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  • 1.
    Abels, M.
    et al.
    Lund Univ, Ctr Diabet, Malmo, Sweden..
    Riva, M.
    Lund Univ, Ctr Diabet, Malmo, Sweden..
    Poon, W.
    Lund Univ, Ctr Diabet, Malmo, Sweden..
    Bennet, H.
    Lund Univ, Ctr Diabet, Malmo, Sweden..
    Nagaraj, V.
    Lund Univ, Ctr Diabet, Malmo, Sweden..
    Dyachok, Oleg
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Isomaa, B.
    Folkhalsan Res Ctr, Helsinki, Finland.;Dept Social Serv & Hlth Care, Jacobstad, Finland..
    Tuomi, T.
    Folkhalsan Res Ctr, Helsinki, Finland.;Dept Med, Helsinki, Finland..
    Ahren, B.
    Lund Univ, Ctr Diabet, Lund, Sweden..
    Tengholm, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Fex, M.
    Lund Univ, Ctr Diabet, Malmo, Sweden..
    Renstrom, E.
    Lund Univ, Ctr Diabet, Malmo, Sweden..
    Groop, L.
    Lund Univ, Ctr Diabet, Malmo, Sweden..
    Lyssenko, V.
    Lund Univ, Ctr Diabet, Malmo, Sweden..
    Wierup, N.
    Lund Univ, Ctr Diabet, Malmo, Sweden..
    CART is a novel glucose-dependent peptide with antidiabetic actions in humans2015Inngår i: Diabetologia, ISSN 0012-186X, E-ISSN 1432-0428, Vol. 58, nr Suppl. 1, s. S279-S280Artikkel i tidsskrift (Annet vitenskapelig)
  • 2.
    Alenkvist, Ida
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Dyachok, Oleg
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Tian, Geng
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Li, Jia
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Mehrabanfar, Saba
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Jin, Yang
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för neurovetenskap.
    Birnir, Bryndis
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för neurovetenskap, Fysiologi.
    Tengholm, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Welsh, Michael
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Absence of Shb impairs insulin secretion by elevated FAK activity in pancreatic islets2014Inngår i: Journal of Endocrinology, ISSN 0022-0795, E-ISSN 1479-6805, Vol. 223, nr 3, s. 267-275Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 3.
    Alenkvist, Ida
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Gandasi, Nikhil R
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Barg, Sebastian
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Tengholm, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Recruitment of Epac2A to Insulin Granule Docking Sites Regulates Priming for Exocytosis2017Inngår i: Diabetes, ISSN 0012-1797, E-ISSN 1939-327X, Vol. 66, nr 10, s. 2610-2622Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Epac is a cAMP-activated guanine nucleotide exchange factor that mediates cAMP signaling in various types of cells, including -cells, where it is involved in the control of insulin secretion. Upon activation, the protein redistributes to the plasma membrane, but the underlying molecular mechanisms and functional consequences are unclear. Using quantitative high-resolution microscopy, we found that cAMP elevation caused rapid binding of Epac2A to the -cell plasma membrane, where it accumulated specifically at secretory granules and rendered them more prone to undergo exocytosis. cAMP-dependent membrane binding required the high-affinity cyclic nucleotide-binding (CNB) and Ras association domains, but not the disheveled-Egl-10-pleckstrin domain. Although the N-terminal low-affinity CNB domain (CNB-A) was dispensable for the translocation to the membrane, it was critical for directing Epac2A to the granule sites. Epac1, which lacks the CNB-A domain, was recruited to the plasma membrane but did not accumulate at granules. We conclude that Epac2A controls secretory granule release by binding to the exocytosis machinery, an effect that is enhanced by prior cAMP-dependent accumulation of the protein at the plasma membrane.

  • 4.
    Chowdhury, Azazul Islam
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Dyachok, Oleg
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Tengholm, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Sandler, Stellan
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Bergsten, Peter
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Functional differences between aggregated and dispersed insulin-producing cells2013Inngår i: Diabetologia, ISSN 0012-186X, E-ISSN 1432-0428, Vol. 56, nr 7, s. 1557-1568Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Beta cells situated in the islet of Langerhans respond more vigorously to glucose than do dissociated beta cells. Mechanisms for this discrepancy were studied by comparing insulin-producing MIN6 cells aggregated into pseudoislets with MIN6 monolayer cells and mouse and human islets. MIN6 monolayers, pseudoislets and mouse and human islets were exposed to glucose, alpha-ketoisocaproic acid (KIC), pyruvate, KIC plus glutamine and the phosphatidylinositol 3-kinase (PI3K) inhibitors LY294002 or wortmannin. Insulin secretion (ELISA), cytoplasmic Ca2+ concentration ([Ca2+](c); microfluorometry), glucose oxidation (radiolabelling), the expression of genes involved in mitochondrial metabolism (PCR) and the phosphorylation of insulin receptor signalling proteins (western blotting) were measured. Insulin secretory responses to glucose, pyruvate, KIC and glutamine were higher in pseudoislets than monolayers and comparable to those of human islets. Glucose oxidation and genes for mitochondrial metabolism were upregulated in pseudoislets compared with single cells and monolayers, respectively. Phosphorylation at the inhibitory S636/639 site of IRS-1 was significantly higher in monolayers and dispersed human and mouse cells than pseudoislets and intact human and mouse islets. PI3K inhibition only slightly attenuated glucose-stimulated insulin secretion from monolayers, but substantially reduced that from pseudoislets and human and mouse islets without suppressing the glucose-induced [Ca2+](c) response. We propose that islet architecture is critical for proper beta cell mitochondrial metabolism and IRS-1 signalling, and that PI3K regulates insulin secretion at a step distal to the elevation of [Ca2+](c).

  • 5. Dezaki, Katsuya
    et al.
    Damdindorj, Boldbaatar
    Sone, Hideyuki
    Dyachok, Oleg
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Tengholm, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Gylfe, Erik
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Kurashina, Tomoyuki
    Yoshida, Masashi
    Kakei, Masafumi
    Yada, Toshihiko
    Ghrelin Attenuates cAMP-PKA Signaling to Evoke Insulinostatic Cascade in Islet beta-Cells2011Inngår i: Diabetes, ISSN 0012-1797, E-ISSN 1939-327X, Vol. 60, nr 9, s. 2315-2324Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    OBJECTIVE-Ghrelin reportedly restricts insulin release in islet beta-cells via the G alpha(i2) subtype of G-proteins and thereby regulates glucose homeostasis. This study explored whether ghrelin regulates cAMP signaling and whether this regulation induces insulinostatic cascade in islet beta-cells. RESEARCH DESIGN AND METHODS-Insulin release was measured in rat perfused pancreas and isolated islets and cAMP production in isolated islets. Cytosolic cAMP concentrations ([cAMP](i)) were monitored in mouse MIN6 cells using evanescent-wave fluorescence imaging. In rat single beta-cells, cytosolic protein kinase-A activity ([PKA](i)) and Ca(2+) concentration ([Ca(2+)](i)) were measured by DR-II and fura-2 microfluorometry, respectively, and whole cell currents by patch-clamp technique. RESULTS-Ghrelin suppressed glucose (8.3 mmol/L)-induced insulin release in rat perfused pancreas and isolated islets, and these effects of ghrelin were blunted in the presence of cAMP analogs or adenylate cyclase inhibitor. Glucose-induced cAMP production in isolated islets was attenuated by ghrelin and enhanced by ghrelin receptor antagonist and anti-ghrelin antiserum, which counteract endogenous islet-derived ghrelin. Ghrelin inhibited the glucose-induced [cAMP](i) elevation and [PKA](i) activation in MIN6 and rat beta-cells, respectively. Furthermore, ghrelin potentiated voltage-dependent K(+) (Kv) channel currents without altering Ca(2+) channel currents and attenuated glucose-induced [Ca(2+)](i) increases in rat beta-cells in a PKA-dependent manner. CONCLUSIONS-Ghrelin directly interacts with islet beta-cells to attenuate glucose-induced cAMP production and PKA activation, which lead In activation of Kv channels and suppression of glucose-induced [Ca(2+)](i) increase and insulin release.

