Regulation of Peroxisome Proliferator–Activated Receptor γ Activity by Losartan Metabolites
Two active metabolites of the angiotensin type 1 (AT1) receptor blocker losartan have been described previously, EXP3174 and EXP3179. Whereas EXP3174 is the main antihypertensive AT1 receptor–blocking metabolite, the role of EXP3179 is widely unknown. Recently, a subgroup of AT1 receptor blockers has been identified as ligands for the peroxisome proliferator–activated receptor γ (PPAR-γ). Here we characterize the PPAR-γ–activating properties of the 2 active losartan metabolites. PPAR-γ activity was measured with a chimeric Gal4-DNA–binding domain–hPPARγ-ligand–binding domain (LBD) fusion protein on a Gal4-dependent luciferase reporter system. EXP3179 prominently induced the activation of the PPAR-γ–LBD reaching a maximum at 100 μmol/L with a 7.1±1-fold induction (P<0.05 versus vehicle-treated cells). Maximum PPAR-γ–LBD activation by EXP3179 reached 51% of the maximum response induced by the full PPAR-γ agonist pioglitazone, identifying EXP3179 as a partial PPAR-γ agonist. EXP3174 did not induce PPAR-γ–LBD activation. EC50 values were calculated for PPAR-γ–LBD activity (pioglitazone EC50: 0.88 μmol/L; EXP3179 EC50: 17.1 μmol/L; losartan EC50: >50 μmol/L). Consistent with the activation of PPAR-γ, EXP3179 potently induced 3T3-L1 adipocyte differentiation, a typical PPAR-γ–dependent cell function, and markedly stimulated PPAR-γ target gene expression. EXP3174 failed to regulate differentiation or PPAR-γ target gene expression. The present study characterizes the active losartan metabolite EXP3179 as a partial PPAR-γ agonist. PPAR-γ activation by EXP3179 may help us to understand the beneficial metabolic effects of losartan observed in clinical trials.
The Losartan Intervention For End point reduction in hypertension (LIFE) study has shown that hypertensive patients receiving the angiotensin type 1 receptor blocker (ARB) losartan have a 25% lower rate of new-onset diabetes than patients treated with the β-blocker atenolol.1 Although these data suggest a possible antidiabetic action of losartan, the molecular mechanisms are widely unknown.
We and others recently demonstrated that a subset of ARBs, including losartan, induces the activity of a nuclear hormone receptor named peroxisome proliferator–activated receptor γ (PPAR-γ) by partial agonism.2,3 The direct activation of the ligand-binding domain of PPAR-γ by ARBs is independent of their angiotensin type 1 receptor (AT1R) blocking actions.3 PPAR-γ functions as a transcriptional regulator in adipose tissue where it regulates multiple genes involved in lipid and glucose metabolism.4 Activated by synthetic full agonists like thiazolidinediones/glitazones, PPAR-γ markedly improves whole-body insulin sensitivity resulting in decreased levels of fasting plasma glucose, fasting plasma insulin, and plasma triglycerides.5 Thus, PPAR-γ activation by ARBs presents a promising molecular mechanism for metabolic actions of these compounds.
Losartan induced PPAR-γ activity only at high concentrations in vitro.3 Losartan is hepatically metabolized by the cytochrome-P450 pathway and exerts its antihypertensive actions in vivo predominantly by its main metabolite, EXP3174 (Figure 1).6,7 During hepatic metabolization of losartan, additional active metabolites are produced, including EXP3179 (Figure 1). This metabolite has a significant molecular homology with indomethacin, an antiinflammatory cyclooxygenase (COX) inhibitor, and mediates a variety of AT1R-independent, pleiotropic functions (eg, inhibition of platelet aggregation, endothelial adhesion molecule expression, etc).7 Indomethacin has been also identified as an activator of PPAR-γ.8 Given the structural homology of indomethacin and EXP3179, it is likely that EXP3179 has also PPAR-γ–binding properties. We hypothesized that the PPAR-γ–activating properties of losartan in vivo might be enhanced by its active metabolites and that PPAR-γ activation by losartan metabolites may provide a potential mechanism of the antidiabetic actions of losartan observed in clinical trials.
3T3-L1 adipocytes were differentiated as described previously in the absence of 3-isobutyl-1-methylxanthine.3 After 9 days, Oil-Red-O staining was performed to assess lipid accumulation. COS-7 cells were purchased from American Type Culture Collection.
Synthesis of EXP3179
The in vitro synthesis of EXP3179 has been described previously.7 In brief, EXP3179 was synthesized from losartan by incubation with RuCl3 and H2O2 in MeCN followed by liquid chromatography purification.
