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  • 1.
    Alenkvist, Ida
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Gandasi, Nikhil R
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Barg, Sebastian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Tengholm, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Recruitment of Epac2A to Insulin Granule Docking Sites Regulates Priming for Exocytosis2017In: Diabetes, ISSN 0012-1797, E-ISSN 1939-327X, Vol. 66, no 10, p. 2610-2622Article in journal (Refereed)
    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.

  • 2. Barg, S
    et al.
    Galvanovskis, J
    Göpel, S O
    Rorsman, P
    Eliasson, L
    Tight coupling between electrical activity and exocytosis in mouse glucagon-secreting alpha-cells.2000In: Diabetes, ISSN 0012-1797, E-ISSN 1939-327X, Vol. 49, no 9, p. 1500-10Article in journal (Refereed)
    Abstract [en]

    alpha-Cells were identified in preparations of dispersed mouse islets by immunofluorescence microscopy. A high fraction of alpha-cells correlated with a small cell size measured as the average cell diameter (10 microm) and whole-cell capacitance (<4 pF). The alpha-cells generated action potentials at a low frequency (1 Hz) in the absence of glucose. These action potentials were reversibly inhibited by elevation of the glucose concentration to 20 mmol/l. The action potentials originated from a membrane potential more negative than -50 mV, had a maximal upstroke velocity of 5 V/s, and peaked at +1 mV. Voltage-clamp experiments revealed the ionic conductances underlying the generation of action potentials. alpha-Cells are equipped with a delayed tetraethyl-ammonium-blockable outward current (activating at voltages above -20 mV), a large tetrodotoxin-sensitive Na+ current (above -30 mV; peak current 200 pA at +10 mV), and a small Ca2+ current (above -50 mV; peak current 30 pA at +10 mV). The latter flowed through omega-conotoxin GVIA (25%)- and nifedipine-sensitive (50%) Ca(2+)-channels. Mouse alpha-cells contained, on average, 7,300 granules, which undergo Ca(2+)-induced exocytosis when the alpha-cell is depolarized. Three functional subsets of granules were identified, and the size of the immediately releasable pool was estimated as 80 granules, or 1% of the total granule number. The maximal rate of exocytosis (1.5 pF/s) was observed 21 ms after the onset of the voltage-clamp depolarization, which is precisely the duration of Ca(2+)-influx during an action potential. Our results suggest that the secretory machinery of the alpha-cell is optimized for maximal efficiency in the use of Ca2+ for exocytosis.

  • 3. Barg, Sebastian
    Mechanisms of exocytosis in insulin-secreting B-cells and glucagon-secreting A-cells.2003In: Pharmacology and Toxicology, ISSN 0901-9928, E-ISSN 1600-0773, Vol. 92, no 1, p. 3-13Article in journal (Refereed)
    Abstract [en]

    In pancreatic B- and A-cells, metabolic stimuli regulate biochemical and electrical processes that culminate in Ca2+-influx and release of insulin or glucagon, respectively. Like in other (neuro)endocrine cells, Ca2+-influx triggers the rapid exocytosis of hormone-containing secretory granules. Only a small fraction of granules (<1% in insulin-secreting B-cells) can be released immediately, while the remainder requires translocation to the plasma membrane and further "priming" for release by several ATP- and Ca2+-dependent reactions. Such functional organization may account for systemic features such as the biphasic time course of glucose-stimulated insulin secretion. Since this release pattern is altered in type-2 diabetes mellitus, it is conceivable that disturbances in the exocytotic machinery underlie the disease. Here I will review recent data from our laboratory relevant for the understanding of these processes in insulin-secreting B-cells and glucagon-secreting A-cells and for the identification of novel targets for antidiabetic drug action. Two aspects are discussed in detail: 1) The importance of a tight interaction between L-type Ca2+-channels and the exocytotic machinery for efficient secretion; and 2) the role of intragranular acidification for the priming of secretory granules and its regulation by a granular 65-kDa sulfonylurea-binding protein.

  • 4.
    Barg, Sebastian
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Copello, J A
    Fleischer, S
    Different interactions of cardiac and skeletal muscle ryanodine receptors with FK-506 binding protein isoforms1997In: American Journal of Physiology, ISSN 0002-9513, E-ISSN 2163-5773, Vol. 272, no 5 Pt 1, p. C1726-C1733Article in journal (Refereed)
    Abstract [en]

    In the present study, we compare functional consequences of dissociation and reconstitution of binding proteins FKBP12 and FKBP12.6 with ryanodine receptors from cardiac (RyR2) and skeletal muscle (RyR1). The skeletal muscle RyR1 channel became activated on removal of endogenously bound FKBP12, consistent with previous reports. Both FKBP12 and FKBP12.6 rebind to FKBP-depleted RyR1 and restore its quiescent channel behavior by altering ligand sensitivity, as studied by single-channel recordings in planar lipid bilayers, and macroscopic behavior of the channels (ryanodine binding and net energized Ca2- uptake). By contrast, removal of FKBP12.6 from the cardiac RyR2 did not modulate the function of the channel using the same types of assays as for RyR1. FKBP12 or FKBP12.6 had no effect on channel activity of FKBP12.6-depleted cardiac RyR2, although FKBP12.6 rebinds. Our studies reveal important differences between the two ryanodine receptor isoforms with respect to their functional interaction with FKBP12 and FKBP12.6.

  • 5. Barg, Sebastian
    et al.
    Eliasson, Lena
    Renström, Erik
    Rorsman, Patrik
    A subset of 50 secretory granules in close contact with L-type Ca2+ channels accounts for first-phase insulin secretion in mouse beta-cells.2002In: Diabetes, ISSN 0012-1797, E-ISSN 1939-327X, Vol. 51 Suppl 1, p. S74-82Article in journal (Refereed)
    Abstract [en]

    Capacitance measurements were applied to mouse pancreatic beta-cells to elucidate the cellular mechanisms underlying biphasic insulin secretion. We report here that only <50 of the beta-cell's >10,000 granules are immediately available for release. The releasable granules tightly associate with the voltage-gated alpha(1C) Ca(2+) channels, and it is proposed that the release of these granules accounts for first-phase insulin secretion. Subsequent replenishment of the releasable pool by priming of previously nonreleasable granules is required for second-phase insulin secretion. The latter reaction depends on intragranular acidification due to the concerted action of granular bafilomycin-sensitive v-type H(+)-ATPase and 4,4-diisothiocyanostilbene-2,2-disulfonate--blockable ClC-3 Cl(-) channels. Lowering the cytoplasmic ATP/ADP ratio prevents granule acidification, granule priming, and refilling of the releasable pool. The latter finding provides an explanation to the transient nature of insulin secretion elicited by, for example, high extracellular K(+) in the absence of metabolizable fuels.

