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Petrenko, V., Gandasi, N., Sage, D., Tengholm, A., Barg, S. & Dibner, C. (2020). In pancreatic islets from type 2 diabetes patients, the dampened circadian oscillators lead to reduced insulin and glucagon exocytosis. Proceedings of the National Academy of Sciences of the United States of America, 117(5), 2484-2495
Open this publication in new window or tab >>In pancreatic islets from type 2 diabetes patients, the dampened circadian oscillators lead to reduced insulin and glucagon exocytosis
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2020 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 117, no 5, p. 2484-2495Article in journal (Refereed) Published
Abstract [en]

Circadian clocks operative in pancreatic islets participate in the regulation of insulin secretion in humans and, if compromised, in the development of type 2 diabetes (T2D) in rodents. Here we demonstrate that human islet alpha- and beta-cells that bear attenuated clocks exhibit strongly disrupted insulin and glucagon granule docking and exocytosis. To examine whether compromised clocks play a role in the pathogenesis of T2D in humans, we quantified parameters of molecular clocks operative in human T2D islets at population, single islet, and single islet cell levels. Strikingly, our experiments reveal that islets from T2D patients contain clocks with diminished circadian amplitudes and reduced in vitro synchronization capacity compared to their nondiabetic counterparts. Moreover, our data suggest that islet clocks orchestrate temporal profiles of insulin and glucagon secretion in a physiological context. This regulation was disrupted in T2D subjects, implying a role for the islet cell-autonomous clocks in T2D progression. Finally, Nobiletin, an agonist of the core-clock proteins ROR alpha/gamma, boosted both circadian amplitude of T2D islet clocks and insulin secretion by these islets. Our study emphasizes a link between the circadian clockwork and T2D and proposes that clock modulators hold promise as putative therapeutic agents for this frequent disorder.

Place, publisher, year, edition, pages
NATL ACAD SCIENCES, 2020
Keywords
circadian clock, exocytosis, human pancreatic islet, type 2 diabetes, real-time bioluminescence
National Category
Endocrinology and Diabetes
Identifiers
urn:nbn:se:uu:diva-406908 (URN)10.1073/pnas.1916539117 (DOI)000512340900040 ()31964806 (PubMedID)
Funder
Swedish Research Council, 2017-00956Swedish Research Council, 2018-02871
Available from: 2020-03-20 Created: 2020-03-20 Last updated: 2020-03-20Bibliographically approved
Xu, Y., Schwede, F., Wienk, H., Tengholm, A. & Rehmann, H. (2019). A membrane permeable prodrug of S223 for selective Epac2 activation in living cells. Cells, 8(12), Article ID 1589.
Open this publication in new window or tab >>A membrane permeable prodrug of S223 for selective Epac2 activation in living cells
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2019 (English)In: Cells, E-ISSN 2073-4409, Vol. 8, no 12, article id 1589Article in journal (Refereed) Published
Abstract [en]

Signalling by cyclic adenosine monophosphate (cAMP) occurs via various effector proteins, notably protein kinase A and the guanine nucleotide exchange factors Epac1 and Epac2. These proteins are activated by cAMP binding to conserved cyclic nucleotide binding domains. The specific roles of the effector proteins in various processes in different types of cells are still not well defined, but investigations have been facilitated by the development of cyclic nucleotide analogues with distinct selectivity profiles towards a single effector protein. A remaining challenge in the development of such analogues is the poor membrane permeability of nucleotides, which limits their applicability in intact living cells. Here, we report the synthesis and characterisation of S223-AM, a cAMP analogue designed as an acetoxymethyl ester prodrug to overcome limitations of permeability. Using total internal reflection imaging with various fluorescent reporters, we show that S223-AM selectively activates Epac2, but not Epac1 or protein kinase A, in intact insulin-secreting β-cells, and that this effect was associated with pronounced activation of the small G-protein Rap. A comparison of the effects of different cAMP analogues in pancreatic islet cells deficient in Epac1 and Epac2 demonstrates that cAMP-dependent Rap activity at the β-cell plasma membrane is exclusively dependent on Epac2. With its excellent selectivity and permeability properties, S223-AM should get broad utility in investigations of cAMP effector involvement in many different types of cells.

