| 1. |
- Braun, Matthias, et al.
(författare)
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Corelease and differential exit via the fusion pore of GABA, serotonin, and ATP from LDCV in rat pancreatic beta cells
- 2007
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Ingår i: Journal of General Physiology. - : Rockefeller University Press. - 0022-1295 .- 1540-7748. ; 129:3, s. 221-231
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Tidskriftsartikel (refereegranskat)abstract
- The release gamma-aminobutyric acid (GABA) and ATP from rat beta cells was monitored using an electrophysiological assay based on overexpression GABAA or P2X2 receptor ion channels. Exocytosis of LDCVs, detected by carbon fiber amperometry of serotonin, correlated strongly (similar to 80%) with ATP release. The increase in membrane capacitance per ATP release event was 3.4 fF, close to the expected capacitance of an individual LDCV with a diameter of 0.3 mu m. ATP and GABA were coreleased with serotonin with the same probability. Immunogold electron microscopy revealed that similar to 15% of the LDCVs contain GABA. Prespike "pedestals," reflecting exit of granule constituents via the fusion pore, were less frequently observed for ATP than for serotonin or GABA and the relative amplitude (amplitude of foot compared to spike) was smaller: in some cases the ATP-dependent pedestal was missing entirely. An inward tonic current, not dependent on glucose and inhibited by the GABAA receptor antagonist SR95531, was observed in beta cells in clusters of islet cells. Noise analysis indicated that it was due to the activity of individual channels with a conductance of 30 pS, the same as expected for individual GABA(A) Cl- channels with the ionic gradients used. We conclude that (a) LDCVs accumulate ATP and serotonin; (b) regulated release of GABA can be accounted for by exocytosis of a subset of insulin-containing LDCVs; (c) the fusion pore of LDCVs exhibits selectivity and compounds are differentially released depending on their chemical properties (including size); and (d) a glucose-independent nonvesicular form of GABA release exists in beta cells.
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| 2. |
- Eliasson, Lena, et al.
(författare)
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Novel aspects of the molecular mechanisms controlling insulin secretion
- 2008
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Ingår i: Journal of Physiology. - : Wiley. - 1469-7793 .- 0022-3751. ; 586:14, s. 3313-3324
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Forskningsöversikt (refereegranskat)abstract
- Pancreatic beta-cells secrete insulin by Ca2+-dependent exocytosis of secretory granules. beta-cell exocytosis involves SNARE (soluble NSF-attachment protein receptor) proteins similar to those controlling neurotransmitter release and depends on the close association of L-type Ca2+ channels and granules. In most cases, the secretory granules fuse individually but there is ultrastructural and biophysical evidence of multivesicular exocytosis. Estimates of the secretory rate in beta-cells in intact islets indicate a release rate of similar to 15 granules per beta-cell per second, 100-fold higher than that observed in biochemical assays. Single-vesicle capacitance measurements reveal that the diameter of the fusion pore connecting the granule lumen with the exterior is similar to 1.4 nm. This is considerably smaller than the size of insulin and membrane fusion is therefore not obligatorily associated with release of the cargo, a feature that may contribute to the different rates of secretion detected by the biochemical and biophysical measurements. However, small molecules like ATP and GABA, which are stored together with insulin in the granules, are small enough to be released via the narrow fusion pore, which accordingly functions as a molecular sieve. We finally consider the possibility that defective fusion pore expansion accounts for the decrease in insulin secretion observed in pathophysiological states including long-term exposure to lipids.
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| 3. |
- Galvanovskis, Juris, et al.
