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Wuttke, Anne
Publications (10 of 16) Show all publications
Wuttke, A. (2015). Lipid Signalling Dynamics at the beta-cell Plasma Membrane. Basic & Clinical Pharmacology & Toxicology, 116(4), 281-290
Open this publication in new window or tab >>Lipid Signalling Dynamics at the beta-cell Plasma Membrane
2015 (English)In: Basic & Clinical Pharmacology & Toxicology, ISSN 1742-7835, E-ISSN 1742-7843, Vol. 116, no 4, p. 281-290Article, review/survey (Refereed) Published
Abstract [en]

Pancreatic -cells are clustered in islets of Langerhans and secrete insulin in response to increased concentrations of circulating glucose. Insulin in turn acts on liver, muscle and fat tissue to store energy and normalize the blood glucose level. Inappropriate insulin release may lead to impaired glucose tolerance and diabetes. In addition to glucose, other nutrients, neural stimuli and hormonal stimuli control insulin secretion. Many of these signals are perceived at the plasma membrane, which is also the site where insulin granules undergo exocytosis. Therefore, it is not surprising that membrane lipids play an important role in the regulation of insulin secretion. -cells release insulin in a pulsatile fashion. Signalling lipids integrate the nutrient and neurohormonal inputs to fine-tune, shape and co-ordinate the pulsatility. An important group of signalling lipids are phosphoinositides and their downstream messengers. This MiniReview will discuss new insights into lipid signalling dynamics in -cells obtained from live-cell imaging experiments with fluorescent translocation biosensors. The plasma membrane concentration of several phosphoinositides and of their downstream messengers changes rapidly upon nutrient or neurohormonal stimulation. Glucose induces the most complex spatio-temporal patterns, typically involving oscillations of messenger concentrations, which sometimes are locally restricted. The tightly controlled levels of lipid messengers can mediate specific binding of downstream effectors to the plasma membrane, contributing to the appropriate regulation of insulin secretion.

National Category
Pharmacology and Toxicology
Identifiers
urn:nbn:se:uu:diva-252390 (URN)10.1111/bcpt.12369 (DOI)000351367600001 ()25529872 (PubMedID)
Available from: 2015-05-06 Created: 2015-05-06 Last updated: 2018-01-11Bibliographically approved
Tengholm, A. & Wuttke, A. (2013). Autocrine signals mediate plasma membrane translocation of protein kinase C in insulin-secreting cells. Paper presented at 49th Annual Meeting of the European-Association-for-the-Study-of-Diabetes (EASD), SEP 23-27, 2013, Barcelona, SPAIN. Diabetologia, 56, S207-S207
Open this publication in new window or tab >>Autocrine signals mediate plasma membrane translocation of protein kinase C in insulin-secreting cells
2013 (English)In: Diabetologia, ISSN 0012-186X, E-ISSN 1432-0428, Vol. 56, p. S207-S207Article in journal, Meeting abstract (Other academic) Published
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-218011 (URN)000329196901157 ()
Conference
49th Annual Meeting of the European-Association-for-the-Study-of-Diabetes (EASD), SEP 23-27, 2013, Barcelona, SPAIN
Available from: 2014-02-07 Created: 2014-02-06 Last updated: 2017-12-06Bibliographically approved
Mokhtari, D., Al-Amin, A., Turpaev, K., Li, T., Idevall-Hagren, O., Li, J., . . . Welsh, N. (2013). Imatinib mesilate-induced phosphatidylinositol 3-kinase signalling and improved survival in insulin-producing cells: role of Src homology 2-containing inositol 5'-phosphatase interaction with c-Abl. Diabetologia, 56(6), 1327-1338
Open this publication in new window or tab >>Imatinib mesilate-induced phosphatidylinositol 3-kinase signalling and improved survival in insulin-producing cells: role of Src homology 2-containing inositol 5'-phosphatase interaction with c-Abl
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2013 (English)In: Diabetologia, ISSN 0012-186X, E-ISSN 1432-0428, Vol. 56, no 6, p. 1327-1338Article in journal (Refereed) Published
Abstract [en]

