| 1. |
- Barg, Sebastian, et al.
(författare)
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Fast exocytosis with few Ca(2+) channels in insulin-secreting mouse pancreatic B cells
- 2001
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Ingår i: Biophysical Journal. - 1542-0086 .- 0006-3495. ; 81:6, s. 3308-3323
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Tidskriftsartikel (refereegranskat)abstract
- The association of L-type Ca(2+) channels to the secretory granules and its functional significance to secretion was investigated in mouse pancreatic B cells. Nonstationary fluctuation analysis showed that the B cell is equipped with <500 alpha1(C) L-type Ca(2+) channels, corresponding to a Ca(2+) channel density of 0.9 channels per microm(2). Analysis of the kinetics of exocytosis during voltage-clamp depolarizations revealed an early component that reached a peak rate of 1.1 pFs(-1) (approximately 650 granules/s) 25 ms after onset of the pulse and is completed within approximately 100 ms. This component represents a subset of approximately 60 granules situated in the immediate vicinity of the L-type Ca(2+) channels, corresponding to approximately 10% of the readily releasable pool of granules. Experiments involving photorelease of caged Ca(2+) revealed that the rate of exocytosis was half-maximal at a cytoplasmic Ca(2+) concentration of 17 microM, and concentrations >25 microM are required to attain the rate of exocytosis observed during voltage-clamp depolarizations. The rapid component of exocytosis was not affected by inclusion of millimolar concentrations of the Ca(2+) buffer EGTA but abolished by addition of exogenous L(C753-893), the 140 amino acids of the cytoplasmic loop connecting the 2(nd) and 3(rd) transmembrane region of the alpha1(C) L-type Ca(2+) channel, which has been proposed to tether the Ca(2+) channels to the secretory granules. In keeping with the idea that secretion is determined by Ca(2+) influx through individual Ca(2+) channels, exocytosis triggered by brief (15 ms) depolarizations was enhanced 2.5-fold by the Ca(2+) channel agonist BayK8644 and 3.5-fold by elevating extracellular Ca(2+) from 2.6 to 10 mM. Recordings of single Ca(2+) channel activity revealed that patches predominantly contained no channels or many active channels. We propose that several Ca(2+) channels associate with a single granule thus forming a functional unit. This arrangement is important in a cell with few Ca(2+) channels as it ensures maximum usage of the Ca(2+) entering the cell while minimizing the influence of stochastic variations of the Ca(2+) channel activity.
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| 2. |
- Barg, Sebastian, 1969-, et al.
(författare)
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Granule docking and cargo release in pancreatic β-cells
- 2008
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Ingår i: Biochemical Society Transactions. - 0300-5127 .- 1470-8752. ; 36:Pt 3, s. 294-299
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Tidskriftsartikel (refereegranskat)abstract
- Biphasic insulin secretion is required for proper insulin action and is observed not only in vivo, but also in isolated pancreatic islets and even single beta-cells. Late events in the granule life cycle are thought to underlie this temporal pattern. In the last few years, we have therefore combined live cell imaging and electrophysiology to study insulin secretion at the level of individual granules, as they approach the plasma membrane, undergo exocytosis and finally release their insulin cargo. In the present paper, we review evidence for two emerging concepts that affect insulin secretion at the level of individual granules: (i) the existence of specialized sites where granules dock in preparation for exocytosis; and (ii) post-exocytotic regulation of cargo release by the fusion pore.
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| 3. |
- Barg, Sebastian, et al.
(författare)
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Priming of insulin granules for exocytosis by granular Cl(-) uptake and acidification
- 2001
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Ingår i: Journal of Cell Science. - 0021-9533 .- 1477-9137. ; 114:Pt 11, s. 2145-54
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Tidskriftsartikel (refereegranskat)abstract
- ATP-dependent priming of the secretory granules precedes Ca(2+)-regulated neuroendocrine secretion, but the exact nature of this reaction is not fully established in all secretory cell types. We have further investigated this reaction in the insulin-secreting pancreatic B-cell and demonstrate that granular acidification driven by a V-type H(+)-ATPase in the granular membrane is a decisive step in priming. This requires simultaneous Cl(-) uptake through granular ClC-3 Cl(-) channels. Accordingly, granule acidification and priming are inhibited by agents that prevent transgranular Cl(-) fluxes, such as 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and an antibody against the ClC-3 channels, but accelerated by increases in the intracellular ATP:ADP ratio or addition of hypoglycemic sulfonylureas. We suggest that this might represent an important mechanism for metabolic regulation of Ca(2+)-dependent exocytosis that is also likely to be operational in other secretory cell types.
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| 4. |
- Ivarsson, Rosita, et al.
(författare)
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Temperature-Sensitive Random Insulin Granule Diffusion is a Prerequisite for Recruiting Granules for Release.