(författare)
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Probability of Exocytosis in Pancreatic β-Cells : Dependence on Ca2+ Sensing Latency Times, Ca2+ Channel Kinetic Parameters, and Channel Clustering
- 2008
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Ingår i: Biosimulation in Drug Development. - Weinheim, Germany : Wiley-VCH Verlag GmbH & Co. KGaA. - 9783527316991 ; , s. 299-311
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Bokkapitel (refereegranskat)abstract
- The fusion of secretory vesicles and granules with the cell membrane prior to the release of their content into the extracellular space requires a transient increase of free Ca2+ concentration in the vicinity of the fusion site. Usually there is a short temporal delay in the onset of the actual fusion of membranes with reference to the rising free Ca2+ levels. This delay is described as a latency time of the Ca2+-sensing system of the secretory machinery and has been observed in several cell types, including pancreatic β-cells. The presence of a delay time of a finite length inherent to the secretory machinery of the cell has an essential effect on the probability for a certain granule to fuse with the cell membrane and to release its contents into the extracellular space during the action potential. We investigate here, theoretically and by numerical simulations, the extent of this influence and its dependence on the parameters of Ca2+ channels, channel clustering, the Ca2+-sensing system, and the length of depolarizing pulses.We use a linear probabilistic model for a random opening and closing of channels that yields an explicit expression for the Laplace transforms of the waiting time distributions for an event that at least one channel is open during the latency time. This allows one in principle to calculate the probability that a vesicle will fuse with the cell membrane during the action potential. We compare our theoretical results with numerical simulatio
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| 4. |
- Kanno, Takahiro, et al.
(författare)
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Chaotic electrical activity of living beta-cells in the mouse pancreatic islet
- 2007
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Ingår i: Physica D: Nonlinear Phenomena. - : Elsevier BV. - 0167-2789. ; 226:2, s. 107-116
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Tidskriftsartikel (refereegranskat)abstract
- To test for chaotic dynamics of the insulin producing beta-cell and explore its biological role, we observed the action potentials with the perforated patch clamp technique, for isolated cells as well as for intact cells of the mouse pancreatic islet. The time series obtained were analyzed using nonlinear diagnostic algorithms associated with the surrogate method. The isolated cells exhibited short-term predictability and visible determinism, in the steady state response to 10 mM glucose, while the intact cells did not. In the latter case, determinism became visible after the application of a gap junction inhibitor. This tendency was enhanced by the stimulation with tolbutamide. Our observations suggest that, thanks to the integration of individual chaotic dynamics via gap junction coupling, the beta-cells will lose memory of fluctuations occurring at any instant in their electrical activity more rapidly with time. This is likely to contribute to the functional stability of the islet against uncertain perturbations. (c) 2006 Elsevier B.V All rights reserved.
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| 5. |
- MacDonald, Patrick, et al.
(författare)
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Regulated Exocytosis and Kiss-and-Run of Synaptic-Like Microvesicles in INS-1 and Primary Rat {beta}-Cells.
- 2005
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Ingår i: Diabetes. - : American Diabetes Association. - 1939-327X .- 0012-1797. ; 54:3, s. 736-743
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Tidskriftsartikel (refereegranskat)abstract
- We have applied cell-attached capacitance measurements to investigate whether synaptic-like microvesicles (SLMVs) undergo regulated exocytosis in insulinoma and primary pancreatic beta-cells. SLMV and large dense-core vesicle (LDCV) exocytosis was increased 1.6- and 2.4-fold upon stimulation with 10 mmol/l glucose in INS-1 cells. Exocytosis of both types of vesicles was coupled to Ca(2+) entry through l-type channels. Thirty percent of SLMV exocytosis in INS-1 and rat beta-cells was associated with transient capacitance increases consistent with kiss-and-run. Elevation of intracellular cAMP (5 micromol/l forskolin) increased SLMV exocytosis 1.6-fold and lengthened the duration of kiss-and-run events in rat beta-cells. Experiments using isolated inside-out patches of INS-1 cells revealed that the readily releasable pool (RRP) of SLMVs preferentially undergoes kiss-and-run exocytosis (67%), is proportionally larger than the LDCV RRP, and is depleted more quickly upon Ca(2+) stimulation. We conclude that SLMVs undergo glucose-regulated exocytosis and are capable of high turnover. Following kiss-and-run exocytosis, the SLMV RRP may be reloaded with gamma-aminobutyric acid and undergo several cycles of exo- and endocytosis. Our observations support a role for beta-cell SLMVs in a synaptic-like function of rapid intra-islet signaling.