  • 6.
    Dyachok, O
    et al.
    Uppsala universitet, Medicinska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Sagetorp, J
    Uppsala universitet, Medicinska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Isakov, Y
    Uppsala universitet, Medicinska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Tengholm, A
    Uppsala universitet, Medicinska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    cAMP oscillations restrict protein kinase A redistribution in insulin-secreting cells.2006Inngår i: Biochem Soc Trans, ISSN 0300-5127, Vol. 34, nr Pt 4, s. 498-501Artikkel i tidsskrift (Fagfellevurdert)
  • 7.
    Dyachok, Oleg
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Idevall-Hagren, Olof
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Sågetorp, Jenny
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Tian, Geng
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Wuttke, Anne
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Arrieumerlou, Cecile
    Infection Biology, Biozentrum, University of Basel, Switzerland.
    Akusjärvi, Göran
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Gylfe, Erik
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Tengholm, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Glucose-induced cyclic AMP oscillations regulate pulsatile insulin secretion2008Inngår i: Cell Metabolism, ISSN 1550-4131, E-ISSN 1932-7420, Vol. 8, nr 1, s. 26-37Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 8.
    Dyachok, Oleg
    et al.
    Uppsala universitet, Medicinska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Isakov, Yegor
    Uppsala universitet, Medicinska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Sågetorp, Jenny
    Uppsala universitet, Medicinska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Tengholm, Anders
    Uppsala universitet, Medicinska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Oscillations of cyclic AMP in hormone-stimulated insulin-secreting beta-cells.2006Inngår i: Nature, ISSN 1476-4687, Vol. 439, nr 7074, s. 349-52Artikkel i tidsskrift (Fagfellevurdert)
  • 9.
    Gucek, Alenka
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Gandasi, Nikhil R
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Omar-Hmeadi, Muhmmad
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Bakke, Marit
    Univ Bergen, Dept Biomed, Bergen, Norway.
    Doskeland, Stein O.
    Univ Bergen, Dept Biomed, Bergen, Norway.
    Tengholm, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Barg, Sebastian
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Fusion pore regulation by cAMP/Epac2 controls cargo release during insulin exocytosis2019Inngår i: eLIFE, E-ISSN 2050-084X, Vol. 8, artikkel-id e41711Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Regulated exocytosis establishes a narrow fusion pore as initial aqueous connection to the extracellular space, through which small transmitter molecules such as ATP can exit. Co-release of polypeptides and hormones like insulin requires further expansion of the pore. There is evidence that pore expansion is regulated and can fail in diabetes and neurodegenerative disease. Here, we report that the cAMP-sensor Epac2 (Rap-GEF4) controls fusion pore behavior by acutely recruiting two pore-restricting proteins, amisyn and dynamin-1, to the exocytosis site in insulin-secreting beta-cells. cAMP elevation restricts and slows fusion pore expansion and peptide release, but not when Epac2 is inactivated pharmacologically or in Epac2(-/-) (Rapgef4(-/-)) mice. Consistently, overexpression of Epac2 impedes pore expansion. Widely used antidiabetic drugs (GLP-1 receptor agonists and sulfonylureas) activate this pathway and thereby paradoxically restrict hormone release. We conclude that Epac2/cAMP controls fusion pore expansion and thus the balance of hormone and transmitter release during insulin granule exocytosis.

  • 10.
    Gylfe, Erik
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Tengholm, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Neurotransmitter control of islet hormone pulsatility2014Inngår i: Diabetes, obesity and metabolism, ISSN 1462-8902, E-ISSN 1463-1326, Vol. 16, nr S1, s. 102-110Artikkel, forskningsoversikt (Fagfellevurdert)
    Abstract [en]

    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 -cells couple via gap junctions to generate synchronized insulin pulses. In contrast, - and -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 -cell-intrinsic mechanism/s. Further glucose elevation will stimulate pulsatile insulin release and co-secretion of neurotransmitters. Excitatory ATP may synchronize -cells with -cells to generate coinciding pulses of insulin and somatostatin. Inhibitory neurotransmitters from - and -cells can then generate antiphase pulses of glucagon release. Neurotransmitters released from intrapancreatic ganglia are required to synchronize -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.

