| 21. |
- Cervin, Camilla, et al.
(författare)
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Characterization of a naturally occurring mutation (L107I) in the HNF1 alpha (MODY3) gene.
- 2002
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Ingår i: Diabetologia. - : Springer Science and Business Media LLC. - 1432-0428 .- 0012-186X. ; 45:12, s. 1703-1708
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Tidskriftsartikel (refereegranskat)abstract
- AIMS/HYPOTHESIS: Maturity onset diabetes of the young type 3 (MODY3) is a monogenic form of diabetes mellitus caused by mutations in the gene encoding for hepatocyte nuclear factor 1 alpha, HNF1 alpha. In this study we have examined the in vivo and in vitro effects of a mutation (L107I) outside the DNA binding and dimerization domains in the N terminal part of the HNF1 alpha gene. METHODS: Beta-cell function of the affected family members was assessed by an oral glucose tolerance test. Functional tests were carried out to explain the role of the mutation in vitro by transcriptional activity assay, Western blotting, DNA-binding assays and subcellular localization experiments. RESULTS: Affected family members showed an 86% decreased insulin response to glucose when compared to age-matched healthy control subjects. In vitro the mutation showed a 79% decrease in transcriptional activity as compared to wild type HNF1 alpha in HeLa cells lacking HNF1 alpha. The transcriptional activity was not suppressed when the mutant was co-expressed with wild type HNF1 alpha suggesting that the decreased activity was not mediated by a dominant negative mechanism. The L107I/HNF1alpha protein showed normal nuclear targeting but impaired binding to an HNF1 alpha consensus sequence. CONCLUSION/INTERPRETATION: Our results suggest that the L107I substitution represents a MODY3 mutation which impairs beta-cell function by a loss-of-function mechanism.
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| 22. |
- Chen, Xi, et al.
(författare)
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Release of the styryl dyes from single synaptic vesicles in hippocampal neurons.
- 2008
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Ingår i: The Journal of neuroscience : the official journal of the Society for Neuroscience. - 1529-2401. ; 28:8, s. 1894-1903
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Tidskriftsartikel (refereegranskat)abstract
- In small presynaptic boutons in brain, synaptic vesicles are thought not to merge with the plasma membrane when they release transmitter, but instead to close their fusion pores and survive intact for future use (kiss-and-run exocytosis). The strongest evidence for this idea is the slow and incomplete release of the fluorescent membrane marker, FM1-43 [N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide], from single vesicles. We investigated the release of FM1-43 from sparse cultures of hippocampal neurons grown on coverslips with no glia. This allowed presynaptic boutons to be imaged at favorable signal-to-noise ratio. Sparingly stained boutons were imaged at high time resolution, while high-frequency electrical stimulation caused exocytosis. The release of FM1-43 was quantal and occurred in abrupt steps, each representing a single fusion event. The fluorescence of vesicle clusters traveling along axons had a distribution with the same quantal size, indicating that a vesicle releases all the dye it contains. In most fusion events, the time constant of dye release was <100 ms, and slower release was rarely observed. After exocytosis, no FM1-43 could be detected in the axon to either side of a bouton, indicating that dye was released before it could spread. Our results are consistent with synaptic vesicles fusing fully with the plasma membrane during high-frequency stimulation.
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| 23. |
- Cortese, Giuliana, et al.
(författare)
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Statistical Frailty Modeling for Quantitative Analysis of Exocytotic Events Recorded by Live Cell Imaging : Rapid Release of Insulin-Containing Granules Is Impaired in Human Diabetic beta-cells
- 2016
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 11:12
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Tidskriftsartikel (refereegranskat)abstract
- Hormones and neurotransmitters are released when secretory granules or synaptic vesicles fuse with the cell membrane, a process denoted exocytosis. Modern imaging techniques, in particular total internal reflection fluorescence (TIRF) microscopy, allow the investigator to monitor secretory granules at the plasma membrane before and when they undergo exocytosis. However, rigorous statistical approaches for temporal analysis of such exocytosis data are still lacking. We propose here that statistical methods from time-to-event (also known as survival) analysis are well suited for the problem. These methods are typically used in clinical settings when individuals are followed over time to the occurrence of an event such as death, remission or conception. We model the rate of exocytosis in response to pulses of stimuli in insulin-secreting pancreatic beta-cell from healthy and diabetic human donors using piecewise-constant hazard modeling. To study heterogeneity in the granule population we exploit frailty modeling, which describe unobserved differences in the propensity to exocytosis. In particular, we insert a discrete frailty in our statistical model to account for the higher rate of exocytosis in an immediately releasable pool (IRP) of insulin-containing granules. Estimates of parameters are obtained from maximum-likelihood methods. Since granules within the same cell are correlated, i.e., the data are clustered, a modified likelihood function is used for log-likelihood ratio tests in order to perform valid inference. Our approach allows us for example to estimate the size of the IRP in the cells, and we find that the IRP is deficient in diabetic cells. This novel application of time-to-event analysis and frailty modeling should be useful also for the study of other well-defined temporal events at the cellular level.
