Leptin-stimulated Endothelial Nitric-Oxide Synthase via an AMPK/Akt Signalling Pathway is Attenuated by Interaction with C Reactive Protein.

The AMP-activated protein kinase (AMPK) lies upstream of Akt in the pathway leading to endothelial NO synthase (eNOS) activation. Whether leptin promotes eNOS activation via AMPK-dependent activation of Akt, and which of the two AMPKalpha catalytic subunits is involved, it remains unknown. Leptin-resistance may be partly attributed to interaction between leptin and C reactive protein (CRP). We hypothesized that leptin effect on eNOS activation in human aortic endothelial cells (HAECs) might be blunted by direct interaction with hrCRP. siRNAs were used to knockdown expression of alpha1- or alpha2- AMPK in transient transfection assay to evaluate which is involved in this pathway and whether leptin effect on eNOS activation in HAECs might be blunted by direct interaction with human CRP. siRNA-mediated down-regulation of AMPKalpha1, but not AMPKalpha2, abolished leptin-induced Akt-Ser(473) phosphorylation, eNOS-Ser(1177) phosphorylation, eNOS activation and cGMP accumulation. By contrast, siRNA-mediated knockdown of Akt1 did not affect AMPKalpha1 phosphorylation, but it abolished leptin-induced phosphorylation of Akt-Ser(473) and eNOS-Ser(1177), suggesting that Akt functions downstream of AMPKalpha1. Pre-incubation of leptin with hrCRP impaired leptin-induced AMPK activation, eNOS-Ser(1177) phosphorylation, eNOS activity, and intracellular cGMP accumulation. The data are consistent with a model implicating an AMPKalpha1-->Akt-->eNOS pathway leading to NO production in response to leptin supporting to the idea that interaction between leptin and CRP may have a role in impairing leptin effect on eNOS activation suggesting a link between leptin-resistance, low grade inflammation, and endothelial dysfunction.