  • 11. Hinke, Simon A.
    et al.
    Navedo, Manuel F.
    Ulman, Allison
    Whiting, Jennifer L.
    Nygren, Patrick J.
    Tian, Geng
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Jimenez-Caliani, Antonio J.
    Langeberg, Lorene K.
    Cirulli, Vincenzo
    Tengholm, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Dell'Acqua, Mark L.
    Santana, L. Fernando
    Scott, John D.
    Anchored phosphatases modulate glucose homeostasis2012Inngår i: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 31, nr 20, s. 3991-4004Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 12. Hoivik, Erling A.
    et al.
    Witsoe, Solveig L.
    Bergheim, Inger R.
    Xu, Yunjian
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Jakobsson, Ida
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Tengholm, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Doskeland, Stein Ove
    Bakke, Marit
    DNA Methylation of Alternative Promoters Directs Tissue Specific Expression of Epac2 Isoforms2013Inngår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, nr 7, s. e67925-Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 13.
    Hägerkvist, Robert
    et al.
    Uppsala universitet, Medicinska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Mokhtari, Dariush
    Uppsala universitet, Medicinska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Myers, Jason W
    Tengholm, Anders
    Uppsala universitet, Medicinska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Welsh, Nils
    Uppsala universitet, Medicinska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    siRNA Produced by Recombinant Dicer Mediates Efficient Gene Silencing in Islet Cells.2005Inngår i: Ann N Y Acad Sci, ISSN 0077-8923, Vol. 1040, s. 114-22Artikkel i tidsskrift (Fagfellevurdert)
  • 14.
    Idevall Hagren, Olof
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Barg, Sebastian
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Gylfe, Erik
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Tengholm, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    cAMP Mediators of Pulsatile Insulin Secretion from Glucose-stimulated Single β-Cells2010Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 285, nr 30, s. 23005-23016Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 15.
    Idevall Hagren, Olof
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Tengholm, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Glucose and insulin synergistically activate phosphatidylinositol 3-kinase to trigger oscillations of phosphatidylinositol 3,4,5-trisphosphate in beta-cells2006Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 281, nr 51, s. 39121-39127Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 16.
    Idevall-Hagren, Olof
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Jakobsson, Ida
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Xu, Yunjian
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Tengholm, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Spatial Control of Epac2 Activity by cAMP and Ca2+-Mediated Activation of Ras in Pancreatic beta Cells2013Inngår i: Science Signaling, ISSN 1945-0877, E-ISSN 1937-9145, Vol. 6, nr 273, s. ra29-Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 17.
    Korol, Sergiy V
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för neurovetenskap, Fysiologi.
    Jin, Zhe
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för neurovetenskap, Fysiologi.
    Jin, Yang
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för neurovetenskap, Fysiologi.
    Bhandage, Amol K.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för neurovetenskap, Fysiologi.
    Tengholm, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Gandasi, Nikhil R.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för neurovetenskap, Fysiologi.
    Barg, Sebastian
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Espes, Daniel
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Carlsson, Per-Ola
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Laver, Derek
    University of Newcastle, Callaghan, Australia.
    Birnir, Bryndis
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för neurovetenskap, Fysiologi.
    Functional Characterization of Native, High-Affinity GABAA Receptors in Human Pancreatic β Cells2018Inngår i: EBioMedicine, ISSN 0360-0637, E-ISSN 2352-3964, Vol. 30Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    In human pancreatic islets, the neurotransmitter γ-aminobutyric acid (GABA) is an extracellular signaling molecule synthesized by and released from the insulin-secreting β cells. The effective, physiological GABA concentration range within human islets is unknown. Here we use native GABAA receptors in human islet β cells as biological sensors and reveal that 100-1000nM GABA elicit the maximal opening frequency of the single-channels. In saturating GABA, the channels desensitized and stopped working. GABA modulated insulin exocytosis and glucose-stimulated insulin secretion. GABAA receptor currents were enhanced by the benzodiazepine diazepam, the anesthetic propofol and the incretin glucagon-like peptide-1 (GLP-1) but not affected by the hypnotic zolpidem. In type 2 diabetes (T2D) islets, single-channel analysis revealed higher GABA affinity of the receptors. The findings reveal unique GABAA receptors signaling in human islets β cells that is GABA concentration-dependent, differentially regulated by drugs, modulates insulin secretion and is altered in T2D.

  • 18.
    Li, Jia
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Shuai, Hongyan
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Gylfe, Erik
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Tengholm, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Oscillations of sub-membrane ATP in glucose-stimulated beta cells depend on negative feedback from Ca2+2013Inngår i: Diabetologia, ISSN 0012-186X, E-ISSN 1432-0428, Vol. 56, nr 7, s. 1577-1586Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    ATP 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. The 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. Glucose dose-dependently increased [ATP](pm) with half-maximal and maximal effects at 5.2 and 9 mmol/l, respectively. Additional elevations of glucose to 11 to 20 mmol/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). In 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.

  • 19.
    Li, Jia
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Yu, Qian
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Ahooghalandari, Parvin
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Gribble, Fiona M.
    Reimann, Frank
    Tengholm, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Gylfe, Erik
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Submembrane ATP and Ca2+ kinetics in alpha-cells: unexpected signaling for glucagon secretion2015Inngår i: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 29, nr 8, s. 3379-3388Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Cytoplasmic ATP and Ca2+ are implicated in current models of glucose's control of glucagon and insulin secretion from pancreatic alpha- and beta-cells, respectively, but little is known about ATP and its relation to Ca2+ in alpha-cells. We therefore expressed the fluorescent ATP biosensor Perceval in mouse pancreatic islets and loaded them with a Ca2+ indicator. With total internal reflection fluorescence microscopy, we recorded subplasma membrane concentrations of Ca2+ and ATP ([Ca2+](pm); [ATP](pm)) in superficial alpha- and beta-cells of intact islets and related signaling to glucagon and insulin secretion by immunoassay. Consistent with ATP's controlling glucagon and insulin secretion during hypo- and hyperglycemia, respectively, the dose-response relationship for glucoseinduced [ATP](pm) generation was left shifted in alpha-cells compared to beta-cells. Both cell types showed [Ca2+](pm) and [ATP](pm) oscillations in opposite phase, probably reflecting energy-consuming Ca2+ transport. Although pulsatile insulin and glucagon release are in opposite phase, [Ca2+](pm) synchronized in the same phase between alpha- and beta-cells. This paradox can be explained by the overriding of Ca2+ stimulation by paracrine inhibition, because somatostatin receptor blockade potently stimulated glucagon release with little effect on Ca2+. The data indicate that an alpha-cell-intrinsic mechanism controls glucagon in hypoglycemia and that paracrine factors shape pulsatile secretion in hyperglycemia.

  • 20.
    Li, Jia
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Yu, Qian
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Ahooghalandari, Parvin
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Gribble, Fiona M
    Cambridge Institute for Medical Research, Addenbrooke’s Hospital, Cambridge, United Kingdom.
    Reimann, Frank
    Cambridge Institute for Medical Research, Addenbrooke’s Hospital, Cambridge, United Kingdom.
    Tengholm, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Gylfe, Erik
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Submembrane ATP and Ca2+ kinetics in α‑cells: unexpected signaling for glucagon secretion2015Inngår i: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 29, nr 8, s. 3379-3388Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Cytoplasmic ATP and Ca(2+) are implicated in current models of glucose's control of glucagon and insulin secretion from pancreatic α- and β-cells, respectively, but little is known about ATP and its relation to Ca(2+) in α-cells. We therefore expressed the fluorescent ATP biosensor Perceval in mouse pancreatic islets and loaded them with a Ca(2+) indicator. With total internal reflection fluorescence microscopy, we recorded subplasma membrane concentrations of Ca(2+) and ATP ([Ca(2+)]pm; [ATP]pm) in superficial α- and β-cells of intact islets and related signaling to glucagon and insulin secretion by immunoassay. Consistent with ATP's controlling glucagon and insulin secretion during hypo- and hyperglycemia, respectively, the dose-response relationship for glucose-induced [ATP]pm generation was left shifted in α-cells compared to β-cells. Both cell types showed [Ca(2+)]pm and [ATP]pm oscillations in opposite phase, probably reflecting energy-consuming Ca(2+) transport. Although pulsatile insulin and glucagon release are in opposite phase, [Ca(2+)]pm synchronized in the same phase between α- and β-cells. This paradox can be explained by the overriding of Ca(2+) stimulation by paracrine inhibition, because somatostatin receptor blockade potently stimulated glucagon release with little effect on Ca(2+). The data indicate that an α-cell-intrinsic mechanism controls glucagon in hypoglycemia and that paracrine factors shape pulsatile secretion in hyperglycemia.

