Mutational analysis of VAMP domains implicated in Ca2+-induced insulin exocytosis.

Vesicle-associated membrane protein-2 (VAMP-2) and cellubrevin are associated with the membrane of insulin-containing secretory granules and of gamma-aminobutyric acid (GABA)-containing synaptic-like vesicles of pancreatic beta-cells. We found that a point mutation in VAMP-2 preventing targeting to synaptic vesicles also impairs the localization on insulin-containing secretory granules, suggesting a similar requirement for vesicular targeting. Tetanus toxin (TeTx) treatment of permeabilized HIT-T15 cells leads to the proteolytic cleavage of VAMP-2 and cellubrevin and causes the inhibition of Ca2+-triggered insulin exocytosis. Transient transfection of HIT-T15 cells with VAMP-1, VAMP-2 or cellubrevin made resistant to the proteolytic action of TeTx by amino acid replacements in the cleavage site restored Ca2+-stimulated secretion. Wild-type VAMP-2, wild-type cellubrevin or a mutant of VAMP-2 resistant to TeTx but not targeted to secretory granules were unable to rescue Ca2+-evoked insulin release. The transmembrane domain and the N-terminal region of VAMP-2 were not essential for the recovery of stimulated exocytosis, but deletions preventing the binding to SNAP-25 and/or to syntaxin I rendered the protein inactive in the reconstitution assay. Mutations of putative phosphorylation sites or of negatively charged amino acids in the SNARE motif recognized by clostridial toxins had no effect on the ability of VAMP-2 to mediate Ca2+-triggered secretion. We conclude that: (i) both VAMP-2 and cellubrevin can participate in the exocytosis of insulin; (ii) the interaction of VAMP-2 with syntaxin and SNAP-25 is required for docking and/or fusion of secretory granules with the plasma membrane; and (iii) the phosphorylation of VAMP-2 is not essential for Ca2+-stimulated insulin exocytosis.     

A tetanus toxin sensitive protein other than VAMP 2 is required for exocytosis in the pancreatic acinar cell

The neurotoxin sensitivity of regulated exocytosis in the pancreatic acinar cell was investigated using streptolysin-O permeabilized pancreatic acini. Treatment of permeabilized acini with botulinum toxin B (BoNT/B) or botulinum toxin D (BoNT/D) had no detectable effect on Ca(2+)-dependent amylase secretion but did result in the complete cleavage of VAMP 2. In comparison, tetanus toxin (TeTx) treatment both significantly inhibited Ca(2+)-dependent amylase secretion and cleaved VAMP 2. These results indicate that regulated exocytosis in the pancreatic acinar cell requires a tetanus toxin sensitive protein(s) other than VAMP 2.     

The glucose transporter (GLUT-4) and vesicle-associated membrane protein-2 (VAMP-2) are segregated from recycling endosomes in insulin- sensitive cells

Insulin stimulates glucose transport in adipocytes by translocation of the glucose transporter (GLUT-4) from an intracellular site to the cell surface. We have characterized different synaptobrevin/vesicle- associated membrane protein (VAMP) homologues in adipocytes and studied their intracellular distribution with respect to GLUT-4. VAMP-1, VAMP- 2, and cellubrevin cDNAs were isolated from a 3T3-L1 adipocyte expression library. VAMP-2 and cellubrevin were: (a) the most abundant isoforms in adipocytes, (b) detectable in all insulin responsive tissues, (c) translocated to the cell surface in response to insulin, and (d) found in immunoadsorbed GLUT-4 vesicles. To further define their intracellular distribution, 3T3-L1 adipocytes were incubated with a transferrin/HRP conjugate (Tf/HRP) and endosomes ablated following addition of DAB and H2O2. While this resulted in ablation of > 90% of the transferrin receptor (TfR) and cellubrevin found in intracellular membranes, 60% of GLUT-4 and 90% of VAMP-2 was not ablated. Immuno-EM on intracellular vesicles from adipocytes revealed that VAMP-2 was colocalized with GLUT-4, whereas only partial colocalization was observed between GLUT-4 and cellubrevin. These studies show that two different v-SNAREs, cellubrevin and VAMP-2, are partially segregated in different intracellular compartments in adipocytes, implying that they may define separate classes of secretory vesicles in these cells. We conclude that a proportion of GLUT-4 is found in recycling endosomes in nonstimulated adipocytes together with cellubrevin and the transferrin receptor. In addition, GLUT-4 and VAMP-2 are selectively enriched in a postendocytic compartment. Further study is required to elucidate the function of this latter compartment in insulin-responsive cells.     