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| 24. |
- da Silva Xavier, Gabriela, et al.
(författare)
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TCF7L2 regulates late events in insulin secretion from pancreatic islet beta-cells
- 2009
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Ingår i: Diabetes. - : American Diabetes Association. - 0012-1797 .- 1939-327X. ; 58:4, s. 894-905
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE: Polymorphisms in the human TCF7L2 gene are associated with reduced insulin secretion and an increased risk of type 2 diabetes. However, the mechanisms by which TCF7L2 affect insulin secretion are still unclear. We define the effects of TCF7L2 expression level on mature beta-cell function and suggest a potential mechanism for its actions. RESEARCH DESIGN AND METHODS: TCF7L2 expression in rodent islets and beta-cell lines was altered using RNAi or adenoviral transduction. Beta-cell gene profiles were measured by quantitative real-time PCR and the effects on intracellular signaling and exocytosis by live cell imaging, electron microscopy, and patch clamp electrophysiology. RESULTS: Reducing TCF7L2 expression levels by RNAi decreased glucose- but not KCl-induced insulin secretion. The glucose-induced increments in both ATP/ADP ratio and cytosolic free Ca2+ concentration ([Ca2+]i) were increased compared with controls. Overexpression of TCF7L2 exerted minor inhibitory effects on glucose-regulated changes in [Ca2+]i and insulin release. Gene expression profiling in TCF7L2-silenced cells revealed increased levels of mRNA encoding syntaxin 1A but decreased Munc18–1 and ZnT8 mRNA. Whereas the number of morphologically docked vesicles was unchanged by TCF7L2 suppression, secretory granule movement increased and capacitance changes decreased, indicative of defective vesicle fusion. CONCLUSION: TCF7L2 is involved in maintaining expression of beta-cell genes regulating secretory granule fusion. Defective insulin exocytosis may thus underlie increased diabetes incidence in carriers of the at-risk TCF7L2 alleles.
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| 25. |
- Eliasson, Lena, et al.
(författare)
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SUR1 Regulates PKA-independent cAMP-induced Granule Priming in Mouse Pancreatic B-cells.
- 2003
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Ingår i: Journal of General Physiology. - : Rockefeller University Press. - 0022-1295 .- 1540-7748. ; 121:3, s. 181-197
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Tidskriftsartikel (refereegranskat)abstract
- Measurements of membrane capacitance were applied to dissect the cellular mechanisms underlying PKA-dependent and -independent stimulation of insulin secretion by cyclic AMP. Whereas the PKA-independent (Rp-cAMPS–insensitive) component correlated with a rapid increase in membrane capacitance of ~80 fF that plateaued within ~200 ms, the PKA-dependent component became prominent during depolarizations >450 ms. The PKA-dependent and -independent components of cAMP-stimulated exocytosis differed with regard to cAMP concentration dependence; the Kd values were 6 and 29 µM for the PKA-dependent and -independent mechanisms, respectively. The ability of cAMP to elicit exocytosis independently of PKA activation was mimicked by the selective cAMP-GEFII agonist 8CPT-2Me-cAMP. Moreover, treatment of B-cells with antisense oligodeoxynucleotides against cAMP-GEFII resulted in partial (50%) suppression of PKA-independent exocytosis. Surprisingly, B-cells in islets isolated from SUR1-deficient mice (SUR1-/- mice) lacked the PKA-independent component of exocytosis. Measurements of insulin release in response to GLP-1 stimulation in isolated islets from SUR1-/- mice confirmed the complete loss of the PKA-independent component. This was not attributable to a reduced capacity of GLP-1 to elevate intracellular cAMP but instead associated with the inability of cAMP to stimulate influx of Cl- into the granules, a step important for granule priming. We conclude that the role of SUR1 in the B cell extends beyond being a subunit of the plasma membrane KATP-channel and that it also plays an unexpected but important role in the cAMP-dependent regulation of Ca2+-induced exocytosis.
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| 26. |
- Gandasi, Nikhil R., et al.
(författare)
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Ca2+ channel clustering with insulin-containing granules is disturbed in type 2 diabetes
- 2017
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Ingår i: Journal of Clinical Investigation. - 0021-9738 .- 1558-8238. ; 127:6, s. 2353-2364
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Tidskriftsartikel (refereegranskat)abstract
- Loss of first-phase insulin secretion is an early sign of developing type 2 diabetes (T2D). Ca2+ entry through voltage-gated L-type Ca2+ channels triggers exocytosis of insulin-containing granules in pancreatic β cells and is required for the postprandial spike in insulin secretion. Using high-resolution microscopy, we have identified a subset of docked insulin granules in human β cells and rat-derived clonal insulin 1 (INS1) cells for which localized Ca2+ influx triggers exocytosis with high probability and minimal latency. This immediately releasable pool (IRP) of granules, identified both structurally and functionally, was absent in β cells from human T2D donors and in INS1 cells cultured in fatty acids that mimic the diabetic state. Upon arrival at the plasma membrane, IRP granules slowly associated with 15 to 20 L-type channels. We determined that recruitment depended on a direct interaction with the synaptic protein Munc13, because expression of the II-III loop of the channel, the C2 domain of Munc13-1, or of Munc13-1 with a mutated C2 domain all disrupted L-type channel clustering at granules and ablated fast exocytosis. Thus, rapid insulin secretion requires Munc13-mediated recruitment of L-type Ca2+ channels in close proximity to insulin granules. Loss of this organization underlies disturbed insulin secretion kinetics in T2D.
