FGF21 increases SIRT1 and Nampt and the level of NAD+in human dopaminergic neurons. Human dopaminergic neurons were treated with 50 ng/ml FGF21 for 24 h as indicated below. (A-B) Immunoblots. β-actin was used as a control. The levels of SIRT1 (A) and Nampt (B) were increased by FGF21. Left, immunoblots. Right, quantifications using ImageJ. Values are means ± SD, n = 4, ***p < 0.001 and *p < 0.05 for FGF21-treated vs C. (C) The ratio of NAD+ to NADH in the dopaminergic neurons was measured as described in Methods. FGF21 increased NAD+ levels in the neurons. Values are means ± SD, n = 4, *p < 0.05 for FGF21 vs C. (D) Immunoprecipitation experiments. Cells were stimulated with FGF21 for 24 h in the absence or presence of 20 μM NAM to inhibit SIRT1. The degree of acetylation of PGC-1α was analyzed as above. Left, immunoblot. Right, quantification. FGF21 decreased PGC-1α acetylation in control cells but not in NAM treated cells. There was also an increase in acetylated PGC-1α due to SIRT1 inhibition as compared with controls. Values are means ± SD, n = 4, *p < 0.05 for FGF21 vs C.