Pancreatic acinar cells express vesicle-associated membrane protein 2- and 8-specific populations of zymogen granules with distinct and overlapping roles in secretion

Previous studies have demonstrated roles for vesicle-associated membrane protein 2 (VAMP 2) and VAMP 8 in Ca(2+)-regulated pancreatic acinar cell secretion, however, their coordinated function in the secretory pathway has not been addressed. Here we provide evidence using immunofluorescence microscopy, cell fractionation, and SNARE protein interaction studies that acinar cells contain two distinct populations of zymogen granules (ZGs) expressing either VAMP 2 or VAMP 8. Further, VAMP 8-positive granules also contain the synaptosome-associated protein 29, whereas VAMP 2-expressing granules do not. Analysis of acinar secretion by Texas red-dextran labeling indicated that VAMP 2-positive ZGs mediate the majority of exocytotic events during constitutive secretion and also participate in Ca(2+)-regulated exocytosis, whereas VAMP 8-positive ZGs are more largely involved in Ca(2+)-stimulated secretion. Previously undefined functional roles for VAMP and syntaxin isoforms in acinar secretion were established by introducing truncated constructs of these proteins into permeabilized acini. VAMP 2 and VAMP 8 constructs each attenuated Ca(2+)-stimulated exocytosis by 50%, whereas the neuronal VAMP 1 had no effects. In comparison, the plasma membrane SNAREs syntaxin 2 and syntaxin 4 each inhibited basal exocytosis, but only syntaxin 4 significantly inhibited Ca(2+)-stimulated secretion. Syntaxin 3, which is expressed on ZGs, had no effects. Collectively, these data demonstrate that individual acinar cells express VAMP 2- and VAMP 8-specific populations of ZGs that orchestrate the constitutive and Ca(2+)-regulated secretory pathways.     

Soluble NSF attachment protein receptors (SNAREs) in RBL-2H3 mast cells: functional role of syntaxin 4 in exocytosis and identification of a vesicle-associated membrane protein 8-containing secretory compartment

Mast cells upon stimulation through high affinity IgE receptors massively release inflammatory mediators by the fusion of specialized secretory granules (related to lysosomes) with the plasma membrane. Using the RBL-2H3 rat mast cell line, we investigated whether granule secretion involves components of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) machinery. Several isoforms of each family of SNARE proteins were expressed. Among those, synaptosome-associated protein of 23 kDa (SNAP23) was central in SNARE complex formation. Within the syntaxin family, syntaxin 4 interacted with SNAP23 and all vesicle-associated membrane proteins (VAMPs) examined, except tetanus neurotoxin insensitive VAMP (TI-VAMP). Overexpression of syntaxin 4, but not of syntaxin 2 nor syntaxin 3, caused inhibition of FcepsilonRI-dependent exocytosis. Four VAMP proteins, i.e., VAMP2, cellubrevin, TI-VAMP, and VAMP8, were present on intracellular membrane structures, with VAMP8 residing mainly on mediator-containing secretory granules. We suggest that syntaxin 4, SNAP23, and VAMP8 may be involved in regulation of mast cell exocytosis. Furthermore, these results are the first demonstration that the nonneuronal VAMP8 isoform, originally localized on early endosomes, is present in a regulated secretory compartment.     

