Tadao Shibasaki

cAMP-GEFII is a direct target of cAMP in regulated exocytosis

Although cAMP is well known to regulate exocytosis in many secretory cells, its direct target in the exocytotic machinery is not known. Here we show that cAMP-GEFII, a cAMP sensor, binds to Rim (Rab3-interacting molecule, Rab3 being a small G protein) and to a new isoform, Rim2, both of which are putative regulators of fusion of vesicles to the plasma membrane. We also show that cAMP-GEFII, through its interaction with Rim2, mediates cAMP-induced, Ca2+-dependent secretion that is not blocked by an inhibitor of cAMP-dependent protein kinase (PKA). Accordingly, cAMP-GEFII is a direct target of cAMP in regulated exocytosis and is responsible for cAMP-dependent, PKA-independent exocytosis.

Mouse model of Prinzmetal angina by disruption of the inward rectifier Kir6.1

The inwardly rectifying K+ channel Kir6.1 forms K+ channels by coupling with a sulfonylurea receptor in reconstituted systems, but the physiological roles of Kir6.1-containing K+ channels have not been determined. We report here that mice lacking the gene encoding Kir6.1 (known as Kcnj8) have a high rate of sudden death associated with spontaneous ST elevation followed by atrioventricular block as seen on an electrocardiogram. The K+ channel opener pinacidil did not induce K+ currents in vascular smooth-muscle cells of Kir6.1-null mice, and there was no vasodilation response to pinacidil. The administration of methylergometrine, a vasoconstrictive agent, elicited ST elevation followed by cardiac death in Kir6.1-null mice but not in wild-type mice, indicating a phenotype characterized by hypercontractility of coronary arteries and resembling Prinzmetal (or variant) angina in humans. The Kir6.1-containing K+ channel is critical in the regulation of vascular tonus, especially in the coronary arteries, and its disruption may cause Prinzmetal angina.

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.

GLP-1 receptor activation and Epac2 link atrial natriuretic peptide secretion to control of blood pressure

Glucagon-like peptide-1 receptor (GLP-1R) agonists exert antihypertensive actions through incompletely understood mechanisms. Here we demonstrate that cardiac Glp1r expression is localized to cardiac atria and that GLP-1R activation promotes the secretion of atrial natriuretic peptide (ANP) and a reduction of blood pressure. Consistent with an indirect ANP-dependent mechanism for the antihypertensive effects of GLP-1R activation, the GLP-1R agonist liraglutide did not directly increase the amount of cyclic GMP (cGMP) or relax preconstricted aortic rings; however, conditioned medium from liraglutide-treated hearts relaxed aortic rings in an endothelium-independent, GLP-1R–dependent manner. Liraglutide did not induce ANP secretion, vasorelaxation or lower blood pressure in Glp1r−/− or Nppa−/− mice. Cardiomyocyte GLP-1R activation promoted the translocation of the Rap guanine nucleotide exchange factor Epac2 (also known as Rapgef4) to the membrane, whereas Epac2 deficiency eliminated GLP-1R–dependent stimulation of ANP secretion. Plasma ANP concentrations were increased after refeeding in wild-type but not Glp1r−/− mice, and liraglutide increased urine sodium excretion in wild-type but not Nppa−/− mice. These findings define a gut-heart GLP-1R–dependent and ANP–dependent axis that regulates blood pressure.

Regulation of Ca2+ channel expression at the cell surface by the small G-protein kir/Gem.

Voltage-dependent calcium (Ca2+) channels are involved in many specialized cellular functions, and are controlled by intracellular signals such as heterotrimeric G-proteins, protein kinases and calmodulin (CaM). However, the direct role of small G-proteins in the regulation of Ca2+ channels is unclear. We report here that the GTP-bound form of kir/Gem, identified originally as a Ras-related small G-protein that binds CaM, inhibits high-voltage-activated Ca2+ channel activities by interacting directly with the β-subunit. The reduced channel activities are due to a decrease in α1-subunit expression at the plasma membrane. The binding of Ca2+/CaM to kir/Gem is required for this inhibitory effect by promoting the cytoplasmic localization of kir/Gem. Inhibition of L-type Ca2+ channels by kir/Gem prevents Ca2+-triggered exocytosis in hormone-secreting cells. We propose that the small G-protein kir/Gem, interacting with β-subunits, regulates Ca2+ channel expression at the cell surface.

