Atherosclerosis 224 (2012) 377e383. Contents lists available at SciVerse ScienceDirect. Atherosclerosis

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1 Atherosclerosis 224 (2012) 377e383 Contents lists available at SciVerse ScienceDirect Atherosclerosis journal homepage: Decaffeinated green tea extract improves hypertension and insulin resistance in a rat model of metabolic syndrome Sang-Hyun Ihm a, Sung-Won Jang a, Ok-Ran Kim a, Kiyuk Chang a, Min-Ho Oak b, Jung-Ok Lee c, Dong-Yoon Lim d, Jae-Hyung Kim a, * a Division of Cardiology, Department of Internal Medicine, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea b College of Pharmacy, Mokpo National University, Muan, Jeonnam, Republic of Korea c UMR CNRS 7213, Laboratoire de Biophotonique et Pharmacologie, Faculté de Pharmacie, Université de Strasbourg, Illkirch, France d Department of Pharmacology, College of Medicine, Chosun University, Gwangju , Korea article info abstract Article history: Received 24 February 2012 Received in revised form 3 June 2012 Accepted 5 July 2012 Available online 15 July 2012 Keywords: Green tea extract Caffeine Hypertension Endothelial function Insulin resistance Background: Oxidative stress and endothelial dysfunction are closely associated with hypertension and insulin resistance (IR) in metabolic syndrome (MetS). It is still controversial whether green tea extract (GTE) may have blood pressure (BP) lowering effect. Decaffeinated GTE might be presumed to have strong antioxidative effect and BP-lowering effect as compared with catechins. Thus we investigated whether decaffeinated-gte could attenuate hypertension and IR by improving endothelial dysfunction and reducing oxidative stress in a rat model of MetS. Methods and Results: 20 Otsuka Long-Evans Tokushima Fatty (OLETF) rats at 13 weeks old, MetS rats, were randomized into a saline treated group (OLETF; n ¼ 10) and a group treated with decaffeinated-gte (25 mg/kg/day) (GTEeOLETF; n ¼ 10). Intraperitoneal glucose tolerance tests and BP measurements were performed at 13 and 25 weeks. Decaffeinated-GTE significantly reduced BP (OLETF vs. GTEeOLETF; vs mmhg, p ¼ 0.01), fasting/postprandial 2 h glucose (141 18/ vs / mg/dl, p ¼ 0.009/0.002) and insulin levels ( vs ng/ml, p < 0.001). Decaffeinated-GTE significantly reduced vascular reactive oxygen species (ROS) formation and NADPH oxidase activity, and improved endothelium dependent relaxation in the thoracic aorta of OLETF rats. Decaffeinated-GTE also suppressed the expression of p47 and p22phox (NADPH oxidase subunits) in the immunohistochemical staining, and stimulated phosphorylation of endothelial nitric oxide synthase (enos) and Akt in the immunoblotting of aortas. Conclusions: Decaffeinated-GTE reduced the formation of ROS and NADPH oxidase activity and stimulated phosphorylation of enos and Akt in the aorta of a rat model of MetS, which resulted in improved endothelial dysfunction and IR, and eventually lowered BP. Ó 2012 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Metabolic syndrome (MetS), a cluster of medical disorders that poses a risk of diabetes and cardiovascular disease [1], has become a significant public health problem worldwide. Hypertension and insulin resistance (IR) are the principal determinants of endothelial dysfunction and oxidative stress in patients with MetS [2]. Green tea is a rich source of polyphenols, particularly flavonoids. Clinical studies have indicated that regular intake of green tea reduces the risk of the cardiovascular disease [3]. While animal * Corresponding author. St Paul s Hospital, Jeonnong-Dong, Dongdaemoon-Gu, Seoul , Republic of Korea. Tel.: þ ; fax: þ address: jhkim480@catholic.ac.kr (J.-H. Kim). studies and epidemiologic studies have shown that green tea ingestion had a favorable effect on hypertension [4e7], other studies have not shown blood pressure (BP)-lowering effect [8]. The BP lowering effect of green tea is still controversial. Although catechins, one of major flavonoids of green tea, have received a great deal of attention due to its strong antioxidant and important biological properties, green tea also contains xanthic bases (caffeine and theophylline), essential oils, vitamins, minerals and certain phytochemical compounds in addition to catechins. Whereas vitamins and minerals in green tea can increase the antioxidant potentials of the catechin, caffeine may have a potential to reverse these benefits. Furthermore, some studies have reported that caffeine increases BP in human and animal studies [9e13]. The Otsuka Long-Evans Tokushima Fatty (OLETF) rat is known to be a distinct model of MetS and type2 diabetes, displaying /$ e see front matter Ó 2012 Elsevier Ireland Ltd. All rights reserved.

