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.

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.

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.

Imaging analysis reveals mechanistic differences between first- and second-phase insulin exocytosis.

The mechanism of glucose-induced biphasic insulin release is unknown. We used total internal reflection fluorescence (TIRF) imaging analysis to reveal the process of first- and second-phase insulin exocytosis in pancreatic beta cells. This analysis showed that previously docked insulin granules fused at the site of syntaxin (Synt)1A clusters during the first phase; however, the newcomers fused during the second phase external to the Synt1A clusters. To reveal the function of Synt1A in phasic insulin exocytosis, we generated Synt1A-knockout (Synt1A(-/-)) mice. Synt1A(-/-) beta cells showed fewer previously docked granules with no fusion during the first phase; second-phase fusion from newcomers was preserved. Rescue experiments restoring Synt1A expression demonstrated restoration of granule docking status and fusion events. Inhibition of other syntaxins, Synt3 and Synt4, did not affect second-phase insulin exocytosis. We conclude that the first phase is Synt1A dependent but the second phase is not. This indicates that the two phases of insulin exocytosis differ spatially and mechanistically.

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.

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.

Characterization of the taste receptor-related G-protein, α-gustducin, in pancreatic ß-cells.

AIMS/INTRODUCTION: Taste receptors, T1rs and T2rs, and the taste-selective G-protein, α-gustducin, are expressed outside the taste-sensing system, such as enteroendocrine L cells. Here, we examined whether α-gustducin also affects nutrition sensing and insulin secretion by pancreatic ß-cells. MATERIALS AND METHODS: The expression of α-gustducin and taste receptors was evaluated in ß-cell lines, and in rat and mouse islets either by quantitative polymerase chain reaction or fluorescence immunostaining. The effects of α-gustducin knockdown on insulin secretion and on cyclic adenosine monophosphate and intracellular Ca2+ levels in rat INS-1 cells were estimated. Sucralose (taste receptor agonist)-induced insulin secretion was investigated in INS-1 cells with α-gustducin suppression and in islets from mouse disease models. RESULTS: The expression of Tas1r3 and α-gustducin was confirmed in ß-cell lines and pancreatic islets. Basal levels of cyclic adenosine monophosphate, intracellular calcium and insulin secretion were significantly enhanced with α-gustducin knockdown in INS-1 cells. The expression of α-gustducin was decreased in high-fat diet-fed mice and in diabetic db/db mice. Sucralose-induced insulin secretion was not attenuated in INS-1 cells with α-gustducin knockdown or in mouse islets with decreased expression of α-gustducin. CONCLUSIONS: α-Gustducin is involved in the regulation of cyclic adenosine monophosphate, intracellular calcium levels and insulin secretion in pancreatic ß-cells in a manner independent of taste receptor signaling. α-Gustducin might play a novel role in ß-cell physiology and the development of type 2 diabetes.

Pattern of rise in subplasma membrane Ca2+ concentration determines type of fusing insulin granules in pancreatic beta cells.

We simultaneously analyzed insulin granule fusion with insulin fused to green fluorescent protein and the subplasma membrane Ca2+ concentration ([Ca2+](PM)) with the Ca2+ indicator Fura Red in rat beta cells by dual-color total internal reflection fluorescence microscopy. We found that rapid and marked elevation in [Ca2+](PM) caused insulin granule fusion mostly from previously docked granules during the high KCl-evoked release and high glucose-evoked first phase release. In contrast, the slow and sustained elevation in [Ca2+](PM) induced fusion from newcomers translocated from the internal pool during the low KCl-evoked release and glucose-evoked second phase release. These data suggest that the pattern of the [Ca2+](PM) rise directly determines the types of fusing granules.

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.

Insulin exocytosis in Goto-Kakizaki rat beta-cells subjected to long-term glinide or sulfonylurea treatment.

