Dig Dis Sci (2007) 52:3455–3464 DOI 10.1007/s10620-007-9741-4
ORIGINAL PAPER
Telmisartan, An Angiotensin II Type 1 Receptor Blocker, Controls Progress of Nonalcoholic Steatohepatitis in Rats Koji Fujita · Masato Yoneda · Koichiro Wada · Hironori Mawatari · Hirokazu Takahashi · Hiroyuki Kirikoshi · Masahiko Inamori · Yuichi Nozaki · Shiro Maeyama · Satoru Saito · Tomoyuki Iwasaki · Yasuo Terauchi · Atsushi Nakajima
Received: 20 November 2006 / Accepted: 1 January 2007 / Published online: 5 April 2007 C Springer Science+Business Media, LLC 2007
Abstract The term nonalcoholic steatohepatitis (NASH) has recently been proposed to identify a fatty liver disease accompanied by diffuse fatty infiltration and inflammation. However, no drug therapy has been established for NASH as yet. In the present study, we demonstrate the effect of the angiotensin II type 1 receptor antagonist telmisartan on the development of NASH in a rat model. Telmisartan, but not the angiotensin receptor antagonist valsartan, markedly attenuated hepatic steatosis, inflammation, and fibrosis in these rats. The quantitative parameters of steatosis, inflammation, and fibrosis were also ameliorated by treatment with telmisartan. Compared with telmisartan, the peroxisome proliferator-activated receptor-γ agonist pioglitazone attenuated hepatic steatosis and fibrosis of the liver to a similar degree. However, telmisartan, but not pioglitazone, dramatically decreased both subcutaneous and visceral fat. In conclusion, these results indicated that telmisartan should K. Fujita · M. Yoneda · H. Mawatari · H. Takahashi · H. Kirikoshi · M. Inamori · Y. Nozaki · S. Saito · A. Nakajima () Division of Gastroenterology, Yokohama City University Graduate School of Medicine, 3-9 Fuku-ura, Kanazawa-ku, Yokohama, Japan e-mail:
[email protected] K. Wada Department of Pharmacology, Graduate School of Dentistry, Osaka University, 1-8 Yamadaoka, Suita, Osaka, Japan S. Maeyama Kitakashiwa Rehabilitation Hospital, 265 Kitakashiwa, Chiba, Japan T. Iwasaki · Y. Terauchi Division of Endocrinology and Metabolism, Yokohama City University Graduate School of Medicine, 3-9 Fuku-ura, Kanazawa-ku, Yokohama, Japan
be the drug of first choice for the treatment of patients with NASH. Keywords Steatosis . Fibrosis . Inflammation . AT-II blockade . PPAR-γ activation Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver injury, and represents a spectrum of conditions that are histologically characterized by macrovesicular hepatic steatosis [1, 2]. NAFLD affects individuals who have not consumed alcohol in amounts considered harmful to the liver. The spectrum of NAFLD is broad, extending from simple steatosis through nonalcoholic steatohepatitis (NASH) to cirrhosis and liver failure [3, 4]. Among them, the term nonalcoholic steatohepatitis has recently been proposed to identify a fatty liver disease that is accompanied with diffuse fatty infiltration and inflammation [5, 6]. Although the pathologic findings in NASH and alcoholic liver disease are similar, the exact mechanisms that cause NASH are still unclear. Recently, a “2-hit hypothesis” was proposed to explain the development of NASH. Namely, steatosis as the first hit followed by a second hit that includes inflammation, oxidative damage, and fibrosis [5]. Many factors, such as cytokines, endotoxin, oxidative stress, and insulin resistance, have been proposed as the causes for the second hits of NASH [5, 6]. Several therapies, including diet [7] and antioxidants [8], have been tried to treat patients with NASH. However, no therapeutic strategy has been established for NASH as yet. Two drug therapies have been recently proposed. One is peroxisome proliferator-activated receptor gamma (PPARγ ) ligand therapy to improve insulin resistance [9]. Because NASH is a metabolic disease, it is reasonable to assume that an improvement of insulin resistance by PPAR-γ ligand will result in the amelioration of NASH. Another therapy is the Springer
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use of angiotensin II type I receptor antagonists. Recently, angiotensin II, the principal effector of the renin–angiotensin system, has been reported to have a crucial role in the pathogenesis of hepatic steatosis [10] and hepatic fibrosis [11] in a rat model of NASH. In addition, administration of the angiotensin II type 1 receptor antagonist losartan, was reported to improve liver biochemical indices of hepatic necroinflammation in NASH patients [12]. However, the mechanism of action of angiotensin II type 1 receptor antagonists has not been fully investigated yet. To clarify various questions about the recently proposed drug therapies, we investigated the inhibitory effect of angiotensin II type 1 receptor antagonists on the development of NASH in a choline-deficient, l-amino acid-defined diet (CDAA) animal model. In the present study, we have clarified 2 important points: (1) Whether the inhibitory effect on the development of NASH can be obtained
with any angiotensin II type 1 receptor antagonist; and (2) whether amelioration of NASH is due to a blockade of the angiotensin II type 1 receptor, or other mechanisms such as the activation of PPAR-γ . Our findings may contribute to the establishment of a therapy for patients with NASH in the near future.
