Enhanced Rap1 activation and insulin secretagogue properties of an acetoxymethyl ester of an Epac-selective cyclic AMP analog in rat INS-1 cells: studies with 8-pCPT-2'-O-Me-cAMP-AM

Abstract

To ascertain the identities of cyclic nucleotide-binding proteins that mediate the insulin secretagogue action of cAMP, the possible contributions of the exchange protein directly activated by cAMP (Epac) and protein kinase A (PKA) were evaluated in a pancreatic beta cell line (rat INS-1 cells). Assays of Rap1 activation, CREB phosphorylation, and PKA-dependent gene expression were performed in combination with live cell imaging and high throughput screening of a fluorescence resonance energy transfer-based cAMP sensor (Epac1-camps) to validate the selectivity with which acetoxymethyl esters (AM-esters) of cAMP analogs preferentially activate Epac or PKA. Selective activation of Epac or PKA was achieved following exposure of INS-1 cells to 8-pCPT-2'-O-Me-cAMP-AM or Bt(2)cAMP-AM, respectively. Both cAMP analogs exerted dose-dependent and glucose metabolism-dependent actions to stimulate insulin secretion, and when each was co-administered with the other, a supra-additive effect was observed. Because 2.4-fold more insulin was secreted in response to a saturating concentration (10 microm) of Bt(2)cAMP-AM as compared with 8-pCPT-2'-O-Me-cAMP-AM, and because the action of Bt(2)cAMP-AM but not 8-pCPT-2'-O-Me-cAMP-AM was nearly abrogated by treatment with 3 microm of the PKA inhibitor H-89, it is concluded that for INS-1 cells, it is PKA that acts as the dominant cAMP-binding protein in support of insulin secretion. Unexpectedly, 10-100 microm of the non-AM-ester of 8-pCPT-2'-O-Me-cAMP failed to stimulate insulin secretion and was a weak activator of Rap1 in INS-1 cells. Moreover, 10 microm of the AM-ester of 8-pCPT-2'-O-Me-cAMP stimulated insulin secretion from mouse islets, whereas the non-AM-ester did not. Thus, the membrane permeability of 8-pCPT-2'-O-Me-cAMP in insulin-secreting cells is so low as to limit its biological activity. It is concluded that prior reports documenting the failure of 8-pCPT-2'-O-Me-cAMP to act in beta cells, or other cell types, need to be re-evaluated through the use of the AM-ester of this cAMP analog.

FIGURE 1.

8-pCPT-2′-O-Me-cAMP-AM activates the cAMP reporter Epac1-camps. A, expression of a fusion protein of Epac1 and EGFP in stably transfected INS-1 cells (calibration bar, 0.8 μm). B, stable expression of Epac1-camps in INS-1 cells (calibration bar, 3 μm). C, FRET-based live cell imaging of Epac1-camps in INS-1 cells exposed to 8-pCPT-2′-O-Me-cAMP-AM (20 μm; arrows indicate duration of exposure). D, single cell population study summarizing the action of 8-pCPT-2′-O-Me-cAMP-AM (ESCA-AM; 20 μm) to activate Epac1-camps in INS-1 cells. Note that both the non-AM-ester of 8-pCPT-2′-O-Me-cAMP (ESCA, no AM;20 μm) and acetoxymethyl ester phosphate (Phosphate-AM₃; 6.6 μm) were without significant effect. All solutions were administered in SES containing 0.1% dimethyl sulfoxide. Values above each histogram bar indicate the numbers of cells (n values) assayed for each experimental condition. For panels C and D, the experiment was repeated 3 times with similar results.

FIGURE 2.

High throughput assay for activation of Epac1-camps by cAMP analogs. A, concentration-response relationship for the activation of Epac1-camps by 0.3–3.0 μm 8-pCPT-2′-O-Me-cAMP-AM (ESCA-AM). The vehicle solution was comprised of SES containing 0.1% dimethyl sulfoxide. A vertical arrow indicates the time at which the test solutions were injected. An increase of 485/535 nm emission ratio signifies the activation of Epac1-camps. B, differential potencies of 8-pCPT-2′-O-Me-cAMP-AM (ESCA-AM, blue line and symbols) and 8-pCPT-2′-O-Me-cAMP (ESCA-No-AM, red line and symbols) to activate Epac1-camps. For panels A and B, each data point corresponds to the mean of 24–36 wells, and each experiment was repeated 3 times with similar results.

FIGURE 3.

