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Glucose-induced cyclic AMP oscillations regulate pulsatile insulin secretion
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. (Gylfe Tengholm)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. (Gylfe Tengholm)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. (Gylfe Tengholm)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. (Gylfe Tengholm)
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2008 (English)In: Cell Metabolism, ISSN 1550-4131, E-ISSN 1932-7420, Vol. 8, no 1, p. 26-37Article in journal (Refereed) Published
Abstract [en]

Cyclic AMP (cAMP) and Ca2+ are key regulators of exocytosis in many cells, including insulin-secreting β-cells. Glucose-stimulated insulin secretion from β cells is pulsatile and involves oscillations of the cytoplasmic Ca2+ concentration ([Ca2+]i), but little is known about the detailed kinetics of cAMP signalling. Using evanescent-wave fluorescence imaging we found that glucose induces pronounced oscillations of cAMP in the sub-membrane space of single MIN6-cells and primary mouse β-cells. These oscillations were preceded and enhanced by elevations of [Ca2+]i. However, conditions raising cytoplasmic ATP could trigger cAMP elevations without accompanying [Ca2+]i rise, indicating that adenylyl cyclase activity may be controlled also by the substrate concentration. The cAMP oscillations correlated with pulsatile insulin release. Whereas elevation of cAMP enhanced secretion, inhibition of adenylyl cyclases suppressed both cAMP oscillations and pulsatile insulin release. We conclude that cell metabolism directly controls cAMP, and that glucose-induced cAMP oscillations regulate the magnitude and kinetics of insulin exocytosis.

Place, publisher, year, edition, pages
Cell Press , 2008. Vol. 8, no 1, p. 26-37
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-86694DOI: 10.1016/j.cmet.2008.06.003ISI: 000257339600007OAI: oai:DiVA.org:uu-86694DiVA, id: diva2:127005
Available from: 2008-12-03 Created: 2008-11-27 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Cyclic AMP Oscillations in Insulin-Secreting Cells
Open this publication in new window or tab >>Cyclic AMP Oscillations in Insulin-Secreting Cells
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cyclic AMP is an intracellular messenger that regulates numerous processes in various types of cells. In pancreatic β-cells, cAMP potentiates the secretion of insulin by promoting Ca2+ signals and by amplifying Ca2+-triggered exocytosis. Whereas Ca2+ signals have been extensively characterized, little is known about the kinetics of cAMP signals. To enable measurements of the cAMP concentration beneath the plasma membrane ([cAMP]pm) of individual cells, a translocation biosensor was created based on fluorescent-protein-tagged subunits of protein kinase A (PKA). Evanescent wave microscopy imaging of biosensor-expressing clonal β-cells revealed that the insulinotropic hormones glucagon and GLP-1 triggered pronounced oscillations in [cAMP]pm. Simultaneous measurements of the intracellular Ca2+ concentration showed that cAMP and Ca2+ oscillations were synchronized and interdependent. [cAMP]pm oscillations were also triggered in clonal and primary mouse β-cells by an elevation of the glucose concentration from 3 to 11 mM. These oscillations were preceded and enhanced by elevations of Ca2+. However, conditions raising cytoplasmic ATP could trigger cAMP elevations also without accompanying Ca2+ changes, indicating that adenylyl cyclase activity may be directly controlled by the substrate concentration. Experiments with 3-isobutylmethylxanthine (IBMX) and various family-selective phosphodiesterase (PDE) inhibitors indicated that [cAMP]pm oscillations are generated by periodic formation of the messenger by adenylyl cyclases. PDE1 and PDE3 as well as IBMX-insensitive mechanisms shape [cAMP]pm, but no single PDE isoform was required for glucose generation of [cAMP]pm oscillations. Recordings of single-cell insulin secretion kinetics with a fluorescent biosensor that reports formation of the phospholipid PIP3 in the plasma membrane in response to autocrine insulin receptor activation showed that [cAMP]pm oscillations were paralleled by pulsatile insulin release. Whereas adenylyl cyclase inhibition suppressed both [cAMP]pm oscillations and pulsatile insulin release, elevation of [cAMP]pm enhanced secretion. Investigation of the effects of different temporal patterns of [cAMP]pm showed that brief [cAMP]pm elevation is sufficient to trigger cytoplasmic responses, whereas sustained elevation is required to induce translocation of the PKA catalytic subunit into the nucleus. In conclusion, these studies demonstrate for the first time in mammalian cells that [cAMP]pm oscillates in response to physiological stimuli. The glucose-induced [cAMP]pm oscillations are generated by periodic cAMP production mediated by interplay between ATP and Ca2+ in the sub-membrane space, and may contribute to both triggering and amplifying pathways of insulin secretion. Apart from regulating the precise kinetics of insulin exocytosis, temporal encoding of cAMP signals might constitute a basis for differential regulation of downstream cellular targets.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2009. p. 60
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 423
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-9563 (URN)978-91-554-7421-8 (ISBN)
Public defence
2009-03-21, B21, BMC, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2009-02-27 Created: 2009-02-12 Last updated: 2018-01-13Bibliographically approved
2. Oscillatory Signaling and Insulin Secretion from Single ß-cells
Open this publication in new window or tab >>Oscillatory Signaling and Insulin Secretion from Single ß-cells
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