Transfection and Luciferase Assay
Transient transfection and luciferase assays were performed as described previously.3 COS-7 cells were transfected using Lipofectamine 2000 (Invitrogen) with pGal4-human [h] PPARγDEF (hPPAR-γ ligand-binding domain [LBD] fused to Gal4 DBD) and pGal5-TK-pGL3 kindly provided by Bart Staels (UR 545 INSERM), and 10 ng pRL-CMV, a renilla luciferase control reporter vector. After 4 hours, transfection medium was replaced by 10% FBS DMEM plus the indicated compounds or vehicle (dimethylsulfoxide), and luciferase activity was measured after 24 hours.
Quantitative Real-Time PCR
Real-time PCR was performed as described previously with an ABI 7000 sequence detection system.3 Day-8 adipocytes were serum starved overnight, incubated with compounds for 24 hours, and RNA was isolated. Mouse 18S ribosomal RNA was chosen as endogenous controls (housekeeping genes).
ANOVA and t test were performed for statistical analysis as appropriate. Statistical significance was designated at P<0.05. Values are expressed as mean±SD.
EXP3179 Enhances 3T3-L1 Adipocyte Differentiation
To examine whether losartan metabolites regulate a PPAR-γ–mediated cell function, differentiation of 3T3-L1 adipocytes was studied in the presence and absence of losartan, EXP3174, EXP3179, and pioglitazone. EXP3179 (10 μmol/L) potently promoted 3T3-L1 adipocyte differentiation as indicated by an increased lipid accumulation assessed with Oil-Red-O staining (Figure 2A and 2B). In similar concentrations, losartan (10 μmol/L) weakly induced lipid accumulation, and the losartan metabolite EXP3174 had no effect (Figure 2A and 2B). Concentration-response experiments revealed that EXP3179-mediated 3T3-L1 adipocyte differentiation started between 1 and 10 μmol/L (Figure 2B). Losartan markedly enhanced the differentiation process only at high concentrations (100 μmol/L; Figure 2B).
EXP3179 Induces PPAR-γ Target Gene Expression
Consistent with the stimulation of adipocyte differentiation, EXP3179 (10 μmol/L) induced mRNA expression of the adipogenic marker and PPAR-γ target gene, adipose protein 2, in 3T3-L1 adipocytes (Figure 3A). Losartan and EXP3174 at 10-μmol/L concentrations had no effect on adipose protein 2 mRNA expression.
A specific characteristic of agonists for PPAR-γ is the downregulation of the receptor on mRNA and/or the protein level in adipocytes on ligand activation in an autoregulatory manner.9 To additionally characterize EXP3179 as a PPAR-γ activator, we studied the regulation of PPAR-γ2 mRNA expression in 3T3-L1 adipocytes under stimulation with losartan metabolites. In line with pioglitazone-mediated PPAR-γ downregulation, EXP3179 significantly downregulated PPAR-γ2 mRNA expression in 3T3-L1 adipocytes, whereas losartan and EXP3174 had no effect (Figure 3B).
EXP3179 Activates the PPAR-γ Ligand-Binding Domain
To prove that EXP3179 activates PPAR-γ, we assessed its ability to directly activate the PPAR-γ LBD by using a chimeric Gal4-DBD-hPPARγ-LBD fusion protein on a Gal4-dependent luciferase reporter. EXP3179 prominently induced the activation of the PPAR-γ LBD reaching a maximum at 100 μmol/L with a 7.1±1-fold induction (P<0.05 versus vehicle-treated cells; Figure 4). Maximum PPAR-γ LBD activation by EXP3179 reached 51% of the maximum response induced by the full PPAR-γ agonist pioglitazone, identifying EXP3179 as a partial PPAR-γ agonist (Figure 4). EXP3174 did not induce PPAR-γ LBD activation (Figure 4). EC50 values were calculated for PPAR-γ LBD activity (pioglitazone EC50: 0.88 μmol/L; EXP3179 EC50: 17.1 μmol/L; and losartan EC50: >50 μmol/L).
This study identifies the active losartan metabolite EXP3179 as a partial PPAR-γ agonist. EXP3179 markedly promotes 3T3-L1 adipocyte differentiation, induces PPAR-γ target gene expression, and directly activates the PPAR-γ LBD. PPAR-γ activation by EXP3179 may provide a mechanism for the beneficial antidiabetic actions of losartan observed in clinical trials.