  • 6.
    Barg, Sebastian
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Gandasi, Nikhil
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Quantitative analysis of t-SNARE and Ca2+-channel clusters near secretory granules2010In: Diabetologia, ISSN 0012-186X, E-ISSN 1432-0428, Vol. 53, no Suppl. 1, p. S46-S46Article in journal (Other academic)
  • 7.
    Barg, Sebastian
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Gucek, Alenka
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    How Kiss-and-Run Can Make Us Sick: SOX4 Puts a Break on the Pore2016In: Diabetes, ISSN 0012-1797, E-ISSN 1939-327X, Vol. 65, no 7, p. 1791-1793Article in journal (Other academic)
  • 8.
    Barg, Sebastian
    et al.
    Department of Physiological Sciences, Lund University, Lund.
    Huang, Ping
    University of Chicago, Department of Neurobiology, Pharmacology and Physiology, Chicago.
    Eliasson, Lena
    Department of Physiological Sciences, Lund University, Lund.
    Nelson, Deborah J
    University of Chicago, Department of Neurobiology, Pharmacology and Physiology, Chicago.
    Obermüller, Stefanie
    Department of Physiological Sciences, Lund University, Lund.
    Rorsman, Patrik
    Department of Physiological Sciences, Lund University, Lund.
    Thévenod, Frank
    Physiologisches Institut, Universität des Saarlandes, Homburg/Saar.
    Renström, Erik
    Department of Physiological Sciences, Lund University, Lund.
    Priming of insulin granules for exocytosis by granular Cl(-) uptake and acidification2001In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 114, no Pt 11, p. 2145-54Article in journal (Refereed)
    Abstract [en]

    ATP-dependent priming of the secretory granules precedes Ca(2+)-regulated neuroendocrine secretion, but the exact nature of this reaction is not fully established in all secretory cell types. We have further investigated this reaction in the insulin-secreting pancreatic B-cell and demonstrate that granular acidification driven by a V-type H(+)-ATPase in the granular membrane is a decisive step in priming. This requires simultaneous Cl(-) uptake through granular ClC-3 Cl(-) channels. Accordingly, granule acidification and priming are inhibited by agents that prevent transgranular Cl(-) fluxes, such as 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and an antibody against the ClC-3 channels, but accelerated by increases in the intracellular ATP:ADP ratio or addition of hypoglycemic sulfonylureas. We suggest that this might represent an important mechanism for metabolic regulation of Ca(2+)-dependent exocytosis that is also likely to be operational in other secretory cell types.

  • 9.
    Barg, Sebastian
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Knowles, M. K.
    Chen, X.
    Midorikawa, M.
    Almers, Wolfhard
    Syntaxin clusters assemble reversibly at sites of secretory granules in live cells2010In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 107, no 48, p. 20804-20809Article in journal (Refereed)
    Abstract [en]

    Syntaxin resides in the plasma membrane, where it helps to catalyze membrane fusion during exocytosis. The protein also forms clusters in cell-free and granule-free plasma-membrane sheets. We imaged the interaction between syntaxin and single secretory granules by two-color total internal reflection microscopy in PC12 cells. Syntaxin-GFP assembled in clusters at sites where single granules had docked at the plasma membrane. Clusters were intermittently present at granule sites, as syntaxin molecules assembled and disassembled in a coordinated fashion. Recruitment to granules required the N-terminal domain of syntaxin, but not the entry of syntaxin into SNARE complexes. Clusters facilitated exocytosis and disassembled once exocytosis was complete. Syntaxin cluster formation defines an intermediate step in exocytosis.

  • 10. Barg, Sebastian
    et al.
    Lindqvist, Anders
    Obermüller, Stefanie
    Granule docking and cargo release in pancreatic β-cells2008In: Biochemical Society Transactions, ISSN 0300-5127, E-ISSN 1470-8752, Vol. 36, no Pt 3, p. 294-299Article in journal (Refereed)
    Abstract [en]

    Biphasic insulin secretion is required for proper insulin action and is observed not only in vivo, but also in isolated pancreatic islets and even single beta-cells. Late events in the granule life cycle are thought to underlie this temporal pattern. In the last few years, we have therefore combined live cell imaging and electrophysiology to study insulin secretion at the level of individual granules, as they approach the plasma membrane, undergo exocytosis and finally release their insulin cargo. In the present paper, we review evidence for two emerging concepts that affect insulin secretion at the level of individual granules: (i) the existence of specialized sites where granules dock in preparation for exocytosis; and (ii) post-exocytotic regulation of cargo release by the fusion pore.

  • 11.
    Barg, Sebastian
    et al.
    Department of Molecular and Cellular Physiology, Institute of Physiological Sciences, Lund University, Lund.
    Ma, Xiaosong
    Department of Molecular and Cellular Physiology, Institute of Physiological Sciences, Lund University, Lund.
    Eliasson, Lena
    Department of Molecular and Cellular Physiology, Institute of Physiological Sciences, Lund University, Lund.
    Galvanovskis, Juris
    Department of Molecular and Cellular Physiology, Institute of Physiological Sciences, Lund University, Lund.
    Göpel, Sven O
    Department of Molecular and Cellular Physiology, Institute of Physiological Sciences, Lund University, Lund.
    Obermüller, Stefanie
    Department of Molecular and Cellular Physiology, Institute of Physiological Sciences, Lund University, Lund.
    Platzer, Josef
    Institut für Biochemische Pharmakologie, Innsbruck.
    Renström, Erik
    Department of Molecular and Cellular Physiology, Institute of Physiological Sciences, Lund University, Lund.
    Trus, Michel
    Department of Biological Chemistry, The Hebrew University of Jerusalem.
    Atlas, Daphne
    Department of Biological Chemistry, The Hebrew University of Jerusalem.
    Striessnig, Jörg
    Institut für Biochemische Pharmakologie, Innsbruck.
    Rorsman, Patrik
    Department of Molecular and Cellular Physiology, Institute of Physiological Sciences, Lund University, Lund.
    Fast exocytosis with few Ca(2+) channels in insulin-secreting mouse pancreatic B cells2001In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 81, no 6, p. 3308-23Article in journal (Refereed)
    Abstract [en]

    The association of L-type Ca(2+) channels to the secretory granules and its functional significance to secretion was investigated in mouse pancreatic B cells. Nonstationary fluctuation analysis showed that the B cell is equipped with <500 alpha1(C) L-type Ca(2+) channels, corresponding to a Ca(2+) channel density of 0.9 channels per microm(2). Analysis of the kinetics of exocytosis during voltage-clamp depolarizations revealed an early component that reached a peak rate of 1.1 pFs(-1) (approximately 650 granules/s) 25 ms after onset of the pulse and is completed within approximately 100 ms. This component represents a subset of approximately 60 granules situated in the immediate vicinity of the L-type Ca(2+) channels, corresponding to approximately 10% of the readily releasable pool of granules. Experiments involving photorelease of caged Ca(2+) revealed that the rate of exocytosis was half-maximal at a cytoplasmic Ca(2+) concentration of 17 microM, and concentrations >25 microM are required to attain the rate of exocytosis observed during voltage-clamp depolarizations. The rapid component of exocytosis was not affected by inclusion of millimolar concentrations of the Ca(2+) buffer EGTA but abolished by addition of exogenous L(C753-893), the 140 amino acids of the cytoplasmic loop connecting the 2(nd) and 3(rd) transmembrane region of the alpha1(C) L-type Ca(2+) channel, which has been proposed to tether the Ca(2+) channels to the secretory granules. In keeping with the idea that secretion is determined by Ca(2+) influx through individual Ca(2+) channels, exocytosis triggered by brief (15 ms) depolarizations was enhanced 2.5-fold by the Ca(2+) channel agonist BayK8644 and 3.5-fold by elevating extracellular Ca(2+) from 2.6 to 10 mM. Recordings of single Ca(2+) channel activity revealed that patches predominantly contained no channels or many active channels. We propose that several Ca(2+) channels associate with a single granule thus forming a functional unit. This arrangement is important in a cell with few Ca(2+) channels as it ensures maximum usage of the Ca(2+) entering the cell while minimizing the influence of stochastic variations of the Ca(2+) channel activity.

  • 12.
    Barg, Sebastian
    et al.
    Department of Cell Biology, Division of Medicine, Imperial College, London, UK.
    Machado, Jose David
    Compensatory endocytosis in chromaffin cells2008In: Acta Physiologica, ISSN 1748-1708, E-ISSN 1748-1716, Vol. 192, no 2, p. 195-201Article, review/survey (Refereed)
    Abstract [en]

    Exocytosis occurs via fusion of secretory granules with the cell membrane, whereupon the granule content is at least partially released and the granule membrane is temporarily added to the plasma membrane. Exocytosis is balanced by compensatory endocytosis to achieve net equilibrium of the cell surface area and to recycle and redistribute components of the exocytosis machinery. The underlying molecular mechanisms remain a matter of debate. In this review, we summarize and discuss recent progress in the understanding of compensatory endocytosis, with the focus on chromaffin cells as a useful model for studying mechanisms of regulated secretion.

