Adiponectin induces insulin secretion in vitro and in vivo at a low glucose concentration.

AIMS/HYPOTHESIS: A decrease in plasma adiponectin levels has been shown to contribute to the development of diabetes. However, it remains uncertain whether adiponectin plays a role in the regulation of insulin secretion. In this study, we investigated whether adiponectin may be involved in the regulation of insulin secretion in vivo and in vitro. METHODS: The effect of adiponectin on insulin secretion was measured in vitro and in vivo, along with the effects of adiponectin on ATP generation, membrane potentials, Ca2+ currents, cytosolic calcium concentration and state of 5'-AMP-activated protein kinase (AMPK). In addition, insulin granule transport was measured by membrane capacitance and total internal reflection fluorescence (TIRF) analysis. RESULTS: Adiponectin significantly stimulated insulin secretion from pancreatic islets to approximately 2.3-fold the baseline value in the presence of a glucose concentration of 5.6 mmol/l. Although adiponectin had no effect on ATP generation, membrane potentials, Ca2+ currents, cytosolic calcium concentrations or activation status of AMPK, it caused a significant increase of membrane capacitance to approximately 2.3-fold the baseline value. TIRF analysis revealed that adiponectin induced a significant increase in the number of fusion events in mouse pancreatic beta cells under 5.6 mmol/l glucose loading, without affecting the status of previously docked granules. Moreover, intravenous injection of adiponectin significantly increased insulin secretion to approximately 1.6-fold of baseline in C57BL/6 mice. CONCLUSIONS/INTERPRETATION: The above results indicate that adiponectin induces insulin secretion in vitro and in vivo.

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

AIMS/HYPOTHESIS: The 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. METHODS: Primary 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. RESULTS: The 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/INTERPRETATION: There 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.

Adenovirus-mediated preproinsulin gene transfer into adipose tissues ameliorates hyperglycemia in obese diabetic KKA(y) mice.

We investigated whether adenovirus-mediated preproinsulin gene transfer into insulin target tissues (adipocytes) ameliorates hyperglycemia in diabetic mice. KKA(y) mice, a genetically obese type 2 diabetic animal model, were treated with a single subcutaneous injection of recombinant adenovirus, Adex1CA-human preproinsulin (Adex1CA-pchi), into the epididymal fat pads. pchi mRNA was expressed only in adipose tissue in which mature insulin was produced. Three days after virus injection these mice showed a marked decrease of blood glucose levels (from about 400 to 200 mg/dl), and an intraperitoneal glucose tolerance test revealed the markedly improved glucose tolerance. There was no significant difference in serum insulin levels between control and recombinant adenovirus-treated KKA(y) mice. The normalized glucose levels in diabetic mice were maintained for at least 2 weeks after the virus injection. This strategy could provide a novel and, most importantly, a simple and convenient gene therapy for obese type 2 diabetes patients.

Mastoparan stimulates GABA release from MIN6 cells: relationship between SNARE proteins and mastoparan action.

We examined the action of mastoparan on beta cell exocytosis. Mastoparan stimulated GABA and insulin release from MIN6 beta cells. On the other hand, mastopraran-induced GABA release was decreased by expressing the tetanus toxin C1 light chain in MIN6 cells. We have then investigated the relationship between SNARE proteins and mastoparan action using adenovirus-mediated gene transfer system. Overexpression of t-SNAREs, syntaxin 1A, and SNAP-25 inhibited the mastoparan-induced insulin release by approximately half-fold of control levels, however, the mastoparan-induced GABA release was not affected by these t-SNAREs overexpression. The overexpression of mutant alpha-SNAP (1-285), which inhibits the wild-type alpha-SNAP function in a dominant negative manner, did not influence either mastoparan-induced GABA or insulin release in spite of its marked inhibition of glucose-stimulated insulin release. Our data indicate that mastoparan stimulates GABA exocytosis via vesicular transport; however, SNARE proteins are differently involved in the exocytosis of insulin and GABA.

Protein kinase C controls the priming step of regulated exocytosis in adrenal chromaffin cells.

