Anders Tengholm

Oscillations of cyclic AMP in hormone-stimulated insulin-secreting beta-cells.

Cyclic AMP is a ubiquitous second messenger that transduces signals from a variety of cell surface receptors to regulate diverse cellular functions, including secretion, metabolism and gene transcription. In pancreatic β-cells, cAMP potentiates Ca2+-dependent exocytosis and mediates the stimulation of insulin release exerted by the hormones glucagon and glucagon-like peptide-1 (GLP-1) (refs 4, 5–6). Whereas Ca2+ signals have been extensively characterized and shown to involve oscillations important for the temporal control of insulin secretion, the kinetics of receptor-triggered cAMP signals is unknown. Here we introduce a new ratiometric evanescent-wave-microscopy approach to measure cAMP concentration beneath the plasma membrane, and show that insulin-secreting β-cells respond to glucagon and GLP-1 with marked cAMP oscillations. Simultaneous measurements of intracellular Ca2+ concentration revealed that the two messengers are interlinked and reinforce each other. Moreover, cAMP oscillations are capable of inducing rapid on–off Ca2+ responses, but only sustained elevation of cAMP concentration induces nuclear translocation of the catalytic subunit of the cAMP-dependent protein kinase. Our results establish a new signalling mode for cAMP and indicate that temporal encoding of cAMP signals might constitute a basis for differential regulation of downstream cellular targets.

Glucose-induced cyclic AMP oscillations regulate pulsatile insulin secretion

Cyclic AMP (cAMP) and Ca(2+) are key regulators of exocytosis in many cells, including insulin-secreting beta cells. Glucose-stimulated insulin secretion from beta cells is pulsatile and involves oscillations of the cytoplasmic Ca(2+) concentration ([Ca(2+)](i)), but little is known about the detailed kinetics of cAMP signaling. Using evanescent-wave fluorescence imaging we found that glucose induces pronounced oscillations of cAMP in the submembrane space of single MIN6 cells and primary mouse beta cells. These oscillations were preceded and enhanced by elevations of [Ca(2+)](i). However, conditions raising cytoplasmic ATP could trigger cAMP elevations without accompanying [Ca(2+)](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.

Glucose- and Hormone-Induced cAMP Oscillations in α- and β-Cells Within Intact Pancreatic Islets

OBJECTIVE cAMP is a critical messenger for insulin and glucagon secretion from pancreatic β- and α-cells, respectively. Dispersed β-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 α- and β-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 α- and β-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 β-cells. Whereas glucagon also induced [cAMP]pm oscillations in most α-cells, <20% of the α-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 α- and β-cells. These cAMP oscillations were coordinated with those of the cytoplasmic Ca2+ concentration ([Ca2+]i) in the β-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-β-triol perturbed cell metabolism and lacked effect, respectively. CONCLUSIONS Oscillatory [cAMP]pm signaling in secretagogue-stimulated β-cells is maintained within intact islets and depends on transmembrane AC activity. The discovery of glucose- and glucagon-induced [cAMP]pm oscillations in α-cells indicates the involvement of cAMP in the regulation of pulsatile glucagon secretion.

cAMP Mediators of Pulsatile Insulin Secretion from Glucose-stimulated Single β-Cells

Pulsatile insulin release from glucose-stimulated β-cells is driven by oscillations of the Ca2+ and cAMP concentrations in the subplasma membrane space ([Ca2+]pm and [cAMP]pm). To clarify mechanisms by which cAMP regulates insulin secretion, we performed parallel evanescent wave fluorescence imaging of [cAMP]pm, [Ca2+]pm, and phosphatidylinositol 3,4,5-trisphosphate (PIP3) in the plasma membrane. This lipid is formed by autocrine insulin receptor activation and was used to monitor insulin release kinetics from single MIN6 β-cells. Elevation of the glucose concentration from 3 to 11 mm induced, after a 2.7-min delay, coordinated oscillations of [Ca2+]pm, [cAMP]pm, and PIP3. Inhibitors of protein kinase A (PKA) markedly diminished the PIP3 response when applied before glucose stimulation, but did not affect already manifested PIP3 oscillations. The reduced PIP3 response could be attributed to accelerated depolarization causing early rise of [Ca2+]pm that preceded the elevation of [cAMP]pm. However, the amplitude of the PIP3 response after PKA inhibition was restored by a specific agonist to the cAMP-dependent guanine nucleotide exchange factor Epac. Suppression of cAMP formation with adenylyl cyclase inhibitors reduced already established PIP3 oscillations in glucose-stimulated cells, and this effect was almost completely counteracted by the Epac agonist. In cells treated with small interfering RNA targeting Epac2, the amplitudes of the glucose-induced PIP3 oscillations were reduced, and the Epac agonist was without effect. The data indicate that temporal coordination of the triggering [Ca2+]pm and amplifying [cAMP]pm signals is important for glucose-induced pulsatile insulin release. Although both PKA and Epac2 partake in initiating insulin secretion, the cAMP dependence of established pulsatility is mediated by Epac2.

