Cell signalling in insulin secretion: the molecular targets of ATP, cAMP and sulfonylurea
Clarification of the molecular mechanisms of insulin secretion is crucial for understanding the pathogenesis and pathophysiology of diabetes and for development of novel therapeutic strategies for the disease. Insulin secretion is regulated by various intracellular signals generated by nutrients and hormonal and neural inputs. In addition, a variety of glucose-lowering drugs including sulfonylureas, glinide-derivatives, and incretin-related drugs such as dipeptidyl peptidase IV (DPP-4) inhibitors and glucagon-like peptide 1 (GLP-1) receptor agonists are used for glycaemic control by targeting beta cell signalling for improved insulin secretion. There has been a remarkable increase in our understanding of the basis of beta cell signalling over the past two decades following the application of molecular biology, gene technology, electrophysiology and bioimaging to beta cell research. This review discusses cell signalling in insulin secretion, focusing on the molecular targets of ATP, cAMP and sulfonylurea, an essential metabolic signal in glucose-induced insulin secretion (GIIS), a critical signal in the potentiation of GIIS, and the commonly used glucose-lowering drug, respectively.
KeywordsATP cAMP Epac Incretin Insulin secretion KATP channel Review Sulfonylurea
Intracellular calcium concentration
Dipeptidyl peptidase IV
Enhanced cyan fluorescent protein
Exchange protein activated by cAMP
Exocyst complex component 3-like
Enhanced yellow fluorescent protein
Fluorescence resonance energy transfer
Guanine nucleotide exchange factor
Green fluorescent protein
Glucose-induced insulin secretion
Glucose-dependent insulinotropic polypeptide
Glucagon-like peptide 1
- KATP channel
ATP-sensitive K+ channel
Persistent hyperinsulinaemic hypoglycaemia of infancy
Protein kinase A
Readily releasable pool
Total internal reflection fluorescence microscopy
Transient receptor potential
The blood glucose level is tightly controlled by insulin secretion from pancreatic beta cells and insulin action in target tissues such as liver, muscle and adipose tissue. Pancreatic beta cells secrete an appropriate amount of insulin in a process that is precisely regulated temporally to maintain glucose homeostasis. Insulin secretion is regulated by various factors, including nutrients and hormonal and neural inputs to the beta cells, among which glucose is the most important physiological regulator. Beta cell dysfunction impairs normal regulation of insulin secretion and leads to diabetes or hypoglycaemia. The mechanisms of insulin secretion have been studied extensively both in vivo and in vitro in the 50 years since the establishment of the radioimmunoassay for insulin . Our understanding of the mechanisms of insulin secretion was deepened but remained incomplete. By the early 1980s, the major intracellular signals in pancreatic beta cells for insulin secretion had been identified by pharmacological, physiological and biochemical methods. These include Ca2+, ATP, cAMP and phospholipid-derived molecules such as diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3) [2, 3, 4].
Dynamics of insulin secretion
Insulin secretion is a highly dynamic process. Glucose induces insulin secretion in a biphasic pattern: there is an initial component (first phase) that develops rapidly but lasts only a few minutes, and this is followed by a progressively increasing or sustained component (second phase) [14, 19, 20]. Loss of first phase secretion and reduced second phase secretion are characteristic features of type 2 diabetes. It is known that there is a decrease in the first phase of GIIS in the early stage of type 2 diabetes and in impaired glucose tolerance .
By analogy with the exocytosis of neurotransmitters in neurons , insulin granule exocytosis is thought to involve several steps, including recruitment, docking, priming and fusion . It has been suggested that secretory vesicles in pancreatic beta cells exist in functionally distinct pools and that the sequential release of these pools underlies the separable components in the dynamics of exocytosis . Pancreatic beta cells contain at least two pools of insulin secretory granules that differ in release competence: a reserve pool (RP) accounting for the vast majority of granules, and a readily releasable pool (RRP) accounting for the remaining <5%. A current hypothesis maintains that the first phase of GIIS is caused by release of RRP granules and that the second phase of GIIS represents a subsequent supply of new granules mobilised from the RP [14, 20, 24].
Investigation of insulin granule dynamics has recently been refined by use of the total internal reflection fluorescence microscopy (TIRFM) system [25, 26, 27, 28]. TIRF is a technology that provides a means of selectively exciting fluorophores in an aqueous or cellular environment very near a solid surface (within 100 nm) without exciting fluorescence from regions further from the surface . This unique feature of TIRFM analysis has has led to its application in various different areas of biochemistry and cell biology. A previous TIRFM study reported that insulin granule exocytosis occurs in two modes . In one mode (mode 1), fusion events are caused by granules that are predocked to the plasma membrane (referred to as ‘previously docked granules’ in  and ‘old face’ in ). In the other mode, fusion events are caused by granules that are newly recruited to the plasma membrane (‘newcomer’). Detailed analyses of insulin granule dynamics induced by various stimuli using primary cultured pancreatic beta cells show that ‘newcomer’ can be classified into two modes: one mode (mode 2), in which granules are newly recruited and immediately fused to the plasma membrane without docking (a docking state that can barely be detected by TIRFM) (‘restless newcomer’), and another mode (mode 3) in which granules are newly recruited, docked and then fused to the plasma membrane (‘resting newcomer’)  (Fig. 1b). The three modes of insulin granule exocytosis have been confirmed by other studies [28, 30]. Unlike the original model of GIIS, in which the first phase results from the RRP comprising predocked granules and the second phase from RP, a new model in which both phases of GIIS are caused by ‘restless newcomer’ has been proposed (Fig. 1c) .
