P2250P66Shc mediates the influence of Sirt-1 on age and obesity related endothelial function

Abstract

Abstract Background Sirtuins are histone deacetylase downregulated in conditions of excessive calory intake, with important roles in controlling cellular ageing pathways. Among their different isoforms, Sirt-1 is the most studied and experimental models suggest it could contribute to the downregulation of the ageing protein p66Shc in conditions of reduced energy intake, ultimately resulting in a reduced mitochondrial free radical production (mtROS). Purpose We assessed the combined influence of ageing and obesity (a condition of excessive calory intake) on the expression and functional activity of Sirt-1 in humans small resistance arteries, as well as whether the effects of Sirt-1 on endothelial function might depend from the regulation of p66Shc activity and its influence on mtROS. Methods Small resistance arteries were isolated from subcutaneous tissue of obese subjects and healthy controls undergoing bariatric surgery of inguinal hernia repair, respectively. The obese and control groups were further divided into young (<40 years old) and old (>40 years old) subjects. Arteries were mounted on a pressurised myograph and endothelial function was assessed using dose-response curves to acetylcholine before and after incubation with SRT1720 (selective agonist of Sirt-1), L-NAME (eNOS inhibitor), MitoTEMPO (mtROS scavenger) and gp91ds-tat (NADPH oxidase inhibitor), alone or in combination. The expression of Sirt-1 and p66Shc in the vascular wall of obese and control subjects was assessed by qPCR while the ChIP assay was used to assess the binding of Sirt-1 to the promoter region of p66Shc. Results Endothelial function was lower in older than younger and in obese than control groups (p<0.001). Incubation with SRT1720 did not affect endothelial function in young controls, slightly improved endothelial function in young obese (p<0.01) and induced a substantial improvement in endothelial function in both the old control and old obese groups (p<0.001). Pre-incubation with L-NAME suggested that the effects of Sirt-1 were dependent from an improved vascular NO availability. Incubation with gp91ds-tat improved endothelial function in both obese groups and the old control group (p<0.01), but to a lesser extent than SRT1720. Conversely, incubation with MitoTEMPO induced the same improvement of endothelial function than SRT1720 in all study groups and, when vessels were preincubated with MitoTEMPO, the addition of Sirt-1 did not produce further improvement of endothelial function. The expression of Sirt-1 and p66Shc was increased in the vessels from obese subjects compared to the non-obese group (p<0.001), while the ChIP assay revealed a lower binding of Sirt-1 to the promoter region of p66Shc in obese than controls subjects (p<0.001). Conclusions Our results suggest that Sirt-1 has an important role in the obesity and age-related endothelial dysfunction, throughout epigenetic regulation of p66Shc and, thus, controlling the production of mtROS. Acknowledgement/Funding Ministero dell'istruzione, dell'università e della ricerca