AMPK activation increases the expression and activity of NADSYN1. (A) ChIP assay illustrating the level of FOXO3a binding to the NADSYN1 promoter following treatment with AMPK activator, 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide (AICAR, 1 mM), for 8 h. (B) qRT-PCR results illustrating the NADSYN1 mRNA abundance following transfection with control, AMPK, or FOXO3a siRNA and then treated with AICAR or left untreated. (C) Immunoblot of AMPK+/+ and AMPK−/− MEFs transfected with control, AMPK, or FOXO3a siRNA and treated with AICAR or left untreated. (D) Immunoblotting of lysates from cells transfected with control, AMPK, or FoxO3a siRNA then treated with or without AICAR. [NAD+] measured in AMPK+/+ and AMPK−/− MEFs (dashed line represents CTRL no treatment) (E) or C2C12 cells (F) transfected with control, AMPK, or NADSYN1 siRNA and treated with AICAR with or without the NAMPT inhibitor FK688. Data analyzed using Wilcoxon signed-rank test or Mann–Whitney U test. *p < 0.05, all experiments were repeated at least 3 times.