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Regulation of docking and priming in pancreatic α- and β-cells
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. (Sebastian Barg)ORCID iD: 0000-0001-8893-7348
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The secretion of islet hormones from endocrine cells of the pancreas plays vital roles in maintaining glucose homeostasis. Dysfunction of these cells leads to diabetes, a devastating metabolic disorder affecting millions worldwide, but underlying mechanisms remain poorly understood. In hyperglycemic conditions, β-cells secrete insulin, whereas α-cells secrete an increased amount of glucagon in hypoglycemic conditions. Both insulin and glucagon are stored in secretory granules preceding their release by regulated exocytosis. This process involves several steps, including tethering, docking, priming, and finally, a fusion of the granules with the plasma membrane. Soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) proteins and phosphoinositides (PIs) drive pancreatic hormone exocytosis and secretion, which follows a biphasic time course. Biphasic secretion is thought to reflect the vastly different release probabilities of individual granules, but direct evidence for this is still lacking.  Therefore, this thesis investigates exocytosis in the two main pancreatic cell types with a particular focus on preceding steps docking and priming, to identify rate-limiting steps in health and type-2 diabetes (T2D). Our data indicated that granule docking is critical for sustained secretion in α- and β-cells. Glucagon granule exocytosis had a U-shaped sensitivity to glucose in both healthy and T2D α-cells. However, T2D α-cells exhibited a marginal decrease in exocytosis, as well as docking, and they were markedly insensitive to somatostatin and insulin. T2D β-cells reduced exocytosis dramatically, and docking was compromised and no longer responsive to glucose, which correlated with reduced insulin secretion and elevated donor HbA1c. These results were further strengthened by the finding that expression of a group of genes that are involved explicitly in granule docking was reduced (by RNAseq of islets from over 200 human donors), and overexpression of the corresponding proteins increased granule docking in human β-cells.

We further aimed to study the basis for the recruitment of these proteins to the docking site. Here we tested the hypothesis that highly charged lipids mainly PIs act as a hotspot to interact with SNARE proteins that initiate docking. We showed the homogenous distribution of all PIs markers in the plasma membrane, with no PIs microdomains at the exocytotic site during granule docking. However, rapid and local PI(4,5)P2 signaling at fusion sites was crucial for stabilizing fusion pore by binding to proteins related to the release site. These results suggested a role of PI(4,5)P2 in priming and fusion regulation rather than docking. Overall, this work gives new insights into the mechanisms underlying pancreatic hormone secretion in both healthy and diabetic conditions.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. , p. 54
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1586
Keywords [en]
Type-2 diabetes, glucagon, insulin, somatostatin, exocytosis
National Category
Cell and Molecular Biology Cell Biology Endocrinology and Diabetes
Identifiers
URN: urn:nbn:se:uu:diva-388354ISBN: 978-91-513-0704-6 (print)OAI: oai:DiVA.org:uu-388354DiVA, id: diva2:1340194
Public defence
2019-09-11, B42 BMC, BMC, Husargatan 1, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2019-08-20 Created: 2019-08-02 Last updated: 2019-09-17
List of papers
1. Glucose-Dependent Granule Docking Limits Insulin Secretion and Is Decreased in Human Type 2 Diabetes
Open this publication in new window or tab >>Glucose-Dependent Granule Docking Limits Insulin Secretion and Is Decreased in Human Type 2 Diabetes
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2018 (English)In: Cell Metabolism, ISSN 1550-4131, E-ISSN 1932-7420, Vol. 27, no 2, p. 470-478Article in journal (Refereed) Published
Abstract [en]

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.

Keywords
GLP-1, biphasic secretion, dense core vesicle, docking, exocytosis, genome-wide association, insulin secretion, priming, somatostatin, type 2 diabetes
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-341518 (URN)10.1016/j.cmet.2017.12.017 (DOI)000424465200021 ()29414688 (PubMedID)
Funder
Swedish Research CouncilSwedish Diabetes AssociationSwedish Society for Medical Research (SSMF)The Swedish Brain FoundationNovo NordiskErnfors Foundation
Available from: 2018-02-09 Created: 2018-02-09 Last updated: 2019-08-02Bibliographically approved
2. Paracrine control of α-cell glucagon exocytosis is compromised in human type-2 diabetes
Open this publication in new window or tab >>Paracrine control of α-cell glucagon exocytosis is compromised in human type-2 diabetes
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(English)Manuscript (preprint) (Other academic)
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-389793 (URN)
Available from: 2019-07-27 Created: 2019-07-27 Last updated: 2019-08-02
3. PtdIns(4,5)P2 is not required for secretory granule docking
Open this publication in new window or tab >>PtdIns(4,5)P2 is not required for secretory granule docking
2018 (English)In: Traffic: the International Journal of Intracellular Transport, ISSN 1398-9219, E-ISSN 1600-0854, Vol. 19, no 6, p. 436-445Article in journal (Refereed) Published
Abstract [en]

Phosphoinositides (PtdIns) play important roles in exocytosis and are thought to regulate secretory granule docking by co-clustering with the SNARE protein syntaxin to form a docking receptor in the plasma membrane. Here we tested this idea by high-resolution total internal reflection imaging of EGFP-labeled PtdIns markers or syntaxin-1 at secretory granule release sites in live insulin-secreting cells. In intact cells, PtdIns markers distributed evenly across the plasma membrane with no preference for granule docking sites. In contrast, syntaxin-1 was found clustered in the plasma membrane, mostly beneath docked granules. We also observed rapid accumulation of syntaxin-1 at sites where granules arrived to dock. Acute depletion of plasma membrane phosphatidylinositol (4,5) bisphosphate (PtdIns(4,5)P-2) by recruitment of a 5-phosphatase strongly inhibited Ca2+-dependent exocytosis, but had no effect on docked granules or the distribution and clustering of syntaxin-1. Cell permeabilization by -toxin or formaldehyde-fixation caused PtdIns marker to slowly cluster, in part near docked granules. In summary, our data indicate that PtdIns(4,5)P-2 accelerates granule priming, but challenge a role of PtdIns in secretory granule docking or clustering of syntaxin-1 at the release site.

Keywords
exocytosis, insulin, live cell imaging, phosphoinositides, PtdIns(4, 5)P-2, syntaxin clustering, vesicle docking
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-356858 (URN)10.1111/tra.12562 (DOI)000432037000005 ()29542271 (PubMedID)
Funder
Swedish Research CouncilSwedish Child Diabetes FoundationSwedish Society for Medical Research (SSMF)Novo NordiskThe Swedish Brain FoundationErnfors Foundation
Available from: 2018-08-15 Created: 2018-08-15 Last updated: 2019-08-02Bibliographically approved
4. Fusion pore regulation by transient local generation of PI(4,5)P2 in pancreatic in β-cells
Open this publication in new window or tab >>Fusion pore regulation by transient local generation of PI(4,5)P2 in pancreatic in β-cells
(English)Manuscript (preprint) (Other academic)
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-389794 (URN)
Available from: 2019-07-27 Created: 2019-07-27 Last updated: 2019-08-02

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