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On the Generation of cAMP Oscillations and Regulation of the Ca2+ Store-operated Pathway in Pancreatic Islet α- and 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]

Insulin and glucagon are released in pulses from pancreatic β- and α-cells, respectively. Both cell types are electrically excitable, and elevation of the cytoplasmic Ca2+ concentration ([Ca2+]i) due to depolarization with voltage-dependent entry of the cation is the main trigger of hormone secretion. Store-operated Ca2+ entry  (SOCE) also contributes to the [Ca2+]i elevation and this process has been suggested to be particularly important for glucagon secretion. cAMP is another important messenger that amplifies Ca2+-triggered secretion of both hormones, but little is known about cAMP dynamics in islet cells. In type-2 diabetes, there is deteriorated β-cell function associated with elevated concentrations of fatty acids, but the underlying mechanisms are largely unknown. To clarify the processes that regulate insulin and glucagon secretion, cAMP signalling and the store-operated pathway were investigated in β- and α-cells, primarily within their natural environment in intact mouse and human islets of Langerhans. Fluorescent biosensors and total internal reflection microscopy were used to investigate signalling specifically at the plasma membrane (PM). Adrenaline increased and decreased the sub-PM cAMP concentration ([cAMP]pm) in immuno-identified α-cells and β-cells, respectively, which facilitated cell identification. Glucagon elicited [cAMP]pm oscillations in α- and β-cells, demonstrating both auto- and paracrine effects of the hormone. Whereas glucagon-like peptide 1 (GLP-1) consistently elevated [cAMP]pm in β-cells, only few α-cells responded, indicating that GLP-1 regulates glucagon secretion without changes of α-cell [cAMP]pm. Both α- and β-cells responded to glucose with pronounced oscillations of [cAMP]pm that were partially Ca2+-dependent and synchronized among islet β-cells. The glucose-induced cAMP formation was mediated by plasma membrane-bound adenylyl cyclases. Several phosphodiesterases (PDEs), including the PDE1, -3, -4, and -8 families, were required for shaping the [cAMP]pm signals and pulsatile insulin secretion. Prolonged exposure of islets to the fatty acid palmitate deteriorated glucose-stimulated insulin secretion with loss of pulsatility. This defect was associated with impaired cAMP generation, while [Ca2+]i signalling was essentially unaffected. Stromal interacting molecule 1 (STIM1) is critical for activation of SOCE by sensing the Ca2+ concentration in the endoplasmic reticulum (ER). ER Ca2+ depletion caused STIM1 aggregation, co-clustering with the PM Ca2+ channel protein Orai1 and SOCE activation. Glucose, which inhibits SOCE by filling the ER with Ca2+, reversed the PM association of STIM1. Consistent with a role of the store-operated pathway in glucagon secretion, this effect was maximal at the low glucose concentrations that inhibit glucagon release, whereas considerably higher concentrations were required in β-cells. Adrenaline induced STIM1 translocation to the PM in α-cells and the reverse process in β-cells, partially reflecting the opposite effects of adrenaline on cAMP in the two cell types. However, cAMP-induced STIM1 aggregates did not co-cluster with Orai1 or activate SOCE, indicating that STIM1 translocation can occur independently of Orai1 clustering and SOCE.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. , 64 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 860
Keyword [en]
cAMP, oscillations, adrenaline, GLP-1, phosphodiesterase, palmitate, STIM1, Orai1, store-operated calcium entry, insulin secretion, glucagon secretion, β-cell, α-cell
National Category
Cell and Molecular Biology
Research subject
Medical Cell Biology
Identifiers
URN: urn:nbn:se:uu:diva-191852ISBN: 978-91-554-8584-9 (print)OAI: oai:DiVA.org:uu-191852DiVA: diva2:589662
Public defence
2013-03-07, B22, BMC, Uppsala University, Husargatan 3, 751 23, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2013-02-13 Created: 2013-01-14 Last updated: 2013-04-02Bibliographically approved
List of papers
1. Glucose- and Hormone-Induced cAMP Oscillations in α- and β-Cells Within Intact Pancreatic Islets
Open this publication in new window or tab >>Glucose- and Hormone-Induced cAMP Oscillations in α- and β-Cells Within Intact Pancreatic Islets
2011 (English)In: Diabetes, ISSN 0012-1797, E-ISSN 1939-327X, Vol. 60, no 5, 1535-1543 p.Article in journal (Refereed) Published
Abstract [en]

OBJECTIVE

cAMP is a critical messenger for insulin and glucagon secretion from pancreatic beta- and alpha-cells, respectively. Dispersed beta-cells show cAMP oscillations, but the signaling kinetics in cells within intact islets of Langerhans is unknown.

