Figure 5. β-hydroxybutyrate (HB) regulates insulin-induced expression of FoxO1. (A) Levels of p-FoxO1, total FoxO1, and PGC-1α were noticeably diminished after treatment with 0.1–2 mM HB for 3 h, followed by incubation with or without 100 nM of insulin for 2 h. (B) Expressions of genes encoding cytokines such as IL-1β, TNFα, and IL-6 were analyzed using qRT–PCR (n = 3 each). Results were normalized to GAPDH mRNA levels. #p < 0.05, ##p < 0.01 vs. Normal; *p < 0.05, vs. insulin treated group. (C) HEK293T cells were transiently transfected with a catalase and catalase-containing plasmid linked to the luciferase gene, pre-incubated with HB (0.25–1 mM) for 4 h, and then treated with insulin for 24 h. Results are presented in relative luminescence units (RLU). Results were obtained using one-factor ANOVA: $$$p<0.001 vs. pcDNA transduced cells; ##p<0.01 vs. catalase-luciferase transduced cells; *p<0.05, **p<0.01 vs. insulin with catalase-luciferase transduced cells. (D) HEK293T cells were pretreated with or without 0.5 mM of HB for 3 h and then treated with insulin (100 nM) for 10 min. Cells were immunostained using rabbit anti-FoxO1 antibody followed by IgG conjugated with fluorescein isothiocyanate (green). Bar = 50 µm.