AMPK as a redox sensor and modulator

说明(申请人提供)：AMPK是一种丝氨酸/苏氨酸蛋白激酶，参与细胞和生物代谢的调节。AMPK系统作为细胞能量状态的传感器，在所有真核细胞中都是保守的。AMPK被认为是通过检测AMP/ATP比率的变化来作为细胞内的能量计。AMPK激活的结果包括抑制ATP消耗过程和激活ATP产生过程。申请人的小组首先证明了生理上相关浓度的ONOO可增加AMPK活性及其下游酶，如eNOS和乙酰辅酶A羧基酶(ACC)。此外，我们还证明了ONOO依赖的AMPK激活在缺氧-复氧、二甲双胍刺激的内皮细胞、血栓素受体刺激和尼古丁处理的脂肪细胞中起作用。同样，我们发现香烟烟雾的主要成分尼古丁以ROS依赖的方式激活分化的3T3L1脂肪细胞中的AMPK。有趣的是，由药理学AMPK激活剂(AICAR和二甲双胍)、生理刺激(葡萄糖耗竭和渗透压应激)或遗传操作(编码固有活性AMPK的腺病毒)激活AMPK可以保护内皮免受尼古丁的不利影响。同时，药物或遗传抑制AMPK显著增加培养的内皮细胞、分化的3T3L1脂肪细胞和原代培养的大鼠肝细胞的ROS、NF？B和炎症基因(ICAM-1、VCAM-1和E-选择素)。同时，在支持AMPK作为氧化应激抑制因子的同时，我们获得的初步证据表明，缺血预适应(IPC)激活AMPK有效地阻止了缺氧/复氧触发的氧化应激。AMPK减轻氧化应激的最确凿证据是，IPC未能改变AMPK 11基因敲除(KO)小鼠的氧化应激标志物和内皮功能。我们一直发现，与野生型相比，从AMPK 11或12 KO小鼠分离的主动脉表现出内皮松弛受损，同时O2.-和ONOO-的检测增加。同样，与C57BL6野生型相比，AMPK12 KO小鼠的NADPH氧化酶亚基gp91Phox(NOX2)、NOx-4、p22Phox、p47Phox和p67Phox的水平更高，NAD(P)H氧化酶活性也更高。重要的是，Apoynin，一种有效的NAD(P)H氧化酶抑制剂，恢复了AMPK 12KO小鼠由乙酰胆碱引起的内皮依赖的松弛，进一步表明NAD(P)H氧化酶具有功能活性，并与AMPK 12KO小鼠的内皮功能受损有关。最后，在不改变血脂(胆固醇和甘油三酯)的情况下，与Apo-E/AMPK11或AMPK/AMPK 12双KO小鼠相比，Apo-E/AMPK11或AMPK/AMPK 12双KO小鼠分离的主动脉显示出氧化应激标志物的检测增加，动脉粥样硬化病变增加，促炎症黏附分子的表达增加。这项应用的目的是确定：(A)AMPK通过其对AMP水平非常微小的变化做出反应的能力，可能是细胞近端的“氧化应激传感器”；(B)AMPK的激活可能触发生理反应，以抑制产生氧化剂(调节剂)的过程，或增加抗氧化防御系统；以及(C)AMPK通过减少氧化应激，维持血管细胞中非血管生成、非炎性和抗动脉粥样硬化的表型。与公共健康相关：这项提议的一个基本前提是，AMPK的激活可以通过启动减少氧化应激的活动来保护细胞和整个生物体免受尼古丁等心血管危险因素的不利影响。为了清晰和集中于当前的应用，我们使用尼古丁作为与心血管疾病相关的病理刺激。由于越来越多的证据表明，氧化应激参与了包括高胆固醇血症、糖尿病和高血压在内的心血管危险因素的血管效应，因此，这一建议的意义超出了尼古丁的范围，并将适用于其他心血管疾病。目前的应用解决了生物学和医学中的一个基本问题，即氧化应激源在健康和疾病中是如何被感知和调节的。因此，这项应用的完成可能会为AMPK在应激反应中的作用增加一个全新的维度，并在氧化应激、能量代谢和心血管生物学之间提供一个接口。拟议研究的完成将为AMPK是否是吸烟和常见疾病(包括衰老、肥胖、糖尿病、高血压和动脉粥样硬化)治疗的潜在靶点提供新的见解。

DESCRIPTION (provided by applicant): AMPK is a serine/threonine protein kinase involved in the regulation of cellular and organismal metabolism. The AMPK system acts as a sensor of cellular energy status that is conserved in all eukaryotic cells. AMPK has been suggested to serve as an energy gauge in cells, by detecting changes in the ratios of AMP to ATP. The consequences of AMPK activation include inhibition of ATP-consuming processes and activation of an ATP-producing process. The applicant's group was the first to demonstrate that physiologically relevant concentrations of ONOO- increased AMPK activity as well as its downstream enzymes such as eNOS and acetyl Co-A carboxylase (ACC). In addition, we have also demonstrated that ONOO--dependent AMPK activation is operative in hypoxia-reoxygenation, metformin- stimulated endothelial cells, thromboxane receptor stimulation, and nicotine-treated adipocytes. Similarly, we found that nicotine, a major constituent of cigarette smoke, activates AMPK in differentiated 3T3L1 adipocytes in a ROS-dependent fashion. Interestingly, AMPK activation by pharmacological AMPK activators (AICAR and metformin), physiological stimuli (glucose-depletion and osmotic stress), or genetic manipulation (adenoviruses encoding constitutively active AMPK) protects the endothelium against the adverse effects of nicotine. Concomitantly, pharmacological or genetic inhibition of AMPK markedly increased ROS, NF?B, and inflammatory genes (ICAM-1, VCAM-1, and E-selectin) in cultured endothelial cells, differentiated 3T3L1 adipocytes, and cultured primary rat hepatocytes. In parallel, in supporting that AMPK