Activation of cAMP/PKA/CREB pathway contributes to NAM-induced Sirt1 upregulation. HepG2 cells were treated with 5 mM NAM, with or without 2-hour H89 pretreatment, for 2 hours and PKA and CREB activations were determined. A. PKA and CREB phosphorylations. B. Nuclear CREB protein abundance. C-E. Both H89 pretreatment (C) and CREB siRNA transfection (D & E) abolishes NAM-induced Sirt1 upregulation. HepG2 cells were treated with 5 mM NAM with or without a 2-hour pretreatment of H89, a specific PKA inhibitor, or overnight transfection of siRNA for CREB. CREB and Sirt1 protein abundance after an 8-hour NAM treatment were determined by Western blot. F. Sirt1 protein abundance in primary mouse hepatocytes. Mouse primary hepatocytes were treated with NAM, with or without a 2-hour H89 pretreatment, for 8 hours. Sirt1 protein abundance were determined by Western blot.