Cleavage of SNAP-25 and VAMP-2 impairs store-operated Ca2+ entry in mouse pancreatic acinar cells

We recently reported that store-operated Ca²⁺ entry (SOCE) in nonexcitable cells is likely to be mediated by a reversible interaction between Ca²⁺ channels in the plasma membrane and the endoplasmic reticulum, a mechanism known as "secretion-like coupling." As for secretion, in this model the actin cytoskeleton plays a key regulatory role. In the present study we have explored the involvement of the secretory proteins synaptosome-associated protein (SNAP-25) and vesicle-associated membrane protein (VAMP) in SOCE in pancreatic acinar cells. Cleavage of SNAP-25 and VAMPs by treatment with botulinum toxin A (BoNT A) and tetanus toxin (TeTx), respectively, effectively inhibited amylase secretion stimulated by the physiological agonist CCK-8. BoNT A significantly reduced Ca²⁺ entry induced by store depletion using thapsigargin or CCK-8. In addition, treatment with BoNT A once SOCE had been activated reduced Ca²⁺ influx, indicating that SNAP-25 is needed for both the activation and maintenance of SOCE in pancreatic acinar cells. VAMP-2 and VAMP-3 are expressed in mouse pancreatic acinar cells. Both proteins associate with the cytoskeleton upon Ca²⁺ store depletion, although only VAMP-2 seems to be sensitive to TeTx. Treatment of pancreatic acinar cells with TeTx reduced the activation of SOCE without affecting its maintenance. These findings support a role for SNAP-25 and VAMP-2 in the activation of SOCE in pancreatic acinar cells and show parallels between this process and secretion in a specialized secretory cell type. synaptosome-associated protein; vesicle-associated membrane protein; pancreatic acinar cells; cytoskeleton; calcium entry     

Identification of the amino acid residues rendering TI-VAMP insensitive toward botulinum neurotoxin B

Botulinum neurotoxins types B, D, F, and G, and tetanus neurotoxin inhibit vesicular fusion via proteolytic cleavage of VAMP/Synaptobrevin, a core component of the membrane fusion machinery. Thus, these neurotoxins became widely used tools for investigating vesicular trafficking routes. Except for VAMP-1, VAMP-2, and Cellubrevin, no other member of the VAMP family represents a substrate for these neurotoxins. The molecular basis for this discrepancy is not known. A 34 amino acid residue segment of VAMP-2 was previously suggested to mediate the interaction with botulinum neurotoxin B, but the validity of the data was later questioned. To check whether this segment alone controls the susceptibility toward botulinum neurotoxin B, it was used to replace the corresponding segment in TI-VAMP. The resulting VAMP hybrid and VAMP-2 were hydrolysed at virtually identical rates. Resetting the VAMP-2 portion in the hybrid from either end to TI-VAMP residues gradually reduced the cleavability. A hybrid encompassing merely the VAMP-2 segment 71-80 around the Gln76/Phe77 scissile bond was still hydrolysed, albeit at a approximately tenfold lower cleavage rate. The contribution of each non-conserved amino acid of the whole 34-mer segment to the interaction was investigated employing VAMP-2. We find that the eight non-conserved residues of the 71-80 segment are all necessary for efficient cleavage. Mutation of an additional six residues located upstream and downstream of this segment affects substrate hydrolysis as well. Vice versa, a readily cleavable TI-VAMP molecule requires at the least the replacement of Ile158, Thr161, and the section 165-174 by Asp64, Ala67, and the 71-80 segment of VAMP-2, respectively. However, the insensitivity of TI-VAMP to botulinum neurotoxin B relies on at least 12 amino acid changes versus VAMP-2. These are scattered along an interface of 22 amino acid residues in length.     

