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  • 1.
    Alenkvist, Ida
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Dyachok, Oleg
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Tian, Geng
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Li, Jia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Mehrabanfar, Saba
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Jin, Yang
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Birnir, Bryndis
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Physiology.
    Tengholm, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Welsh, Michael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Absence of Shb impairs insulin secretion by elevated FAK activity in pancreatic islets2014In: Journal of Endocrinology, ISSN 0022-0795, E-ISSN 1479-6805, Vol. 223, no 3, p. 267-275Article in journal (Refereed)
    Abstract [en]

    The Src homology-2 domain containing protein B (SHB) has previously been shown to function as a pleiotropic adapter protein, conveying signals from receptor tyrosine kinases to intracellular signaling intermediates. The overexpression of Shb in β-cells promotes β-cell proliferation by increased insulin receptor substrate (IRS) and focal adhesion kinase (FAK) activity, whereas Shb deficiency causes moderate glucose intolerance and impaired first-peak insulin secretion. Using an array of techniques, including live-cell imaging, patch-clamping, immunoblotting, and semi-quantitative PCR, we presently investigated the causes of the abnormal insulin secretory characteristics in Shb-knockout mice. Shb-knockout islets displayed an abnormal signaling signature with increased activities of FAK, IRS, and AKT. β-catenin protein expression was elevated and it showed increased nuclear localization. However, there were no major alterations in the gene expression of various proteins involved in the β-cell secretory machinery. Nor was Shb deficiency associated with changes in glucose-induced ATP generation or cytoplasmic Ca(2) (+) handling. In contrast, the glucose-induced rise in cAMP, known to be important for the insulin secretory response, was delayed in the Shb-knockout compared with WT control. Inhibition of FAK increased the submembrane cAMP concentration, implicating FAK activity in the regulation of insulin exocytosis. In conclusion, Shb deficiency causes a chronic increase in β-cell FAK activity that perturbs the normal insulin secretory characteristics of β-cells, suggesting multi-faceted effects of FAK on insulin secretion depending on the mechanism of FAK activation.

  • 2.
    Dyachok, Oleg
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Idevall-Hagren, Olof
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Sågetorp, Jenny
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Tian, Geng
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Wuttke, Anne
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Arrieumerlou, Cecile
    Infection Biology, Biozentrum, University of Basel, Switzerland.
    Akusjärvi, Göran
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Gylfe, Erik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Tengholm, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Glucose-induced cyclic AMP oscillations regulate pulsatile insulin secretion2008In: Cell Metabolism, ISSN 1550-4131, E-ISSN 1932-7420, Vol. 8, no 1, p. 26-37Article in journal (Refereed)
    Abstract [en]

    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. Hinke, Simon A.
    et al.
    Navedo, Manuel F.
    Ulman, Allison
    Whiting, Jennifer L.
    Nygren, Patrick J.
    Tian, Geng
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Jimenez-Caliani, Antonio J.
    Langeberg, Lorene K.
    Cirulli, Vincenzo
    Tengholm, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Dell'Acqua, Mark L.
    Santana, L. Fernando
    Scott, John D.
    Anchored phosphatases modulate glucose homeostasis2012In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 31, no 20, p. 3991-4004Article in journal (Refereed)
    Abstract [en]

    Endocrine release of insulin principally controls glucose homeostasis. Nutrient-induced exocytosis of insulin granules from pancreatic beta-cells involves ion channels and mobilization of Ca2+ and cyclic AMP (cAMP) signalling pathways. Whole-animal physiology, islet studies and live-beta-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 beta-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.

  • 4.
    Tian, Geng
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    On the Generation of cAMP Oscillations and Regulation of the Ca2+ Store-operated Pathway in Pancreatic Islet α- and β-cells2013Doctoral 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.

    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, p. 1535-1543Article 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, p. 5084-5095Article 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.

    Keywords
    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, p. 904-915Article 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: 2018-01-11Bibliographically 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, p. 9862-9872Article 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: 2018-01-12Bibliographically approved
  • 5.
    Tian, Geng
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Sandler, Stellan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Gylfe, Erik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Tengholm, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Glucose- and Hormone-Induced cAMP Oscillations in α- and β-Cells Within Intact Pancreatic Islets2011In: Diabetes, ISSN 0012-1797, E-ISSN 1939-327X, Vol. 60, no 5, p. 1535-1543Article in journal (Refereed)
    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.

