-AR stimulation enhances the activity of AMPK, indicating an important role of α 1

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1 Acta Physiologica Sinica, April 25, 2007, 59 (2): Research Paper α 1 -adrenergic receptors activate AMP-activated protein kinase in rat hearts XU Ming 1, ZHAO Yan-Ting 2, SONG Yao 1, HAO Tian-Pao 1, LU Zhi-Zhen 1, HAN Qi-De 1, WANG Shi-Qiang 2, ZHANG You-Yi 1,* 1 Institute of Vascular Medicine, Peking University Third Hospital and Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education, Beijing , China; 2 College of Life Sciences, National Key Laboratory of Biomembrane and Membrane Biotechnology, Peking University, Beijing , China Abstract: To test the hypothesis that AMP-activated protein kinase (AMPK) is possibly the downstream signaling molecule of certain subtypes of adrenergic receptor (AR) in the heart, we evaluated AMPK activation mediated by ARs in H9C2 cells, a rat cardiac source cell line, and rat hearts. The AMPK-α subunit and the phosphorylation level of Thr 172 -AMPK-α subunit were subjected to Western blot analysis. Osmotic minipumps filled with norepinephrine (NE), phenylephrine (PE) or vehicle [0.01% (W/V) vitamin C solution] were implanted into male Sprague-Dawley rats subcutaneously. The pumps delivered NE or PE continuously at the rate of 0.2 mg/kg per hour. After 7-day infusion, the activity of AMPK was examined following immunoprecipitation with anti-ampk-α antibody. At the cellular level, we found that NE elevated AMPK phosphorylation level in a dose- and time-dependent manner, with the maximal effect at 10 μmol/l NE after 10-minute treatment. This effect was insensitive to propranolol, a specific β-ar antagonist, but abolished by prazosin, an α 1 -AR antagonist, suggesting that α 1 -AR but not β-ar mediated the phosphorylation of AMPK. Moreover, the results from rat models of 7-day-infusion of AR agonists demonstrated that the activity of AMPK was significantly higher in NE (7.4-fold) and PE (6.0-fold) infusion groups than that in the vehicle group (P<0.05, n=6). On the other hand, no obvious cardiac hypertrophy and tissue fibrosis changes were observed in PE-infused rats. Taken together, our results demonstrate that α 1 -AR stimulation enhances the activity of AMPK, indicating an important role of α 1 -AR stimulation in the regulation of AMPK in the heart. Understanding the activation of AMPK mediated by α 1 -AR might have clinical implications in the therapy of heart failure. Key words: adrenergic receptor; AMP-activated protein kinase; heart α 1 - 肾上腺素受体激活大鼠心脏腺苷酸活化蛋白激酶徐明 1, 赵燕婷 2, 宋 1, 郝天袍 1, 吕志珍 1, 韩启德 1, 王世强 2 1,*, 张幼怡 1 北京大学第三医院血管医学研究所, 分子心血管教育部重点实验室, 北京 ; 2 生命科学学院, 生物膜与膜生物工程国家重点实验室, 北京 摘要 : 为了验证心脏腺苷酸活化蛋白激酶 (AMP-activated protein kinase, AMPK) 是否为肾上腺素受体 (adrenergic receptor, AR) 的下游信号分子, 本实验在大鼠心室肌源细胞和大鼠心脏中观察了 α 1 -AR 对 AMPK 的激活作用, 利用 Western blot 检测了 AMPK-α 总蛋白表达量及其 172 位苏氨酸磷酸化水平 雄性 Sprague-Dawley 大鼠皮下植入去甲肾上腺素 (norepinephrine, NE), 苯肾上腺素 (phenylephrine, PE) 或者溶剂载体 [0.01% (W/V) 维生素 C] 的缓释微泵 (osmotic minipump) NE 或 PE 以每小时 0.2 mg/kg 的速率持续输注,7 d 后用 AMPK-α 抗体免疫沉淀处理样本并测定 AMPK 的活性 结果显示, 在细胞水平,NE 引起的 AMPK 磷酸化水平增高具有时间依赖和剂量依赖特点, NE 处理细胞 10 min 后 AMPK 磷酸化水平达到最高峰 ;NE 引起的这种效应对 β-ar 的拮抗剂普萘洛尔 (propranolol) 不敏感, 但是可以被 α 1 -AR 拮抗剂哌唑嗪 (prazosin) 所阻断 结果提示,α 1 -AR 介导 AMPK 的磷酸化, 但 β-ar 无此作用 AR 激动剂持续灌注 7 d 后,AMPK 的活性在 NE (7.4 倍 ) 和 PE (6.0 倍 ) 灌注组较对照组显著增 Received Accepted This work was supported by the National Natural Science Foundation of China (No , ), the National Basic Research Development Program of China (No. 2006CB503806) and the Special Fund for Promotion of Education, Ministry of Education of China. * Corresponding author. Tel: ; Fax: ; zhangyy@bjmu.edu.cn

