Fig. 4.
Menin increases FOXO1 levels and a menin inhibitor increases GLP1R agonist-induced and PKA-mediated phosphorylation of FOXO1 and CREB and decreases FOXO1 levels. Menin-complemented MEF cells (MEF-Men1Δ/Δ+Menin) express higher levels of FOXO1 mRNA (n = 2) (A) and FOXO1 protein (B), compared with vector-transfected menin-null (MEF-Men1Δ/Δ) MEF cells (representative blots of three similar experiments). C: MIN6 cells were starved in low-glucose, serum-free DMEM for 3 h, at which time, the cells were treated with either vehicle or MI-2-2 (500 nM) for a total of 48 h before treatment with exendin-4 (2 nM) for an additional 30 min. PKA inhibitor (H-89) pretreatment was initiated 30 min before exendin-4 treatment. Menin inhibitor (MI-2-2) treatment of MIN6 cells for 48 h decreases FOXO1 transcript levels in a concentration-dependent manner, both in the presence and absence of exendin-4 (2 nM, 30 min) (two experiments). Values are expressed as means ± SE. Data were analyzed using Student’s t-test (*P < 0.05 and $P = 0.08) or two-way ANOVA for effect of MI-2-2 on FOXO1 mRNA (P < 0.001, MI-2-2 vs. vehicle). D: menin inhibitor (MI-2-2, 500 nM) increases both basal and exendin-4 (2 nM)-induced FOXO1 and CREB phosphorylation levels (pFOXO1-S253 and pCREB-S133, respectively) and decreases FOXO1 protein levels (lanes 5 and 6) compared with vehicle alone and exendin-4 alone treatment groups (lanes 1 and 2, respectively). The effect of exendin-4 and/or menin inhibitor is reversed by PKA inhibitor, H-89 (lanes 3, 4, 7, and 8) (representative plots). Quantitation of pFOXO1/FOXO1 (E) and pCREB/CREB ratios (F) from three similar experiments for lanes 1, 2, 5, and 6. Values are expressed as means ± SE. Data were analyzed using one-way ANOVA followed by Tukey’s multiple-comparisons test (*P < 0.05 vs. DMSO, §P = 0.07 vs. Exendin, and &P = 0.09 vs. DMSO).