Glinide, but Not Sulfonylurea, Can Evoke Insulin Exocytosis by Repetitive Stimulation: Imaging Analysis of Insulin Exocytosis by Secretagogue-Induced Repetitive Stimulations

To investigate the different effects between sulfonylurea (SU) and glinide drugs in insulin secretion, pancreatic β-cells were repeatedly stimulated with SU (glimepiride) or glinide (mitiglinide). Total internal reflection fluorescent (TIRF) microscopy revealed that secondary stimulation with glimepiride, but not glucose and mitiglinide, failed to evoke fusions of insulin granules although primary stimulation with glucose, glimepiride, and mitiglinide induced equivalent numbers of exocytotic responses. Glimepiride, but not glucose and mitiglinide, induced abnormally sustained [Ca2+]i elevations and reductions of docked insulin granules on the plasma membrane. Our data suggest that the effect of glinide on insulin secretory mechanisms is similar to that of glucose

Deletion of CDKAL1 Affects Mitochondrial ATP Generation and First-Phase Insulin Exocytosis

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.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 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.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, K(ATP) channel responsiveness and the subsequent activity of Ca(2+) channels through pathways other than CDK5-mediated regulation

The effect of Akt inhibitor on dynamics of intracellular insulin granules.

<p>(A, B) Control (A) and 1 µM Akti-1/2 treated β-cells (B) expressing insulin-GFP were observed under TIRF microscopy with PD = 150 nm and the motions of insulin granules for 1 min were manually tracked (red lines). (C, D) Cumulative plots of mean square displacement for 1min observed in TIRF microscopy with PD = 45 nm (C; n = 79 and 101 for control and Akti-1/2, respectively) and PD = 150 nm (D; n = 154 and 149 for control and Akti-1/2, respectively).</p

ELKS, a Protein Structurally Related to the Active Zone-associated Protein CAST, Is Expressed in Pancreatic β 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 Ca(2+)-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 β 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 β cells

Acute treatment with PIK-75 selectively enhanced the fusions from newcomer granules during the second phase.

<p>(A) Pancreatic β-cells expressing insulin-GFP were stimulated with 22 mM glucose at time 0 and the exocytotic responses (events per 200 µm²) detected within every 1-min were counted. Histograms show the numbers of fusion events from control (left; n = 10) and 0.5 µM PIK-75-treated cells (right; n = 16). The black column shows fusions from previously docked granules and the white column shows fusions from newcomer granules. (B) Quantitative analysis of the total numbers of fusion events from previously docked (black column) and newcomer (white column) granules detected during the 0–6 min (left) and 6–17 min (right). (C) Control (open triangle; n = 6) and 0.5 µM PIK-75 treated (filled square; n = 6) islets were perifused with 22 mM glucose. (D) [Ca²⁺]_i was measured by microfluorometry in Fura-2 loaded β-cells. Control cells (open triangle; n = 31) and cells treated with 0.1 µM wortmannin (open diamond; n = 29) or 0.5 µM PIK-75 (filled square; n = 29) were stimulated with 22 mM glucose at 3 min. The [Ca²⁺]_i responses are represented as ratios of the fluorescence intensity. Results are represented as mean ± S.E.M. *; p<0.01.</p

Acute Inhibition of PI3K-PDK1-Akt Pathway Potentiates Insulin Secretion through Upregulation of Newcomer Granule Fusions in Pancreatic β-Cells

<div><p>In glucose-induced insulin secretion from pancreatic β-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.</p> </div

The constitutive active mutant of PDK1counteracted the potentiating effect of PIK-75 on the glucose-induced secretion.

<p>(A) COS7 cells transfected with mock or PDK1(A280V) were treated with 0.5 µM PIK-75 for 30 min and subjected to immunoblotting using anti-Pi-Akt and α-tubulin antibodies. Primary cultured islets transfected with PDK1(A280V) (B; n = 6 for each group), Arf6(Q67L) (E; n = 6 for each group) and Rac1(V12) (F; n = 6 for each group) pretreated with or without 0.5 µM PIK-75 for 30 min were stimulated with 16 mM glucose. (C) Cultured islets pretreated with UCN-01 or UCN-02 for 30 min were subjected to immunoblotting using anti-Pi-Akt and Pan-Akt antibodies. (D) Cultured islets pretreated with 1.0 µM UCN-01 or UCN-02 for 30 min were stimulated with 2.2 or 16 mM glucose (n = 8 for each group). Results are represented as mean ± S.E.M. *; p<0.01, N.S.; p>0.3.</p

Chronic treatment of isolated islets with PIK-75 reduced the glucose-induced insulin secretion.

<p>(A) Pancreatic islets cultured for 48 h with PIK-75 at the indicated concentrations were subjected to immunoblotting using anti-Pi-Akt and Pan-Akt antibodies. (B) Pancreatic islets cultured for 48 h with PIK-75 at the indicated concentrations were stimulated with 16 mM glucose with or without 0.5 µM PIK-75 for 30 min. The amount of secreted insulin was normalized to that from the PIK-75 untreated islets of each group (n = 8 for each group). (C) Freshly isolated islets or those cultured with or without 1 µM PIK-75 for 24, 48 and 72 h were stimulated with 16 mM glucose supplemented with or without 0.5 µM PIK-75 for 30 min. The amount of secreted insulin was normalized to that from the PIK-75 untreated islets of each group (n = 8 for each group). Data are the means ± S.E.M. (*, p<0.01; **, p<0.03).</p