Yoko Nakamichi

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 β 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−/− β 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.

Expression and functional role of syntaxin 1/HPC-1 in pancreatic beta cells. Syntaxin 1A, but not 1B, plays a negative role in regulatory insulin release pathway.

Syntaxin 1/HPC-1 is an integral membrane protein, which is thought to be implicated in the regulation of synaptic neurotransmitter release. We investigated syntaxin 1 expression in pancreatic β cells and the functional role of syntaxin 1 in the insulin release mechanism. Expression of syntaxin 1A, but not 1B, was detected in mouse isolated islets by the reverse transcriptase-polymerase chain reaction procedure. An immunoprecipitation study of metabolically labeled islets with an anti-rat syntaxin 1/HPC-1 antibody demonstrated syntaxin 1A protein with an apparent molecular mass of ∼35 kDa. Immunohistochemistry of the mouse pancreas demonstrated that syntaxin 1/HPC-1 was present in the plasma membranes of the islets of Langerhans. In order to determine the functional role of syntaxin 1 in pancreatic β-cells, rat syntaxin 1A or 1B was overexpressed in mouse βTC3 cells using the transient transfection procedure. Transfection of βTC3 cells with either syntaxin 1 resulted in approximately 7-fold increases in their immunodetectable protein levels. Glucose-stimulated insulin release by syntaxin 1A-overexpressing cells was suppressed to about 50% of the level in control cells, whereas insulin release by syntaxin 1B-overexpressing and control cells did not differ. Next, we established stable βTC3 cell lines that overexpressed syntaxin 1A and used them to evaluate the effect of syntaxin 1A on the regulatory insulin release pathway. Two insulin secretogogues, 4-β-phorbol 12-myristate 13-acetate or forskolin, increased insulin release by untransfected βTC3 cells markedly, but their effects were diminished in syntaxin 1A-overexpressing βTC3 cells. Glucose-unstimulated insulin release and the proinsulin biosynthetic rate were not affected by syntaxin 1A overexpression, indicating a specific role of syntaxin 1A in the regulatory insulin release pathway. Finally, in vitro binding assays showed that syntaxin 1A binds to insulin secretory granules, indicating an inhibitory role of syntaxin 1A in insulin exocytosis via its interaction with vesicular proteins. These results demonstrate that syntaxin 1A is expressed in the islets of Langerhans and functions as a negative regulator in the regulatory insulin release pathway.

Site of docking and fusion of insulin secretory granules in live MIN6 β cells analyzed by TAT-conjugated anti-syntaxin 1 antibody and total internal reflection fluorescence microscopy

To determine the site of insulin exocytosis in the pancreatic β cell plasma membrane, we analyzed the interaction between the docking/fusion of green fluorescent protein-tagged insulin granules and syntaxin 1 labeled by TAT-conjugated Cy3-labeled antibody (Ab) using total internal reflection fluorescence microscopy (TIRFM). Monoclonal Ab against syntaxin 1 was labeled with Cy3 then conjugated with the protein transduction domain of HIV-1 TAT. TAT-conjugated Cy3-labeled anti-syntaxin 1 Ab was transduced rapidly into the subplasmalemmal region in live MIN6 β cells, which enabled us to observe the spatial organization and distribution of endogenous syntaxin 1. TIRFM imaging revealed that syntaxin 1 is distributed in numerous separate clusters in the intact plasma membrane, where insulin secretory granules were docked preferentially to the sites of syntaxin 1 clusters, colocalizing with synaptosomal-associated protein of 25 kDa (SNAP-25) clusters. TIRFM imaging analysis of the motion of single insulin granules demonstrated that the fusion of insulin secretory granules stimulated by 50 mm KCl occurred exclusively at the sites of the syntaxin 1 clusters. Cholesterol depletion by methyl-β-cyclodextrin treatment, in which the syntaxin 1 clusters were disintegrated, decreased the number of docked insulin granules, and, eventually the number of fusion events was significantly reduced. Our results indicate that 1) insulin exocytosis occurs at the site of syntaxin 1 clusters; 2) syntaxin 1 clusters are essential for the docking and fusion of insulin granules in MIN6 β cells; and 3) the sites of syntaxin 1 clusters are distinct from flotillin-1 lipid rafts.

