Harumi Takahashi

Essential role of Epac2/Rap1 signaling in regulation of insulin granule dynamics by cAMP

cAMP is well known to regulate exocytosis in various secretory cells, but the precise mechanism of its action remains unknown. Here, we examine the role of cAMP signaling in the exocytotic process of insulin granules in pancreatic beta cells. 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. Interestingly, all granules responsible for this potentiation are newly recruited and immediately fused to the plasma membrane without docking (restless newcomer). Importantly, cAMP-potentiated fusion events in the first phase of glucose-induced exocytosis are markedly reduced in mice lacking the cAMP-binding protein Epac2 (Epac2ko/ko). In addition, the small GTPase Rap1, which is activated by cAMP specifically through Epac2 in pancreatic beta cells, mediates cAMP-induced insulin secretion in a protein kinase A-independent manner. We also have developed a simulation model of insulin granule movement in which potentiation of the first phase is associated with an increase in the insulin granule density near the plasma membrane. Taken together, these data indicate that Epac2/Rap1 signaling is essential in regulation of insulin granule dynamics by cAMP, most likely by controlling granule density near the plasma membrane.

The cAMP Sensor Epac2 Is a Direct Target of Antidiabetic Sulfonylurea Drugs

Expanding Sulfonylurea Mechanisms Sulfonylureas are important drugs used for treatment of diabetes that act through adenosine triphosphate–sensitive potassium channels to promote secretion of insulin from the pancreas. Zhang et al. (p. 607) present another mechanism by which the beneficial effects of sulfonylureas may also be obtained. Sulfonylureas were identified in a screen for substances that modify the activity of Epac2, a guanine nucleotide exchange factor for the small guanosine triphosphatase Rap1. Mice lacking Epac2 were less responsive to sulfonylureas, which may suggest that Epac2 would be a useful target for development of drugs for treatment of diabetes. A drug used to enhance insulin secretion in diabetes has a previously unrecognized protein target. Epac2, a guanine nucleotide exchange factor for the small guanosine triphosphatase Rap1, is activated by adenosine 3′,5′-monophosphate. Fluorescence resonance energy transfer and binding experiments revealed that sulfonylureas, widely used antidiabetic drugs, interact directly with Epac2. Sulfonylureas activated Rap1 specifically through Epac2. Sulfonylurea-stimulated insulin secretion was reduced both in vitro and in vivo in mice lacking Epac2, and the glucose-lowering effect of the sulfonylurea tolbutamide was decreased in these mice. Epac2 thus contributes to the effect of sulfonylureas to promote insulin secretion. Because Epac2 is also required for the action of incretins, gut hormones crucial for potentiating insulin secretion, it may be a promising target for antidiabetic drug development.

Roles of cAMP signalling in insulin granule exocytosis.

Insulin secretion is regulated by a series of complex events generated by various intracellular signals including Ca2+, ATP, cAMP and phospholipid‐derived signals. Glucose‐stimulated insulin secretion is the principal mode of insulin secretion, and the mechanism potentiating the secretion is critical for physiological responses. Among the various intracellular signals involved, cAMP is particularly important for amplifying insulin secretion. Recently, glucagon‐like peptide‐1 (GLP‐1) analogues and dipeptidyl peptidase‐IV (DPP‐IV) inhibitors have been developed as new antidiabetic drugs. These drugs all act through cAMP signalling in pancreatic β‐cells. Until recently, cAMP was generally thought to potentiate insulin secretion through protein kinase A (PKA) phosphorylation of proteins associated with the secretory process. However, it is now known that in addition to PKA, cAMP has other targets such as Epac (also referred to as cAMP‐GEF). The variety of the effects mediated by cAMP signalling may be linked to cAMP compartmentation in the pancreatic β‐cells.

Rim2α Determines Docking and Priming States in Insulin Granule Exocytosis

Insulin secretion is essential for maintenance of glucose homeostasis, but the mechanism of insulin granule exocytosis, the final step of insulin secretion, is largely unknown. Here, we investigated the role of Rim2alpha in insulin granule exocytosis, including the docking, priming, and fusion steps. We found that interaction of Rim2alpha and Rab3A is required for docking, which is considered a brake on fusion events, and that docking is necessary for K(+)-induced exocytosis, but not for glucose-induced exocytosis. Furthermore, we found that dissociation of the Rim2alpha/Munc13-1 complex by glucose stimulation activates Syntaxin1 by Munc13-1, indicating that Rim2alpha primes insulin granules for fusion. Thus, Rim2alpha determines docking and priming states in insulin granule exocytosis depending on its interacting partner, Rab3A or Munc13-1, respectively. Because Rim2alpha(-/-) mice exhibit impaired secretion of various hormones stored as dense-core granules, including glucose-dependent insulinotropic polypeptide, growth hormone, and epinephrine, Rim2alpha plays a critical role in exocytosis of these dense-core granules.