Losartan is a prodrug that is actively metabolized by the cytochrome P450 isoenzyme CYP2C9 on first liver passage to its main antihypertensive metabolite, EXP3174.6 EXP3174 is 10-fold to 40-fold more potent compared with losartan and mediates most of the AT1R-blocking effects of losartan.6 Recently, an important intermediate aldehyde metabolite of losartan, EXP3179, has been identified. In contrast to EXP3174, EXP3179 has very little AT1R-blocking activity and has been hypothesized to mediate pleiotropic actions of losartan observed in clinical and animal studies.6 Kramer et al7 demonstrated that EXP3179 potently inhibits the expression of endothelial COX-2, thereby exerting potent antiinflammatory actions. In addition, Watanabe et al10 showed that EXP3179 stimulates endothelial NO synthase phosphorylation and suppresses endothelial cell apoptosis induced by tumor necrosis factor α independent of AT1R-mediated signaling. The molecular mechanism underlying these pleiotropic EXP3179 actions are unknown. Ligand-activated PPAR-γ exerts potent antiinflammatory actions by inhibiting the action of proinflammatory transcription factors, such as AP-1 and nuclear factor κB.11 Activation of PPAR-γ has also been shown to repress COX-2 promoter activity and mRNA expression by interacting with the c-jun component of the AP-1 complex.12 EXP3179-mediated activation of PPAR-γ may, therefore, provide a new mechanism of the observed antiinflammatory actions of this compound.
EXP3179 induced PPAR-γ activation as a partial agonist, which implies the consideration of whether the compound may antagonize the actions of a full glitazone agonist during cotreatment. We performed experiments with another PPAR-γ–activating ARB, telmisartan, in the absence and presence of pioglitazone analyzing the activation of the PPAR-γ LBD in vitro (M. Schupp and U. Kintscher, unpublished data, 2005). These results demonstrated that telmisartan attenuates pioglitazone-induced PPAR-γ activation only at concentrations >10 μmol/L, a concentration that is usually not achieved in patients treated with antihypertensive doses. Because telmisartan is the most potent PPAR-γ–activating ARB, antagonistic actions of these substances on glitazone-induced PPAR-γ activation are unlikely to play a role in vivo or in clinical routine. Furthermore, we demonstrated recently that PPAR-γ–activating ARBs, such as telmisartan and irbesartan, act like selective PPAR-γ modulators compared with the full agonist pioglitazone involving distinct PPAR-γ coactivator binding and induction of distinct gene expression profiles in adipocytes.13 EXP3179 behaves similar to telmisartan and irbesartan in differentiation assays, adipocytic gene regulation, and transactivation assays, which implicates that EXP3179 may also exert selective PPAR-γ modulator activity.
In clinical trials, losartan has been shown to mediate prominent antidiabetic actions, such as a marked reduction of new-onset diabetes.1,14,15 The molecular mechanism of these metabolic actions is still far from being understood. It is now well known that blockade of AT1R results in multiple beneficial effects on insulin and glucose metabolism mediated via an improvement of muscular and pancreatic blood flow or an inhibition of deleterious angiotensin II actions on insulin signaling.14 We and others recently demonstrated that certain ARBs act like activators of PPAR-γ, which might contribute to their antidiabetic effects.2,3 However, because PPAR-γ activation by losartan was only achieved at very high concentrations, it appears unlikely that PPAR-γ is responsible for the antidiabetic actions of this compound.3 In contrast, the losartan metabolite EXP3179 induced PPAR-γ activation more potently compared with losartan identifying this metabolite as a possible mediator of the antidiabetic properties of losartan.
The question remains as to whether EXP3179-mediated PPAR-γ activation plays a role in the antidiabetic actions of losartan observed in clinical studies. Kramer et al7 reported that after a single oral dose of losartan (100 mg), maximum serum concentrations of EXP3179 between 0.1 and 1 μmol/L were achieved. In the present study, PPAR-γ–mediated adipocyte differentiation, as well as activation of the PPAR-γ LBD by EXP3179, started between 1 and 10 μmol/L. In consideration of the high lipophilicity of EXP3179 rendering it receptive for tissue accumulation and in consideration of an additional increase of serum levels under chronic losartan treatment, EXP3179 concentrations required for PPAR-γ activation may well be reached under losartan treatment. However, rapid hepatic metabolization of EXP3179 has to be taken into account, and additional studies are required to assess whether stable serum and tissue concentrations of EXP3179 are achieved to activate PPAR-γ in patients treated with different doses of losartan.
PPAR-γ activation by EXP3179 may provide a new mechanism of antidiabetic actions induced by losartan. In addition, the identification of an additional PPAR-γ–activating compound with a chemical structure equal to ARBs helps us to understand the characteristic of such substances and supports the development of new dual ARB/PPAR-γ ligands for the treatment of patients experiencing hypertension, insulin resistance, or diabetes.
We thank Merck (Rahway, NJ) for kindly providing losartan. T.U. and U.K. are supported by the Deutsche Forschungsgemeinschaft (GK 754). We thank Bart Staels for kindly providing constructs.
T.U. has been a member of the advisory boards and speakers bureau of Boehringer, MSD, Abbot, Novartis, Sanofi-Aventis, and Takeda. T.U. has received research grants from Boehringer, MSD, Novartis, and Takeda. U.K. has received research grants from Boehringer, and Glaxo-Smith-Kline. B.S. has received research grants from Novartis and Schering AG.
- Received September 28, 2005.
- Revision received October 11, 2005.
- Accepted November 2, 2005.
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