| 1. |
- Fatsis-Kavalopoulos, Nikos, et al.
(author)
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Formation of precisely composed cancer cell clusters using a cell assembly generator (CAGE) for studying paracrine signaling at single-cell resolution
- 2019
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In: Lab on a Chip. - : ROYAL SOC CHEMISTRY. - 1473-0197 .- 1473-0189. ; 19:6, s. 1071-1081
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Journal article (peer-reviewed)abstract
- The function and behaviour of any given cell in a healthy tissue, or in a tumor, is affected by interactions with its neighboring cells. It is therefore important to create methods that allow for reconstruction of tissue niches in vitro for studies of cell-cell signaling and associated cell behaviour. To this end we created the cell assembly generator (CAGE), a microfluidic device which enables the organization of different cell types into precise cell clusters in a flow chamber compatible with high-resolution microscopy. In proof-of-concept paracrine signalling experiments, 4-cell clusters consisting of one pancreatic -cell and three breast cancer cells were formed. It has previously been established that extracellular ATP induces calcium (Ca2+) release from the endoplasmic reticulum (ER) to the cytosol before it is cleared back into the ER via sarcoplasmic/ER Ca2+ ATPase (SERCA) pumps. Here, ATP release from the -cell was stimulated by depolarization, and dynamic changes in Ca2+ levels in the adjacent cancer cells measured using imaging of the calcium indicator Fluo-4. We established that changes in the concentration of cytosolic Ca2+ in the cancer cells were proportional to the distance from the ATP-releasing -cell. Additionally, we established that the relationship between distance and cytosolic calcium changes were dependent on Ca2+-release from the ER using 5-cell clusters composed of one -cell, two untreated cancer cells and two cancer cells pretreated with Thapsigargin (to deplete the ER of Ca2+). These experiments show that the CAGE can be used to create exact cell clusters, which affords precise control for reductionist studies of cell-cell signalling and permits the formation of heterogenous cell models of specific tissue niches.
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| 2. |
- Griesche, Nadine, et al.
(author)
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Cortical mitochondria regulate insulin secretion by local Ca2+ buffering in rodent beta cells
- 2019
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In: Journal of Cell Science. - : COMPANY BIOLOGISTS LTD. - 0021-9533 .- 1477-9137. ; 132:9
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Journal article (peer-reviewed)abstract
- Mitochondria play an essential role in regulating insulin secretion from beta cells by providing the ATP needed for the membrane depolarization that results in voltage-dependent Ca2+ influx and subsequent insulin granule exocytosis. Ca2+, in turn, is also rapidly taken up by the mitochondria and exerts important feedback regulation of metabolism. The aim of this study was to determine whether the distribution of mitochondria within beta cells is important for the secretory capacity of these cells. We find that cortically localized mitochondria are abundant in rodent beta cells, and that these mitochondria redistribute towards the cell interior following depolarization. The redistribution requires Ca2+-induced remodeling of the cortical F-actin network. Using light-regulated motor proteins, we increased the cortical density of mitochondria twofold and found that this blunted the voltage-dependent increase in cytosolic Ca2+ concentration and suppressed insulin secretion. The activity-dependent changes in mitochondria distribution are likely to be important for the generation of Ca2+ microdomains required for efficient insulin granule release.
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| 3. |
- Idevall Hagren, Olof, 1980-, et al.
(author)
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cAMP Mediators of Pulsatile Insulin Secretion from Glucose-stimulated Single β-Cells
- 2010
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In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 285:30, s. 23005-23016
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Journal article (peer-reviewed)abstract
- Pulsatile insulin release from glucose-stimulated beta-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 beta-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.
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| 4. |
- Idevall Hagren, Olof, 1980-, et al.