Fig. 1 Choline-deficient, l-amino acid-defined diet (CDAA), NASH animal model. (a) Typical photographs of tissue sections from rats fed a CDAA diet or a CSAA control diet, showing steatosis, inflammation, oxidative stress, and fibrosis. Steatosis, inflammation, oxidative stress, and fibrosis were evaluated by Oil red O (red), hematoxylin and eosin, nitrotyrosine (brown), and Masson trichrome (blue) staining, respectively (original magnification, ×100). (b) Quantitative parameters of steatosis, inflammation, and fibrosis. The tissue content of triglycerides (TG), inflammatory cell number, and percentage of collagen area were
measured using quantitative parameters of steatosis, inflammation, or fibrosis, respectively. Data are expressed as the mean values ± SEM from 10 samples. (c) Comparison of other parameters in CDAA and CSAA control rats. Expression of collagen I and α-SMA protein in the liver from CDAA rats was evaluated by Western blot analysis and expressed as fold increase compared to that in CSAA control rats. Serum levels of TGF-β, TNF-α, ALT, leptin, and adiponectin were measured and expressed as the mean values ± SEM (n = 10)
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Materials and methods Reagents and diets PPAR-γ ligand, pioglitazone, and angiotensin II type I receptor antagonists, telmisartan and valsartan, were kind gifts from Takeda Pharmaceutical Co. (Tokyo, Japan), Nippon Boehringer Ingelheim Co. (Tokyo), and Novartis Pharma Co.
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Fig. 1 Continued.
(Tokyo), respectively. The CDAA and choline-sufficient, lamino acid-defined diet (CSAA) were obtained in powdered form from Dyets (Bethlehem, PA; product Nos. 518753 and 518754) [13]. Animal treatment and experimental procedure All animals were treated humanely according to the National Institutes of Health guidelines and the AERI-BBRI Animal Care and Use Committee guidelines. All animal experiments were approved by the institutional animal care and use committee of Yokohama City University School of Medicine.
Briefly, male Fischer-344 rats (6 weeks, body weight 150– 160 g) were purchased from Japan SLC Inc. (Hamamatsu, Shizuoka, Japan). They were quarantined for 1 week and then housed in stainless steel, mesh cages under controlled conditions of temperature (25 ± 2◦ C), humidity (50 ± 10%), and lighting (12-hour light/dark cycle). The animals were allowed free access to food and tap water throughout the acclimatization and experimental periods. After acclimatization for 1 week, the rats were divided into 7 experimental groups. Animals in groups 1–6 were given the CDAA diet throughout the experiment, whereas those in group 7 were given the CSAA diet as the control. Group 2 was given pioglitazone by gavage once a day at a dose of Springer
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Fig. 2 Effects of angiotensin II type 1 receptor antagonists and a PPAR-γ agonist on liver steatosis. (a) Typical photographs of liver tissue sections stained with Oil red O staining, as a marker of steatosis (original magnification, ×100). (b) Content of triglycerides (TG) in the livers of rats treated with pioglitazone (Pio, 1.875 mg/kg per day), valsartan (Val, 5 mg/kg per day), telmisartan (Tel, 5 mg/kg per day), or vehicle (Control). TG content is expressed as mg/g tissue, and data are expressed as mean values ± SEM of 10 animals
1.875 mg/kg per day [14, 15]. Group 3 was given valsartan at a dose of 5 mg/kg per day. Groups 4, 5, and 6 were given telmisartan at a dose of 1.7, 5, and 15 mg/kg per day, respectively [16]. After 12 weeks, the animals were humanely killed under ether anesthesia and samples were collected.
a TGF-β1 ELISA system (Amersham Biosciences Co, Piscataway, NJ).