In contrast, most K+-induced insulin granule exocytosis that occurs immediately and transiently after stimulation represents the release of predocked granules (‘old face’) [27, 32]. The dynamics of insulin granule exocytosis vary according to whether stimulation is due to K+ or glucose. As K+ stimulation elicits only Ca2+ influx and glucose stimulation generates various metabolic signals such as ATP in addition to Ca2+ influx in pancreatic beta cells, this difference in intracellular signal may underlie the distinct modes of exocytosis.
Various proteins associated with insulin granule exocytosis have been identified [23, 33], among which Rab-interacting molecule 2 (Rim2, Rim2α) was identified as a molecule interacting with exchange protein activated by cAMP (Epac) 2A (cAMP-GEFII) . In addition to Epac2A, Rim2α interacts with various exocytosis-related molecules, at least in vitro, including Rab3 , Munc13-1 , Rab8 , ELKS [37, 38], Piccolo , and synaptotagmin 1 . Although synaptotagmin 1 is produced in insulinoma cells, the synaptotagmin genes expressed in primary mouse beta cells are those encoding synaptotagmin 7  and 9 . Rim2α null (Rim2α−/−) mice exhibit a marked impairment in glucose tolerance . Analysis by TIRFM shows that both K+-induced insulin granule exocytosis and glucose-induced insulin granule exocytosis, especially the first phase, are severely impaired in pancreatic beta cells of Rim2α null mice . Rim2α has been found to determine the docking and priming states depending on interaction with Rab3 or Munc13-1, respectively.
The exocyst is an octameric protein complex that ensures spatial docking or tethering of exocytotic vesicles to fusion sites of the plasma membrane . Eight subunits of the exocyst complex are expressed in both pancreatic islets and MIN6 cells. Exocyst complex component 3-like (Exoc3l), an isoform of Sec6, the core subunit of the exocyst complex, was identified by in silico screening . Exoc3l forms tertiary complexes consisting of Sec5, Sec8 and Sec10, all of which are binding partners of Sec6. Exoc3l is suggested to be involved in the regulated exocytosis of insulin granules through formation of the exocyst complex.
The role of cAMP signalling in insulin granule exocytosis has also been investigated by TIRFM. The cAMP analogue 8-bromo-cAMP alone did not cause either significant docking or fusion events of insulin granules. However, 8-bromo-cAMP clearly enhanced the frequency of glucose-induced fusion events in both the first phase and the second phase. 8-bromo-cAMP promoted fusion events by increasing only ‘restless newcomer’. Comparison of the fusion sites induced by glucose stimulation and those induced by 8-bromo-cAMP stimulation showed that new fusion sites appeared upon 8-bromo-cAMP stimulation, suggesting that cAMP signaling also participates in the spatial regulation of insulin granule exocytosis .
The KATP channel as a target of ATP and sulfonylurea
Studies of various KATP genetically engineered mice [61, 62, 63, 64] clearly show the essential role of the KATP channel for GIIS and sulfonylurea-induced insulin secretion (Fig. 3c). Under certain conditions in Kir6.2 null mice [63, 65], GIIS was detected to some extent, indicating that the metabolic amplifying pathway contributes at least in part to GIIS. However, during stimulation with glucose alone, the metabolic amplifying pathway is ineffective as long as the triggering pathway is inoperative. The triggering and metabolic amplifying pathways in GIIS have recently been reviewed in detail . Mutations of the beta cell KATP channels cause neonatal diabetes or persistent hyperinsulinaemic hypoglycaemia of infancy (PHHI), depending on gain-of function (activating) mutation or loss-of-function mutation, respectively. The pathophysiology of PHHI and neonatal diabetes owing to mutations of Kir6.2 and SUR1 have been discussed in detail in recent review articles [66, 67, 68].
The ventromedial hypothalamus (VMH) has been shown to possess the highest density of glucose-responsive (GR) neurons, which play a critical role in glucose homeostasis and are involved in glucagon secretion during hypoglycaemia . The VMH KATP channel has been found to consist of Kir6.2 and SUR1 , which is identical to the beta cell KATP channel. Recovery from systemic hypoglycaemia induced by insulin injection was severely impaired in Kir6.2 null mice due to a marked reduction in glucagon secretion in vivo in these mice. Glucagon secretion in response to low glucose concentrations in isolated pancreatic islets from Kir6.2 null mice to that islets from wild-type mice, indicating a normal response of glucagon secretion from alpha cells . Administration of 2-deoxyglucose (2DG) into the intracerebroventricle, which is known to induce neuroglucopenia in the hypothalamus and to stimulate glucagon secretion through activation of autonomic neurons, produced an increase in glucagon secretion in normal mice but not in Kir6.2 null mice. Thus, the KATP channels in the VMH function as glucose sensors for glucagon secretion during hypoglycemia. Beta-cell and VMH KATP channels act in concert as peripheral and central glucose sensors in the maintenance of glucose homeostasis.
