Leptin-stimulated endothelial nitric-oxide synthase via an adenosine 5'-monophosphate-activated protein kinase/Akt signaling 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, 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 might be blunted by direct interaction with human recombinant CRP. Small interfering RNAs (siRNAs) were used to knock down 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 human aortic endothelial cells 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. Preincubation of leptin with human recombinant CRP 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 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.
Department of Experimental and Clinical Medicine, University Magna Graecia of Catanzaro, 88100 Catanzaro, Italy.
Cristina
C
Procopio
Francesco
F
Andreozzi
Emanuela
E
Laratta
Angela
A
Cassese
Francesco
F
Beguinot
Franco
F
Arturi
Marta Letizia
ML
Hribal
Francesco
F
Perticone
Giorgio
G
Sesti
D055372:AMP-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D000818:Animals; D002097:C-Reactive Protein / Q000378:metabolism*; D002460:Cell Line; D006152:Cyclic GMP / Q000378:metabolism; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology; D005260:Female; D006801:Humans; D020738:Leptin / Q000378:metabolism* / Q000494:pharmacology; D051379:Mice; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D034741:RNA, Small Interfering; D015398:Signal Transduction / Q000187:drug effects
D020738:Leptin; D034741:RNA, Small Interfering; D002097:C-Reactive Protein; D052250:Nitric Oxide Synthase Type III; C494918:AKT1 protein, human; C526181:PRKAA2 protein, human; D051057:Proto-Oncogene Proteins c-akt; D055372:AMP-Activated Protein Kinases; C526180:PRKAA1 protein, human; D006152:Cyclic GMP