Place, publisher, year, edition, pages
Basel: , 2019
National Category
Cell and Molecular Biology
Research subject
Medical Cell Biology
Identifiers
urn:nbn:se:uu:diva-404599 (URN)10.3390/cells8121589 (DOI)000506643500117 ()31817822 (PubMedID)
Funder
Ernfors FoundationNovo NordiskSwedish Diabetes AssociationSwedish Research Council
Available from: 2020-02-24 Created: 2020-02-24 Last updated: 2020-03-10Bibliographically approved
Tengholm, A. (2019). Cyclic AMP links glucose stimulation to somatostatin secretion in delta-cells. The Journal of General Physiology, 151(9), 1062-1065
Open this publication in new window or tab >>Cyclic AMP links glucose stimulation to somatostatin secretion in delta-cells
2019 (English)In: The Journal of General Physiology, ISSN 0022-1295, E-ISSN 1540-7748, Vol. 151, no 9, p. 1062-1065Article in journal, Editorial material (Other academic) Published
Place, publisher, year, edition, pages
ROCKEFELLER UNIV PRESS, 2019
National Category
Physiology
Identifiers
urn:nbn:se:uu:diva-394720 (URN)10.1085/jgp.201912417 (DOI)000484022600003 ()31413066 (PubMedID)
Available from: 2019-10-11 Created: 2019-10-11 Last updated: 2019-10-11Bibliographically approved
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.
Open this publication in new window or tab >>Fusion pore regulation by cAMP/Epac2 controls cargo release during insulin exocytosis
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2019 (English)In: eLIFE, E-ISSN 2050-084X, Vol. 8, article id e41711Article in journal (Refereed) 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.

Place, publisher, year, edition, pages
ELIFE SCIENCES PUBLICATIONS LTD, 2019
National Category
Cell Biology
Identifiers
urn:nbn:se:uu:diva-389824 (URN)10.7554/eLife.41711 (DOI)000471123300001 ()31099751 (PubMedID)
Funder
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
Available from: 2019-07-29 Created: 2019-07-29 Last updated: 2019-07-29Bibliographically approved
Yu, Q., Shuai, H., Ahooghalandari, P., Gylfe, E. & Tengholm, A. (2019). Glucose controls glucagon secretion by directly modulating cAMP in alpha cells. Diabetologia, 62(7), 1212-1224
Open this publication in new window or tab >>Glucose controls glucagon secretion by directly modulating cAMP in alpha cells
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2019 (English)In: Diabetologia, ISSN 0012-186X, E-ISSN 1432-0428, Vol. 62, no 7, p. 1212-1224Article in journal (Refereed) Published
Abstract [en]

Aims/hypothesis

Glucagon is critical for normal glucose homeostasis and aberrant secretion of the hormone aggravates dysregulated glucose control in diabetes. However, the mechanisms by which glucose controls glucagon secretion from pancreatic alpha cells remain elusive. The aim of this study was to investigate the role of the intracellular messenger cAMP in alpha-cell-intrinsic glucose regulation of glucagon release.

Methods

Subplasmalemmal cAMP and Ca2+ concentrations were recorded in isolated and islet-located alpha cells using fluorescent reporters and total internal reflection microscopy. Glucagon secretion from mouse islets was measured using ELISA.

Results

Glucose induced Ca2+-independent alterations of the subplasmalemmal cAMP concentration in alpha cells that correlated with changes in glucagon release. Glucose-lowering-induced stimulation of glucagon secretion thus corresponded to an elevation in cAMP that was independent of paracrine signalling from insulin or somatostatin. Imposed cAMP elevations stimulated glucagon secretion and abolished inhibition by glucose elevation, while protein kinase A inhibition mimicked glucose suppression of glucagon release.

Conclusions/interpretation

Glucose concentrations in the hypoglycaemic range control glucagon secretion by directly modulating the cAMP concentration in alpha cells independently of paracrine influences. These findings define a novel mechanism for glucose regulation of glucagon release that underlies recovery from hypoglycaemia and may be disturbed in diabetes.

Keywords
Ca2+, Cyclic AMP, Glucagon release, Hypoglycaemia, Insulin, Pancreatic alpha cell, Protein kinase A, Somatostatin
National Category
Endocrinology and Diabetes
Identifiers
urn:nbn:se:uu:diva-388762 (URN)10.1007/s00125-019-4857-6 (DOI)000471176200012 ()30953108 (PubMedID)
Funder
Swedish Research CouncilErnfors FoundationNovo NordiskSwedish Diabetes AssociationEXODIAB - Excellence of Diabetes Research in Sweden
Available from: 2019-08-13 Created: 2019-08-13 Last updated: 2019-08-14Bibliographically approved
Pietilä, I., van Mourik, D., Tamelander, A., Kriz, V., Claesson-Welsh, L., Tengholm, A. & Welsh, M. (2019). Temporal Dynamics of VEGFA-Induced VEGFR2/FAK Co-Localization Depend on SHB. Cells, 8(12), Article ID 1645.
Open this publication in new window or tab >>Temporal Dynamics of VEGFA-Induced VEGFR2/FAK Co-Localization Depend on SHB
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2019 (English)In: Cells, ISSN 2073-4409, Vol. 8, no 12, article id 1645Article in journal (Refereed) Published
Abstract [en]

