Imeglimin mitigates the accumulation of dysfunctional mitochondria to restore insulin secretion and suppress apoptosis of pancreatic ß-cells from db/db mice.

Mitochondrial dysfunction in pancreatic ß-cells leads to impaired glucose-stimulated insulin secretion (GSIS) and type 2 diabetes (T2D), highlighting the importance of autophagic elimination of dysfunctional mitochondria (mitophagy) in mitochondrial quality control (mQC). Imeglimin, a new oral anti-diabetic drug that improves hyperglycemia and GSIS, may enhance mitochondrial activity. However, chronic imeglimin treatment's effects on mQC in diabetic ß-cells are unknown. Here, we compared imeglimin, structurally similar anti-diabetic drug metformin, and insulin for their effects on clearance of dysfunctional mitochondria through mitophagy in pancreatic ß-cells from diabetic model db/db mice and mitophagy reporter (CMMR) mice. Pancreatic islets from db/db mice showed aberrant accumulation of dysfunctional mitochondria and excessive production of reactive oxygen species (ROS) along with markedly elevated mitophagy, suggesting that the generation of dysfunctional mitochondria overwhelmed the mitophagic capacity in db/db ß-cells. Treatment with imeglimin or insulin, but not metformin, reduced ROS production and the numbers of dysfunctional mitochondria, and normalized mitophagic activity in db/db ß-cells. Concomitantly, imeglimin and insulin, but not metformin, restored the secreted insulin level and reduced ß-cell apoptosis in db/db mice. In conclusion, imeglimin mitigated accumulation of dysfunctional mitochondria through mitophagy in diabetic mice, and may contribute to preserving ß-cell function and effective glycemic control in T2D.

A new beta cell-specific mitophagy reporter mouse shows that metabolic stress leads to accumulation of dysfunctional mitochondria despite increased mitophagy.

AIMS/HYPOTHESIS: Mitophagy, the selective autophagy of mitochondria, is essential for maintenance of mitochondrial function. Recent studies suggested that defective mitophagy in beta cells caused diabetes. However, because of technical difficulties, the development of a convenient and reliable method to evaluate mitophagy in beta cells in vivo is needed. The aim of this study was to establish beta cell-specific mitophagy reporter mice and elucidate the role of mitophagy in beta cell function under metabolically stressed conditions induced by a high-fat diet (HFD). METHODS: Mitophagy was assessed using newly generated conditional mitochondrial matrix targeting mitophagy reporter (CMMR) mice, in which mitophagy can be visualised specifically in beta cells in vivo using a fluorescent probe sensitive to lysosomal pH and degradation. Metabolic stress was induced in mice by exposure to the HFD for 20 weeks. The accumulation of dysfunctional mitochondria was examined by staining for functional/total mitochondria and reactive oxygen species (ROS) using specific fluorescent dyes and antibodies. To investigate the molecular mechanism underlying mitophagy in beta cells, overexpression and knockdown experiments were performed. HFD-fed mice were examined to determine whether chronic insulin treatment for 6 weeks could ameliorate mitophagy, mitochondrial function and impaired insulin secretion. RESULTS: Exposure to the HFD increased the number of enlarged (HFD-G) islets with markedly elevated mitophagy. Mechanistically, HFD feeding induced severe hypoxia in HFD-G islets, which upregulated mitophagy through the hypoxia-inducible factor 1-ɑ (Hif-1ɑ)/BCL2 interacting protein 3 (BNIP3) axis in beta cells. However, HFD-G islets unexpectedly showed the accumulation of dysfunctional mitochondria due to excessive ROS production, suggesting an insufficient capacity of mitophagy for the degradation of dysfunctional mitochondria. Chronic administration of insulin ameliorated hypoxia and reduced ROS production and dysfunctional mitochondria, leading to decreased mitophagy and restored insulin secretion. CONCLUSIONS/INTERPRETATION: We demonstrated that CMMR mice enabled the evaluation of mitophagy in beta cells. Our results suggested that metabolic stress induced by the HFD caused the aberrant accumulation of dysfunctional mitochondria, which overwhelmed the mitophagic capacity and was associated with defective maintenance of mitochondrial function and impaired insulin secretion.

Role of the active zone protein, ELKS, in insulin secretion from pancreatic ß-cells.