2 378 S.-H. Ihm et al. / Atherosclerosis 224 (2012) 377e383 characteristic features such as hypertension, a late onset of hyperglycemia (after 18 weeks of age), hyperinsulinemia, obesity and IR [14]. In this regard, the prediabetic or early stage of OLETF rats can be used as an animal model for MetS. We investigated whether the decaffeinated GTE could decrease BP and improve blood glucose and insulin sensitivity by reducing oxidative stress and ameliorating endothelial dysfunction of the vasculature in a rat model of MetS. 2. Materials and methods This investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No , revised 1996). All procedures were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee in School of Medicine (CUMC ), The Catholic University of Korea, Seoul, Republic of Korea In vivo treatment of rats Twenty male Otsuka Long-Evans Tokushima Fatty (OLETF) rats were used for the MetS rat model and 10 male Long-Evans Tokushima Otsuka (LETO) rats were used for the matched controls. The rats age was 5 weeks old at the start of the experiment. The rats were caged individually and they received normal rat chow and tap water in a temperature-controlled environment under a 12-h artificial light and dark cycle. At 13 weeks old, a total of 20 OLETF rats were randomized into two groups for treatment with either decaffeinated-gte (powdered form: 25 mg/kg/day; GTEeOLETF, n ¼ 10) or saline (the control group; OLETF, n ¼ 10) on a daily basis. Then the rats were treated orally with decaffeinated GTE dissolved in the saline or saline for 12 weeks. A total of 10 LETO rats were used for the matched non-mets controls (LETO, n ¼ 10) Preparation of decaffeinated-gte The preparation of decaffeinated-gte (Total catechins: 83.12%, Epigallocatechin Gallate (EGCG) : 45.60% and Caffeine: 0.67%) was previously described [4]. Powdered green tea (Thea sinensis) leaves were extracted at 100 C for 1 h and after cooling at 4 C for 12 h, the precipitate was removed by centrifugation at 5000 g for 30 min. Evaporation of the filtrate was done in a dryer and then the filtrate was ground into powder. Finally, this powder was shaken with 95% ethanol for 10 h, and then after removing the ethanol layer the supernatant was vaporized in the spray-dryer to give a water-soluble fraction into powdered form Measurement of the blood pressure, plasma glucose and insulin At the age of 13 weeks, prior to drug administration, the body weight, blood pressure and heart rate were determined with the rats in a 12-h fasting state. The blood pressure was measured every 2 weeks using tail-cuff plethysmography (BP-2000, Visitech system, Apex, NC, USA). An intraperitoneal glucose tolerance test (IP-GTT) was performed at 8 a.m. after a 12-h fasting period. A baseline fasting blood sample was drawn. The 2 h sample was drawn after a 25% glucose solution, at a dose of 2 g/kg, was injected intraperitoneally. The plasma glucose level was measured using the Beckman Glucose Analyzer (Beckman Instrument Co., Palo Alto, CA, USA). At the age of 25 weeks, all the measurements were performed again. The fasting plasma insulin levels were determined at 25 weeks of age. Fasting plasma insulin levels were determined using commercially available enzyme immunoassay kits (Rat Insulin, DRG Diagnostic, Mountainside, NJ, USA) Vascular reactivity studies After treatment, rats at the age of 25 weeks were anesthetized by intraperitoneal injection of pentobarbital (60 mg/kg, Halim Pharm. Co., Ltd, Republic of Korea.), and the adequacy of anesthesia was confirmed with the absence of pedal reflex. Thereafter, the aorta was excised, cleaned of connective tissue, and cut into rings (3e4 mm in length) and placed into Krebs solution (ph 7.4) containing 119 mmol/l NaCl, 4.7 mmol/l KCl, 1.17 mmol/l MgSO 4, 1.18 mmol/l KH 2 PO 4, 1.25 mmol/l CaCl 2, 25 mmol/l NaHCO 3 and 11 mmol/l glucose. As indicated the endothelium was removed by rubbing the intimal surface of rings with a pair of forceps. Changes in isometric tension were recorded using