  • 6.
    Tian, Geng
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Sol, E. -RM.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Xu, Yunjian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Tengholm, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Prolonged exposure to palmitate deteriorates glucose-induced cAMP generation and pulsatile insulin secretion2013In: Diabetologia, ISSN 0012-186X, E-ISSN 1432-0428, Vol. 56, p. S194-S194Article in journal (Other academic)
  • 7.
    Tian, Geng
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Sol, Eri Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Xu, Yunjian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Shuai, Hongyan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Tengholm, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Impaired cAMP generation contributes to defective glucose-stimulated insulin secretion after long-term exposure to palmitate2015In: Diabetes, ISSN 0012-1797, E-ISSN 1939-327X, Vol. 64, no 3, p. 904-915Article in journal (Refereed)
    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.

  • 8.
    Tian, Geng
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Sågetorp, Jenny
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Xu, Yunjian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Shuai, Hongyan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Degerman, Eva
    Tengholm, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Role of phosphodiesterases in the shaping of sub-plasma-membrane cAMP oscillations and pulsatile insulin secretion2012In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 125, no 21, p. 5084-5095Article in journal (Refereed)
    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.

  • 9.
    Tian, Geng
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Tepikin, Alexei V.
    Tengholm, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Gylfe, Erik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    cAMP Induces Stromal Interaction Molecule 1 (STIM1) Puncta but neither Orai1 Protein Clustering nor Store-operated Ca2+ Entry (SOCE) in Islet Cells2012In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 13, p. 9862-9872Article in journal (Refereed)
    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.

  • 10.
    Zang, Guangxiang
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Christoffersson, Gustaf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology, Integrative Physiology.
    Tian, Geng
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Harun-Or-Rashid, Mohammad
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Neuroscience.
    Vågesjö, Evelina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology, Integrative Physiology.
    Phillipson, Mia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology, Integrative Physiology.
    Barg, Sebastian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Tengholm, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Welsh, Michael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Aberrant association between vascular endothelial growth factor receptor-2 and VE-cadherin in response to vascular endothelial growth factor-a in Shb-deficient lung endothelial cells2013In: Cellular Signalling, ISSN 0898-6568, E-ISSN 1873-3913, Vol. 25, no 1, p. 85-92Article in journal (Refereed)
    Abstract [en]

    Vascular permeability is a hallmark response to the main angiogenic factor VEGF-A and we have previously described a reduction of this response in Shb knockout mice. To characterize the molecular mechanisms responsible for this effect, endothelial cells were isolated from lungs and analyzed in vitro. Shb deficient endothelial cells exhibited less migration in a scratch wound-healing assay both under basal conditions and after vascular endothelial growth factor-A (VEGF-A) stimulation, suggesting a functional impairment of these cells in vitro. Staining for VE-cadherin and vascular endothelial growth factor receptor-2 (VEGFR-2) showed co-localization in adherens junctions and in intracellular sites such as the perinuclear region in wild-type and Shb knockout cells. VEGF-A decreased the VE-cadherin/VEGFR-2 co-localization in membrane structures resembling adherens junctions in wild-type cells whereas no such response was noted in the Shb knockout cells. VE-cadherin/VEGFR-2 co-localization was also recorded using spinning-disc confocal microscopy and VEGF-A caused a reduced association in the wild-type cells whereas the opposite pattern was observed in the Shb knockout cells. The latter expressed slightly more of cell surface VEGFR-2. VEGF-A stimulated extracellular-signal regulated kinase, Akt and Rac1 activities in the wild-type cells whereas no such responses were noted in the knockout cells. We conclude that aberrant signaling characteristics with respect to ERK, Akt and Rac1 are likely explanations for the observed altered pattern of VE-cadherin/VEGFR-2 association. The latter is important for understanding the reduced in vivo vascular permeability response in Shb knockout mice, a phenomenon that has patho-physiological relevance.

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