Focal adhesion kinase (FAK) is essential for vascular endothelial growth factor-A (VEGFA)/VEGF receptor-2 (VEGFR2)-stimulated angiogenesis and vascular permeability. We have previously noted that presence of the Src homology-2 domain adapter protein B (SHB) is of relevance for VEGFA-stimulated angiogenesis in a FAK-dependent manner. The current study was conducted in order address the temporal dynamics of co-localization between these components in HEK293 and primary lung endothelial cells (EC) by total internal reflection fluorescence microscopy (TIRF). An early (<2.5 min) VEGFA-induced increase in VEGFR2 co-localization with SHB was dependent on tyrosine 1175 in VEGFR2. VEGFA also enhanced SHB co-localization with FAK. FAK co-localization with VEGFR2 was dependent on SHB since it was significantly lower in SHB deficient EC after VEGFA addition. Absence of SHB also resulted in a gradual decline of VEGFR2 co-localization with FAK under basal (prior to VEGFA addition) conditions. A similar basal response was observed with expression of the Y1175F-VEGFR2 mutant in wild type EC. The distribution of focal adhesions in SHB-deficient EC was altered with a primarily perinuclear location. These live cell data implicate SHB as a key component regulating FAK activity in response to VEGFA/VEGFR2.

Place, publisher, year, edition, pages
Basel, Switzerland: MDPI, 2019
Keywords
VEGFR2, FAK, SHB, TIRF, focal adhesions, angiogenesis
National Category
Basic Medicine Cell Biology
Research subject
Molecular Cellbiology
Identifiers
urn:nbn:se:uu:diva-400532 (URN)10.3390/cells8121645 (DOI)000506643500173 ()
Funder
Swedish Research CouncilSwedish Cancer SocietyErnfors FoundationEXODIAB - Excellence of Diabetes Research in Sweden
Available from: 2019-12-23 Created: 2019-12-23 Last updated: 2020-02-27Bibliographically approved
Mojtaba Ghiasi, S., Dahlby, T., Hede Andersen, C., Haataja, L., Petersen, S., Omar-Hmeadi, M., . . . Tomasz Marzec, M. (2019). The Endoplasmic Reticulum Chaperone Glucose-Regulated Protein 94 is Essential for Proinsulin Handling. Diabetes, 68(4), 747-760
Open this publication in new window or tab >>The Endoplasmic Reticulum Chaperone Glucose-Regulated Protein 94 is Essential for Proinsulin Handling
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2019 (English)In: Diabetes, ISSN 0012-1797, E-ISSN 1939-327X, Vol. 68, no 4, p. 747-760Article in journal (Refereed) Published
Abstract [en]

Although endoplasmic reticulum (ER) chaperone binding to mutant proinsulin has been reported, the role of protein chaperones in the handling of wild-type proinsulin is under-investigated. Here, we have explored the importance of glucose regulated protein 94 (GRP94), a prominent ER chaperone known to fold insulin-like growth factors, in proinsulin handling within β-cells. We found that GRP94 co-immunoprecipitated with proinsulin and that inhibition of GRP94 function and/or expression reduced glucose-dependent insulin secretion, shortened proinsulin half-life and lowered intracellular proinsulin and insulin levels. This phenotype was accompanied by post-ER proinsulin misprocessing and higher numbers of enlarged insulin granules that contained amorphic material with reduced immunogold staining for mature insulin. Insulin granule exocytosis was two-fold accelerated but the secreted insulin had diminished bioactivity. Moreover, GRP94 knockdown or knockout in β-cells selectively activated Protein Kinase R-like Endoplasmic Reticulum Kinase (PERK), without increasing apoptosis levels. Finally, GRP94 mRNA was overexpressed in islets from T2D patients. We conclude that GRP94 is a chaperone crucial for proinsulin handling and insulin secretion.