Sulfonylurea and glinide drugs display different effects on insulin granule motion in single beta-cells in vitro. We therefore investigated the different effects that these drugs manifest towards insulin release in an in vivo long-term treatment model. Diabetic GK (Goto-Kakizaki) rats were treated with nateglinide, glibenclamide or insulin for 6 weeks. Insulin granule motion in single beta-cells and the expression of SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) proteins were then analysed. Perifusion studies showed that decreased first-phase insulin release was partially recovered when GK rats were treated with nateglinide or insulin for 6 weeks, whereas no first-phase release occurred with glibenclamide treatment. In accord with the perifusion results, TIRF (total internal reflection fluorescence) imaging of insulin exocytosis showed restoration of the decreased number of docked insulin granules and the fusion events from them during first-phase release for nateglinide or insulin, but not glibenclamide, treatment; electron microscopy results confirmed the TIRF microscopy data. Relative to vehicle-treated GK beta-cells, an increased number of SNARE clusters were evident in nateglinide- or insulin-treated cells; a lesser increase was observed in glibenclamide-treated cells. Immunostaining for insulin showed that nateglinide treatment better preserved pancreatic islet morphology than did glibenclamide treatment. However, direct exposure of GK beta-cells to these drugs could not restore the decreased first-phase insulin release nor the reduced numbers of docked insulin granules. We conclude that treatment of GK rats with nateglinide and glibenclamide varies in long-term effects on beta-cell functions; nateglinide treatment appears overall to be more beneficial.

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.

TDP-43 regulates early-phase insulin secretion via CaV1.2-mediated exocytosis in islets.

TAR DNA-binding protein 43 kDa (TDP-43), encoded by TARDBP, is an RNA-binding protein, the nuclear depletion of which is the histopathological hallmark of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder affecting both upper and lower motor neurons. Besides motor symptoms, patients with ALS often develop nonneuronal signs including glucose intolerance, but the underlying pathomechanism is still controversial, i.e., whether it is impaired insulin secretion and/or insulin resistance. Here, we showed that ALS subjects reduced early-phase insulin secretion and that the nuclear localization of TDP-43 was lost in the islets of autopsied ALS pancreas. Loss of TDP-43 inhibited exocytosis by downregulating CaV1.2 calcium channels, thereby reducing early-phase insulin secretion in a cultured ß cell line (MIN6) and ß cell-specific Tardbp knockout mice. Overexpression of CaV1.2 restored early-phase insulin secretion in Tardbp knocked-down MIN6 cells. Our findings suggest that TDP-43 regulates cellular exocytosis mediated by L-type voltage-dependent calcium channels and thus plays an important role in the early phase of insulin secretion by pancreatic islets. Thus, nuclear loss of TDP-43 is implicated in not only the selective loss of motor neurons but also in glucose intolerance due to impaired insulin secretion at an early stage of ALS.

Rab27a mediates the tight docking of insulin granules onto the plasma membrane during glucose stimulation.

The monomeric small GTPase Rab27a is specifically localized on both secretory granules and lysosome-related organelles. Although natural mutations of the Rab27a gene in human Griscelli syndrome and in ashen mice cause partial albinism and immunodeficiency reflecting the dysfunction of lysosome-related organelles, phenotypes resulting from the defective exocytosis of secretory granules have not been reported. To explore the roles of Rab27a in secretory granules, we analyzed insulin secretion profiles in ashen mice. Ashen mice showed glucose intolerance after a glucose load without signs of insulin resistance in peripheral tissues or insulin deficiency in the pancreas. Insulin secretion from isolated islets was decreased specifically in response to high glucose concentrations but not other nonphysiological secretagogues such as high K+ concentrations, forskolin, or phorbol ester. Neither the intracellular Ca2+ concentration nor the dynamics of fusion pore opening after glucose stimulation were altered. There were, however, marked reductions in the exocytosis from insulin granules predocked on the plasma membrane and in the replenishment of docked granules during glucose stimulation. These results provide the first genetic evidence to our knowledge for the role of Rab27a in the exocytosis of secretory granules and suggest that the Rab27a/effector system mediates glucose-specific signals for the exocytosis of insulin granules in pancreatic beta cells.

