational changes of the receptor upon ligand-binding followed by a higher resistance to proteolytic degradation in protease digestion assays provides one line of evidence for direct ligand-receptor interactions. Although this in vitro assay usually requires high ligand concentrations, which do not correlate to concentrations required for receptor activation in transactivation assays, it has been shown that the full agonist rosiglitazone induces a conformational change in PPAR, leading to a more compact structure of the receptor protecting the protein from trypsin digestion (22,23,24).
To test whether the activation of the PPAR LBD by ARBs is mediated via a direct interaction of ARBs with the PPAR protein, we compared the PPAR2 digestion pattern of rosiglitazone with that of telmisartan and irbesartan. Due to two starting sites, the plasmid gave rise to a double band of labeled human PPAR2. Rosiglitazone protected a 20- and 25-kDa band in a time-dependent digestion with trypsin compared with DMSO-incubated protein (Fig. 2A). In the presence of telmisartan, the resistant 25-kDa band was not protected but rather more susceptible to the protease. In contrast, incubation with irbesartan did not substantially influence the digestion pattern in the used concentration, which might result from the limited sensitivity of the digestion assay (Fig. 2B). Studying different concentrations of telmisartan, we observed that only concentrations >10 e蘭ol/l showed significant effects in regard to stability of the 25-kDa band (Fig. 2C). The results of the protease digestion suggest a direct binding of telmisartan to PPAR2, which induces a conformational change different to that elicited by rosiglitazone.
PPAR-activating ARBs change PPAR cofactor specificity.
Conformational changes of PPAR after ligand binding result in dissociation of corepressors and formation of coactivator complexes (25). We therefore tested the capacity of telmisartan and irbesartan to release the corepressor NCoR and to recruit the coactivators DRIP205 and TIF-2 to the PPAR LBD in GST pull-down assays. With increasing concentrations of TZDs, the amount of PPAR-bound NCoR decreased, and DRIP205 and TIF-2 were recruited (Fig. 3A). In contrast, ARBs exhibited only a slight effect on NCoR release and enhanced the interactions with DRIP205 with a lower potency than TZDs. Surprisingly, recruitment of TIF-2 was absent with ARB treatment in GST pull-down assays (Fig. 3A). To confirm these results, we performed FRET assays with peptides of the same cofactors. Rosiglitazone showed release of NCoR and strong recruitment of DRIP205 and TIF-2 (Fig. 3B). The slight release of NCoR by ARBs in the GST pull-down experiments was confirmed showing dissociation of NCoR from PPAR starting at 3 e蘭ol/l up to a maximum of 50% of the rosiglitazone-induced effect at 100 e蘭ol/l. Both compounds potently recruited DRIP205, reaching a plateau at 10 e蘭ol/l. Consistent with the pull-down experiments, telmisartan was not able to induce recruitment of TIF-2, whereas irbesartan had a modest effect on TIF-2 recruitment at high concentrations (Fig. 3B). In line with a distinct conformational change of the receptor, characteristics of the ARBs in terms of cofactor recruitment differ from the TZD moiety with less efficiency to release NCoR and no, or rather slight, recruitment of TIF-2 to the PPAR LBD. These results suggest that PPAR-activating ARBs induce a conformation of PPAR that is less permissive for dissociation of NCoR and configured for association with DRIP205, but not with TIF-2, characterizing these compounds as agonists inducing selective cofactor docking.
Differential recruitment of TIF-2 is functionally relevant.
To prove functional relevance of the observed ligand specificity for TIF-2, COS-7 cells were transiently transfected with PPAR2, a PPRE-dependent luciferase reporter vector, and increasing amounts of TIF-2 followed by stimulation with the ligands. In the absence of ectopic cofactor, pioglitazone (1 e蘭ol/l), but also telmisartan and irbesartan (both 10 e蘭ol/l), increased transcriptional activity of PPAR (Fig. 4). Transfection of increasing amounts of TIF-2 led to an increase of basal ligand-independent activation, which was potentiated by pioglitazone and by irbesartan at the highest concentrations. Transcritptional activity induced by telmisartan did not benefit from ectopic cofactor expression and was equal to ligand-independent activation (Fig. 4). These data demonstrate the functional relevance of selective cofactor docking by ARBs.
Divergent gene expression induced by pioglitazone and the ARBs in 3T3-L1 adipocytes.
Given the selective agonism with distinct cofactor docking by ARBs, we hypothesized that ARBs elicit a different gene expression profile compared with TZDs in 3T3-L1 adipocytes. Cells were treated with pioglitazone, telmisartan, or irbesartan for 24 h, and pooled total RNA was hybridized on Agilent oligonucleotide microarrays. Genes with a false discovery rate of 1% (significance analysis of microarrays), when drug treatment and controls were compared, were considered as significantly deregulated. On the basis of functional relevance in regard to PPAR and fat-cell physiology, a selection of these genes with a significant deregulation in at least one drug treatment was hierarchically clustered (26). Differential expression was confirmed by real-time PCR analysis for six different genes (Fig. 5B). Although in different magnitudes, most of the genes with identified PPREs in their promoters were similarly regulated by pioglitazone and the ARBs (Figs. 5A and B). Consistently with distinctive cofactor recruitment by ARBs, numerous genes involved in adipocyte function were differentially regulated by the compounds compared with the full agonist pioglitazone (Figs. 5A and B). Interestingly, there were also differences in the regulation of genes between irbesartan and telmisartan, suggesting additional differences in PPAR activation among these ARBs. These data implicate that selective cofactor binding induced by ARBs results in distinctive gene expression profiles identifying these compounds as selective PPAR ligands.