Neuron-specific glucose transporter (NSGT): CNS distribution of GLUT3 rat glucose transporter (RGT3) in rat central neurons.

The identity of the glucose transporters (GLUT) expressed in neurons in situ has yet to be fully established. In the present study we have isolated the GLUT3 (RGT3) cDNA and produced anti RGT3 polyclonal antibody allowing us to investigate the cellular localization and tissue distributions of RGT3 mRNA and protein in the central nervous system of the rat by the methods of in situ hybridization and immunohistochemistry. Here we demonstrate the direct evidence that RGT3 is present in neurons in adult rat brain. In situ hybridization showed the expression of RGT3 mRNA mostly in the regions of hippocampus, cerebral cortex, striatum, and the granule cell layer of the cerebellum, indicating that RGT3 mRNA is predominantly expressed within neurons. Immunohistochemistry showed that RGT3 protein is widely distributed in the rat brain, and concentrated on the plasma membrane of neurons. Double labeling studies with anti‐RGT3, glial fibrillary acidic protein (GFAP), and neuron specific enolase (NSE) antibodies revealed the specific expression of RGT3 protein in neurons. Thus, RGT3 is indicated to be a neuron specific glucose transporter isoform (NSGT), and suggested to play a functionally significant role in rat central neurons.

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

Significance During pregnancy, maternal insulin secretion increases markedly. This increase is not simply a response to increased demand, as it precedes the insulin resistance that develops late in pregnancy, nor is it solely a result of increased β cell mass, as secretion per beta cell increases as well. Here we show that the increased islet serotonin induced by pregnancy signals through the 5-HT3 receptor (Htr3) to increase insulin secretion dramatically. Htr3 signaling increases the excitability of the β cell membrane, thereby decreasing the threshold for insulin secretion. These studies elucidate the mechanism for pregnancy-induced increase in insulin release. In preparation for the metabolic demands of pregnancy, β 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 β 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 β 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 β cells, which increased Ca2+ uptake and insulin exocytosis in response to glucose. Thus, our data indicate that serotonin, acting in a paracrine/autocrine manner through Htr3, lowers the β cell threshold for glucose and plays an essential role in the increased GSIS of pregnancy.

Monitoring of exocytosis and endocytosis of insulin secretory granules in the pancreatic β-cell line MIN6 using pH-sensitive green fluorescent protein (pHluorin) and confocal laser microscopy

The dynamics of exocytosis/endocytosis of insulin secretory granules in pancreatic beta-cells remains to be clarified. In the present study, we visualized and analysed the motion of insulin secretory granules in MIN6 cells using pH-sensitive green fluorescent protein (pHluorin) fused to either insulin or the vesicle membrane protein, phogrin. In order to monitor insulin exocytosis, pHluorin, which is brightly fluorescent at approximately pH 7.4, but not at approximately pH 5.0, was attached to the C-terminus of insulin. To monitor the motion of insulin secretory granules throughout exocytosis/endocytosis, pHluorin was inserted between the third and fourth amino acids after the identified signal-peptide cleavage site of rat phogrin cDNA. Using this method of cDNA construction, pHluorin was located in the vesicle lumen, which may enable discrimination of the unfused acidic secretory granules from the fused neutralized ones. In MIN6 cells expressing insulin-pHluorin, time-lapse confocal laser scanning microscopy (5 or 10 s intervals) revealed the appearance of fluorescent spots by depolarization after stimulation with 50 mM KCl and 22 mM glucose. The number of these spots in the image at the indicated times was counted and found to be consistent with the results of insulin release measured by RIA during the time course. In MIN6 cells expressing phogrin-pHluorin, data showed that fluorescent spots appeared following high KCl stimulation and remained stationary for a while, moved on the plasma membrane and then disappeared. Thus we demonstrate the visualized motion of insulin granule exocytosis/endocytosis using the pH-sensitive marker, pHluorin.