  • 13.
    Barg, Sebastian
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Olofsson, Charlotta S
    Schriever-Abeln, Jenny
    Wendt, Anna
    Gebre-Medhin, Samuel
    Renström, Erik
    Rorsman, Patrik
    Delay between fusion pore opening and peptide release from large dense-core vesicles in neuroendocrine cells2002In: Neuron, ISSN 0896-6273, E-ISSN 1097-4199, Vol. 33, no 2, p. 287-299Article in journal (Refereed)
    Abstract [en]

    Peptidergic neurotransmission is slow compared to that mediated by classical neurotransmitters. We have studied exocytotic membrane fusion and cargo release by simultaneous capacitance measurements and confocal imaging of single secretory vesicles in neuroendocrine cells. Depletion of the readily releasable pool (RRP) correlated with exocytosis of 10%-20% of the docked vesicles. Some remaining vesicles became releasable after recovery of RRP. Expansion of the fusion pore, seen as an increase in luminal pH, occurred after approximately 0.3 s, and peptide release was delayed by another 1-10 s. We conclude that (1) RRP refilling involves chemical modification of vesicles already in place, (2) the release of large neuropeptides via the fusion pore is negligible and only proceeds after complete fusion, and (3) sluggish peptidergic transmission reflects the time course of vesicle emptying.

  • 14.
    Barg, Sebastian
    et al.
    Department of Physiological Sciences, Lund University, Lund.
    Renström, Erik
    Department of Physiological Sciences, Lund University, Lund.
    Berggren, Per-Olof
    The Rolf Luft Center for Diabetes Research, Department of Molecular Medicine, Karolinska Institute, Stockholm.
    Bertorello, Alejandro
    The Rolf Luft Center for Diabetes Research, Department of Molecular Medicine, Karolinska Institute, Stockholm.
    Bokvist, Krister
    Novo Nordisk AyS, Bagsvaerd.
    Braun, Matthias
    Physiologisches Institut, Universität des Saarlandes, HomburgySaar.
    Eliasson, Lena
    Department of Physiological Sciences, Lund University, Lund.
    Holmes, William E
    Novo Nordisk AyS, Bagsvaerd.
    Köhler, Martin
    The Rolf Luft Center for Diabetes Research, Department of Molecular Medicine, Karolinska Institute, Stockholm.
    Rorsman, Patrik
    Department of Physiological Sciences, Lund University, Lund.
    Thévenod, Frank
    Physiologisches Institut, Universität des Saarlandes, HomburgySaar.
    The stimulatory action of tolbutamide on Ca2+-dependent exocytosis in pancreatic beta cells is mediated by a 65-kDa mdr-like P-glycoprotein1999In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 96, no 10, p. 5539-44Article in journal (Refereed)
    Abstract [en]

    Intracellular application of the sulfonylurea tolbutamide during whole-cell patch-clamp recordings stimulated exocytosis >5-fold when applied at a cytoplasmic Ca2+ concentration of 0.17 microM. This effect was not detectable in the complete absence of cytoplasmic Ca2+ and when exocytosis was elicited by guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). The stimulatory action could be antagonized by the sulfonamide diazoxide, by the Cl--channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), by intracellular application of the antibody JSB1 [originally raised against a 170-kDa multidrug resistance (mdr) protein], and by tamoxifen (an inhibitor of the mdr- and volume-regulated Cl- channels). Immunocytochemistry and Western blot analyses revealed that JSB1 recognizes a 65-kDa protein in the secretory granules. This protein exhibited no detectable binding of sulfonylureas and is distinct from the 140-kDa sulfonylurea high-affinity sulfonylurea receptors also present in the granules. We conclude that (i) tolbutamide stimulates Ca2+-dependent exocytosis secondary to its binding to a 140-kDa high-affinity sulfonylurea receptor in the secretory granules; and (ii) a granular 65-kDa mdr-like protein mediates the action. The processes thus initiated culminate in the activation of a granular Cl- conductance. We speculate that the activation of granular Cl- fluxes promotes exocytosis (possibly by providing the energy required for membrane fusion) by inducing water uptake and an increased intragranular hydrostatic pressure.

  • 15. Barg, Sebastian
    et al.
    Rorsman, Patrik
    Insulin secretion: a high-affinity Ca2+ sensor after all?2004In: The Journal of General Physiology, ISSN 0022-1295, E-ISSN 1540-7748, Vol. 124, no 6, p. 623-5Article in journal (Refereed)
  • 16. Cervin, C
    et al.
    Orho-Melander, M
    Ridderstråle, M
    Lehto, M
    Barg, S
    Groop, L
    Cilio, C M
    Characterization of a naturally occurring mutation (L107I) in the HNF1 alpha (MODY3) gene.2002In: Diabetologia, ISSN 0012-186X, E-ISSN 1432-0428, Vol. 45, no 12, p. 1703-8Article in journal (Refereed)
    Abstract [en]

    AIMS/HYPOTHESIS: Maturity onset diabetes of the young type 3 (MODY3) is a monogenic form of diabetes mellitus caused by mutations in the gene encoding for hepatocyte nuclear factor 1 alpha, HNF1 alpha. In this study we have examined the in vivo and in vitro effects of a mutation (L107I) outside the DNA binding and dimerization domains in the N terminal part of the HNF1 alpha gene.

    METHODS: Beta-cell function of the affected family members was assessed by an oral glucose tolerance test. Functional tests were carried out to explain the role of the mutation in vitro by transcriptional activity assay, Western blotting, DNA-binding assays and subcellular localization experiments.

    RESULTS: Affected family members showed an 86% decreased insulin response to glucose when compared to age-matched healthy control subjects. In vitro the mutation showed a 79% decrease in transcriptional activity as compared to wild type HNF1 alpha in HeLa cells lacking HNF1 alpha. The transcriptional activity was not suppressed when the mutant was co-expressed with wild type HNF1 alpha suggesting that the decreased activity was not mediated by a dominant negative mechanism. The L107I/HNF1alpha protein showed normal nuclear targeting but impaired binding to an HNF1 alpha consensus sequence.

    CONCLUSION/INTERPRETATION: Our results suggest that the L107I substitution represents a MODY3 mutation which impairs beta-cell function by a loss-of-function mechanism.

  • 17.
    Chen, Xi
    et al.
    Vollum Institute, Oregon Health & Sciences University, Portland, Oregon.
    Barg, Sebastian
    Department of Cell Biology, Division of Medicine, Sir Alexander Fleming Building, Imperial College London, London.
    Almers, Wolfhard
    Vollum Institute, Oregon Health & Sciences University, Portland, Oregon.
    Release of the styryl dyes from single synaptic vesicles in hippocampal neurons.2008In: The Journal of neuroscience : the official journal of the Society for Neuroscience, ISSN 1529-2401, Vol. 28, no 8, p. 1894-1903Article in journal (Refereed)
    Abstract [en]

    In small presynaptic boutons in brain, synaptic vesicles are thought not to merge with the plasma membrane when they release transmitter, but instead to close their fusion pores and survive intact for future use (kiss-and-run exocytosis). The strongest evidence for this idea is the slow and incomplete release of the fluorescent membrane marker, FM1-43 [N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide], from single vesicles. We investigated the release of FM1-43 from sparse cultures of hippocampal neurons grown on coverslips with no glia. This allowed presynaptic boutons to be imaged at favorable signal-to-noise ratio. Sparingly stained boutons were imaged at high time resolution, while high-frequency electrical stimulation caused exocytosis. The release of FM1-43 was quantal and occurred in abrupt steps, each representing a single fusion event. The fluorescence of vesicle clusters traveling along axons had a distribution with the same quantal size, indicating that a vesicle releases all the dye it contains. In most fusion events, the time constant of dye release was <100 ms, and slower release was rarely observed. After exocytosis, no FM1-43 could be detected in the axon to either side of a bouton, indicating that dye was released before it could spread. Our results are consistent with synaptic vesicles fusing fully with the plasma membrane during high-frequency stimulation.

