Genetic deletion of p66Sh protects against obesity-induced mitochondrial dysfunction, oxidative stress and vascular inflammation

Introduction: Leptin deficiency in mice (LepOb/Ob) triggers high caloric intake leading to obesity/insulin resistance and activates several reactive oxygen species (ROS) generating pathways. The mitochondrial adaptor p66Shc is a crucial player in ROS-mediated vascular damage. Hypothesis: The present study investigates p66Shc role in this model. Methods: Three groups of male mice were studied (C57/B6, 4-6 months old, n=10/group): 1) wild-type (WT), 2)leptin-deficient (LepOb/Ob), 3) double-mutant (p66Shc-/--LepOb/Ob). Plasma 8-iso-prostaglandin-F2α (8-iso-PGF2α) was measured as a 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 aortic 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 also observed. Genetic disruption of p66Shc in obese mice prevented vascular activation of NF-kB signalling and upregulation of VCAM-1, ICAM-1, IL-6 and MCP-1. Conclusions: Our results suggest that p66Shc is critically involved in obesity-induced oxidative stress, endothelial dysfunction and vascular inflammation. A better understanding of the molecular mechanism responsible for ROS production may provide a novel target to reduce cardiovascular health burden in this setting.