Purpose: Leptin deficiency in mice (LepOb/Ob) triggers high caloric intake leading to obesity/insulin resistance and activates several reactive oxygen species (ROS) generating pathways. Recent evidence suggest that mitochondrial adaptor p66Shc is a crucial player in ROS-mediated vascular damage. The present study investigates p66Shc role in obesity-driven vascular dysfunction. Methods: Three groups of male mice were studied (C57/B6, 4-6 months old, n=14 per group): 1) wild-type (WT), 2) leptin-deficient (p66Shc-LepOb/Ob), 3) double-mutant (p66Shc-/-/LepOb/Ob;). Plasma 8-iso-prostaglandin-F2α (8-iso-PGF2α was measured as a systemic marker of oxidative stress. mRNA and protein expression were assessed by real-time PCR and immunoblotting. ESR spectroscopy assessment of superoxide anion (O2-) levels and mitochondrial swelling were performed in mitochondria isolated from mouse aortas. The interaction between p66Shc and cytochrome c was investigated by pull-down assays. Organ chamber experiments were performed to assess endothelium-dependent relaxations to acetylcholine (Ach, 10-9-10-6 mol/L). NF-kB activity was determined as p65 nuclear translocation. Results: Plasma 8-iso-PGF2α as well as mitochondrial O2- levels were significantly higher in LepOb/Ob than WT. This increase was abolished in p66Shc-/-LepOb/Ob mice. Oxidative stress in LepOb/Ob mice was associated with p66Shc upregulation, mitochondrial translocation and swelling. Consistently, co-immunoprecipitation of p66Shc with cytochrome c and impaired Ach-induced relaxations were observed in the aortas of LepOb/Ob but not in WT mice. By contrast, in p66Shc-/-/LepOb/Ob mice endothelial dysfunction and mitochondrial swelling did not occur. In agreement with these findings, a preserved activation of Akt/eNOS pathway was observed. Genetic disruption of p66Shc in obese mice also prevented vascular activation of NF-kB signalling and upregulation of VCAM-.1, ICAM-1, IL-6, IL-8 and MCP-1 mRNAs. Conclusions: Our results suggest that p66Shc is critically involved in obesity-induced oxidative stress and vascular dysfunction. A better understanding of the molecular mechanism responsible for ROS production may provide a novel target to reduce cardiovascular health burden in this setting.
The lifespan determinant p66shc drives obesity-induced oxidative stress, mitochondrial dysfunction and vascular inflammation / F., Paneni; Capretti, Giuliana; S., Costantino; S., Chiandotto; A., Akhmedov; E., Di Stasio; B., Rocca; T. F., Luscher; Volpe, Massimo; Cosentino, Francesco. - In: EUROPEAN HEART JOURNAL. - ISSN 0195-668X. - 34:(2013). (Intervento presentato al convegno ESC Congress 2013 tenutosi a Amsterdam - Netherlands nel 31 agosto - 04 settembre 2013).
The lifespan determinant p66shc drives obesity-induced oxidative stress, mitochondrial dysfunction and vascular inflammation
CAPRETTI, GIULIANA;VOLPE, Massimo;COSENTINO, Francesco
2013
Abstract
Purpose: Leptin deficiency in mice (LepOb/Ob) triggers high caloric intake leading to obesity/insulin resistance and activates several reactive oxygen species (ROS) generating pathways. Recent evidence suggest that mitochondrial adaptor p66Shc is a crucial player in ROS-mediated vascular damage. The present study investigates p66Shc role in obesity-driven vascular dysfunction. Methods: Three groups of male mice were studied (C57/B6, 4-6 months old, n=14 per group): 1) wild-type (WT), 2) leptin-deficient (p66Shc-LepOb/Ob), 3) double-mutant (p66Shc-/-/LepOb/Ob;). Plasma 8-iso-prostaglandin-F2α (8-iso-PGF2α was measured as a systemic marker of oxidative stress. mRNA and protein expression were assessed by real-time PCR and immunoblotting. ESR spectroscopy assessment of superoxide anion (O2-) levels and mitochondrial swelling were performed in mitochondria isolated from mouse aortas. The interaction between p66Shc and cytochrome c was investigated by pull-down assays. Organ chamber experiments were performed to assess endothelium-dependent relaxations to acetylcholine (Ach, 10-9-10-6 mol/L). NF-kB activity was determined as p65 nuclear translocation. Results: Plasma 8-iso-PGF2α as well as mitochondrial O2- levels were significantly higher in LepOb/Ob than WT. This increase was abolished in p66Shc-/-LepOb/Ob mice. Oxidative stress in LepOb/Ob mice was associated with p66Shc upregulation, mitochondrial translocation and swelling. Consistently, co-immunoprecipitation of p66Shc with cytochrome c and impaired Ach-induced relaxations were observed in the aortas of LepOb/Ob but not in WT mice. By contrast, in p66Shc-/-/LepOb/Ob mice endothelial dysfunction and mitochondrial swelling did not occur. In agreement with these findings, a preserved activation of Akt/eNOS pathway was observed. Genetic disruption of p66Shc in obese mice also prevented vascular activation of NF-kB signalling and upregulation of VCAM-.1, ICAM-1, IL-6, IL-8 and MCP-1 mRNAs. Conclusions: Our results suggest that p66Shc is critically involved in obesity-induced oxidative stress and vascular dysfunction. A better understanding of the molecular mechanism responsible for ROS production may provide a novel target to reduce cardiovascular health burden in this setting.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