Evidence for a vesicle-mediated maintenance of store-operated calcium channels in a human embryonic kidney cell line

Direct microinjection of the clostridial neurotoxins botulinum neurotoxin A light chain or tetanus neurotoxin into cells of a human embryonic kidney cell line significantly reduced calcium entry after depletion of internal calcium stores by cyclopiazonic acid, a reversible inhibitor of the sarcoplasmic-endoplasmic reticular calcium-ATPases. Botulinum neurotoxin A light chain specifically hydrolyzes a synaptosomal-associated protein of 25 kilodaltons (SNAP-25), and tetanus neurotoxin specifically hydrolyzes synaptobrevin-2 (vesicle-associated membrane protein 2, VAMP-2) and cellubrevin (vesicle-associated membrane protein 3, VAMP-3). Since these substrate proteins are required for vesicle docking and fusion, inhibition of store-operated calcium entry by botulinum neurotoxin A light chain and tetanus neurotoxin supports a model in which vesicle fusion is a prerequisite for activation of store-operated calcium entry. Brefeldin A, a fungal metabolite that interferes with vesicle traffic, partially reduced calcium entry following store depletion. The size of the reserve pool of vesicles or parallel vesicle recycling pathways employing brefeldin A-sensitive and brefeldin A-insensitive ADP-ribosylation factors may explain the failure of brefeldin A to completely inhibit store-operated calcium entry.     

Oleic acid interacts with GPR40 to induce Ca2+ signaling in rat islet beta-cells: mediation by PLC and L-type Ca2+ channel and link to insulin release

It has long been thought that long-chain free fatty acids (FFAs) stimulate insulin secretion via mechanisms involving their metabolism in pancreatic beta-cells. Recently, it was reported that FFAs function as endogenous ligands for GPR40, a G protein-coupled receptor, to amplify glucose-stimulated insulin secretion in an insulinoma cell line and rat islets. However, signal transduction mechanisms for GPR40 in beta-cells are little known. The present study was aimed at elucidating GPR40-linked Ca(2+) signaling mechanisms in rat pancreatic beta-cells. We employed oleic acid (OA), an FFA that has a high affinity for the rat GPR40, and examined its effect on cytosolic Ca(2+) concentration ([Ca(2+)](i)) in single beta-cells by fura 2 fluorescence imaging. OA at 1-10 microM concentration-dependently increased [Ca(2+)](i) in the presence of 5.6, 8.3, and 11.2 mM, but not 2.8 mM, glucose. OA-induced [Ca(2+)](i) increases at 11.2 mM glucose were inhibited in beta-cells transfected with small interfering RNA targeted to rat GPR40 mRNA. OA-induced [Ca(2+)](i) increases were also inhibited by phospholipase C (PLC) inhibitors, U73122 and neomycin, Ca(2+)-free conditions, and an L-type Ca(2+) channel blocker, nitrendipine. Furthermore, OA increased insulin release from isolated islets at 8.3 mM glucose, and it was markedly attenuated by PLC and L-type Ca(2+) channel inhibitors. These results demonstrate that OA interacts with GPR40 to increase [Ca(2+)](i) via PLC- and L-type Ca(2+) channel-mediated pathway in rat islet beta-cells, which may be link to insulin release.     

Depolarizing stimuli reduce Ca(2+)/calmodulin-dependent protein kinase II activity in islets of Langerhans

Elevations in intracellular Ca(2+) ([Ca(2+)](i)) initiate insulin secretion from pancreatic beta-cells, but the secretory responses become rapidly desensitised to maintained elevations in [Ca(2+)](i). We have investigated the mechanisms underlying the Ca(2+) desensitization of insulin secretion using electrically permeabilized rat islets of Langerhans. Measurements of Ca(2+)/calmodulin-dependent protein kinase II (CaMK II) enzyme activity and immunoreactivity in permeabilized islets demonstrated Ca(2+)-induced reductions in enzyme activity which could not be attributed to reductions in CaMK II immunoreactive protein. Measurements in intact islets demonstrated that the Ca(2+)-induced reduction of CaMK II activity was also operative in intact cells, suggesting that this mechanism may have pathophysiological implications for beta-cell function.     