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| 6. |
- Meidute, Sandra, et al.
(författare)
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Palmitate-induced beta-cell dysfunction is associated with excessive NO pro-duction and is reversed by thiazolidinedione-mediated inhibition of GPR40 transduction mechanisms
- 2008
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). ; 3:5
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Tidskriftsartikel (populärvet., debatt m.m.)abstract
- Background: Type 2 diabetes often displays hyperlipidemia. We examined palmitate effects on pancreatic islet function in relation to FFA receptor GPR40, NO generation, insulin release, and the PPARgama agonistic thiazolidinedione, rosiglitazone. Principal findings: Rosiglitazone suppressed acute palmitate-stimulated GPR40-transduced PI hydrolysis in HEK293 cells and insulin release from MIN6c cells and mouse islets. Culturing islets 24 h with palmitate at 5 mmol/l glucose induced beta-cell iNOS expression as revealed by confocal microscopy and in-creased the activities of ncNOS and iNOS associated with suppression of glucose-stimulated insulin response. Rosiglitazone reversed these effects. The expression of iNOS after high-glucose culturing was unaffected by rosiglitazone. Downregulation of GPR40 by antisense treatment abrogated GPR40 expression and suppressed palmitate-induced iNOS activity and insulin release. Conclusion: We conclude that, in addition to mediating acute FFA-stimulated insulin release, GPR40 is an important regulator of iNOS expression and dysfunctional insulin release during long-term exposure to FFA. The adverse effects of palmitate were counteracted by rosiglitazone at GPR40, suggesting that thiazolidinediones are beneficial for beta-cell function in hyperlipidemic type 2 diabetes.
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| 7. |
- Obermüller, Stefanie, et al.
(författare)
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Selective nucleotide-release from dense-core granules in insulin-secreting cells.
- 2005
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Ingår i: Journal of Cell Science. - : The Company of Biologists. - 0021-9533 .- 1477-9137. ; 118:Pt 18, s. 4271-4282
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Tidskriftsartikel (refereegranskat)abstract
- Secretory granules of insulin-secreting cells are used to store and release peptide hormones as well as low-molecular-weight compounds such as nucleotides. Here we have compared the rate of exocytosis with the time courses of nucleotide and peptide release by a combination of capacitance measurements, electrophysiological detection of ATP release and single-granule imaging. We demonstrate that the release of nucleotides and peptides is delayed by similar to 0.1 and similar to 2 seconds with respect to membrane fusion, respectively. We further show that in up to 70% of the cases exocytosis does not result in significant release of the peptide cargo, likely because of a mechanism that leads to premature closure of the fusion pore. Release of nucleotides and protons occurred regardless of whether peptides were secreted or not. These observations suggest that insulin-secreting cells are able to use the same secretory vesicles to release small molecules either alone or together with the peptide hormone.
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| 8. |
- Olofsson, Charlotta S, 1971, et al.
(författare)
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Impaired insulin exocytosis in neural cell adhesion molecule-/- mice due to defective reorganization of the submembrane F-actin network.
