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Published online January 9, 2008
Diabetes 57:1405-1413, 2008
DOI: 10.2337/db07-0839
© 2008 by the American Diabetes Association
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Liver-Specific Peroxisome Proliferator–Activated Receptor {alpha} Target Gene Regulation by the Angiotensin Type 1 Receptor Blocker Telmisartan

Markus Clemenz1, Nikolaj Frost1, Michael Schupp2, Sandrine Caron3, Anna Foryst-Ludwig1, Christian Böhm1, Martin Hartge1, Ronald Gust4, Bart Staels4, Thomas Unger1, and Ulrich Kintscher1

1 Center for Cardiovascular Research, Institute of Pharmacology, Charité-Universitätsmedizin Berlin, Berlin, Germany
2 Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania
3 Unité de Recherche 545 Institut National de la Santé et de la Recherche Médicale, Institute Pasteur de Lille, Université de Lille 2, Lille, France
4 Institute of Pharmacy, Free University of Berlin, Berlin, Germany

Corresponding author: Prof. Ulrich Kintscher, MD, Center for Cardiovascular Research, Institute of Pharmacology, Charité-Universitätsmedizin Berlin, Hessische Str. 3-4, 10115 Berlin, Germany. E-mail: ulrich.kintscher{at}charite.de

Key Words: ACSL1, acyl-CoA synthetase long-chain family member 1 • ALT, alanine aminotransferase • ARB, angiotensin type 1 receptor blocker • AST, aspartate aminotransferase • CPT1A, carnitine palmitoyl transferase 1A • hPPAR{alpha}, human peroxisome proliferator–activated receptor {alpha} • LBD, ligand binding domain; NASH, nonalcoholic steatohepatitis • PPAR, peroxisome proliferator–activated receptor • siRNA, small interfering RNA


   ABSTRACT
TOP
 ABSTRACT
RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
OBJECTIVE—The angiotensin type 1 receptor blocker (ARB) and peroxisome proliferator–activated receptor (PPAR) {gamma} modulator telmisartan has been recently demonstrated to reduce plasma triglycerides in nondiabetic and diabetic hypertensive patients. The present study investigates the molecular mechanisms of telmisartans hypolipidemic actions, in particular its effect on the PPAR{alpha} pathway.

RESEARCH DESIGN AND METHODS—Regulation of PPAR{alpha} target genes by telmisartan was studied by real-time PCR and Western immunoblotting in vitro and in vivo in liver/skeletal muscle of mice with diet-induced obesity. Activation of the PPAR{alpha} ligand binding domain (LBD) was investigated using transactivation assays.

RESULTS—Telmisartan significantly induced the PPAR{alpha} target genes carnitine palmitoyl transferase 1A (CPT1A) in human HepG2 cells and acyl-CoA synthetase long-chain family member 1 (ACSL1) in murine AML12 cells in the micromolar range. Telmisartan-induced CPT1A stimulation was markedly reduced after small interfering RNA–mediated knockdown of PPAR{alpha}. Telmisartan consistently activated the PPAR{alpha}-LBD as a partial PPAR{alpha} agonist. Despite high in vitro concentrations required for PPAR{alpha} activation, telmisartan (3 mg · kg–1 · day–1) potently increased ACSL1 and CPT1A expression in liver from diet-induced obese mice associated with a marked decrease of hepatic and serum triglycerides. Muscular CPT1B expression was not affected. Tissue specificity of telmisartan-induced PPAR{alpha} target gene induction may be the result of previously reported high hepatic concentrations of telmisartan.

CONCLUSIONS—The present study identifies the ARB/PPAR{gamma} modulator telmisartan as a partial PPAR{alpha} agonist. As a result of its particular pharmacokinetic profile, PPAR{alpha} activation by telmisartan seems to be restricted to the liver. Hepatic PPAR{alpha} activation may provide an explanation for telmisartan's antidyslipidemic actions observed in recent clinical trials.

Angiotensin type 1 receptor blockers (ARBs) are commonly used in the treatment of hypertension and related cardiovascular and organ damage (1). Recently, a distinct subgroup of ARBs has been identified as partial agonists for the peroxisome proliferator–activated receptor (PPAR) {gamma} with selective PPAR{gamma} modulating properties (2–4). In contrast to full glitazone agonists, PPAR{gamma}-activating ARBs exert selective recruitment of nuclear cofactors resulting in in vivo insulin sensitization in the absence of weight gain in obese insulin-resistant mice (3). Among the ARBs, telmisartan has been shown to be the most potent PPAR{gamma} modulator (3,4). Based on these in vitro results and data from animal experiments, a number of clinical studies (5–9) have been conducted in which the metabolic actions of the PPAR{gamma}-activating ARB telmisartan have been intensively investigated. When compared with ARBs that do not exert PPAR{gamma}-activating properties, telmisartan not only improves insulin sensitivity but also induces beneficial actions on serum lipid levels such as a reduction of serum triglycerides. (5,10,11)

PPARs are ligand-activated transcription factors belonging to the superfamily of nuclear receptors. PPAR{gamma} is abundantly expressed in adipose tissue and a major regulator of insulin and glucose metabolism (12). In contrast, PPAR{alpha} is highly expressed in tissues displaying a high metabolic rate of fatty acids, such as the liver and skeletal muscle (13). PPAR{alpha} modulates intracellular lipid metabolism by transcriptional regulation of genes involved in fatty acid uptake, mitochondrial fatty acid oxidation, and triglyceride catabolism (13,14). Natural PPAR{alpha} ligands comprise mono- and polyunsaturated fatty acids as well as eicosanoids (15). In addition, PPAR{alpha} is also the molecular target of lipid-lowering fibrates such as gemfibrozil, bezafibrate, clofibrate, and fenofibrate. These substances are used to treat dyslipidemia and cardiovascular disease. (13,15)

It has been reported that certain PPAR{gamma} activators such as pioglitazone are also able to activate PPAR{alpha}. Furthermore, it has been proposed that the positive actions of pioglitazone on diabetic dyslipidemia might at least in part be mediated by its PPAR{alpha}-activating abilities (16,17). To understand the underlying mechanism of telmisartan's lipid-lowering actions we studied the effect of telmisartan on major PPAR{alpha} target genes involved in fatty acid oxidation in the human hepatoma cell line HepG2, the murine hepatic cell line AML12, and in liver/skeletal muscle of diet-induced obese mice treated with telmisartan. Furthermore, activation of the PPAR{alpha} ligand binding domain (LBD) and regulation of PPAR{alpha} protein/mRNA expression by telmisartan was studied.