Measurement of serum biochemical markers
Liver samples were homogenized in 50 mmol Tris/HCl buffer, pH 7.4, containing 150 mmol NaCl, 1 mmol EDTA, and 1 µmol PMSF. Triglycerides were analyzed enzymatically using a diagnostic kit [17] (Infinity, Thermo DMA, Arlington, TX) and measured by spectrophotometry (Beckman Coulter Inc, Fullerton, CA).
Serum alanine aminotransferase (ALT) was measured using Spotchem SP-4410 (Arklay Co, Kyoto, Japan). Leptin, adiponectin, fasting blood sugar, and fasting insulin levels were determined by radioimmunoassay using commercially available kits (Linco Research Inc., St. Charles, MO). Hepatic tumor necrosis factor (TNF)-α was measured by ELISA using the Quantikine Rat ELISA kit (R&D Systems, Minneapolis, MN). Transforming growth factor (TGF)-β1 expression level was measured using
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Measurement of liver triglycerides content
Marker protein expressions in liver Expression of actin α smooth muscle (α-SMA) and collagen I was determined by Western blot analysis as described pre-
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Fig. 3 Effects of angiotensin II type 1 receptor antagonists and a PPAR-γ agonist on liver inflammation. (a) Typical photographs of liver tissue sections stained with hematoxylin and eosin staining (original magnification, ×100). (b) Quantitative parameters of inflammation in control (Cont), pioglitazone-treated (Pio, 1.875 mg/kg per day), valsartan-treated (Val, 5 mg/kg per day), or telmisartan-treated (Tel, 5 mg/kg per day) rats. Serum alanine aminotransferase (ALT, a marker of liver inflammation) and TNF-α were measured and expressed as the mean values ± SEM of 10 animals. The number of infiltrating inflammatory cells in the liver was measured and expressed as the mean value for 10 animals
viously [17]. Briefly, liver samples were homogenized in a lysis buffer (50 mmol Tris/HCl, pH7.4, containing 150 mmol NaCl, 25 mmol EDTA, 5 mmol EGTA, 0.25% sodium deoxycholate, 1% Nonidet P-40, and 1 mmol DTT), and were resolved by SDS-PAGE and immunoblotting [18]. α-SMA expression was determined using mouse monoclonal antiactin α-SMA (Sigma, Saint Louis, MO). Collagen type I expression was determined using goat polyclonal antibody raised against a peptide mapping at the carboxy terminus of mature collagen type of human origin (Santa Cruz Biotechnology, Santa Cruz, CA) [19–21]. Histopathologic and immunohistochemical evaluations Liver samples were excised and embedded in Tissue-Tek OCT compound (Sakura Finetek U.S.A Inc, Torrance, CA) and paraffin for histologic analysis. Five-micrometer-thick sections of formalin-fixed and paraffin-embedded sections were processed routinely for hematoxylin and eosin staining. The presence of collagen, as an index of fibrosis in the lesions, was examined in Masson’s trichrome-stained preparations. Serial cryostat sections were used for staining