- 2004
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Ingår i: Traffic: the International Journal of Intracellular Transport. - : Wiley. - 1398-9219. ; 5:10, s. 750-762
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Tidskriftsartikel (refereegranskat)abstract
- Glucose-evoked insulin secretion exhibits a biphasic time course and is associated with accelerated intracellular granule movement. We combined live confocal imaging of EGFP-labelled insulin granules with capacitance measurements of exocytosis in clonal INS-1 cells to explore the relation between distinct random and directed modes of insulin granule movement, as well as exocytotic capacity. Reducing the temperature from 34 °C to 24 °C caused a dramatic 81% drop in the frequency of directed events, but reduced directed velocities by a mere 25%. The much stronger temperature sensitivity of the frequency of directed events (estimated energy of activation ~ 135 kJ/mol) than that of the granule velocities (~ 22 kJ/mol) suggests that cooling-induced suppression of insulin granule movement is attributable to factors other than reduced motor protein adenosine 5'-triphosphatase activity. Indeed, cooling suppresses random granule diffusion by ~ 50%. In the single cell, the number of directed events depends on the extent of granule diffusion. Finally, single-cell exocytosis exhibits a biphasic pattern corresponding to that observed in vivo, and only the component reflecting 2nd phase insulin secretion is affected by cooling. We conclude that random diffusive movement is a prerequisite for directed insulin granule transport and for the recruitment of insulin granules released during 2nd phase insulin secretion.
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| 5. |
- Jimenez, Javier, et al.
(författare)
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Insulin feedback actions: complex effects involving isoforms of islet nitric oxide synthase.
- 2004
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Ingår i: Regulatory Peptides. - : Elsevier BV. - 1873-1686 .- 0167-0115. ; 122:2, s. 109-118
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Tidskriftsartikel (refereegranskat)abstract
- The present study examined the effects of exogenous insulin on C-peptide release in relation to islet activities of neural constitutive nitric oxide synthase (ncNOS) and inducible NOS (iNOS). The dose–response curves for glucose-stimulated insulin and C-peptide release from isolated islets were practically identical: 0.05–0.1 nmol/l insulin stimulated, 1–100 nmol/l had no effect, whereas concentrations ≥250 nmol/l (“high insulin”), inhibited C-peptide release. Both the stimulatory and inhibitory effects were abolished by the phosphatidylinositol 3′-kinase inhibitor wortmannin. Addition of a NOS inhibitor partially reversed the inhibitory action of high insulin, but had no effect on the stimulatory action of low insulin (0.1 nmol/l). Moreover, high insulin markedly increased islet ncNOS activity and induced a strong iNOS activity. As shown biochemically and with confocal microscopy, the stimulatory action of high insulin on NOS activities and the associated inhibition of C-peptide release were reversed by raising cyclic AMP through addition of either glucagon-like peptide 1 (GLP-1) or dibutyryl cyclic AMP (Bt2cAMP) to the incubated islets. We conclude that the positive feedback mechanisms of action of insulin are independent of islet NOS activities and remain unclear. The negative feedback action of insulin, however, can be explained by its ability to stimulate both islet ncNOS activity and the expression and activity of iNOS. The effects on iNOS are most likely transduced through phosphatidylinositol 3′-kinase and are counteracted by raising islet cyclic AMP levels.
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| 6. |
- 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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| 7. |
- Obermüller, Stefanie, et al.
(författare)
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Defective secretion of islet hormones in chromogranin-B deficient mice
- 2010
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 5:1, s. e8936-
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Tidskriftsartikel (refereegranskat)abstract
- Granins are major constituents of dense-core secretory granules in neuroendocrine cells, but their function is still a matter of debate. Work in cell lines has suggested that the most abundant and ubiquitously expressed granins, chromogranin A and B (CgA and CgB), are involved in granulogenesis and protein sorting. Here we report the generation and characterization of mice lacking chromogranin B (CgB-ko), which were viable and fertile. Unlike neuroendocrine tissues, pancreatic islets of these animals lacked compensatory changes in other granins and were therefore analyzed in detail. Stimulated secretion of insulin, glucagon and somatostatin was reduced in CgB-ko islets, in parallel with somewhat impaired glucose clearance and reduced insulin release, but normal insulin sensitivity in vivo. CgB-ko islets lacked specifically the rapid initial phase of stimulated secretion, had elevated basal insulin release, and stored and released twice as much proinsulin as wildtype (wt) islets. Stimulated release of glucagon and somatostatin was reduced as well. Surprisingly, biogenesis, morphology and function of insulin granules were normal, and no differences were found with regard to beta-cell stimulus-secretion coupling. We conclude that CgB is not required for normal insulin granule biogenesis or maintenance in vivo, but is essential for adequate secretion of islet hormones. Consequentially CgB-ko animals display some, but not all, hallmarks of human type-2 diabetes. However, the molecular mechanisms underlying this defect remain to be determined.
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| 8. |
- 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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| 10. |
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