SNARE proteins are critical for regulated exocytosis of ECP from human eosinophils

The SNARE hypothesis, describing a protein assembly-disassembly pathway, was recently proposed for the sequential steps of synaptic vesicle docking, activation and fusion. To determine if SNARE proteins are involved in regulated exocytosis in eosinophils, the presence and functional role of SNAREs was examined in human blood eosinophils. Immunoblotting, subcellular fractionation, and immunocytochemistry documented that vesicle-associated membrane protein-2 (VAMP-2), a vesicle-SNARE, was expressed in human eosinophils. Syntaxin 4 and SNAP-25 were also detected. Sequencing of cloned RT-PCR products amplified from a domain conserved among VAMP isoforms revealed identity only to VAMP-2 but not to VAMP-1 or cellubrevin. Functional experiments revealed that tetanus toxin pretreatment, which cleaved VAMP-2 in eosinophils, significantly inhibited both IgE receptor- and phorbol ester-mediated exocytosis of eosinophil cationic protein (ECP) from streptolysin-O-permeabilized eosinophils. Thus, these results strongly suggest a critical role of SNAREs in regulated exocytosis in eosinophils.     

Inhibition of calpain blocks pancreatic beta-cell spreading and insulin secretion

In addition to promoting insulin secretion, an increase in cytosolic Ca(2+) triggered by glucose has been shown to be crucial for spreading of beta-cells attached on extracellular matrix (804G matrix). Calpains are Ca(2+)-dependent cysteine proteases involved in an extended spectrum of cellular responses, including cytoskeletal rearrangements and vesicular trafficking. The present work aimed to assess whether calpain is also implicated in the process of Ca(2+)-induced insulin secretion and spreading of rat pancreatic beta-cells. The results indicate calpain dependency of beta-cell spreading on 804G matrix. Indeed, treatment with three distinct calpain inhibitors (N-Ac-Leu-Leu-norleucinal, calpeptin, and ethyl(+)-(2S,3S)-3-[(S)-3-methyl-1-(3-methylbutylcarbamoyl)butyl-carbamoyl]-2-ox-iranecarboxylate) inhibited cell spreading induced by glucose and KCl, whereas cell attachment was not significantly modified. Calpain inhibitors also suppressed glucose- and KCl-stimulated insulin secretion without affecting insulin synthesis. Washing the inhibitor out of the cell culture restored spreading on 804G matrix and insulin secretory response after 24 h. In addition, incubation with calpeptin did not affect insulin secretory response to mastoparan that acts on exocytosis downstream of intracellular calcium [Ca(2+)]i. Finally, calpeptin was shown to affect the [Ca(2+)]i response to glucose but not to KCl. In summary, the results show that inhibition of calpain blocks spreading and insulin secretion of primary pancreatic beta-cells. It is therefore suggested that calpain could be a mediator of Ca(2+)-induced-insulin secretion and beta-cell spreading.     

Spatial organization of Ca(2+) entry and exocytosis in mouse pancreatic beta-cells

Secretion from single pancreatic beta-cells was imaged using a novel technique in which Zn(2+), costored in secretory granules with insulin, was detected by confocal fluorescence microscopy as it was released from the cells. Using this technique, it was observed that secretion from beta-cells was limited to an active region that comprised approximately 50% of the cell perimeter. Using ratiometric imaging with indo-1, localized increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) evoked by membrane depolarization were also observed. Using sequential measurements of secretion and [Ca(2+)](i) at single cells, colocalization of exocytotic release sites and Ca(2+) entry was observed when cells were stimulated by glucose or K(+). Treatment of cells with the Ca(2+) ionophore 4-Br-A23187 induced large Ca(2+) influx around the entire cell circumference. Despite the nonlocalized increase in [Ca(2+)](i), secretion evoked by 4-Br-A23187 was still localized to the same region as that evoked by secretagogues such as glucose. It is concluded that Ca(2+) channels activated by depolarization are localized to specific membrane domains where exocytotic release also occurs; however, localized secretion is not exclusively regulated by localized increases in [Ca(2+)](i), but instead involves spatial localization of other components of the exocytotic machinery.     