  • 21.
    Mojtaba Ghiasi, Seyed
    et al.
    Univ Copenhagen, Dept Biomed Sci, Copenhagen, Denmark.
    Dahlby, Tina
    Univ Copenhagen, Dept Biomed Sci, Copenhagen, Denmark.
    Hede Andersen, Caroline
    Univ Copenhagen, Dept Biomed Sci, Copenhagen, Denmark.
    Haataja, Leena
    Univ Michigan, Div Metab Endocrinol & Diabet, Med Ctr, Ann Arbor, MI USA.
    Petersen, Sólrun
    Univ Copenhagen, Dept Biomed Sci, Copenhagen, Denmark.
    Omar-Hmeadi, Muhmmad
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Yang, Mingyu
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Pihl, Celina
    Univ Copenhagen, Dept Biomed Sci, Copenhagen, Denmark.
    Emilie Bresson, Sophie
    Univ Copenhagen, Dept Biomed Sci, Copenhagen, Denmark.
    Saad Khilji, Muhammad
    Univ Copenhagen, Dept Biomed Sci, Copenhagen, Denmark.
    Klindt, Kristian
    Univ Copenhagen, Dept Biomed Sci, Copenhagen, Denmark.
    Cheta, Oana
    Univ Copenhagen, Dept Biomed Sci, Copenhagen, Denmark.
    Perone, Marcelo J
    Univ Copenhagen, Dept Biomed Sci, Copenhagen, Denmark;Polo Cient Tecnol, Max Planck Soc, Partner Inst, CONICET,Inst Invest Biomed Buenos Aires IBioBA, Buenos Aires, DF, Argentina.
    Tyrberg, Björn
    AstraZeneca, IMED Biotech Unit, Translat Sci Cardiovasc Renal & Metab, Gothenburg, Sweden.
    Prats, Clara
    Univ Copenhagen, Dept Biomed Sci, Ctr Hlth Ageing, Copenhagen, Denmark.
    Barg, Sebastian
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Tengholm, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Arvan, Peter
    Univ Michigan, Div Metab Endocrinol & Diabet, Med Ctr, Ann Arbor, MI USA.
    Mandrup-Poulsen, Thomas
    Univ Copenhagen, Dept Biomed Sci, Copenhagen, Denmark.
    Tomasz Marzec, Michal
    Univ Copenhagen, Dept Biomed Sci, Copenhagen, Denmark.
    The Endoplasmic Reticulum Chaperone Glucose-Regulated Protein 94 is Essential for Proinsulin Handling2019Inngår i: Diabetes, ISSN 0012-1797, E-ISSN 1939-327X, Vol. 68, nr 4, s. 747-760Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Although endoplasmic reticulum (ER) chaperone binding to mutant proinsulin has been reported, the role of protein chaperones in the handling of wild-type proinsulin is under-investigated. Here, we have explored the importance of glucose regulated protein 94 (GRP94), a prominent ER chaperone known to fold insulin-like growth factors, in proinsulin handling within β-cells. We found that GRP94 co-immunoprecipitated with proinsulin and that inhibition of GRP94 function and/or expression reduced glucose-dependent insulin secretion, shortened proinsulin half-life and lowered intracellular proinsulin and insulin levels. This phenotype was accompanied by post-ER proinsulin misprocessing and higher numbers of enlarged insulin granules that contained amorphic material with reduced immunogold staining for mature insulin. Insulin granule exocytosis was two-fold accelerated but the secreted insulin had diminished bioactivity. Moreover, GRP94 knockdown or knockout in β-cells selectively activated Protein Kinase R-like Endoplasmic Reticulum Kinase (PERK), without increasing apoptosis levels. Finally, GRP94 mRNA was overexpressed in islets from T2D patients. We conclude that GRP94 is a chaperone crucial for proinsulin handling and insulin secretion.

  • 22.
    Mokhtari, Dariush
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Al-Amin, Abdullah
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Turpaev, Kyrill
    Li, Tingting
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Idevall-Hagren, Olof
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Li, Jia
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Wuttke, Anne
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Fred, Rikard G
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Ravassard, Philippe
    Scharfmann, R
    Tengholm, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Welsh, Nils
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    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-Abl2013Inngår i: Diabetologia, ISSN 0012-186X, E-ISSN 1432-0428, Vol. 56, nr 6, s. 1327-1338Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    AIMS/HYPOTHESIS: It 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. METHODS: Human 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. RESULTS: Imatinib 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/INTERPRETATION: Imatinib inhibition of c-Abl in beta cells decreases SHIP2 activity, which results in enhanced signalling downstream of PI3 kinase.

  • 23.
    Omar-Hmeadi, Muhmmad
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Lund, Per-Eric
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Gandasi, Nikhil
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Tengholm, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Barg, Sebastian
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Paracrine control of α-cell glucagon exocytosis is compromised in human type-2 diabetesManuskript (preprint) (Annet vitenskapelig)
  • 24.
    Pietilä, Ilkka
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    van Mourik, Djenolan
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Tamelander, Andreas
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Kriz, Vitezslav
    Czech Academy of Sciences.
    Claesson-Welsh, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi.
    Tengholm, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Welsh, Michael
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Temporal Dynamics of VEGFA-Induced VEGFR2/FAK Co-Localization Depend on SHB2019Inngår i: Cells, ISSN 2073-4409, Vol. 8, artikkel-id 1645Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Focal adhesion kinase (FAK) is essential for vascular endothelial growth factor-A (VEGFA)/VEGF receptor-2 (VEGFR2)-stimulated angiogenesis and vascular permeability. We have previously noted that presence of the Src homology-2 domain adapter protein B (SHB) is of relevance for VEGFA-stimulated angiogenesis in a FAK-dependent manner. The current study was conducted in order address the temporal dynamics of co-localization between these components in HEK293 and primary lung endothelial cells (EC) by total internal reflection fluorescence microscopy (TIRF). An early (<2.5 min) VEGFA-induced increase in VEGFR2 co-localization with SHB was dependent on tyrosine 1175 in VEGFR2. VEGFA also enhanced SHB co-localization with FAK. FAK co-localization with VEGFR2 was dependent on SHB since it was significantly lower in SHB deficient EC after VEGFA addition. Absence of SHB also resulted in a gradual decline of VEGFR2 co-localization with FAK under basal (prior to VEGFA addition) conditions. A similar basal response was observed with expression of the Y1175F-VEGFR2 mutant in wild type EC. The distribution of focal adhesions in SHB-deficient EC was altered with a primarily perinuclear location. These live cell data implicate SHB as a key component regulating FAK activity in response to VEGFA/VEGFR2.

