Fig. 8: Time-of-day dictates efficiency of NAD⁺ treatment of diet-induced metabolic disease through synchronizing the hepatic circadian clock and adjusting coordination between intracellular signaling and gene expression.

a RT-qPCR from clock genes in the SCN at ZT0 and ZT12 (n = 3 biological replicates and 2 technical replicates). b Representative double-plotted actograms of locomotion measured using infrared sensors in a 12-h light/12-h dark cycle. c Average 24-h activity profile from the indicated groups of mice. Average was calculated for five days after NAD⁺ treatment. n = 7 mice for HF and HFN, and 6 for HFN23. d Food intake was measured over 24 h e Average food intake during light phase (day) and dark phase (night). For the boxplots, the top and bottom lines of each box represent the 75th and 25th percentiles, respectively. The line inside each box represents the median. The lines above and below the boxes are the whiskers f At ZT12, NAD⁺ sustains the alignment of the hepatic molecular clock while reinforcing circadian oscillation in the activity of nutritional sensors such as AMPK, AKT or mTOR. Transcriptional responses are adjusted accordingly to suppress inflammation probably through inhibition of NF-κB transcription factor, and to increase amplitude in lipolytic gene expression and fatty acid oxidation, with a peak during the active phase. Membrane trafficking and ULK activity are indicative of active autophagy as a specific response to NAD⁺ treatment. At ZT0, increased NAD⁺ in the liver inverts the phase of the molecular clock, imposing antiphase rhythms in clock gene and protein expression. This in turn inverts the phase of clock-controlled genes and uncouples transcriptional responses. CD control diet fed mice, HF high-fat diet-fed mice, HFN high-fat diet fed, NAD⁺ treated mice at ZT11, HFN23 high-fat diet fed, NAD⁺ treated mice at ZT23. The data represent means ± SE. *p < 0.05, **p < 0.01, ***p < 0.001, Two-way RM ANOVA followed by Sidak’s posttest. Symbol key for multiple comparisons: ^#HF vs HFN, X HF vs HFN23, ^$HFN vs HFN23. See also Supplementary Fig. S8.