It was suggested that some sulfonylureas enhance glucose uptake in skeletal muscles [72, 73]. The glucose-lowering effect by insulin injection at a relatively low dose is significantly increased in Kir6.2 null mice compared with that in normal mice, suggesting that insulin sensitivity is enhanced in Kir6.2 null mice . In fact, glucose uptake in some skeletal muscles is increased in Kir6.2 null mice . The involvement of the KATP channels in glucose uptake in skeletal muscles also is indicated by a study of SUR2 null (SUR2−/−) mice . Thus, closure of the KATP channels in skeletal muscles can enhance glucose transport. KATP channels in pancreatic beta cells, hypothalamus and skeletal muscle are critically involved in the maintenance of glucose homeostasis .
Epac2A (cAMP-GEFII) as a target of cAMP
Since the discovery of cAMP as an intracellular second messenger, it has been shown to mediate a variety of cellular responses. Various hormones and neurotransmitters, including GLP-1 [77, 78, 79], GIP [77, 79], vasoactive intestinal polypeptide (VIP)  and pituitary adenylate cyclase-activating polypeptide (PACAP) , potentiate insulin secretion by promoting cAMP generation in pancreatic beta cells. Eight adenylyl cyclase isoforms (types I–VIII) are expressed in pancreatic islets and beta cell lines [81, 82]. In fact, MDL12330A, an adenylyl cyclase inhibitor, completely blocks both GLP-1- and GIP-induced cAMP production in islets and also markedly reduces both GLP-1- and GIP-potentiated insulin secretions .
Until recently, the action of cAMP in insulin secretion was thought to primarily be mediated by protein kinase A (PKA), which phosphorylates various proteins associated with the secretory process . Kir6.2, the pore-forming subunit of KATP channels, and the α-subunit of the voltage-dependent Ca2+ channel can be phosphorylated by PKA on stimulation in beta cell lines [85, 86]. GLUT2 can also be phosphorylated by GLP-1 in purified beta bcells . Although phosphorylation of these proteins influences their activities [85, 86, 87], a direct effect of phosphorylation on insulin secretion has not been established. We recently found that in MIN6 cells, Rip11, an effector of the small G-protein Rab11, participates in the potentiation of exocytosis by cAMP plus glucose stimulation but not in that of glucose stimulation alone . In addition, Rip11 was found to be phosphorylated by PKA in MIN6 cells. These findings indicate that Rip11, as a substrate of PKA, is involved in the regulation of insulin secretion potentiated by cAMP in pancreatic beta cells.
Studies of Epac2A null (Epac2a−/−) mice and Rap1 knockdown in clonal mouse beta cells indicate that Epac2A/Rap1 signalling is required for first phase potentiation of glucose-induced insulin granule exocytosis by cAMP . It has been proposed that activation of Epac2A/Rap1 signalling increases the size of the RRP and/or recruitment of insulin granules from the RRP, while PKA signalling increases the size of the RP and/or recruitment of insulin granules from the RP (Fig. 4c) . Rim2α has been found to be essential for Epac2A-mediated potentiation of GIIS by cAMP [43, 83]. Epac2A has also been shown to be involved in mobilisation of Ca2+ from intracellular Ca2+ stores in pancreatic beta cells . The effect of Epac2A on Ca2+ mobilisation has been shown to be mediated by ryanodine receptors and IP3 receptors by studying ryanodine receptor null mice and phospholipase Cε null mice, respectively .
cAMP signals are known to be compartmentalised in different regions of cardiac myocytes . Such cAMP compartmentalisation is thought to underlie the distinct biological responses mediated by the different cAMP-increasing ligands. By analogy with the effects of cAMP in cardiac myocytes, cAMP compartmentalisation has also been proposed in pancreatic beta cells .
Epac2A as a target of sulfonylurea
Sulfonylureas stimulate insulin secretion by closing KATP channels through binding to SUR1, as mentioned above. Although sulfonylureas were also suggested to act intracellularly to stimulate insulin granule exocytosis [100, 101, 102], the direct target was not identified.
This review was presented in partial form as the Albert Renold Prize Lecture at the European Association for the Study of Diabetes (EASD) 2010, Stockholm, Sweden. I thank present and former colleagues in our laboratory and our collaborators for their involvement in the studies in this review. I am especially grateful to T. Shibasaki, N. Yokoi, and K Minami for critical reading of the manuscript along with their helpful suggestions and G. H. Honkawa for her assistance in preparing the manuscript. The studies of our laboratory were supported by a CREST grant from the Japan Science and Technology Agency and Grant-in-Aid for Scientific Research.
The author was responsible for the conception, design and drafting of the manuscript, and approved the final version for publication.