  • 25.
    Shuai, Hongyan
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Constitutive GLP-1-receptor signaling contributes to basal and glucose-stimulated cAMP formation in β-cells: Constitutive GLP-1 receptor signaling in β-cellsInngår i: Artikkel i tidsskrift (Annet (populærvitenskap, debatt, mm))
  • 26.
    Shuai, Hongyan
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Tengholm, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Effects of Ca2+ and autocrine signals on cAMP dynamics in β-cellsManuskript (preprint) (Annet (populærvitenskap, debatt, mm))
  • 27.
    Shuai, Hongyan
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Tengholm, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Glucagon and GLP-1 evoke cAMP elevations in sub-populations of mouse and human α-cellsManuskript (preprint) (Annet (populærvitenskap, debatt, mm))
  • 28.
    Tengholm, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Cyclic AMP dynamics in the pancreatic beta-cell2012Inngår i: Upsala Journal of Medical Sciences, ISSN 0300-9734, E-ISSN 2000-1967, Vol. 117, nr 4, s. 355-369Artikkel, forskningsoversikt (Fagfellevurdert)
    Abstract [en]

    Insulin secretion from pancreatic beta-cells is tightly regulated by glucose and other nutrients, hormones, and neural factors. The exocytosis of insulin granules is triggered by an elevation of the cytoplasmic Ca2+ concentration ([Ca2+](i)) and is further amplified by cyclic AMP (cAMP). Cyclic AMP is formed primarily in response to glucoincretin hormones and other G(s)-coupled receptor agonists, but generation of the nucleotide is critical also for an optimal insulin secretory response to glucose. Nutrient and receptor stimuli trigger oscillations of the cAMP concentration in beta-cells. The oscillations arise from variations in adenylyl cyclase-mediated cAMP production and phosphodiesterase-mediated degradation, processes controlled by factors like cell metabolism and [Ca2+](i). Protein kinase A and the guanine nucleotide exchange factor Epac2 mediate the actions of cAMP in beta-cells and operate at multiple levels to promote exocytosis and pulsatile insulin secretion. The cAMP signaling system contains important targets for pharmacological improvement of insulin secretion in type 2 diabetes.

  • 29.
    Tengholm, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Cyclic AMP links glucose stimulation to somatostatin secretion in delta-cells2019Inngår i: The Journal of General Physiology, ISSN 0022-1295, E-ISSN 1540-7748, Vol. 151, nr 9, s. 1062-1065Artikkel i tidsskrift (Annet vitenskapelig)
  • 30.
    Tengholm, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Purinergic P2Y1 receptors take centre stage in autocrine stimulation of human beta cells2014Inngår i: Diabetologia, ISSN 0012-186X, E-ISSN 1432-0428, Vol. 57, nr 12, s. 2436-2439Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Insulin secretory vesicles contain high concentrations of adenine nucleotides, which are co-released with insulin during exocytosis. There is strong evidence that ATP and ADP serve as autocrine messengers in pancreatic beta cells, but the functional effects and detailed mechanisms of action are under debate. In this issue of Diabetologia, Khan and colleagues (DOI: 10.1007/s00125-014-3368-8 ) present the results of their study of autocrine purinergic signalling in isolated human beta cells. Using a combination of electrophysiological techniques, Ca(2+) imaging and measurements of insulin secretion, it is demonstrated that voltage-dependent Ca(2+) influx triggers release of ATP/ADP, which activates purinergic receptors of the Gq/11-coupled P2Y1 isoform. Activation of these receptors leads to membrane depolarisation and phospholipase C-mediated mobilisation of Ca(2+) from endoplasmic reticulum stores, which amplifies the exocytosis-triggering Ca(2+) signal. In contrast, there is little evidence for involvement of ionotropic P2X receptors in the autocrine stimulation of human beta cells. This commentary discusses these findings as well as various functional and therapeutic implications of the complex purinergic signalling network in the pancreatic islet.

  • 31.
    Tengholm, Anders
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Gylfe, Erik
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    cAMP signalling in insulin and glucagon secretion2017Inngår i: Diabetes, obesity and metabolism, ISSN 1462-8902, E-ISSN 1463-1326, Vol. 19, s. 42-53Artikkel, forskningsoversikt (Fagfellevurdert)
    Abstract [en]

    The second messenger archetype cAMP is one of the most important cellular signalling molecules with central functions including the regulation of insulin and glucagon secretion from the pancreatic - and -cells, respectively. cAMP is generally considered as an amplifier of insulin secretion triggered by Ca2+ elevation in the -cells. Both messengers are also positive modulators of glucagon release from -cells, but in this case cAMP may be the important regulator and Ca2+ have a more permissive role. The actions of cAMP are mediated by protein kinase A (PKA) and the guanine nucleotide exchange factor Epac. The present review focuses on how cAMP is regulated by nutrients, hormones and neural factors in - and -cells via adenylyl cyclase-catalysed generation and phosphodiesterase-mediated degradation. We will also discuss how PKA and Epac affect ion fluxes and the secretory machinery to transduce the stimulatory effects on insulin and glucagon secretion. Finally, we will briefly describe disturbances of the cAMP system associated with diabetes and how cAMP signalling can be targeted to normalize hypo- and hypersecretion of insulin and glucagon, respectively, in diabetic patients.

  • 32.
    Tengholm, Anders
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Idevall-Hagren, Olof
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Imaging sub-plasma membrane cAMP dynamics with fluorescent translocation reporters2015Inngår i: cAMP Signaling: Methods And Protocols, Springer-Verlag New York, 2015, Vol. 1294, s. 85-101Kapittel i bok, del av antologi (Fagfellevurdert)
    Abstract [en]

    Imaging cAMP dynamics in single cells and tissues can provide important insights into the regulation of a variety of cellular processes. In recent years, a large number of tools for cAMP measurements have been developed. While most cAMP reporters are designed to undergo changes in fluorescence resonance energy transfer (FRET), there are alternative techniques with advantages for certain applications. Here, we describe protocols for cAMP measurements in the sub-plasma membrane space based on the detection of the cAMP-induced translocation of engineered fluorescent protein-tagged subunits of protein kinase A between the cytoplasm and the plasma membrane. Total internal reflection fluorescence (TIRF) imaging of the changes in reporter localization yields robust signal changes and has contributed to the discovery of cAMP oscillations in the sub-plasma membrane space of insulin-secreting β-cells stimulated with glucose and gluco-incretin hormones. We also demonstrate how the technique can be combined with measurements of the cytosolic Ca2+ concentration or with recordings of the subcellular localization of the cAMP effector protein Epac2. The translocation reporter approach provides a valuable complement to other methods for imaging sub-membrane cAMP dynamics in various types of cells.