  • 18. Copello, J A
    et al.
    Barg, S
    Onoue, H
    Fleischer, S
    Heterogeneity of Ca2+ gating of skeletal muscle and cardiac ryanodine receptors.1997In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 73, no 1, p. 141-56Article in journal (Refereed)
    Abstract [en]

    The single-channel activity of rabbit skeletal muscle ryanodine receptor (skeletal RyR) and dog cardiac RyR was studied as a function of cytosolic [Ca2+]. The studies reveal that for both skeletal and cardiac RyRs, heterogeneous populations of channels exist, rather than a uniform behavior. Skeletal muscle RyRs displayed two extremes of behavior: 1) low-activity RyRs (LA skeletal RyRs, approximately 35% of the channels) had very low open probability (Po < 0.1) at all [Ca2+] and remained closed in the presence of Mg2+ (2 mM) and ATP (1 mM); 2) high-activity RyRs (HA skeletal RyRs) had much higher activity and displayed further heterogeneity in their Po values at low [Ca2+] (< 50 nM), and in their patterns of activation by [Ca2+]. Hill coefficients for activation (nHa) varied from 0.8 to 5.2. Cardiac RyRs, in comparison, behaved more homogeneously. Most cardiac RyRs were closed at 100 nM [Ca2+] and activated in a cooperative manner (nHa ranged from 1.6 to 5.0), reaching a high Po (> 0.6) in the presence and absence of Mg2+ and ATP. Heart RyRs were much less sensitive (10x) to inhibition by [Ca2+] than skeletal RyRs. The differential heterogeneity of heart versus skeletal muscle RyRs may reflect the modulation required for calcium-induced calcium release versus depolarization-induced Ca2+ release.

  • 19. Copello, J A
    et al.
    Barg, S
    Sonnleitner, A
    Porta, M
    Diaz-Sylvester, P
    Fill, M
    Schindler, H
    Fleischer, S
    Differential activation by Ca2+, ATP and caffeine of cardiac and skeletal muscle ryanodine receptors after block by Mg2+.2002In: Journal of Membrane Biology, ISSN 0022-2631, E-ISSN 1432-1424, Vol. 187, no 1, p. 51-64Article in journal (Refereed)
    Abstract [en]

    The block of rabbit skeletal ryanodine receptors (RyR1) and dog heart RyR2 by cytosolic [Mg2+], and its reversal by agonists Ca2+, ATP and caffeine was studied in planar bilayers. Mg2+ effects were tested at submaximal activating [Ca2+] (5 microM). Approximately one third of the RyR1s had low open probability ("LA channels") in the absence of Mg2+. All other RyR1s displayed higher activity ("HA channels"). Cytosolic Mg2+ (1 mM) blocked individual RyR1 channels to varying degrees (32 to 100%). LA channels had residual P(o) <0.005 in 1 mM Mg2+ and reactivated poorly with [Ca2+] (100 microM), caffeine (5 mM), or ATP (4 mM; all at constant 1 mM Mg2+). HA channels had variable activity in Mg2+ and variable degree of recovery from Mg2+ block with Ca2+, caffeine or ATP application. Nearly all cardiac RyR2s displayed high activity in 5 microM [Ca2+]. They also had variable sensitivity to Mg2+. However, the RyR2s consistently recovered from Mg2+ block with 100 microM [Ca2+] or caffeine application, but not when ATP was added. Thus, at physiological [Mg2+], RyR2s behaved as relatively homogeneous Ca2+/caffeine-gated HA channels. In contrast, RyR1s displayed functional heterogeneity that arises from differential modulatory actions of Ca2+ and ATP. These differences between RyR1 and RyR2 function may reflect their respective roles in muscle physiology and excitation-contraction coupling.

  • 20.
    Cortese, Giuliana
    et al.
    Univ Padua, Dept Stat Sci, Padua, Italy.
    Gandasi, Nikhil R.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Barg, Sebastian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Pedersen, Morten Gram
    Univ Padua, Dept Informat Engn, Padua, Italy.
    Statistical Frailty Modeling for Quantitative Analysis of Exocytotic Events Recorded by Live Cell Imaging: Rapid Release of Insulin-Containing Granules Is Impaired in Human Diabetic beta-cells2016In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 12, article id e0167282Article in journal (Refereed)
    Abstract [en]

    Hormones and neurotransmitters are released when secretory granules or synaptic vesicles fuse with the cell membrane, a process denoted exocytosis. Modern imaging techniques, in particular total internal reflection fluorescence (TIRF) microscopy, allow the investigator to monitor secretory granules at the plasma membrane before and when they undergo exocytosis. However, rigorous statistical approaches for temporal analysis of such exocytosis data are still lacking. We propose here that statistical methods from time-to-event (also known as survival) analysis are well suited for the problem. These methods are typically used in clinical settings when individuals are followed over time to the occurrence of an event such as death, remission or conception. We model the rate of exocytosis in response to pulses of stimuli in insulin-secreting pancreatic beta-cell from healthy and diabetic human donors using piecewise-constant hazard modeling. To study heterogeneity in the granule population we exploit frailty modeling, which describe unobserved differences in the propensity to exocytosis. In particular, we insert a discrete frailty in our statistical model to account for the higher rate of exocytosis in an immediately releasable pool (IRP) of insulin-containing granules. Estimates of parameters are obtained from maximum-likelihood methods. Since granules within the same cell are correlated, i.e., the data are clustered, a modified likelihood function is used for log-likelihood ratio tests in order to perform valid inference. Our approach allows us for example to estimate the size of the IRP in the cells, and we find that the IRP is deficient in diabetic cells. This novel application of time-to-event analysis and frailty modeling should be useful also for the study of other well-defined temporal events at the cellular level.

  • 21. da Silva Xavier, Gabriela
    et al.
    Loder, Merewyn K
    McDonald, Angela
    Tarasov, Andrei I
    Carzaniga, Raffaella
    Kronenberger, Katrin
    Barg, Sebastian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Rutter, Guy A
    TCF7L2 regulates late events in insulin secretion from pancreatic islet beta-cells2009In: Diabetes, ISSN 0012-1797, E-ISSN 1939-327X, Vol. 58, no 4, p. 894-905Article in journal (Refereed)
    Abstract [en]

    OBJECTIVE:

    Polymorphisms in the human TCF7L2 gene are associated with reduced insulin secretion and an increased risk of type 2 diabetes. However, the mechanisms by which TCF7L2 affect insulin secretion are still unclear. We define the effects of TCF7L2 expression level on mature beta-cell function and suggest a potential mechanism for its actions.

    RESEARCH DESIGN AND METHODS:

    TCF7L2 expression in rodent islets and beta-cell lines was altered using RNAi or adenoviral transduction. Beta-cell gene profiles were measured by quantitative real-time PCR and the effects on intracellular signaling and exocytosis by live cell imaging, electron microscopy, and patch clamp electrophysiology.

    RESULTS:

    Reducing TCF7L2 expression levels by RNAi decreased glucose- but not KCl-induced insulin secretion. The glucose-induced increments in both ATP/ADP ratio and cytosolic free Ca2+ concentration ([Ca2+]i) were increased compared with controls. Overexpression of TCF7L2 exerted minor inhibitory effects on glucose-regulated changes in [Ca2+]i and insulin release. Gene expression profiling in TCF7L2-silenced cells revealed increased levels of mRNA encoding syntaxin 1A but decreased Munc18–1 and ZnT8 mRNA. Whereas the number of morphologically docked vesicles was unchanged by TCF7L2 suppression, secretory granule movement increased and capacitance changes decreased, indicative of defective vesicle fusion.

    CONCLUSION:

    TCF7L2 is involved in maintaining expression of beta-cell genes regulating secretory granule fusion. Defective insulin exocytosis may thus underlie increased diabetes incidence in carriers of the at-risk TCF7L2 alleles.

