Adiponetin and AMPK Biosensor

描述（由申请方提供）：脂联素（Adipo）作用于多种组织类型，发挥胰岛素增敏和心血管保护作用。生理和/或临床意义是Adipo的血浆水平与肥胖、胰岛素抵抗和心血管疾病负相关，但体重减轻或增加运动显著升高糖尿病或肥胖个体中的Adipo水平。在血管内皮中，Adipo与Adipo受体结合，激发AMP激活的蛋白激酶（AMPK）磷酸化/激活内皮型一氧化氮合酶（eNOS），从而提高NO的生物利用度。与野生型人Adipo相比，2个氨基酸的突变导致细菌和哺乳动物细胞系统的高表达和在盐水中的高溶解度。事实上，这种新产生的重组蛋白，命名为Adipo 3962，可以大量生产，体外和体内生物利用度与野生型Adipo相当。使用Adipo 3962，我们发现Ca 2+？依赖性蛋白激酶激酶（CaMKK）是响应Adipo的磷酸化AMPK的上游激酶。此外，我们开发了一个原型的荧光共振能量转移（FRET）为基础的AMPK活性报告（AMPKAR）来测量AMPK活性。根据这些初步结果，我们提出了这项R21试点研究，重点是两个具体目标：具体目标1将建立一个基于细胞的系统来评估AMPK激活。我们将建立稳定表达AMPKAR的血管内皮细胞（EC）系。EC-AMPKAR细胞中CaMKK-AMPK轴的激活将通过FRET响应来评估。具体目标2将研究脂肪激活的CaMKK-AMPK和小鼠血管中的相关有益作用。我们将向Adipo-/-、CaMKK 2-/-和AMPK 11-/-小鼠给予临床剂量的Adipo 3962。小鼠主动脉中的AMPK活性和随后的血管张力（即，血压、NO生物利用度、主动脉环松弛）。因此，该R21提案的目标是改进AMPKAR原型，以准确测量体外和体内AMPK活性，并表征CaMKK-AMPK信号传导在受益于Adipo的内皮功能中的作用。公共卫生相关性：近年来，与代谢性疾病相关的心血管损害已达到流行病的程度。由于脂联素和腺苷酸活化激酶（AMPK）在调节血管功能和代谢中的重要作用，我们建议建立一种AMPK生物传感器，该传感器可以准确地测量AMPK活性对各种药物的响应，包括大量生产的重组脂联素。这一建议将增加我们对脂联素激活AMPK的机制以及重组脂联素在心血管保护中的效用的理解。

DESCRIPTION (provided by applicant): Acting on multiple tissue types, adiponectin (Adipo) exerts insulin-sensitizing and cardiovascular protective effects. The physiological and/or clinical implications are that the plasma level of Adipo is negatively related to adiposity, insulin resistance, and cardiovascular disease, but weight reduction or increased exercise significantly elevates Adipo levels in diabetic or obese individuals. In vascular endothelium, Adipo binding to the Adipo receptors elicits AMP-activated protein kinase (AMPK) to phosphorylate/activate endothelial nitric oxide synthase (eNOS), which enhances NO bioavailability. Compared with the wild-type human Adipo, mutation of 2 amino acids resulted in high expression by both bacterial- and mammalian cell-based systems and high solubility in saline. Indeed, this newly created recombinant protein, designated Adipo3962, could be produced in large quantity, with in vitro and in vivo bioavailability comparable to that of the wild-type Adipo. Using Adipo3962, we found that Ca2????dependent protein kinase kinase (CaMKK) is the upstream kinase phosphorylating AMPK responding to Adipo. In addition, we developed a prototype of a fluorescence resonance energy transfer (FRET)-based AMPK activity reporter (AMPKAR) to measure AMPK activity. From these preliminary results, we propose this R21 pilot study, with emphasis on two specific aims: Specific Aim 1 will establish a cell-based system to assess the AMPK activation. We will create a vascular endothelial cell (EC) line stably expressing AMPKAR. Activation of the CaMKK-AMPK axis in EC-AMPKAR cells will be assessed by the FRET response. Specific Aim 2 will investigate the Adipo-activated CaMKK-AMPK and the associated beneficial effects in mouse vessels. We will administer clinical doses of Adipo3962 to Adipo-/-, CaMKK2-/-, and AMPK11-/- mice. The AMPK activity in the mouse aorta and the consequent vascular tone (i.e., blood pressure, NO bioavailability, aortic ring relaxation) will be investigated. Thus, the goal of this R21 proposal is to improve the AMPKAR prototype to accurately measure AMPK activity in vitro and in vivo and to characterize the role of CaMKK-AMPK signaling in endothelial functions benefiting from Adipo. PUBLIC HEALTH RELEVANCE: Cardiovascular impairment associated with metabolic diseases has reached epidemic proportions in recent years. Because of the important role of adiponectin and AMP-activated kinase (AMPK) in regulating vascular function and metabolism, we propose to establish an AMPK biosensor that can accurately measure AMPK activity responding to various pharmacological agents, including a mass-produced recombinant adiponectin. The proposal will increase our understanding of the mechanisms by which adiponectin activates AMPK and the utility of the recombinant adiponectin in cardiovascular protection.