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.
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.
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