Oscillation of NAMPT-mediated NAD⁺ biosynthesis. (A) Relative expression levels of Nampt RNA in liver and white adipose tissue (WAT) across the 24 hr light:dark cycle. Gray shading indicates dark period (n=4-6 mice/genotype/time point). (B) Western blots showing NAMPT and GAPDH at indicated ZTs. Quantitation of NAMPT normalized to GAPDH. (C) Relative expression levels of Nampt RNA across 48 hrs in liver of WT and Clock^Δ19 mice in constant darkness. Shading indicates where light and dark periods would normally occur under 12:12 LD conditions. (n=2 WT, n=1 Clock^Δ19/time point); (p<0.01, one-way ANOVA for WT oscillation). (D) NAD⁺ levels across 48 hrs in liver of WT mice in constant darkness. (n=2 WT/time point). (E) NAD⁺ levels in WT and Clock^Δ19 mutant liver at ZT2 and 14 (n=3) (upper panel). Relative expression levels of Nampt RNA (middle panels) and NAD⁺ (lower panels) in Bmal1^-/-, Cry1^-/-/Cry2^-/-, and WT liver at ZT7 (n=4-13). *p<0.05; **p<0.01; ***p<0.001. All data is presented as mean ± SEM.

NAMPT, NAD⁺, and SIRT1 regulate CLOCK:BMAL1 activity. (A-B) NAD⁺ levels (A) and relative expression levels of Pepck, G6Pase, and Igf-1 RNA (B) in primary hepatocytes incubated with 200nM FK866 for 24 hours (n=3/group). (Pepck, phosphoenolpyruvate carboxykinase; G6Pase, glucose-6-phosphatase; Igf-1, insulin-like growth factor-1). (C-E) Relative luciferase activity of Per2-luciferase in the presence of: (C) 10nM FK866 for 24 or 48 hours, (D) increasing doses of Sirt1 (200, 400, 800 ng), and (E) Sirt1 (800 ng) alone or Sirt1 (800 ng) and 10nM FK866 for 48 hours. *p<0.05; **p<0.01; ***p<0.001; NS not significant. All data is presented as mean ± SEM.

NAMPT/NAD⁺-driven feedback loop through SIRT1/CLOCK:BMAL1. (A) ChIP assays in primary hepatocytes for Per2 with rIgG control (left), SIRT1 antibody (middle), and input (right). Primer locations are schematically shown above. (B-C) Relative expression levels of Per2 RNA in primary hepatocytes following serum shock. Hepatocytes were either (B) incubated with 200nM FK866 or (C) infected with SIRT1- and GFP (control)-expressing adenovirus (average of 2 independent experiments). (D) ChIP assays in liver isolated from mice at CT6 and CT15 for Nampt with no Ab control (left), CLOCK antibody (middle), and input (right). Primer locations are shown schematically above. The black box indicates a canonical E-box, while gray circles represent non-canonical E-boxes. Control primers are located ∼5kb upstream of the Nampt transcriptional start site. Per2 E-box primers are included as a positive control. (E) Relative Nampt expression levels in adenovirally GFP- and Clock/Bmal1-infected mouse embryonic fibroblasts (average of 2 independent experiments).