(author)
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Dynamic control of Epac2 localization by cAMP and Ca2+-mediated activation of Ras
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Other publication (other academic/artistic)abstract
- Epac2, a cAMP-regulated guanine nucleotide exchange factor for the small GTPases Rap1 and Rap2, is an important mediator of a variety of cAMP-regulated cellular processes, including insulin secretion from pancreatic β-cells. Epac2 has been suggested to associate with the plasma membrane by interacting with active Ras (Ras-GTP), but the dynamics and regulation of membrane binding is unknown. Using real-time confocal and total internal reflection fluorescence microscopy we demonstrate that cAMP-elevating agents cause rapid translocation of GFP-tagged Epac2 from the cytoplasm to the plasma membrane in insulin-secreting MIN6 β-cells. Glucose concentrations that stimulate insulin secretion often triggered oscillatory translocation of GFP-Epac2 following oscillations of the sub-membrane concentrations of cAMP and Ca2+ ([cAMP]pm and [Ca2+]pm). The translocation was suppressed after inhibition of adenylyl cyclases or removal of extracellular Ca2+. GFP-Epac2 translocation by rise of [Ca2+]pm required concomitant elevation of [cAMP]pm and cAMP-induced translocation was enhanced by moderate [Ca2+]pm elevations. However the effect of Ca2+ was dual since translocation was inhibited by high [Ca2+]pm spikes. Epac2 mutants lacking the cAMP-binding or Ras-association domains were unable to translocate and localized constitutively to the plasma membrane and cytoplasm, respectively. Ras activity monitored with a fluorescent Ras-GTP binding reporter was tightly correlated with the translocation of Epac2. It is concluded that Epac2 localization is dynamically controlled by cAMP as well as by Ca2+-mediated activation of Ras, and that reversible translocation of Epac2 between the cytoplasm and plasma membrane requires both Ras-association and cAMP-binding domains. Spatio-temporal control of Epac2 in β-cells has implications for the understanding of its involvement in insulin secretion kinetics by Rap GTPases and other downstream effectors at the plasma membrane.
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| 5. |
- Idevall Hagren, Olof, 1980-, et al.
(author)
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Keeping pace : the primary cilium as the conducting baton of the islet
- 2024
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In: Diabetologia. - : Springer. - 0012-186X .- 1432-0428. ; 67, s. 773-782
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Research review (peer-reviewed)abstract
- Primary cilia are rod-like sensory organelles that protrude from the surface of most mammalian cells, including the cells of the islet, and mounting evidence supports important roles of these structures in the regulation of beta cell function and insulin secretion. The sensory abilities of the cilium arise from local receptor activation that is coupled to intrinsic signal transduction, and ciliary signals can propagate into the cell and influence cell function. Here, we review recent advances and studies that provide insights into intra-islet cues that trigger primary cilia signalling; how second messenger signals are generated and propagated within cilia; and how ciliary signalling affects beta cell function. We also discuss the potential involvement of primary cilia and ciliary signalling in the development and progression of type 2 diabetes, identify gaps in our current understanding of islet cell cilia function and provide suggestions on how to further our understanding of this intriguing structure.
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| 6. |
- Idevall Hagren, Olof, 1980-, et al.
(author)
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Metabolic regulation of calcium signaling in beta cells
- 2020
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In: Seminars in Cell and Developmental Biology. - : ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD. - 1084-9521 .- 1096-3634. ; 103, s. 20-30
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Research review (peer-reviewed)abstract
- The cytoplasmic Ca2+ concentration ([Ca2+](cyt)) regulates a vast number of cellular functions, including insulin secretion from beta cells. The major physiological insulin secretagogue, glucose, triggers [Ca2+](cyt) oscillations in beta cells. Synchronization of the oscillations among the beta cells within an islet underlies the generation of pulsatile insulin secretion. This review describes the mechanisms generating [Ca2+](cyt) oscillations, the interactions between [Ca2+](cyt) and cell metabolism, as well as the contribution of various organelles to the shaping of [Ca2+](cyt) signals and insulin secretion. It also discusses how Ca2+ signals are coordinated and spread throughout the islets and data indicating that altered Ca2+ signaling is associated with beta cell dysfunction and development of type 2 diabetes.
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| 7. |
- Idevall Hagren, Olof, 1980-
(author)
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Oscillatory Signaling and Insulin Secretion from Single ß-cells
- 2010
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Doctoral thesis (other academic/artistic)abstract
- 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.
