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Lipid Signalling Dynamics in Insulin-secreting β-cells
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Certain membrane lipids are involved in intracellular signalling processes, among them phosphoinositides and diacylglycerol (DAG). They mediate a variety of functions, including the effects of nutrients and neurohormonal stimuli on insulin secretion from pancreatic β-cells. To ensure specificity of the signal, their concentrations are maintained under tight spatial and temporal control. Here, live-cell imaging techniques were employed to investigate spatio-temporal aspects of lipid signalling in the plasma membrane of insulin-secreting β-cells. The concentration of phosphatidylinositol 4-phosphate [PtdIns(4)P] increased after stimulation with glucose or Gq protein-coupled receptor agonists. The glucose effect was Ca2+-dependent, whereas the receptor response was mediated by isoforms of novel protein kinase C (PKC). The increases in PtdIns(4)P were paralleled by lowerings of the phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] concentration. This relationship was not caused by conversion of PtdIns(4,5)P2 to PtdIns(4)P but rather reflected independent regulation of the two lipids. Stimulation of β-cells with glucose or a high K+ concentration induced pronounced, repetitive increases in plasma-membrane DAG concentration, which were locally restricted and lasted only for a few seconds. This pattern was caused by exocytotic release of ATP, which feedback-activates purinergic P2Y1-receptors and stimulates local phospholipase C-mediated DAG generation. Despite their short durations the DAG spikes triggered local activation of PKC. Novel PKCs were recruited to the plasma membrane both after glucose and muscarinic receptor stimulation. While the glucose-induced translocation was synchronized with DAG spiking, muscarinic stimulation induced sustained elevation of the DAG concentration and stable membrane association of the kinase. Also conventional PKCs translocated to the membrane after glucose and receptor stimulation. The glucose-induced response was complex with sustained membrane association mirroring the cytoplasmic Ca2+ concentration, and superimposed brief recurring translocations caused by DAG. Interruption of the purinergic feedback loop underlying DAG spiking suppressed insulin secretion. Since the DAG spikes reflected exocytosis events, a single-cell secretion assay was established, which allowed continuous recording of secretion dynamics from many cells in parallel over extended periods of time. With this approach it was possible to demonstrate that insulin exerts negative feedback on its own release via a phosphatidylinositol 3,4,5-trisphosphate-dependent mechanism.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. , 71 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 892
Keyword [en]
ATP, β-cell, diacylglycerol, insulin secretion, oscillations, PtdIns(4)P, PtdIns(4, 5)P2, protein kinase C, P2Y receptor
National Category
Cell and Molecular Biology
Research subject
Medical Cell Biology
Identifiers
URN: urn:nbn:se:uu:diva-198046ISBN: 978-91-554-8644-0 (print)OAI: oai:DiVA.org:uu-198046DiVA: diva2:615087
Public defence
2013-05-23, B41, Biomedical Centre, Husargatan 3, 75123 Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2013-04-30 Created: 2013-04-08 Last updated: 2013-09-02Bibliographically approved
List of papers
1. Distinct plasma-membrane PtdIns(4)P and PtdIns(4,5)P2 dynamics in secretagogue-stimulated β-cells
Open this publication in new window or tab >>Distinct plasma-membrane PtdIns(4)P and PtdIns(4,5)P2 dynamics in secretagogue-stimulated β-cells
2010 (English)In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 123, no 9, 1492-1502 p.Article in journal (Refereed) Published
Abstract [en]

