Npas4 directly regulates insulin gene expression in β-cells. Enforced adenoviral expression of Npas4 in MIN6 cells (A–C) significantly decreased expression of insulin 1 (A) and insulin 2 (B) message levels and total cell content (n = 3) (C). Adenoviral-driven Npas4 in mouse pancreatic islets (D–F) significantly decreased expression of insulin 1 (n = 4) (D) and insulin 2 (n = 3) (E) as well as significantly reduced total islet content of insulin (n = 6) (F). Forty-eight hours after transfection with siRNAs targeting Npas4, increases in both insulin 1 (G) and insulin 2 (H) expression were observed in MIN6 cells (n = 4). Npas4 reduced RIP1-driven luciferase transcription in cotransfected MIN6 cells (n = 3) (I). Npas4 overexpression in MIN6 did not alter Pdx-1 (J) or NeuroD1 (K) mRNA expression but significantly decreased both at the protein level (n = 3) (M–P). Npas4 also increased the expression of MafA at the message (L) and protein (Q and R) level (n = 3). S: To determine if Npas4 binds directly to both insulin promoters in MIN6 cells, cells were depolarized for 2 h with 40 mmol/L
KCl to induce Npas4, fixed, and chromatin fragmented, and immunoprecipitation was carried out using antibodies specific for
Npas4 under both induced (black bars) and control (white bars) conditions. Cross-links were then reverse transcribed, and
Taqman PCR was used to assess enrichment at the insulin promoters. Using this approach, there was between 10- and 30-fold
enrichment at both promoters (n = 3). T: Illustrations of quantitative primers and probe binding sites on the insulin 1 and insulin 2 promoter (modified from LeRoith
et al. ). Significance was determined using two-tailed Student t tests. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.