Alterations in deoxyglucose uptake due to specific gene expression profile.
Next, we wanted to know whether selective PPAR activation by ARBs retains cellular metabolic functions of receptor activation. Therefore, we measured insulin-stimulated and insulin-independent incorporation of 3H-labeled deoxyglucose in 3T3-L1 adipocytes treated with the full agonist pioglitazone, the selective ARB ligands telmisartan and irbesartan, or the noneCPPAR-activating ARB eprosartan (Fig. 6). After 72 h of incubation, pioglitazone, telmisartan, and irbesartan significantly increased insulin-dependent and -independent deoxyglucose uptake, whereas eprosartan had no effect (Fig. 6).
Telmisartan improves insulin sensitivity in diet-induced obese mice without weight gain.
To assess the effects of PPAR-activating ARBs on insulin sensitivity, glucose tolerance, and body weight, we treated diet-induced obese C57BL/6J mice for 10 weeks with vehicle, telmisartan (3 mg · kgeC1 · dayeC1) or the full PPAR agonist pioglitazone (10 mg · kgeC1 · dayeC1). Consistent with the PPAR-modulating activity of telmisartan, animals treated with telmisartan gained significantly less weight compared with mice receiving vehicle or pioglitazone (Fig. 7A). Food intake was unaffected by telmisartan and pioglitazone (Fig. 7B). Mean arterial blood pressure was lower in the telmisartan group (vehicle: 89 ± 2.1 mmHg, telmisartan: 62.6 ± 2.5 mmHg [P < 0.05 vs.vehicle], pioglitazone: 90.8 ± 1.8 mmHg). Both treatment regimens significantly lowered fasting insulin and glucose levels compared with vehicle (insulin: vehicle: 4.2 ± 0.7 ng/ml, telmisartan: 2.5 ± 0.4 ng/ml [P < 0.05 vs. vehicle], pioglitazone: 1.3 ± 0.2 ng/ml [P < 0.01 vs. vehicle]; glucose: vehicle: 209 ± 18 mg/dl, telmisartan: 178 ± 5 mg/dl, pioglitazone: 154 ± 13 mg/dl [both P < 0.05 vs. vehicle]). Glucose tolerance and insulin sensitivity were significantly improved in the group treated with the ARB/SPPARM telmisartan and in the pioglitazone-treated mice (Figs. 7C and D). Analysis of body composition by nuclear magnetic resonance revealed that differences in body weight are predominantly mediated by changes in body fat. Telmisartan decreased body fat content, whereas pioglitazone treatment augmented fat accumulation (Fig. 7E). Together, these data demonstrate that telmisartan improves insulin sensitivity in the absence of weight gain, providing in vivo evidence for its SPPARM activity.
DISCUSSION
Treatment of obese and insulin-resistant type 2 diabetic patients using PPAR agonists such as rosiglitazone or pioglitazone leads to insulin sensitization and improved glycemic control. However, in parallel, exacerbation of weight gain and a number of adverse effects are frequently encountered. Data from hemizygous PPAR-null mice, TZD-treated wild-type animals, and humans carrying PPAR mutations have suggested that neither full agonism nor full antagonism of PPAR provide the optimal approach to treat metabolic disorders (27,28,29). Therefore, the identification and development of improved PPAR ligands with a moderate and more specific mode of activation remains an important objective.
A promising new group of such ligands are SPPARMs, which are compounds that, based on receptor-cofactor interactions, activate only a subset of the functions induced by cognate ligands or act in a cell-type selective manner. Recently, Berger et al. (23) described a non-TZD partial agonist as a new SPPARM in preclinical studies. In accordance with the non-TZD partial agonist, we identified certain ARBs as partial agonists, which induce distinctive conformational receptor changes after binding. Irbesartan, in concentrations similar to telmisartan, failed to induce major changes of the PPAR protein digestion pattern, which might be a result of the limited sensitivity of the protease assay. However, irbesartan prominently regulated PPAR cofactor binding and induced selective gene expression, implicating that both ARBs, telmisartan and irbesartan, interact with PPAR and exert SPPARM activity.
In line with the specific receptor conformation, ARBs selectively regulated adipocyte gene expression. Comparisons with gene expression patterns induced by the full agonist pioglitazone demonstrated that ARB-mediated regulation of genes involved in adipocyte lipid metabolism was moderate compared with that of the glitazone. Consistenly with different effects of the ARBs on the conformational change, there was also distinct gene regulation between telmisartan and irbesartan. Some of these genes were chosen for further quantitative analysis. The prostacyclin receptor is an important inducer of adipogenesis (30,31). Microarray and quantitative PCR analysis demonstrated that pioglitazone, but not the ARBs, potently enhanced expression of the prostacyclin receptor. Another important mediator of adipocytic lipid storage is glycerol kinase, which is potently stimulated by TZDs (32). Stimulation of glycerol kinase by ARBs was much weaker compared with that of pioglitazone. Together, we identified an important characteristic of the distinctive gene expression pattern induced by PPAR-activating ARBs in adipocytes, which comprises a modest induction of adipogenic genes compared with the full glitazone agonists. This kind of selective gene regulation attenuates PPAR-mediated adipogenesis, which may prevent weight gain during ligand therapy.