a force-displacement transducer (K30 force transducer, Hugo sachs elektronik-harvard apparatus, March-Hugstetten, Germany) connected to a polygraph recording system (iox2, Emka Technologies, Paris, France). Following equilibration for 90 min under a resting tension of 2 g, rings were contracted with phenylephrine (1 mm) to about 80% of the maximal contraction reached by increasing concentrations of phenylephrine. After washout and a 1-h equilibration period, rings were contracted again with phenylephrine (1 mm) before a concentrationerelaxation curve either to acetylcholine or sodium nitroprusside was constructed Determining the formation of vascular reactive oxygen species The oxidative fluorescent dye dihydroethidine (DHE, SigmaeAldrich, Milwaukee, WI, USA) was used to evaluate the in situ formation of reactive oxygen species as previously described [15]. The thoracic aortas from the LETO, OLETF and GTEeOLETF groups were embedded in OCT compound (O.C.T. Tissue-Tek, Sakura Finetek, Torrance, CA, USA) and then frozen in a liquid nitrogen bath for the cryostat sections. These unfixed frozen aortic rings were cut into sections of 5 mm thickness and dihydroethidine (2.5 mm) was applied to all the tissue sections, which were then incubated in a light-protected humidified chamber for 30 min at 37 C. The sections were examined using a confocal microscope (LSM 510 META Carl Zeiss Inc., Overkochen, Germany) with a 20 epifluorescence objective. After excitation at 543 nm, the emission signal was recorded with a Zeiss 560e615 nm filter. The mean intensities are expressed as arbitrary densitometry units Determination of vascular NADPH oxidase activity The lucigenin-derived enhanced chemiluminescence assay was used to determine NADPH oxidase activity in aortic tissues as previously detailed [16]. Aortic rings (3e4 mm length) from LETO, OLETF, OLETF-GTE groups were washed with HBSS (Hanks Balanced Salt Solution, Gibco, Grand Island, NY, USA) and incubated for 15 min at 37 C. Mixture of NADPH (200 mm) and lucigenin (25 mm) was added to aortic rings in HBSS. This concentration of lucigenin does not appear to be involved in redox cycling and specifically detects $O 2. Luminescence was measured for 15 min using a luminometer (VitorTM Light, PerkinElmer Life and Analytical Sciences Inc., Boston, MA, USA). Endothelium was removed by gently rubbing the lumen of the aorta. In addition, the lucigenin-derived enhanced chemiluminescence assay was used to determine NADPH oxidase activity in endothelium denuded aortic tissues Immunohistochemical determination for NADPH oxidase subunits Sections (5 mm) of thoracic aortas were cut from 4% paraformaldehyde-fixed, paraffin embedded tissue blocks,

3 S.-H. Ihm et al. / Atherosclerosis 224 (2012) 377e mounted on polylysin-coated slides. The antigen was retrieved by heating the sections in a 10 mm citrate buffer (60 Covenovernight). The cooled sections were incubated with 3% H 2 O 2 for 10 min to block endogenous peroxidases. For p22phox immunostaining, the primary antibody was the anti-p22phox rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), used at a 1:50 dilution. The secondary antibody was coupled to alexa fluor 488 (Invitrogen Corp., Carlsbad, CA, USA) used at a 1:2000 dilution. For p47phox immunostaining, sections were incubated at 4 C overnight with the rabbit polyclonal anti-p47phox antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a 1:50 dilution in 0.5 M TBST. The secondary antibody was coupled to alexa fluor 555 (Invitrogen Corp., Carlsbad, CA, USA) used at a 1:2000 dilution. Nuclear counterstaining was performed with DAPI at a 1:10000 dilution. Sections were kept in the dark until the fluorescence was studied using a confocal microscope (LSM 510 META Carl Zeiss, Inc., Overkochen, Germany) Endothelial nitric oxide synthase (enos) and Akt activation enos and Akt activation were determined by measuring the level of phosphorylation in the cytosolic fraction by western blot. The membranes were exposed to the primary antibodies against enos, phospho-enos, Akt and phospho-akt. The optical densities of the bands on the film were quantified using densitometry. The band density of phosphorylated protein was normalized to the corresponding total protein expression Statistical analysis All the values were expressed as a mean SD. Differences in the measured values among multiple groups were analyzed by ANOVA, and Bonferoni s multiple comparison test was used for post-hoc analysis. All the comparisons were two-sided, and a p-value of less than 0.05 was considered statistically significant. 