  • 33.
    Tengholm, Anders
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Wuttke, Anne
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Autocrine signals mediate plasma membrane translocation of protein kinase C in insulin-secreting cells2013Inngår i: Diabetologia, ISSN 0012-186X, E-ISSN 1432-0428, Vol. 56, s. S207-S207Artikkel i tidsskrift (Annet vitenskapelig)
  • 34.
    Thore, Sophia
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Dyachok, Oleg
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Gylfe, Erik
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Tengholm, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Feedback activation of phospholipase C via intracellular mobilization and store-operated influx of Ca2+ in insulin-secreting β-cells2005Inngår i: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 118, nr Pt 19, s. 4463-4471Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Phospholipase C (PLC) regulates various cellular processes by catalyzing the formation of inositol-1,4,5-trisphosphate (IP3) and diacylglycerol from phosphatidylinositol-4,5-bisphosphate (PIP2). Here, we have investigated the influence of Ca2+ on receptor-triggered PLC activity in individual insulin-secreting β-cells. Evanescent wave microscopy was used to record PLC activity using green fluorescent protein (GFP)-tagged PIP2/IP3-binding pleckstrin homology domain from PLCδ1, and the cytoplasmic Ca2+ concentration ([Ca2+]i) was simultaneously measured using the indicator Fura Red. Stimulation of MIN6 β-cells with the muscarinic-receptor agonist carbachol induced rapid and sustained PLC activation. By contrast, only transient activation was observed after stimulation in the absence of extracellular Ca2+ or in the presence of the non-selective Ca2+ channel inhibitor La3+. The Ca2+-dependent sustained phase of PLC activity did not require voltage-gated Ca2+ influx, as hyperpolarization with diazoxide or direct Ca2+ channel blockade with nifedipine had no effect. Instead, the sustained PLC activity was markedly suppressed by the store-operated channel inhibitors 2-APB and SKF96365. Depletion of intracellular Ca2+ stores with the sarco(endo)plasmic reticulum Ca2+-ATPase inhibitors thapsigargin or cyclopiazonic acid abolished Ca2+ mobilization in response to carbachol, and strongly suppressed the PLC activation in Ca2+-deficient medium. Analogous suppressions were observed after loading cells with the Ca2+ chelator BAPTA. Stimulation of primary mouse pancreatic β-cells with glucagon elicited pronounced [Ca2+]i spikes, reflecting protein kinase A-mediated activation of Ca2+-induced Ca2+ release via IP3 receptors. These [Ca2+]i spikes were found to evoke rapid and transient activation of PLC. Our data indicate that receptor-triggered PLC activity is enhanced by positive feedback from Ca2+ entering the cytoplasm from intracellular stores and via store-operated channels in the plasma membrane. Such amplification of receptor signalling should be important in the regulation of insulin secretion by hormones and neurotransmitters.

  • 35.
    Thore, Sophia
    et al.
    Uppsala universitet, Medicinska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Dyachok, Oleg
    Uppsala universitet, Medicinska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Tengholm, Anders
    Uppsala universitet, Medicinska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Oscillations of Phospholipase C Activity Triggered by Depolarizatio and Ca2+ Influx in Insulin-secreting Cells2004Inngår i: The Journal of Biological Chemistry (JBC), Vol. 279, nr 19, s. 19396-19400Artikkel i tidsskrift (Fagfellevurdert)
  • 36.
    Tian, Geng
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Sandler, Stellan
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Gylfe, Erik
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Tengholm, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Glucose- and Hormone-Induced cAMP Oscillations in α- and β-Cells Within Intact Pancreatic Islets2011Inngår i: Diabetes, ISSN 0012-1797, E-ISSN 1939-327X, Vol. 60, nr 5, s. 1535-1543Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 37.
    Tian, Geng
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Sol, E. -RM.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Xu, Yunjian
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Tengholm, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Prolonged exposure to palmitate deteriorates glucose-induced cAMP generation and pulsatile insulin secretion2013Inngår i: Diabetologia, ISSN 0012-186X, E-ISSN 1432-0428, Vol. 56, s. S194-S194Artikkel i tidsskrift (Annet vitenskapelig)
  • 38.
    Tian, Geng
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Sol, Eri Maria
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Xu, Yunjian
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Shuai, Hongyan
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Tengholm, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Impaired cAMP generation contributes to defective glucose-stimulated insulin secretion after long-term exposure to palmitate2015Inngår i: Diabetes, ISSN 0012-1797, E-ISSN 1939-327X, Vol. 64, nr 3, s. 904-915Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 39.
    Tian, Geng
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Sågetorp, Jenny
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Xu, Yunjian
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Shuai, Hongyan
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Degerman, Eva
    Tengholm, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Role of phosphodiesterases in the shaping of sub-plasma-membrane cAMP oscillations and pulsatile insulin secretion2012Inngår i: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 125, nr 21, s. 5084-5095Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 40.
    Tian, Geng
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi. Binzhou Med Univ, Yantai, Peoples R China..
    Tengholm, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Prolonged exposure to palmitate deteriorates glucose-induced cAMP generation and pulsatile insulin secretion2015Inngår i: Diabetes/Metabolism Research Reviews, ISSN 1520-7552, E-ISSN 1520-7560, Vol. 31, s. 9-10Artikkel i tidsskrift (Annet vitenskapelig)
  • 41.
    Tian, Geng
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Tepikin, Alexei V.
    Tengholm, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Gylfe, Erik
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    cAMP Induces Stromal Interaction Molecule 1 (STIM1) Puncta but neither Orai1 Protein Clustering nor Store-operated Ca2+ Entry (SOCE) in Islet Cells2012Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, nr 13, s. 9862-9872Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 42.
    Tuomi, Tiinamaija
    et al.
    Helsinki Univ Hosp, Abdominal Ctr, Endocrinol, FI-00014 Helsinki, Finland.;Folkhalsan Res Ctr, FI-00250 Helsinki, Finland.;Univ Helsinki, Res Programs Unit, Diabet & Obes Res Program, FI-00014 Helsinki, Finland.;Univ Helsinki, Finnish Inst Mol Med, FI-00014 Helsinki, Finland..
    Nagorny, Cecilia L. F.
    Lund Univ, Ctr Diabet, Unit Mol Metab, SE-20502 Lund, Sweden..
    Singh, Pratibha
    Lund Univ, Ctr Diabet, Unit Mol Metab, SE-20502 Lund, Sweden..
    Bennet, Hedvig
    Lund Univ, Ctr Diabet, Unit Diabet & Celiac Dis, SE-20502 Lund, Sweden..
    Yu, Qian
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Alenkvist, Ida
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Isomaa, Bo
    Folkhalsan Res Ctr, FI-00250 Helsinki, Finland.;Univ Helsinki, Finnish Inst Mol Med, FI-00014 Helsinki, Finland.;Dept Social Serv & Hlth Care, FI-68601 Pietarsaari, Finland..
    Ostman, Bjarne
    Folkhalsan Res Ctr, FI-00250 Helsinki, Finland..
    Soderstrom, Johan
    Folkhalsan Res Ctr, FI-00250 Helsinki, Finland.;Univ Helsinki, Finnish Inst Mol Med, FI-00014 Helsinki, Finland..
    Pesonen, Anu-Katriina
    Univ Helsinki, Inst Behav Sci, FI-00014 Helsinki, Finland..
    Martikainen, Silja
    Univ Helsinki, Inst Behav Sci, FI-00014 Helsinki, Finland..
    Raikkonen, Katri
    Univ Helsinki, Inst Behav Sci, FI-00014 Helsinki, Finland..
    Forsen, Tom
    Folkhalsan Res Ctr, FI-00250 Helsinki, Finland..
    Hakaste, Liisa
    Helsinki Univ Hosp, Abdominal Ctr, Endocrinol, FI-00014 Helsinki, Finland.;Folkhalsan Res Ctr, FI-00250 Helsinki, Finland.;Univ Helsinki, Res Programs Unit, Diabet & Obes Res Program, FI-00014 Helsinki, Finland..
    Almgren, Peter
    Univ Helsinki, Inst Behav Sci, FI-00014 Helsinki, Finland.;Lund Univ, Ctr Diabet, Unit Diabet & Endocrinol, SE-20502 Lund, Sweden..
    Storm, Petter
    Lund Univ, Ctr Diabet, Unit Diabet & Endocrinol, SE-20502 Lund, Sweden..
    Asplund, Olof
    Lund Univ, Ctr Diabet, Unit Diabet & Endocrinol, SE-20502 Lund, Sweden..
    Shcherbina, Liliya
    Lund Univ, Ctr Diabet, Unit Neuroendocrine Cell Biol, SE-20502 Lund, Sweden..
    Fex, Malin
    Lund Univ, Ctr Diabet, Unit Diabet & Celiac Dis, SE-20502 Lund, Sweden..
    Fadista, Joao
    Lund Univ, Ctr Diabet, Unit Diabet & Endocrinol, SE-20502 Lund, Sweden..
    Tengholm, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Wierup, Nils
    Lund Univ, Ctr Diabet, Unit Neuroendocrine Cell Biol, SE-20502 Lund, Sweden..
    Groop, Leif
    Univ Helsinki, Finnish Inst Mol Med, FI-00014 Helsinki, Finland.;Lund Univ, Ctr Diabet, Unit Diabet & Endocrinol, SE-20502 Lund, Sweden..
    Mulder, Hindrik
    Lund Univ, Ctr Diabet, Unit Mol Metab, SE-20502 Lund, Sweden..
    Increased Melatonin Signaling Is a Risk Factor for Type 2 Diabetes2016Inngår i: Cell Metabolism, ISSN 1550-4131, E-ISSN 1932-7420, Vol. 23, nr 6, s. 1067-1077Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Type 2 diabetes (T2D) is a global pandemic. Genome-wide association studies (GWASs) have identified >100 genetic variants associated with the disease, including a common variant in the melatonin receptor 1 b gene (MTNR1B). Here, we demonstrate increased MTNR1B expression in human islets from risk G-allele carriers, which likely leads to a reduction in insulin release, increasing T2D risk. Accordingly, in insulin-secreting cells, melatonin reduced cAMP levels, and MTNR1B overexpression exaggerated the inhibition of insulin release exerted by melatonin. Conversely, mice with a disruption of the receptor secreted more insulin. Melatonin treatment in a human recall-by-genotype study reduced insulin secretion and raised glucose levels more extensively in risk G-allele carriers. Thus, our data support a model where enhanced melatonin signaling in islets reduces insulin secretion, leading to hyperglycemia and greater future risk of T2D. The findings also imply that melatonin physiologically serves to inhibit nocturnal insulin release.