2 176 Acta Physiologica Sinica, April 25, 2007, 59 (2): 高 (P<0.05, n=6) PE 持续灌注组大鼠与对照组相比无明显的心肌肥厚和组织纤维化变化 本文证明 α 1 -AR 激动剂可以增强 AMPK 的活性, 揭示了心脏中 α 1 -AR 激动在调控 AMPK 活性方面的重要作用 深入了解 α 1 -AR 介导的 AMPK 激活可能在心衰治疗中具有重要的临床意义 关键词 : 肾上腺素受体 ; 腺苷酸活化蛋白激酶 ; 心脏中图分类号 :Q591;R AMP-activated protein kinase (AMPK) is a key regulator of cellular and systemic energy homeostasis [1]. As a sensor of energy gauge, AMPK not only regulates the energyrelated substance metabolism [2], but also has extensive influence on the regulation of cell functions, including the regulation of gene transcription [3,4], protein synthesis [5], and cellular environment homeostasis [6,7]. A recent study [8] indicated that the activation of AMPK plays a critical role in the regulation of energy metabolism during the pathological cardiac remodeling. Both eccentric and concentric cardiac hypertrophies are associated with abnormal myocardial energetics [9] that energy and oxygen supplies are relatively insufficient. AMPK is activated when the energy and oxygen supplies are restricted. Once activated, the enzyme switches off ATP-consuming anabolic pathways and switches on ATP-producing catabolic pathways, such as inhibition of key biosynthetic pathways [10] and promotion of fatty acid (FA) oxidation [11]. In addition to its direct role in promoting FA oxidation, AMPK-mediated recruitment of lipoprotein lipase to the coronary lumen represents an immediate compensatory response by the heart to guarantee FA supply [12]. A diminished AMPK signaling in the myocardium was one of the major determinants in the progression of enhanced concentric cardiac hypertrophy and increased mortality in pressure-overload model of adiponectin-deficient mice [13]. Therefore, understanding the mechanisms involved in the regulation of AMPK is very important for developing clinical therapy against the development of heart failure. Sympathetic nervous activation is a crucial compensatory mechanism in the transition from hypertrophy to heart failure. The catecholamine release matches the need of cardiac contraction during hypertrophy. However, an excess of catecholamine may induce cardiac dysfunction and adrenergic desensitization [14]. It has been previously reported that the activation of adrenergic receptors (ARs) mediated by plasma norepinephrine (NE) plays a critical role in controlling the development of heart failure [15,16]. However, besides the ratios of AMP/ATP or PCr/Cr [11,17], whether the regulation of AMPK is subject to the upstream regulation mediated by adrenergic stimulation in the heart remains unknown. Furthermore, the functional complexity of ARs in the heart is that endogenous catecholamine, noradrenaline, activates both α 1 - and β-ars in the heart and there is a crosstalk between α 1 - and β-ars [18,19]. Thus, here come the following questions: whether the activation of AMPK is subject to a specific adrenergic stimulation? If so, whether the activation of AMPK mediated by the activation of different ARs results in different extent of cardiac hypertrophy The objective of the present study was to investigate the effects of adrenergic stimulation on the regulation of AMPK in rat cardiac myoblast cell line (H9C2 cells) and hearts. 