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| 8. |
- Li, Hanbing, et al.
(author)
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Dual Function of PI(4,5)P2 in Insulin-Regulated Exocytic Trafficking of GLUT4 in Adipocytes
- 2020
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In: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 432:16, s. 4341-4357
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Journal article (peer-reviewed)abstract
- Phosphoinositides are important signaling molecules involved in the regulation of vesicular trafficking. It has been implicated that phosphatidylinositol 4,5-bisphosphate [PI(4,5)P-2] is involved in insulin-regulated GLUT4 translocation in adipocytes. However, it remains unclear where and how PI(4,5)P-2 regulates discrete steps of GLUT4 vesicle translocation in adipocytes, especially on the exocytic arm of regulation. Here, we employed optogenetic tools to acutely control the PI(4,5)P-2 metabolism in living cells. By combination of TIRFM imaging, we were able to monitor the temporal-spatial-dependent PI(4,5)P-2 regulation on discrete steps of GLUT4 translocation in adipocytes. We found that the plasma membrane localized PI(4,5)P-2 is crucial for proper insulin signaling propagation and for insulin-stimulated GLUT4 vesicle translocation in 3T3-L1 adipocytes. Global depletion of PI(4,5)P-2 on the cell surface blunted insulin-stimulated Akt phosphorylation and abolished insulin effects in promotion of the docking and fusion of GLUT4 vesicle with the plasma membrane. Furthermore, by development of a novel optogenetic module to selectively modulate PI(4,5)P-2 levels on the GLUT4 vesicle docking site, we identified an important regulatory role of PI(4,5)P-2 in controlling of vesicle docking process. Local depletion of PI(4,5)P-2 at the vesicle docking site promoted GLUT4 vesicle undocking, diminished insulin-stimulated GLUT4 vesicle docking and fusion, but without perturbation of insulin signaling propagation in adipocytes. Our results provide strong evidence that cell surface PI(4,5)P-2 plays two distinct functions on regulation of the exocytic trafficking of GLUT4 in adipocytes. PI(4,5)P-2 not only regulates the proper activation of insulin signaling in general but also controls GLUT4 vesicle docking process at the vesicle-membrane contact sites.
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| 9. |
- Mousavy Gharavy, S. Neda, et al.
(author)
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Sexually dimorphic roles for the type 2 diabetes-associated C2cd4b gene in murine glucose homeostasis
- 2021
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In: Diabetologia. - : Springer Nature. - 0012-186X .- 1432-0428. ; 64:4, s. 850-864
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Journal article (peer-reviewed)abstract
- Aims/hypothesis Variants close to the VPS13C/C2CD4A/C2CD4B locus are associated with altered risk of type 2 diabetes in genome-wide association studies. While previous functional work has suggested roles for VPS13C and C2CD4A in disease development, none has explored the role of C2CD4B. Methods CRISPR/Cas9-induced global C2cd4b-knockout mice and zebrafish larvae with c2cd4a deletion were used to study the role of this gene in glucose homeostasis. C2 calcium dependent domain containing protein (C2CD)4A and C2CD4B constructs tagged with FLAG or green fluorescent protein were generated to investigate subcellular dynamics using confocal or near-field microscopy and to identify interacting partners by mass spectrometry. Results Systemic inactivation of C2cd4b in mice led to marked, but highly sexually dimorphic changes in body weight and glucose homeostasis. Female C2cd4b mice displayed unchanged body weight compared with control littermates, but abnormal glucose tolerance (AUC, p = 0.01) and defective in vivo, but not in vitro, insulin secretion (p = 0.02). This was associated with a marked decrease in follicle-stimulating hormone levels as compared with wild-type (WT) littermates (p = 0.003). In sharp contrast, male C2cd4b null mice displayed essentially normal glucose tolerance but an increase in body weight (p < 0.001) and fasting blood glucose (p = 0.003) after maintenance on a high-fat and -sucrose diet vs WT littermates. No metabolic disturbances were observed after global inactivation of C2cd4a in mice, or in pancreatic beta cell function at larval stages in C2cd4a null zebrafish. Fasting blood glucose levels were also unaltered in adult C2cd4a-null fish. C2CD4B and C2CD4A were partially localised to the plasma membrane, with the latter under the control of intracellular Ca2+. Binding partners for both included secretory-granule-localised PTPRN2/phogrin. Conclusions/interpretation Our studies suggest that C2cd4b may act centrally in the pituitary to influence sex-dependent circuits that control pancreatic beta cell function and glucose tolerance in rodents. However, the absence of sexual dimorphism in the impact of diabetes risk variants argues for additional roles for C2CD4A or VPS13C in the control of glucose homeostasis in humans. Data availability The datasets generated and/or analysed during the current study are available in the Biorxiv repository (www. biorxiv.org/content/10.1101/2020.05.18.099200v1). RNA-Seq (GSE152576) and proteomics (PXD021597) data have been deposited to GEO (www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE152576) and ProteomeXchange (www.ebi.ac.uk/pride/ archive/projects/PXD021597) repositories, respectively.