Protein kinase C-dependent and -independent inhibition of Ca(2+) influx by phorbol ester in rat pancreatic beta-cells

Phorbol esters were used to investigate the action of protein kinase C (PKC) on insulin secretion from pancreatic beta-cells. Application of 80 nM phorbol 12-myristate 13-acetate (PMA), a PKC-activating phorbol ester, had little effect on glucose (15 mM)-induced insulin secretion from intact rat islets. In islets treated with bisindolylmaleimide (BIM), a PKC inhibitor, PMA significantly reduced the glucose-induced insulin secretion. PMA decreased the level of intracellular Ca(2+) concentration ([Ca(2+)](i)) elevated by the glucose stimulation when tested in isolated rat beta-cells. This inhibitory effect of PMA was not prevented by BIM. PMA inhibited glucose-induced action potentials, and this effect was not prevented by BIM. Further, 4alpha-phorbol 12,13-didecanoate (4alpha-PDD), a non-PKC-activating phorbol ester, produced an effect similar to PMA. In the presence of nifedipine, the glucose stimulation produced only depolarization, and PMA applied on top of glucose repolarized the cell. When applied at the resting state, PMA hyperpolarized beta-cells with an increase in the membrane conductance. Recorded under the voltage-clamp condition, PMA reduced the magnitude of Ca(2+) currents through L-type Ca(2+) channels. BIM prevented the PMA inhibition of the Ca(2+) currents. These results suggest that activation of PKC maintains glucose-stimulated insulin secretion in pancreatic beta-cells, defeating its own inhibition of the Ca(2+) influx through L-type Ca(2+) channels. PKC-independent inhibition of electrical excitability by phorbol esters was also demonstrated.     

Dephosphorylation of beta2-syntrophin and Ca2+/mu-calpain-mediated cleavage of ICA512 upon stimulation of insulin secretion

Islet cell autoantigen (ICA) 512 is a receptor-tyrosine phosphatase-like protein associated with the secretory granules of neuroendocrine cells, including pancreatic beta-cells. Binding of its cytoplasmic tail to beta2-syntrophin suggests that ICA512 connects secretory granules to the utrophin complex and the actin cytoskeleton. Here we show that stimulation of insulin secretion from INS-1 cells triggers the biosynthesis of pro-ICA512 and the degradation of its mature form. Inhibition of calpain, which is activated upon stimulation of insulin secretion, prevents the Ca2+-dependent proteolysis of ICA512. In vitro mu-calpain cleaves ICA512 between a putative PEST domain and the beta2-syntrophin binding site, whereas binding of ICA512 to beta2-syntrophin protects the former from cleavage. beta2-syntrophin and its F-actin-binding protein utrophin are enriched in subcellular fractions containing secretory granules. ICA512 preferentially binds phospho-beta2-syntrophin and stimulation of insulin secretion induces the Ca2+-dependent, okadaic acid-sensitive dephosphorylation of beta2-syntrophin. Similarly to calpeptin, okadaic acid inhibits ICA512 proteolysis and insulin secretion. Thus, stimulation of insulin secretion might promote the mobilization of secretory granules by inducing the dissociation of ICA512 from beta2-syntrophin-utrophin complexes and the cleavage of the ICA512 cytoplasmic tail by mu-calpain.     

Disruption of pancreatic beta-cell lipid rafts modifies Kv2.1 channel gating and insulin exocytosis

In pancreatic beta-cells, the predominant voltage-gated Ca(2+) channel (Ca(V)1.2) and K(+) channel (K(V)2.1) are directly coupled to SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor) proteins. These SNARE proteins modulate channel expression and gating and closely associate these channels with the insulin secretory vesicles. We show that K(V)2.1 and Ca(V)1.2, but not K(V)1.4, SUR1, or Kir6.2, target to specialized cholesterol-rich lipid raft domains on beta-cell plasma membranes. Similarly, the SNARE proteins syntaxin 1A, SNAP-25, and VAMP-2, but not Munc-13-1 or n-Sec1, are associated with lipid rafts. Disruption of the lipid rafts by depleting membrane cholesterol with methyl-beta-cyclodextrin shunts K(V)2.1, Ca(V)1.2, and SNARE proteins out of lipid rafts. Furthermore, methyl-beta-cyclodextrin inhibits K(V)2.1 but not Ca(V)1.2 channel activity and enhances single-cell exocytic events and insulin secretion. Membrane compartmentalization of ion channels and SNARE proteins in lipid rafts may be critical for the temporal and spatial coordination of insulin release, forming what has been described as the excitosome complex.     

