Fig. (5). SIRT1 activates Akt1 and maintains inhibitory phosphorylation of p-FoxO3a and relies upon p-FoxO3a inhibition for EC protection during elevated D-glucose.

In A and B, primary EC protein extracts (50 µg/lane) were immunoblotted with anti-phosphorylated-Akt1 (p-Akt1, Ser⁴⁷³) or anti-total Akt1 at 6, 24, and 48 hours following administration of elevated D-glucose (HG = high glucose, 20 mM). Phosphorylated (active) Akt1 (p-Akt1) expression is initially increased at 6 hours following elevated D-glucose, but subsequently is reduced and returns to control levels at 24 hours and 48 hours after elevated D-glucose (*P<0.01 vs. control). Total Akt1 is not altered. In C, D, E, and F, primary EC protein extracts (50 µg/lane) were immunoblotted with anti-phosphorylated-FoxO3a (p-FoxO3a, Ser²⁵³) or anti-total FoxO3a at 6, 24, and 48 hours following administration of elevated D-glucose (HG = high glucose, 20 mM). Quantification of western band intensity was performed using the public domain NIH Image program (http://rsb.info.nih.gov/nih-image). During elevated D-glucose and correlating with Akt1 activity, phosphorylated (inactive) FoxO3a (p-FoxO3a) expression is initially increased at 6 hours but then significantly reduced at 24 hours and at 48 hours (C and D) following elevated D-glucose but total FoxO3a expression (E) is not affected (*P<0.01 vs. control). Activation of SIRT1 with SIRT1 protein (2 µM) or with resveratrol (RES) (15 µM) maintained the expression of p-FoxO3a at 6, 24, and 48 hours to a greater degree than during elevated D-glucose (20 mM) exposure alone. In contrast, inhibition of SIRT1 activity with EX527 (2 µM) sharply reduced p-FoxO3a expression below levels for elevated D-glucose alone at 6 and 24 hours and at similar levels to elevated D-glucose alone at 48 hours (A and B). In E and F, gene knockdown of SIRT1 with siRNA significantly reduced p-FoxO3a expression at 6 hours below levels observed with elevated D-glucose alone, but expression of total FoxO3a remained unchanged, suggesting that the loss of SIRT1 protein only affected phosphorylation of FoxO3a and that the total FoxO3a protein was not degraded. In G, transfection with FoxO3a siRNA increased EC survival during elevated D-glucose (HG = high glucose, 20 mM) alone and also produced equal protective capacity and increased survival in ECs assessed by trypan blue uptake during either SIRT1 protein (2 µM) or resveratrol (RES) (15 µM) application at 48 hours after elevated D-glucose. Gene knockdown of FoxO3a also reversed EC injury during SIRT1 inhibition with EX527 (2 µM) (*P<0.01 vs. HG without FoxO3a siRNA; †P<0.01 vs. HG without FoxO3a siRNA). Control = untreated cells and each data point represents the mean and SEM from 6 experiments.