The present study demonstrates that telmisartan induces the PPAR{alpha} target gene carnitine palmitoyl transferase 1 (CPT1A) in HepG2 cells and acyl-CoA synthetase long-chain family member 1 (ACSL1) in AML12 cells. Consistently, telmisartan acts as a partial PPAR{alpha} agonist in PPAR{alpha} transactivation assays and induces PPAR{alpha} expression. High-fat diet–fed mice treated with telmisartan showed a pronounced induction of hepatic ACSL1 and CPT1A, which was associated with a significant decrease of hepatic and serum triglycerides. Interestingly, CPT1B expression in skeletal muscle was not affected by telmisartan. Tissue specificity of telmisartan-induced PPAR{alpha} target gene induction may result from high hepatic telmisartan concentrations that have been documented in rodents during early preclinical studies (18).

In summary, the present study identifies the ARB telmisartan as a partial PPAR{alpha} agonist. Based on its specific pharmacokinetic profile, PPAR{alpha} activation by telmisartan appears to be liver specific. Hepatic induction of PPAR{alpha} target genes involved in mitochondrial fatty acid oxidation might contribute to the antidyslipidemic actions of telmisartan.


   RESEARCH DESIGN AND METHODS
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture.
HepG2, AML12, and COS7 cells were cultured following the manufacturer's guidelines. Cells were serum deprived for 16 h before stimulation with the vehicle (DMSO) or different effectors.

Quantitative real-time PCR.
Real-time PCR was performed as previously described using an ABI 7000 and Stratagene 3000 MXP PCR cycler with either the SybrGreen or FAM-TAMRA detection system (3). Primers and probes are shown in the online appendix (available at http://dx.doi.org/10.2337/db07-0839).

Transfection and luciferase assay.
Transient transfection and luciferase assays were performed as previously described (3). Cos7 cells were transfected with the use of Lipofectamine 2000 (Invitrogen), with pGal4-hPPAR (human PPAR{alpha}) {alpha}DEF (hPPAR{alpha} LBD fused to Gal4 DBD [DNA binding domain]) and pGal5-TK-pGL3, and 10 ng pRL-CMV (Renilla-Cytomegalovirus), a renilla luciferase control reporter vector. After 4 h, transfection medium was replaced by 10% fetal bovine serum DMEM plus the indicated ARBs, Wy14.643, fenofibric acid, or vehicle (DMSO), and luciferase activity was measured after 24 h.

Western immunoblotting.
Electrophoresis and blotting following protein isolation from murine liver tissue was performed as previously described (3). Blots were incubated with an anti-PPAR{alpha} (Sigma) or anti-ACSL1 (kindly provided by Rosalind A. Coleman, Chapel Hill, NC) antibody.

Gene silencing with small interfering RNA.
The small interfering RNA (siRNA) targeting human PPAR{alpha} was purchased from Dharmacon, and two sequences (D-003434–01 and D-003434–02) were used simultaneously according to the manufacturer's instructions. The siRNA negative control from Dharmacon (D-001810–01) was used to test nonspecific effects on gene expression. Overnight-starved HepG2 cells were transfected using HiPerfect (Qiagen), according to the manufacturer's instructions, in 24-well plates containing 105 cells/well with 5 nmol/l siRNA/well (each sequence 2.5 nmol/l). Thirty minutes after the start of transfection, cells were treated for 48 h with telmisartan (50 µmol/l; 0.5% serum) before RNA analysis.

Animals.
Male C57BL/6J mice were treated as previously described (3). Mice aged 4–5 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) for 16 weeks, followed by randomization to either a vehicle-treated (n = 6) (0.5% Tween 80/H2O), a telmisartan-treated (n = 6) (3 mg · kg–1 · day–1), or a pioglitazone-treated (n = 6) (10 mg · kg–1 · day–1) group. Telmisartan was provided by Boehringer Ingelheim, and piogliatzone was extracted from tablets. Animals were treated by oral gavage for 10 weeks. Before and after treatment, blood samples were collected from overnight-fasted animals by retroorbital venous puncture under isoflurane anesthesia for analysis of serum triglycerides (enzymatic-colorimetric test; Cypress Diagnostics). After the experiment, animals were killed and organs were dissected. All animal procedures were in accordance with institutional guidelines and were approved.

Triglyceride content in liver was measured as described previously (19). Briefly, tissues were homogenized in liquid nitrogen and treated with ice-cold chloroform/methanol/water mixture (2:1:0.8) for 2 min. After centrifugation, the aqueous layer was removed and the chloroform layer was decanted. The mixture was incubated at 70°C for chloroform clearance, and the residues were dissolved in isopropanol and assessed for the triglyceride content using an enzymatic-colorimetric test (Cypress Diagnostics), according to the manufacturer's instructions. For immunohistochemical studies, organs were fixed in 4% formalin, embedded in paraffin, and stained with hematoxylin/eosin.

Statistical analysis.
ANOVA, followed by multiple comparison testing or t test, was performed for statistical analysis as appropriate. Statistical significance was designated at P < 0.05. Values are expressed as means ± SE or as indicated.