1 MATERIALS AND METHODS 1.1 Materials NE, phenylephrine (PE), isoproterenol (ISO), propranolol (Prop), prazosin (Praz) and vitamin C (VitC) were purchased from Sigma; goat anti-rabbit-hrp IgG was the product of Bio-Rad, rabbit anti-ampk-α antibody and antiphospho-ampk-α/thr 172 antibody were from Cell Signaling, anti-ampk-α 2 antibody were the product of US- BIO, SAMS peptide (HMRSAMSGLHLVKRR) were purchased from US-BIO, [γ- 32 P]ATP was from Amersham- Pharmacia Biotech. All other reagents were of biochemical grade. 1.2 Animal model Male Sprague-Dawley (SD) rats ( g) aged 12 weeks were obtained from the Medical Experimental Animal Center of Peking University. Rats were free access to diet and tap water ad libitum and housed at a constant room temperature [(23±1) C] in12-hour light/12-hour dark cycle. On the surgery day, rats were anesthetized with a ketaminexylazine mixture (5:3, 1.32 mg/kg, i.p.). Osmotic minipumps (model 2002, Alzet Corp., Palo Alto, CA, USA) filled with NE, PE or vehicle [0.01% (W/V) VitC solution] were implanted subcutaneously as described [20-22]. In the present study, the pumps delivered NE or PE at the rate of 0.2 mg/kg per hour. After 7-day infusion, rats were sacrificed by cervical dislocation, and hearts were rapidly removed, freeze-clamped at liquid nitrogen, and then stored

3 XU Ming et al: Adrenergic Receptor-mediated Activation of AMPK at 80 C until analysis. 1.3 Tissue homogenization Myocardial samples were weighed and then homogenized using a polytron (Brinkmann Instrument) in ice-cold Tris buffer (mmol/l): Tris-HCl 125, EDTA 10, EGTA 10, mannitol 250, dithiothreitol 1, benzamidine 1, phenylmethylsulfonyl fluoride 0.1, 4 µg/ml trypsin inhhibitor, 2 µg/ml aprotinin, 2 µg/ml leupeptin, ph 7.5 as previously described [23]. The homogenate was centrifuged at g for 5 min and the supernatant was subjected to polyethylene glycol (PEG) precipitation. The 2.5%-6% PEG fraction was suspended in homogenization buffer without mannitol for assay of AMPK activity. All procedures were performed at 4 C. Protein concentration was determined spectrophotometrically using the Bio- Rad reagent. 1.4 Cell culture and treatment H9C2 cells were cultured as previously described [24]. Cells were treated with 100 µmol/l VitC (control) or different agonists of ARs: NE (10 µmol/l), PE (1 µmol/l) or ISO (1 µmol/l) for 10 min, unless time course experiment, in serum-free DMEM (with 4.5 g/l glucose) medium, and different antagonists of ARs: the antagonist of α 1 -AR, Praz (1 µmol/l), antagonist of β-ar, Prop (10 µmol/l), were pre-incubated with cells for 30 min before the incubation of agonists. H9C2 cells were lysed with the buffer (mmol/l): Tris-HCl 20, NaCl 150, EDTA 2.5, NaF 50, Na 4 P 2 O 7 0.1, Na 3 VO 4 1, 1% Triton X-100, 10% glycerol, 1% SDS, 1% deoxycholic acid, phenylmethylsulfonyl fluoride 1 and 1 µg/ml aprotinin, ph 7.4. After 10-minute centrifugation at g, the supernatant was subjected to the Western blot analysis. 