Dynamics of insulin secretion and the clinical implications for obesity and diabetes.

Insulin secretion is a highly dynamic process regulated by various factors including nutrients, hormones, and neuronal inputs. The dynamics of insulin secretion can be studied at different levels: the single β cell, pancreatic islet, whole pancreas, and the intact organism. Studies have begun to analyze cellular and molecular mechanisms underlying dynamics of insulin secretion. This review focuses on our current understanding of the dynamics of insulin secretion in vitro and in vivo and discusses their clinical relevance.

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.

GLP-1 Inhibits and Adrenaline Stimulates Glucagon Release by Differential Modulation of N- and L-Type Ca2+ Channel-Dependent Exocytosis

Glucagon secretion is inhibited by glucagon-like peptide-1 (GLP-1) and stimulated by adrenaline. These opposing effects on glucagon secretion are mimicked by low (1-10 nM) and high (10 muM) concentrations of forskolin, respectively. The expression of GLP-1 receptors in alpha cells is <0.2% of that in beta cells. The GLP-1-induced suppression of glucagon secretion is PKA dependent, is glucose independent, and does not involve paracrine effects mediated by insulin or somatostatin. GLP-1 is without much effect on alpha cell electrical activity but selectively inhibits N-type Ca(2+) channels and exocytosis. Adrenaline stimulates alpha cell electrical activity, increases [Ca(2+)](i), enhances L-type Ca(2+) channel activity, and accelerates exocytosis. The stimulatory effect is partially PKA independent and reduced in Epac2-deficient islets. We propose that GLP-1 inhibits glucagon secretion by PKA-dependent inhibition of the N-type Ca(2+) channels via a small increase in intracellular cAMP ([cAMP](i)). Adrenaline stimulates L-type Ca(2+) channel-dependent exocytosis by activation of the low-affinity cAMP sensor Epac2 via a large increase in [cAMP](i).

Interaction of ATP Sensor, cAMP Sensor, Ca2+ Sensor, and Voltage-dependent Ca2+ Channel in Insulin Granule Exocytosis

ATP, cAMP, and Ca2+ are the major signals in the regulation of insulin granule exocytosis in pancreatic β cells. The sensors and regulators of these signals have been characterized individually. The ATP-sensitive K+ channel, acting as the ATP sensor, couples cell metabolism to membrane potential. cAMP-GEFII, acting as a cAMP sensor, mediates cAMP-dependent, protein kinase A-independent exocytosis, which requires interaction with both Piccolo as a Ca2+ sensor and Rim2 as a Rab3 effector. l-type voltage-dependent Ca2+ channels (VDCCs) regulate Ca2+ influx. In the present study, we demonstrate interactions of these molecules. Sulfonylurea receptor 1, a subunit of ATP-sensitive K+ channels, interacts specifically with cAMP-GEFII through nucleotide-binding fold 1, and the interaction is decreased by a high concentration of cAMP. Localization of cAMP-GEFII overlaps with that of Rim2 in plasma membrane of insulin-secreting MIN6 cells. Localization of Rab3 co-incides with that of Rim2. Rim2 mutant lacking the Rab3 binding region, when overexpressed in MIN6 cells, is localized exclusively in cytoplasm, and impairs cAMP-dependent exocytosis in MIN6 cells. In addition, Rim2 and Piccolo bind directly to the α11.2-subunit of VDCC. These results indicate that ATP sensor, cAMP sensor, Ca2+ sensor, and VDCC interact with each other, which further suggests that ATP, cAMP, and Ca2+ signals in insulin granule exocytosis are integrated in a specialized domain of pancreatic β cells to facilitate stimulus-secretion coupling.

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.