   RESULTS
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Telmisartan induces PPAR{alpha} target gene expression in human and murine hepatocytes.
To evaluate whether telmisartan regulates "classical" PPAR{alpha} target genes, mRNA expression of CPT1A, the rate-limiting enzyme of fatty acid oxidation, in HepG2 cells was examined. Telmisartan induced CPT1A mRNA expression after 48 h in a dose-dependent manner starting at 10 µmol/l (1.5 ± 0.2–fold vs. vehicle-treated cells, P < 0.05) and reaching a maximum at 50 µmol/l telmisartan (2.84 ± 0.63–fold vs. vehicle-treated cells, P < 0.001 vs. control) (Fig. 1A and B). Treatment of HepG2 cells with a classical PPAR{alpha} agonist Wy-14643 for 24 h resulted in a 6.6 ± 1.6–fold induction of CPT1A mRNA (100 µmol/l; P < 0.05 vs. vehicle-treated cells; data not shown).


Figure 1
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  		FIG. 1. Telmisartan induces PPAR{alpha} target gene expression in human and murine hepatocytes. A: HepG2 cells were serum deprived for 16 h and incubated with 50 µmol/l telmisartan. mRNA expression of CPT1A was determined by real-time PCR after the indicated time points. ***P < 0.001 vs. vehicle control. B: mRNA expression of CPT1A in HepG2 cells with incubation of telmisartan (0.1, 1, 10, and 50 µmol/l) for 48 h. *P < 0.05; ***P < 0.001 vs. vehicle control. C: HepG2 cells were serum deprived for 16 h and transfected with PPAR{alpha} siRNA or control siRNA followed by incubation with 50 µmol/l telmisartan for 48 h. mRNA expression of CPT1A was determined by real-time PCR. *P < 0.05 and ***P < 0.001 vs. vehicle control with control siRNA; ##P < 0.01 vs. telmisartan-treated cells with control siRNA (small graph: PPAR{alpha} mRNA expression in HepG2 cells with control/PPAR{alpha} siRNA. ***P < 0.001 vs. control siRNA; data are shown as % of PPAR{alpha} mRNA expression in control siRNA-transfected cells.) D: Murine AML12 cells were incubated with telmisartan (10 µmol/l), Wy-14643 (10 µmol/l), or fenofibric acid (100 µmol/l). After 48 h of incubation ACSL1 mRNA was determined. ***P < 0.001; ** P < 0.01; *P < 0.05 vs. vehicle control. Expression was normalized to 18S expression. Experiments were repeated four times and results are presented as x-fold induction over vehicle-treated cells. Means ± SE is shown.

 
To prove that telmisartan mediates its actions via PPAR{alpha} activation, HepG2 cells were transfected with PPAR{alpha}-specific siRNA. After siRNA treatment of HepG2 cells, PPAR{alpha} mRNA expression was significantly reduced (Fig. 1C, small graph). This siRNA knockdown of PPAR{alpha} resulted in a significant reduction of telmisartan-induced CPT1A mRNA expression compared with control siRNA, indicating a PPAR{alpha}-dependent mechanism of action (Fig. 1C).

To determine whether PPAR{alpha} target gene regulation by telmisartan was gene or species specific, we next studied the expression of ACSL1, the key player in lipid biosynthesis and fatty acid degradation, in the murine hepatic cell line AML12. ACSL1 mRNA was markedly induced by telmisartan (Fig. 1D). In contrast to CPT1A induction in HepG2 cells, maximum ACSL1 mRNA upregulation in AML12 cells was already achieved at 10 µmol/l 2.4 ± 0.1–fold after 48 h incubation with telmisartan 10 µmol/l (P < 0.001 vs. vehicle) (Fig. 1D). Wy-14643 (10 µmol/l) resulted in a 1.46 ± 0.12–fold (P < 0.01 vs. vehicle), and fenofibrate (100 µmol/l) in a 1.55 ± 0.38–fold, induction of ACSL1 mRNA (P < 0.05 vs. vehicle) (Fig. 1D). The present data demonstrate that telmisartan induces the PPAR{alpha} target genes CPT1A in HepG2 cells and ACSL1 in AML12 cells.

Telmisartan activates the PPAR{alpha} LBD and acts like a partial PPAR{alpha} agonist.
In order to prove whether the induction of hepatic PPAR{alpha} target genes by telmisartan resulted from a direct activation of PPAR{alpha}, we examined its ability to directly activate the PPAR{alpha} LBD by using a chimeric Gal4-DBD-hPPAR{alpha}-LBD fusion protein on a Gal4-dependent luciferase reporter. Telmisartan induced the activation of the PPAR{alpha} LBD in a concentration-dependent manner, reaching a maximum at 50 µmol/l (Fig. 2), with 22.5% of the maximum response induced by the reference PPAR{alpha} agonist Wy-14643 identifying telmisartan as a partial PPAR{alpha} agonist. No activation of the PPAR{alpha} LBD was achieved with the ARBs irbesartan or losartan (Fig. 2). The computed EC50 values for activation are as follows: telmisartan EC50, 21.8 µmol/l; fenofibric acid EC50, 18.2 µmol/l; and Wy-14643 EC50, 6.4 µmol/l. Here, we identify telmisartan as a partial PPAR{alpha} agonist inducing activation in the micromolar range.


Figure 2
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  		FIG. 2. Telmisartan activates the PPAR{alpha} LBD and acts like a partial PPAR{alpha} agonist. COS7 cells were transiently transfected with the pGal4-h PPAR{alpha} LBD and pGal5-Tk-pGL3 reporter followed by stimulation with the ARBs, fenofibric acid (Feno Acid), and Wy-14643 (Wy14643) as indicated. Firefly luciferase activity was measured after 24 h and normalized with activity of cotransfected renilla luciferase. Experiments were repeated three times. Results are presented as means ± SD.