3. Results 3.1. Decaffeinated-GTE lowers blood pressure in the OLEFT rats (animal model of metabolic syndrome) The body weight at 13 and 25 weeks was significantly greater in the OLETF and GTEeOLETF groups than that in the LETO group. There was no significant difference in the initial and final body weight between the OLETF and GTEeOLETF groups. At the beginning of treatment, the systolic BP was similar in the OLETF and GTEeOLETF groups, and these values were significantly higher than that of the LETO group (Table 1). From 13 until 25 weeks of age, the systolic BP had increased. At the age of 25 weeks, the systolic BP of the GTEeOLETF group was significantly lower than that of the OLETF group. A significant BP lowering effect was observed after 4 weeks of treatment with decaffeinated GTE Decaffeinated-GTE decreases plasma glucose and insulin levels in the OLETF rats At the age of 13 weeks, the plasma fasting glucose levels were not significantly different among three groups, but the postprandial 2-h glucose levels of the OLETF and GTEeOLETF groups were significantly higher than those of the LETO group. At 25 weeks old, the plasma fasting glucose levels of the OLETF and GTEeOLETF group were significantly higher than those of the LETO group. The postprandial 2-h glucose levels of the OLETF and GTEeOLETF groups were significantly higher than those of the LETO group. Treatment with decaffeinated-gte resulted in Table 1 Body weight, systolic blood pressure and serum glucose and insulin levels. Characteristics LETO (n ¼ 10) OLETF (n ¼ 10) GTEeOLETF (n ¼ 10) Body weight (g) Initial (13 weeks) * * Final (25 weeks) * * Blood pressure (mm Hg) Initial (13 weeks) * 114 8* Final (25 weeks) * 121 3* y Fasting glucose (mg/dl) Initial (13 weeks) Final (25 weeks) * 115 7* y PP 2 h glucose (mg/dl) Initial (13 weeks) * * Final (25 weeks) * * y Fasting insulin (ng/ml) Final (25 weeks) * * y PP 2 h: postprandial 2 h, GTEeOLETF: Decaffeinated-green tea extract treated OLETF. Data are expressed as mean S.D. *p < 0.05 vs. LETO rats. y p < 0.05 vs. OLETF rats. a significant reduction in the fasting and the postprandial 2-h glucose levels. In addition, the postprandial insulin levels of the GTEeOLETF group were significantly lower than those of the OLETF group (Table 1) Decaffeinated-GTE improves endothelial dysfunction in the OLETF rats The relaxation to acetylcholine was significantly reduced in the cut aortic rings derived from the OLETF rats as compared with those from the LETO rats, indicating impaired endothelium-dependent vascular relaxation (Fig. 1A). The reduced vascular reactivity to acetylcholine in the OLETF rats was significantly improved by the treatment with decaffeinated-gte. Relaxation of the aortic rings to sodium nitroprusside (SNP), a nitric oxide (NO) donor, was not different among the three groups (Fig. 1B), suggesting that the reactivity of the vascular smooth muscle towards NO remained normal in the OLETF rats Decaffeinated-GTE activates the expression of phosphorylated enos and Akt To obtain direct evidence that decaffeinated-gte causes the PI3 kinase/akt dependent activation of enos, levels of phosphorylated/ total enos and Akt were assessed in the aortic ring from LETO, OLETF and GTEeOLETF by immunoblotting. OLETF rats had a low level of phosphorylated enos (Fig. 2A, C) and phosphorylated Akt (Fig. 2B,D). However decaffeinated-gte induced a concentration increase in the phosphorylation levels