  • 22. Eliasson, Lena
    et al.
    Ma, Xiaosong
    Renström, Erik
    Barg, Sebastian
    Berggren, Per-Olof
    Galvanovskis, Juris
    Gromada, Jesper
    Jing, Xingjun
    Lundquist, Ingmar
    Salehi, Albert
    Sewing, Sabine
    Rorsman, Patrik
    SUR1 regulates PKA-independent cAMP-induced granule priming in mouse pancreatic B-cells.2003In: The Journal of General Physiology, ISSN 0022-1295, E-ISSN 1540-7748, Vol. 121, no 3, p. 181-97Article in journal (Refereed)
    Abstract [en]

    Measurements of membrane capacitance were applied to dissect the cellular mechanisms underlying PKA-dependent and -independent stimulation of insulin secretion by cyclic AMP. Whereas the PKA-independent (Rp-cAMPS-insensitive) component correlated with a rapid increase in membrane capacitance of approximately 80 fF that plateaued within approximately 200 ms, the PKA-dependent component became prominent during depolarizations >450 ms. The PKA-dependent and -independent components of cAMP-stimulated exocytosis differed with regard to cAMP concentration dependence; the K(d) values were 6 and 29 micro M for the PKA-dependent and -independent mechanisms, respectively. The ability of cAMP to elicit exocytosis independently of PKA activation was mimicked by the selective cAMP-GEFII agonist 8CPT-2Me-cAMP. Moreover, treatment of B-cells with antisense oligodeoxynucleotides against cAMP-GEFII resulted in partial (50%) suppression of PKA-independent exocytosis. Surprisingly, B-cells in islets isolated from SUR1-deficient mice (SUR1(-/-) mice) lacked the PKA-independent component of exocytosis. Measurements of insulin release in response to GLP-1 stimulation in isolated islets from SUR1(-/-) mice confirmed the complete loss of the PKA-independent component. This was not attributable to a reduced capacity of GLP-1 to elevate intracellular cAMP but instead associated with the inability of cAMP to stimulate influx of Cl(-) into the granules, a step important for granule priming. We conclude that the role of SUR1 in the B cell extends beyond being a subunit of the plasma membrane K(ATP)-channel and that it also plays an unexpected but important role in the cAMP-dependent regulation of Ca(2+)-induced exocytosis.

  • 23.
    Gandasi, Nikhil R
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Barg, Sebastian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Contact-induced clustering of syntaxin and munc18 docks secretory granules at the exocytosis site2014In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 5, article id 3914Article in journal (Refereed)
    Abstract [en]

    Docking of secretory vesicles at the plasma membrane is a poorly understood prerequisite for exocytosis. Current models propose raft-like clusters containing syntaxin as docking receptor, but direct evidence for this is lacking. Here we provide quantitative measurements of several exocytosis proteins (syntaxin, SNAP25, munc18, munc13 and rab3) at the insulin granule release site and show that docking coincides with rapid de novo formation of syntaxin1/munc18 clusters at the nascent docking site. Formation of such clusters prevents undocking and is not observed during failed docking attempts. Overexpression of syntaxins' N-terminal Habc-domain competitively interferes with both cluster formation and successful docking. SNAP25 and munc13 are recruited to the docking site more than a minute later, consistent with munc13's reported role in granule priming rather than docking. We conclude that secretory vesicles dock by inducing syntaxin1/munc18 clustering in the target membrane, and find no evidence for preformed docking receptors.

  • 24.
    Gandasi, Nikhil R.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Barg, Sebastian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Quantitative Imaging of the Exocytosis Machinery Assembly2015In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 108, no 2, p. 102A-102AArticle in journal (Other academic)
  • 25.
    Gandasi, Nikhil R.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Vesto, Kim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Helou, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Yin, Peng
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Saras, Jan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Barg, Sebastian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Survey of Red Fluorescence Proteins as Markers for Secretory Granule Exocytosis2015In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 6, article id e0127801Article in journal (Refereed)
    Abstract [en]

    Fluorescent proteins (FPs) have proven to be valuable tools for high-resolution imaging studies of vesicle transport processes, including exo- and endocytosis. Since the pH of the vesicle lumen changes between acidic and neutral during these events, pH-sensitive FPs with near neutral pKa, such as pHluorin, are particularly useful. FPs with pKa>6 are readily available in the green spectrum, while red-emitting pH-sensitive FPs are rare and often not well characterized as reporters of exo- or endocytosis. Here we tested a panel of ten orange/red and two green FPs in fusions with neuropeptide Y (NPY) for use as secreted vesicle marker and reporter of dense core granule exocytosis and release. We report relative brightness, bleaching rate, targeting accuracy, sensitivity to vesicle pH, and their performance in detecting exocytosis in live cells. Tandem dimer (td)-mOrange2 was identified as well-targeted, bright, slowly bleaching and pH-sensitive FP that performed similar to EGFP. Single exocytosis events were readily observed, which allowed measurements of fusion pore lifetime and the dynamics of the exocytosis protein syntaxin at the release site during membrane fusion and cargo release.

  • 26.
    Gandasi, Nikhil R
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Yin, Peng
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Omar-Hmeadi, Muhmmad
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Ottosson Laakso, Emilia
    Lund University Diabetes Centre, Malmö, Sweden.
    Vikman, Petter
    Lund University Diabetes Centre, Malmö, Sweden.
    Barg, Sebastian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Glucose-Dependent Granule Docking Limits Insulin Secretion and Is Decreased in Human Type 2 Diabetes2018In: Cell Metabolism, ISSN 1550-4131, E-ISSN 1932-7420, Vol. 27, no 2, p. 470-478Article in journal (Refereed)
    Abstract [en]

    Glucose-stimulated insulin secretion is biphasic, with a rapid first phase and a slowly developing sustained second phase; both are disturbed in type 2 diabetes (T2D). Biphasic secretion results from vastly different release probabilities of individual insulin granules, but the morphological and molecular basis for this is unclear. Here, we show that human insulin secretion and exocytosis critically depend on the availability of membrane-docked granules and that T2D is associated with a strong reduction in granule docking. Glucose accelerated granule docking, and this effect was absent in T2D. Newly docked granules only slowly acquired release competence; this was regulated by major signaling pathways, but not glucose. Gene expression analysis indicated that key proteins involved in granule docking are downregulated in T2D, and overexpression of these proteins increased granule docking. The findings establish granule docking as an important glucose-dependent step in human insulin secretion that is dysregulated in T2D.

  • 27.
    Gandasi, Nikhil R.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Yin, Peng
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Riz, Michela
    Univ Padua, Dept Informat Engn, Padua, Italy..
    Chibalina, Margarita V
    Univ Oxford, Oxford Ctr Diabet Endocrinol & Metab, Oxford, England..
    Cortese, Giuliana
    Univ Padua, Dept Stat Sci, Padua, Italy..
    Lund, Per-Eric
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Matveev, Victor
    New Jersey Inst Technol, Dept Math Sci, Newark, NJ 07102 USA..
    Rorsman, Patrik
    Univ Oxford, Oxford Ctr Diabet Endocrinol & Metab, Oxford, England..
    Sherman, Arthur
    NIDDK, Lab Biol Modeling, NIH, Bethesda, MD 20892 USA..
    Pedersen, Morten G
    Univ Padua, Dept Informat Engn, Padua, Italy..
    Barg, Sebastian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Ca2+ channel clustering with insulin-containing granules is disturbed in type 2 diabetes2017In: Journal of Clinical Investigation, ISSN 0021-9738, E-ISSN 1558-8238, Vol. 127, no 6, p. 2353-2364Article in journal (Refereed)
    Abstract [en]

    Loss of first-phase insulin secretion is an early sign of developing type 2 diabetes (T2D). Ca2+ entry through voltage-gated L-type Ca2+ channels triggers exocytosis of insulin-containing granules in pancreatic β cells and is required for the postprandial spike in insulin secretion. Using high-resolution microscopy, we have identified a subset of docked insulin granules in human β cells and rat-derived clonal insulin 1 (INS1) cells for which localized Ca2+ influx triggers exocytosis with high probability and minimal latency. This immediately releasable pool (IRP) of granules, identified both structurally and functionally, was absent in β cells from human T2D donors and in INS1 cells cultured in fatty acids that mimic the diabetic state. Upon arrival at the plasma membrane, IRP granules slowly associated with 15 to 20 L-type channels. We determined that recruitment depended on a direct interaction with the synaptic protein Munc13, because expression of the II-III loop of the channel, the C2 domain of Munc13-1, or of Munc13-1 with a mutated C2 domain all disrupted L-type channel clustering at granules and ablated fast exocytosis. Thus, rapid insulin secretion requires Munc13-mediated recruitment of L-type Ca2+ channels in close proximity to insulin granules. Loss of this organization underlies disturbed insulin secretion kinetics in T2D.