 
Telmisartan induces hepatic PPAR{alpha} target gene expression in diet-induced obese mice.
It has been shown previously that after a single oral administration of telmisartan at a dose of 1 mg/kg in rats, telmisartan prominently concentrates in the liver (tissue distribution of 14C-telmisartan 4 h after application: liver, 10,673.06 ± 1,274.93 ng eq/ml; plasma, 218.85 ± 6.08 ng eq/ml; and skeletal muscle, 17.55 ± 1.18 ng eq/ml), implicating that the high concentrations required for PPAR{alpha} activation and target gene regulation observed in vitro might be achieved in vivo (18). To demonstrate that hepatic PPAR{alpha} activation by telmisartan occurs in vivo and translates into metabolic changes, high-fat diet–fed obese mice were treated with telmisartan 3 mg · kg–1 · day–1 or vehicle for 10 weeks and hepatic/muscular PPAR{alpha} target gene expression, hepatic triglyceride accumulation, and serum triglyceride levels were determined.

ACSL1 protein expression in liver tissue was markedly increased in telmisartan-treated mice compared with the vehicle-treated animals (Fig. 3A). Accordingly, relative ACSL1 mRNA expression increased 2.5 ± 0.3–fold in livers of telmisartan-treated animals (P < 0.01 vs. vehicle) (Fig. 3B). In consonance, telmisartan treatment led to a 3.2 ± 0.4–fold increase of hepatic CPT1A mRNA compared with vehicle-treated mice (P < 0.01 vs. vehicle) (Fig. 3B). Interestingly, telmisartan had no effect on PPAR{alpha} target gene expression in skeletal muscle (Fig. 3C). Together, these results indicate that telmisartan markedly induces hepatic PPAR{alpha} target genes involved in fatty acid catabolism in obese mice.


Figure 3
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  		FIG. 3. Telmisartan induces hepatic PPAR{alpha} target gene expression in diet-induced obese mice. C57BL/6J mice were fed a high-fat diet (HFD) (60% kcal from fat) for 16 weeks followed by 10 weeks treatment with vehicle, telmisartan (3 mg · kg–1 · day–1), or pioglitazone (10 mg · kg–1 · day–1). At the end of the treatment period, animals were dissected and organs were shock frozen in liquid nitrogen. A: Hepatic ACSL1 protein expression. A representative Western immunoblot is shown. B: Relative hepatic mRNA expression of ACSL1 and CPT1A in telmisartan-treated animals. **P < 0.01 vs. HFD vehicle. C: mRNA expression of CPT1B in skeletal muscle tissue of telmisartan-treated animals. n.s., statistically not significant versus HFD vehicle animals. D: Relative hepatic mRNA expression of CD36 and PPAR{gamma}2 in telmisartan-treated animals. n.s., statistically not significant versus HFD vehicle animals. E: Relative hepatic mRNA expression of CPT1A and CD36 in pioglitazone-treated animals. n.s., statistically not significant versus HFD vehicle animals. **P < 0.01 vs. HFD vehicle. Expression was normalized to 18S expression. Experiments were repeated four times and results are presented as x-fold induction over vehicle-treated cells. Mean ± SE is shown.

 
Previously, telmisartan has been characterized as a partial PPAR{gamma} agonist (4). To evaluate the role of hepatic PPAR{gamma} activation in the actions of telmisartan, mRNA expression of the PPAR{gamma} target gene CD36 and PPAR{gamma}2 was analyzed in liver tissue from telmisartan-treated mice (Fig. 3D). Hepatic CD36 and PPAR{gamma}2 mRNA levels were not significantly regulated by telmisartan treatment, suggesting the absence of major hepatic PPAR{gamma} activation by telmisartan (Fig. 3D). Furthermore, we studied the effect of the full PPAR{gamma} agonist pioglitazone in livers from high-fat diet–fed mice. Mice treated with pioglitazone (10 mg · kg–1 · day–1) for 10 weeks exhibited no regulation of hepatic CPT1A mRNA expression, whereas the PPAR{gamma} target gene CD36 was significantly induced by 2.8 ± 0.3–fold compared with vehicle-treated high-fat diet–fed mice (P < 0.01 vs. vehicle-treated high-fat diet–fed mice) (Fig. 3E).

Telmisartan reduces hepatic and serum triglycerides in diet-induced obese mice.
Increased expression of hepatic CPT1A and ACSL1, and subsequent higher rates of hepatic fatty acid oxidation in telmisartan-treated animals, should result in decreased accumulation of triglycerides in liver and serum. Telmisartan treatment prominently reduced liver triglyceride content in obese mice (29.7 ± 11.7µmol/g wet wt) when compared with vehicle-treated mice (79.5 ± 17.4 µmol/g wet wt, P < 0.005) (Fig. 4A and B). In accordance, serum triglycerides in high-fat diet–fed mice declined from 122.3 ± 28.4 mg/dl before treatment to 53.9 ± 4.5 mg/dl after telmisartan treatment (P < 0.005) (Fig. 4C), implicating that hepatic PPAR{alpha} gene induction by telmisartan translates into a lowering of systemic and local triglyceride level.


Figure 4
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  		FIG. 4. Telmisartan reduces hepatic and serum triglycerides in diet-induced obese mice. A: Liver triglyceride content of telmisartan- and vehicle-treated animals on high-fat diet (HFD) in µmol/g wet weight. ***P < 0.005. B: Hematoxylin-eosin staining of representative liver sections from vehicle and telmisartan HFD-fed mice. C: Serum triglycerides in HFD-fed mice before and after telmisartan treatment. ***P < 0.005 pre- versus posttreatment.

 
In humans, aspartate aminotransferase (AST)-to-alanine aminotrasferase (ALT) ratios are indicative for the extent and etiology of liver damage (20). Whereas mild liver disease like nonalcoholic fatty liver disease and uncomplicated virus hepatitis are affiliated with ratios <1, severe liver disease like chronic hepatitis, liver cirrhosis, and alcoholic fatty liver disease result in increased ratios >1 (20). First, we compared AST-to-ALT ratios in mice on a low-fat diet (10% kcal from fat) with high-fat diet–fed animals. The mean AST-to-ALT ratio in low-fat diet–fed control mice was 1.1 ± 0.1, which was significantly decreased by high-fat diet to 0.5 ± 0.1 (P < 0.05). In line with the reduction of liver steatosis, telmisartan treatment restored impaired the AST-to-ALT ratio to normal levels (1.1 ± 0.3), indicating that reduction in hepatic triglycerides by telmisartan also improved high-fat diet–mediated deterioration of liver function.