New insights concerning the glucose-dependent insulin secretagogue action of glucagon-like peptide-1 in pancreatic beta-cells.

The GLP-1 receptor is a Class B heptahelical G-protein-coupled receptor that stimulates cAMP production in pancreatic beta-cells. GLP-1 utilizes this receptor to activate two distinct classes of cAMP-binding proteins: protein kinase A (PKA) and the Epac family of cAMP-regulated guanine nucleotide exchange factors (cAMPGEFs). Actions of GLP-1 mediated by PKA and Epac include the recruitment and priming of secretory granules, thereby increasing the number of granules available for Ca(2+)-dependent exocytosis. Simultaneously, GLP-1 promotes Ca(2+) influx and mobilizes an intracellular source of Ca(2+). GLP-1 sensitizes intracellular Ca(2+) release channels (ryanodine and IP (3) receptors) to stimulatory effects of Ca(2+), thereby promoting Ca(2+)-induced Ca(2+) release (CICR). In the model presented here, CICR activates mitochondrial dehydrogenases, thereby upregulating glucose-dependent production of ATP. The resultant increase in cytosolic [ATP]/[ADP] concentration ratio leads to closure of ATP-sensitive K(+) channels (K-ATP), membrane depolarization, and influx of Ca(2+) through voltage-dependent Ca(2+) channels (VDCCs). Ca(2+) influx stimulates exocytosis of secretory granules by promoting their fusion with the plasma membrane. Under conditions where Ca(2+) release channels are sensitized by GLP-1, Ca(2+) influx also stimulates CICR, generating an additional round of ATP production and K-ATP channel closure. In the absence of glucose, no "fuel" is available to support ATP production, and GLP-1 fails to stimulate insulin secretion. This new "feed-forward" hypothesis of beta-cell stimulus-secretion coupling may provide a mechanistic explanation as to how GLP-1 exerts a beneficial blood glucose-lowering effect in type 2 diabetic subjects.     

Synchronization of Ca(2+)-signals within insulin-secreting pseudoislets: effects of gap-junctional uncouplers

The secretory response of the intact islet is greater than the response of individual beta-cells in isolation, and functional coupling between cells is critical in insulin release. The changes in intracellular Ca(2+)([Ca(2+)](i)) which initiate insulin secretory responses are synchronized between groups of cells within the islet, and gap-junctions are thought to play a central role in coordinating signalling events. We have used the MIN6 insulin-secreting cell line, to examine whether uncoupling gap-junctions alters the synchronicity of nutrient- and non-nutrient-evoked Ca(2+)oscillations, or affects insulin secretion. MIN6 cells express mRNA species that can be amplified using PCR primers for connexin 36. A commonly used gap-junctional inhibitor, heptanol, inhibited glucose- and tolbutamide-induced Ca(2+)-oscillations to basal levels in MIN6 cell clusters at concentrations of 0.5 mM and greater, and it had similar effects in pseudoislets when used at 2.5 mM. Lower heptanol concentrations altered the frequency of Ca(2+)transients without affecting their synchronicity, in both monolayers and pseudoislets. Heptanol also had effects on insulin secretion from MIN6 pseudoislets such that 1 mM enhanced secretion while 2.5 mM was inhibitory. These data suggest that heptanol has multiple effects in pancreatic beta-cells, none of which appears to be related to uncoupling of synchronicity of Ca(2+)signalling between cells. A second gap-junction uncoupler, 18 alpha-glycyrrhetinic acid, also failed to uncouple synchronized Ca(2+)-oscillations, and it had no effect on insulin secretion. These data provide evidence that Ca(2+)signalling events occur simultaneously across the bulk mass of the pseudoislet, and suggest that gap-junctions are not required to coordinate the synchronicity of these events, nor is communication via gap junctions essential for integrated insulin secretory responses.