BACKGROUND: Insulin is stored within large dense-core granules in pancreatic beta (ß)-cells and is released by Ca2+-triggered exocytosis with increasing blood glucose levels. Polarized and targeted secretion of insulin from ß-cells in pancreatic islets into the vasculature has been proposed; however, the mechanisms related to cellular and molecular localization remain largely unknown. Within nerve terminals, the Ca2+-dependent release of a polarized transmitter is limited to the active zone, a highly specialized area of the presynaptic membrane. Several active zone-specific proteins have been characterized; among them, the CAST/ELKS protein family members have the ability to form large protein complexes with other active zone proteins to control the structure and function of the active zone for tight regulation of neurotransmitter release. Notably, ELKS but not CAST is also expressed in ß-cells, implying that ELKS may be involved in polarized insulin secretion from ß-cells. SCOPE OF REVIEW: This review provides an overview of the current findings regarding the role(s) of ELKS and other active zone proteins in ß-cells and focuses on the molecular mechanism underlying ELKS regulation within polarized insulin secretion from islets. MAJOR CONCLUSIONS: ELKS localizes at the vascular-facing plasma membrane of ß-cells in mouse pancreatic islets. ELKS forms a potent insulin secretion complex with L-type voltage-dependent Ca2+ channels on the vascular-facing plasma membrane of ß-cells, enabling polarized Ca2+ influx and first-phase insulin secretion from islets. This model provides novel insights into the functional polarity observed during insulin secretion from ß-cells within islets at the molecular level. This active zone-like region formed by ELKS at the vascular side of the plasma membrane is essential for coordinating physiological insulin secretion and may be disrupted in diabetes.

ELKS/Voltage-Dependent Ca2+ Channel-ß Subunit Module Regulates Polarized Ca2+ Influx in Pancreatic ß Cells.

Pancreatic ß cells secrete insulin by Ca2+-triggered exocytosis. However, there is no apparent secretory site similar to the neuronal active zones, and the cellular and molecular localization mechanism underlying polarized exocytosis remains elusive. Here, we report that ELKS, a vertebrate active zone protein, is used in ß cells to regulate Ca2+ influx for insulin secretion. ß cell-specific ELKS-knockout (KO) mice showed impaired glucose-stimulated first-phase insulin secretion and reduced L-type voltage-dependent Ca2+ channel (VDCC) current density. In situ Ca2+ imaging of ß cells within islets expressing a membrane-bound G-CaMP8b Ca2+ sensor demonstrated initial local Ca2+ signals at the ELKS-localized vascular side of the ß cell plasma membrane, which were markedly decreased in ELKS-KO ß cells. Mechanistically, ELKS directly interacted with the VDCC-ß subunit via the GK domain. These findings suggest that ELKS and VDCCs form a potent insulin secretion complex at the vascular side of the ß cell plasma membrane for polarized Ca2+ influx and first-phase insulin secretion from pancreatic islets.

VAMP7 Regulates Autophagosome Formation by Supporting Atg9a Functions in Pancreatic ß-Cells From Male Mice.

Dysfunctional mitochondria are observed in ß-cells of diabetic patients, which are eventually removed by autophagy. Vesicle-associated membrane protein (VAMP)7, a vesicular SNARE protein, regulates autophagosome formation to maintain mitochondrial homeostasis and control insulin secretion in pancreatic ß-cells. However, its molecular mechanism is largely unknown. In this study, we investigated the molecular mechanism of VAMP7-dependent autophagosome formation using VAMP7-deficient ß-cells and ß-cell-derived Min6 cells. VAMP7 localized in autophagy-related (Atg)9a-resident vesicles of recycling endosomes (REs), which contributed to autophagosome formation, and it interacted with Hrb, Syntaxin16, and SNAP-47. Hrb recruited VAMP7 and Atg9a from the plasma membrane to REs. Syntaxin16 and SNAP-47 mediated autophagosome formation at a step later than the proper localization of VAMP7 to Atg9a-resident vesicles. Knockdown of Hrb, Syntaxin16, and SNAP-47 resulted in defective autophagosome formation, accumulation of dysfunctional mitochondria, and impairment of glucose-stimulated insulin secretion. Our data indicate that VAMP7 and Atg9a are initially recruited to REs to organize VAMP7 and Atg9a-resident vesicles in an Hrb-dependent manner. Additionally, VAMP7 forms a SNARE complex with Syntaxin16 and SNAP-47, which may cause fusions of Atg9a-resident vesicles during autophagosome formation. Thus, VAMP7 participates in autophagosome formation by supporting Atg9a functions that contribute to maintenance of mitochondrial quality.