Telmisartan induces PPAR{alpha} expression in vivo and in vitro.
To explore additional mechanisms of PPAR{alpha} activation by telmisartan, which might be additive to LBD-dependent activation, we investigated the regulation of PPAR{alpha} by telmisartan. Liver PPAR{alpha} protein expression markedly increased with telmisartan treatment (Fig. 5A). In accordance, hepatic PPAR{alpha} mRNA was upregulated 1.9 ± 0.2–fold in the telmisartan-treated animals compared with vehicle-treated mice (P < 0.01) (Fig. 5B). Moreover, PPAR{alpha} mRNA induction by telmisartan was observed in HepG2 cells, with a maximum induction of 3.4 ± 0.4–fold (P < 0.05 vs. vehicle) after 48 h at high concentrations of telmisartan (50 µmol/l) (Fig. 5C). The ARB eprosartan had no effect on PPAR{alpha} mRNA expression (Fig. 5C). These data show that, in addition to LBD activation, telmisartan is capable of inducing PPAR{alpha} expression, which might contribute to the observed target gene regulation.


Figure 5
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  		FIG. 5. Telmisartan induces PPAR{alpha} expression in vivo and in vitro. A: PPAR{alpha} protein expression from liver protein extracts of HFD-fed mice treated with telmisartan or vehicle control. A representative Western immunoblot is shown. B: PPAR{alpha} mRNA expression in livers from telmisartan- and vehicle-treated HFD animals. **P < 0.01 vs. HFD vehicle-treated control. C: PPAR{alpha} mRNA induction by telmisartan in HepG2 cells. Cells were incubated for 48 h with 1, 10, and 50 µmol/l telmisartan, 100 µmol/l eprosartan, or vehicle. *P < 0.05 vs. vehicle control. Results are presented as x-fold induction over vehicle-treated mice/cells. Means ± SE is shown.

 

   DISCUSSION
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present data demonstrate that the ARB telmisartan induces PPAR{alpha} target genes in human and murine hepatic cells and acts as a partial PPAR{alpha} agonist in the higher micromolar range in vitro. As a result of its particular pharmacokinetic profile with high concentrations in liver, telmisartan potently induces hepatic PPAR{alpha} target genes involved in fatty acid catabolism in obese mice, which was associated with a significant reduction of systemic and local triglyceride level.

Derosa et al. (5) could demonstrate that telmisartan, when compared with eprosartan, significantly reduced serum triglycerides in hypertensive type 2 diabetic patients by 24.8% compared with baseline, an effect that may not be fully explained by its AT1-blocking/PPAR{gamma}-modulating actions. Additional studies (10,11) in diabetic and nondiabetic hypertensive patients have confirmed significant lower plasma triglyceride levels after telmisartan treatment. In the present study, we identify telmisartan as a weak PPAR{alpha} agonist in vitro. Furthermore, telmisartan treatment in vivo induced PPAR{alpha}-regulated genes involved in hepatic fatty acid oxidation at relatively low doses, suggesting that hepatic PPAR{alpha} activation by telmisartan might be clinically relevant. It is well known that treatment of hypertriglyceridemic patients with fibrates results in potent lowering of triglyceride levels, which is, at least in part, mediated via hepatic PPAR{alpha} activation, subsequent induction of fatty acid oxidation in the liver, and decreased VLDL particle production and plasma triglycerides (21). In consonance with this, telmisartan also significantly reduced circulating triglyceride levels in our animal model, which is consistent with recent data observed in the leptin receptor–deficient Zucker rat treated with telmisartan (22). Together, these results underscore that PPAR{alpha} activation in the liver may contribute to telmisartans action on dyslipidemia in patients.

Telmisartan has been recently characterized as a selective PPAR{gamma} modulator (3,4). Hepatic activation of PPAR{gamma} contributes to the actions of PPAR{gamma} agonists on lipid and glucose metabolism in mice (23–25). To characterize the relevance of hepatic PPAR{gamma} activation for telmisartan's effects, CD36 and PPAR{gamma}2 expression were investigated and compared with the full PPAR{gamma} agonist pioglitazone. Telmisartan did not regulate CD36 or PPAR{gamma}2 in livers from high-fat diet–fed mice, excluding a major role of hepatic PPAR{gamma} in telmisartan's action. In contrast, pioglitazone failed to induce CPT1A but stimulated CD36 mRNA expression in liver. These data are consistent with previous findings of distinct gene expression profiles in adipocytes treated with telmisartan or piogliatzone (3). In liver, telmisartan may mainly act as a partial PPAR{alpha} agonist, whereas pioglitazone also activates PPAR{gamma} pathways.

Telmisartan reduced hepatic triglyceride content in high-fat diet–fed mice. Nonalcoholic steatohepatitis (NASH) frequently develops during obesity as a result of insulin resistance and subsequent hepatic triglyceride overload (26). NASH is considered a hepatic component of the metabolic syndrome, leading to liver fibrosis and cirrhosis (26). Currently, no drug therapy has been established for the treatment of NASH. Recently, a number of small clinical trials (27–29) have demonstrated that the PPAR{gamma} agonists rosiglitazone and pioglitazone improve liver histology and aminotransferase levels in patients with NASH. PPAR{alpha} activation by fibrates has been recently reported to reduce the development of NASH in different animal models (30,31). Therefore, combination of hepatic PPAR{alpha} activation and systemic PPAR{gamma} modulation by telmisartan, together with the observed reduction of liver triglyceride content, may provide a new therapeutic option for the future treatment of NASH. Telmisartan's action on hepatic pathology has already been described by Sugimoto et al. (32). In rats fed a high-fat, high-carbohydrate diet, telmisartan, but not valsartan, significantly reduced hepatic triglyceride (32). Along this line, Fujita et al. (33) could show that telmisartan application to a rat model of NASH improved numerous pathological features of the disease, including liver steatosis, liver inflammation, and liver fibrosis, underlining the potential role of telmisartan for NASH therapy. The hepatic actions of telmisartan will gain clinical importance in light of previous data demonstrating an increased prevalence of fatty liver disease in hypertensive patients (34). The antihypertensive actions combined with hepatoprotective actions of telmisartan could be of added clinical value in these patients.

