epac2 insulin

Experiments with cAMP addition to permeabilized cells showed a left-shifted dose-response relationship for Epac2A translocation after deletion of the CNB-A domain. Copyright © 2020 Elsevier B.V. or its licensors or contributors. n = 26 cells from 5 experiments for wt, 12 cells from 4 experiments for ΔCNB-A, and 14 cells from 5 experiments for ΔDEP. A: TIRF images of MIN6 cells and human islet cells (from normoglycemic donors or donors with type 2 diabetes) coexpressing NPY-mEGFP (granule) and mCherry-Epac2A-wt under basal conditions (Ctrl) and after exposure to 10 nmol/L GLP-1. Blue arrows indicate incident laser excitation light and green arrows fluorescent light.

[15][16], Several Epac-selective cAMP analogs have been developed to clarify the functional roles of Epacs as well those of the Epac-dependent signaling pathway distinct from the PKA-dependent signaling pathway. These results indicate that activation of cAMP signaling enhances fusion events by increasing the number of restless newcomer.
Epacs (Epac1 and Epac2) are recently discovered cAMP-binding proteins (9, 17, 18).

Pull-down assay for GTP-Rap1 was performed as described in ref. Clipboard, Search History, and several other advanced features are temporarily unavailable. This conformational change enables access of Rap1 to the catalytic region and allows activation. Your opinions are important to us. Although activation of cAMP signaling alone does not cause fusion of the granules to the plasma membrane, it clearly potentiates both the first phase (a prompt, marked, and transient increase) and the second phase (a moderate and sustained increase) of glucose-induced fusion events.

Data represent 10–13 cells/condition from 2 experiments. Isolated pancreatic islets were infected with adenovirus carrying β-galactosidase (open bars) or mRFP-Rap1GAP1 (filled bars) at an MOI of 200. Effects of cAMP on insulin granule dynamics. It is not clear how Epac2A promotes priming and exocytosis, but it is probably related to its interaction with RIM2α (37) or other components of the secretory machinery, such as the SUR1 subunit of the KATP channel (2), the granular sulphonylurea receptor (21), the active zone protein piccolo (38), or SNAP25 (39). Right panels: Granules that fail to undergo exocytosis show stable fluorescence and a ΔF/S signal for Epac2 significantly lower than that for exocytosing granules (P = 0.002 for averaged period before and P = 0.03 for that after time 0).

These results demonstrate that Rap1 is activated in insulin-secreting cells in a cAMP-dependent, PKA-independent manner.

Data were obtained from six independent experiments (n = 2–5 for each) and expressed as means ± SE. Before that, insulin molecules are stored in tiny granules inside the β-cells; these granules must fuse with the cells’ surface membranes to release their contents.  |  ) were generated by placing tandemly oriented lox P sites around a 1.3-kb DNA fragment containing a part of exon 1 and intron 1 of the mouse Epac2 gene, and subsequently removing them with Cre recombinase (SI Fig.

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After preincubation with 2.8 mM glucose, MIN6 cells were incubated with 2.8 mM glucose plus 10 nM GIP, 10 nM GLP-1, or 1 mM 8-Bromo-cAMP for 15 min. ( A…, NLM The best studied process is insulin secretion by islet beta cells. cAMP binding to the CNB domain(s) results in a conformational change that releases the autoinhibition and exposes the catalytic region (14,18,19). Figure 8A shows that forskolin amplified secretion several-fold in cells expressing mCherry-Epac2A-wt. C). 3) (10). 8, SI Table 1, and SI Movie 5). Scale bar, 10 µm. While a direct role for Epac2 in SU-induced insulin secretion may be disputed by the observation that SUs are incapable of inducing insulin secretion in SUR1−/−mice (Seghers, Nakazaki, et al, 2000), it is important to note that pancreatic islets isolated from these mice also demonstrate impaired cAMP-mediated, PKA-independent insulin secretion, indicative of disrupted Epac2 signaling …  |  As shown in Fig. (Right) Effects of Rap1GAP1 on 8-Bromo-cAMP-potentiated insulin secretion in isolated pancreatic islets. This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Image credit: Noah Whiteman (University of California, Berkeley, CA).

In Figs. Binding of Epac2A to granule docking sites promotes exocytosis. In this review, we summarize the structure and function of Epac2. N.R.G. 7A). The first phase was counted as fusion events during the first 5 min after stimulation, and the second phase was counted as fusion events occurring thereafter.