In vivo regulation of glycogen synthase kinase 3ß activity in neurons and brains.

Glycogen synthase kinase 3ß (GSK3ß) is a multifunctional protein kinase involved in many cellular activities including development, differentiation and diseases. GSK3ß is thought to be constitutively activated by autophosphorylation at Tyr216 and inactivated by phosphorylation at Ser9. The GSK3ß activity has previously been evaluated by inhibitory Ser9 phosphorylation, but it does not necessarily indicate the kinase activity itself. Here, we applied the Phos-tag SDS-PAGE technique to the analysis of GSK3ß phosphoisotypes in cells and brains. There were three phosphoisotypes of GSK3ß; double phosphorylation at Ser9 and Tyr216, single phosphorylation at Tyr216 and the nonphosphorylated isotype. Active GSK3ß with phosphorylation at Tyr216 represented half or more of the total GSK3ß in cultured cells. Although levels of phospho-Ser9 were increased by insulin treatment, Ser9 phosphorylation occurred only in a minor fraction of GSK3ß. In mouse brains, GSK3ß was principally in the active form with little Ser9 phosphorylation, and the phosphoisotypes of GSK3ß changed depending on the regions of the brain, age, sex and disease conditions. These results indicate that the Phos-tag SDS-PAGE method provides a simple and appropriate measurement of active GSK3ß in vivo, and the activity is regulated by the mechanism other than phosphorylation on Ser9.

Opposing roles for SNAP23 in secretion in exocrine and endocrine pancreatic cells.

The membrane fusion of secretory granules with plasma membranes is crucial for the exocytosis of hormones and enzymes. Secretion disorders can cause various diseases such as diabetes or pancreatitis. Synaptosomal-associated protein 23 (SNAP23), a soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor (SNARE) molecule, is essential for secretory granule fusion in several cell lines. However, the in vivo functions of SNAP23 in endocrine and exocrine tissues remain unclear. In this study, we show opposing roles for SNAP23 in secretion in pancreatic exocrine and endocrine cells. The loss of SNAP23 in the exocrine and endocrine pancreas resulted in decreased and increased fusion of granules to the plasma membrane after stimulation, respectively. Furthermore, we identified a low molecular weight compound, MF286, that binds specifically to SNAP23 and promotes insulin secretion in mice. Our results demonstrate opposing roles for SNAP23 in the secretion mechanisms of the endocrine and exocrine pancreas and reveal that the SNAP23-binding compound MF286 may be a promising drug for diabetes treatment.

VAMP7 Regulates Autophagy to Maintain Mitochondrial Homeostasis and to Control Insulin Secretion in Pancreatic ß-Cells.

VAMP7 is a SNARE protein that mediates specific membrane fusions in intracellular trafficking and was recently reported to regulate autophagosome formation. However, its function in pancreatic ß-cells is largely unknown. To elucidate the physiological role of VAMP7 in ß-cells, we generated pancreatic ß-cell-specific VAMP7 knockout (Vamp7(flox/Y);Cre) mice. VAMP7 deletion impaired glucose-stimulated ATP production and insulin secretion, though VAMP7 was not localized to insulin granules. VAMP7-deficient ß-cells showed defective autophagosome formation and reduced mitochondrial function. p62/SQSTM1, a marker protein for defective autophagy, was selectively accumulated on mitochondria in VAMP7-deficient ß-cells. These findings suggest that accumulation of dysfunctional mitochondria that are degraded by autophagy caused impairment of glucose-stimulated ATP production and insulin secretion in Vamp7(flox/Y);Cre ß-cells. Feeding a high-fat diet to Vamp7(flox/Y);Cre mice exacerbated mitochondrial dysfunction, further decreased ATP production and insulin secretion, and consequently induced glucose intolerance. Moreover, we found upregulated VAMP7 expression in wild-type mice fed a high-fat diet and in db/db mice, a model for diabetes. Thus our data indicate that VAMP7 regulates autophagy to maintain mitochondrial quality and insulin secretion in response to pathological stress in ß-cells.

A Gain-of-Function Mutation in NALCN in a Child with Intellectual Disability, Ataxia, and Arthrogryposis.