1. To investigate the mechanism whereby protein kinase C enhances secretory function in adrenal chromaffin cells, we examined the effects of 12-O-tetradecanoylphorbor-13-acetate (TPA) on Ca(2+)-induced catecholamine release from digitonin-permeabilized cells, resolving the release into a MgATP-dependent priming step and a MgATP-independent Ca(2+)-triggered step. Treatment with TPA selectively potentiated the priming activity of MgATP, with little increase in the MgATP-independent release. The potentiation by TPA of the MgATP-dependent priming was blocked by [Ser25]protein kinase C(19-31), a specific substrate of protein kinase C. Gö 6976, an inhibitor selective for protein kinase C alpha and beta isoforms, also blocked the potentiation by TPA. These results suggest that activation of protein kinase C, probably the alpha isoform, potentiates the MgATP-dependent priming step. 2. The antibody raised against GAP-43, a known substrate of protein kinase C, also potentiated the MgATP-dependent priming. The effect of TPA and that of the anti-GAP-43 antibody were not additive. Calmodulin, which binds to GAP-43 and inhibits its phosphorylation by protein kinase C, abolished the effect of TPA. Thus, the present results suggest that protein kinase C potentiates MgATP-dependent priming, at least in part, through phosphorylation of GAP-43.

Regulation of the priming of exocytosis and the dissociation of SNAP-25 and VAMP-2 in adrenal chromaffin cells.

The MgATP-dependent priming step of exocytosis has been suggested to be regulated negatively by GTP-binding protein G0 in permeabilized adrenal chromaffin cells. We have reported that synaptosomal-associated protein of 25 kDa (SNAP-25) and vesicle-associated membrane protein 2 (VAMP-2) form a complex in chromaffin cells, and the complex dissociates during MgATP-dependent priming. In this study, we examined whether G0 controls such dissociation of the SNAP-25/VAMP-2 complex in the regulation of priming. In digitonin-permeabilized cells, MgATP-gamma-S which can be a phosphate donor for protein phosphorylation failed to cause priming and dissociation of the SNAP-25/VAMP-2 complex. Mastoparan, which directly activates G0, selectively inhibited priming and blocked dissociation of the SNAP-25/VAMP-2 complex. These results suggest that ATP hydrolysis and dissociation of the SNAP-25/VAMP-2 complex are responsible for priming. These results also suggest that dissociation of the complex is one of the sequential steps for priming controlled by G0.

Distinct roles of C2A and C2B domains of synaptotagmin in the regulation of exocytosis in adrenal chromaffin cells.

Synaptotagmin that contains two repeats of C2 regulatory domains is considered to be involved in neurotransmitter release. To reveal the roles of synaptotagmin in the regulation of exocytosis, we examined the effects of antibodies against C2A and C2B domains on Ca2+-evoked catecholamine (CA) release from digitonin-permeabilized adrenal chromaffin cells, resolving the Ca2+-evoked release into ATP-dependent priming and ATP-independent Ca2+-triggered steps. Anti-C2A antibody clearly reduced the ATP-independent release, suggesting that the C2A domain directly facilitate or promote Ca2+-triggered step, vesicular fusion. In contrast, anti-C2B antibody did not affect Ca2+-evoked release by itself, but significantly increased the spontaneous Ca2+-independent release. In addition, inositol high-polyphosphate series (IHPS) that bind the C2B domain inhibited both the ATP-independent Ca2+-evoked release and the spontaneous release in a dose-dependent manner. The inhibition by IHPS was totally reversed by anti-C2B antibody and significantly reversed by high concentration of Ca2+. These results suggest that IHPS binding to C2B domain arrests membrane fusion by presumably preventing interaction of synaptotagmin with phospholipids or with proteins of plasma membrane. Thus, IHPS binding to the C2B domain might keep the docked or primed vesicles away from spontaneous fusion at resting level of intracellular Ca2+. Binding of the increased intracellular Ca2+ to the C2A domain may facilitate or trigger the vesicular fusion by releasing this suppression by IHPS.

Dissociation of SNAP-25 and VAMP-2 by MgATP in permeabilized adrenal chromaffin cells.