Selective cofactor dissociation/binding provides the molecular link between ligand-induced conformational changes of PPAR and selective gene regulation, which then triggers specific metabolic in vitro/in vivo effects (23,33,34). The release of the corepressor NCoR at saturating concentrations was less pronounced with ARBs than with TZDs, which points toward their weaker adipogenic potential due to the inhibitory effect of NCoR in the differentiation process (35). Mice deficient for the PPAR coactivator TIF-2 are protected against diet-induced obesity with an improvement of whole-body insulin sensitivity (36). Altered gene expression patterns as a result of TIF-2 deficiency were associated with a reduction of fatty acid storage in adipose tissue (36). These data demonstrate that TIF-2 is a PPAR coactivator mainly involved in the adipogenic/lipogenic actions of the receptor and suggests that a TIF-2 independent activation of PPAR has beneficial effects on body weight and metabolic parameters. Attenuated TIF-2 recruitment by ARBs may provide the molecular explanation for the modest induction of adipogenic genes in the gene expression analysis.
To prove the relevance of the SPPARM activity of ARBs in vivo, diet-induced obese mice were treated with telmisartan or pioglitazone. Both substances attenuated the obesity-mediated insulin resistance. However, only telmisartan was able to lower body weight in these animals, which was mainly mediated by a decrease in total body fat. These data are consistent with the study by Berger et al. (23), in which a newly identified SPPARM significantly improved parameters of insulin sensitivity without promotion of weight gain in diet-induced obese mice.
A number of clinical studies have already been conducted to provide clinical evidence for the beneficial metabolic effects of PPAR-modulating ARBs in type 2 diabetic patients. Recently, Miura et al. (37) showed that by switching hypertensive type 2 diabetic patients from ARBs with no clinically relevant PPAR activity to the PPAR-modulating ARB telmisartan, a significant improvement of multiple metabolic parameters could be observed, including a decrease in fasting insulin and elevation of serum adiponectin levels. Body weight gain and edema were not detected in this study (37). In addition, in a double-blind, placebo-controlled trial in 119 diabetic patients with mild hypertension, only telmisartan significantly lowered plasma triglyceride levels, whereas the noneCPPAR-activating ARB eprosartan had no effect (10). However, in this study no change in glucose metabolism could be observed in the treatment groups (10). Together, these studies demonstrate that PPAR-modulating ARBs are capable of retaining the beneficial metabolic PPAR effects in vivo and in diabetic patients, which is associated with the circumvention of adverse effects such as weight gain and edema.
In conclusion, the present study characterizes two PPAR-activating ARBs, telmisartan and irbesartan, as new SPPARMs. Selective PPAR modulation by ARBs includes a distinct conformational change of the receptor associated with selective cofactor recruitment and a distinctive gene expression profile with a modest adipogenic pattern. Selective PPAR modulation by ARBs may reduce the weight-promoting effects of PPAR activation and in parallel retains PPAR-mediated metabolic efficacy, providing a new therapeutic option for patients suffering from diabetes or the metabolic syndrome. Furthermore, the molecular characterization of these compounds will open new perspectives to develop future substances acting as SPPARM-ARBs.
ACKNOWLEDGMENTS
M.C., T.U., and U.K. are supported by the German Research Society (DFG-GK 754-II).
We thank H.J. Mollenkopf and J. Angermann (Max Planck Institute for Infection Biology) for help with the microarray experiments and A. Foryst-Ludwig, M. Hartge, and C. Sprang for their technical help with the animal experiments.
FOOTNOTES
M.S. and M.C. contributed equally to this article.
M.S. has received honoraria from Bayer. T.U. has been on an advisory panel for Boehringer Ingelheim, Sanofi-Aventis, and Takeda; has received honoraria from Bayer, Boehringer Ingelheim, Sanofi-Aventis, and Takeda; and has received grant/research support from Bayer, Boehringer Ingelheim, Sanofi-Aventis, and Takeda. U.K. has received honoraria from Bayer Boehringer Ingelheim, Sanofi-Aventis, and Takeda and has received grant/research support from Boehringer Ingelheim.
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2 GENFIT, Parc Eurasante, Loos, France
3 Max-Planck Institute for Molecular Genetics, Berlin, Germany
4 Franz Volhard Clinic, HELIOS Hospital Berlin, Charitee Campus Buch, Charitee-Universittsmedizin Berlin, Berlin, Germany
5 Department of Atherosclerose, Faculty of Pharmacy, Institute Pasteur Lille, University Lille II, UR 545 Institut National de la Santee et de la Recherche Meedicale, Lille, France
ARB, angiotensin type 1 receptor blocker; AT1R, angiotensin type 1 receptor; FRET, fluorescence resonance energy transfer; GST, glutathione S-transferase; ITT, insulin tolerance test; LBD, ligand binding domain; NCoR, nuclear receptor corepressor; PPAR, peroxisome proliferatoreCactivated receptor; PPRE, PPAR response element; RPE, R-phycoerythrin; SPPARM, selective PPAR modulators; TIF-2, transcriptional intermediary factor-2; TZD, thiazolidinedione
ABSTRACT
Selective peroxisome proliferatoreCactivated receptor (PPAR) modulation is a new pharmacological approach that, based on selective receptor-cofactor interactions and target gene regulation, should result in potent insulin sensitization in the absence of PPAR-mediated adverse effects. Here, we characterize two angiotensin receptor blockers (ARBs), telmisartan and irbesartan, as new selective PPAR modulators (SPPARMs). Analysis of PPAR protein conformation using protease protection showed that telmisartan directly interacts with the receptor, producing a distinct conformational change compared with a glitazone. Glutathione S-transferase pull-down and fluorescence resonance energy transfer assays revealed selective cofactor binding by the ARBs compared with glitazones with an attenuated release of the nuclear receptor corepressor and absence of transcriptional intermediary factor 2 recruitment by ARBs. Consistently, selective cofactor binding resulted in differential gene expression profiles in adipocytes (ARB versus glitazone treated) assessed by oligo microarray analysis. Finally, telmisartan improved insulin sensitivity in diet-induced obese mice in the absence of weight gain. The present study identifies two ARBs as new SPPARMs. SPPARM activity by ARBs could retain the metabolic efficacy of PPAR activation with reduction in adverse effects exerting in parallel AT1 receptor blockade. This may provide a new therapeutic option for better cardiovascular risk management in metabolic diseases and may initiate the development of new classes of drugs combining potent antihypertensive and antidiabetic actions.