- 2009
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Ingår i: Endocrinology. - : The Endocrine Society. - 1945-7170 .- 0013-7227. ; 150:7, s. 3067-75
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Tidskriftsartikel (refereegranskat)abstract
- The neural cell adhesion molecule (NCAM) is required for cell type segregation during pancreatic islet organogenesis. We have investigated the functional consequences of ablating NCAM on pancreatic beta-cell function. In vivo, NCAM(-/-) mice exhibit impaired glucose tolerance and basal hyperinsulinemia. Insulin secretion from isolated NCAM(-/-) islets is enhanced at glucose concentrations below 15 mM but inhibited at higher concentrations. Glucagon secretion from pancreatic alpha-cells evoked by low glucose was also severely impaired in NCAM(-/-) islets. The diminution of insulin secretion is not attributable to defective glucose metabolism or glucose sensing (documented as glucose-induced changes in intracellular Ca(2+) and K(ATP)-channel activity). Resting K(ATP) conductance was lower in NCAM(-/-) beta-cells than wild-type cells, and this difference was abolished when F-actin was disrupted by cytochalasin D (1 muM). In wild-type beta-cells, the submembrane actin network disassembles within 10 min during glucose stimulation (30 mM), an effect not seen in NCAM(-/-) beta-cells. Cytochalasin D eliminated this difference and normalized insulin and glucagon secretion in NCAM(-/-) islets. Capacitance measurements of exocytosis indicate that replenishment of the readily releasable granule pool is suppressed in NCAM(-/-) alpha- and beta-cells. Our data suggest that remodeling of the submembrane actin network is critical to normal glucose regulation of both insulin and glucagon secretion.
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| 9. |
- Shimomura, Kenju, et al.
(författare)
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Insulin secretion from beta-cells is affected by deletion of nicotinamide nucleotide transhydrogenase.
- 2009
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Ingår i: Methods in enzymology. - 1557-7988. ; 457, s. 451-80
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Tidskriftsartikel (refereegranskat)abstract
- Nicotinamide nucleotide transhydrogenase (NNT) is an inner mitochondrial membrane transmembrane protein involved in regenerating NADPH, coupled with proton translocation across the inner membrane. We have shown that a defect in Nnt function in the mouse, and specifically within the beta-cell, leads to a reduction in insulin secretion. This chapter describes methods for examining Nnt function in the mouse. This includes generating in vivo models with point mutations and expression of Nnt by transgenesis, and making in vitro models, by silencing of gene expression. In addition, techniques are described to measure insulin secretion, calcium and hydrogen peroxide concentrations, membrane potential, and NNT activity. These approaches and techniques can also be applied to other genes of interest.
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| 10. |
- Zhang, Quan, et al.
(författare)
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Cell coupling in mouse pancreatic beta-cells measured in intact islets of Langerhans
- 2008
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Ingår i: Philosophical Transactions of the Royal Society A: Mathematical, Physical and Engineering Science. - : The Royal Society. - 1364-503X .- 1471-2962. ; 366:1880, s. 3503-3523
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Tidskriftsartikel (refereegranskat)abstract
- The perforated whole-cell configuration of the patch-clamp technique was applied to functionally identified beta-cells in intact mouse pancreatic islets to study the extent of cell coupling between adjacent beta-cells. Using a combination of current- and voltage-clamp recordings, the total gap junctional conductance between beta-cells in an islet was estimated to be 1.22 nS. The analysis of the current waveforms in a voltage-clamped cell ( due to the. ring of an action potential in a neighbouring cell) suggested that the gap junctional conductance between a pair of beta-cells was 0.17 nS. Subthreshold voltage-clamp depolarization (to -55 mV) gave rise to a slow capacitive current indicative of coupling between beta-cells, but not in non-beta-cells, with a time constant of 13.5 ms and a total charge movement of 0.2 pC. Our data suggest that a superficial beta-cell in an islet is in electrical contact with six to seven other beta-cells. No evidence for dye coupling was obtained when cells were dialysed with Lucifer yellow even when electrical coupling was apparent. The correction of the measured resting conductance for the contribution of the gap junctional conductance indicated that the whole-cell K-ATP channel conductance (G(K,ATP)) falls from approximately 2.5 nS in the absence of glucose to 0.1 nS at 15 mM glucose with an estimated IC50 of approximately 4 mM. Theoretical considerations indicate that the coupling between beta-cells within the islet is sufficient to allow propagation of [Ca2+](i) waves to spread with a speed of approximately 80 mu m s(-1), similar to that observed experimentally in confocal [Ca2+](i) imaging.
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