Oscillations of sub-membrane ATP in glucose-stimulated beta cells depend on negative feedback from Ca2+

Aims/hypothesisATP links changes in glucose metabolism to electrical activity, Ca2+ signalling and insulin secretion in pancreatic beta cells. There is evidence that beta cell metabolism oscillates, but little is known about ATP dynamics at the plasma membrane, where regulation of ion channels and exocytosis occur.MethodsThe sub-plasma-membrane ATP concentration ([ATP]pm) was recorded in beta cells in intact mouse and human islets using total internal reflection microscopy and the fluorescent reporter Perceval.ResultsGlucose dose-dependently increased [ATP]pm with half-maximal and maximal effects at 5.2 and 9xa0mmol/l, respectively. Additional elevations of glucose to 11 to 20xa0mmol/l promoted pronounced [ATP]pm oscillations that were synchronised between neighbouring beta cells. [ATP]pm increased further and the oscillations disappeared when voltage-dependent Ca2+ influx was prevented. In contrast, K+-depolarisation induced prompt lowering of [ATP]pm. Simultaneous recordings of [ATP]pm and the sub-plasma-membrane Ca2+ concentration ([Ca2+]pm) during the early glucose-induced response revealed that the initial [ATP]pm elevation preceded, and was temporarily interrupted by the rise of [Ca2+]pm. During subsequent glucose-induced oscillations, the increases of [Ca2+]pm correlated with lowering of [ATP]pm.Conclusions/interpretationIn beta cells, glucose promotes pronounced oscillations of [ATP]pm, which depend on negative feedback from Ca2+. The bidirectional interplay between these messengers in the sub-membrane space generates the metabolic and ionic oscillations that underlie pulsatile insulin secretion.

Anchored phosphatases modulate glucose homeostasis

Endocrine release of insulin principally controls glucose homeostasis. Nutrient‐induced exocytosis of insulin granules from pancreatic β‐cells involves ion channels and mobilization of Ca2+ and cyclic AMP (cAMP) signalling pathways. Whole‐animal physiology, islet studies and live‐β‐cell imaging approaches reveal that ablation of the kinase/phosphatase anchoring protein AKAP150 impairs insulin secretion in mice. Loss of AKAP150 impacts L‐type Ca2+ currents, and attenuates cytoplasmic accumulation of Ca2+ and cAMP in β‐cells. Yet surprisingly AKAP150 null animals display improved glucose handling and heightened insulin sensitivity in skeletal muscle. More refined analyses of AKAP150 knock‐in mice unable to anchor protein kinase A or protein phosphatase 2B uncover an unexpected observation that tethering of phosphatases to a seven‐residue sequence of the anchoring protein is the predominant molecular event underlying these metabolic phenotypes. Thus anchored signalling events that facilitate insulin secretion and glucose homeostasis may be set by AKAP150 associated phosphatase activity.

cAMP Induces Stromal Interaction Molecule 1 (STIM1) Puncta but neither Orai1 Protein Clustering nor Store-operated Ca2+ Entry (SOCE) in Islet Cells

Background: Regulation of the ER Ca2+ sensor STIM1 and its association with the plasma membrane channel Orai1 to activate store-operated Ca2+ entry (SOCE) remains incompletely understood. Results: cAMP induced plasma membrane translocation of STIM1 in islet cells without concomitant SOCE activation. Conclusion: STIM1 translocation alone is insufficient to activate SOCE. Significance: This study describes novel interaction between the components of cAMP and Ca2+ signaling cascades. 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 β-cells and glucagon-secreting α-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 α- than in β-cells consistent with involvement of SOCE in the regulation of glucagon secretion. Epinephrine stimulated subplasmalemmal translocation of STIM1 in α-cells and retranslocation in β-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.

Neurotransmitter control of islet hormone pulsatility

Pulsatile secretion is an inherent property of hormone‐releasing pancreatic islet cells. This secretory pattern is physiologically important and compromised in diabetes. Neurotransmitters released from islet cells may shape the pulses in auto/paracrine feedback loops. Within islets, glucose‐stimulated β‐cells couple via gap junctions to generate synchronized insulin pulses. In contrast, α‐ and δ‐cells lack gap junctions, and glucagon release from islets stimulated by lack of glucose is non‐pulsatile. Increasing glucose concentrations gradually inhibit glucagon secretion by α‐cell‐intrinsic mechanism/s. Further glucose elevation will stimulate pulsatile insulin release and co‐secretion of neurotransmitters. Excitatory ATP may synchronize β‐cells with δ‐cells to generate coinciding pulses of insulin and somatostatin. Inhibitory neurotransmitters from β‐ and δ‐cells can then generate antiphase pulses of glucagon release. Neurotransmitters released from intrapancreatic ganglia are required to synchronize β‐cells between islets to coordinate insulin pulsatility from the entire pancreas, whereas paracrine intra‐islet effects still suffice to explain coordinated pulsatile release of glucagon and somatostatin. The present review discusses how neurotransmitters contribute to the pulsatility at different levels of integration.

DNA Methylation of Alternative Promoters Directs Tissue Specific Expression of Epac2 Isoforms

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.

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

Aims/hypothesisIt 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.MethodsHuman 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.ResultsImatinib 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/interpretationImatinib inhibition of c-Abl in beta cells decreases SHIP2 activity, which results in enhanced signalling downstream of PI3 kinase.