1.5 AMPK immunoprecipitation The AMPK activity associated with specific α subunit isoforms was examined following immunoprecipitation with anti-ampk-α 2 antibody. The immunoprecipitation was performed as described previously [25]. The antibodies were pre-incubated in excess to Protein G-Sepharose for 4 h and then incubated with homogenization of PEG precipitation overnight at 4 C. The immunoprecipitates were extensively washed with immunoprecipitation buffer (mmol/ L): Tris-HCl 50, NaCl 150, NaF 50, Na-pyrophosphate 5, EDTA 1, EGTA 1, dithiothreitol 1, benzamidine 0.1, phenylmethylsulfonyl fluoride 0.1, 5 µg/ml trypsin inhibitor, ph 7.4, and then washed with assay buffer (mmol/ L): Na-HEPES 62.5, NaCl 62.5, NaF 62.5, Na-pyrophosphate 6.25, EDTA 1.25, EGTA 1.25, dithiothreitol 1, benzamidine 1, phenylmethylsulfonyl fluoride 1 and 5 µg/ 177 ml trypsin inhibitor, ph 7.0, prior to the activity assay. 1.6 AMPK activity assay AMPK activity was measured as described [25,26]. Kinase reactions were performed in 40 mmol/l HEPES (ph 7.0), 0.2 mmol/l AMPK, 80 mmol/l NaCl, 0.8 mmol/l dithiothreitol, 5 mmol/l MgCl 2, 0.2 mmol/l ATP (containing 2 µci [γ- 32 P]ATP) and 0.2 mmol/l SAMS in a final volume of 25 µl for 20 min at 30 C. The reaction was terminated by adding 12.5 µl 40% trichloroacetic acid. Then a 20-µL aliquot was spotted on Whatmann filter paper (P81). The filter paper was washed 6 times with 1% phosphoric acid and once with acetone. The papers were air-dried and radioactivity was counted in 4 ml of scintillation liquid. AMPK activity was expressed as incorporated ATP picomoles per milligram of PEG precipitated protein per minute. 1.7 Immunoblotting Western blot analyses of AMPK-α subunit and phosphorylation of Thr 172 -AMPK-α subunit in H9C2 cells were performed by the method described [23]. Equivalent protein loading for the phosphorylated protein of AMPK was verified by reprobing with antiserum raised against the component of eif Statistical analysis Results were expressed as mean±sd. The statistical significance of the differences was determined by one-way ANOVA followed by a post hoc comparison using the Fisher s least significant difference method, significance was accepted at P< RESULTS 2.1 ARs-mediated changes in phosphorylation level of AMPK-α subunit in H9C2 cells The total protein expression of AMPK-α subunit was not significantly changed by the incubation with different concentrations of NE within 1 h in H9C2 cells (n=3, P>0.05. Fig.1). But 10 µmol/l NE induced AMPK activation to the peak after 10-minute treatment. Furthermore, the phosphorylation level of AMPK-α subunit was significantly increased to1.5 folds by the treatment of PE, an α 1 -AR agonist, for 10 min (P<0.05, n=3. Fig.2). And PE-induced elevation of AMPK phosphorylation level was time-dependent within 50 min (n=3, P<0.05, P<0.01. Fig.3). On the contrary, the phosphorylation level of AMPK-α subunit was not significantly different between the control group and ISO treatment group in 10 min (Fig.2). The increased phosphorylation level induced by NE was not inhibited by pre-incubation with 10 µmol/l Prop, an antagonist of b-ar,

4 178 Acta Physiologica Sinica, April 25, 2007, 59 (2): Fig. 2. Effects of different agonists and antagonists of ARs on the phosphorylation level of AMPK-α subunit. H9C2 cells were treated with 100 µmol/l vitamin C (control) or different agonists of ARs: NE (10 µmol/l), PE (1 µmol/l) and ISO (1 µmol/ L) for 10 min in serum-free DMEM medium, and different antagonists of ARs: the antagonist of α 1 -AR, Praz (1 µmol/l), antagonist of β-ar, Prop (10 µmol/l), were pre-incubated with cells for 30 min before the incubation of AR agonists. Protein levels of AMPK-α subunit and p-thr 172 -AMPK-α subunit were analyzed by immunoblotting