cAMP and Ca2+ are key regulators of exocytosis in many cells, including insulin-secreting pancreatic β-cells. Glucose-stimulated insulin secretion from β-cells is pulsatile and driven by oscillations of the cytoplasmic Ca2+ concentration ([Ca2+]i), but little is known about the kinetics of cAMP signaling and the mechanisms of cAMP action. Evanescent wave microscopy and fluorescent translocation biosensors were used to monitor plasma membrane-related signaling events in single MIN6-cells and primary mouse β-cells. Glucose stimulation of insulin secretion resulted in pronounced oscillations of the membrane phospholipid PIP3 caused by autocrine activation of insulin receptors. Glucose also triggered oscillations of the sub-plasma membrane cAMP concentration ([cAMP]pm). These oscillations were preceded and enhanced by elevations of [Ca2+]i, but conditions raising cytoplasmic ATP triggered [cAMP]pm elevations without accompanying changes in [Ca2+]i. The [cAMP]pm oscillations were also synchronized with PIP3 oscillations and both signals were suppressed after inhibition of adenylyl cyclases. Protein kinase A (PKA) was important for promoting concomitant initial elevations of [cAMP]pm and [Ca2+]i, and PKA inhibitors diminished the PIP3 response when applied before glucose stimulation, but did not affect already manifested PIP3 oscillations. The glucose-induced PIP3 oscillations were markedly suppressed in cells treated with siRNA against the cAMP-dependent guanine nucleotide exchange factor Epac2. Pharmacological activation of Epac restored PIP3 responses after adenylyl cyclase or PKA inhibition. Glucose and other cAMP-elevating stimuli induced redistribution of fluorescence-tagged Epac2 from the cytoplasm to the plasma membrane. This translocation was modulated by [Ca2+]i and depended on intact cyclic nucleotide-binding and Ras-association domains. In conclusion, glucose generates cAMP oscillations in β-cells via a concerted action of Ca2+ and metabolically generated ATP. The oscillations are important for the magnitude and kinetics of insulin secretion. While both protein kinase A and Epac is required for initiation of insulin secretion the cAMP-dependence of established pulsatility is mediated by Epac2.

Place, publisher, year, edition, pages
Uppsala: A U U, 2010. p. 71
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 520
Keywords
cAMP, Ca2+, oscillations, beta-cell, insulin secretion, evanescent wave microscopy, PIP3, PKA, Epac
National Category
Cell and Molecular Biology
Research subject
Medical Cell Biology
Identifiers
urn:nbn:se:uu:diva-113686 (URN)978-91-554-7718-9 (ISBN)
Public defence
2010-03-19, B21, BMC, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2010-02-25 Created: 2010-02-02 Last updated: 2018-01-12Bibliographically approved

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Dyachok, OlegTian, GengWuttke, AnneAkusjärvi, GöranGylfe, ErikTengholm, Anders
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