National Category
Cell and Molecular Biology Endocrinology and Diabetes
Identifiers
urn:nbn:se:uu:diva-378915 (URN)10.2337/db18-0671 (DOI)000462053100007 ()30670477 (PubMedID)
Funder
Swedish Research Council
Available from: 2019-03-08 Created: 2019-03-08 Last updated: 2019-05-06Bibliographically approved
Wang, X., Jiang, L., Wallerman, O., Younis, S., Yu, Q., Klaesson, A., . . . Andersson, L. (2019). ZBED6 negatively regulates insulin production, neuronal differentiation, and cell aggregation in MIN6 cells. The FASEB Journal, 33(1), 88-100
Open this publication in new window or tab >>ZBED6 negatively regulates insulin production, neuronal differentiation, and cell aggregation in MIN6 cells
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2019 (English)In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 33, no 1, p. 88-100Article in journal (Refereed) Published
Abstract [en]

Zinc finger BED domain containing protein 6 (Zbed6) has evolved from a domesticated DNA transposon and encodes a transcription factor unique to placental mammals. The aim of the present study was to investigate further the role of ZBED6 in insulin-producing cells, using mouse MIN6 cells, and to evaluate the effects of Zbed6 knockdown on basal -cell functions, such as morphology, transcriptional regulation, insulin content, and release. Zbed6-silenced cells and controls were characterized with a range of methods, including RNA sequencing, chromatin immunoprecipitation sequencing, insulin content and release, subplasma membrane Ca2+ measurements, cAMP determination, and morphologic studies. More than 700 genes showed differential expression in response to Zbed6 knockdown, which was paralleled by increased capacity to generate cAMP, as well as by augmented subplasmalemmal calcium concentration and insulin secretion in response to glucose stimulation. We identified >4000 putative ZBED6-binding sites in the MIN6 genome, with an enrichment of ZBED6 sites at upregulated genes, such as the -cell transcription factors v-maf musculoaponeurotic fibrosarcoma oncogene homolog A and Nk6 homeobox 1. We also observed altered morphology/growth patterns, as indicated by increased cell clustering, and in the appearance of axon-like Neurofilament, medium polypeptide and tubulin 3, class III-positive protrusions. We conclude that ZBED6 acts as a transcriptional regulator in MIN6 cells and that its activity suppresses insulin production, cell aggregation, and neuronal-like differentiation.Wang, X., Jiang, L., Wallerman, O., Younis, S., Yu, Q., Klaesson, A., Tengholm, A., Welsh, N., Andersson, L. ZBED6 negatively regulates insulin production, neuronal differentiation, and cell aggregation in MIN6 cells.

Place, publisher, year, edition, pages
FEDERATION AMER SOC EXP BIOL, 2019
Keywords
-cells, cell adhesion, transcriptome analysis, ChIP-seq
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-377365 (URN)10.1096/fj.201600835R (DOI)000457401500007 ()29957057 (PubMedID)
Funder
Swedish Research Council, 80576801Swedish Research Council, 70374401Swedish Research CouncilKnut and Alice Wallenberg FoundationSwedish Child Diabetes FoundationNovo NordiskErnfors FoundationSwedish Diabetes Association
Available from: 2019-02-19 Created: 2019-02-19 Last updated: 2019-02-19Bibliographically approved
Yang, M., Idevall-Hagren, O., Gylfe, E. & Tengholm, A. (2018). A genetically encoded low-affinity Ca2+ sensor unmasks autocrine purinergic signalling in beta cells. Paper presented at 54th Annual Meeting of the European-Association-for-the-Study-of-Diabetes (EASD), OCT 01-05, 2018, Berlin, GERMANY. Diabetologia, 61, S196-S197
Open this publication in new window or tab >>A genetically encoded low-affinity Ca2+ sensor unmasks autocrine purinergic signalling in beta cells
2018 (English)In: Diabetologia, ISSN 0012-186X, E-ISSN 1432-0428, Vol. 61, p. S196-S197Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
Springer, 2018
National Category
Endocrinology and Diabetes
Identifiers
urn:nbn:se:uu:diva-367135 (URN)000443556002189 ()
Conference
54th Annual Meeting of the European-Association-for-the-Study-of-Diabetes (EASD), OCT 01-05, 2018, Berlin, GERMANY
Available from: 2018-11-29 Created: 2018-11-29 Last updated: 2018-11-29Bibliographically approved
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
Open this publication in new window or tab >>Functional Characterization of Native, High-Affinity GABAA Receptors in Human Pancreatic β Cells
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2018 (English)In: EBioMedicine, ISSN 0360-0637, E-ISSN 2352-3964, Vol. 30Article in journal (Refereed) 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.

Keywords
GABA, GABA(A) receptor, Pancreatic islet, Type 2 diabetes
National Category
Other Medical Sciences not elsewhere specified Endocrinology and Diabetes
Identifiers
urn:nbn:se:uu:diva-348267 (URN)10.1016/j.ebiom.2018.03.014 (DOI)000430303000032 ()29606630 (PubMedID)
Funder
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
Note

De 2 första författarna delar förstaförfattarskapet.

Available from: 2018-04-11 Created: 2018-04-11 Last updated: 2018-06-19Bibliographically approved
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ORCID iD: ORCID iD iconorcid.org/0000-0003-4508-0836

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