with anti-ampk-α and anti-p- Thr 172 -AMPK-α antibodies, respectively. mean±sd, n=3. * P< 0.01 vs control. Δ P<0.05 vs NE. Fig. 1. Effect of NE on the phosphorylation level of AMPK-α subunit. H9C2 cells were treated with 10 µmol/l NE for different time (A) and with different concentrations of NE for 10 min (B), respectively. mean±sd, n=3. * P<0.05 vs control. but abolished by 1 µmol/l Praz, an a 1 -AR antagonist, suggesting that NE-induced elevated phosphorylation level of AMPK was mainly attributed to the activation of a 1 -AR (Fig.2). 2.2 Effects of AR agonist infusion on activities of AMPK-a 2 subunit in hearts Results from rat with 7-day-infusion of AR agonists showed that the activities of AMPK-a 2 subunit were significantly higher in NE (7.4-fold) and PE (6.0-fold) infusion groups than that in the vehicle group, respectively (P< 0.05, n=6. Fig.4). These results implied that adrenergic stimulation mediated by NE or PE may result in different extent of AMPK activation. Fig. 3. Effect of PE on the phosphorylation level of AMPK-α subunit. Cells were treated with 100 µmol/l vitamin C (control) or PE (1 µmol/l) for different time in serum-free DMEM medium. mean±sd, n=3. * P<0.05, ** P<0.01 vs control.

5 XU Ming et al: Adrenergic Receptor-mediated Activation of AMPK 179 Table 1. Effects of AR agonist infusion on LVW/BW and WHW/BW ratios in rat hearts Vehicle NE PE LVW/BW 2.16± ±0.07 * 2.16±0.07 WHW/BW 2.79± ±0.11 * 2.75±0.04 Fig.4. Effects of AR agonist infusion on the activity of AMPK-α subunits. Rats were treated with NE (0.2 mg/kg per hour), PE (0.2 mg/kg per hour) or vechicle [0.01% (W/V) VitC] infusion for 7 d, respectively. The activity of AMPK associated with specific α subunit isoforms was examined following immunoprecipitation with anti-ampk-α 2 antibody. * P<0.05 vs the vehicle group. n= Effects of AR agonist infusion on cardiac hypertrophy and structure Upon continuous infusion of NE or PE, the ratios of both left ventricular weight (LVW) and whole heart weight (WHW) to body weight (BW) were increased significantly in NE infusion group compared with that in the vehicle group (P<0.01, n=6), but PE infusion did not significantly Rats were treated with NE (0.2 mg/kg per hour), PE (0.2 mg/kg per hour) or vechicle [0.01% (W/V) VitC] infusion for 7 d, respectively. BW, body weight; LVW, left ventricular weight; WHW, whole heart weight. * P<0.01 vs the vehicle group. means±sem, n=6. affect the ratios (Table 1). Thus, it s suggested that the different AR agonist infusion might lead to the different extent of cardiac hypertrophy. This was further identified by the histologic examination that abundant Hematoxylin Eosin (HE)-stained nuclei distributed in endocardium and enhanced Sirius-Red-stained collagen I located in cardiac interstitium in NE infusion group. Compared to the vehicle infusion, the averaged across area of cardiomyocytes in the middle of heart muscle was obviously increased by NE infusion (Fig.5). Much less histologic change was noticed in PE infusion group than that in NE infusion group (Fig.5). Fig. 5. Effects of AR agonist infusion on cardiac hypertrophy of rats. Rats were treated with NE (0.2 mg/kg per hour) or PE (0.2 mg/kg per hour) infusion for 7 d, respectively. The section of left ventricular of rat was stained with Hematoxylin-Eosin (HE) and Sirius-Red. The location of endocardium was indicated by arrows. Scale bar, 400 nm.