RESEARCH DESIGN AND METHODS

The subplasma-membrane cAMP concentration ([cAMP](pm)) was recorded in alpha-and beta-cells in the mantle of intact mouse pancreatic islets using total internal reflection microscopy and a fluorescent translocation biosensor. Cell identification was based on the opposite effects of adrenaline on cAMP in alpha- and beta-cells.

RESULTS

In islets exposed to 3 mmol/L glucose, [cAMP](pm) was low and stable. Glucagon and glucagon-like peptide-1(7-36)-amide (GLP-1) induced dose-dependent elevation of [cAMP](pm), often with oscillations synchronized among beta-cells. Whereas glucagon also induced [cAMP](pm) oscillations in most alpha-cells, < 20% of the alpha-cells responded to GLP-1. Elevation of the glucose concentration to 11-30 mmol/L in the absence of hormones induced slow [cAMP](pm) oscillations in both alpha- and beta-cells. These cAMP oscillations were coordinated with those of the cytoplasmic Ca2+ concentration ([Ca2+](i)) in the beta-cells but not caused by the changes in [Ca2+](i) . The transmembrane adenylyl cyclase (AC) inhibitor 2'5'-dideoxyadenosine suppressed the glucose- and hormone-induced [cAMP](pm) elevations, whereas the preferential inhibitors of soluble AC, KH7, and 1,3,5(10)-estratrien-2,3,17-beta-triol perturbed cell metabolism and lacked effect, respectively.

CONCLUSIONS

Oscillatory [cAMP](pm) signaling in secretagogue-stimulated beta-cells is maintained within intact islets and depends on transmembrane AC activity. The discovery of glucose- and glucagon-induced [cAMP](pm) oscillations in alpha-cells indicates the involvement of cAMP in the regulation of pulsatile glucagon secretion.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-154114 (URN)10.2337/db10-1087 (DOI)000290349700021 ()21444924 (PubMedID)
Available from: 2011-05-26 Created: 2011-05-26 Last updated: 2017-12-11Bibliographically approved
2. Role of phosphodiesterases in the shaping of sub-plasma-membrane cAMP oscillations and pulsatile insulin secretion
Open this publication in new window or tab >>Role of phosphodiesterases in the shaping of sub-plasma-membrane cAMP oscillations and pulsatile insulin secretion
Show others...
2012 (English)In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 125, no 21, 5084-5095 p.Article in journal (Refereed) Published
Abstract [en]

Specificity and versatility in cyclic AMP (cAMP) signalling are governed by the spatial localisation and temporal dynamics of the signal. Phosphodiesterases (PDEs) are important for shaping cAMP signals by hydrolyzing the nucleotide. In pancreatic β-cells, glucose triggers sub-plasma-membrane cAMP oscillations, which are important for insulin secretion, but the mechanisms underlying the oscillations are poorly understood. Here, we investigated the role of different PDEs in the generation of cAMP oscillations by monitoring the concentration of cAMP in the sub-plasma-membrane space ([cAMP](pm)) with ratiometric evanescent wave microscopy in MIN6 cells or mouse pancreatic β-cells expressing a fluorescent translocation biosensor. The general PDE inhibitor IBMX increased [cAMP](pm), and whereas oscillations were frequently observed at 50 µM IBMX, 300 µM-1 mM of the inhibitor caused a stable increase in [cAMP](pm). The [cAMP](pm) was nevertheless markedly suppressed by the adenylyl cyclase inhibitor 2',5'-dideoxyadenosine, indicating IBMX-insensitive cAMP degradation. Among IBMX-sensitive PDEs, PDE3 was most important for maintaining a low basal level of [cAMP](pm) in unstimulated cells. After glucose induction of [cAMP](pm) oscillations, inhibitors of PDE1, PDE3 and PDE4 inhibitors the average cAMP level, often without disturbing the [cAMP](pm) rhythmicity. Knockdown of the IBMX-insensitive PDE8B by shRNA in MIN6 cells increased the basal level of [cAMP](pm) and prevented the [cAMP](pm)-lowering effect of 2',5'-dideoxyadenosine after exposure to IBMX. Moreover, PDE8B-knockdown cells showed reduced glucose-induced [cAMP](pm) oscillations and loss of the normal pulsatile pattern of insulin secretion. It is concluded that [cAMP](pm) oscillations in β-cells are caused by periodic variations in cAMP generation, and that several PDEs, including PDE1, PDE3 and the IBMX-insensitive PDE8B, are required for shaping the sub-membrane cAMP signals and pulsatile insulin release.