of enos and Akt Decaffeinated-GTE prevents excessive vascular formation of reactive oxygen species by inhibiting NADPH oxidase activity and expression in the OLETF rats A marked fluorescence signal throughout the entire aortic wall was observed in the OLETF group, whereas only a slight signal was observed in the LETO group (Fig. 3A). The intake of decaffeinated-gte markedly reduced the fluorescent signal in the OLETF group (Fig. 3B). In addition, the activity of NADPH oxidase using enhanced chemiluminescence in endothelium-intact aortic segments was significantly increased in the OLETF group compared to the LETO group and this effect was abolished by the intake of decaffeinated GTE (Fig. 4A). However decaffeinated-gte did not inhibit NADPH oxidase activity in endothelium-denuded aortic segment from OLETF group (Fig. 4B). In the immunohistochemical staining for NADPH oxidase subunits,

4 380 S.-H. Ihm et al. / Atherosclerosis 224 (2012) 377e383 Fig. 1. Decaffeinated-GTE prevents the endothelial dysfunction in the OLETF rats. The concentrationerelaxation curve shows that vascular relaxation to acetylcholine (Ach) in the aortic rings with endothelium is improved in the GTE treatment group (A). Relaxation to sodium nitroprusside (SNP) without endothelium was not different among the three groups (B). The results are shown as means S.E.M. of 5 different rats. *p < 0.05 compared with the OLETF group. SNP; sodium nitroprusside. a low fluorescent signal for p22phox (red fluorescence) and p47phox (red fluorescence) was detected throughout the aortic wall in LETO group (Fig. 4C, E). Both of these signals were markedly increased in the aortic rings from the OLETF group but not in those from OLETF rats receiving decaffeinated-gte (Fig. 4D, F). 4. Discussion This is the first study to investigate the effects of decaffeinated- GTE on hypertension and IR by improving endothelial dysfunction and oxidative stress in a rat model of MetS. In this study, we found that endothelium-dependent relaxation induced by acetylcholine was significantly impaired in aortic rings of OLETF rats compared with LETO rats. The impaired endothelium-dependent relaxation was associated with an excessive vascular oxidative stress, an increased expression of several NADPH oxidase subunits including p22phox and p47phox and a decreased expression of phosphorylated Akt and enos. These findings suggest that the beneficial effect of decaffeinated-gte may be due to inhibition of NADPH oxidase activity and activation of Akt and enos pathway in the environment of MetS, and are in good agreement with previous studies indicating that vascular NADPH oxidase is involved in the impaired endothelium-dependent relaxation in OLETF rats [17,18] and reduced NO production, and that endothelial dysfunction are mediated via the inhibition of phosphorylated enos [19,20]. Furthermore, we have observed that BP, glucose and insulin values were normalized by decaffeinated-gte treatment (powdered form: 25 mg/kg/day). We chose 25 mg/kg/day (total catechins: mg/ kg/day and EGCG: mg/kg/day) as the concentration of decaffeinated-gte based on the study of catechins on humans [21]. Green tea catechins (585 mg/day, about 10 mg/kg in adult) improved body fat percentage, BP, and cholesterol [21]. Therefore, our dose of decaffeinated-gte effectively ameliorated MetS and endothelial dysfunction in vasculature. High BP and IR are the principal determinants of endothelial dysfunction and oxidative stress in patients with MetS [2]. Some Fig. 2. Activation of enos (A,C) and Akt (B,D) in isolated aortic rings from LETO, OLETF and decaffeinated-gte treated OLETF (GTEeOLETF). Representative immunoblots of phosphorylated-enos (p-enos), total enos, phosphorylated-akt (p-akt) and total Akt proteins (A,B). Densitometric analysis of enos phosphorylation (C) and Akt phosphorylation (D). Data are expressed by the phosphorylated: total protein ratios. Results are shown as mean S.E.M. of 8 different rats. *p < 0.05 vs LETO group, #p < 0.05 vs OLETF group.