  • 28. Gromada, J
    et al.
    Bokvist, K
    Ding, W G
    Barg, S
    Buschard, K
    Renström, E
    Rorsman, P
    Adrenaline stimulates glucagon secretion in pancreatic A-cells by increasing the Ca2+ current and the number of granules close to the L-type Ca2+ channels.1997In: The Journal of General Physiology, ISSN 0022-1295, E-ISSN 1540-7748, Vol. 110, no 3, p. 217-28Article in journal (Refereed)
    Abstract [en]

    We have monitored electrical activity, voltage-gated Ca2+ currents, and exocytosis in single rat glucagon-secreting pancreatic A-cells. The A-cells were electrically excitable and generated spontaneous Na+- and Ca2+-dependent action potentials. Under basal conditions, exocytosis was tightly linked to Ca2+ influx through omega-conotoxin-GVIA-sensitive (N-type) Ca2+ channels. Stimulation of the A-cells with adrenaline (via beta-adrenergic receptors) or forskolin produced a greater than fourfold PKA-dependent potentiation of depolarization-evoked exocytosis. This enhancement of exocytosis was due to a 50% enhancement of Ca2+ influx through L-type Ca2+ channels, an effect that accounted for <30% of the total stimulatory action. The remaining 70% of the stimulation was attributable to an acceleration of granule mobilization resulting in a fivefold increase in the number of readily releasable granules near the L-type Ca2+ channels.

  • 29. Gromada, J
    et al.
    Ding, W G
    Barg, S
    Renström, E
    Rorsman, P
    Multisite regulation of insulin secretion by cAMP-increasing agonists: evidence that glucagon-like peptide 1 and glucagon act via distinct receptors.1997In: Pflügers Archiv: European Journal of Physiology, ISSN 0031-6768, E-ISSN 1432-2013, Vol. 434, no 5, p. 515-24Article in journal (Refereed)
    Abstract [en]

    The mechanisms by which glucagon-like peptide 1(7-36)amide (GLP-1[7-36]amide) potentiates insulin secretion were investigated by measurements of whole-cell K+ and Ca2+ currents, membrane potential, the cytoplasmic Ca2+ concentration ([Ca2+]i) and exocytosis in mouse pancreatic B-cells. GLP-1(7-36)amide (10 nM) stimulated glucose-induced (10 mM) electrical activity in intact pancreatic islets. The effect was manifested as a 34% increase in the duration of the bursts of action potentials and a corresponding 28% shortening of the silent intervals. GLP-1(7-36)amide had no effect on the electrical activity at subthreshold glucose concentrations (< or = 6.5 mM). In cultured B-cells, GLP-1(7-36)amide produced a decrease of the whole-cell ATP-sensitive K+ (KATP) conductance remaining at 5 mM glucose by approximately 30%. This effect was associated with membrane depolarization and the initiation of electrical activity. GLP-1(7-36)amide produced a protein-kinase-A-(PKA-) and glucose-dependent fourfold potentiation of Ca(2+)-induced exocytosis whilst only increasing the Ca2+ current marginally. The stimulatory action of GLP-1(7-36)amide on exocytosis was mimicked by the pancreatic hormone glucagon and exendin-4, a GLP-1 receptor agonist. Whereas the stimulatory action of GLP-1(7-36)amide could be antagonized by exendin-(9-39), this peptide did not interfere with the ability of glucagon to stimulate exocytosis. We suggest that GLP-1(7-36)amide and glucagon stimulate insulin secretion by binding to distinct receptors. The GLP-1(7-36)amide-induced stimulation of electrical activity and Ca2+ influx can account for (maximally) a doubling of insulin secretion. The remainder of its stimulatory action results from a cAMP/PKA-dependent potentiation of Ca(2+)-dependent exocytosis exerted at a stage distal to the elevation of [Ca2+]i.

  • 30.
    Gucek, Alenka
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Gandasi, Nikhil R
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Omar-Hmeadi, Muhmmad
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Bakke, Marit
    Univ Bergen, Dept Biomed, Bergen, Norway.
    Doskeland, Stein O.
    Univ Bergen, Dept Biomed, Bergen, Norway.
    Tengholm, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Barg, Sebastian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Fusion pore regulation by cAMP/Epac2 controls cargo release during insulin exocytosis2019In: eLIFE, E-ISSN 2050-084X, Vol. 8, article id e41711Article in journal (Refereed)
    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.

  • 31. Göpel, S
    et al.
    Kanno, T
    Barg, S
    Galvanovskis, J
    Rorsman, P
    Voltage-gated and resting membrane currents recorded from B-cells in intact mouse pancreatic islets.1999In: Journal of Physiology, ISSN 0022-3751, E-ISSN 1469-7793, Vol. 521 Pt 3, p. 717-28Article in journal (Refereed)
    Abstract [en]

    1. The perforated patch whole-cell configuration of the patch-clamp technique was applied to superficial cells in intact pancreatic islets. Immunostaining in combination with confocal microscopy revealed that the superficial cells consisted of 35 % insulin-secreting B-cells and 65 % non-B-cells (A- and D-cells). 2. Two types of cell, with distinct electrophysiological properties, could be functionally identified. One of these generated oscillatory electrical activity when the islet was exposed to 10 mM glucose and had the electrophysiological characteristics of isolated B-cells maintained in tissue culture. 3. The Ca2+ current recorded from B-cells in situ was 80 % larger than that of isolated B-cells. It exhibited significant (70 %) inactivation during 100 ms depolarisations. The inactivation was voltage dependent and particularly prominent during depolarisations evoking the largest Ca2+ currents. 4. Voltage-dependent K+ currents were observed during depolarisations to membrane potentials above -20 mV. These currents inactivated little during a 200 ms depolarisation and were unaffected by varying the holding potential between -90 and -30 mV. 5. The maximum resting conductance in the absence of glucose, which reflects the conductance of ATP-regulated K+ (KATP) channels, amounted to approximately 4 nS. Glucose produced a concentration-dependent reduction of KATP channel conductance with half-maximal inhibition observed with 5 mM glucose. 6. Combining voltage- and current-clamp recording allowed the estimation of the gap junction conductance between different B-cells. These experiments indicated that the input conductance of the B-cell at stimulatory glucose concentrations ( approximately 1 nS) is almost entirely accounted for by coupling to neighbouring B-cells.

  • 32. Göpel, S O
    et al.
    Kanno, T
    Barg, S
    Eliasson, L
    Galvanovskis, J
    Renström, E
    Rorsman, P
    Activation of Ca(2+)-dependent K(+) channels contributes to rhythmic firing of action potentials in mouse pancreatic beta cells.1999In: The Journal of General Physiology, ISSN 0022-1295, E-ISSN 1540-7748, Vol. 114, no 6, p. 759-70Article in journal (Refereed)
    Abstract [en]

    We have applied the perforated patch whole-cell technique to beta cells within intact pancreatic islets to identify the current underlying the glucose-induced rhythmic firing of action potentials. Trains of depolarizations (to simulate glucose-induced electrical activity) resulted in the gradual (time constant: 2.3 s) development of a small (<0.8 nS) K(+) conductance. The current was dependent on Ca(2+) influx but unaffected by apamin and charybdotoxin, two blockers of Ca(2+)-activated K(+) channels, and was insensitive to tolbutamide (a blocker of ATP-regulated K(+) channels) but partially (>60%) blocked by high (10-20 mM) concentrations of tetraethylammonium. Upon cessation of electrical stimulation, the current deactivated exponentially with a time constant of 6.5 s. This is similar to the interval between two successive bursts of action potentials. We propose that this Ca(2+)-activated K(+) current plays an important role in the generation of oscillatory electrical activity in the beta cell.