Keyword
cAMP, PDE, palmitate, STIM1, insulin secretion, total internal reflection fluorescence microscopy, beta-cell, alpha-cell, glucagon secretion, oscillations
National Category
Endocrinology and Diabetes
Identifiers
urn:nbn:se:uu:diva-192307 (URN)10.1242/jcs.107201 (DOI)000312984300016 ()22946044 (PubMedID)
Available from: 2013-01-17 Created: 2013-01-17 Last updated: 2017-12-06Bibliographically approved
3. Impaired cAMP generation contributes to defective glucose-stimulated insulin secretion after long-term exposure to palmitate
Open this publication in new window or tab >>Impaired cAMP generation contributes to defective glucose-stimulated insulin secretion after long-term exposure to palmitate
Show others...
2015 (English)In: Diabetes, ISSN 0012-1797, E-ISSN 1939-327X, Vol. 64, no 3, 904-915 p.Article in journal (Refereed) Published
Abstract [en]

Chronic palmitate exposure impairs glucose-stimulated insulin secretion and other aspects of β-cell function but the underlying mechanisms are not known. Using various live-cell fluorescence imaging approaches we show here that long-term palmitate treatment influences cAMP signaling in pancreatic β-cells. Glucose stimulation of mouse and human β-cells induced oscillations of the sub-plasma-membrane cAMP concentration but after 48 h exposure to palmitate, most β-cells failed to increase cAMP in response to glucose. In contrast, GLP-1-triggered cAMP formation and glucose- and depolarization-induced increases in cytoplasmic Ca2+ concentration were unaffected by the fatty acid treatment. Insulin secretion from control β-cells was pulsatile but the response deteriorated after long-term palmitate exposure. Palmitate-treated mouse islets showed reduced expression of adenylyl cyclase 9 and knockdown of this protein in insulinoma cells reduced the glucose-stimulated cAMP response and insulin secretion. We conclude that impaired glucose-induced generation of cAMP is an important determinant of defective insulin secretion after chronic palmitate exposure.

National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-192309 (URN)10.2337/db14-1036 (DOI)000350235900031 ()25281428 (PubMedID)
Funder
Swedish Research Council
Available from: 2013-01-17 Created: 2013-01-17 Last updated: 2017-12-06Bibliographically approved
4. cAMP Induces Stromal Interaction Molecule 1 (STIM1) Puncta but neither Orai1 Protein Clustering nor Store-operated Ca2+ Entry (SOCE) in Islet Cells
Open this publication in new window or tab >>cAMP Induces Stromal Interaction Molecule 1 (STIM1) Puncta but neither Orai1 Protein Clustering nor Store-operated Ca2+ Entry (SOCE) in Islet Cells
2012 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 13, 9862-9872 p.Article in journal (Refereed) Published
Abstract [en]

The events leading to the activation of store-operated Ca2+ entry (SOCE) involve Ca2+ depletion of the endoplasmic reticulum (ER) resulting in translocation of the transmembrane Ca2+ sensor protein, stromal interaction molecule 1 (STIM1), to the junctions between ER and the plasma membrane where it binds to the Ca2+ channel protein Orai1 to activate Ca2+ influx. Using confocal and total internal reflection fluorescence microscopy, we studied redistribution kinetics of fluorescence-tagged STIM1 and Orai1 as well as SOCE in insulin-releasing beta-cells and glucagon-secreting alpha-cells within intact mouse and human pancreatic islets. ER Ca2+ depletion triggered accumulation of STIM1 puncta in the subplasmalemmal ER where they co-clustered with Orai1 in the plasma membrane and activated SOCE. Glucose, which promotes Ca2+ store filling and inhibits SOCE, stimulated retranslocation of STIM1 to the bulk ER. This effect was evident at much lower glucose concentrations in alpha-than in beta-cells consistent with involvement of SOCE in the regulation of glucagon secretion. Epinephrine stimulated subplasmalemmal translocation of STIM1 in beta-cells and retranslocation in beta-cells involving raising and lowering of cAMP, respectively. The cAMP effect was mediated both by protein kinase A and exchange protein directly activated by cAMP. However, the cAMP-induced STIM1 puncta did not co-cluster with Orai1, and there was no activation of SOCE. STIM1 translocation can consequently occur independently of Orai1 clustering and SOCE.

National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-173662 (URN)10.1074/jbc.M111.292854 (DOI)000302167200019 ()
Available from: 2012-05-02 Created: 2012-05-02 Last updated: 2017-12-07Bibliographically approved

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