Fig. 2.

VU0119498 (PAM) promotes ACh-stimulated insulin secretion in isolated mouse and human islets in an M3R-dependent fashion. (A) Insulin secretion studies with isolated islets from WT mice and whole-body M3R KO mice. All assays were carried out in the presence of 16.7 mM glucose. ACh-induced increases in insulin release were studied in the absence or presence of 20 μM PAM. The amount of insulin secreted into the medium during the 1-h incubation period was normalized to the total insulin content of each well (islets plus medium). Data are given as mean ± SEM of at least 3 independent experiments, each carried out in duplicate or triplicate. **P < 0.01, 2-way ANOVA followed by Tukey’s post hoc test. (B) Insulin release studies with perifused human islets. Perifused human islets were incubated with of 4 mM and 8 mM glucose (G4 and G8, respectively) in either the absence or presence of 5 μM PAM. During the 8G perifusion period, islets were incubated with increasing concentrations of ACh (0 to 1 μM). The bar diagram to the right demonstrates the stimulatory effect of PAM on ACh-induced insulin release at 8G (AUC, area under the curve). Experimental details are provided in SI Appendix, Methods. Each curve represents the mean ± SEM of 3 independent perfusion experiments (180 human islets per group and perifusion). **P < 0.001, 2-tailed Student’s t test.