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| 27. |
- Gandasi, Nikhil R, et al.
(författare)
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Contact-induced clustering of syntaxin and munc18 docks secretory granules at the exocytosis site
- 2014
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 5
-
Tidskriftsartikel (refereegranskat)abstract
- Docking of secretory vesicles at the plasma membrane is a poorly understood prerequisite for exocytosis. Current models propose raft-like clusters containing syntaxin as docking receptor, but direct evidence for this is lacking. Here we provide quantitative measurements of several exocytosis proteins (syntaxin, SNAP25, munc18, munc13 and rab3) at the insulin granule release site and show that docking coincides with rapid de novo formation of syntaxin1/munc18 clusters at the nascent docking site. Formation of such clusters prevents undocking and is not observed during failed docking attempts. Overexpression of syntaxins' N-terminal Habc-domain competitively interferes with both cluster formation and successful docking. SNAP25 and munc13 are recruited to the docking site more than a minute later, consistent with munc13's reported role in granule priming rather than docking. We conclude that secretory vesicles dock by inducing syntaxin1/munc18 clustering in the target membrane, and find no evidence for preformed docking receptors.
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| 28. |
- Gandasi, Nikhil R, et al.
(författare)
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Glucose-Dependent Granule Docking Limits Insulin Secretion and Is Decreased in Human Type 2 Diabetes
- 2018
-
Ingår i: Cell Metabolism. - : Elsevier BV. - 1550-4131 .- 1932-7420. ; 27:2, s. 470-478
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Tidskriftsartikel (refereegranskat)abstract
- Glucose-stimulated insulin secretion is biphasic, with a rapid first phase and a slowly developing sustained second phase; both are disturbed in type 2 diabetes (T2D). Biphasic secretion results from vastly different release probabilities of individual insulin granules, but the morphological and molecular basis for this is unclear. Here, we show that human insulin secretion and exocytosis critically depend on the availability of membrane-docked granules and that T2D is associated with a strong reduction in granule docking. Glucose accelerated granule docking, and this effect was absent in T2D. Newly docked granules only slowly acquired release competence; this was regulated by major signaling pathways, but not glucose. Gene expression analysis indicated that key proteins involved in granule docking are downregulated in T2D, and overexpression of these proteins increased granule docking. The findings establish granule docking as an important glucose-dependent step in human insulin secretion that is dysregulated in T2D.
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| 29. |
- Gandasi, Nikhil R.
(författare)
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Molecular mechanisms of biphasic insulin secretion
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Pancreatic beta-cells secrete insulin in response to increase in blood glucose concentration with a rapid first phase and slower, sustained second phase. This secretion pattern is similar in entire pancreas, isolated islets of Langerhans and single beta-cells and it is disrupted in type 2-diabetes. Insulin stored in secretory vesicles has to undergo preparatory steps upon translocation to the plasma membrane which include docking and priming before being released by exocytosis. A better understanding of the molecules involved in these steps is required to determine the rate limiting factors for sustained secretion. Here these processes were studied in real time using total internal reflection fluorescence microscopy, which enables observation of insulin granules localized at the plasma membrane. A pool of granules morphologically docked at the plasma membrane was found to be depleted upon repeated stimulations. Recovery of the docked pool of granules took tens of minutes and became rate limiting for sustained secretion. Shorter depolarization stimuli did not deplete the docked pool and allowed rapid recovery of releasable granules. When a new granule arrived at the plasma membrane, docking was initiated by de novo formation of syntaxin/munc18 clusters at the docking site. Two-thirds of the granules which arrived at the plasma membrane failed to recruit these proteins and hence failed to dock. Priming involved recruitment of several other proteins including munc13, SNAP25 and Cav1.2 channels. Exocytosing granules were in close proximity to Ca2+ influx sites with high degree of association with Cav1.2 channels. This is because of the association of these channels to exocytosis site through syntaxin and SNAP25. During exocytosis the assembled release machinery disintegrated and the proteins at the release site dispersed. Syntaxin dispersal was initiated already during fusion pore formation rather than after release during exocytosis. This was studied using a newly developed red fluorescent probe - NPY-tdmOrange2 which was the most reliable pH sensitive red granule marker to label insulin granules. Overall these data give new insights into the molecular mechanisms involved in biphasic insulin secretion. Disturbances in the secretion at the level of granule docking and fusion may contribute to the early manifestations of type-2 diabetes.
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| 30. |
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