Adiponectin-Induced Endothelial Nitric Oxide Synthase Activation and Nitric Oxide Production Are Mediated by APPL1 in Endothelial Cells

Adiponectin-Induced Endothelial Nitric Oxide Synthase Activation and Nitric Oxide Production Are Mediated by APPL1 in Endothelial Cells Kenneth K.Y. Cheng 1 2 , Karen S.L. Lam 1 2 , Yu Wang 3 , Yu Huang 4 , David Carling 5 , Donghai Wu 6 , Chiwai Wong 6 and Aimin Xu 1 2 6 1 Department of Medicine, University of Hong Kong, Hong Kong, China 2 Research Center of Heart, Brain Hormone and Healthy Aging, University of Hong Kong, Hong Kong, China 3 Genome Research Center and Department of Biochemistry, University of Hong Kong, Hong Kong, China 4 Department of Physiology, Chinese University of Hong Kong, Hong Kong, China 5 Cellular Stress Group, Medical Research Council (MRC) Clinical Sciences Centre, Imperial College, U.K 6 Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China Address correspondence and reprint requests to Aimin Xu, PhD, Department of Medicine, University of Hong Kong, L8-43, New Laboratory Block, 21 Sassoon Road, Hong Kong. E-mail: amxu{at}hkucc.hku.hk Abstract Adiponectin protects the vascular system partly through stimulation of endothelial nitric oxide (NO) production and endothelium-dependent vasodilation. The current study investigated the role of two recently identified adiponectin receptors, AdipoR1 and -R2, and their downstream effectors in mediating the endothelium actions of adiponectin. In human umbilical vein endothelial cells, adiponectin-induced phosphorylation of endothelial NO synthase (eNOS) at Ser 1177 and NO production were abrogated when expression of AdipoR1 and -R2 were simultaneously suppressed. Proteomic analysis demonstrated that the cytoplasmic tails of both AdipoR1 and -R2 interacted with APPL1, an adaptor protein that contains a PH (pleckstrin homology) domain, a PTB (phosphotyrosine-binding) domain, and a Leucine zipper motif. Suppression of APPL1 expression by RNA interference significantly attenuated adiponectin-induced phosphorylation of AMP-activated protein kinase (AMPK) at Thr 172 and eNOS at Ser 1177 , and the complex formation between eNOS and heat shock protein 90, resulting in a marked reduction of NO production. Adenovirus-mediated overexpression of a constitutively active version of AMPK reversed these changes. In db/db diabetic mice, both APPL1 expression and adiponectin-induced vasodilation were significantly decreased compared with their lean littermates. Taken together, these results suggest that APPL1 acts as a common downstream effector of AdipoR1 and -R2, medi