6 180 3 DISCUSSION The activation of ARs induced by enhanced activity of endogenous catecholamine was believed to be an important mediator of cardiac hypertrophy, following the change of energy homeostasis in the heart [9], while AMPK plays a critical role in regulating cellular metabolism for sustaining energy homeostasis [27]. A question was raised concerning the effect of the activation of ARs on the regulation of AMPK. Whether the activation of AMPK is a result of chronic changes of myocardial energetics in hypertrophied heart [8,28] or a feedback of the activation of hypertrophic signaling pathways mediated by the activation of ARs [29] remains elusive. In the present study, the effect of the activation of AR on the regulation of AMPK was evaluated in both rat H9C2 cells and hearts. At the cellular level, we firstly identified a pattern of the activation of AMPK mediated by NE (Fig.1). The results showed that the elevation of phosphorylation level of Thr 172 -AMPK-α subunit induced by NE was in a doseand time-dependent manner. Furthermore, NE-induced activation was mainly attributed to the activation of α 1 -AR (Fig.2 and 3). This result was different from the previous report in adipocytes that NE caused phosphorylation of AMPK via β-ar but not α 1 - or α 2 -AR [30]. The mechanism underlying this difference might be associated with the functional complexity of the ARs that the relative amount of ARs is different in respective tissues and the distribution of ARs is different in different tissues. To seek the physiological importance of ARs-mediated activation of AMPK in heart, we firstly evaluated 7-day infusion of AR agonists on the activities of AMPK-α 2 subunit, since AMPK-α 2 subunit plays crucially regulatory roles in the heart [27,31]. The activities of AMPK-α 2 subunit were significantly higher in both NE (7.4-fold) and PE (6.0-fold) infusion groups than that in the vehicle group (P< 0.05, n=6. Fig.4). However, no obvious cardiac hypertrophy and tissue fibrosis changes were observed in PEinfused rats, compared to that in NE-infused rats (Fig.5). It might be possible that the activation of AMPK-α 2 subunit induced by PE contributes to suppressing cardiac protein synthesis and reducing tissue fibrosis. Since the significant increase of AMPK-α 2 activity was associated with decreased activation of protein kinases in the mammalian target of rapamycin (mtor) signal transduction pathway in skeletal muscle [5], we proposed that different extent of hypertrophy mediated by NE or PE might be due to the effect of AMPK activation on mtor signaling pathway. The exact mechanism remains to be defined. Acta Physiologica Sinica, April 25, 2007, 59 (2): To understand the regulation of AMPK activation, both direct substrate (AMP/ATP and PCr/Cr) regulation and upstream regulation (kinase or phosphatase) need to be considered. We found that the activation of AMPK induced by 5-aminoimidazole-4-carboxamide (AICA) riboside, which mimics the effect of AMP, could be further activated by NE treatment (data not shown). To identify the effects of AMP/ATP and PCr/Cr on NE-induced AMPK activation, the concentrations of AMP/ATP and PCr/Cr need to be determined in further studies. It has been reported previously that AMPK kinase (AMPKK) [32,33] and phosphatase [34] are both involved in the upstream regulation of AMPK. Taken together, a specific upstream mechanism other than the change of AMP/ATP ratio might be involved in the regulation of the activity of AMPK mediated by the activation of ARs. A recent study [35] reported that Ca 2+ as a regulator of AMPK activation is responsible to a physiologic stimulation and Ca 2+ /calmodulin-dependent protein kinase kinase beta (CaMKKβ) is the responsible AMPKK in endothelial cells. Whether the activation of AMPK is Ca 2+ / CaMKKβ-dependent or AMP/LKB1-dependent in the heart remains to be elucidated. AMPK activation promotes glycolysis and FA oxidation conserving ATP level in the heart. Therefore, understanding the different effects of ARs on the activation of AMPK is important in maintaining energy homeostasis during cardiac remodeling. AR agonist or antagonist treatment may affect the status of energy metabolism in heart failure. How to maintain the healthy status of energy metabolism in heart failure is yet to be known. In the present study, our results firstly demonstrate that α 1 -AR stimulation enhances the activity of AMPK, indicating an important role of α 1 -AR stimulation on the regulation of AMPK in the heart. 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