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. 2023 Apr 8;14:1994. doi: 10.1038/s41467-023-37517-6

Fig. 6. eNAMPT inhibits hypothalamic FOXO1 via SIRT2−mediated deacetylation.

Fig. 6

a Molecular interaction of SIRT2 and FOXO1 in C57 mouse hypothalamic protein extracts. IP: immunoprecipitation, IB immunoblotting. b Effect of Sirt2 overexpression on FOXO1 acetylation in hypothalamic N1 cells. Ac-K: acetylated lysine. a, b Two independent replicates were performed. c Antagonizing effect of SIRT2 overexpression on FOXO1 overexpression-induced changes in the POMC and AGRP promoter activities (POMC SIRT2 alone, n = 4; POMC other groups, n = 5; AGRP all groups, n = 3 cell wells). One-way ANOVA followed by Fisher’s LSD test. d Comparison of the effect of FOXO1 wild type (WT) and deacetylation mutant (6KR) overexpression in the regulatory ability of POMC and AGRP transcription (POMC, n = 5; AGRP Vector, n = 3; AGRP WT and 6KR, n = 4 cell wells). One-way ANOVA followed by Fisher’s LSD test. n.s. not significant. c, d Three independent replicates were performed. e Effect of i.c.v. administration of NAMPT (100 ng) on hypothalamic FOXO1 expression (n = 3 mice). Unpaired t-test (two-sided). f Circadian rhythms of hypothalamic FOXO1 expression levels in normal mice (ZT22, n = 2; other groups, n = 3 mice). P value was determined using JTK-cycle algorithm. The shaded area in the figures represents the lights-off period. g Effect of hypothalamic Sirt2 knockdown on i.c.v. NAMPT-induced decreases in hypothalamic FOXO1 expression (n = 4 mice). One-way ANOVA followed by Fisher’s LSD test. n.s. not significant. h Effect of hypothalamic Foxo1 overexpression by Foxo1-Ad injection on i.c.v. NAMPT (100 ng)-induced changes on LMA, EE, and hypothalamic Pomc and Agrp expression (EE and LMA, n = 5; Pomc and Agrp, n = 4 mice). One-way ANOVA followed by Fisher’s LSD test. e-h Two independent experiments were performed to validate the results. Data represent the mean ± SEM.