Resting newcomer: granules that are newly recruited, docked, and fused to the plasma membrane by stimulation. [33] In contrast, chronic stimulation of Epac2 may be a cause of cardiac arrhythmia through CaMKII-dependent diastolic sarcoplasmic reticulum (SR) Ca2+ release in mice. C). Exchange protein directly activated by cAMP 2 (EPAC2) is encoded by Rapgef4 gene. *, P < 0.05 (Student's unpaired t test). 6). Fluorescence at 530-nm/50-nm half-bandwidth (EGFP) and 620-nm long-pass (mCherry) was detected with a back-illuminated EMCCD camera (iXON DU897; Andor Technology) controlled by MetaFluor software (Molecular Devices, Downingtown, PA). The analysis was restricted to cells that showed clustered Epac2A fluorescence. The granular accumulation did not require cAMP concentrations corresponding to the very high Kd (87 μmol/L) of the CNB-A domain, because it was observed with GLP-1, and this stimulation did not seem to trigger cAMP elevations >30 μmol/L (compare data in Figs. No potential conflicts of interest relevant to this article were reported. Copyright © 2015 Elsevier B.V. All rights reserved.

Thank you for your interest in spreading the word about Diabetes. *, P < 0.005 (Student's unpaired t test). (.

Funding. Freely available online through the PNAS open access option. The catalytic region, which is responsible for the activation of Rap, consists of a CDC25 homology domain (CDC25-HD), a Ras exchange motif (REM) domain, and a Ras association (RA) domain. SI Methods Thirty seconds after acquisition of image, the primary cultured cells were stimulated by 60 mM K+ (B) or 16.7 mM glucose (C). J Biol Chem. Number of cells from at least three experiments is shown for each bar. (Scale bar, 10 μm.) Thus, Epac2 is involved in the pathogenesis and pathophysiology of various diseases, and represents a promising therapeutic target. Epub 2020 Jan 16. This finding indicates that the RRP involved in the first phase does not necessarily comprise predocked granules, and that some granules a distance away from the plasma membrane are immediately releasable in pancreatic beta cells, as has been found in the neuromuscular junction in frogs (16).
Your email address is used only to let the recipient know who sent the email. Under this condition, whereas glucose-induced insulin secretion was similar in control and Rap1 knockdown MIN6 cells, potentiation of insulin secretion by 8-Bromo-cAMP in Rap1 knockdown cells was decreased by ≈40% compared with control (Fig. We also showed that the localization process regulates granule priming and exocytosis. Primary cultured pancreatic beta cells were preincubated with Hepes-KRB containing 2.8 mM glucose at 37°C for 30 min. The low-affinity domain seemed to contribute to membrane binding only at very high (>30 μmol/L) cAMP concentrations, but it is questionable whether this effect has any physiological relevance, because the translocation response to a high concentration of GLP-1 in intact cells was not reduced by the CNB-A deletion.

How much and when insulin is released depends on a complex system of messenger molecules and proteins that is not well understood. Pharmacological stimulation of the processes that localize Epac2A to the vesicles may provide a new strategy for improving insulin secretion in type 2 diabetes. Although activation of cAMP signaling alone does not cause fusion of the granules to the plasma membrane, it clearly potentiates both the first phase (a prompt, marked, and transient increase) and the second phase (a moderate and sustained increase) of glucose-induced fusion events.

Online ISSN 1091-6490. The CNB-A domain is only present in the Epac2A splice variant, which is the dominating version in secretory cells, like neurons and β-cells (11). Both signaling branches are considered to increase insulin release from ß-cells when these are stimulated by increasing blood glucose levels. A: Simultaneous TIRF recording of submembrane cAMP concentration (blue trace) and Epac2A membrane association (black trace) in a single MIN6 cell stimulated with 1 nmol/L GLP-1 in the presence of 3 mmol/L glucose. A.T. is the guarantor of this work and, as such, had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. In the course of our study of the Epac2-mediated mechanisms of insulin secretion, we developed a fluorescence resonance energy transfer (FRET)-based Epac2 sensor (termed C-Epac2-Y) in which the full-length Epac2 is fused amino-terminally to enhanced cyan fluorescent protein (ECFP) and carboxyl-terminally to enhanced yellow fluorescent protein (EYFP). ***P < 0.001 for difference from wt control. Although not required for the translocation of Epac2A to the plasma membrane, the CNB-A domain was responsible for this clustering near granules. Characteristics of insulin granule dynamics in pancreatic beta cells. Sulfonylurea drugs (SUs), widely used for the treatment of type 2 diabetes through stimulation of insulin secretion from pancreatic β-cells, have also been shown to specifically activate Epac2. G: Confocal images of MIN6 cells coexpressing GFP-Epac2A-wt and either mCherry-Epac2A-ΔCNB-A (representative for 12 cells in 4 experiments) or mCherry-Epac2A-ΔDEP (representative for 14 cells in 4 experiments).

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