The peroxisome proliferatoreCactivated receptors (PPARs) are members of the nuclear receptor superfamily of ligand-activated transcription factors (1). A series of coactivators/corepressors are involved in ligand-induced transcription of genes allowing a tissue- and ligand-specific activation of these target genes by PPARs (2). The most abundant isoform in adipose tissue, PPAR, plays an important role in the regulation of insulin sensitivity (3). Glitazones or thiazolidinediones (TZDs) are high-affinity ligands for this receptor and are currently used in the treatment of type 2 diabetes (4). Together with an improvement of insulin sensitivity and glucose tolerance, they improve lipid profiles (5). Given the side effects of glitazones, like weight gain, edema, and fluid retention, the characterization of new PPAR ligands that retain metabolic efficacy without exerting adverse actions plays a central role in the development of new therapeutic strategies for insulin resistance and type 2 diabetes (6).
Angiotensin type 1 receptor (AT1R) blockers (ARBs) prevent the binding of angiotensin II to the AT1R and are widely used for the treatment of hypertension and related cardiovascular and organ damage (7). Recent clinical trials revealed a substantial lower risk in developing type 2 diabetes for patients treated with ARBs in comparison with other hypertensive treatment (8). In addition, the ARB telmisartan has been shown to improve metabolic parameters in rodents and type 2 diabetic patients (9,10,11). Recently, a subgroup of ARBs has been characterized as PPAR ligands independent of their AT1R blocking actions (11,12,13). Although designed to bind to the AT1R, these ARBs have been shown to activate the PPAR ligand binding domain (LBD) in the e蘭ol/l range (11,13).
The underlying molecular mechanisms of PPAR activation by ARBs are unknown. Here, we characterize the molecular interactions between PPAR and two PPAR-activating ARBs, telmisartan and irbesartan. Glutathione S-transferase (GST) pull-down and fluorescence resonance energy transfer (FRET) assays revealed selective cofactor recruitment by ARBs. Oligo microarray analysis of RNA from 3T3-L1 adipocytes treated with the ARBs or the full agonist pioglitazone demonstrated qualitative differences in gene expression profiles. Finally, telmisartan improved insulin sensitivity in diet-induced obese mice in the absence of weight gain. Taken together, we have identified two ARBs, telmisartan and irbesartan, that are already widely in clinical use as new selective PPAR modulators (SPPARMs).
RESEARCH DESIGN AND METHODS
3T3-L1 preadipocytes and COS-7 cells were purchased from the American Type Culture Collection. Cell culture reagents and lipofectamine 2000 were from Invitrogen. Trypsin and DMSO were purchased from Sigma. 35S-labeled methionine was purchased from Amersham. Irbesartan, telmisartan, eprosartan, pioglitazone, and rosiglitazone were kindly provided by the manufacturers. The plasmids DR1eC6 x PPAR response element (PPRE)-TkpGl3basic, hPPAR2-pSG5, and murine transcriptional intermediary factor-2 (TIF-2)-pSG5 were described elsewhere (14). Antibodies were purchased from Santa Cruz (PPAR [E-8] sc7273) and Abcam (glyceraldehyde-3-phosphate dehydrogenase, ab9485).
Cell culture and differentiation.
Mouse 3T3-L1 preadipocytes were cultured as described elsewhere (13). Adipocytes were serum deprived for 16 h and then treated with vehicle or various effectors as outlined. The isolation and differentiation of human preadipocytes is described elsewhere (15).
Quantitative real-time PCR.
RNA isolation, reverse transcription, and quantitation of gene expression were performed as previously described using an ABI 7000 sequence detection system for real-time PCR (13). Human glyceraldehyde-3-phosphate dehydrogenase and mouse mitochondrial subunit 18s RNA were chosen for real-time PCR as endogenous controls (housekeeping genes). Sequences of primers and probes are shown in the supplemental data.
Protein analysis.
Protein isolation was performed as previously described (16). Proteins were separated in 10% SDS polyacrylamide gels and transferred to polyvinylidene difluoride membrane (Amersham). After incubation with the primary antibody for PPAR at a 1:200 dilution, a secondary horseradish-conjugated antibody (Amersham) was added, and an enhanced chemiluminescent substrate kit (Amersham) was used for detection.
Assessment of receptor conformation by partial protease digestion.
Two microliters of [35S]-methionineeClabeled, full-length, human PPAR2 synthesized using the rabbit reticulocyte lysate and T7 RNA polymerase system (Promega) were preincubated with DMSO or the ligands for 60 min at 25°C in 40 e蘬 digestion buffer (20 mmol/l KCl, 10 mmol/l Tris-HCl, pH 8.0, and 10 mmol/l dithiothreitol). Samples were removed before and 10, 30, and 60 min after adding trypsin to 75 e蘥/ml final concentration. The protease digestion was immediately terminated by the addition of denaturing gel loading buffer and boiling for 10 min. The products of digestion were separated by electrophoresis through a 10% SDS polyacrylamide gel. After drying, the gel was analyzed by autoradiography.
GST pull-down.