functions as a suppressor of oxidant stress, we have obtained preliminary evidence suggesting that AMPK activation by ischemic preconditioning (IPC) effectively blocked hypoxia/reoxygenation-triggered oxidant stress. The most conclusive evidence that AMPK reduced oxidant stress is that IPC failed to alter both the markers of oxidant stress and endothelial function in the AMPK 11 knockout (KO) mice. Consistently, we have found that compared to the wild types, aortas isolated from AMPK 11 or 12 KO mice exhibited impaired endothelial relaxation together with increased detections of both O2.- and ONOO-. Similarly, there was greater levels of NADPH oxidase subunits including gp91phox (NOX2), NOX-4, p22phox, p47phox, and p67phox together with increased NAD(P)H oxidase activity in AMPK12 KO mice than those in C57BL6 wild types. Importantly, apocynin, a potent NAD(P)H oxidase inhibitor, restored acetylcholine-induced endothelium-dependent relaxation in AMPK 12 KO mice, further suggesting NAD(P)H oxidase is functionally active and is responsible for impaired endothelial function in AMPK 12KO mice. Finally, without altering plasma lipids (cholesterol and triglyceride), the aortas isolated from Apo-E/AMPK11 or AMPK/AMPK 12 dual KO mice exhibited increased detection of oxidant stress markers, increased atherosclerotic lesions, and increased expression of proinflammatory adhesion molecules when compared to those in Apo-E KO mice. The goal of this application is to establish (a) that AMPK, through its ability to respond to very small changes in AMP levels, may be the proximal "oxidant stress-sensor" of the cell; (b) that AMPK activation may trigger physiological responses to suppress processes that generate oxidants (modulator) and or increase anti-oxidant defense systems; and (c) AMPK, via a reduction of oxidant stress, maintains the non-angiogenic, non-inflammatory, and atherosclerosis-resistant phenotypes in vascular cells. PUBLIC HEALTH RELEVANCE: A basic premise of this proposal is that AMPK activation could protect cells and the whole organism against the adverse effects of cardiovascular risk factors such as nicotine by setting in motion events that decrease oxidant stress. For the sake of clarity and being focused of the current application we use nicotine as a pathological stimulus relevant to cardiovascular diseases. As there is growing evidence that oxidant stress is involved in the vascular effects of cardiovascular risk factors including hypercholesterolemia, diabetes, and hypertension, thus, the significance of this proposal is beyond nicotine and will be applicable to other cardiovascular diseases. The current application addresses a fundamental question in biology and medicine, i.e., how oxidant stressors are sensed and modulated in health and disease. Completion of this application, thus, may add an entirely new dimension to the role of AMPK in stress responses and provide an interface between oxidant stress, energy metabolism, and cardiovascular biology. Completion of the proposed studies will provide novel insights into whether AMPK is a potential target for therapy in smoking and common diseases including aging, obesity, diabetes, hypertension, and atherosclerosis.