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- Barg, Sebastian
(författare)
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Mechanisms of insulin exocytosis and release
- 2001
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Endocrine cells as well as neurons release their hormones and transmitters by regulated exocytosis. In the pancreatic B-cell, stimuli like glucose initiate biochemical and electrical processes that culminate in influx of Ca2+, which then triggers exocytosis of insulin-containing granules. Fusion of the secretory vesicles occurs rapidly upon Ca2+-influx but requires a granule to be ?primed? by an ATP-, Ca2+- and temperature-dependent reaction. Only a small fraction of the B-cell's granules (~ 0.5 %) are in the primed state and can undergo exocytosis immediately upon Ca2+-influx. These granules are referred to as the readily releasable pool (RRP), whereas the remaining granules form a ?reserve? pool. The functional organization of the granules in a reserve pool and a readily releasable pool could account for the fact that glucose-stimulated insulin secretion follows a biphasic time course, with the early rapid component (1st phase secretion) corresponding to RRP release and the second slower component reflecting time- and ATP-dependent mobilization of granules from the reserve pool. The commonest form of human diabetes (type-2 diabetes) is associated with disturbances in this release pattern. In this thesis, electrophysiology, fluorescence microscopy and biochemistry were combined to explore mechanisms of granule trafficking, priming, exocytosis, and release in insulin-secreting B-cells. Three aspects are discussed in detail: 1) The importance of a tight interaction between L-type Ca2+-channels and the exocytotic machinery for efficient secretion; 2) A novel ATP-dependent priming reaction that is regulated by a granular 65-kDa sulfonylurea-binding protein, and involves granule acidification and ClC-3 chloride channels; and 3) A previously overlooked delay between fusion of the granule with the plasma membrane and insulin release. Since regulated secretion is very similar in all (neuro)-endocrine cells, the data obtained are likely to be relevant for peptide secretion in general.
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- 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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