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Gucek, A., Gandasi, N. R., Omar-Hmeadi, M., Bakke, M., Doskeland, S. O., Tengholm, A. & Barg, S. (2019). Fusion pore regulation by cAMP/Epac2 controls cargo release during insulin exocytosis. eLIFE, 8, Article ID e41711.
Åpne denne publikasjonen i ny fane eller vindu >>Fusion pore regulation by cAMP/Epac2 controls cargo release during insulin exocytosis
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2019 (engelsk)Inngår i: eLIFE, E-ISSN 2050-084X, Vol. 8, artikkel-id e41711Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Regulated exocytosis establishes a narrow fusion pore as initial aqueous connection to the extracellular space, through which small transmitter molecules such as ATP can exit. Co-release of polypeptides and hormones like insulin requires further expansion of the pore. There is evidence that pore expansion is regulated and can fail in diabetes and neurodegenerative disease. Here, we report that the cAMP-sensor Epac2 (Rap-GEF4) controls fusion pore behavior by acutely recruiting two pore-restricting proteins, amisyn and dynamin-1, to the exocytosis site in insulin-secreting beta-cells. cAMP elevation restricts and slows fusion pore expansion and peptide release, but not when Epac2 is inactivated pharmacologically or in Epac2(-/-) (Rapgef4(-/-)) mice. Consistently, overexpression of Epac2 impedes pore expansion. Widely used antidiabetic drugs (GLP-1 receptor agonists and sulfonylureas) activate this pathway and thereby paradoxically restrict hormone release. We conclude that Epac2/cAMP controls fusion pore expansion and thus the balance of hormone and transmitter release during insulin granule exocytosis.

sted, utgiver, år, opplag, sider
ELIFE SCIENCES PUBLICATIONS LTD, 2019
HSV kategori
Identifikatorer
urn:nbn:se:uu:diva-389824 (URN)10.7554/eLife.41711 (DOI)000471123300001 ()31099751 (PubMedID)
Forskningsfinansiär
Novo NordiskSwedish Research Council, 2014-02575Swedish Research Council, 2017-00956Swedish Research Council, 2018-02871Ernfors FoundationSwedish Society for Medical Research (SSMF)The Research Council of NorwaySwedish Diabetes AssociationEXODIAB - Excellence of Diabetes Research in SwedenThe Swedish Brain Foundation
Tilgjengelig fra: 2019-07-29 Laget: 2019-07-29 Sist oppdatert: 2019-07-29bibliografisk kontrollert
Korol, S. V., Jin, Z., Jin, Y., Bhandage, A. K., Tengholm, A., Gandasi, N. R., . . . Birnir, B. (2018). Functional Characterization of Native, High-Affinity GABAA Receptors in Human Pancreatic β Cells. EBioMedicine, 30
Åpne denne publikasjonen i ny fane eller vindu >>Functional Characterization of Native, High-Affinity GABAA Receptors in Human Pancreatic β Cells
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2018 (engelsk)Inngår i: EBioMedicine, ISSN 0360-0637, E-ISSN 2352-3964, Vol. 30Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

In human pancreatic islets, the neurotransmitter γ-aminobutyric acid (GABA) is an extracellular signaling molecule synthesized by and released from the insulin-secreting β cells. The effective, physiological GABA concentration range within human islets is unknown. Here we use native GABAA receptors in human islet β cells as biological sensors and reveal that 100-1000nM GABA elicit the maximal opening frequency of the single-channels. In saturating GABA, the channels desensitized and stopped working. GABA modulated insulin exocytosis and glucose-stimulated insulin secretion. GABAA receptor currents were enhanced by the benzodiazepine diazepam, the anesthetic propofol and the incretin glucagon-like peptide-1 (GLP-1) but not affected by the hypnotic zolpidem. In type 2 diabetes (T2D) islets, single-channel analysis revealed higher GABA affinity of the receptors. The findings reveal unique GABAA receptors signaling in human islets β cells that is GABA concentration-dependent, differentially regulated by drugs, modulates insulin secretion and is altered in T2D.

Emneord
GABA, GABA(A) receptor, Pancreatic islet, Type 2 diabetes
HSV kategori
Identifikatorer
urn:nbn:se:uu:diva-348267 (URN)10.1016/j.ebiom.2018.03.014 (DOI)000430303000032 ()29606630 (PubMedID)
Forskningsfinansiär
Swedish Research Council, 521-2009-4021EXODIAB - Excellence of Diabetes Research in SwedenSwedish Child Diabetes FoundationSwedish Diabetes AssociationNovo NordiskSwedish Society for Medical Research (SSMF)Swedish Research Council, 521-2012-1789Swedish Research Council, 2015-02417Swedish Research Council, 2017-00956Swedish Research Council, 2014-2575
Merknad

De 2 första författarna delar förstaförfattarskapet.

