Abstract 1149: Endothelial Dysfunction in AMP-activated Kinase α2 Knockout Mice: Role of NAD(P)H oxidase-derived Superoxide Anions
Objective: AMP-activated kinase (AMPK) is a serine/threonine kinase and is activated by oxidant stress. Whether or not AMPK activation alters the balance of oxidants/antioxidant is unknown. The aim of this study was to determine if a lack of AMPK impairs vascular function by altering the equilibrium of oxidants/antioxidants in vivo.
Methods and Results: The thoracic aortas of 8 –12 weeks old male AMPKα2 knockout mice (AMPK-KO) and genetic control Wildtype (WT) mice were isolated and endothelium-dependent and independent relaxation were assayed in organ baths. Superoxide anions were assayed by using 10 μM dihydroethidine in HPLC. The protein levels of SOD, catalase and NAD(P)H oxidase subunits were detected by Western Blot. Aortas were preconstricted with U46619 (30 nM) and endothelium-dependent relaxation was induced by acetylcholine (ACh). Compared to the WT mice, ACh-induced endothelium-dependent relaxation was significantly impaired in AMPK-KO mice (60.9±5.5% vs 85.5±10.5%, n=7, P<0.05). In contrast, endothelium-independent relaxations induced by SNP were similar in both WT and AMPK-KO mice. Compared to WT mice, the levels of total biopterins and tetrahydrobiopterin (BH4) were reduced in AMPK-KO mice. Further, pretreatment of aortas with superoxide anion scavengers, tempol (1 mM) and PEG-SOD (100 U/ml), or a BH4 precursor, sepiapterin (10 μM), for 24 hours improved endothelium-dependent relaxation in AMPK-KO mice (73.82±9.50%, 83.6±.60%, 79.4736.82 vs 60.935.5%, n=7, P<0.05). In addition, the basal levels of superoxide anions were markedly elevated in AMPK-KO mice compared to WT mice whereas the activity and protein expression of superoxide anion-scavenging enzymes, SOD and catalase, were similar in the aortas of WT and AMPK-KO mice. Furthermore, compared with WT mice, the expressions of NAD(P)H oxidase subunits, gp91phox, p22phox, p47phox, and p67phox were significantly increased in AMPK-KO mice aortas. Finally, inhibition of NAD(P)H oxidase with apocynin (100 μM), a selective NAD(P)H oxidase inhibitor, dramatically increased ACh-induced endothelium-dependent relaxation in AMPK-KO mice.
Conclusion: We conclude that AMPKα2 might exert vasoprotective effects by a suppression of NAD(P)H oxidase-derived superoxide anions.