NALCN and its homologues code for the ion channel responsible for half of background Na(+) -leak conductance in vertebrate and invertebrate neurons. Recessive mutations in human NALCN cause intellectual disability (ID) with hypotonia. Here, we report a de novo heterozygous mutation in NALCN affecting a conserved residue (p.R1181Q) in a girl with ID, episodic and persistent ataxia, and arthrogryposis. Interestingly, her episodes of ataxia were abolished by the administration of acetazolamide, similar to the response observed in episodic ataxia associated with other ion channels. Introducing the analogous mutation in the Caenorhabditis elegans homologue nca-1 induced a coiling locomotion phenotype, identical to that obtained with previously characterized C. elegans gain-of-function nca alleles, suggesting that p.R1181Q confers the same property to NALCN. This observation thus suggests that dominant mutations in NALCN can cause a neurodevelopmental phenotype that overlaps with, while being mostly distinct from that associated with recessive mutations in the same gene.

Serotonin regulates glucose-stimulated insulin secretion from pancreatic ß cells during pregnancy.

In preparation for the metabolic demands of pregnancy, ß cells in the maternal pancreatic islets increase both in number and in glucose-stimulated insulin secretion (GSIS) per cell. Mechanisms have been proposed for the increased ß cell mass, but not for the increased GSIS. Because serotonin production increases dramatically during pregnancy, we tested whether flux through the ionotropic 5-HT3 receptor (Htr3) affects GSIS during pregnancy. Pregnant Htr3a(-/-) mice exhibited impaired glucose tolerance despite normally increased ß cell mass, and their islets lacked the increase in GSIS seen in islets from pregnant wild-type mice. Electrophysiological studies showed that activation of Htr3 decreased the resting membrane potential in ß cells, which increased Ca(2+) uptake and insulin exocytosis in response to glucose. Thus, our data indicate that serotonin, acting in a paracrine/autocrine manner through Htr3, lowers the ß cell threshold for glucose and plays an essential role in the increased GSIS of pregnancy.

Calmodulin-dependent regulation of neurotransmitter release differs in subsets of neuronal cells.

The purpose of this study was to determine whether calmodulin (CaM) plays a role in neurotransmitter release by examining the effect that ophiobolin A (OBA), a CaM antagonist, on neurotransmitter release from clonal rat pheochromocytoma PC12 cells, primary cortical neurons, and primary cerebellar granule cells. OBA inhibited Ca²âº/CaM-dependent phosphorylation of cAMP response element binding protein in all cell types tested. Moreover, Ca²âº-dependent release of dopamine and acetylcholine from PC12 cells were remarkably reduced by OBA in a dose-dependent and temporal manner, but neurotransmitter release partially recovered with the addition of CaM in membrane permeabilized PC12 cells. OBA and several synthetic CaM antagonists suppressed Ca²âº-dependent glutamate release from cerebral cortical neurons, but not from cerebellar granule cells. Myosin Va, a CaM binding protein, localized to synaptic vesicles of PC12 cells and cerebral cortical neurons, but not in cerebellar granule cells. OBA suppressed Ca²âº-induced myosin Va dissociation from secretory vesicles, and inhibited secretory vesicle motility in PC12 cells. These results suggest that CaM, although not essential, regulates neurotransmitter release in a subset of neurons and secretory cells, and myosin Va is a possible target of OBA in this process.

NLF-1 delivers a sodium leak channel to regulate neuronal excitability and modulate rhythmic locomotion.

A cation channel NCA/UNC-79/UNC-80 affects neuronal activity. We report here the identification of a conserved endoplasmic reticulum protein NLF-1 (NCA localization factor-1) that regulates neuronal excitability and locomotion through the NCA channel. In C. elegans, the loss of either NLF-1 or NCA leads to a reduced sodium leak current, and a hyperpolarized resting membrane potential in premotor interneurons. This results in a decreased premotor interneuron activity that reduces the initiation and sustainability of rhythmic locomotion. NLF-1 promotes axonal localization of all NCA reporters. Its mouse homolog mNLF-1 functionally substitutes for NLF-1 in C. elegans, interacts with the mammalian sodium leak channel NALCN in vitro, and potentiates sodium leak currents in primary cortical neuron cultures. Taken together, an ER protein NLF-1 delivers a sodium leak channel to maintain neuronal excitability and potentiates a premotor interneuron network critical for C. elegans rhythmic locomotion.

Acute inhibition of PI3K-PDK1-Akt pathway potentiates insulin secretion through upregulation of newcomer granule fusions in pancreatic ß-cells.