Tilgjengelig fra: 2018-04-11 Laget: 2018-04-11 Sist oppdatert: 2018-06-19bibliografisk kontrollert
Gandasi, N. R., Yin, P., Omar-Hmeadi, M., Ottosson Laakso, E., Vikman, P. & Barg, S. (2018). Glucose-Dependent Granule Docking Limits Insulin Secretion and Is Decreased in Human Type 2 Diabetes. Cell Metabolism, 27(2), 470-478
Åpne denne publikasjonen i ny fane eller vindu >>Glucose-Dependent Granule Docking Limits Insulin Secretion and Is Decreased in Human Type 2 Diabetes
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2018 (engelsk)Inngår i: Cell Metabolism, ISSN 1550-4131, E-ISSN 1932-7420, Vol. 27, nr 2, s. 470-478Artikkel i tidsskrift (Fagfellevurdert) 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.

Emneord
GLP-1, biphasic secretion, dense core vesicle, docking, exocytosis, genome-wide association, insulin secretion, priming, somatostatin, type 2 diabetes
HSV kategori
Identifikatorer
urn:nbn:se:uu:diva-341518 (URN)10.1016/j.cmet.2017.12.017 (DOI)000424465200021 ()29414688 (PubMedID)
Forskningsfinansiär
Swedish Research CouncilSwedish Diabetes AssociationSwedish Society for Medical Research (SSMF)The Swedish Brain FoundationNovo NordiskErnfors Foundation
Tilgjengelig fra: 2018-02-09 Laget: 2018-02-09 Sist oppdatert: 2019-08-02bibliografisk kontrollert
Omar-Hmeadi, M., Gandasi, N. R. & Barg, S. (2018). PtdIns(4,5)P2 is not required for secretory granule docking. Traffic: the International Journal of Intracellular Transport, 19(6), 436-445
Åpne denne publikasjonen i ny fane eller vindu >>PtdIns(4,5)P2 is not required for secretory granule docking
2018 (engelsk)Inngår i: Traffic: the International Journal of Intracellular Transport, ISSN 1398-9219, E-ISSN 1600-0854, Vol. 19, nr 6, s. 436-445Artikkel i tidsskrift (Fagfellevurdert) 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.

Emneord
exocytosis, insulin, live cell imaging, phosphoinositides, PtdIns(4, 5)P-2, syntaxin clustering, vesicle docking
HSV kategori
Identifikatorer
urn:nbn:se:uu:diva-356858 (URN)10.1111/tra.12562 (DOI)000432037000005 ()29542271 (PubMedID)
Forskningsfinansiär
Swedish Research CouncilSwedish Child Diabetes FoundationSwedish Society for Medical Research (SSMF)Novo NordiskThe Swedish Brain FoundationErnfors Foundation
Tilgjengelig fra: 2018-08-15 Laget: 2018-08-15 Sist oppdatert: 2019-08-02bibliografisk kontrollert
Yin, P., Gandasi, N. R., Arora, S., Omar-Hmeadi, M., Saras, J. & Barg, S. (2018). Syntaxin clusters at secretory granules in a munc18-bound conformation. Molecular Biology of the Cell, 29(22), 2700-2708
Åpne denne publikasjonen i ny fane eller vindu >>Syntaxin clusters at secretory granules in a munc18-bound conformation
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2018 (engelsk)Inngår i: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 29, nr 22, s. 2700-2708Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Syntaxin (stx)-1 is an integral plasma membrane protein that is crucial for two distinct steps of regulated exocytosis, docking of secretory granules at the plasma membrane and membrane fusion. During docking, stx1 clusters at the granule docking site, together with the S/M protein munc18. Here we determined features of stx1 that contribute to its clustering at granules. In live insulin-secreting cells, stx1 and stx3 (but not stx4 or stx11) accumulated at docked granules, and stx1 (but not stx4) rescued docking in cells expressing botulinum neurotoxin-C. Using a series of stx1 deletion mutants and stx1/4 chimeras, we found that all four helical domains (Ha, Hb, Hc, SNARE) and the short N-terminal peptide contribute to recruitment to granules. However, only the Hc domain confers specificity, and it must be derived from stx1 for recruitment to occur. Point mutations in the Hc or the N-terminal peptide designed to interfere with binding to munc18-1 prevent stx1 from clustering at granules, and a mutant munc18 deficient in binding to stx1 does not cluster at granules. We conclude that stx1 is recruited to the docking site in a munc18-1-bound conformation, providing a rationale for the requirement for both proteins for granule docking.