  • 33. Göpel, S O
    et al.
    Kanno, T
    Barg, S
    Rorsman, P
    Patch-clamp characterisation of somatostatin-secreting -cells in intact mouse pancreatic islets.2000In: Journal of Physiology, ISSN 0022-3751, E-ISSN 1469-7793, Vol. 528, no Pt 3, p. 497-507Article in journal (Refereed)
    Abstract [en]

    The perforated patch whole-cell configuration of the patch-clamp technique was applied to superficial cells in intact mouse pancreatic islets. Three types of electrical activity were observed corresponding to alpha-, beta- and delta-cells. The delta-cells were electrically active in the presence of glucose but lacked the oscillatory pattern seen in the beta-cells. By contrast, the alpha-cells were electrically silent at high glucose concentrations but action potentials could be elicited by removal of the sugar. Both alpha- and beta-cells contained transient voltage-activated K+ currents. In the delta-cells, the K+ currents activated above -20 mV and were completely blocked by TEA (20 mM). The alpha-cells differed from the delta-cells in possessing a TEA-resistant K+ current activating already at -40 mV. Immunocytochemistry revealed the presence of Kv3.4 channels in delta-cells and TEA-resistant Kv4.3 channels in alpha-cells. Thus the presence of a transient TEA-resistant current can be used to functionally separate the delta- and alpha-cells. A TTX-sensitive Na+ current developed in delta-cells during depolarisations beyond -30 mV and reached a peak amplitude of 350 pA. Steady-state inactivation of this current was half-maximal at -28 mV. The delta-cells were also equipped with a sustained Ca2+ current that activated above -30 mV and reached a peak of 60 pA when measured at 2.6 mM extracellular Ca2+. A tolbutamide-sensitive KATP channel conductance was observed in delta-cells exposed to glucose-free medium. Addition of tolbutamide (0.1 mM) depolarised the delta-cell and evoked electrical activity. We propose that the KATP channels in delta-cells serve the same function as in the beta-cell and couple an elevation of the blood glucose concentration to stimulation of hormone release.

  • 34. Göpel, S O
    et al.
    Kanno, T
    Barg, S
    Weng, X G
    Gromada, J
    Rorsman, P
    Regulation of glucagon release in mouse -cells by KATP channels and inactivation of TTX-sensitive Na+ channels.2000In: Journal of Physiology, ISSN 0022-3751, E-ISSN 1469-7793, Vol. 528, no Pt 3, p. 509-20Article in journal (Refereed)
    Abstract [en]

    The perforated patch whole-cell configuration of the patch-clamp technique was applied to superficial glucagon-secreting alpha-cells in intact mouse pancreatic islets. alpha-cells were distinguished from the beta- and delta-cells by the presence of a large TTX-blockable Na+ current, a TEA-resistant transient K+ current sensitive to 4-AP (A-current) and the presence of two kinetically separable Ca2+ current components corresponding to low- (T-type) and high-threshold (L-type) Ca2+ channels. The T-type Ca2+, Na+ and A-currents were subject to steady-state voltage-dependent inactivation, which was half-maximal at -45, -47 and -68 mV, respectively. Pancreatic alpha-cells were equipped with tolbutamide-sensitive, ATP-regulated K+ (KATP) channels. Addition of tolbutamide (0.1 mM) evoked a brief period of electrical activity followed by a depolarisation to a plateau of -30 mV with no regenerative electrical activity. Glucagon secretion in the absence of glucose was strongly inhibited by TTX, nifedipine and tolbutamide. When diazoxide was added in the presence of 10 mM glucose, concentrations up to 2 microM stimulated glucagon secretion to the same extent as removal of glucose. We conclude that electrical activity and secretion in the alpha-cells is dependent on the generation of Na+-dependent action potentials. Glucagon secretion depends on low activity of KATP channels to keep the membrane potential sufficiently negative to prevent voltage-dependent inactivation of voltage-gated membrane currents. Glucose may inhibit glucagon release by depolarising the alpha-cell with resultant inactivation of the ion channels participating in action potential generation.

  • 35. Hoppa, M B
    et al.
    Jones, E
    Karanauskaite, J
    Ramracheya, R
    Braun, M
    Collins, S C
    Zhang, Q
    Clark, A
    Eliasson, L
    Genoud, C
    Macdonald, P E
    Monteith, A G
    Barg, Sebastian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Galvanovskis, J
    Rorsman, P
    Multivesicular exocytosis in rat pancreatic beta cells2012In: Diabetologia, ISSN 0012-186X, E-ISSN 1432-0428, Vol. 55, no 4, p. 1001-1012Article in journal (Refereed)
    Abstract [en]

    AIMS/HYPOTHESIS

    To establish the occurrence, modulation and functional significance of compound exocytosis in insulin-secreting beta cells.

    METHODS

    Exocytosis was monitored in rat beta cells by electrophysiological, biochemical and optical methods. The functional assays were complemented by three-dimensional reconstruction of confocal imaging, transmission and block face scanning electron microscopy to obtain ultrastructural evidence of compound exocytosis.

    RESULTS

    Compound exocytosis contributed marginally (<5% of events) to exocytosis elicited by glucose/membrane depolarisation alone. However, in beta cells stimulated by a combination of glucose and the muscarinic agonist carbachol, 15–20% of the release events were due to multivesicular exocytosis, but the frequency of exocytosis was not affected. The optical measurements suggest that carbachol should stimulate insulin secretion by ∼40%, similar to the observed enhancement of glucose-induced insulin secretion. The effects of carbachol were mimicked by elevating [Ca2+]i from 0.2 to 2 μmol/l Ca2+. Two-photon sulforhodamine imaging revealed exocytotic events about fivefold larger than single vesicles and that these structures, once formed, could persist for tens of seconds. Cells exposed to carbachol for 30 s contained long (1–2 μm) serpentine-like membrane structures adjacent to the plasma membrane. Three-dimensional electron microscopy confirmed the existence of fused multigranular aggregates within the beta cell, the frequency of which increased about fourfold in response to stimulation with carbachol.

    CONCLUSIONS/INTERPRETATION

    Although contributing marginally to glucose-induced insulin secretion, compound exocytosis becomes quantitatively significant under conditions associated with global elevation of cytoplasmic calcium. These findings suggest that compound exocytosis is a major contributor to the augmentation of glucose-induced insulin secretion by muscarinic receptor activation.