The pharmacokinetic profile of telmisartan seems to be highly important for its PPAR{alpha}-activating properties. In preclinical studies with telmisartan, Shimasaki et al. (18) have shown that telmisartan prominently concentrates in the liver with ~40 times higher levels compared with plasma and skeletal muscle, which opens the possibility that the high concentrations required for PPAR{alpha} activation and target gene regulation observed in vitro might be achieved in vivo in a tissue-specific manner. The high liver content of telmisartan is likely caused by binding of the compound to the glutathione-S-transferase type 1–1 (ligandin) protein, which is present at high concentrations in the liver (35). Hepatic storage may be further supported by the lipophilic characteristic of telmisartan. Compared with the liver, telmisartan concentrations in skeletal muscle are extremely low 4 h after administration, which makes it unlikely that concentrations required for muscular PPAR{alpha} activation are reached (18). In accordance with the pharmacokinetic data, telmisartan did not affect PPAR{alpha} target gene expression in skeletal muscle of obese mice, strongly supporting a tissue-specificity for telmisartan-induced PPAR{alpha} activity.

The liver specificity of telmisartan-mediated PPAR{alpha} activation does not only provide a mechanism for its beneficial effects on triglyceride levels but also plays a major role for potential PPAR{alpha}-mediated side effects. One of the most common toxic side effect during fibrate therapy are myopathies associated with myalgias, in particular in combination with statin therapy (36). Since muscular PPAR{alpha} target genes are not activated by telmisartan, and telmisartan concentrations in skeletal muscle are minor, the occurrence of PPAR{alpha}-mediated muscular side effects under telmisartan therapy are very unlikely. Moreover, the unique pharmacokinetic profile of telmisartan allows beneficial liver-specific PPAR{alpha} activation in the absence of common PPAR{alpha}-mediated side effects.

In addition to activation of the PPAR{alpha} LBD, telmisartan-mediated induction of protein and mRNA expression of PPAR{alpha} could be detected in vitro and in vivo. This data are in accordance with previously published studies, demonstrating a stabilization of the PPAR{alpha} protein after ligand binding (37). Here, we identify that also PPAR{alpha} mRNA expression is positively regulated by telmisartan, suggesting an additional transcriptional mechanism of ligand (telmisartan)-mediated receptor regulation. This is in line with previous reports demonstrating an induction of hepatic PPAR{alpha} mRNA expression by fibrate treatment in rodents and human hepatocytes (38–40). Nevertheless, future studies are required to investigate whether PPAR{alpha} mRNA regulation by telmisartan depends on telmisartan PPAR{alpha} LBD interactions or whether this might be mediated via blockade of the AT1 receptor. As yet, it seems that telmisartan positively regulates the PPAR{alpha} pathway by two different mechanisms: 1) LBD activation and 2) receptor upregulation.

In summary, the present study identifies the ARB/PPAR{gamma} modulator telmisartan as a partial PPAR{alpha} agonist. As a result of its particular pharmacokinetic profile with high concentration in liver, PPAR{alpha} activation by telmisartan seems to be restricted to the liver. Hepatic PPAR{alpha} activation by telmisartan may provide an explanation for its antidyslipidemic actions observed in clinical trials and prevents simultaneously potential danger from systemic PPAR{alpha}-mediated adverse effects. The multimodal mechanism of action of telmisartan, including AT1-receptor blockade/PPAR{gamma} modulation and hepatic PPAR{alpha} activation, characterizes this compound as a therapeutic option for the treatment of patients suffering from multiple cardiometabolic disorders such as hypertension, glucose intolerance, and dyslipidemia.


   ACKNOWLEDGMENTS
 
This study was supported by Bayer Schering Pharma and Boehringer Ingelheim Pharma. M.C. is supported by the Deutsche Forschungsgemeinschaft (DFG-KI 712/3-1). C.B. is supported by the Deutsche Forschungsgemeinschaft (DFG-GK 754 III). R.G. is supported by the Deutsche Forschungsgemeinschaft (DFG-GU-285/7-1). T.U. is supported by the Deutsche Forschungsgemeinschaft (DFG-GK 754-III, DFG-GK 865-II). U.K. is supported by the Deutsche Forschungsgemeinschaft (DFG-GK 754-III, DFG-GK 865-II, DFG-KI 712/3-1).

We thank Rosalind A. Coleman (Department of Nutrition, University of North Carolina, Chapel Hill, NC) for providing the anti mouse ACSL1 antibody. We are deeply in debt to Christiane Sprang for excellent technical assistance.


   FOOTNOTES
 
Published ahead of print at http://diabetes.diabetesjournals.org on 9 January 2008. DOI: 10.2337/db07-0839.

Additional information for this article can be found in an online appendix at http://dx.doi.org/10.2337/db07-0839.

M.C. and N.F. contributed equally to this article.