Phosphoinositides regulate numerous processes in various subcellular compartments. Whereas many stimuli trigger changes in the plasma-membrane PtdIns(4,5)P-2 concentration, little is known about its precursor, PtdIns(4)P, in particular whether there are stimulus-induced alterations independent of those of PtdIns(4,5)P-2. We investigated plasma-membrane PtdIns(4)P and PtdIns(4,5)P-2 dynamics in insulin-secreting MIN6 cells using fluorescent translocation biosensors and total internal reflection microscopy. Loss of PtdIns(4,5)P-2 induced by phospholipase C (PLC)-activating receptor agonists or stimulatory glucose concentrations was paralleled by increased PtdIns(4)P levels. In addition, glucose-stimulated cells regularly showed anti-synchronous oscillations of the two lipids. Whereas glucose-induced PtdIns(4)P elevation required voltage-gated Ca2+ entry and was mimicked by membrane-depolarizing stimuli, the receptor-induced response was Ca2+ independent, but sensitive to protein kinase C (PKC) inhibition and mimicked by phorbol ester stimulation. We conclude that glucose and PLC-activating receptor stimuli trigger Ca2+- and PKC-dependent changes in the plasma-membrane PtdIns(4)P concentration that are independent of the effects on PtdIns(4,5)P-2. These findings indicate that enhanced formation of PtdIns(4)P, apart from ensuring efficient replenishment of the PtdIns(4,5)P-2 pool, might serve an independent signalling function by regulating the association of PtdIns(4)P-binding proteins with the plasma membrane.

Keyword
Ca2+, Glucose, Insulin-secreting cells, Oscillations, PI4-kinase, PtdIns(4)P, PtdIns(4, 5)P-2
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-136347 (URN)10.1242/jcs.060525 (DOI)000276912300013 ()
Available from: 2010-12-11 Created: 2010-12-11 Last updated: 2017-12-11Bibliographically approved
2. P2Y1 receptor-dependent diacylglycerol signaling microdomains in β cells promote insulin secretion
Open this publication in new window or tab >>P2Y1 receptor-dependent diacylglycerol signaling microdomains in β cells promote insulin secretion
2013 (English)In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 27, no 4, 1610-1620 p.Article in journal (Refereed) Published
Abstract [en]

Diacylglycerol (DAG) controls numerous cell functions by regulating the localization of C1-domain-containing proteins, including protein kinase C (PKC), but little is known about the spatiotemporal dynamics of the lipid. Here, we explored plasma membrane DAG dynamics in pancreatic beta cells and determined whether DAG signaling is involved in secretagogue-induced pulsatile release of insulin. Single MIN6 cells, primary mouse beta cells, and human beta cells within intact islets were transfected with translocation biosensors for DAG, PKC activity, or insulin secretion and imaged with total internal reflection fluorescence microscopy. Muscarinic receptor stimulation triggered stable, homogenous DAG elevations, whereas glucose induced short-lived (7.1 +/- 0.4 s) but high-amplitude elevations (up to 109 +/- 10% fluorescence increase) in spatially confined membrane regions. The spiking was mimicked by membrane depolarization and suppressed after inhibition of exocytosis or of purinergic P2Y(1), but not P2X receptors, reflecting involvement of autocrine purinoceptor activation after exocytotic release of ATP. Each DAG spike caused local PKC activation with resulting dissociation of its substrate protein MARCKS from the plasma membrane. Inhibition of spiking reduced glucose-induced pulsatile insulin secretion. Thus, stimulus-specific DAG signaling patterns appear in the plasma membrane, including distinctmicrodomains, which have implications for the kinetic control of exocytosis and other membrane-associated processes.-Wuttke, A., Idevall-Hagren, O., Tengholm, A. P2Y(1) receptor-dependent diacylglycerol signaling microdomains in beta cells promote insulin secretion. 

National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-198038 (URN)10.1096/fj.12-221499 (DOI)000316940800031 ()
Available from: 2013-04-08 Created: 2013-04-08 Last updated: 2017-12-06Bibliographically approved
3. Autocrine signalling underlies fast repetitive plasma membrane translocation of protein kinase C in β-cells
Open this publication in new window or tab >>Autocrine signalling underlies fast repetitive plasma membrane translocation of protein kinase C in β-cells
(English)Manuscript (preprint) (Other academic)
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-198040 (URN)
Available from: 2013-04-08 Created: 2013-04-08 Last updated: 2013-09-02
4. Continuous imaging of exocytosis in β-cells reveals negative feedback of insulin
Open this publication in new window or tab >>Continuous imaging of exocytosis in β-cells reveals negative feedback of insulin
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-198041 (URN)
Available from: 2013-04-08 Created: 2013-04-08 Last updated: 2013-10-21

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