The [35S]-methionineeClabeled cofactors TIF-2 (AA 412-813), the coactivators vitamin D interacting protein (DRIP205, AA 529-790), and NCoR (AA 1990-2310) were synthesized by using a Quick T7 TnT kit (Promega). After bacterial expression of GST-PPAR LBD (AA 209-505) fusion protein and subsequent lysis, supernatants were incubated with glutathione-Sepharose 4B beads (Amersham), and proteins were quantified by sodium SDS gel electrophoresis and coomassie blue staining. A total of 15 e蘥 GST-PPAR fusion protein were incubated with 4 e蘬 [35S]-methionineeClabeled nuclear receptor interacting domain cofactors in the presence of increasing amounts of compound or vehicle for 2 h at room temperature in GST binding buffer. Bound proteins were washed four times and boiled and separated by SDS-PAGE. Bands were visualized by autoradiography and quantified by a FLA-3000 PhosphorImager (Fujifilm).
FRET.
The cofactor peptides were synthesized and bound to biotin. GST-PPAR and cofactor peptides were labeled with allophycocyanin coupled to anti-GST antibody and R-phycoerythrin (RPE) coupled to streptavidine, respectively. Increasing amounts of compounds were incubated in the assay buffer with 35 nmol/l GST-PPAR, 26.3 nmol/l allophycocyanin-labeled anti-GST antibody, 1.25 nmol/l RPE strepavidine, and 5eC30 nmol/l of biotinylated cofactor peptide at 4°C for 4 h in 384-well plates. RPE was excited at 495 nm, and emission was measured at 635 nm (RPE emission) and at 670 nm (allophycocyanin emission). Fluorescence intensities were measured with a Genesis Freedom 200 (Tecan). Plots of fluorescence intensity (intensity at 670 nm/intensity at 635 nm) versus ligand concentration were calculated. Dose-response curves were done in triplicate using each cofactor.
Transfection and luciferase reporter assays.
Transient transfection and luciferase assays were performed as previously described (16). COS-7 cells were plated in 24-well dishes and transfected using lipofectamine 2000 with 150 ng DR1eC6 x PPRE-TkpGl3 basic; 20 ng hPPAR2-pSG5; 0, 50, 100, or 200 ng mTIF-2-pSG5; or the corresponding empty control vector up to 200 ng and 3 ng pRL-CMV, a renilla luciferase reporter vector. After 3 h, the medium was replaced by 10% fetal bovine serum Dulbecco’s modified Eagle’s medium plus the indicated ARBs, pioglitazone or vehicle (DMSO), and luciferase activities were measured after 36 h using the Dual-Luciferase Reporter Assay System (Promega). Transfection experiments were performed in triplicate and repeated at least three times.
Gene expression analysis.
After starvation of 3T3-L1 adipocytes for 16 h (Dulbecco’s modified Eagle’s medium with 0.5% fetal bovine serum), cells were incubated with the different ligands for 24 h. Isolated total RNA of three independent experiments was pooled. cRNA targets were amplified and fluorescently labeled with cyanine 3eCor cyanine 5eCCTP (Perkin Elmer/NEN Life Science) starting from 1 e蘥 of total RNA in each reaction using a Low RNA Input Fluorescent Linear Amplification Kit (Agilent). Yields of cRNA were determined by ultraviolet spectrometry (NanoDrop ND1000). Aliquots (1.2 e蘥) of labeled cRNA were combined and hybridized to Agilent mouse oligo microarrays (G4121A; Agilent) following the manufacturer’s protocol. Four independent measurements were carried out for each treatment using exchanged dye-labeled RNA probes (dye swap). After washing, the microarray slides were scanned using an Agilent microarray scanner (G2565BA), and image analysis was performed with Feature Extraction software 7.1.1 (Agilent). The array data are available as the NCBI Gene Expression Omnibus series "GSE2486."
Glucose uptake.
Day 8 adipocytes were incubated for 72 h with the indicated ligands. After washing, cells were incubated for 30 min ± 100 nmol/l insulin followed by addition of deoxy-D-glucose (Sigma) and 0.5 e藽i/ml deoxy-D-[2,6;3H] glucose (Amersham) in a final concentration of 0.1 mmol/l for the last 6 min. The uptake was stopped by adding ice-cold PBS containing 20 e蘥/ml cytochalasin B (Sigma). After cell lysis, radioactivity was measured. The glucose transporter independent uptake was assessed by performing the same procedure, having 10 e蘥/ml cytochalasin B present as a background value.
Animals.
Male C57BL/6J mice, aged 4eC5 weeks, were purchased from Harlan Winkelmann (Borchen, Germany). All mice were housed in a temperature-controlled (25°C) facility with a 12-h light/dark cycle. Mice were fed with a high-fat diet (60% kcal from fat) (17) for 16 weeks followed by randomization to either a vehicle-treated (0.5% Tween80/H2O), a pioglitazone-treated (10 mg · kgeC1 · dayeC1), or a telmisartan-treated (3 mg · kgeC1 · dayeC1) group. Animals were treated by oral gavage for 10 weeks. Body weight and food intake were determined throughout the experiment (twice a week). At the start and end of treatment, body composition was determined by nuclear magnetic resonance (Bruker’s Minispec MQ10). After 10 weeks’ treatment, blood samples were collected from overnight-fasted animals by retroorbital venous puncture under isofluran anesthesia for analysis of serum insulin (mouse-insulin ELISA; Linco Research, St. Charles, MO) and glucose (colorimetric glucose test; Cypress Diagnostics, Langdorp, Belgium). An oral glucose tolerance test was performed using a dose of 2 g/kg body wt glucose. After 1 week, an insulin tolerance test (ITT) was performed by intraperitoneally injecting 0.5 units/kg body wt insulin (Actrapid; Novo Nordisk). Tail vein blood was used for glucose quantification with a Glucometer (Precision Xtra; Abbott) during the oral glucose tolerance test and ITT. One week after the ITT, blood pressure was invasively measured in the abdominal aorta using a solid-state pressure transducer catheter (Micro-Tip 3F; Millar Instruments) under isoflurane anesthesia. Afterward, animals were killed and organs were dissected. All animal procedures were in accordance with institutional guidelines and were approved.
