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.

Signaling cascades involved in neuroprotection by subpicomolar pituitary adenylate cyclase-activating polypeptide 38.

In neuronal/glial cocultures, pituitary adenylate cyclase-activating polypeptide 38 (PACAP38) prevented neuronal death induced by gp120, lipopolysaccharide (LPS), or other toxic agents, but the dose response of the neuroprotective effect is bimodal, with a peak at a subpicomolar concentration and another peak at a subnanomolar to nanomolar concentration. Although the signaling cascade involved in neuroprotection by nanomolar concentration of the peptide has been shown to be mediated by activation of cAMP-dependent protein kinase and subsequent activation of mitogen-activated protein kinase (MAPK), the mechanism for neuroprotection by a subpicomolar level of PACAP38 remains elusive. In the present study, the signaling involved in neuroprotection by subpicomolar PACAP38 was studied in rat neuronal/glial cocultures. Addition of PACAP38 stimulated expression and activation of extracellular signal-related kinase-type MAPK with a peak response at 10-13 M; greater concentrations of the peptide induced lesser response. cAMP production also increased at subpicomolar levels of PACAP38, but the level remained unchanged at a level four to five times higher than the base level at concentrations below 10-11 M. cAMP then started increasing again dose-dependently in a range >10-11 M PACAP38. Lipopolysaccharide (LPS)-induced neuronal death, indicated by increased release of neuron-specific enolase, was suppressed by PACAP38 in a bimodal fashion. Neuroprotection by 10-12 M PACAP38 was completely abolished by a MAPK kinase-1 inhibitor, PD98059, and also partially suppressed by Rp-cAMP, a cAMP-dependent protein kinase inhibitor. Moreover, neuroprotection by a nanomolar level of PACAP38 was completely suppressed by Rp-cAMP but not affected by PD98059. We conclude that neuroprotection by subpicomolar PACAP38 is mainly mediated by the signaling pathway involving MAPK activation and partially regulated by cAMP-dependent protein kinase activation. Furthermore, PACAP38 stimulated expression of activity- dependent neuroprotective protein (ADNP), with a peak at 10-13 M. Greater doses of the peptide induced lesser response. However, 10-13 M PACAP38-stimulated expression of ADNP was not affected by PD98059. This suggests that neuroprotection by subpicomolar PACAP38 might be mediated partially by expression of ADNP, but the major events for neuroprotection by subpicomolar PACAP38 remain to be identified.

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.

Docking and fusion of insulin secretory granules in SUR1 knock out mouse beta-cells observed by total internal reflection fluorescence microscopy.

To explore how the sulfonylurea receptor (SUR1) is involved in docking and fusion of insulin granules, dynamic motion of single insulin secretory granules near the plasma membrane was examined in SUR1 knock-out (Sur1KO) beta-cells by total internal reflection fluorescence microscopy. Sur1KO beta-cells exhibited a marked reduction in the number of fusion events from previously docked granules. However, the number of docked granules declined during stimulation as a consequence of the release of docked granules into the cytoplasm vs. fusion with the plasma membrane. Thus, the impaired docking and fusion results in decreased insulin exocytosis from Sur1KO beta-cells.

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.

The cytokine interleukin-1beta reduces the docking and fusion of insulin granules in pancreatic beta-cells, preferentially decreasing the first phase of exocytosis.

The prediabetic period in type I diabetes mellitus is characterized by the loss of first phase insulin release. This might be due to islet infiltration mediated by mononuclear cells and local release of cytokines, but the mechanisms involved are unknown. To determine the role of cytokines in insulin exocytosis, we have presently utilized total internal reflection fluorescence microscopy (TIRFM) to image and analyze the dynamic motion of single insulin secretory granules near the plasma membrane in live beta-cells exposed for 24 h to interleukin (IL)-1beta or interferon (IFN)-gamma. Immunohistochemistry observed via TIRFM showed that the number of docked insulin granules was decreased by 60% in beta-cells treated with IL-1beta, while it was not affected by exposure to IFN-gamma. This effect of IL-1beta was paralleled by a 50% reduction in the mRNA and the number of clusters of SNAP-25 in the plasma membrane. TIRF images of single insulin granule motion during a 15-min stimulation by 22 mm glucose in IL-1beta-treated beta-cells showed a marked reduction in the fusion events from previously docked granules during the first phase insulin release. Fusion from newcomers, however, was well preserved during the second phase of insulin release of IL-1beta-treated beta-cells. The present observations indicate that IL-1beta, but not IFN-gamma, has a preferential inhibitory effect on the first phase of glucose-induced insulin release, mostly via an action on previously docked granules. This suggests that beta-cell exposure to immune mediators during the course of insulitis might be responsible for the loss of first phase insulin release.

Functions of pancreatic beta cells and adipocytes in bombesin receptor subtype-3-deficient mice.

