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Jakobsson, Ida
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Publications (7 of 7) Show all publications
Alenkvist, I. (2016). Epac2 signaling at the β-cell plasma membrane. (Doctoral dissertation). Uppsala: Acta Universitatis Upsaliensis
Open this publication in new window or tab >>Epac2 signaling at the β-cell plasma membrane
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Secretion of appropriate amounts of insulin from pancreatic β-cells is crucial for glucose homeostasis. The β-cells release insulin in response to glucose and other nutrients, hormones and neurotransmitters, which trigger intracellular signaling cascades, that result in exocytotic fusion of insulin-containing vesicles with the plasma membrane. Increases of the intracellular concentration of calcium ions ([Ca2+]i) trigger exocytosis, whereas the messenger cyclic adenosine monophosphate (cAMP) amplifies various steps of the secretion process. The protein Epac2 mediates some effects of cAMP, but little is known about its regulation in β-cells. In this study, the spatio-temporal dynamics of Epac2 was investigated in insulin-secreting MIN6-cells and primary β-cells using various cell signaling biosensors and live-cell fluorescence microscopy approaches. Increases in the cAMP concentration triggered translocation of Epac2 from the cytoplasm to the plasma membrane. Oscillations of cAMP induced by glucose and the insulin-releasing hormone GLP-1 were associated with cyclic translocation of Epac2. Analyses of Epac2 mutants showed that the high-affinity cyclic nucleotide-binding domain and Ras-association domains were crucial for the translocation, whereas neither the DEP domain, nor the low-affinity cAMP-binding domain were required for membrane binding. However, the latter domain targeted Epac2 to insulin granules at the plasma membrane, which promoted their priming for exocytosis. Depolarization-induced elevations of [Ca2+]i also stimulated Epac2 translocation, but the effects were complex and in the presence of high cAMP concentrations, [Ca2+]i increases often reduced membrane binding. The stimulatory effect of Ca2+ was mediated by increased Ras activity, while the inhibitory effect reflected reduced concentrations of the membrane phospholipid PtdIns(4,5)P2. Anti-diabetic drugs of the sulfonylurea class, suggested to directly activate Epac2, induced translocation indirectly by depolarizing β-cells to increase [Ca2+]i. Epac2 is an activator of Rap GTPases, and its translocation increased Rap activity at the plasma membrane. It is concluded that the subcellular localization of Epac2 is controlled by a complex interplay between cAMP, Ca2+ and PtdIns(4,5)P2 and that the protein controls insulin release by binding to the exocytosis machinery. These results provide new insights into the regulation of β-cell function and may facilitate the development of new anti-diabetic drugs that amplify insulin secretion.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. p. 51
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1227
Keywords
β-cell, insulin secretion, cAMP, Ca2+, Epac, Rap, exocytosis, sulfonylurea
National Category
Cell and Molecular Biology
Research subject
Medical Cell Biology
Identifiers
urn:nbn:se:uu:diva-284638 (URN)978-91-554-9580-0 (ISBN)
Public defence
2016-06-08, A1:107a, BMC, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2016-05-17 Created: 2016-04-19 Last updated: 2018-01-10
Alenkvist, I., Dyachok, O., Tian, G., Li, J., Mehrabanfar, S., Jin, Y., . . . Welsh, M. (2014). Absence of Shb impairs insulin secretion by elevated FAK activity in pancreatic islets. Journal of Endocrinology, 223(3), 267-275
Open this publication in new window or tab >>Absence of Shb impairs insulin secretion by elevated FAK activity in pancreatic islets
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2014 (English)In: Journal of Endocrinology, ISSN 0022-0795, E-ISSN 1479-6805, Vol. 223, no 3, p. 267-275Article in journal (Refereed) Published
Abstract [en]

The Src homology-2 domain containing protein B (SHB) has previously been shown to function as a pleiotropic adapter protein, conveying signals from receptor tyrosine kinases to intracellular signaling intermediates. The overexpression of Shb in β-cells promotes β-cell proliferation by increased insulin receptor substrate (IRS) and focal adhesion kinase (FAK) activity, whereas Shb deficiency causes moderate glucose intolerance and impaired first-peak insulin secretion. Using an array of techniques, including live-cell imaging, patch-clamping, immunoblotting, and semi-quantitative PCR, we presently investigated the causes of the abnormal insulin secretory characteristics in Shb-knockout mice. Shb-knockout islets displayed an abnormal signaling signature with increased activities of FAK, IRS, and AKT. β-catenin protein expression was elevated and it showed increased nuclear localization. However, there were no major alterations in the gene expression of various proteins involved in the β-cell secretory machinery. Nor was Shb deficiency associated with changes in glucose-induced ATP generation or cytoplasmic Ca(2) (+) handling. In contrast, the glucose-induced rise in cAMP, known to be important for the insulin secretory response, was delayed in the Shb-knockout compared with WT control. Inhibition of FAK increased the submembrane cAMP concentration, implicating FAK activity in the regulation of insulin exocytosis. In conclusion, Shb deficiency causes a chronic increase in β-cell FAK activity that perturbs the normal insulin secretory characteristics of β-cells, suggesting multi-faceted effects of FAK on insulin secretion depending on the mechanism of FAK activation.

