Fig. 6: Distinct impact of NAD⁺ treatment at ZT11 versus ZT23 on hepatic nutrient and insulin sensing pathways.

a Expression of AKT, p-AKT(S473), AMPK and p-AMPK(T172) along the day in the liver from mice treated with NAD⁺ either at ZT11 (HFN) or at ZT23 (HFN23) determined by western blot. Uncropped blots in Source Data. b Quantification of western blots from n = 5 biological samples, normalized to GAPDH loading control. Data from CD at ZT0 was set to 1. c Expression of proteins and phosphor-proteins in the mTOR pathway along the day in the liver from mice treated with NAD⁺ assessed by western blot. d Quantification of western blots from n = 5 biological samples. Measurements were normalized to p84 or GAPDH loading controls. Data from CD at ZT0 was set to 1. e–g RT-qPCR determined expression of rate-limiting and regulatory enzymes involved in mitochondrial (e) or peroxisomal (f) β-oxidation, and (g) ω-oxidation (n = 5 mice for CD and HF, 6 for HFN and 5 for HFN23 except at ZT6, where n = 6). h ¹⁴C-palmitic acid oxidation was assessed by quantifying ¹⁴CO₂ release ex vivo from livers of HF and HFN mice (n = 4 mice per group with three independent measurements. Two experimental replicates were performed with comparable results). Two-way ANOVA i Oxygen consumption rate (OCR) was determined using Seahorse XF analyzer to assess CPT-1-dependent mitochondrial respiration from n = 6 mice with seven technical replicates each. j Mitochondrial bioenergetic parameters were calculated from extracellular flux analyses. One-way ANOVA with Siddak´s posttest. 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. Data points at ZT24 are duplicates from ZT0, replotted to show 24-h trends. The data represent means ± SE. *p < 0.05, **p < 0.01, ***p < 0.001, Two-way ANOVA followed by Bonferroni’s (b, d) or Tukey’s (e–g) posttest. Statistical details and exact p values are provided in Supplementary Data 1.Symbol key for multiple comparisons: *HFN vs HFN23; ^#HF vs HFN. See also Supplementary Fig. S6.