Fig. 1: A NAD⁺ chronotherapy at ZT11 improves the pathophysiology of diet-induced obesity.

a Schematic diagram of the study design. Mice were fed either a normocaloric diet (CD) or a high-fat diet for 11 weeks. At week 8, a subgroup of high-fat-fed mice was supplied a chronotherapy with NAD⁺, consisting of a daily intraperitoneal injection of 50 mg/Kg of NAD⁺ at ZT11 for three weeks (HFN). The rest of the mice were injected with vehicle (saline solution). b Weekly body weight (n = 20 mice for CD and HFN and 17 for HF). Red arrow indicates the period of treatment with NAD⁺ or saline at ZT11. (c) Hepatic NAD⁺ content measured by HPLC along the day at the indicated times for all groups after the experimental paradigm (n = 5 biological replicates per time point and group, and three technical replicates). d Serum levels of insulin along the day at indicated times (n = 5 biological replicates per time point, and 2 technical replicates). AUC: area under the curve. e–h Glucose (e, f GTT) and insulin (g,h; ITT) tolerance tests were performed at both the rest (ZT4) and the active (ZT16) period at the indicated days (10 and 20) after the beginning of treatments (n = 5 mice per point, except for HFN in f, where n = 6). AUC: area under the curve. CD control diet fed mice, HF high-fat diet fed mice, HFN high-fat diet fed, NAD⁺ treated mice at ZT11. Data represent mean ± SEM and were analyzed by two-way ANOVA using Tukey posttest, except when comparing AUC, where one-way ANOVA followed by Tukey’s posttest was used. *p < 0.05, **p < 0.01, ***p < 0.001. p values are provided in Supplementary Data 1. Points at ZT24 are duplicates of ZT0 replotted to show 24-h trends. Symbol key for comparisons: *CD vs HF; ⁺CD vs HFN; ^#HF vs HFN. See also Supplementary Fig. S1.