nitrotyrosine, as an index of oxidative stress, using a polyclonal rabbit anti-nitrotyrosine antibody (Upstate Biotechnology Inc, Iowa city, IA) diluted 1:2000 as previously described [22, 23]. As a negative control, normal mouse or rabbit IgG was used instead of the primary antibody. The OCT-embedded samples were serially sectioned at 4 µm. For the evaluation of fatty deposition, the liver tissues were stained with oil red O. For quantification, 50 fields were microscopically examined at a 40 × magnification using a grid of 0.0625 mm2 with 100 points. Values were expressed as the number of stained points per 100 points. The rest of liver tissues was frozen and stored in liquid nitrogen or at − 70◦ C until use for molecular and biochemical determinations. Measurement of PPAR-γ activation The PPAR-γ response element (PPRE) was transfected into 70% confluent Huh7 cells using Lipofectamin 2000 (Invitrogen, Carlsbad, CA). The cells were treated with 50 nmol PPRE for 24 hours before measurement by reporter assay. We used control medium as the control. To perform the reporter assay, Huh7 cells were seeded at a density of 1 × 104 Springer
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Fig. 4 Effects of angiotensin II type 1 receptor antagonists and PPAR-γ agonist on hepatic fibrosis. (a) Typical photographs of liver tissue sections stained with Masson trichrome staining, as a marker of fibrosis (original magnification, ×100). (b) Quantitative parameters of fibrosis in control (Cont), pioglitazone-treated (Pio, 1.875 mg/kg per day), valsartan-treated (Val, 5 mg/kg per day), or telmisartan-treated (Tel, 5 mg/kg per day) rats. Collagen and α-SMA protein levels were detected by Western blot analysis and were expressed as fold increase in comparison with those in CSAA rats (nonfibrosis liver) The serum level of TGF-β was also measured. Data are expressed as mean values for 10 animals
cells per well in 24-well plates and incubated at 37◦ C for 24 hours before transfection. Transfection was performed as indicated by the manufacturer using 3 µL lipofectamin 2000, 2 µg of Firefly luciferase reporter DNA (Upstate Biotechnology Inc), 0.5 µg phRL-TK-Renilla luciferase (Promega, Madison, WI) as the control for transfection efficiency in 200 µL of Opti-mem (Invitrogen). Twenty-four hours after transfection, the media was removed and cells were treated with medium containing 5 and 50 µmol of pioglitazone, valsartan, telmisartan, and vehicle for 8 hours. Then, the media was removed and Firefly and Renilla luciferase activities were sequentially measured using the Dual-Glo buffer system (Promega). Experiments were performed in triplicate and each set was repeated 3 times. To normalize the data, the Firefly luciferase value of each well was divided by the Renilla luciferase value. Statistical analysis All results are expressed as mean values ± SE. Statistical comparisons were made using Student’s t-test or Scheff´e’s
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multiple comparison test after the analysis of variances. The results were considered significantly different at P < .05.
Results Animal model of NASH In the pathogenesis of human NASH, hepatic steatosis followed by inflammation, oxidative damage and fibrosis are observed [4, 24, 25]. As shown in Fig. 1a, steatosis, inflammation, oxidative stress, and fibrosis were observed in rats fed the CDAA. In contrast, the rats fed the CSAA diet developed no steatosis (Fig. 1a). In addition, other biochemical, inflammatory, and fibrosis markers showed a pattern similar to that observed in human NASH (Fig. 1b, c). Therefore, rats fed the CDAA diet were considered a good animal model of NASH [13, 15]. There were no significant differences between the CDAA and CSAA diet groups in the total amount of calories consumed nor in body weight (data not shown).