AIMS/HYPOTHESIS: It is not clear how small tyrosine kinase inhibitors, such as imatinib mesilate, protect against diabetes and beta cell death. The aim of this study was to determine whether imatinib, as compared with the non-cAbl-inhibitor sunitinib, affects pro-survival signalling events in the phosphatidylinositol 3-kinase (PI3K) pathway. METHODS: Human EndoC-βH1 cells, murine beta TC-6 cells and human pancreatic islets were used for immunoblot analysis of insulin receptor substrate (IRS)-1, Akt and extracellular signal-regulated kinase (ERK) phosphorylation. Phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] plasma membrane concentrations were assessed in EndoC-βH1 and MIN6 cells using evanescent wave microscopy. Src homology 2-containing inositol 5'-phosphatase 2 (SHIP2) tyrosine phosphorylation and phosphatase and tensin homologue deleted on chromosome 10 (PTEN) serine phosphorylation, as well as c-Abl co-localisation with SHIP2, were studied in HEK293 and EndoC-βH1 cells by immunoprecipitation and immunoblot analysis. Gene expression was assessed using RT-PCR. Cell viability was measured using vital staining. RESULTS: Imatinib stimulated ERK(thr202/tyr204) phosphorylation in a c-Abl-dependent manner. Imatinib, but not sunitinib, also stimulated IRS-1(tyr612), Akt(ser473) and Akt(thr308) phosphorylation. This effect was paralleled by oscillatory bursts in plasma membrane PI(3,4,5)P3 levels. Wortmannin induced a decrease in PI(3,4,5)P3 levels, which was slower in imatinib-treated cells than in control cells, indicating an effect on PI(3,4,5)P3-degrading enzymes. In line with this, imatinib decreased the phosphorylation of SHIP2 but not of PTEN. c-Abl co-immunoprecipitated with SHIP2 and its binding to SHIP2 was largely reduced by imatinib but not by sunitinib. Imatinib increased total β-catenin levels and cell viability, whereas sunitinib exerted negative effects on cell viability. CONCLUSIONS/INTERPRETATION: Imatinib inhibition of c-Abl in beta cells decreases SHIP2 activity, which results in enhanced signalling downstream of PI3 kinase.

National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-196342 (URN)10.1007/s00125-013-2868-2 (DOI)000318787600015 ()23462796 (PubMedID)
Available from: 2013-03-08 Created: 2013-03-08 Last updated: 2018-01-11Bibliographically approved
Wuttke, A. (2013). Lipid Signalling Dynamics in Insulin-secreting β-cells. (Doctoral dissertation). Uppsala: Acta Universitatis Upsaliensis
Open this publication in new window or tab >>Lipid Signalling Dynamics in Insulin-secreting β-cells
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. p. 71
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 892
Keywords
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:nbn:se:uu:diva-198046 (URN)978-91-554-8644-0 (ISBN)
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: 2018-01-11Bibliographically approved
Wuttke, A., Idevall-Hagren, O. & Tengholm, A. (2013). P2Y1 receptor-dependent diacylglycerol signaling microdomains in β cells promote insulin secretion. The FASEB Journal, 27(4), 1610-1620
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, p. 1610-1620Article 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: 2018-01-11Bibliographically approved
Bergenholtz, S. S., Wessman, P., Wuttke, A. & Håkansson, S. (2012). A case study on stress preconditioning of a Lactobacillus strain prior to freeze-drying. Cryobiology, 64(3), 152-159
Open this publication in new window or tab >>A case study on stress preconditioning of a Lactobacillus strain prior to freeze-drying
2012 (English)In: Cryobiology, ISSN 0011-2240, E-ISSN 1090-2392, Vol. 64, no 3, p. 152-159Article in journal (Refereed) Published
Abstract [en]

Freeze-drying of bacterial cells with retained viability and activity after storage requires appropriate formulation, i.e. mixing of physiologically adapted cell populations with suitable protective agents, and control of the freeze-drying process. Product manufacturing may alter the clinical effects of probiotics and it is essential to identify and understand possible factor co-dependencies during manufacturing. The physical solid-state behavior of the formulation and the freeze-drying parameters are critical for bacterial survival and thus process optimization is important, independent of strain. However, the maximum yield achievable is also strain-specific and strain survival is governed by e.g. medium, cell type, physiological state, excipients used, and process. The use of preferred compatible solutes for cross-protection of Lactobacilli during industrial manufacturing may be a natural step to introduce robustness, but knowledge is lacking on how compatible solutes, such as betaine, influence formulation properties and cell survival. This study characterized betaine formulations, with and without sucrose, and tested these with the model lactic acid bacteria Lactobacillus coryniformis Si3. Betaine alone did not act as a lyo-protectant and thus betaine import prior to freeze-drying should be avoided. Differences in protective agents were analyzed by calorimetry, which proved to be a suitable tool for evaluating the characteristics of the freeze-dried end products.