  • 43.
    Wan Saudi, Wan Salman
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för neurovetenskap, Fysiologi.
    Halim, Mohammed Abdul
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Gastroenterologi/hepatologi.
    Gillberg, Linda
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Gastroenterologi/hepatologi.
    Rudholm-Feldreich, Tobias
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Gastroenterologi/hepatologi.
    Rosenqvist, Evelina
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för neurovetenskap, Fysiologi.
    Tengholm, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Sundbom, Magnus
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för kirurgiska vetenskaper, Gastrointestinalkirurgi.
    Karlbom, Urban
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för kirurgiska vetenskaper, Kolorektalkirurgi.
    Näslund, Erik
    Webb, Dominic-Luc
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Gastroenterologi/hepatologi.
    Sjöblom, Markus
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för neurovetenskap, Fysiologi.
    Hellström, Per M
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Gastroenterologi/hepatologi.
    Neuropeptide S inhibits gastrointestinal motility and increases mucosal permeability through nitric oxide2015Inngår i: American Journal of Physiology - Gastrointestinal and Liver Physiology, ISSN 0193-1857, E-ISSN 1522-1547, Vol. 309, nr 8, s. G625-G634Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Neuropeptide S (NPS) receptor (NPSR1) polymorphisms are associated with enteral dysmotility and inflammatory bowel disease (IBD). This study investigated the role of NPS in conjunction with nitrergic mechanisms in the regulation of intestinal motility and mucosal permeability. In rats, small intestinal myoelectric activity and luminal pressure changes in small intestine and colon, along with duodenal permeability were studied. In human intestine, NPS and NPSR1 were localized by immunostaining. Pre- and postprandial plasma NPS was measured by ELISA in healthy and active IBD humans. Effects and mechanisms of NPS were studied in human intestinal muscle strips. In rats, NPS 100-4000 pmol/kg·min had effects on the small intestine and colon. Low doses of NPS increased myoelectric spiking (p<0.05). Higher doses reduced spiking and prolonged the cycle length of the migrating myoelectric complex, reduced intraluminal pressures (p<0.05-0.01) and increased permeability (p<0.01) through NO-dependent mechanisms. In human intestine, NPS localized at myenteric nerve cell bodies and fibers. NPSR1 was confined to nerve cell bodies. Circulating NPS in humans was tenfold below the ~0.3 nmol/l dissociation constant (Kd) of NPSR1, with no difference between healthy and IBD subjects. In human intestinal muscle strips pre-contracted by bethanechol, NPS 1-1000 nmol/l induced NO-dependent muscle relaxation (p<0.05) that was sensitive also to tetrodotoxin (p<0.01). In conclusion, NPS inhibits motility and increases permeability in neurocrine fashion acting through NO in the myenteric plexus in rats and humans. Aberrant signaling and up-regulation of NPSR1 could potentially exacerbate dysmotility and hyperpermeability by local mechanisms in gastrointestinal functional and inflammatory reactions.

  • 44.
    Wang, Xuan
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Jiang, L.
    Chinese Acad Agr Sci, Inst Anim Sci, Beijing, Peoples R China..
    Wallerman, Ola
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Yu, Qian
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Klaesson, Axel
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Molekylära verktyg.
    Tengholm, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Andersson, Leif
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Welsh, Nils
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    ZBED6 negatively regulates insulin content, glucose-stimulated insulin secretion, neuronal differentiation and cell aggregation in MIN6 cells2016Inngår i: Diabetologia, ISSN 0012-186X, E-ISSN 1432-0428, Vol. 59, s. S214-S215Artikkel i tidsskrift (Fagfellevurdert)
  • 45.
    Wang, Xuan
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Jiang, Lin
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab. Chinese Acad Agr Sci, Inst Anim Sci, Key Lab Farm Anim Genet Resources & Utilizat, Minist Agr China, Beijing, Peoples R China.
    Wallerman, Ola
    Swedish Univ Agr Sci, Dept Anim Breeding & Genet, Uppsala, Sweden.
    Younis, Shady
    Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Ain Shams Univ, Dept Anim Prod, Cairo, Egypt.
    Yu, Qian
    Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Klaesson, Axel
    Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Tengholm, Anders
    Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Welsh, Nils
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Andersson, Leif
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab. Swedish Univ Agr Sci, Dept Anim Breeding & Genet, Uppsala, Sweden.
    ZBED6 negatively regulates insulin production, neuronal differentiation, and cell aggregation in MIN6 cells2019Inngår i: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 33, nr 1, s. 88-100Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Zinc finger BED domain containing protein 6 (Zbed6) has evolved from a domesticated DNA transposon and encodes a transcription factor unique to placental mammals. The aim of the present study was to investigate further the role of ZBED6 in insulin-producing cells, using mouse MIN6 cells, and to evaluate the effects of Zbed6 knockdown on basal -cell functions, such as morphology, transcriptional regulation, insulin content, and release. Zbed6-silenced cells and controls were characterized with a range of methods, including RNA sequencing, chromatin immunoprecipitation sequencing, insulin content and release, subplasma membrane Ca2+ measurements, cAMP determination, and morphologic studies. More than 700 genes showed differential expression in response to Zbed6 knockdown, which was paralleled by increased capacity to generate cAMP, as well as by augmented subplasmalemmal calcium concentration and insulin secretion in response to glucose stimulation. We identified >4000 putative ZBED6-binding sites in the MIN6 genome, with an enrichment of ZBED6 sites at upregulated genes, such as the -cell transcription factors v-maf musculoaponeurotic fibrosarcoma oncogene homolog A and Nk6 homeobox 1. We also observed altered morphology/growth patterns, as indicated by increased cell clustering, and in the appearance of axon-like Neurofilament, medium polypeptide and tubulin 3, class III-positive protrusions. We conclude that ZBED6 acts as a transcriptional regulator in MIN6 cells and that its activity suppresses insulin production, cell aggregation, and neuronal-like differentiation.Wang, X., Jiang, L., Wallerman, O., Younis, S., Yu, Q., Klaesson, A., Tengholm, A., Welsh, N., Andersson, L. ZBED6 negatively regulates insulin production, neuronal differentiation, and cell aggregation in MIN6 cells.