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Fig. 5 Effects of angiotensin II type 1 receptor antagonists and a PPAR-γ agonist on lipid metabolism and organ fat accumulation in the whole body. Food intake (a), liver triglycerides content (b; Liver TG), subcutaneous (c), and visceral (d) fat in control (Cont), pioglitazone-treated (Pio, 1.875 mg/kg per day), valsartan-treated (Val, 5 mg/kg per day), or telmisartan-treated (Tel, 5 mg/kg per day) rats. Data are expressed as mean values ± SEM of 10 animals
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Effect of angiotensin II type 1 receptor antagonists and a PPAR-γ agonist on liver steatosis We used 2 angiotensin II type 1 receptor antagonists, valsartan and telmisartan, and the PPAR-γ agonist pioglitazone, to treat rats fed the CDAA diet. Hepatic steatosis was markedly suppressed by telmisartan in comparison to the control (Fig. 2a). The effect of telmisartan on hepatic steatosis was dose dependent (1.7–15 mg/kg). The content of triglycerides (TG) in the liver was also suppressed by telmisartan (Fig. 2b). A similar effect was observed when CDAA rats were treated with pioglitazone. In contrast, valsartan exerted a weak suppressive effect on the development of hepatic steatosis. These results indicate that angiotensin II type 1 receptor antagonists suppress steatosis but their efficacy varies greatly. Effect of angiotensin II type 1 receptor antagonists and a PPAR-γ agonist on liver inflammation We next investigated the effect of the angiotensin II type 1 receptor antagonists and a PPAR-γ agonist on liver inflammation. As shown in Fig. 3a and b, the number of infiltrating inflammatory cells in the liver of rats treated with pioglitazone, valsartan, and telmisartan was significantly reduced compared to that in the control liver. However, no differ-
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ence was found among the treatment groups. Other indexes of inflammation, such as ALT and TNF-α levels, were also significantly inhibited by pioglitazone, valsartan, and telmisartan. Treatment with pioglitazone or telmisartan inhibited ALT and TNF-α levels more effectively than valsartan; this difference was statically significant. Effect of angiotensin II type 1 receptor antagonists and a PPAR-γ agonist on hepatic fibrosis Hepatic fibrosis was markedly suppressed by valsartan and telmisartan as well as by pioglitazone (Fig. 4a). The indexes of fibrosis, namely, increases in collagen and α-SMA, were significantly suppressed by valsartan, telmisartan, and pioglitazone (Fig. 4b). Pioglitazone or telmisartan inhibited the increases in collagen and α-SMA levels more effectively than valsartan. A similar effect was observed on the TGF-β level. Effect of angiotensin II type 1 receptor antagonists and a PPAR-γ agonist on lipid metabolism and fat accumulation in the whole body Food intake was similar in all groups (Fig. 5a). The liver TG content was significantly suppressed by pioglitazone Springer
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and telmisartan, but not by valsartan (Fig. 5b). In addition, the serum level of TG rats was lower in telmisartantreated rats than in valsartan-treated rats (420 ± 48 versus 562 ± 63 mg/dL). Subcutaneous fat accumulation was significantly suppressed by valsartan and telmisartan. However, pioglitazone did not inhibit subcutaneous fat accumulation (Fig. 5c). In contrast, visceral fat accumulation was significantly suppressed by pioglitazone and telmisartan, but not by valsartan (Fig. 5d). These results indicate that telmisartan suppresses hepatic steatosis more effectively than valsartan, and that the effect of telmisartan might be due to an improvement of lipid metabolism in the whole body. In fact, the serum level of leptin that is related to fat accumulation, was significantly reduced by pioglitazone and telmisartan, but not by valsartan (Fig. 6b). The serum level of adiponectin, which is an anti-inflammatory and anti–insulin-resistant adipokine, tended to increase in pioglitazone and telmisartan-treated animals (Fig. 6b). Effect of angiotensin II type 1 receptor antagonists and a PPAR-γ agonist on PPRE-dependent transcription We next examined the effect of angiotensin II type 1 antagonists on PPRE-dependent transcription in cultured hepatic cells Huh7 strain, because PPRE-dependent transcripSpringer
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Fig. 6 Effects of angiotensin II type 1 receptor antagonists and a PPAR-γ agonist on PPRE-dependent transcription and serum parameters. (a) Effect of angiotensin II type 1 receptor antagonists and a PPAR-γ agonist on PPRE-dependent transcription. Huh7 cells were transfected with a PPAR-γ -responsive element-luciferase reporter construct, and luciferase activity was measured and expressed as relative PPRE activity (fold increase). Data are expressed as mean values ± SEM of 3 independent experiments. (b) Serum levels of the metabolic syndrome-related molecules leptin and adiponectin, and TNF-α in control (Cont), pioglitazone-treated (Pio, 1.875 mg/kg per day), valsartan-treated (Val, 5 mg/kg per day), or telmisartan-treated (Tel, 5 mg/kg per day) rats. The serum levels of leptin, adiponectin, and TNF-α were measured and expressed as mean values ± SEM
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tion is one of the most important factors involved in lipid metabolism and liver steatosis [26–28]. Valsartan-treated cells showed no significant alterations of PPRE-dependent transcription compared with the control (Fig. 6a). In contrast, PPRE-dependent transcription was significantly activated in telmisartan-treated cells, and an almost similar effect was observed in pioglitazone-treated cells. These results indicate that telmisartan is a potent PPAR-γ agonist and, therefore, it is different from other angiotensin II type 1 receptor antagonists such as valsartan.
