FIGURE 6.

Incretins increase protein-protein interaction between acetyl-histone H3 and phospho-CREB or TORC2 in the nucleus. A–C, co-immunoprecipitation between acetylated histone H3 and phospho-CREB. INS-1 (832/13) cells were serum-starved in 3 mm glucose RPMI containing 0.1% BSA overnight and stimulated with 100 nm GIP or GLP-1. Nuclear extracts were isolated from each sample and immunoprecipitated (IP) with antibodies against acetyl-histone H3 at Lys-9 (A), Lys-18 (B), or phospho-histone H3 at Ser-10 (C) followed by immunoblotting (IB) for phospho-CREB (Ser-133). D–F, co-immunoprecipitation between acetylated Histone H3 and TORC2. INS-1 (832/13) cells were treated as described above. Nuclear extracts were isolated and IP with acetyl-histone H3 at Lys-9 (D), Lys-18 (E), or phospho-histone H3 at Ser-10 (F) followed by IB for TORC2. Input represents one-tenth of total nuclear extract used in the co-immunoprecipitation assay. G, effect of histone H3 modulation on incretin-mediated Bcl-2 gene transcription. INS-1 (832/13) cells were serum-starved in 3 mm glucose RPMI containing 0.1% BSA overnight, and GIP or GLP-1 (100 nm) was added in the presence or absence of HAT inhibitor II (40 μm) for 24 h. Total RNA was isolated from each sample, and real-time reverse transcription-PCR was performed to quantify Bcl-2 mRNA expression levels; shown as the -fold difference versus control normalized to 18 S rRNA levels. Significance was tested using ANOVA with a Newman-Keuls post hoc test; ^**, p < 0.05 versus control; ^##, p < 0.05 versus GIP; and ^§§, p < 0.05 versus GLP-1.