EPAC1 has a small role in GSIS potentiation. EPAC2A KO islets lack GSIS potentiation in response to pharmacologic secretagogue
stimulation. A: Quantitative RT-PCR of mouse islets reveals predominant expression of EPAC2A in islets. EPAC1 and EPAC2B isoforms are expressed
at low levels and are similar in WT and EPAC2A deficient islets. B: shRNA-mediated EPAC1 knockdown in WT and EPAC2A KO islets. IB of islet protein samples shows EPAC1 immunoreactivity in both
WT (left) and EPAC2A KO (right) islets. EPAC2A is present in WT controls and lacking in EPAC2A KO islets. Specific shRNA knockdown specifically reduces
EPAC1 immunoreactivity, while no changes were detected with scrambled shRNA. Signals on IB were detected at the expected position
corresponding to the molecular weight (Supplementary Table 2). Representative IB is shown with triplicate samples from three different mice for each group. C: Insulin-secretion rates during static incubation of EPAC2A KO islets. shRNA-mediated EPAC1 knockdown in EPAC2A KO islets
does not impair E4-potentiated GSIS. Left: E4 has no effect on insulin secretion at low (3 mmol/L) glucose levels. Right: At 10 mmol/L glucose, both scrambled and EPAC1 shRNA-treated islets show similar insulin secretion rates and similarly augment
GSIS under E4 (10 nmol/L) exposure. D: WT islets secrete insulin in a glucose-dependent manner. Stimulation with incretin hormone analog E4, PKA-selective activator
6BZN, EPAC2-selective activator ESCA, as well as GPR40-selective activator PMAP augment GSIS at 10 but not 3 mmol/L glucose.
E: EPAC2A KO islets secrete insulin in a glucose-dependent manner. Stimulation with incretin hormone analog E4, PKA-selective
activator 6BZN, as well as GPR40-selective activator PMAP augment GSIS. E4 and PMAP augment GSIS to a lesser degree in EPAC2A
KO islets (C) as compared with WT islets (D). EPAC2-selective activator ESCA fails to augment GSIS in EPAC2A KO islets. Vehicle controls: for E4, PBS; for ESCA and PMAP,
DMSO. Results are shown as mean ± SEM of triplicate studies. *P < 0.05 vs. vehicle, #P < 0.05 vs. E4; !P < 0.05 for respective treatment EPAC2A KO vs. WT islet (C vs. D) as determined by two-way ANOVA and Bonferroni posttest analysis. scr, scrambled.