Statistical analysis.
Results from real-time PCR of cell lines, transfections, glucose uptakes, and animal experiments were analyzed by ANOVA followed by multiple comparison testing or with paired/unpaired t tests, as appropriate. Data are expressed as means ± SD or as indicated. Human data are presented as box and whisker plots and analyzed by an H test (Kruskal-Wallis) or U test (Mann-Whitney) as appropiate. Results were considered to be statistically significant at P < 0.05. For statistical analysis of microarray data, we used the significance analysis of microarrays (18).
RESULTS
PPAR-activating ARBs negatively autoregulate PPAR expression in murine and human adipocytes.
A specific property of agonists for certain nuclear receptors is the downregulation of the receptor on mRNA and/or protein level upon ligand activation (19,20,21). We therefore tested the ability of ARBs to downregulate PPAR in 3T3-L1 and human adipocytes. Telmisartan and irbesartan downregulated PPAR1 and 2 mRNA levels in 3T3-L1 adipocytes (Figs. 1A and B). Western blotting experiments revealed modestly reduced PPAR protein levels after telmisartan and irbesartan treatment (Fig. 1C). Eprosartan did not affect PPAR mRNA or protein expression (Figs. 1A and C). Consistently, human adipocytes showed reduced levels of PPAR mRNA levels after incubation with pioglitazone, telmisartan, and irbesartan (Fig. 1D). The noneCPPAR-activating ARB eprosartan had no effect (Fig. 1D). Together, these data demonstrate that PPAR-activating ARBs moderately downregulate PPAR expression, a typical characteristic of PPAR receptor ligands.
Telmisartan induces different PPAR conformation changes compared with a TZD-full PPAR agonist.
Conformational changes of the receptor upon ligand-binding followed by a higher resistance to proteolytic degradation in protease digestion assays provides one line of evidence for direct ligand-receptor interactions. Although this in vitro assay usually requires high ligand concentrations, which do not correlate to concentrations required for receptor activation in transactivation assays, it has been shown that the full agonist rosiglitazone induces a conformational change in PPAR, leading to a more compact structure of the receptor protecting the protein from trypsin digestion (22,23,24).
To test whether the activation of the PPAR LBD by ARBs is mediated via a direct interaction of ARBs with the PPAR protein, we compared the PPAR2 digestion pattern of rosiglitazone with that of telmisartan and irbesartan. Due to two starting sites, the plasmid gave rise to a double band of labeled human PPAR2. Rosiglitazone protected a 20- and 25-kDa band in a time-dependent digestion with trypsin compared with DMSO-incubated protein (Fig. 2A). In the presence of telmisartan, the resistant 25-kDa band was not protected but rather more susceptible to the protease. In contrast, incubation with irbesartan did not substantially influence the digestion pattern in the used concentration, which might result from the limited sensitivity of the digestion assay (Fig. 2B). Studying different concentrations of telmisartan, we observed that only concentrations >10 e蘭ol/l showed significant effects in regard to stability of the 25-kDa band (Fig. 2C). The results of the protease digestion suggest a direct binding of telmisartan to PPAR2, which induces a conformational change different to that elicited by rosiglitazone.
PPAR-activating ARBs change PPAR cofactor specificity.
Conformational changes of PPAR after ligand binding result in dissociation of corepressors and formation of coactivator complexes (25). We therefore tested the capacity of telmisartan and irbesartan to release the corepressor NCoR and to recruit the coactivators DRIP205 and TIF-2 to the PPAR LBD in GST pull-down assays. With increasing concentrations of TZDs, the amount of PPAR-bound NCoR decreased, and DRIP205 and TIF-2 were recruited (Fig. 3A). In contrast, ARBs exhibited only a slight effect on NCoR release and enhanced the interactions with DRIP205 with a lower potency than TZDs. Surprisingly, recruitment of TIF-2 was absent with ARB treatment in GST pull-down assays (Fig. 3A). To confirm these results, we performed FRET assays with peptides of the same cofactors. Rosiglitazone showed release of NCoR and strong recruitment of DRIP205 and TIF-2 (Fig. 3B). The slight release of NCoR by ARBs in the GST pull-down experiments was confirmed showing dissociation of NCoR from PPAR starting at 3 e蘭ol/l up to a maximum of 50% of the rosiglitazone-induced effect at 100 e蘭ol/l. Both compounds potently recruited DRIP205, reaching a plateau at 10 e蘭ol/l. Consistent with the pull-down experiments, telmisartan was not able to induce recruitment of TIF-2, whereas irbesartan had a modest effect on TIF-2 recruitment at high concentrations (Fig. 3B). In line with a distinct conformational change of the receptor, characteristics of the ARBs in terms of cofactor recruitment differ from the TZD moiety with less efficiency to release NCoR and no, or rather slight, recruitment of TIF-2 to the PPAR LBD. These results suggest that PPAR-activating ARBs induce a conformation of PPAR that is less permissive for dissociation of NCoR and configured for association with DRIP205, but not with TIF-2, characterizing these compounds as agonists inducing selective cofactor docking.
Differential recruitment of TIF-2 is functionally relevant.