Treating diabetes today: a matter of selectivity of sulphonylureas

It is well known that sulphonylureas (SUs), commonly used in the treatment of type 2 diabetes mellitus, stimulate insulin secretion by closing ATP‐sensitive K+ (KATP) channels in pancreatic β‐cells by binding to the SU receptor SUR1. SUs are now known also to activate cAMP sensor Epac2 (cAMP‐GEFII) to Rap1 signalling, which promotes insulin granule exocytosis. For SUs to exert their full effects in insulin secretion, they are required to activate Epac2 as well as to inhibit the β‐cell KATP channels. As Epac2 is also necessary for potentiation of glucose‐induced insulin secretion by cAMP‐increasing agents, such as incretin, Epac2 is a target of both cAMP and SUs. The distinct effects of various SUs appear to be because of their different actions on Epac2/Rap1 signalling as well as KATP channels. Differently from other SUs, gliclazide is unique in that it is specific for β‐cell KATP channel and does not activate Epac2.

Actin Dynamics Regulated by the Balance of Neuronal Wiskott-Aldrich Syndrome Protein (N-WASP) and Cofilin Activities Determines the Biphasic Response of Glucose-induced Insulin Secretion

Background: Actin dynamics is involved in insulin secretion, but the mechanism is unknown. Results: The G-actin predominant or F-actin remodeling state in pancreatic β-cells, which is regulated by the balance of N-WASP and cofilin activities, determines the biphasic glucose-induced insulin secretion (GIIS). Conclusion: Actin dynamics regulated by N-WASP and cofilin underlie the biphasic GIIS. Significance: The regulation of actin dynamics in β-cells and its role in GIIS are clarified. Actin dynamics in pancreatic β-cells is involved in insulin secretion. However, the molecular mechanisms of the regulation of actin dynamics by intracellular signals in pancreatic β-cells and its role in phasic insulin secretion are largely unknown. In this study, we elucidate the regulation of actin dynamics by neuronal Wiskott-Aldrich syndrome protein (N-WASP) and cofilin in pancreatic β-cells and demonstrate its role in glucose-induced insulin secretion (GIIS). N-WASP, which promotes actin polymerization through activation of the actin nucleation factor Arp2/3 complex, was found to be activated by glucose stimulation in insulin-secreting clonal pancreatic β-cells (MIN6-K8 β-cells). Introduction of a dominant-negative mutant of N-WASP, which lacks G-actin and Arp2/3 complex-binding region VCA, into MIN6-K8 β-cells or knockdown of N-WASP suppressed GIIS, especially the second phase. We also found that cofilin, which severs F-actin in its dephosphorylated (active) form, is converted to the phosphorylated (inactive) form by glucose stimulation in MIN6-K8 β-cells, thereby promoting F-actin remodeling. In addition, the dominant-negative mutant of cofilin, which inhibits activation of endogenous cofilin, or knockdown of cofilin reduced the second phase of GIIS. However, the first phase of GIIS occurs in the G-actin predominant state, in which cofilin activity predominates over N-WASP activity. Thus, actin dynamics regulated by the balance of N-WASP and cofilin activities determines the biphasic response of GIIS.

Antidiabetic Sulfonylureas and cAMP Cooperatively Activate Epac2A

Identification of the binding site of Epac2A for an antidiabetic drug may enable the development of improved therapies. Cooperation Between cAMP and Antidiabetic Drugs A class of drugs called sulfonylureas increase insulin secretion from pancreatic β cells in patients with type 2 diabetes. One sulfonylurea mechanism for enhancing insulin secretion is through stimulation of the guanine nucleotide exchange factor Epac2A. Takahashi et al. used molecular docking simulation to predict the sulfonylurea binding site on Epac2A and verified in cells with site-directed mutants that sulfonylureas bound to one of two cyclic nucleotide–binding domains and cooperated with the endogenous Epac activator cyclic adenosine monophosphate to promote Epac2A function and stimulate the Epac target Rap1. The identification of specific amino acids in Epac2A that mediate interactions with sulfonylureas could help to develop drugs for treating diabetes. Sulfonylureas are widely used drugs for treating insulin deficiency in patients with type 2 diabetes. Sulfonylureas bind to the regulatory subunit of the pancreatic β cell potassium channel that controls insulin secretion. Sulfonylureas also bind to and activate Epac2A, a member of the Epac family of cyclic adenosine monophosphate (cAMP)–binding proteins that promote insulin secretion through activation of the Ras-like guanosine triphosphatase Rap1. Using molecular docking simulation, we identified amino acid residues in one of two cyclic nucleotide–binding domains, cNBD-A, in Epac2A predicted to mediate the interaction with sulfonylureas. We confirmed the importance of the identified residues by site-directed mutagenesis and analysis of the response of the mutants to sulfonylureas using two assays: changes in fluorescence resonance energy transfer (FRET) of an Epac2A-FRET biosensor and direct sulfonylurea-binding experiments. These residues were also required for the sulfonylurea-dependent Rap1 activation by Epac2A. Binding of sulfonylureas to Epac2A depended on the concentration of cAMP and the structures of the drugs. Sulfonylureas and cAMP cooperatively activated Epac2A through binding to cNBD-A and cNBD-B, respectively. Our data suggest that sulfonylureas stabilize Epac2A in its open, active state and provide insight for the development of drugs that target Epac2A.

