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Ca2+ channel clustering with insulin-containing granules is disturbed in type 2 diabetes
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. (Barg)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. (Barg)
Univ Padua, Dept Informat Engn, Padua, Italy..
Univ Oxford, Oxford Ctr Diabet Endocrinol & Metab, Oxford, England..
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2017 (English)In: Journal of Clinical Investigation, ISSN 0021-9738, E-ISSN 1558-8238, Vol. 127, no 6, p. 2353-2364Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2017. Vol. 127, no 6, p. 2353-2364
National Category
Cell and Molecular Biology
Research subject
Molecular Cellbiology
Identifiers
URN: urn:nbn:se:uu:diva-321935DOI: 10.1172/JCI88491ISI: 000402620800029PubMedID: 28481223OAI: oai:DiVA.org:uu-321935DiVA, id: diva2:1095360
Funder
Swedish Research CouncilSwedish Diabetes AssociationThe Swedish Brain FoundationSwedish Child Diabetes FoundationEXODIAB - Excellence of Diabetes Research in SwedenNovo NordiskAvailable from: 2017-05-12 Created: 2017-05-12 Last updated: 2018-03-11Bibliographically approved
In thesis
1. Architecture and function of the insulin granule secretion machinery
Open this publication in new window or tab >>Architecture and function of the insulin granule secretion machinery
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Insulin is released into the blood stream to normalize elevated blood glucose, for example after a meal. The hormone is synthesized by β-cells in the endocrine pancreas, and stored in small vesicles, known as secretory granules, until required. When glucose is elevated, these granules undergo regulated exocytosis and thereby secrete the hormone. The primary trigger for this is a glucose-dependent elevation in cytosolic Ca2+, which enters the cell through voltage-gated Ca2+ channels. Glucose-stimulated insulin secretion follows a biphasic timecourse, with a rapid 1st phase that lasts for a few minutes, followed by a slowly developing sustained 2nd phase. Compromised 1st phase secretion is an early sign of developing type-2 diabetes. Biphasic secretion is thought reflect the vastly different probabilities of individual insulin granules, but direct evidence for this is still lacking. In this thesis, high resolution TIRF microscopy was used to identify rate limiting steps for insulin granule exocytosis in health and in type-2 diabetes, and to understand these steps at the molecular level. It is shown that granule docking is critical for sustained insulin secretion. In β-cells from type-2 diabetic donors, docking is compromised and no longer responsive to glucose. Expression analysis in a large donor cohort suggests that this is due to decreased expression of proteins involved in the docking step. One of these proteins, the SNARE protein syntaxin-1, is well-known to cluster at the site of docked granules, which initiates the formation of functional release sites. Analysis using a series of syntaxin-1 mutations indicates that this clustering depends on specific features in its N-terminal Habc domain and involves binding of the S/M protein munc-18. The data suggest that the closed conformation of syntaxin-1 mediates the interaction between granule and plasma membrane. Finally, it is shown that voltage-gated L-type Ca2+ channels are slowly recruited to the sites of docked granules, which depends on interaction with the granule priming factor Munc13. This arrangement leads to localized the Ca2+ influx near a subset of the docked granules, which dramatically increases their release probability. Importantly, the interaction between Ca2+ channels and granules fails in type-2 diabetic β cells. In summary, the thesis highlights the importance of the spatial organization of the secretory machinery for adequate insulin secretion, and suggests that defects in this process partly underlie the disturbed blood glucose regulation in type-2 diabetes.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2018. p. 34
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1440
Keywords
Docking, Syntaxin-1A, Voltage-gated Ca2+ Channels, Exocytosis, Type-2 Diabetes
National Category
Cell and Molecular Biology Endocrinology and Diabetes
Identifiers
urn:nbn:se:uu:diva-344789 (URN)978-91-513-0264-5 (ISBN)
Public defence
2018-04-23, A3:117a, BMC, Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2018-03-26 Created: 2018-03-11 Last updated: 2018-04-24

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