Fig. 4.

Sirt2 deacetylates GKRP. a–d Immunoblot analysis (IB) with the indicated antibodies of total cell lysate and precipitates after immunoprecipitation (IP) with anti-acetylated lysine (AcK) antibodies from HEK293 cells expressing Flag-tagged GKRP and Flag-tagged Sirt2 with and without nicotinamide (NAM), AGK2, Ex527, or trichostatin A (TSA) (a), from Sirt2 knockdown primary hepatocytes cultured in low- or high-glucose medium (b), from db/db mouse-derived hepatocytes with and without adenovirus-mediated overexpression of Sirt2 cultured in low- or high-glucose medium (c), and from db/db mouse-derived hepatocytes with NMN treatment and with and without Sirt2 knockdown cultured in high-glucose medium (d). The quantification of acetylated GKRP levels is normalized to GKRP expression in the right graph (b–d, n = 3 each). e, f Acetylated GKRP levels in the liver of mice fed normal chow (NC) or a high-fat diet (HFD) with adenovirus-mediated hepatic overexpression of Sirt2 after 16-h fasting (e) and acetylated GKRP and keratin 8 levels in the liver of Sirt2 knockdown lean mice after 16-h fasting (f). g, h Successive comparison of lysine acetylation levels of keratin 8 and GKRP in the liver of NC- or HFD-fed mice after NMN injection after 16-h fasting (g) and in the liver of NC-fed mice treated with siSirt2 at 30 min after administration of glucose (4 g/kg) after 16-h fasting (h). *P < 0.05; one-way ANOVA with the Fisher’s PLSD post-hoc test (b–d). All data are representative of at least three independent experiments. Error bars show s.e.m