| 1. |
|
|
| 2. |
- Dyachok, Oleg, 1965-, et al.
(author)
-
Glucose-induced cyclic AMP oscillations regulate pulsatile insulin secretion
- 2008
-
In: Cell Metabolism. - : Cell Press. - 1550-4131 .- 1932-7420. ; 8:1, s. 26-37
-
Journal article (peer-reviewed)abstract
- Cyclic AMP (cAMP) and Ca2+ are key regulators of exocytosis in many cells, including insulin-secreting β-cells. Glucose-stimulated insulin secretion from β cells is pulsatile and involves oscillations of the cytoplasmic Ca2+ concentration ([Ca2+]i), but little is known about the detailed kinetics of cAMP signalling. Using evanescent-wave fluorescence imaging we found that glucose induces pronounced oscillations of cAMP in the sub-membrane space of single MIN6-cells and primary mouse β-cells. These oscillations were preceded and enhanced by elevations of [Ca2+]i. However, conditions raising cytoplasmic ATP could trigger cAMP elevations without accompanying [Ca2+]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.
|
|
| 3. |
- Fatsis-Kavalopoulos, Nikos, et al.
(author)
-
Formation of precisely composed cancer cell clusters using a cell assembly generator (CAGE) for studying paracrine signaling at single-cell resolution
- 2019
-
In: Lab on a Chip. - : ROYAL SOC CHEMISTRY. - 1473-0197 .- 1473-0189. ; 19:6, s. 1071-1081
-
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.
|
|
| 4. |
- Griesche, Nadine, et al.
(author)
-
Cortical mitochondria regulate insulin secretion by local Ca2+ buffering in rodent beta cells
- 2019
-
In: Journal of Cell Science. - : COMPANY BIOLOGISTS LTD. - 0021-9533 .- 1477-9137. ; 132:9
-
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.
|
|
| 5. |
- Hafizi, Sassan, et al.
(author)
-
Tensin2 reduces intracellular phosphatidylinositol 3,4,5-trisphosphate levels at the plasma membrane
- 2010
-
In: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 399:3, s. 396-401
-
Journal article (peer-reviewed)abstract
- Tensins are proposed cytoskeleton-regulating proteins. However, Tensin2 additionally inhibits Akt signalling and cell survival. Structural modelling of the Tensin2 phosphatase (PTPase) domain revealed an active site-like pocket receptive towards phosphoinositides. Tensin2-expressing HEK293 cells displayed negligible levels of plasma membrane phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P-3) under confocal microscopy. However, mock-transfected cells, and Tensin2 cells harbouring a putative phosphatase-inactivating mutation, exhibited significant PtdIns(3,4,5)P-3 levels, which decreased upon phosphatidylinositol 3-kinase inhibition with LY294002. In contrast, wtTensin3, mock and mutant cells were identical in membrane PtdIns(3,4,5)P-3 and Akt phosphorylation. In vitro lipid PTPase activity was however undetectable in isolated recombinant PTPase domains of both Tensins, indicating a possible loss of structural stability when expressed in isolation. In summary, we provide evidence that Tensin2, in addition to regulating cytoskeletal dynamics, influences phosphoinositide-Akt signalling through its PTPase domain.
|
|
| 6. |
- Idevall Hagren, Olof, 1980-, et al.
(author)
-
cAMP Mediators of Pulsatile Insulin Secretion from Glucose-stimulated Single β-Cells
- 2010
-
In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 285:30, s. 23005-23016
-
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.
|
|
| 7. |
- Idevall-Hagren, Olof, et al.
(author)
-
Detection and manipulation of phosphoinositides
- 2015
-
In: Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids. - : Elsevier BV. - 1388-1981 .- 1879-2618. ; 1851:6, s. 736-745
-
Research review (peer-reviewed)abstract
- Phosphoinositides (PIs) are minor components of cell membranes, but play key roles in cell function. Recent refinements in techniques for their detection, together with imaging methods to study their distribution and changes, have greatly facilitated the study of these lipids. Such methods have been complemented by the parallel development of techniques for the acute manipulation of their levels, which in turn allow bypassing the long-term adaptive changes implicit in genetic perturbations. Collectively, these advancements have helped elucidate the role of Pis in physiology and the impact of the dysfunction of their metabolism in disease. Combining methods for detection and manipulation enables the identification of specific roles played by each of the Pis and may eventually lead to the complete deconstruction of the PI signaling network. Here, we review current techniques used for the study and manipulation of cellular Pis and also discuss advantages and disadvantages associated with the various methods. This article is part of a Special Issue entitled Phosphoinositides.
|
|
| 8. |
- Idevall Hagren, Olof, 1980-, et al.
(author)
-
Dynamic control of Epac2 localization by cAMP and Ca2+-mediated activation of Ras
-
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.
|
|
| 9. |
- Idevall Hagren, Olof, et al.
(author)
-
Glucose and insulin synergistically activate phosphatidylinositol 3-kinase to trigger oscillations of phosphatidylinositol 3,4,5-trisphosphate in beta-cells
- 2006
-
In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 281:51, s. 39121-39127
-
Journal article (peer-reviewed)abstract
- In insulin-secreting β-cells, activation of phosphatidylinositol 3′-OH-kinase with resulting formation of phosphatidylinositol 3,4,5-trisphosphate (PIP3) has been implicated in the regulation of ion channels, insulin secretion, and gene transcription as well as in cell growth and survival, but the kinetics of PIP3 signals following physiological stimulation of insulin secretion is unknown. Using evanescent wave microscopy and a green fluorescent protein-tagged PIP3-binding protein domain for real-time monitoring of plasma membrane PIP3 concentration in single MIN6 β-cells, we now demonstrate that glucose stimulation of insulin secretion results in pronounced PIP3 oscillations via autocrine stimulation of insulin receptors. Glucose lacked effect when insulin secretion was prevented with the hyperpolarizing agent diazoxide, but the sugar dose dependently enhanced the PIP3 response to maximal insulin stimulation without affecting the rate of PIP3 degradation. We conclude that glucose is an important co-activator of phosphatidylinositol-3′-OH-kinase and that the plasma membrane PIP 3 concentration in β-cells undergoes oscillations due to pulsatile release of insulin.
|
|
| 10. |
- Idevall Hagren, Olof, 1980-, et al.
(author)
-
Keeping pace : the primary cilium as the conducting baton of the islet
- 2024
-
In: Diabetologia. - : Springer. - 0012-186X .- 1432-0428. ; 67, s. 773-782
-
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.
|
|
