Glucose-dependent potentiation of mouse islet insulin secretion by Epac activator 8-pCPT-2'-O-Me-cAMP-AM

Abstract

Epac2 is a cAMP-regulated guanine nucleotide exchange factor (cAMP-GEF) that is proposed to mediate stimulatory actions of the second messenger cAMP on mouse islet insulin secretion. Here we have used methods of islet perifusion to demonstrate that the acetoxymethyl ester (AM-ester) of an Epac-selective cAMP analog (ESCA) penetrates into mouse islets and is capable of potentiating both first and second phases of glucose-stimulated insulin secretion (GSIS). When used at low concentrations (1-10 μM), 8-pCPT-2'-O-Me-cAMP-AM activates Rap1 GTPase but exhibits little or no ability to activate protein kinase A (PKA), as validated in assays of in vitro PKA activity (phosphorylation of Kemptide), Ser (133) CREB phosphorylation status, RIP1-CRE-Luc reporter gene activity, and PKA-dependent AKAR3 biosensor activation. Since quantitative PCR demonstrates Epac2 mRNA to be expressed at levels ca. 5.3-fold greater than that of Epac1, available evidence indicates that Epac2 does in fact mediate stimulatory actions of cAMP on mouse islet GSIS.

Figure 1

Acetoxymethyl esters of Epac and PKA selective cAMP analogs. 8-pCPT-2'-O-Me-cAMP-AM (top left) is hydrolyzed to 8-pCPT-2'-O-Me-cAMP and binds primarily to the high-affinity cAMP binding domain of Epac2 (bottom left). This binding activates the CDC25 homology domain that is responsible for the catalysis of guanyl nucleotide exchange on Rap1. Dibutyryl cAMP-AM (top right; also known as N⁶-2'-DB-cAMP) is hydrolyzed to dibutyryl-cAMP and then to monobutyryl-cAMP which binds to the regulatory subunits of the PKA holoenzyme (bottom right). This binding induces dissociation of the holoenzyme, thereby releasing catalytic subunits with serine/threonine protein kinase activity.

Figure 2

Differential insulin secretagogue properties of Epac-selective cAMP analogs in adult male C57BL/6J mouse islets. (A) The non-AM-ester of 8-pCPT-2'-O-Me-cAMP (ESCA ; 10 µM) failed to potentiate GSIS induced by a step-wise increase of glucose concentration from 3 to 20 mM. (B) GSIS induced by 20 mM glucose was potentiated by the AM-ester of 8-pCPT-2'-O-Me-cAMP (ESCA-AM; 10 µM) whereas phosphate-AM₃ (3.3 µM) was without effect (phosphate-AM₃ liberates 3 mole equivalents of acetic acid and formaldehyde per mole of phosphate when it is hydrolyzed by intracellular esterases). Note that 8-pCPT-2'-O-Me-cAMP-AM was included for 10 min in the KRB perifusate containing 3 mM glucose prior to switching to a perifusate containing 20 mM glucose. Since the initial rate of insulin secretion measured in the presence of 8-pCPT-2'-O-Me-cAMP-AM did not differ from that measured in its absence, it is concluded that 8-pCPT-2'-O-Me-cAMP-AM potentiated GSIS at a high but not a low concentration of glucose. For the methods of analyses, see the Supplementary Data of Chepurny et al. 2009.

Figure 3

QPCR for Epac1 and Epac2 mRNA expression in adult male C57BL/6J mouse islets. (A) QPCR fluorescence growth curves from 100 ng of mouse islet RNA using Quiagen QuantiTect Sybr Green RT-PCR. Ribosomal S18 mRNA was used as the reference target for quantification of Epac1 and Epac2 mRNA. The average threshold crossing value (C_t) for S18 mRNA (19.1 ± 0.3, n = 8; mean ± s.e.m.) is indicated. (B) Comparison of the delta-C_t values (ΔC_t) for Epac1 and Epac2. The ΔC_t value for each Epac isoform was calculated as the difference in threshold cycle number relative to S18. (C) The ΔΔC_t value for Epac1 relative to Epac2 was computed by subtraction of the ΔC_t values for each isoform (left) and the relative abundance of Epac1 and Epac2 mRNA was then calculated to be 1:5.3 (right).

Figure 4

PKA inhibitor H-89 fails to abrogate the action of 8-pCPT-2'-O-Me-cAMP-AM to potentiate GSIS in adult male C57BL/6J mouse islets. Illustrated are findings obtained in static incubation assays using methodologies of islet isolation and solution exposure described previously. Islets were exposed to KRB containing 2.8 mM glucose for 30 min and were then exposed to KRB containing 20 mM glucose with or without added test substances. These test substances included H-89 (10 µM), dibutyryl-cAMP-AM (Db-cAMP-AM; 10 µM), and 8-pCPT-2'-O-Me-cAMP-AM (ESCA-AM; 10 µM). Each test substance was disolved in a vehicle solution comprised of KRB containing 0.1% DMSO and 0.1% BSA. The findings are representative of a single experiment repeated twice. Values of fold-stimulation were calculated by comparing insulin secretion measured in 20 mM glucose KRB relative to KRB containing 2.8 mM glucose. Values are mean ± s.e.m. for duplicate determinations. Statistical significance was evaluated by the t-test (*p < 0.05).