PKA-dependent potentiation of glucose-stimulated insulin secretion by Epac activator 8-pCPT-2'-O-Me-cAMP-AM in human islets of Langerhans

Abstract

Potential insulin secretagogue properties of an acetoxymethyl ester of a cAMP analog (8-pCPT-2'-O-Me-cAMP-AM) that activates the guanine nucleotide exchange factors Epac1 and Epac2 were assessed using isolated human islets of Langerhans. RT-QPCR demonstrated that the predominant variant of Epac expressed in human islets was Epac2, although Epac1 was detectable. Under conditions of islet perifusion, 8-pCPT-2'-O-Me-cAMP-AM (10 microM) potentiated first- and second-phase 10 mM glucose-stimulated insulin secretion (GSIS) while failing to influence insulin secretion measured in the presence of 3 mM glucose. The insulin secretagogue action of 8-pCPT-2'-O-Me-cAMP-AM was associated with depolarization and an increase of [Ca(2+)](i) that reflected both Ca(2+) influx and intracellular Ca(2+) mobilization in islet beta-cells. As expected for an Epac-selective cAMP analog, 8-pCPT-2'-O-Me-cAMP-AM (10 microM) failed to stimulate phosphorylation of PKA substrates CREB and Kemptide in human islets. Furthermore, 8-pCPT-2'-O-Me-cAMP-AM (10 microM) had no significant ability to activate AKAR3, a PKA-regulated biosensor expressed in human islet cells by viral transduction. Unexpectedly, treatment of human islets with an inhibitor of PKA activity (H-89) or treatment with a cAMP antagonist that blocks PKA activation (Rp-8-CPT-cAMPS) nearly abolished the action of 8-pCPT-2'-O-Me-cAMP-AM to potentiate GSIS. It is concluded that there exists a permissive role for PKA activity in support of human islet insulin secretion that is both glucose dependent and Epac regulated. This permissive action of PKA may be operative at the insulin secretory granule recruitment, priming, and/or postpriming steps of Ca(2+)-dependent exocytosis.

Fig. 1.

8-pCPT-2′-O-Me-cAMP-AM potentiates 1st- and 2nd-phase glucose-stimulated insulin secretion (GSIS). A: secretion profile for GSIS under conditions of human islet perifusion. Prior to the 30-min time point, islets were perifused with Krebs-Ringer buffer (KRB) containing 3 mM glucose only. This allowed basal insulin secretion to decline to a stable level (data not shown). The glucose concentration was then raised to 10 mM to evoke 1st- and 2nd-phase insulin secretion. Administration of KRB containing 8-pCPT-2′-O-Me-cAMP-AM [10 μM; exchange protein directly activated by cAMP (Epac)-selective cAMP analog (ESCA-AM)] began at the 30-min time point and was continuous until the 60-min time point. ESCA-AM indicates where 8-pCPT-2’-O-Me-cAMP-AM was applied, here and in subsequent figures. Dimethylsulfoxide (DMSO; vehicle control) was included in the KRB for islets not treated with 8-pCPT-2′-O-Me-cAMP-AM. B: quantitative analysis of GSIS potentiated by 8-pCPT-2′-O-Me-cAMP-AM. Summarized are findings obtained using the experimental design illustrated in A, with 2 batches of islets obtained from 2 donors. Insulin secretion is expressed as the normalized area under the curve (AUC) for 1st- and 2nd-phase GSIS, as defined in A. For B, *P < 0.05, t-test.

Fig. 2.

PKA-dependent potentiation of GSIS by 8-pCPT-2′-O-Me-cAMP-AM. A: human islets were equilibrated in KRB containing 2.8 or 10 mM glucose (2.8 G or 10 G, respectively) with or without added 8-pCPT-2′-O-Me-cAMP-AM (10 μM; ESCA-AM), dibutyryl-cAMP-AM (Bt₂-cAMP-AM; 10 μM), or 6-Bnz-cAMP-AM (10 μM). B: basal insulin secretion measured under conditions in which the KRB contained 2.8 mM glucose with or without added 0.1% DMSO, H-89 (10 μM), or Rp-8-CPT-cAMPS (200 μM). C and D: GSIS measured under conditions in which the KRB contained 10 mM glucose with or without added DMSO, ESCA-AM, phosphate-AM₃ (Phos-AM₃), H-89 (10 μM), or Rp-8-CPT-cAMPS (200 μM). Values of fold stimulation in C and D were calculated by measuring secreted insulin under conditions in which islets were exposed to KRB containing 2.8 or 10 mM glucose. Results obtained in 3 static incubation assays using islets from 3 donors are summarized in A–D. *P < 0.05, t-test. Error bars denote the mean ± SE for triplicate determinations.

Fig. 3.

