Npas4 may attenuate incretin-stimulated insulin secretion through the induction of Rgs2. Rgs2 expression was increased in response to 40 mmol/L KCl in MIN6 cells (n = 3) (A) and glucose in islets (n = 3–8) (B). In MIN6 cells, Rgs2 protein levels were increased in a calcium-dependent manner by KCl as well as forskolin (n = 3) (C). Adenovirally overexpressing Npas4 in MIN6 cells (D) and islets (E) dramatically increased Rgs2 mRNA expression as well as Rgs2 protein levels in MIN6 (n = 3) (F). Knockdown of Npas4 in 25 mmol/L glucose did not alter Rgs2 expression; however, when stimulated with KCl for 2 h, knockdown of Npas4 significantly inhibited the induction of Rgs2 mRNA
levels (n = 3) (G). Npas4 regulates Rgs2 through a direct binding to a putative enhancer (15 kb upstream) as well as to the first intron of Rgs2 as assayed by ChIP (n = 3) (H). Illustration of the quantitative primers and probe binding sites in the Rgs2 gene (I). Significance was established using two-tailed Student t tests or a one-way ANOVA with Dunnett post hoc analysis where applicable. *P ≤ 0.05; **P ≤ 0.01.