In digitonin-permeabilized adrenal chromaffin cells, Ca(2+)-induced catecholamine release can be resolved into at least two sequential steps: a MgATP-dependent priming step and a MgATP-independent Ca(2+)-triggered step. Botulinum neurotoxins types A and E cleaved SNAP-25, and blocked MgATP-independent Ca(2+)-induced catecholamine release from the permeabilized chromaffin cells. When the permeabilized cells were primed by pretreatment with MgATP, the amount of SNAP-25 associated with VAMP-2 decreased, and the fraction of SNAP-25 proteolyzed by the neurotoxins increased. These results suggest that dissociation of SNAP-25 and VAMP-2 occurs during the MgATP-dependent priming step, and SNAP-25 plays some important roles in the subsequent MgATP-independent step.

Essential role of myosin light chain kinase in the mechanism for MgATP-dependent priming of exocytosis in adrenal chromaffin cells.

Ca(2+)-induced exocytosis in chromaffin cells now seems to consist of at least two distinct steps:MgATP-dependent Ca(2+)-dependent priming of the secretory apparatus, and Ca(2+)-dependent MgATP-independent step that triggers exocytosis (Bittner and Holz, 1992). Recently we found that a specific inhibitor of myosin light chain kinase (MLCK), wortmannin, inhibits Ca(2+)-induced catecholamine release from digitonin-permeabilized chromaffin cells, suggesting an implication of MLCK in the mechanisms of Ca(2+)-induced exocytosis (Imaizumi et al., 1992b). To elucidate further the implication of MLCK in the mechanism of exocytosis, we studied the effects of wortmannin and a peptide inhibitor (SM-1) corresponding to the pseudosubstrate domain of MLCK on MgATP-dependent and MgATP-independent release in digitonin-permeabilized chromaffin cells. Ca(2+)-induced exocytosis from the permeabilized cells in the presence of MgATP was inhibited by both SM-1 and wortmannin. Inhibitory effect of wortmannin on the rate of release induced by 10 microM Ca2+ in the presence of MgATP was much prominent in the later phase (1-10 min), although the initial rate was also decreased. SM-1 strongly inhibited ATP-dependent release without affecting Ca(2+)-dependent ATP-independent release at all. In addition, priming effect of MgATP that underlies Ca(2+)-dependent ATP-independent release was remarkably reduced by both wortmannin and SM-1. These results suggest that MLCK plays an essential role in ATP-dependent priming of Ca(2+)-induced exocytosis in chromaffin cells.

Mastoparan evokes exocytosis without Ca2+ movement in adrenal chromaffin cells.

The secretagogue action of mastoparan, a tetradecapeptide from wasp venom, was studied in adrenal chromaffin cells. Pulsatile stimulation with mastoparan evoked a sharp transient release of CA in a dose-dependent manner, as measured by real-time monitoring system. The secretagogue action of mastoparan in these cells was apparently independent of extracellular Ca2+, and may not require the intracellular mobilization of Ca2+ since BAPTA failed to block its secretagogue action. Video-imaging data also suggested that mastoparan evokes CA release without an increase in the intracellular Ca2+. In addition, visualization of exocytotic events with video-enhanced light microscopy demonstrated that CA release evoked by mastoparan at relatively low concentration was indeed mediated by exocytotic process, not by cell lysis.

Effects of imidazole compounds on catecholamine release in adrenal chromaffin cells.

1. Effects of imidazole compounds and guanabenz on the stimulus-evoked release of catecholamine (CA) were studied in cultured bovine adrenal chromaffin cells. 2. Clonidine, oxymetazoline, phentolamine, chlorpheniramine, and guanabenz inhibited acetylcholine (ACh)-evoked CA release in a dose-dependent manner, but not high K(+)-evoked release. 3. The inhibition by these compounds was not antagonized by nonimidazole and nonguanidine alpha 2-antagonists (yohimbine and phenoxybenzamine) but was significantly antagonized by tolazoline (imidazole alpha 2-antagonist) and cimetidine (imidazole H2-antagonist). Moreover, tolazoline by itself augmented the ACh-evoked, but not the high K(+)-evoked, CA release. 4. Although chlorpheniramine and cimetidine are antagonists for H1 and H2 histaminergic receptors, the site of action for these compounds in our results seemed to differ from the histamine receptors. 5. These results suggest that the inhibitory action of imidazole compounds and guanabenz on ACh-evoked CA release in adrenal chromaffin cells is mediated through an imidazole receptor. Adrenal chromaffin cells may contain an endogenous clonidine-displacing substance (CDS) which has been found in adrenal gland and brain as an endogenous ligand for imidazole receptors. Thus, CDS may have a regulatory role in the stimulus-secretion coupling in these cells.