8-pCPT-2′-O-Me-cAMP-AM fails to activate PKA. A: cAMP response element-binding protein (CREB) phosphorylation assay for human islets treated with Bt₂-cAMP-AM, ESCA-AM, 8-CPT-cAMP-AM, forskolin (Fsk), and IBMX. Immunoblot analysis was performed to detect Ser¹³³-phosphorylated CREB (P-CREB) or total CREB. The histogram is based on the average of 2 experiments using 2 islet preparations (*P < 0.05, t-test). B₁ and B₂: PKA activation assay for human islets treated with 6-Bnz-cAMP-AM or 8-pCPT-2′-O-Me-cAMP-AM. Electrophoresis was performed to resolve phosphorylated (P-Kemptide) and nonphosphorylated (Kemptide) forms of PepTag. C₁–C₃: AKAR3 assay for PKA activation in human β-cells. C₁: AKAR3 exhibited an increase of fluorescence resonance energy transfer (FRET) emission ratio in response to forskolin (2 μM) and IBMX (100 μM), and this increase was reversed by H-89 (10 μM). C₂: an increase of FRET emission ratio was also measured in response to 6-Bnz-cAMP-AM (10 μM) but not 8-pCPT-2′-O-Me-cAMP-AM (10 μM). C₃: single-cell population study of AKAR3 responsiveness to cAMP analogs, Phos-AM₃, or forskolin with IBMX using concentrations of test substances indicated for C₁ and C₂. Numbers above each histogram bar indicate the number of cells imaged. Statistical significance was evaluated using the t-test. n.s., Not significant. Findings are representative of results obtained in 2 experiments using islets obtained from 2 donors.

Fig. 4.

Effects of 8-pCPT-2′-O-Me-cAMP-AM on intracellular Ca²⁺ concentration ([Ca²⁺]_i) in a human islet. A₁: ESCA-AM (1 μM; application indicated by the horizontal bar) stimulated an increase of [Ca²⁺]_i in a human islet equilibrated in standard extracellular saline (SES) containing 5.6 mM glucose. A₂: the increase of [Ca²⁺]_i imaged in this same islet at selected time points. Yellow bracket indicates the region from which ratiometric measurements of fura-2 fluorescence were obtained. Calibration bar indicates 65 μm. Findings are representative of results obtained in 3 experiments using 5 islets from 3 donors.

Fig. 5.

8-pCPT-2′-O-Me-cAMP-AM increases [Ca²⁺]_i and depolarizes human β-cells. A₁: 8-pCPT-2′-O-Me-cAMP-AM (10 μM) stimulated an increase of [Ca²⁺]_i in a single human β-cell equilibrated in SES containing 5.6 mM glucose. A₂: population study conducted at the single-cell level compares the action of 8-pCPT-2′-O-Me-cAMP-AM (10 μM) to increase [Ca²⁺]_i in human β-cells under conditions in which the SES contained 2.8 or 5.6 mM glucose or, alternatively, 5.6 mM glucose plus 10 μM H-89, 200 μM diazoxide, 10 μM nifedipine, or 100 μM CdCl₂. Numbers of cells tested (denominator) and the number of cells responding (numerator) are indicated above each histogram bar. A positive response was assigned to cells in which the [Ca²⁺]_i increased to a level >2-fold of the initial starting value (dashed horizontal bar in A₁). B₁ and B₂: simultaneous measurements of membrane potential (B₁) and [Ca²⁺]_i (B₂) in a human β-cell equilibrated in SES containing 5.6 mM glucose and stimulated with 8-pCPT-2′-O-Me-cAMP-AM (10 μM). B is representative of results obtained in 2 experiments using 5 β-cells from 2 donors.

Fig. 6.

8-pCPT-2′-O-Me-cAMP-AM facilitates Ca²⁺-induced Ca²⁺ release (CICR) in human β-cells. A: Ca²⁺ was uncaged by delivering UV excitation light (arrows) to a β-cell loaded with NP-EGTA and equilibrated in SES containing 5.6 mM glucose. UV light was delivered alone (1st flash) or in combination with Phos-AM₃ (3.3 μM, repeated 30-s applications; note y-axis scaling), which failed to facilitate CICR. B: CICR was not observed in response to UV excitation alone (1st flash), whereas CICR was evoked when UV light was delivered in combination with 8-pCPT-2′-O-Me-cAMP-AM (10 μM, 30-s application, 2nd flash; note y-axis scaling). CICR was defined as an increase of [Ca²⁺]_i, the duration of which did not exceed 30 s when measured at the 10% amplitude cutoff. The increase of [Ca²⁺]_i also must have exceeded 300 nM when measured at the 50% amplitude cutoff. C: facilitation of CICR by 8-pCPT-2′-O-Me-cAMP-AM in a human β-cell treated with H-89 (10 μM). D: population study demonstrating the 10 μM H-89-resistant but the 10 μM ryanodine-sensitive action of 10 μM 8-pCPT-2′-O-Me-cAMP-AM to facilitate CICR.

Fig. 7.

RT-quantitative PCR (QPCR) for Epac1 and Epac2. A: RT-QPCR fluorescence growth curves obtained using 100 ng of human islet RNA in a single PCR reaction from islets of a single donor. Ribosomal S18 mRNA was used as the reference target for quantification of Epac1 and Epac2 mRNA. The threshold crossing value (C_T) for S18 mRNA (16.1) is indicated. B: comparison of ΔC_T values obtained after averaging results of PCR reactions run for Epac1 (ΔC_T 11.83 ± 0.31; 10 reactions) and Epac2 (ΔC_T 6.93 + 0.39; 13 reactions). 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 (4.9) 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 1:29.9 (right). Results in B and C are based on averaged data obtained using 2 batches of islets from 2 donors.