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Xu, Yunjian
Publications (6 of 6) Show all publications
Xu, Y., Schwede, F., Wienk, H., Tengholm, A. & Rehmann, H. (2019). A membrane permeable prodrug of S223 for selective Epac2 activation in living cells. Cells, 8(12), Article ID 1589.
Open this publication in new window or tab >>A membrane permeable prodrug of S223 for selective Epac2 activation in living cells
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2019 (English)In: Cells, E-ISSN 2073-4409, Vol. 8, no 12, article id 1589Article in journal (Refereed) Published
Abstract [en]

Signalling by cyclic adenosine monophosphate (cAMP) occurs via various effector proteins, notably protein kinase A and the guanine nucleotide exchange factors Epac1 and Epac2. These proteins are activated by cAMP binding to conserved cyclic nucleotide binding domains. The specific roles of the effector proteins in various processes in different types of cells are still not well defined, but investigations have been facilitated by the development of cyclic nucleotide analogues with distinct selectivity profiles towards a single effector protein. A remaining challenge in the development of such analogues is the poor membrane permeability of nucleotides, which limits their applicability in intact living cells. Here, we report the synthesis and characterisation of S223-AM, a cAMP analogue designed as an acetoxymethyl ester prodrug to overcome limitations of permeability. Using total internal reflection imaging with various fluorescent reporters, we show that S223-AM selectively activates Epac2, but not Epac1 or protein kinase A, in intact insulin-secreting β-cells, and that this effect was associated with pronounced activation of the small G-protein Rap. A comparison of the effects of different cAMP analogues in pancreatic islet cells deficient in Epac1 and Epac2 demonstrates that cAMP-dependent Rap activity at the β-cell plasma membrane is exclusively dependent on Epac2. With its excellent selectivity and permeability properties, S223-AM should get broad utility in investigations of cAMP effector involvement in many different types of cells.

Place, publisher, year, edition, pages
Basel: , 2019
National Category
Cell and Molecular Biology
Research subject
Medical Cell Biology
Identifiers
urn:nbn:se:uu:diva-404599 (URN)10.3390/cells8121589 (DOI)000506643500117 ()31817822 (PubMedID)
Funder
Ernfors FoundationNovo NordiskSwedish Diabetes AssociationSwedish Research Council
Available from: 2020-02-24 Created: 2020-02-24 Last updated: 2020-03-10Bibliographically approved
Tian, G., Sol, E. M., Xu, Y., Shuai, H. & Tengholm, A. (2015). Impaired cAMP generation contributes to defective glucose-stimulated insulin secretion after long-term exposure to palmitate. Diabetes, 64(3), 904-915
Open this publication in new window or tab >>Impaired cAMP generation contributes to defective glucose-stimulated insulin secretion after long-term exposure to palmitate
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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
Hoivik, E. A., Witsoe, S. L., Bergheim, I. R., Xu, Y., Jakobsson, I., Tengholm, A., . . . Bakke, M. (2013). DNA Methylation of Alternative Promoters Directs Tissue Specific Expression of Epac2 Isoforms. PLoS ONE, 8(7), e67925
Open this publication in new window or tab >>DNA Methylation of Alternative Promoters Directs Tissue Specific Expression of Epac2 Isoforms
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2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 7, p. e67925-Article in journal (Refereed) Published
Abstract [en]

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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-209493 (URN)10.1371/journal.pone.0067925 (DOI)000323350700061 ()
Available from: 2013-10-21 Created: 2013-10-21 Last updated: 2017-12-06Bibliographically approved
Tian, G., Sol, E.-R. -., Xu, Y. & Tengholm, A. (2013). Prolonged exposure to palmitate deteriorates glucose-induced cAMP generation and pulsatile insulin secretion. Paper presented at 49th Annual Meeting of the European-Association-for-the-Study-of-Diabetes (EASD), SEP 23-27, 2013, Barcelona, SPAIN. Diabetologia, 56, S194-S194
Open this publication in new window or tab >>Prolonged exposure to palmitate deteriorates glucose-induced cAMP generation and pulsatile insulin secretion
2013 (English)In: Diabetologia, ISSN 0012-186X, E-ISSN 1432-0428, Vol. 56, p. S194-S194Article in journal, Meeting abstract (Other academic) Published
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-218012 (URN)000329196901124 ()
Conference
49th Annual Meeting of the European-Association-for-the-Study-of-Diabetes (EASD), SEP 23-27, 2013, Barcelona, SPAIN
Available from: 2014-02-07 Created: 2014-02-06 Last updated: 2017-12-06Bibliographically approved
Idevall-Hagren, O., Jakobsson, I., Xu, Y. & Tengholm, A. (2013). Spatial Control of Epac2 Activity by cAMP and Ca2+-Mediated Activation of Ras in Pancreatic beta Cells. Science Signaling, 6(273), ra29
Open this publication in new window or tab >>Spatial Control of Epac2 Activity by cAMP and Ca2+-Mediated Activation of Ras in Pancreatic beta Cells
2013 (English)In: Science Signaling, ISSN 1945-0877, E-ISSN 1937-9145, Vol. 6, no 273, p. ra29-Article in journal (Refereed) Published
Abstract [en]

The cAMP (adenosine 3',5'-monophosphate)-activated guanine nucleotide exchange factor (GEF) Epac2 is an important mediator of cAMP-dependent processes in multiple cell types. We used real-time confocal and total internal reflection fluorescence microscopy to examine the spatiotemporal regulation of Epac2, which is a GEF for the guanosine triphosphatase (GTPase) Rap. We demonstrated that increases in the concentration of cAMP triggered the translocation of Epac2 from the cytoplasm to the plasma membrane in insulin-secreting beta cells. Glucose-induced oscillations of the submembrane concentration of cAMP were associated with cyclic translocation of Epac2, and this translocation could be amplified by increases in the cytoplasmic Ca2+ concentration. Analyses of Epac2 mutants identified the high-affinity cAMP-binding and the Ras association domains as crucial for the translocation. Expression of a dominant-negative Ras mutant reduced Epac2 translocation, and Ca2+-dependent oscillations in Ras activity synchronized with Epac2 translocation in single beta cells. The cyclic translocation of Epac2 was accompanied by oscillations of Rap GTPase activity at the plasma membrane, and expression of an inactive Rap1B mutant decreased insulin secretion. Thus, Epac2 localization is dynamically controlled by cAMP as well as by Ca2+-mediated activation of Ras. These results help to explain how oscillating signals can produce pulses of insulin release from pancreatic b cells.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-201253 (URN)10.1126/scisignal.2003932 (DOI)000318350100002 ()
Available from: 2013-06-10 Created: 2013-06-10 Last updated: 2017-12-06Bibliographically approved
Tian, G., Sågetorp, J., Xu, Y., Shuai, H., Degerman, E. & Tengholm, A. (2012). Role of phosphodiesterases in the shaping of sub-plasma-membrane cAMP oscillations and pulsatile insulin secretion. Journal of Cell Science, 125(21), 5084-5095
Open this publication in new window or tab >>Role of phosphodiesterases in the shaping of sub-plasma-membrane cAMP oscillations and pulsatile insulin secretion
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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
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