HSV kategori
Identifikatorer
urn:nbn:se:uu:diva-375859 (URN)10.1091/mbc.E17-09-0541 (DOI)000455641000013 ()30156474 (PubMedID)
Forskningsfinansiär
Swedish Research CouncilSwedish Child Diabetes FoundationSwedish Diabetes AssociationThe Swedish Brain FoundationNovo NordiskGöran Gustafsson Foundation for Research in Natural Sciences and MedicineErnfors FoundationSwedish Society of Medicine
Tilgjengelig fra: 2019-02-01 Laget: 2019-02-01 Sist oppdatert: 2019-02-01bibliografisk kontrollert
Nguyen, P. M., Gandasi, N. R. & Idevall-Hagren, O. (2018). The lipid phosphatase INPP5F regulates insulin secretion. Paper presented at 54th Annual Meeting of the European-Association-for-the-Study-of-Diabetes (EASD), OCT 01-05, 2018, Berlin, GERMANY. Diabetologia, 61, S197-S197
Åpne denne publikasjonen i ny fane eller vindu >>The lipid phosphatase INPP5F regulates insulin secretion
2018 (engelsk)Inngår i: Diabetologia, ISSN 0012-186X, E-ISSN 1432-0428, Vol. 61, s. S197-S197Artikkel i tidsskrift, Meeting abstract (Annet vitenskapelig) Published
sted, utgiver, år, opplag, sider
Springer, 2018
HSV kategori
Identifikatorer
urn:nbn:se:uu:diva-367130 (URN)000443556002190 ()
Konferanse
54th Annual Meeting of the European-Association-for-the-Study-of-Diabetes (EASD), OCT 01-05, 2018, Berlin, GERMANY
Forskningsfinansiär
Swedish Research Council
Tilgjengelig fra: 2018-11-29 Laget: 2018-11-29 Sist oppdatert: 2018-11-29bibliografisk kontrollert
Gandasi, N. R., Yin, P., Riz, M., Chibalina, M. V., Cortese, G., Lund, P.-E., . . . Barg, S. (2017). Ca2+ channel clustering with insulin-containing granules is disturbed in type 2 diabetes. Journal of Clinical Investigation, 127(6), 2353-2364
Åpne denne publikasjonen i ny fane eller vindu >>Ca2+ channel clustering with insulin-containing granules is disturbed in type 2 diabetes
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2017 (engelsk)Inngår i: Journal of Clinical Investigation, ISSN 0021-9738, E-ISSN 1558-8238, Vol. 127, nr 6, s. 2353-2364Artikkel i tidsskrift (Fagfellevurdert) 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.

HSV kategori
Forskningsprogram
Molekylär cellbiologi
Identifikatorer
urn:nbn:se:uu:diva-321935 (URN)10.1172/JCI88491 (DOI)000402620800029 ()28481223 (PubMedID)
Forskningsfinansiär
Swedish Research CouncilSwedish Diabetes AssociationThe Swedish Brain FoundationSwedish Child Diabetes FoundationEXODIAB - Excellence of Diabetes Research in SwedenNovo Nordisk
Tilgjengelig fra: 2017-05-12 Laget: 2017-05-12 Sist oppdatert: 2018-03-11bibliografisk kontrollert
Salunkhe, V. A., Ofori, J. K., Gandasi, N. R., Salo, S. A., Hansson, S., Andersson, M. E., . . . Eliasson, L. (2017). MiR-335 overexpression impairs insulin secretion through defective priming of insulin vesicles. Physiological Reports, 5(21), Article ID e13493.
Åpne denne publikasjonen i ny fane eller vindu >>MiR-335 overexpression impairs insulin secretion through defective priming of insulin vesicles
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2017 (engelsk)Inngår i: Physiological Reports, E-ISSN 2051-817X, Vol. 5, nr 21, artikkel-id e13493Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

MicroRNAs contribute to the maintenance of optimal cellular functions by fine-tuning protein expression levels. In the pancreatic beta-cells, imbalances in the exocytotic machinery components lead to impaired insulin secretion and type 2 diabetes (T2D). We hypothesize that dysregulated miRNA expression exacerbates beta-cell dysfunction, and have earlier shown that islets from the diabetic GK-rat model have increased expression of miRNAs, including miR-335-5p (miR-335). Here, we aim to determine the specific role of miR-335 during development of T2D, and the influence of this miRNA on glucose-stimulated insulin secretion and Ca2+-dependent exocytosis. We found that the expression of miR-335 negatively correlated with secretion index in human islets of individuals with prediabetes. Overexpression of miR-335 in human EndoC-beta H1 and in rat INS-1 832/13 cells (OE335) resulted in decreased glucose-stimulated insulin secretion, and OE335 cells showed concomitant reduction in three exocytotic proteins: SNAP25, Syntaxin-binding protein 1 (STXBP1), and synaptotagmin 11 (SYT11). Single-cell capacitance measurements, complemented with TIRF microscopy of the granule marker NPY-mEGFP demonstrated a significant reduction in exocytosis in OE335 cells. The reduction was not associated with defective docking or decreased Ca2+ current. More likely, it is a direct consequence of impaired priming of already docked granules. Earlier reports have proposed reduced granular priming as the cause of reduced first-phase insulin secretion during prediabetes. Here, we show a specific role of miR-335 in regulating insulin secretion during this transition period. Moreover, we can conclude that miR-335 has the capacity to modulate insulin secretion and Ca2+-dependent exocytosis through effects on granular priming.