  • 46.
    Wuttke, Anne
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Idevall-Hagren, Olof
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Tengholm, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Imaging phosphoinositide dynamics in living cells2010Inngår i: Inositol Phosphates and Lipids: Methods and Protocols / [ed] Christopher J. Barker, Humana Press , 2010, Vol. 645, s. 219-235Kapittel i bok, del av antologi (Annet vitenskapelig)
    Abstract [en]

    To improve our understanding of the important roles played by inositol lipid derivatives in signalling and other cellular processes, it is crucial to measure phosphoinositide concentration changes in individual cells with high spatial and temporal resolution. A number of protein domains that interact with inositol lipids in a specific manner have been identified. Tagged with the green fluorescent protein or its colour variants, these protein modules can be used as probes to visualize various phosphoinositide species in different sub-cellular compartments. Here, we present protocols for fluorescence imaging of phosphoinositide dynamics in single living cells. Total internal reflection fluorescence microscopy is particularly powerful for time-lapse recordings of phosphoinositides in the plasma membrane. We demonstrate how this technique can be used to record phospholipase C- and PI3-kinase-induced changes in inositol lipids in insulin-secreting cells. These procedures should be applicable to studies of the spatio-temporal regulation of phosphoinositide metabolism in many types of cells.

  • 47.
    Wuttke, Anne
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Idevall-Hagren, Olof
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Tengholm, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    P2Y1 receptor-dependent diacylglycerol signaling microdomains in β cells promote insulin secretion2013Inngår i: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 27, nr 4, s. 1610-1620Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Diacylglycerol (DAG) controls numerous cell functions by regulating the localization of C1-domain-containing proteins, including protein kinase C (PKC), but little is known about the spatiotemporal dynamics of the lipid. Here, we explored plasma membrane DAG dynamics in pancreatic beta cells and determined whether DAG signaling is involved in secretagogue-induced pulsatile release of insulin. Single MIN6 cells, primary mouse beta cells, and human beta cells within intact islets were transfected with translocation biosensors for DAG, PKC activity, or insulin secretion and imaged with total internal reflection fluorescence microscopy. Muscarinic receptor stimulation triggered stable, homogenous DAG elevations, whereas glucose induced short-lived (7.1 +/- 0.4 s) but high-amplitude elevations (up to 109 +/- 10% fluorescence increase) in spatially confined membrane regions. The spiking was mimicked by membrane depolarization and suppressed after inhibition of exocytosis or of purinergic P2Y(1), but not P2X receptors, reflecting involvement of autocrine purinoceptor activation after exocytotic release of ATP. Each DAG spike caused local PKC activation with resulting dissociation of its substrate protein MARCKS from the plasma membrane. Inhibition of spiking reduced glucose-induced pulsatile insulin secretion. Thus, stimulus-specific DAG signaling patterns appear in the plasma membrane, including distinctmicrodomains, which have implications for the kinetic control of exocytosis and other membrane-associated processes.-Wuttke, A., Idevall-Hagren, O., Tengholm, A. P2Y(1) receptor-dependent diacylglycerol signaling microdomains in beta cells promote insulin secretion. 

  • 48.
    Wuttke, Anne
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Sågetorp, Jenny
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Tengholm, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Distinct plasma-membrane PtdIns(4)P and PtdIns(4,5)P2 dynamics in secretagogue-stimulated β-cells2010Inngår i: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 123, nr 9, s. 1492-1502Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Phosphoinositides regulate numerous processes in various subcellular compartments. Whereas many stimuli trigger changes in the plasma-membrane PtdIns(4,5)P-2 concentration, little is known about its precursor, PtdIns(4)P, in particular whether there are stimulus-induced alterations independent of those of PtdIns(4,5)P-2. We investigated plasma-membrane PtdIns(4)P and PtdIns(4,5)P-2 dynamics in insulin-secreting MIN6 cells using fluorescent translocation biosensors and total internal reflection microscopy. Loss of PtdIns(4,5)P-2 induced by phospholipase C (PLC)-activating receptor agonists or stimulatory glucose concentrations was paralleled by increased PtdIns(4)P levels. In addition, glucose-stimulated cells regularly showed anti-synchronous oscillations of the two lipids. Whereas glucose-induced PtdIns(4)P elevation required voltage-gated Ca2+ entry and was mimicked by membrane-depolarizing stimuli, the receptor-induced response was Ca2+ independent, but sensitive to protein kinase C (PKC) inhibition and mimicked by phorbol ester stimulation. We conclude that glucose and PLC-activating receptor stimuli trigger Ca2+- and PKC-dependent changes in the plasma-membrane PtdIns(4)P concentration that are independent of the effects on PtdIns(4,5)P-2. These findings indicate that enhanced formation of PtdIns(4)P, apart from ensuring efficient replenishment of the PtdIns(4,5)P-2 pool, might serve an independent signalling function by regulating the association of PtdIns(4)P-binding proteins with the plasma membrane.

  • 49.
    Yang, Mingyu
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Idevall-Hagren, Olof
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Gylfe, Erik
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Tengholm, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    A genetically encoded low-affinity Ca2+ sensor unmasks autocrine purinergic signalling in beta cells2018Inngår i: Diabetologia, ISSN 0012-186X, E-ISSN 1432-0428, Vol. 61, s. S196-S197Artikkel i tidsskrift (Annet vitenskapelig)
  • 50.
    Yu, Qian
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Gylfe, Erik
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Tengholm, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Quantitative assessment of glucose-regulated cAMP signalling and protein kinase A-mediated glucagon secretion.Manuskript (preprint) (Annet (populærvitenskap, debatt, mm))
12 1 - 50 of 59
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