In glucose-induced insulin secretion from pancreatic ß-cells, a population of insulin granules fuses with the plasma membrane without the typical docking process (newcomer granule fusions), however, its mechanism is unclear. In this study, we investigated the PI3K signaling pathways involved in the upregulation of newcomer granule fusions. Acute treatment with the class IA-selective PI3K inhibitors, PIK-75 and PI-103, enhanced the glucose-induced insulin secretion. Total internal reflection fluorescent microscopy revealed that the PI3K inhibitors increased the fusion events from newcomer granules. We developed a new system for transfection into pancreatic islets and demonstrated the usefulness of this system in order for evaluating the effect of transfected genes on the glucose-induced secretion in primary cultured pancreatic islets. Using this transfection system together with a series of constitutive active mutants, we showed that the PI3K-3-phosphoinositide dependent kinase-1 (PDK1)-Akt pathway mediated the potentiation of insulin secretion. The Akt inhibitor also enhanced the glucose-induced insulin secretion in parallel with the upregulation of newcomer granule fusions, probably via increased motility of intracellular insulin granules. These data suggest that the PI3K-PDK1-Akt pathway plays a significant role in newcomer granule fusions, probably through an alteration of the dynamics of the intracellular insulin granules.

DPP-4 inhibitor des-F-sitagliptin treatment increased insulin exocytosis from db/db mice ß cells.

Incretin promotes insulin secretion acutely. Recently, orally-administered DPP-4 inhibitors represent a new class of anti-hyperglycemic agents. Indeed, inhibitors of dipeptidyl peptidase-IV (DPP-4), sitagliptin, has just begun to be widely used as therapeutics for type 2 diabetes. However, the effects of sitagliptin-treatment on insulin exocytosis from single ß-cells are yet unknown. We therefore investigated how sitagliptin-treatment in db/db mice affects insulin exocytosis by treating db/db mice with des-F-sitagliptin for 2 weeks. Perfusion studies showed that 2 weeks-sitagliptin treatment potentiated insulin secretion. We then analyzed insulin granule motion and SNARE protein, syntaxin 1, by TIRF imaging system. TIRF imaging of insulin exocytosis showed the increased number of docked insulin granules and increased fusion events from them during first-phase release. In accord with insulin exocytosis data, des-F-sitagliptin-treatment increased the number of syntaxin 1 clusters on the plasma membrane. Thus, our data demonstrated that 2-weeks des-F-sitagliptin-treatment increased the fusion events of insulin granules, probably via increased number of docked insulin granules and that of syntaxin 1 clusters.

Regulation of resident and newcomer insulin granules by calcium and SNARE proteins.

Insulin, stored in large dense core granules, is biphasically exocytosed by glucose stimulation in pancreatic beta-cells. Several molecules, such as SNARE proteins, and Ca2+ ion are involved in the regulation of insulin exocytosis. Indeed, studies using gene targeting mice revealed critical roles of SNARE proteins and their accessory proteins, which may be associated with diabetes mellitus. In particular, the total internal reflection fluorescent (TIRF) imaging technique shed new light on the molecular mechanism of the insulin exocytotic process. In this review we discuss the mechanism of insulin exocytosis mainly from a point of view of imaging techniques.

PKC-dependent inhibition of CA2+-dependent exocytosis from astrocytes.

Astrocytes release various bioactive substances via Ca(2+) - and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent exocytosis; however the regulatory mechanisms of glial exocytosis are still poorly understood. In the present study, we investigated the effect of protein kinase C (PKC) on exocytosis in glial cells using primary cultured astrocytes and clonal rat glioma C6 cells. Mass spectrometry and Western blot analysis using phospho-specific antibodies revealed that phorbol 12-myristate 13-acetate (PMA) treatment induced the phosphorylation of synaptosomal-associated protein of 23 kDa (SNAP-23) on Ser(95), Ser(120), and Ser(160) in cultured astrocytes and C6 cells. Phosphorylation at these sites was suppressed by treatment with the PKC inhibitor, bisindolylmaleimide I (BIS). In contrast, Ser(110) of SNAP-23 was constitutively phosphorylated in these cells and was dephosphorylated in a PKC-dependent manner. Exogenously expressed human growth hormone (hGH) accumulated in cytoplasmic granular structures in cultured astrocytes, and its release after ATP-treatment was Ca(2+) - and SNARE-dependent. PMA treatment suppressed the ATP-induced hGH release from astrocytes and this inhibition was reversed by BIS. We also observed PMA-dependent suppression and an attenuation of that suppression by BIS in ionomycin-induced hGH release from C6 cells. These results suggest that intracellular activation of PKC suppresses Ca(2+) - and SNARE-dependent exocytosis in astroglial cells.