sted, utgiver, år, opplag, sider
John Wiley & Sons, 2017
Emneord
Beta cell, exocytosis, insulin secretion, microRNA, patch-clamp, SNAP25, STXBP1, TIRF, Type 2 Diabetes
HSV kategori
Identifikatorer
urn:nbn:se:uu:diva-339717 (URN)10.14814/phy2.13493 (DOI)000415351500008 ()
Tilgjengelig fra: 2018-01-26 Laget: 2018-01-26 Sist oppdatert: 2018-01-26bibliografisk kontrollert
Omar-Hmeadi, M., Gandasi, N. R. & Barg, S. (2017). Plasma membrane PI(4,5)P-2 is critical for secretory granule exocytosis. Paper presented at ASCB/EMBO Meeting, DEC 02-06, 2017, Philadelphia, PA.. Molecular Biology of the Cell, 28(26), 3727-3727
Åpne denne publikasjonen i ny fane eller vindu >>Plasma membrane PI(4,5)P-2 is critical for secretory granule exocytosis
2017 (engelsk)Inngår i: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 28, nr 26, s. 3727-3727Artikkel i tidsskrift, Meeting abstract (Annet vitenskapelig) Published
sted, utgiver, år, opplag, sider
Philadelphia: American Society for Cell Biology (ASCB), 2017
HSV kategori
Identifikatorer
urn:nbn:se:uu:diva-352940 (URN)10.1091/mbc.E17-10-0618 (DOI)000426664300402 ()29237772 (PubMedID)
Konferanse
ASCB/EMBO Meeting, DEC 02-06, 2017, Philadelphia, PA.
Merknad

See: Supplementary Material, 2017 ASCB-EMBO Meeting-Poster Abstracts, p. Sunday-258-Sunday-259, Meeting Abstract: P1403.

Tilgjengelig fra: 2018-06-12 Laget: 2018-06-12 Sist oppdatert: 2019-02-01bibliografisk kontrollert
Alenkvist, I., Gandasi, N. R., Barg, S. & Tengholm, A. (2017). Recruitment of Epac2A to Insulin Granule Docking Sites Regulates Priming for Exocytosis. Diabetes, 66(10), 2610-2622
Åpne denne publikasjonen i ny fane eller vindu >>Recruitment of Epac2A to Insulin Granule Docking Sites Regulates Priming for Exocytosis
2017 (engelsk)Inngår i: Diabetes, ISSN 0012-1797, E-ISSN 1939-327X, Vol. 66, nr 10, s. 2610-2622Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Epac is a cAMP-activated guanine nucleotide exchange factor that mediates cAMP signaling in various types of cells, including -cells, where it is involved in the control of insulin secretion. Upon activation, the protein redistributes to the plasma membrane, but the underlying molecular mechanisms and functional consequences are unclear. Using quantitative high-resolution microscopy, we found that cAMP elevation caused rapid binding of Epac2A to the -cell plasma membrane, where it accumulated specifically at secretory granules and rendered them more prone to undergo exocytosis. cAMP-dependent membrane binding required the high-affinity cyclic nucleotide-binding (CNB) and Ras association domains, but not the disheveled-Egl-10-pleckstrin domain. Although the N-terminal low-affinity CNB domain (CNB-A) was dispensable for the translocation to the membrane, it was critical for directing Epac2A to the granule sites. Epac1, which lacks the CNB-A domain, was recruited to the plasma membrane but did not accumulate at granules. We conclude that Epac2A controls secretory granule release by binding to the exocytosis machinery, an effect that is enhanced by prior cAMP-dependent accumulation of the protein at the plasma membrane.

HSV kategori
Identifikatorer
urn:nbn:se:uu:diva-336299 (URN)10.2337/db17-0050 (DOI)000411195800009 ()28679628 (PubMedID)
Merknad

De två första författarna delar förstaförfattarskapet.

Tilgjengelig fra: 2018-01-23 Laget: 2018-01-23 Sist oppdatert: 2018-01-23bibliografisk kontrollert
Organisasjoner
Identifikatorer
ORCID-id: ORCID iD iconorcid.org/0000-0001-9400-6494