(A) Mitochondria isolated from murine skeletal muscle were maintained for 30 min at 37°C with shaking in respiratory state 3 (MirO5 respiration buffer containing 10 mM Pyruvate, 5 mM Malate, 12.5 mM ADP) supplemented with 0.5 mM NAM, NA, or NMN. (N = 2–4). (B) Mitochondria initially held for 30 min in state 2 (MirO5 respiration buffer containing 10 mM Pyruvate, 5 mM Malate; 37°C with shaking) were then supplemented with NMN alone or NMN + ADP and incubated for an additional 30 min at 37°C. (N = 2). (C) Time course of mitochondrial NAD levels before and after addition of NMN or NMN + ADP. (N = 2). (D) Isolated mitochondria were held in state 2 for 30 min before adding ADP to stimulate state 3 respiration for 60 min in the presence of increasing amounts of NMN added concomitantly with ADP. (N = 2). (E) Isolated mitochondria were maintained in state 2 at 37°C with shaking for 30 min and then transitioned to state three in the absence or presence of NMN (0.5 mM), FCCP (4 µM), or rotenone (ROT; 0.5 µM) and incubating for an additional 60 min at 37°C with shaking. (N = 4). (F) Isolated mitochondria were held in state 2 for 30 min before being supplemented with NMN alone, NMN + ADP or NMN + ATP and incubated for an additional 30 min at 37°C (N = 2–4). The data shown are means ± SEM from two or more biological replicates, each measured in technical duplicate and are representative of three independent experiments. (*, p<0.05; **, p<0.001; ***, p<0.0001; 2-tailed, unpaired Student’s t-test).