Ras-related C3 botulinum toxin substrate 1 (RAC1) regulates glucose-stimulated insulin secretion via modulation of F-actin.

Aims/hypothesisThe small G-protein ras-related C3 botulinum toxin substrate 1 (RAC1) plays various roles in mammalian cells, such as in the regulation of cytoskeletal organisation, cell adhesion, migration and morphological changes. The present study examines the effects of RAC1 ablation on pancreatic beta cell function.MethodsIsolated islets from pancreatic beta cell-specific Rac1-knockout (betaRac1−/−) mice and RAC1 knockdown INS-1 insulinoma cells treated with small interfering RNA were used to investigate insulin secretion and cytoskeletal organisation in pancreatic beta cells.ResultsBetaRac1−/− mice showed decreased glucose-stimulated insulin secretion, while there were no apparent differences in islet morphology. Isolated islets from the mice had blunted insulin secretion in response to high glucose levels. In RAC1 knockdown INS-1 cells, insulin secretion was also decreased in response to high glucose levels, consistent with the phenotype of betaRac1−/− mice. Even under high glucose levels, RAC1 knockdown INS-1 cells remained intact with F-actin, which inhibits the recruitment of the insulin granules, resulting in an inhibition of insulin secretion.Conclusions/interpretationIn RAC1-deficient pancreatic beta cells, F-actin acts as a barrier for insulin granules and reduces glucose-stimulated insulin secretion.

Role of Epac2A/Rap1 Signaling in Interplay Between Incretin and Sulfonylurea in Insulin Secretion

Incretin-related drugs and sulfonylureas are currently used worldwide for the treatment of type 2 diabetes. We recently found that Epac2A, a cAMP binding protein having guanine nucleotide exchange activity toward Rap, is a target of both incretin and sulfonylurea. This suggests the possibility of interplay between incretin and sulfonylurea through Epac2A/Rap1 signaling in insulin secretion. In this study, we examined the combinatorial effects of incretin and various sulfonylureas on insulin secretion and activation of Epac2A/Rap1 signaling. A strong augmentation of insulin secretion by combination of GLP-1 and glibenclamide or glimepiride, which was found in Epac2A+/+ mice, was markedly reduced in Epac2A−/− mice. In contrast, the combinatorial effect of GLP-1 and gliclazide was rather mild, and the effect was not altered by Epac2A ablation. Activation of Rap1 was enhanced by the combination of an Epac-selective cAMP analog with glibenclamide or glimepiride but not gliclazide. In diet-induced obese mice, ablation of Epac2A reduced the insulin secretory response to coadministration of the GLP-1 receptor agonist liraglutide and glimepiride. These findings clarify the critical role of Epac2A/Rap1 signaling in the augmenting effect of incretin and sulfonylurea on insulin secretion and provide the basis for the effects of combination therapies of incretin-related drugs and sulfonylureas.

Cooperation between cAMP signalling and sulfonylurea in insulin secretion.

Although glucose is physiologically the most important regulator of insulin secretion, glucose‐induced insulin secretion is modulated by hormonal and neural inputs to pancreatic β‐cells. Most of the hormones and neurotransmitters evoke intracellular signals such as cAMP, Ca2+, and phospholipid‐derived molecules by activating G protein‐coupled receptors (GPCRs). In particular, cAMP is a key second messenger that amplifies insulin secretion in a glucose concentration‐dependent manner. The action of cAMP on insulin secretion is mediated by both protein kinase A (PKA)‐dependent and Epac2A‐dependent mechanisms. Many of the proteins expressed in β‐cells are phosphorylated by PKA in vitro, but only a few proteins in which PKA phosphorylation directly affects insulin secretion have been identified. On the other hand, Epac2A activates the Ras‐like small G protein Rap in a cAMP‐dependent manner. Epac2A is also directly activated by various sulfonylureas, except for gliclazide. 8‐pCPT‐2′‐O‐Me‐cAMP, an Epac‐selective cAMP analogue, and glibenclamide, a sulfonylurea, synergistically activate Epac2A and Rap1, whereas adrenaline, which suppresses cAMP production in pancreatic β‐cells, blocks activation of Epac2A and Rap1 by glibenclamide. Thus, cAMP signalling and sulfonylurea cooperatively activate Epac2A and Rap1. This interaction could account, at least in part, for the synergistic effects of incretin‐related drugs and sulfonylureas in insulin secretion. Accordingly, clarification of the mechanism of Epac2A activation may provide therapeutic strategies to improve insulin secretion in diabetes.