We previously reported that mice lacking bombesin receptor subtype-3 (BRS-3) exhibit mild late-onset obesity and glucose intolerance [Nature 390 (1997) 160]. To examine the mechanism by which glucose intolerance is developed in these mice, we studied insulin release and proinsulin biosynthesis in isolated pancreatic islets and glucose uptake and facilitative glucose transporter (GLUT)-4 translocation in adipose tissues. Although islet insulin contents and the size and number of islets of Langerhans in BRS-3-deficient mice decreased, there was no difference in glucose-stimulated insulin release and proinsulin biosynthesis between BRS-3-deficient and wild-type control mice. In contrast, adipose tissues exhibited a marked difference: the uptake of [(14)C]2-deoxy-D-glucose by adipocytes isolated from BRS-3-deficient mice was not stimulated by 10(-7)M insulin addition, and membrane fractionation analysis showed that GLUT4 was barely detected in the fraction of plasma membrane in BRS-3-deficient mice in the presence of 10(-7)M insulin. Quantitative reverse transcription-PCR (RT-PCR) showed that mRNA levels of GLUT4, insulin receptor, insulin receptor substrate (IRS)-1 and IRS-2, syntaxin 4, SNAP23, and VAMP-2 in adipose tissues of BRS-3-deficient mice were unchanged compared with those in wild-type control mice. We concluded that impaired glucose metabolism observed in BRS-3-deficient mice was mainly caused by impaired GLUT4 translocation in adipocytes.

TIRF imaging of docking and fusion of single insulin granule motion in primary rat pancreatic beta-cells: different behaviour of granule motion between normal and Goto-Kakizaki diabetic rat beta-cells.

We imaged and analysed the motion of single insulin secretory granules near the plasma membrane in live pancreatic beta-cells, from normal and diabetic Goto-Kakizaki (GK) rats, using total internal reflection fluorescence microscopy (TIRFM). In normal rat primary beta-cells, the granules that were fusing during the first phase originate from previously docked granules, and those during the second phase originate from 'newcomers'. In diabetic GK rat beta-cells, the number of fusion events from previously docked granules were markedly reduced, and, in contrast, the fusion from newcomers was still preserved. The dynamic change in the number of docked insulin granules showed that, in GK rat beta-cells, the total number of docked insulin granules was markedly decreased to 35% of the initial number after glucose stimulation. Immunohistochemistry with anti-insulin antibody observed by TIRFM showed that GK rat beta-cells had a marked decline of endogenous insulin granules docked to the plasma membrane. Thus our results indicate that the decreased number of docked insulin granules accounts for the impaired insulin release during the first phase of insulin release in diabetic GK rat beta-cells.

Site of docking and fusion of insulin secretory granules in live MIN6 beta 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 beta 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 beta 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-beta-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 beta cells; and 3) the sites of syntaxin 1 clusters are distinct from flotillin-1 lipid rafts.

PPAR-gamma overexpression suppresses glucose-induced proinsulin biosynthesis and insulin release synergistically with pioglitazone in MIN6 cells.

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) regulates several cellular functions; however, its physiological role in pancreatic beta cell functions remains to be determined. In the present study, we investigated the synergistic effect of PPAR-gamma and its agonist, pioglitazone, on proinsulin biosynthesis and insulin release in a glucose-responsible insulinoma cell line, MIN6 cells. Expression of PPAR-gamma in MIN6 cells was not detectable by RT-PCR and immunoblot analysis. When PPAR-gamma-1 was overexpressed adenovirally in MIN6 cells, glucose-stimulated proinsulin biosynthesis and insulin release were inhibited. Pioglitazone treatment alone had no effects on these parameters of beta cell function in control MIN6 cells, although pioglitazone synergistically augmented the inhibitory effect of PPAR-gamma on proinsulin biosynthesis and insulin release under the condition of PPAR-gamma overexpression. Our results demonstrate that PPAR-gamma plays a negative role in pancreatic beta cells.

Water secretion associated with exocytosis in endocrine cells revealed by micro forcemetry and evanescent wave microscopy.

It has been a long belief that release of substances from the cell to the extracellular milieu by exocytosis is completed by diffusion of the substances from secretory vesicles through the fusion pore. Involvement of any mechanical force that may be superposed on the diffusion to enhance the releasing process has not been elucidated to date. We tackled this problem in cultured bovine chromaffin cells using direct and sensitive methods: the laser-trap forcemetry and the evanescent-wave fluorescence microscopy. With a laser beam, we trapped a micro bead in the vicinity of a cell (with 1 microm of separation) and observed movements of the bead optically. Electrical stimulation of the cell induced many of rapid and transient movements of the bead in a direction away from the cell surface. Upon the same stimulation, secretory vesicles stained with a fluorescent probe, acridine orange, and excited under the evanescent field illumination, showed a flash-like response: a transient increase in fluorescence intensity associated with a diffuse cloud of brightness, followed by a complete disappearance. These mechanical and fluorescence transients indicate a directional flow of substances. Blockers of the Cl(-) channel suppressed the rates of both responses in a characteristic way but not exocytotic fusion itself. Immunocytochemical studies revealed the presence of Cl(-) and K(+) channels on the vesicle membranes. These results suggest that the externalization of hormones or transmitters upon exocytosis of vesicles is augmented by secretion of water from the vesicle membrane through the widened fusion pore, possibly modulating the rate and reach of the hormone or transmitter release and facilitating transport of the signal molecules in intercellular spaces.