To prove functional relevance of the observed ligand specificity for TIF-2, COS-7 cells were transiently transfected with PPAR2, a PPRE-dependent luciferase reporter vector, and increasing amounts of TIF-2 followed by stimulation with the ligands. In the absence of ectopic cofactor, pioglitazone (1 e蘭ol/l), but also telmisartan and irbesartan (both 10 e蘭ol/l), increased transcriptional activity of PPAR (Fig. 4). Transfection of increasing amounts of TIF-2 led to an increase of basal ligand-independent activation, which was potentiated by pioglitazone and by irbesartan at the highest concentrations. Transcritptional activity induced by telmisartan did not benefit from ectopic cofactor expression and was equal to ligand-independent activation (Fig. 4). These data demonstrate the functional relevance of selective cofactor docking by ARBs.
Divergent gene expression induced by pioglitazone and the ARBs in 3T3-L1 adipocytes.
Given the selective agonism with distinct cofactor docking by ARBs, we hypothesized that ARBs elicit a different gene expression profile compared with TZDs in 3T3-L1 adipocytes. Cells were treated with pioglitazone, telmisartan, or irbesartan for 24 h, and pooled total RNA was hybridized on Agilent oligonucleotide microarrays. Genes with a false discovery rate of 1% (significance analysis of microarrays), when drug treatment and controls were compared, were considered as significantly deregulated. On the basis of functional relevance in regard to PPAR and fat-cell physiology, a selection of these genes with a significant deregulation in at least one drug treatment was hierarchically clustered (26). Differential expression was confirmed by real-time PCR analysis for six different genes (Fig. 5B). Although in different magnitudes, most of the genes with identified PPREs in their promoters were similarly regulated by pioglitazone and the ARBs (Figs. 5A and B). Consistently with distinctive cofactor recruitment by ARBs, numerous genes involved in adipocyte function were differentially regulated by the compounds compared with the full agonist pioglitazone (Figs. 5A and B). Interestingly, there were also differences in the regulation of genes between irbesartan and telmisartan, suggesting additional differences in PPAR activation among these ARBs. These data implicate that selective cofactor binding induced by ARBs results in distinctive gene expression profiles identifying these compounds as selective PPAR ligands.
Alterations in deoxyglucose uptake due to specific gene expression profile.
Next, we wanted to know whether selective PPAR activation by ARBs retains cellular metabolic functions of receptor activation. Therefore, we measured insulin-stimulated and insulin-independent incorporation of 3H-labeled deoxyglucose in 3T3-L1 adipocytes treated with the full agonist pioglitazone, the selective ARB ligands telmisartan and irbesartan, or the noneCPPAR-activating ARB eprosartan (Fig. 6). After 72 h of incubation, pioglitazone, telmisartan, and irbesartan significantly increased insulin-dependent and -independent deoxyglucose uptake, whereas eprosartan had no effect (Fig. 6).
Telmisartan improves insulin sensitivity in diet-induced obese mice without weight gain.
To assess the effects of PPAR-activating ARBs on insulin sensitivity, glucose tolerance, and body weight, we treated diet-induced obese C57BL/6J mice for 10 weeks with vehicle, telmisartan (3 mg · kgeC1 · dayeC1) or the full PPAR agonist pioglitazone (10 mg · kgeC1 · dayeC1). Consistent with the PPAR-modulating activity of telmisartan, animals treated with telmisartan gained significantly less weight compared with mice receiving vehicle or pioglitazone (Fig. 7A). Food intake was unaffected by telmisartan and pioglitazone (Fig. 7B). Mean arterial blood pressure was lower in the telmisartan group (vehicle: 89 ± 2.1 mmHg, telmisartan: 62.6 ± 2.5 mmHg [P < 0.05 vs.vehicle], pioglitazone: 90.8 ± 1.8 mmHg). Both treatment regimens significantly lowered fasting insulin and glucose levels compared with vehicle (insulin: vehicle: 4.2 ± 0.7 ng/ml, telmisartan: 2.5 ± 0.4 ng/ml [P < 0.05 vs. vehicle], pioglitazone: 1.3 ± 0.2 ng/ml [P < 0.01 vs. vehicle]; glucose: vehicle: 209 ± 18 mg/dl, telmisartan: 178 ± 5 mg/dl, pioglitazone: 154 ± 13 mg/dl [both P < 0.05 vs. vehicle]). Glucose tolerance and insulin sensitivity were significantly improved in the group treated with the ARB/SPPARM telmisartan and in the pioglitazone-treated mice (Figs. 7C and D). Analysis of body composition by nuclear magnetic resonance revealed that differences in body weight are predominantly mediated by changes in body fat. Telmisartan decreased body fat content, whereas pioglitazone treatment augmented fat accumulation (Fig. 7E). Together, these data demonstrate that telmisartan improves insulin sensitivity in the absence of weight gain, providing in vivo evidence for its SPPARM activity.
DISCUSSION
Treatment of obese and insulin-resistant type 2 diabetic patients using PPAR agonists such as rosiglitazone or pioglitazone leads to insulin sensitization and improved glycemic control. However, in parallel, exacerbation of weight gain and a number of adverse effects are frequently encountered. Data from hemizygous PPAR-null mice, TZD-treated wild-type animals, and humans carrying PPAR mutations have suggested that neither full agonism nor full antagonism of PPAR provide the optimal approach to treat metabolic disorders (27,28,29). Therefore, the identification and development of improved PPAR ligands with a moderate and more specific mode of activation remains an important objective.