Deletion of CDKAL1 affects mitochondrial ATP generation and first-phase insulin exocytosis.

BACKGROUND: A variant of the CDKAL1 gene was reported to be associated with type 2 diabetes and reduced insulin release in humans; however, the role of CDKAL1 in ß cells is largely unknown. Therefore, to determine the role of CDKAL1 in insulin release from ß cells, we studied insulin release profiles in CDKAL1 gene knockout (CDKAL1 KO) mice. PRINCIPAL FINDINGS: Total internal reflection fluorescence imaging of CDKAL1 KO ß cells showed that the number of fusion events during first-phase insulin release was reduced. However, there was no significant difference in the number of fusion events during second-phase release or high K(+)-induced release between WT and KO cells. CDKAL1 deletion resulted in a delayed and slow increase in cytosolic free Ca(2+) concentration during high glucose stimulation. Patch-clamp experiments revealed that the responsiveness of ATP-sensitive K(+) (K(ATP)) channels to glucose was blunted in KO cells. In addition, glucose-induced ATP generation was impaired. Although CDKAL1 is homologous to cyclin-dependent kinase 5 (CDK5) regulatory subunit-associated protein 1, there was no difference in the kinase activity of CDK5 between WT and CDKAL1 KO islets. CONCLUSIONS/SIGNIFICANCE: We provide the first report describing the function of CDKAL1 in ß cells. Our results indicate that CDKAL1 controls first-phase insulin exocytosis in ß cells by facilitating ATP generation, K(ATP) channel responsiveness and the subsequent activity of Ca(2+) channels through pathways other than CDK5-mediated regulation.

Insulin/phosphoinositide 3-kinase pathway accelerates the glucose-induced first-phase insulin secretion through TrpV2 recruitment in pancreatic ß-cells.

Functional insulin receptor and its downstream effector PI3K (phosphoinositide 3-kinase) have been identified in pancreatic ß-cells, but their involvement in the regulation of insulin secretion from ß-cells remains unclear. In the present study, we investigated the physiological role of insulin and PI3K in glucose-induced biphasic insulin exocytosis in primary cultured ß-cells and insulinoma Min6 cells using total internal reflection fluorescent microscopy. The pretreatment of ß-cells with insulin induced the rapid increase in intracellular Ca2+ levels and accelerated the exocytotic response without affecting the second-phase insulin secretion. The inhibition of PI3K not only abolished the insulin-induced rapid development of the exocytotic response, but also potentiated the second-phase insulin secretion. The rapid development of Ca2+ and accelerated exocytotic response induced by insulin were accompanied by the translocation of the Ca2+-permeable channel TrpV2 (transient receptor potential V2) in a PI3K-dependent manner. Inhibition of TrpV2 by the selective blocker tranilast, or the expression of shRNA (short-hairpin RNA) against TrpV2 suppressed the effect of insulin in the first phase, but the second phase was not affected. Thus our results demonstrate that insulin treatment induced the acceleration of the exocytotic response during the glucose-induced first-phase response by the insertion of TrpV2 into the plasma membrane in a PI3K-dependent manner.

Imaging exocytosis of single glucagon-like peptide-1 containing granules in a murine enteroendocrine cell line with total internal reflection fluorescent microscopy.

To analyze the exocytosis of glucagon-like peptide-1 (GLP-1) granules, we imaged the motion of GLP-1 granules labeled with enhanced yellow fluorescent protein (Venus) fused to human growth hormone (hGH-Venus) in an enteroendocrine cell line, STC-1 cells, by total internal reflection fluorescent (TIRF) microscopy. We found glucose stimulation caused biphasic GLP-1 granule exocytosis: during the first phase, fusion events occurred from two types of granules (previously docked granules and newcomers), and thereafter continuous fusion was observed mostly from newcomers during the second phase. Closely similar to the insulin granule fusion from pancreatic beta cells, the regulated biphasic exocytosis from two types of granules may be a common mechanism in glucose-evoked hormone release from endocrine cells.