Discussion In the present study, we clearly demonstrated that telmisartan, but not valsartan, markedly attenuated hepatic steatosis and fibrosis in a rat model of NASH. The suppressive effect of telmisartan and valsartan on the development of steatosis may also be observed with other antagonists, but that of telmisartan seems to be the strongest. From where does the difference between telmisartan and other angiotensin II type 1 receptor antagonists derive? One of the reasons is the difference in chemical structure. The structure of the angiotensin II type 1 receptor antagonists in clinical use today resembles that of valsartan; namely, they
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are biphenyl tetrazole derivatives. In contrast, telmisartan is a non-tetrazole derivative, with a single carboxylic acid group instead of a large tetrazole ring [29]. Thus, telmisartan is structurally quite different from all other angiotensin II antagonists. Recently, several reports have indicated that the structure of telmisartan resembles that of pioglitazone, a thiazolidinedione-type PPAR-γ ligand, and telmisartan is, therefore, considered to act as a partial agonist of PPAR-γ [29–31]. Namely, telmisartan influences the expression of PPAR-γ target genes that are involved in the metabolism of carbohydrates and lipids, as well as in the control of glucose, insulin, and TG levels. The present study clearly shows that telmisartan strongly activates PPAR-γ . This is a member of the nuclear hormone receptor superfamily and acts as a transcription factor that regulates the expression of multiple genes involved in the metabolism of carbohydrates and lipids as well as in inflammation [32–37]. The PPAR-γ agonist effect of telmisartan resulted in a stronger inhibition of NASH progression compared with other angiotensin II antagonists such as valsartan. Activation of the PPAR-γ pathway would improve insulin resistance, dyslipidemia, adipocytokine secretion, inflammation, and cell proliferation, leading mainly to an improvement of hepatic steatosis, NASH first hit. Blockade of the renin–angiotensin pathway would improve oxidative stress, inflammation, and proliferation, leading to an improvement of hepatic fibrosis, NASH second hit. According to the present results and previous reports, both effects of telmisartan as angiotensin II antagonist and PPAR-γ agonist are great advantages for their therapeutic use in patients with NASH and other types of vascular hypertrophy [38, 39]. In recent years, NASH has been considered as a metabolic syndrome affecting mainly the liver, and visceral fat has been considered to play an important role on the progression of NASH. The majority of NASH patients present with complications like diabetes mellitus, hyperlipidemia, hypertension, and obesity. Therefore, many other agents targeting different components of the pathogenesis of NASH have been studied, including insulin-sensitizing agents, weight loss agents, and anti-hyperlipidemic agents [40–44]. Drug therapy for NASH is positively carried out when patients cannot control eating or drinking, or cannot modify their lifestyle with diet and exercise. Pioglitazone has been used to treat NASH and its effectiveness has been reported [11, 45]. Both telmisartan and pioglitazone ameliorated hepatic steatosis, inflammation, and fibrosis to a similar degree. However, from the viewpoint of systemic conditions such as lipid metabolism and body weight, pioglitazone-treated rats showed increases in body weight and subcutaneous inguinal fat; telmisartan-treated rats showed mild loss of body weight and marked decreases in both subcutaneous inguinal and
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epididymal visceral fat. These are the most different and important points of the therapeutic efficacy of pioglitazone and telmisartan on animal NASH. In facts, we gave medication (pioglitazone 15 mg/day, for 6 months) to our 32 outpatients who could not improve liver steatosis and serum ALT levels with exercise and diet. Almost half (n = 15) showed little or no effect on glucose, lipid metabolism, and became worse about insulin resistance and liver steatosis compared with before medication (data not shown). In conclusion, telmisartan should be the drug of first choice for the treatment of patients with NASH in the near future. Acknowledgments Supported in part by a Grant-in-aid from the Ministry of Health, Labor and Welfare of Japan to AN, by a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan Kiban-B to AN, and by a grant from the National Institute of Biomedical Innovation to AN.
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