T.U. is a member of the speakers bureau of and has received grant/research support from Boehringer Ingelheim and Bayer Schering Pharma. U.K. is a member of the speakers bureau of Bayer Schering Pharma and has received grant/research support from Boehringer Ingelheim and Bayer Schering Pharma.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received for publication June 20, 2007 and accepted in revised form January 2, 2008


   REFERENCES
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. de Gasparo M, Catt KJ, Inagami T, Wright JW, Unger T: International union of pharmacology: XXIII. the angiotensin II receptors. Pharmacol Rev 52:415–472, 2000[Abstract/Free Full Text]
  2. Benson SC, Pershadsingh HA, Ho CI, Chittiboyina A, Desai P, Pravenec M, Qi N, Wang J, Avery MA, Kurtz TW: Identification of telmisartan as a unique angiotensin II receptor antagonist with selective PPARgamma-modulating activity. Hypertension 43:993–1002, 2004[Abstract/Free Full Text]
  3. Schupp M, Clemenz M, Gineste R, Witt H, Janke J, Helleboid S, Hennuyer N, Ruiz P, Unger T, Staels B, Kintscher U: Molecular characterization of new selective peroxisome proliferator–activated receptor {gamma} modulators with angiotensin receptor blocking activity. Diabetes 54:3442–3452, 2005[Abstract/Free Full Text]
  4. Schupp M, Janke J, Clasen R, Unger T, Kintscher U: Angiotensin type 1 receptor blockers induce peroxisome proliferator-activated receptor-gamma activity. Circulation 109:2054–2057, 2004[Abstract/Free Full Text]
  5. Derosa G, Ragonesi PD, Mugellini A, Ciccarelli L, Fogari R: Effects of telmisartan compared with eprosartan on blood pressure control, glucose metabolism and lipid profile in hypertensive, type 2 diabetic patients: a randomized, double-blind, placebo-controlled 12-month study. Hypertens Res 27:457–464, 2004[Medline]
  6. Vitale C, Mercuro G, Castiglioni C, Cornoldi A, Tulli A, Fini M, Volterrani M, Rosano GM: Metabolic effect of telmisartan and losartan in hypertensive patients with metabolic syndrome. Cardiovasc Diabetol 4:6, 2005[Medline]
  7. Miura Y, Yamamoto N, Tsunekawa S, Taguchi S, Eguchi Y, Ozaki N, Oiso Y: Replacement of valsartan and candesartan by telmisartan in hypertensive patients with type 2 diabetes: metabolic and antiatherogenic consequences. Diabetes Care 28:757–758, 2005[Free Full Text]
  8. Shimabukuro M, Tanaka H, Shimabukuro T: Effects of telmisartan on fat distribution in individuals with the metabolic syndrome. J Hypertens 25:841–848, 2007[Medline]
  9. Benndorf RA, Rudolph T, Appel D, Schwedhelm E, Maas R, Schulze F, Silberhorn E, Boger RH: Telmisartan improves insulin sensitivity in nondiabetic patients with essential hypertension. Metabolism 55:1159–1164, 2006[Medline]
  10. Inoue T, Morooka T, Moroe K, Ikeda H, Node K: Effect of telmisartan on cholesterol levels in patients with hypertension: Saga Telmisartan Aggressive Research (STAR). Horm Metab Res 39:372–376, 2007[Medline]
  11. Mori Y, Itoh Y, Tajima N: Telmisartan improves lipid metabolism and adiponectin production but does not affect glycemic control in hypertensive patients with type 2 diabetes. Adv Ther 24:146–153, 2007[Medline]
  12. Picard F, Auwerx J: PPAR(gamma) and glucose homeostasis. Annu Rev Nutr 22:167–197, 2002[Medline]
  13. Staels B, Fruchart JC: Therapeutic roles of peroxisome proliferator–activated receptor agonists. Diabetes 54:2460–2470, 2005[Abstract/Free Full Text]
  14. Staels B, Dallongeville J, Auwerx J, Schoonjans K, Leitersdorf E, Fruchart JC: Mechanism of action of fibrates on lipid and lipoprotein metabolism. Circulation 98:2088–2093, 1998[Abstract/Free Full Text]
  15. Forman BM, Chen J, Evans RM: Hypolipidemic drugs, polyunsaturated fatty acids, and eicosanoids are ligands for peroxisome proliferator-activated receptors alpha and delta. Proc Natl Acad Sci U S A 94:4312–4317, 1997[Abstract/Free Full Text]
  16. Goldberg RB, Kendall DM, Deeg MA, Buse JB, Zagar AJ, Pinaire JA, Tan MH, Khan MA, Perez AT, Jacober SJ: A comparison of lipid and glycemic effects of pioglitazone and rosiglitazone in patients with type 2 diabetes and dyslipidemia. Diabetes Care 28:1547–1554, 2005[Abstract/Free Full Text]
  17. Sakamoto J, Kimura H, Moriyama S, Odaka H, Momose Y, Sugiyama Y, Sawada H: Activation of human peroxisome proliferator-activated receptor (PPAR) subtypes by pioglitazone. Biochem Biophys Res Commun 278:704–711, 2000[Medline]
  18. Shimasaki M, Yamashita K, Imanishi RKY, Kuritani M, Oiwa Y, Igarashi T: Pharmakokinetics of 14C-telmisartan: absorption, distribution and protein binding of 14C-telmisartan after a single oral administration to rats. Xenobio Metabol Dispos 14:425–431, 1999
  19. Cheng L, Ding G, Qin Q, Huang Y, Lewis W, He N, Evans RM, Schneider MD, Brako FA, Xiao Y, Chen YE, Yang Q: Cardiomyocyte-restricted peroxisome proliferator-activated receptor-delta deletion perturbs myocardial fatty acid oxidation and leads to cardiomyopathy. Nat Med 10:1245–1250, 2004[Medline]