Differential Rap1 activation properties of cAMP analogs. A, Western blot analysis demonstrating the weak effect of 8-pCPT-2′-O-Me-cAMP (ESCA, left panel) and the strong effect of 8-pCPT-2′-O-Me-cAMP-AM (ESCA-AM, right panel) to activate Rap1 in lysates prepared from monolayers of INS-1 cells transfected with FLAG-Rap1 and Eapc1. Active Rap1 is labeled as Rap1-GTP and the quantity of active Rap1 was assessed by densitometry. For the left panel, treatment with 3.0 μm 8-pCPT-2′-O-Me-cAMP increased the amount of active Rap1 by 1.41-fold relative to the untreated control value. For the right panel, treatment with 3.0 μm 8-pCPT-2′-O-Me-cAMP-AM increased the amount of active Rap1 by 4.79-fold relative to the untreated control value. B, time course of Rap1 activation by 8-pCPT-2′-O-Me-cAMP-AM (ESCA-AM). The exposure time corresponds to the time in which INS-1 cells were exposed to the test solution. C, contrasting actions of PKA-selective Bt₂cAMP-AM (db-cAMP-AM) and Epac-selective 8-pCPT-2′-O-Me-cAMP-AM (ESCA-AM) to activate Rap1 under conditions in which INS-1 cells were or were not pretreated with the PKA inhibitor H-89 (10 μm). For panels A-C, each experiment was repeated 3 times with similar results.

FIGURE 4.

cAMP analogs differentially regulate RIP-CRE-Luc and CREB. A, 8-pCPT-2′-O-Me-cAMP-AM (ESCA-AM) failed to stimulate luciferase activity in lysates prepared from INS-1 cells expressing RIP1-CRE-Luc. Equivalent concentrations of Bt₂cAMP-AM (db-cAMP-AM) stimulated luciferase activity, and this effect was antagonized by 10 μm of the PKA inhibitor H-89 (^*, p < 0.05). Results are the average of three experiments and each data point within a single experiment was the average of two determinations. B, Western blot analysis of untransfected INS-1 cells demonstrated increased Ser¹³³ CREB phosphorylation after treatment of the cells for 30 min with Bt₂cAMP-AM (db-cAMP-AM) or a combination of forskolin (Fsk., 2 μm) and isobutylmethylxanthine (IBMX, 100 μm). No such effect was observed in response to 8-pCPT-2′-O-Me-cAMP-AM. Similar findings were obtained in 4 experiments. C, pretreatment of INS-1 cells with 8-pCPT-2′-O-Me-cAMP-AM did not reduce the stimulatory actions of exendin-4 (Ex-4), forskolin (Fsk.), or a PKA-selective cAMP analog (6-Bn-cAMP) in assays of RIP1-CRE-Luc-dependent luciferase expression in INS-1 cells. Results are the average of three experiments and each data point within a single experiment was the mean of two determinations.

FIGURE 5.

Secretagogue properties of cAMP analogs in INS-1 cells. A, differential actions of 8-pCPT-2′-O-Me-cAMP and 8-pCPT-2′-O-Me-cAMP-AM under conditions in which cells were equilibrated for 30 min in buffer containing 11.1 mm glucose. B, differential actions of Bt₂cAMP-AM (db-cAMP-AM) and 8-pCPT-2′-O-Me-cAMP-AM. Note that the data for 8-pCPT-2′-O-Me-cAMP-AM presented in A were re-scaled on the y axis of panel B. C, synergistic interaction of Bt₂cAMP-AM (db-cAMP-AM) and 8-pCPT-2′-O-Me-cAMP-AM (1 μm each) to stimulate insulin secretion. D, the actions of 8-pCPT-2′-O-Me-cAMP-AM and Bt₂cAMP-AM (db-cAMP-AM) were glucose-dependent. For panels A-C, a y axis value of 1.0 corresponds to basal insulin secretion (15 ng/ml/30 min) occurring in the presence of 11.1 mm glucose and no cAMP analog. For panel D, a y axis value of 1.0 corresponds to insulin secretion that occurred during 30 min in the presence of 0.1 mm glucose (6 ng/ml/min) or 11.1 mm glucose (15 ng/ml/30 min), but in the absence of added cAMP analogs. For panels A–D, ^*, p < 0.05; ^**, p < 0.005; and each determination is the mean of two determinations. These results summarize findings of a single experiment and this experiment was repeated 3 times with similar outcomes.

FIGURE 6.

Contrasting actions of protein kinase inhibitor H-89 on insulin secretion. A, pretreatment of INS-1 cells with H-89 reduced 11.1 mm glucose-stimulated insulin secretion. B, the secretagogue action of Bt₂cAMP-AM (db-cAMP-AM) was antagonized by H-89, whereas the action of 8-pCPT-2′-O-Me-cAMP-AM was slightly augmented. For A and B, the cultures were incubated in 11.1 mm glucose KRBH with or without H-89 for 15 min prior to a 30-min exposure to test solutions containing 11.1 mm glucose and the indicated test substances. For B, a y axis value of 1.0 corresponds to insulin secretion occurring in the presence of 11.1 mm glucose, either with H-89 (11.7 ng/ml/30min) or without H-89 (15 ng/ml/min). For panels A and B, ^*, p < 0.05 and each determination is the mean of two determinations. These results summarize findings of a single experiment and this experiment was repeated 3 times with similar outcomes.