Keywords
Lactobacillus, Betaine, Sucrose, Glass transition, Crystallization, Freeze-drying, Lyophilization, Protection, Survival, Solid-state characterization
National Category
Microbiology Environmental Biotechnology Industrial Biotechnology
Identifiers
urn:nbn:se:uu:diva-169288 (URN)10.1016/j.cryobiol.2012.01.002 (DOI)000305167400003 ()22266474 (PubMedID)
Available from: 2012-02-27 Created: 2012-02-27 Last updated: 2017-12-07Bibliographically approved
Wuttke, A., Idevall-Hagren, O. & Tengholm, A. (2011). Exocytotic release of ATP triggers diacylglycerol spiking in insulin-secreting cells. In: Minutes Of The 46th General Assembly Of The European Association For The Study Of Diabetes: held in Stockholmsmässan, Stockholm, Sweden on 23 September 2010. Paper presented at 47th Annual Meeting of the European-Association-for-the-Study-of-Diabetes, Lisbon, SEP 12-16, 2011 (pp. S194-S195).
Open this publication in new window or tab >>Exocytotic release of ATP triggers diacylglycerol spiking in insulin-secreting cells
2011 (English)In: Minutes Of The 46th General Assembly Of The European Association For The Study Of Diabetes: held in Stockholmsmässan, Stockholm, Sweden on 23 September 2010, 2011, p. S194-S195Conference paper, Published paper (Other academic)
Series
Diabetologia, ISSN 0012-186X ; 54
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-162075 (URN)10.1007/s00125-011-2276-4 (DOI)
Conference
47th Annual Meeting of the European-Association-for-the-Study-of-Diabetes, Lisbon, SEP 12-16, 2011
Available from: 2011-11-23 Created: 2011-11-23 Last updated: 2018-01-12Bibliographically approved
Barig, S., Alisch, R., Nieland, S., Wuttke, A., Graeser, Y., Huddar, M., . . . Stahmann, K.-P. (2011). Monoseptic growth of fungal lipase producers under minimized sterile conditions: Cultivation of Phialemonium curvatum in 350 L scale. Engineering in Life Sciences, 11(4), 387-394
Open this publication in new window or tab >>Monoseptic growth of fungal lipase producers under minimized sterile conditions: Cultivation of Phialemonium curvatum in 350 L scale
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2011 (English)In: Engineering in Life Sciences, ISSN 1618-0240, E-ISSN 1618-2863, Vol. 11, no 4, p. 387-394Article in journal (Refereed) Published
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:uu:diva-162038 (URN)10.1002/elsc.201000219 (DOI)000294263600007 ()
Available from: 2011-11-23 Created: 2011-11-22 Last updated: 2017-12-08
Wuttke, A., Sågetorp, J. & Tengholm, A. (2010). Distinct plasma-membrane PtdIns(4)P and PtdIns(4,5)P2 dynamics in secretagogue-stimulated β-cells. Journal of Cell Science, 123(9), 1492-1502
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, p. 1492-1502Article 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.

Keywords
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: 2018-01-12Bibliographically approved
Wuttke, A., Emanuelsson, H. & Tengholm, A. (2010). Glucose and muscarinic stimulation trigger distinct diacylglycerol signals in pancreatic beta cells. Paper presented at 46th Annual Meeting of the European-Association-for-the- Study-of-Diabetes (EASD), Stockholm, SEP 20-24, 2010. , 53
Open this publication in new window or tab >>Glucose and muscarinic stimulation trigger distinct diacylglycerol signals in pancreatic beta cells
2010 (English)Conference paper, Published paper (Other academic)
Series
Diabetologia, ISSN 0012-186X ; 53
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-138337 (URN)
Conference
46th Annual Meeting of the European-Association-for-the- Study-of-Diabetes (EASD), Stockholm, SEP 20-24, 2010
Note
Meeting AbstractAvailable from: 2010-12-17 Created: 2010-12-17 Last updated: 2012-02-09Bibliographically approved
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