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 β cells is largely unknown. Therefore, to determine the role of CDKAL1 in insulin release from β cells, we studied insulin release profiles in CDKAL1 gene knockout (CDKAL1 KO) mice. Principal Findings Total internal reflection fluorescence imaging of CDKAL1 KO β 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 Ca2+ concentration during high glucose stimulation. Patch-clamp experiments revealed that the responsiveness of ATP-sensitive K+ (KATP) 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 β cells. Our results indicate that CDKAL1 controls first-phase insulin exocytosis in β cells by facilitating ATP generation, KATP channel responsiveness and the subsequent activity of Ca2+ channels through pathways other than CDK5-mediated regulation.

Correlation of syntaxin-1 and SNAP-25 clusters with docking and fusion of insulin granules analysed by total internal reflection fluorescence microscopy

Aims/hypothesisThe interaction of syntaxin-1 and SNAP-25 with insulin exocytosis was examined using the diabetic Goto–Kakizaki (GK) rat and a total internal reflection fluorescence (TIRF) imaging system.MethodsPrimary rat pancreatic beta cells were immunostained with anti-syntaxin-1A, anti-SNAP-25 and anti-insulin antibodies, and then observed by TIRF microscopy. The real-time image of GFP-labelled insulin granules motion was monitored by TIRF.ResultsThe number of syntaxin-1A and SNAP-25 clusters, and the number of docked insulin granules on the plasma membrane were reduced in GK beta cells. When GK rats were treated with daily insulin injection for 2 weeks, the number of syntaxin-1 and SNAP-25 clusters was restored, along with the number of docked insulin granules. The infection of GK beta cells with Adex1CA SNAP-25 increased the number of docked insulin granules. TIRF imaging analysis demonstrated that the decreased number of fusion events from previously docked insulin granules in GK beta cells was restored when the number of docked insulin granules increased by insulin treatment or Adex1CA SNAP-25 infection.Conclusions/interpretationThere was a close correlation between the number of syntaxin-1 and SNAP-25 clusters and the number of docked insulin granules, which is associated with the fusion of insulin granules.

α-Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Is Expressed in Pancreatic β Cells and Functions in Insulin but Not γ-Aminobutyric Acid Secretion

The function of solubleN-ethylmaleimide-sensitive attachment protein-α (α-SNAP) in exocytosis still remains obscure. This study was conducted to determine the physiological role of α-SNAP in the secretion of insulin and γ-aminobutryric acid (GABA) from pancreatic β cells. Reverse transcriptase-polymerase chain reaction analysis of total RNA isolated from rat islets disclosed α-SNAP, but not β-SNAP, mRNA expression, and an immunofluorescence study of rat pancreas showed that α-SNAP was present predominantly in the cytoplasm of the islets of Langerhans. α-SNAP overexpression in rat islets enhanced insulin release relative to the control levels. Anin vitro binding study showed that both wild-type α-SNAP and C-terminal–deleted α-SNAP mutant (1–285) can bind to syntaxin 1A. α-SNAP mutant (1–285) was overexpressed to evaluate its activity as dominant-negative effector on insulin release. Overexpression of α-SNAP mutant (1–285) in rat islets and MIN6 cells decreased glucose-stimulated insulin release to about 50% of the control levels. Suppression of endogeneous α-SNAP in MIN6 cells by treatment with an antisense phosphorothioate oligonucleotide resulted in inhibition of insulin release. In order to examine if α-SNAP functions in exocytosis from synaptic-like microvesicles in pancreatic β cells, the functional role of α-SNAP in GABA release from MIN6 cells was studied. The data showed no effect of α-SNAP mutant (1–285) overexpression on GABA release. We conclude that 1) α-SNAP plays a crucial role in insulin exocytosis via large dense core vesicles, but not GABA released via synaptic-like microvesicles, in pancreatic β cells; and 2) the interaction of α-SNAP and syntaxin 1A may play an important role in the insulin exocytotic process.

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

Functional insulin receptor and its downstream effector PI3K (phosphoinositide 3-kinase) have been identified in pancreatic β-cells, but their involvement in the regulation of insulin secretion from β-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 β-cells and insulinoma Min6 cells using total internal reflection fluorescent microscopy. The pretreatment of β-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.