Npas4 is an activity-regulated transcription factor in pancreatic β-cells. In MIN6 β-cells, a 2-h exposure to 40 mmol/L KCl
(n = 4) (A) but not 25 mmol/L glucose (n = 3) (B) significantly increased Npas4 gene expression in a calcium-dependent manner (n = 3) (C). Further, Npas4 was induced during the immediate early response in MIN6 cells, as CHX was not able to prevent induction (n = 3) (D). In mouse pancreatic islets, Npas4 was significantly increased by glucose in a dose-dependent fashion (n = 3–8) (E) after a 2-h incubation; however, expression levels returned to baseline by 6 h if culture continued in 16 mmol/L glucose
(n = 3–4). Npas4 was not appreciably expressed at the protein level in either MIN6 cells (G) or mouse islets (H) under low glucose (compare G and H); however, depolarization by KCl in MIN6 cells (n = 3) (G) and by both glucose and KCl in mouse islets (n = 3) (H) strongly induced expression. In both MIN6 cells and islets (compare G and H), this induction could be blocked by chelation of calcium with EGTA and by inhibition of protein synthesis (CHX). I: Immunofluorescent staining of MIN6 displayed that under basal conditions, Npas4 is mainly cytoplasmic (arrows), with a small
percentage of cells containing nuclear Npas4 expression (arrowheads). However, under stimulatory conditions, Npas4 is exclusively
nuclear, colocalized with TO-PRO-3 nuclear dye (n = 3). Npas4 expression was induced in human islets by 25 mmol/L glucose after a 2-h stimulation (n = 4) (J). Significance was established using two-tailed Student t tests or a one-way ANOVA with Dunnett post hoc analysis where applicable. n ≥ 3. *P ≤ 0.05; ***P ≤ 0.001.