Regulatory role of the GTP-binding protein, G(o), in the mechanism of exocytosis in adrenal chromaffin cells.

To elucidate the possible involvement of GTP-binding proteins (G proteins) in the mechanism of exocytosis, we studied effects of pertussis toxin (PTX), guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S), and antibodies against the G proteins (Gi and G(o)) on the secretory function of bovine adrenal chromaffin cells. Pretreatment of chromaffin cells with PTX resulted in an increase in acetylcholine-evoked catecholamine release. High K(+)-, histamine-, or gamma-aminobutyric acid-evoked catecholamine release was also potentiated by PTX pretreatment. The concentration of extracellular Ca2+ required for maximal release by 10(-4) M acetylcholine was decreased significantly in PTX-treated cells. In digitonin-permeabilized cells, PTX pretreatment resulted in a decrease of the half-maximal concentration (Km) of Ca2+ required for exocytosis with no significant change in the maximal stimulation (Vmax). Exposure of permeabilized cells to GTP-gamma-S (a nonhydrolyzable GTP analogue) inhibited Ca(2+)-dependent exocytosis by reducing the affinity for Ca2+. The effects of PTX pretreatment were mimicked by treatment of permeabilized cells with polyclonal antibodies selective for the alpha subunit of the PTX-sensitive G protein, G(o). Treatment with similar antibodies against the alpha subunit of Gi had no effect. These findings suggest that G(o) directly controls the Ca(2+)-triggered process in the machinery of exocytosis by lowering the affinity of the unknown target for Ca2+.

Inhibition of Ca(2+)-dependent catecholamine release by myosin light chain kinase inhibitor, wortmannin, in adrenal chromaffin cells.

To elucidate the possible involvement of myosin light chain kinase (MLCK) in the mechanism of exocytosis, we studied effects of MLCK inhibitor, wortmannin, on the secretory function of bovine adrenal chromaffin cells. Preincubation of chromaffin cells with wortmannin inhibited both acetylcholine- and high K(+)-evoked catecholamine (CA) release. The IC50 for high K(+)-evoked CA release was 1 microM. When the cells were permeabilized with digitonin after wortmannin preincubation, Ca(2+)-dependent exocytosis was inhibited in a dose-dependent manner (IC50, 1 microM). These findings suggest the implication of MLCK in the Ca(2+)-triggered process in the machinery of exocytosis.

Dopamine receptor agonists and antagonists enhance ATP-activated currents.

The effects of dopamine and related compounds on ATP-activated channels were investigated in pheochromocytoma PC12 cells. Dopamine (10 microM) enhanced an inward current activated by 100 microM ATP. A similar enhancement of the ATP-activated current was observed with apomorphine (10 microM), a non-selective dopamine receptor agonist, with (+)-SKF-38393 (10 microM), a selective dopamine D1 receptor agonist, and with (-)-quinpirole (10 microM), a selective dopamine D2 receptor agonist. Moreover, (+)-SCH-23390 (30 microM), a dopamine D1 receptor antagonist, and (-)-sulpiride (30 microM), a dopamine D2 receptor antagonist, also enhanced the ATP-activated current. The results suggest that ATP-activated channels are modulated by dopaminergic mechanisms, and that this modulation cannot be attributed to any single class of dopamine receptors.

Inhibition of ion channels by hirsutine in rat pheochromocytoma cells.

Effects of hirsutine, an alkaloid that produces a potent ganglion blocking effect, were investigated using rat pheochromocytoma PC12 cells. Hirsutine (1 to 10 microM) suppressed dopamine-release evoked by 100 microM nicotine. In voltage-clamped cells, hirsutine (1 to 10 microM) inhibited the inward current activated by 100 microM nicotine. Hirsutine was equipotent to hexamethonium in blocking the nicotine-activated current. The voltage-dependency of the nicotine activated current was not modified by hirsutine. Effects of hirustine on other ion channels were tested to determine its selectivity. Inward currents mediated through ATP-activated channels were scarcely affected by hirsutine (up to 100 microM). However, hirustine (10 microM) inhibited Ba currents passing through Ca channels and K currents activated by depolarizing voltage steps. The results suggest that hirsutine potently blocks nicotinic receptor-channels, but hirsutine also inhibits voltage-gated Ca and K channels. Roles of the inhibition of these channels in the pharmacological effects of hirsutine were discussed.