National Category
Endocrinology and Diabetes
Identifiers
urn:nbn:se:uu:diva-236195 (URN)10.1530/JOE-14-0531 (DOI)000345292500007 ()25274988 (PubMedID)
Available from: 2014-11-14 Created: 2014-11-14 Last updated: 2017-12-05Bibliographically approved
Hoivik, E. A., Witsoe, S. L., Bergheim, I. R., Xu, Y., Jakobsson, I., Tengholm, A., . . . Bakke, M. (2013). DNA Methylation of Alternative Promoters Directs Tissue Specific Expression of Epac2 Isoforms. PLoS ONE, 8(7), e67925
Open this publication in new window or tab >>DNA Methylation of Alternative Promoters Directs Tissue Specific Expression of Epac2 Isoforms
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2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 7, p. e67925-Article in journal (Refereed) Published
Abstract [en]

Epac 1 and Epac 2 (Epac1/2; exchange factors directly activated by cAMP) are multidomain proteins that mediate cellular responses upon activation by the signaling molecule cAMP. Epac1 is ubiquitously expressed, whereas Epac2 exhibits a restricted expression pattern. The gene encoding Epac2 gives rise to at least three protein isoforms (Epac2A, Epac2B and Epac2C) that exhibit confined tissue and cell specific expression profiles. Here, we describe alternative promoter usage for the different isoforms of Epac2, and demonstrate that the activity of these promoters depend on the DNA methylation status. Bisulfite sequencing demonstrated that the level of methylation of the promoters in different tissues correlates with Epac2 isoform expression. The presented data indicate that the tissue-specific expression of the Epac2 isoforms is epigenetically regulated, and identify tissue-specific differentially methylated promoter regions within the Epac2 locus that are essential for its transcriptional control.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-209493 (URN)10.1371/journal.pone.0067925 (DOI)000323350700061 ()
Available from: 2013-10-21 Created: 2013-10-21 Last updated: 2017-12-06Bibliographically approved
Idevall-Hagren, O., Jakobsson, I., Xu, Y. & Tengholm, A. (2013). Spatial Control of Epac2 Activity by cAMP and Ca2+-Mediated Activation of Ras in Pancreatic beta Cells. Science Signaling, 6(273), ra29
Open this publication in new window or tab >>Spatial Control of Epac2 Activity by cAMP and Ca2+-Mediated Activation of Ras in Pancreatic beta Cells
2013 (English)In: Science Signaling, ISSN 1945-0877, E-ISSN 1937-9145, Vol. 6, no 273, p. ra29-Article in journal (Refereed) Published
Abstract [en]

The cAMP (adenosine 3',5'-monophosphate)-activated guanine nucleotide exchange factor (GEF) Epac2 is an important mediator of cAMP-dependent processes in multiple cell types. We used real-time confocal and total internal reflection fluorescence microscopy to examine the spatiotemporal regulation of Epac2, which is a GEF for the guanosine triphosphatase (GTPase) Rap. We demonstrated that increases in the concentration of cAMP triggered the translocation of Epac2 from the cytoplasm to the plasma membrane in insulin-secreting beta cells. Glucose-induced oscillations of the submembrane concentration of cAMP were associated with cyclic translocation of Epac2, and this translocation could be amplified by increases in the cytoplasmic Ca2+ concentration. Analyses of Epac2 mutants identified the high-affinity cAMP-binding and the Ras association domains as crucial for the translocation. Expression of a dominant-negative Ras mutant reduced Epac2 translocation, and Ca2+-dependent oscillations in Ras activity synchronized with Epac2 translocation in single beta cells. The cyclic translocation of Epac2 was accompanied by oscillations of Rap GTPase activity at the plasma membrane, and expression of an inactive Rap1B mutant decreased insulin secretion. Thus, Epac2 localization is dynamically controlled by cAMP as well as by Ca2+-mediated activation of Ras. These results help to explain how oscillating signals can produce pulses of insulin release from pancreatic b cells.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-201253 (URN)10.1126/scisignal.2003932 (DOI)000318350100002 ()
Available from: 2013-06-10 Created: 2013-06-10 Last updated: 2017-12-06Bibliographically approved
Alenkvist, I., Idevall-Hagren, O. & Tengholm, A.Localization of Epac2 to the plasma membrane is controlled by an interplay between cAMP, Ca2+ and PtdIns(4,5)P2.
Open this publication in new window or tab >>Localization of Epac2 to the plasma membrane is controlled by an interplay between cAMP, Ca2+ and PtdIns(4,5)P2
(English)Manuscript (preprint) (Other academic)
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-284580 (URN)
Available from: 2016-04-19 Created: 2016-04-18 Last updated: 2018-01-10
Alenkvist, I., Xu, Y. & Tengholm, A.Sulfonylureas trigger Ca2+-dependent Epac2 recruitment and Rap activation at the plasma membrane in β-cells.
Open this publication in new window or tab >>Sulfonylureas trigger Ca2+-dependent Epac2 recruitment and Rap activation at the plasma membrane in β-cells
(English)Manuscript (preprint) (Other academic)
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-284582 (URN)
Available from: 2016-04-19 Created: 2016-04-18 Last updated: 2018-01-10
Alenkvist, I., Gandasi, N. R., Barg, S. & Tengholm, A.Two-step membrane recruitment of Epac2 primes secretory granules for exocytosis.
Open this publication in new window or tab >>Two-step membrane recruitment of Epac2 primes secretory granules for exocytosis
(English)Manuscript (preprint) (Other academic)
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-284568 (URN)
Available from: 2016-04-19 Created: 2016-04-18 Last updated: 2018-01-10
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