| 1. |
- Hoppa, M. B., et al.
(författare)
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Multivesicular exocytosis in rat pancreatic beta cells
- 2012
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Ingår i: Diabetologia. - : Springer Science and Business Media LLC. - 1432-0428 .- 0012-186X. ; 55:4, s. 1001-1012
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Tidskriftsartikel (refereegranskat)abstract
- To establish the occurrence, modulation and functional significance of compound exocytosis in insulin-secreting beta cells. Exocytosis was monitored in rat beta cells by electrophysiological, biochemical and optical methods. The functional assays were complemented by three-dimensional reconstruction of confocal imaging, transmission and block face scanning electron microscopy to obtain ultrastructural evidence of compound exocytosis. Compound exocytosis contributed marginally (< 5% of events) to exocytosis elicited by glucose/membrane depolarisation alone. However, in beta cells stimulated by a combination of glucose and the muscarinic agonist carbachol, 15-20% of the release events were due to multivesicular exocytosis, but the frequency of exocytosis was not affected. The optical measurements suggest that carbachol should stimulate insulin secretion by similar to 40%, similar to the observed enhancement of glucose-induced insulin secretion. The effects of carbachol were mimicked by elevating [Ca2+](i) from 0.2 to 2 mu mol/l Ca2+. Two-photon sulforhodamine imaging revealed exocytotic events about fivefold larger than single vesicles and that these structures, once formed, could persist for tens of seconds. Cells exposed to carbachol for 30 s contained long (1-2 mu m) serpentine-like membrane structures adjacent to the plasma membrane. Three-dimensional electron microscopy confirmed the existence of fused multigranular aggregates within the beta cell, the frequency of which increased about fourfold in response to stimulation with carbachol. Although contributing marginally to glucose-induced insulin secretion, compound exocytosis becomes quantitatively significant under conditions associated with global elevation of cytoplasmic calcium. These findings suggest that compound exocytosis is a major contributor to the augmentation of glucose-induced insulin secretion by muscarinic receptor activation.
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| 2. |
- Barg, Sebastian, et al.
(författare)
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Tight coupling between electrical activity and exocytosis in mouse glucagon-secreting alpha-cells
- 2000
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Ingår i: Diabetes. - 1939-327X .- 0012-1797. ; 49:9, s. 1500-1510
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Tidskriftsartikel (refereegranskat)abstract
- alpha-Cells were identified in preparations of dispersed mouse islets by immunofluorescence microscopy. A high fraction of alpha-cells correlated with a small cell size measured as the average cell diameter (10 microm) and whole-cell capacitance (<4 pF). The alpha-cells generated action potentials at a low frequency (1 Hz) in the absence of glucose. These action potentials were reversibly inhibited by elevation of the glucose concentration to 20 mmol/l. The action potentials originated from a membrane potential more negative than -50 mV, had a maximal upstroke velocity of 5 V/s, and peaked at +1 mV. Voltage-clamp experiments revealed the ionic conductances underlying the generation of action potentials. alpha-Cells are equipped with a delayed tetraethyl-ammonium-blockable outward current (activating at voltages above -20 mV), a large tetrodotoxin-sensitive Na+ current (above -30 mV; peak current 200 pA at +10 mV), and a small Ca2+ current (above -50 mV; peak current 30 pA at +10 mV). The latter flowed through omega-conotoxin GVIA (25%)- and nifedipine-sensitive (50%) Ca(2+)-channels. Mouse alpha-cells contained, on average, 7,300 granules, which undergo Ca(2+)-induced exocytosis when the alpha-cell is depolarized. Three functional subsets of granules were identified, and the size of the immediately releasable pool was estimated as 80 granules, or 1% of the total granule number. The maximal rate of exocytosis (1.5 pF/s) was observed 21 ms after the onset of the voltage-clamp depolarization, which is precisely the duration of Ca(2+)-influx during an action potential. Our results suggest that the secretory machinery of the alpha-cell is optimized for maximal efficiency in the use of Ca2+ for exocytosis.
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| 3. |
- Collins, S. C., et al.
(författare)
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Increased Expression of the Diabetes Gene SOX4 Reduces Insulin Secretion by Impaired Fusion Pore Expansion
- 2016
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Ingår i: Diabetes. - : American Diabetes Association. - 0012-1797 .- 1939-327X. ; 65:7, s. 1952-1961
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Tidskriftsartikel (refereegranskat)abstract
- The transcription factor Sox4 has been proposed to underlie the increased type 2 diabetes risk linked to an intronic single nucleotide polymorphism in CDKAL1. In a mouse model expressing a mutant form of Sox4, glucose-induced insulin secretion is reduced by 40% despite normal intracellular Ca2+ signaling and depolarization-evoked exocytosis. This paradox is explained by a fourfold increase in kiss-and-run exocytosis (as determined by single-granule exocytosis measurements) in which the fusion pore connecting the granule lumen to the exterior expands to a diameter of only 2 nm, which does not allow the exit of insulin. Microarray analysis indicated that this correlated with an increased expression of the exocytosis-regulating protein Stxbp6. In a large collection of human islet preparations (n = 63), STXBP6 expression and glucose induced insulin secretion correlated positively and negatively with SOX4 expression, respectively. Overexpression of SOX4 in the human insulin-secreting cell EndoC-beta H2 interfered with granule emptying and inhibited hormone release, the latter effect reversed by silencing STXBP6. These data suggest that increased SOX4 expression inhibits insulin secretion and increased diabetes risk by the upregulation of STXBP6 and an increase in kiss- and-run exocytosis at the expense of full fusion. We propose that pharmacological interventions promoting fusion pore expansion may be effective in diabetes therapy.
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| 4. |
- Tarasov, A. I., et al.
(författare)
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Monitoring real-time hormone release kinetics: Via high-content 3-D imaging of compensatory endocytosis
- 2018
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Ingår i: Lab on a Chip. - : Royal Society of Chemistry (RSC). - 1473-0197 .- 1473-0189. ; 18:18, s. 2838-2848
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Tidskriftsartikel (refereegranskat)abstract
- High-content real-time imaging of hormone secretion in tissues or cell populations is a challenging task, which is unlikely to be resolved directly, despite immense translational value. We approach this problem indirectly, using compensatory endocytosis, a process that closely follows exocytosis in the cell, as a surrogate read-out for secretion. The tissue is immobilized in an open-air perifusion chamber and imaged using a two-photon microscope. A fluorescent polar tracer, perifused through the experimental circuit, gets trapped into the cells via endocytosis, and is quantified using a feature-detection algorithm. The signal of the tracer that accumulates into the endocytotic system reliably reflects stimulated exocytosis, which is demonstrated via co-imaging of the latter using existing reporters. A high signal-to-noise ratio and compatibility with multisensor imaging affords the real-time quantification of the secretion at the tissue/population level, whereas the cumulative nature of the signal allows imprinting of the “secretory history” within each cell. The technology works for several cell types, reflects disease progression and can be used for human tissue.
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| 5. |
- 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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