5 S.-H. Ihm et al. / Atherosclerosis 224 (2012) 377e Fig. 3. Decaffeinated-GTE prevents the vascular formation of reactive oxygen species. Aortic sections were exposed to the redox-sensitive fluorescent dye dihydroethidine for 30 min at 37 C. Thereafter, the ethidium fluorescence was determined by confocal microscopy. The upper panel (A) represents LETO, OLETF and GTEeOLETF, respectively. The left, middle and right figures represent ethidium bromide staining (red), autofluorescent elastic laminae (bright) and merged images, respectively. The lower panel (B) represents the corresponding cumulative data. The results are shown as means S.E.M. (n ¼ 8) *p < 0.05 vs LETO group, #p < 0.05 vs OLETF group. epidemiologic studies have shown that tea consumption has a favorable effect on BP [7,22] but the other studies have not consistently demonstrated the BP-lowering effect of green tea [8]. Some compounds of GTE can generally increase the antioxidant potentials of the catechins, but caffeine may reverse these beneficial effects, mainly by increasing BP [9,10]. Caffeine was found to induce dose-dependent increases in heart rate and BP, and to elevate the concentration of cardiac catecholamines [11]. The beneficial effects of GTE on BP can be mainly explained by NO. EGCG, which is a potent antioxidant, increased NO production and the expression of enos and decreased the expression of endothelin-1, a potent vasoconstrictor [23,24]. Thus, we hypothesized that a decaffeinated GTE would improve endothelial dysfunction and oxidative stress in a rat model of MetS, leading to lowering of BP and amelioration of IR. The present study showed that administration of decaffeinated- GTE to OLETF rats reduced BP, decreased ROS and ameliorated the endothelium-dependent relaxations in response to acetylcholine. Since NO plays a significant role in the acetylcholine-induced endothelium-dependent relaxations of aortic rings, we assumed that decaffeinated-gte could increase the production of NO and/or decrease its degradation. In OLETF rats, vascular superoxide production is mainly affected by NADPH oxidase inhibitor [18], which implies that NADPH oxidase is the main enzymatic source for superoxide anion in a diabetic animal model. In addition, aortic rings of OLEFT rats have decreased enos activity which is the major source of NO. MetS or IR causes endothelial dysfunction by decreasing Akt kinase activity, resulting in diminished enos phosphorylation and activity [25]. We demonstrated that decaffeinated-gte reduced the production of ROS by inhibiting the activity of NADPH oxidase and Akt/eNOS pathway, and consequently decaffeinated-gte improved endothelial dysfunction. We assumed that BP lowering effect of decaffeinated-gte in our study was attributable to the reduction in ROS production and endothelial dysfunction, because oxidative stress and endothelial dysfunction are main causes of hypertension, diabetes and other cardiovascular disease conditions. We observed that decaffeinated-gte decreased the fasting and postprandial glucose levels, and fasting insulin levels in the OLETF rats. Glucose intolerance is associated with oxidative stress and it has been demonstrated that green tea was already reported to normalize plasma malondialdehyde concentrations, a marker for oxidative stress, and reverse glucose intolerance [26,27]. We assume that decreased ROS by decaffeinated-gte treatment might have contributed to improved insulin sensitivity in our experiment. Our results are consistent with the previous study [28] in which catechin improved the endothelial dysfunction in OLETF rats. In this experiment, we used decaffeinated-gte which not only contains catechin but also other antioxidants. We administered 25 mg/kg/ day of decaffeinated-gte (20.78 mg/kg/day of catechin), and this dose is relatively lower than in the previous study report [28]. However, the beneficial effects were similar or better than previous data in the aspect of endothelial function. Furthermore we demonstrated that decaffeinated-gte stimulated the phosphorylation of enos and Akt in addition. This study has several weaknesses and strengths. 1. We did not directly compare the effect of decaffeinated GTE with caffeinated GTE in this study. However, the concentrationerelaxation curve in response to acetylcholine showed that the slope of the decaffeinated GTE-treated OLETF rats was near identical to that of the LETO rats. When compared with the effect of catechin or other GTEs on endothelial dysfunction in the literature, we assume that decaffeinated-gte might be superior to caffeinated GTE for improving endothelial dysfunction. Further investigation is warranted on this subject. 