Inhibitory action of peripheral-type benzodiazepines on dopamine release from PC12 pheochromocytoma cells.

Characteristics of the benzodiazepine inhibition of dopamine (DA) release in PC12 cells were investigated. Diazepam inhibited DA release evoked by high concentrations of extracellular K+ in a dose-dependent manner (IC50, 10 microM). Ro 5-4864 [7-chloro-1,3-dihydro-1-methyl-5-(p-chlorophenyl)-2H-1,4-benzodiazepine- 2-one], a peripheral-type benzodiazepine, also inhibited DA release effectively. PK 11195 [1-(2-chlorophenyl)-N-methyl-N-(1-methyl-propyl)-3-isoquinoline carboxamide], a benzodiazepine generally considered a peripheral-type benzodiazepine receptor antagonist, did not antagonize the inhibition induced by diazepam, but rather inhibited DA release itself. On the other hand, the central-type benzodiazepines, clonazepam and Ro 15-1788 (ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5a] [1,4]benzodiazepine-3-carboxylate) did not affect the DA release. Diazepam, Ro 5-4864 and PK 11195 also inhibited a Ba(++)-current carried by voltage-gated Ca++ channels, and diazepam suppressed an increase in intracellular Ca++ evoked by 80 mM extracellular K+ as measured by the fura-2 method. These results suggest that the inhibitory action of diazepam and other benzodiazepines on DA release from PC12 cells may be mediated through one type of peripheral-type benzodiazepine receptors which are coupled to voltage-gated Ca++ channels and that these receptors may not necessarily be the same as those in other tissues.

Visualization of exocytotic secretory processes of mast cells by fluorescence techniques.

Secretory processes via exocytosis in rat peritoneal mast cells were visualized by two complementary fluorescence techniques; one staining pre-exocytotic granules with a basic probe and the other staining post-exocytotic granules with acidic probes. Granules within mast cells were selectively stained with acridine orange and emitted orange yellow fluorescence. Upon stimulation with compound 48/80, release of acridine orange from granules was observed both in population and single cell measurements. This release was seen in some localized area of mast cells. Opening of pores between plasma membranes and granule membranes was monitored using acidic fluorescence probes such as 6-carboxyfluorescein or lucifer yellow CH. Not only granules located at peripheral region, but also granules near the core region participated in exocytosis. The existence of junctions between these granules was suggested. TMA-DPH, a lipophilic membrane probe, which was localized at plasma membrane before stimulation, diffused into granule membranes after stimulation. This shows that after stimulation, some constituents of plasma and granule membranes were mixed. Even after extensive degranulation, mast cells extruded acidic probes, indicating the plasma membranes still play a role of barrier. Activation of lateral motion of granules preceding to exocytosis was not observed. It was concluded that the visualization of secretory processes by fluorescence and image processing techniques will be useful for the study of molecular mechanisms underlying exocytosis.

Inhibition of Ca-channels by diazepam compared with that by nicardipine in pheochromocytoma PC12 cells.

The effects of diazepam on voltage-gated Ca channels were studied in PC12 pheochromocytoma cells using whole-cell voltage-clamp techniques. An inward current activated by a depolarizing voltage step to +10 mV from a holding potential of -60 mV in 10.8 mM Ba was larger than that activated in 10.8 mM Ca. The Ba current was completely blocked by a low concentration of Cd (30 microM) and was also sensitive to nicardipine (100 nM to 10 microM). Diazepam (1-100 microM) inhibited the Ba current in a concentration-dependent manner. Neither diazepam nor nicardipine affected the current-voltage relationship or the dependence on holding potentials of the Ba current. Both slightly accelerated the inactivation time course of the Ba current. When diazepam was applied to the cells in combination with nicardipine, the observed inhibition agreed with a value predicted assuming independent blockade by diazepam and by nicardipine. These results suggest that diazepam inhibits Ca channels in a manner similar to nicardipine, but that the binding sites for diazepam are different from those for nicardipine.