A promising new group of such ligands are SPPARMs, which are compounds that, based on receptor-cofactor interactions, activate only a subset of the functions induced by cognate ligands or act in a cell-type selective manner. Recently, Berger et al. (23) described a non-TZD partial agonist as a new SPPARM in preclinical studies. In accordance with the non-TZD partial agonist, we identified certain ARBs as partial agonists, which induce distinctive conformational receptor changes after binding. Irbesartan, in concentrations similar to telmisartan, failed to induce major changes of the PPAR protein digestion pattern, which might be a result of the limited sensitivity of the protease assay. However, irbesartan prominently regulated PPAR cofactor binding and induced selective gene expression, implicating that both ARBs, telmisartan and irbesartan, interact with PPAR and exert SPPARM activity.
In line with the specific receptor conformation, ARBs selectively regulated adipocyte gene expression. Comparisons with gene expression patterns induced by the full agonist pioglitazone demonstrated that ARB-mediated regulation of genes involved in adipocyte lipid metabolism was moderate compared with that of the glitazone. Consistenly with different effects of the ARBs on the conformational change, there was also distinct gene regulation between telmisartan and irbesartan. Some of these genes were chosen for further quantitative analysis. The prostacyclin receptor is an important inducer of adipogenesis (30,31). Microarray and quantitative PCR analysis demonstrated that pioglitazone, but not the ARBs, potently enhanced expression of the prostacyclin receptor. Another important mediator of adipocytic lipid storage is glycerol kinase, which is potently stimulated by TZDs (32). Stimulation of glycerol kinase by ARBs was much weaker compared with that of pioglitazone. Together, we identified an important characteristic of the distinctive gene expression pattern induced by PPAR-activating ARBs in adipocytes, which comprises a modest induction of adipogenic genes compared with the full glitazone agonists. This kind of selective gene regulation attenuates PPAR-mediated adipogenesis, which may prevent weight gain during ligand therapy.
Selective cofactor dissociation/binding provides the molecular link between ligand-induced conformational changes of PPAR and selective gene regulation, which then triggers specific metabolic in vitro/in vivo effects (23,33,34). The release of the corepressor NCoR at saturating concentrations was less pronounced with ARBs than with TZDs, which points toward their weaker adipogenic potential due to the inhibitory effect of NCoR in the differentiation process (35). Mice deficient for the PPAR coactivator TIF-2 are protected against diet-induced obesity with an improvement of whole-body insulin sensitivity (36). Altered gene expression patterns as a result of TIF-2 deficiency were associated with a reduction of fatty acid storage in adipose tissue (36). These data demonstrate that TIF-2 is a PPAR coactivator mainly involved in the adipogenic/lipogenic actions of the receptor and suggests that a TIF-2 independent activation of PPAR has beneficial effects on body weight and metabolic parameters. Attenuated TIF-2 recruitment by ARBs may provide the molecular explanation for the modest induction of adipogenic genes in the gene expression analysis.
To prove the relevance of the SPPARM activity of ARBs in vivo, diet-induced obese mice were treated with telmisartan or pioglitazone. Both substances attenuated the obesity-mediated insulin resistance. However, only telmisartan was able to lower body weight in these animals, which was mainly mediated by a decrease in total body fat. These data are consistent with the study by Berger et al. (23), in which a newly identified SPPARM significantly improved parameters of insulin sensitivity without promotion of weight gain in diet-induced obese mice.
A number of clinical studies have already been conducted to provide clinical evidence for the beneficial metabolic effects of PPAR-modulating ARBs in type 2 diabetic patients. Recently, Miura et al. (37) showed that by switching hypertensive type 2 diabetic patients from ARBs with no clinically relevant PPAR activity to the PPAR-modulating ARB telmisartan, a significant improvement of multiple metabolic parameters could be observed, including a decrease in fasting insulin and elevation of serum adiponectin levels. Body weight gain and edema were not detected in this study (37). In addition, in a double-blind, placebo-controlled trial in 119 diabetic patients with mild hypertension, only telmisartan significantly lowered plasma triglyceride levels, whereas the noneCPPAR-activating ARB eprosartan had no effect (10). However, in this study no change in glucose metabolism could be observed in the treatment groups (10). Together, these studies demonstrate that PPAR-modulating ARBs are capable of retaining the beneficial metabolic PPAR effects in vivo and in diabetic patients, which is associated with the circumvention of adverse effects such as weight gain and edema.
In conclusion, the present study characterizes two PPAR-activating ARBs, telmisartan and irbesartan, as new SPPARMs. Selective PPAR modulation by ARBs includes a distinct conformational change of the receptor associated with selective cofactor recruitment and a distinctive gene expression profile with a modest adipogenic pattern. Selective PPAR modulation by ARBs may reduce the weight-promoting effects of PPAR activation and in parallel retains PPAR-mediated metabolic efficacy, providing a new therapeutic option for patients suffering from diabetes or the metabolic syndrome. Furthermore, the molecular characterization of these compounds will open new perspectives to develop future substances acting as SPPARM-ARBs.
ACKNOWLEDGMENTS
M.C., T.U., and U.K. are supported by the German Research Society (DFG-GK 754-II).
We thank H.J. Mollenkopf and J. Angermann (Max Planck Institute for Infection Biology) for help with the microarray experiments and A. Foryst-Ludwig, M. Hartge, and C. Sprang for their technical help with the animal experiments.
FOOTNOTES
M.S. and M.C. contributed equally to this article.
M.S. has received honoraria from Bayer. T.U. has been on an advisory panel for Boehringer Ingelheim, Sanofi-Aventis, and Takeda; has received honoraria from Bayer, Boehringer Ingelheim, Sanofi-Aventis, and Takeda; and has received grant/research support from Bayer, Boehringer Ingelheim, Sanofi-Aventis, and Takeda. U.K. has received honoraria from Bayer Boehringer Ingelheim, Sanofi-Aventis, and Takeda and has received grant/research support from Boehringer Ingelheim.
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