Regulation of basal cAMP levels by PDEs in human pancreatic β-cells.
A-C) Subtype-selective PDE inhibitors and IBMX raise cAMP levels in pancreatic β-cells dissociated from human islets under basal conditions (1.7 mM glucose). For each experiment, IBMX (100 μM) and either 8MM-IBMX (100 μM) (A), cilostamide (1 μM) (B) or rolipram (10 μM) (C) were perfused onto the cell. Pancreatic β-cells were identified at the end of each experiment by addition of GLP-1 (50 nM) with and without the α2-receptor agonist clonidine (1 μM). Data shown are representative experiments from single human pancreatic β-cells. D) The percent increase in cAMP levels above baseline elicited by PDE inhibitors in human pancreatic β-cells under basal conditions. IBMX, cilostamide and rolipram significantly elevate cAMP levels above baseline. The percent increase in cAMP levels stimulated by 8MM-IBMX and rolipram is significantly less than that of IBMX (***, P < 0.001, *, P < 0.05 compared to baseline; ###, P < 0.001 compared to IBMX; One-way ANOVA, Tukey post-hoc test). Data shown are average ± SE from 15 cells (8MM-IBMX), 7 cells (cilostamide), 9 cells (rolipram) and 33 cells (IBMX), collected from four human islet preparations. One outlier was removed for IBMX and three outliers were removed for rolipram. E) Resting cAMP levels (mTurquoise2/FRET) in β-cells from eight different human donors. Comparison of pooled data from human β-cells (61 total) to resting cAMP levels in INS-1 cells (41 total) revealed a significantly greater resting cAMP level in human β-cells (***, P < 0.001; Student’s unpaired t-test). Individual data points are shown for each cell measured. Lines represent means ± SE.