  20. Cohen JA, Kaplan MM: The SGOT/SGPT ratio: an indicator of alcoholic liver disease. Dig Dis Sci 24:835–838, 1979[Medline]
  21. Lefebvre P, Chinetti G, Fruchart JC, Staels B: Sorting out the roles of PPAR alpha in energy metabolism and vascular homeostasis. J Clin Invest 116:571–580, 2006[Medline]
  22. Ohmura T, Tsunenari I, Seidler R, Chachin M, Hayashi T, Konomi A, Matsumaru T, Sumida T, Hayashi N, Horie Y: Renoprotective effects of telmisartan on renal injury in obese Zucker rats. Acta Diabetol, 2007 (Epub ahead of print)
  23. Gavrilova O, Haluzik M, Matsusue K, Cutson JJ, Johnson L, Dietz KR, Nicol CJ, Vinson C, Gonzalez FJ, Reitman ML: Liver peroxisome proliferator-activated receptor gamma contributes to hepatic steatosis, triglyceride clearance, and regulation of body fat mass. J Biol Chem 278:34268–34276, 2003[Abstract/Free Full Text]
  24. Matsusue K, Haluzik M, Lambert G, Yim SH, Gavrilova O, Ward JM, Brewer B, Jr, Reitman ML, Gonzalez FJ: Liver-specific disruption of PPARgamma in leptin-deficient mice improves fatty liver but aggravates diabetic phenotypes. J Clin Invest 111:737–747, 2003[Medline]
  25. Zhang YL, Hernandez-Ono A, Siri P, Weisberg S, Conlon D, Graham MJ, Crooke RM, Huang LS, Ginsberg HN: Aberrant hepatic expression of PPARgamma2 stimulates hepatic lipogenesis in a mouse model of obesity, insulin resistance, dyslipidemia, and hepatic steatosis. J Biol Chem 281:37603–37615, 2006[Abstract/Free Full Text]
  26. James O, Day C: Non-alcoholic steatohepatitis: another disease of affluence. Lancet 353:1634–1636, 1999[Medline]
  27. Belfort R, Harrison SA, Brown K, Darland C, Finch J, Hardies J, Balas B, Gastaldelli A, Tio F, Pulcini J, Berria R, Ma JZ, Dwivedi S, Havranek R, Fincke C, DeFronzo R, Bannayan GA, Schenker S, Cusi K: A placebo-controlled trial of pioglitazone in subjects with nonalcoholic steatohepatitis. N Engl J Med 355:2297–2307, 2006[Abstract/Free Full Text]
  28. Neuschwander-Tetri BA, Brunt EM, Wehmeier KR, Oliver D, Bacon BR: Improved nonalcoholic steatohepatitis after 48 weeks of treatment with the PPAR-gamma ligand rosiglitazone. Hepatology 38:1008–1017, 2003[Medline]
  29. Promrat K, Lutchman G, Uwaifo GI, Freedman RJ, Soza A, Heller T, Doo E, Ghany M, Premkumar A, Park Y, Liang TJ, Yanovski JA, Kleiner DE, Hoofnagle JH: A pilot study of pioglitazone treatment for nonalcoholic steatohepatitis. Hepatology 39:188–196, 2004[Medline]
  30. Nagasawa T, Inada Y, Nakano S, Tamura T, Takahashi T, Maruyama K, Yamazaki Y, Kuroda J, Shibata N: Effects of bezafibrate, PPAR pan-agonist, and GW501516, PPARdelta agonist, on development of steatohepatitis in mice fed a methionine- and choline-deficient diet. Eur J Pharmacol 536:182–191, 2006[Medline]
  31. Shiri-Sverdlov R, Wouters K, van Gorp PJ, Gijbels MJ, Noel B, Buffat L, Staels B, Maeda N, van Bilsen M, Hofker MH: Early diet-induced non-alcoholic steatohepatitis in APOE2 knock-in mice and its prevention by fibrates. J Hepatol 44:732–741, 2006[Medline]
  32. Sugimoto K, Qi NR, Kazdova L, Pravenec M, Ogihara T, Kurtz TW: Telmisartan but not valsartan increases caloric expenditure and protects against weight gain and hepatic steatosis. Hypertension 47:1003–1009, 2006[Abstract/Free Full Text]
  33. Fujita K, Yoneda M, Wada K, Mawatari H, Takahashi H, Kirikoshi H, Inamori M, Nozaki Y, Maeyama S, Saito S, Iwasaki T, Terauchi Y, Nakajima A: Telmisartan, an angiotensin II type 1 receptor blocker, controls progress of nonalcoholic steatohepatitis in rats. Dig Dis Sci 52:3455–3464, 2007[Medline]
  34. Donati G, Stagni B, Piscaglia F, Venturoli N, Morselli-Labate AM, Rasciti L, Bolondi L: Increased prevalence of fatty liver in arterial hypertensive patients with normal liver enzymes: role of insulin resistance. Gut 53:1020–1023, 2004[Abstract/Free Full Text]
  35. Entzeroth M, Ebner TJS, Hadamovsky S: Distribution of telmisartan in rat liver and binding to cytosol and cytosolic proteins (Abstract). J Hum Hypertens 13:43, 1999
  36. Roberts WC: Safety of fenofibrate: US and worldwide experience. Cardiology 76:169–179, 1989[Medline]
  37. Blanquart C, Barbier O, Fruchart JC, Staels B, Glineur C: Peroxisome proliferator-activated receptor alpha (PPARalpha) turnover by the ubiquitin-proteasome system controls the ligand-induced expression level of its target genes. J Biol Chem 277:37254–37259, 2002[Abstract/Free Full Text]
  38. Gebel T, Arand M, Oesch F: Induction of the peroxisome proliferator activated receptor by fenofibrate in rat liver. FEBS Lett 309:37–40, 1992[Medline]
  39. Pineda Torra I, Jamshidi Y, Flavell DM, Fruchart JC, Staels B: Characterization of the human PPARalpha promoter: identification of a functional nuclear receptor response element. Mol Endocrinol 16:1013–1028, 2002[Abstract/Free Full Text]
  40. Soria A, Gonzalez Mdel C, Vidal H, Herrera E, Bocos C: Triglyceridemia and peroxisome proliferator-activated receptor-alpha expression are not connected in fenofibrate-treated pregnant rats. Mol Cell Biochem 273:97–107, 2005[Medline]

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