2. Although lipid profiles are major component of MetS, we did not measure the lipid profile at baseline. Because the prediabetic OLEFT rats is well-known to be a distinct model of MetS. However we measured triglyceride and total cholesterol after treatment. Although decaffeinated-gte tends to decreased triglyceride levels in OLETF rats, there were no significant differences between OLETF-control and GTE treated OLETF rats. 3. We did not perform the expression of Akt and GLUT4 in skeletal muscles and adipose tissue. Animal studies have shown that green tea ameliorated IR via increasing the glucose transporter IV (GLUT4) content [29] and improving the protein expression of PPARg [30] and adipose tissue function [9]. IR leads to decreased glucose uptake in skeletal muscle and adipose tissues because of downregulation of GLUT4 translocation [25]. In skeletal muscle, phosphorylated Akt kinase stimulates the expression of GLUT4, resulting in glucose uptake and increasing insulin sensitivity. When

6 382 S.-H. Ihm et al. / Atherosclerosis 224 (2012) 377e383 Fig. 4. Decaffeinated-GTE inhibits NADPH oxidase activity in endothelium-intact aortic segments from OLETF rats (A). But decaffeinated-gte does not inhibit NADPH oxidase activity in endothelium-denuded aortic segment from OLETF rats (B). NADPH-induced $O 2 -generation was measured using enhanced lucigenin chemiluminescence. Results are shown as mean S.E.M. of 5 different rats. Decaffeinated-GTE prevents the expression of p22 and p47phox NADPH oxidase subunit in the arterial wall of OLETF rats. The expression levels of p22 and p47phox NADPH oxidase subunit were determined using a purified polyclonal antibody and a fluorescence-tagged secondary antibody by confocal microscopy (C: p22, E: p47). The photographs represent p22 and p47phox immunoreactivity (red, Red F), cell nuclei (blue, Blue F) and autofluorescent elastic laminae (bright). The other panel (D: p22, F: p47) represents corresponding cumulative data. Results are shown as mean S.E.M. of 8 different rats. *p < 0.05 vs LETO group, #p < 0.05 vs OLETF group. (For interpretation of the references to colour in this figure legend,the reader is referred to the web version of this article.) considering increased phosphorylation of Akt kinase in aortic ring, decaffeinated-gte might increase the phosphorylation of Akt kinase in endothelial cell and other tissues including adipose tissue and skeletal muscle. Phosphorylated Akt kinase stimulates the expression of GLUT4, which would result in improved IR or adipose tissue function. Our data demonstrated that decaffeinated-gte did not reduce body weight nor improve the lipid profile. Thus, it is highly possible that decaffeinated GTE might improve adipose tissue function in the absence of weight loss and improvement of two other metabolic syndrome components. 4. ROS was made by NADPH oxidase, enos, mitochondrial system and other enzymes. However we did not evaluate mitochondrial ROS in vivo due to technical problem. We only performed DHE staining to evaluate the formation of ROS. 5. Previous studies using GTEs with or without caffeine administered a liquid form of GTE. However, in the present study, we used a powdered form of GTE. We chose our emphasis on the powdered form because it is easily absorbed by the human body, and it can be taken as a capsule or pill. In addition, we used only 25 mg/kg/d of decaffeinated GTE in a rat model. This amount is an equal to 1.5e2.0 g/d in the adult human and easily accessible dose in humans. In conclusion, this is the first study demonstrating that chronic administration of decaffeinated-gte in a powdered form to OLETF rats reduced BP, plasma glucose and insulin levels by improving endothelial dysfunction and oxidative stress through the dual effects on NADPH oxidase and enos in a rat model of metabolic syndrome.

7 S.-H. Ihm et al. / Atherosclerosis 224 (2012) 377e Decaffeinated-GTE reduced the formation of ROS and NADPH oxidase activity and stimulated phosphorylation of enos and Akt. These beneficial effects of decaffeinated-gte might strengthen NO activity in aortic ring in MetS condition. These results suggest that decaffeinated-gte might be a potential therapeutic agent for the patients with hypertension and IR. Further study will clarify the relationship between decaffeinated-gte intake and changes in hemodynamic and metabolic parameters in humans. 5. Conflict of Interest None declared. Acknowledgement This work was supported by the Institute of Clinical Medicine Research of Bucheon St. Mary s Hospital, Research Fund, BCMC10LA02. Appendix A. Supplementary material Supplementary material associated with this article can be found, in the online version, at atherosclerosis References [1] Isomaa B, Almgren P, Tuomi T, et al. 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