Imaging exocytosis of single insulin secretory granules with TIRF microscopy.

The total internal reflection fluorescence (TIRF) technique is a powerful tool for visualizing the process of insulin exocytosis. This technique allows us to observe the events just beneath the plasma membrane, allowing us to explore the molecular mechanism of insulin exocytotic processes such as docking and fusion. In this review, we describe the method of TIRF for visualizing insulin exocytosis in pancreatic beta-cells.

ELKS, a protein structurally related to the active zone-associated protein CAST, is expressed in pancreatic beta cells and functions in insulin exocytosis: interaction of ELKS with exocytotic machinery analyzed by total internal reflection fluorescence microscopy.

The cytomatrix at the active zone (CAZ) has been implicated in defining the site of Ca2+-dependent exocytosis of neurotransmitters. Here, we demonstrate the expression and function of ELKS, a protein structurally related to the CAZ protein CAST, in insulin exocytosis. The results of confocal and immunoelectron microscopic analysis showed that ELKS is present in pancreatic beta cells and is localized close to insulin granules docked on the plasma membrane-facing blood vessels. Total internal reflection fluorescence microscopy imaging in insulin-producing clonal cells revealed that the ELKS clusters are less dense and unevenly distributed than syntaxin 1 clusters, which are enriched in the plasma membrane. Most of the ELKS clusters were on the docking sites of insulin granules that were colocalized with syntaxin 1 clusters. Total internal reflection fluorescence images of single-granule motion showed that the fusion events of insulin granules mostly occurred on the ELKS cluster, where repeated fusion was sometimes observed. When the Bassoon-binding region of ELKS was introduced into the cells, the docking and fusion of insulin granules were markedly reduced. Moreover, attenuation of ELKS expression by small interfering RNA reduced the glucose-evoked insulin release. These data suggest that the CAZ-related protein ELKS functions in insulin exocytosis from pancreatic beta cells.

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.

Imaging docking and fusion of insulin granules induced by antidiabetes agents: sulfonylurea and glinide drugs preferentially mediate the fusion of newcomer, but not previously docked, insulin granules.

Sulfonylurea and glinide drugs, commonly used for antidiabetes therapies, are known to stimulate insulin release from pancreatic beta-cells by closing ATP-sensitive K+ channels. However, the specific actions of these drugs on insulin granule motion are largely unknown. Here, we used total internal reflection fluorescence (TIRF) microscopy to analyze the docking and fusion of single insulin granules in live beta-cells exposed to either the sulfonylurea drug glibenclamide or the glinide drug mitiglinide. TIRF images showed that both agents caused rapid fusion of newcomer insulin granules with the cell membrane in both control and diabetic Goto-Kakizaki (GK) rat pancreatic beta-cells. However, in the context of beta-cells from sulfonylurea receptor 1 (SUR1) knockout mice, TIRF images showed that only mitiglinide, but not glibenclamide, caused fusion of newcomer insulin granules. Compositely, our data indicate that 1) the mechanism by which both sulfonylurea and glinide drugs promote insulin release entails the preferential fusion of newcomer, rather than previously docked, insulin granules, and that 2) mitiglinide can induce insulin release by a mechanism independent of mitiglinide binding to SUR1.

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.

Insulin exocytotic mechanism by imaging technique.

Insulin is stored in pancreatic beta cell granules, and released biphasically by the exocytotic mechanism induced by nutrient glucose. Insulin exocytosis must be critically regulated to finely control body glucose homeostasis because insulin is the only hormone that can promptly reduce the blood glucose level. Recent advanced techniques in molecular biology and electrophysiology revealed the molecular mechanism of insulin release in the process from glucose entry to increased [Ca(2+)](i). However, the insulin exocytotic process such as translocation, docking and fusion of insulin granules was largely unknown. In order to reveal the molecular mechanism of this process, we utilized a newly innovated imaging technique, TIRF imaging system. Here we review recent results of our studies into docking and fusion of insulin granules analyzed by TIRF system.