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| 10. |
- Ngamjariyawat, Anongnad, 1976-, et al.
(author)
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Metabolic stress-induced human beta-cell death is mediated by increased intracellular levels of adenosine
- 2023
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In: Frontiers in Endocrinology. - : Frontiers Media S.A.. - 1664-2392. ; 14
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Journal article (peer-reviewed)abstract
- Introduction: High intracellular concentrations of adenosine and 2'-deoxyadenosine have been suggested to be an important mediator of cell death. The aim of the present study was to characterize adenosine-induced death in insulin-producing beta-cells, at control and high glucose + palmitate-induced stress conditions.Methods: Human insulin-producing EndoC-betaH1 cells were treated with adenosine, 2'-deoxyadenosine, inosine and high glucose + sodium palmitate, and death rates using flow cytometry were studied.Results: We observed that adenosine and the non-receptor-activating analogue 2-deoxyadenosine, but not the adenosine deamination product inosine, promoted beta-cell apoptosis at concentrations exceeding maximal adenosine-receptor stimulating concentrations. Both adenosine and inosine were efficiently taken up by EndoC-betaH1 cells, and inosine counteracted the cell death promoting effect of adenosine by competing with adenosine for uptake. Both adenosine and 2'-deoxyadenosine promptly reduced insulin-stimulated production of plasma membrane PI(3,4,5)P-3, an effect that was reversed upon wash out of adenosine. In line with this, adenosine, but not inosine, rapidly diminished Akt phosphorylation. Both pharmacological Bax inhibition and Akt activation blocked adenosine-induced beta-cell apoptosis, indicating that adenosine/2'-deoxyadenosine inhibits the PI3K/Akt/BAD anti-apoptotic pathway. High glucose + palmitate-induced cell death was paralleled by increased intracellular adenosine and inosine levels. Overexpression of adenosine deaminase-1 (ADA1) in EndoC-betaH1 cells, which increased Akt phosphorylation, prevented both adenosine-induced apoptosis and high glucose + palmitate-induced necrosis. ADA2 overexpression not only failed to protect against adenosine and high glucose + palmitate-activated cell death, but instead potentiated the apoptosis-stimulating effect of adenosine. In line with this, ADA1 overexpression increased inosine production from adenosine-exposed cells, whereas ADA2 did not. Knockdown of ADA1 resulted in increased cell death rates in response to both adenosine and high glucose + palmitate. Inhibition of miR-30e-3p binding to the ADA1 mRNA 3'-UTR promoted the opposite effects on cell death rates and reduced intracellular adenosine contents.Discussion: It is concluded that intracellular adenosine/2'-deoxyadenosine regulates negatively the PI3K pathway and is therefore an important mediator of beta-cell apoptosis. Adenosine levels are controlled, at least in part, by ADA1, and strategies to upregulate ADA1 activity, during conditions of metabolic stress, could be useful in attempts to preserve beta-cell mass in diabetes.
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