  • 36. Høy, M
    et al.
    Olsen, H L
    Bokvist, K
    Buschard, K
    Barg, S
    Rorsman, P
    Gromada, J
    Tolbutamide stimulates exocytosis of glucagon by inhibition of a mitochondrial-like ATP-sensitive K+ (KATP) conductance in rat pancreatic A-cells.2000In: Journal of Physiology, ISSN 0022-3751, E-ISSN 1469-7793, Vol. 527 Pt 1, p. 109-20Article in journal (Refereed)
    Abstract [en]

    1. Capacitance measurements were used to examine the effects of the sulphonylurea tolbutamide on Ca2+-dependent exocytosis in isolated glucagon-secreting rat pancreatic A-cells. 2. When applied extracellularly, tolbutamide stimulated depolarization-evoked exocytosis 4.2-fold without affecting the whole-cell Ca2+ current. The concentration dependence of the stimulatory action was determined by intracellular application through the recording pipette. Tolbutamide produced a concentration-dependent increase in cell capacitance. Half-maximal stimulation was observed at 33 microM and the maximum stimulation corresponded to a 3.4-fold enhancement of exocytosis. 3. The stimulatory action of tolbutamide was dependent on protein kinase C activity. The action of tolbutamide was mimicked by the general K+ channel blockers TEA (10 mM) and quinine (10 microM). A similar stimulation was elicited by 5-hydroxydecanoate (5-HD; 10 microM), an inhibitor of mitochondrial ATP-sensitive K+ (KATP) channels. 4. Tolbutamide-stimulated, but not TEA-induced, exocytosis was antagonized by the K+ channel openers diazoxide, pinacidil and cromakalim. 5. Dissipating the transgranular K+ gradient with nigericin and valinomycin inhibited tolbutamide- and Ca2+-evoked exocytosis. Furthermore, tolbutamide- and Ca2+-induced exocytosis were abolished by the H+ ionophore FCCP or by arresting the vacuolar (V-type) H+-ATPase with bafilomycin A1 or DCCD. Finally, ammonium chloride stimulated exocytosis to a similar extent to that obtained with tolbutamide. 6. We propose that during granular maturation, a granular V-type H+-ATPase pumps H+ into the secretory granule leading to the generation of a pH gradient across the granular membrane and the development of a positive voltage inside the granules. The pumping of H+ is facilitated by the concomitant exit of K+ through granular K+ channels with pharmacological properties similar to those of mitochondrial KATP channels. Release of granules that have been primed is then facilitated by the addition of K+ channel blockers. The resulting increase in membrane potential promotes exocytosis by unknown mechanisms, possibly involving granular alkalinization.

  • 37.
    Idevall Hagren, Olof
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Barg, Sebastian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Gylfe, Erik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Tengholm, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    cAMP Mediators of Pulsatile Insulin Secretion from Glucose-stimulated Single β-Cells2010In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 285, no 30, p. 23005-23016Article in journal (Refereed)
    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.

  • 38. Iglesias, José
    et al.
    Barg, Sebastian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Vallois, David
    Lahiri, Shawon
    Roger, Catherine
    Yessoufou, Akadiri
    Pradevand, Sylvain
    McDonald, Angela
    Bonal, Claire
    Reimann, Frank
    Gribble, Fiona
    Debril, Marie-Bernard
    Metzger, Daniel
    Chambon, Pierre
    Herrera, Pedro
    Rutter, Guy A
    Prentki, Marc
    Thorens, Bernard
    Wahli, Walter
    PPARβ/δ affects pancreatic β cell mass and insulin secretion in mice2012In: Journal of Clinical Investigation, ISSN 0021-9738, E-ISSN 1558-8238, Vol. 122, no 11, p. 4105-4117Article in journal (Refereed)
    Abstract [en]

    PPARβ/δ protects against obesity by reducing dyslipidemia and insulin resistance via effects in muscle, adipose tissue, and liver. However, its function in pancreas remains ill defined. To gain insight into its hypothesized role in β cell function, we specifically deleted Pparb/d in the epithelial compartment of the mouse pancreas. Mutant animals presented increased numbers of islets and, more importantly, enhanced insulin secretion, causing hyperinsulinemia. Gene expression profiling of pancreatic β cells indicated a broad repressive function of PPARβ/δ affecting the vesicular and granular compartment as well as the actin cytoskeleton. Analyses of insulin release from isolated PPARβ/δ-deficient islets revealed an accelerated second phase of glucose-stimulated insulin secretion. These effects in PPARβ/δ-deficient islets correlated with increased filamentous actin (F-actin) disassembly and an elevation in protein kinase D activity that altered Golgi organization. Taken together, these results provide evidence for a repressive role for PPARβ/δ in β cell mass and insulin exocytosis, and shed a new light on PPARβ/δ metabolic action.

  • 39.
    Jin, Yang
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Physiology.
    Korol, Sergiy V
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Physiology.
    Jin, Zhe
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Physiology.
    Barg, Sebastian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Birnir, Bryndis
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Physiology.
    In Intact Islets Interstitial GABA Activates GABA(A) Receptors That Generate Tonic Currents in alpha-Cells2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 6, p. e67228-Article in journal (Refereed)
    Abstract [en]

    In the rat islets γ-aminobutyric acid (GABA) is produced by the β-cells and, at least, the α-cells express the GABAA receptors (GABAA channels). In this study, we examined in intact islets if the interstitial GABA activated the GABAA receptors. We used the patch-clamp technique to record whole-cell and single-channel currents and single-cell RT-PCR to identify the cell-type we recorded from, in the intact rat islets. We further identified which GABAA receptor subunits were expressed. We determined the cell-type of 43 cells we recorded from and of these 49%, 28% and 7% were α, β and δ-cells, respectively. In the remaining 16% of the cells, mRNA transcripts of more than one hormone gene were detected. The results show that in rat islets interstitial GABA activates tonic current in the α-cells but not in the β-cells. Seventeen different GABAA receptor subunits are expressed with high expression of α1, α2, α4, α6, β3, γ1, δ, ρ1, ρ2 and ρ3 subunits whereas no expression was detected for α5 or ε subunits. The abundance of the GABAA receptor subunits detected suggests that a number of GABAA receptor subtypes are formed in the islets. The single-channel and tonic currents were enhanced by pentobarbital and inhibited by the GABAA receptor antagonist SR-95531. The single-channel conductance ranged from 24 to 105 pS. Whether the single-channel conductance is related to subtypes of the GABAA receptor or variable interstitial GABA concentrations remains to be determined. Our results reveal that GABA is an extracellular signaling molecule in rat pancreatic islets and reaches concentration levels that activate GABAA receptors on the glucagon-releasing α-cells.

  • 40. Kanno, Takahiro
    et al.
    Ma, Xiasong
    Barg, Sebastian
    Eliasson, Lena
    Galvanovskis, Juris
    Göpel, Sven
    Larsson, Max
    Renström, Erik
    Rorsman, Patrik
    Large dense-core vesicle exocytosis in pancreatic beta-cells monitored by capacitance measurements.2004In: Methods, ISSN 1046-2023, E-ISSN 1095-9130, Vol. 33, no 4, p. 302-11Article in journal (Refereed)
    Abstract [en]

    This article discusses the currently used methodologies for monitoring exocytosis as changes in cell capacitance. Details are given on composition of solutions, experimental protocols, and how the observed responses can be interpreted physiologically. The concepts are illustrated by examples from our own work on insulin-releasing pancreatic beta-cells. Finally, we consider the feasibility of applying capacitance measurements to endocrine cells in intact pancreatic islets, where the cells are electrically coupled to each other.

  • 41.
    Kay, Emma I.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Barg, Sebastian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    G-protein coupled receptor dynamics in insulin secreting cells2015In: Diabetologia, ISSN 0012-186X, E-ISSN 1432-0428, Vol. 58, no Suppl. 1, p. S212-S212Article in journal (Other academic)
  • 42. Knowles, M. K.
    et al.
    Barg, Sebastian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Wan, L.
    Midorikawa, M.
    Chen, X.
    Almers, Wolfhard
    Single secretory granules of live cells recruit syntaxin-1 and synaptosomal associated protein 25 (SNAP-25) in large copy numbers2010In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 107, no 48, p. 20810-20815Article in journal (Refereed)
    Abstract [en]

    Before secretory vesicles undergo exocytosis, they must recruit the proteins syntaxin-1 and synaptosomal associated protein 25 (SNAP-25) in the plasma membrane. GFP-labeled versions of both proteins cluster at sites where secretory granules have docked. Single-particle tracking shows that minority populations of both molecules are strongly hindered in their mobility, consistent with their confinement in nanodomains. We measured the fluorescence of granule-associated clusters, the fluorescence of single molecules, and the numbers of unlabeled syntaxin-1 and SNAP-25 molecules per cell. There was a more than 10-fold excess of SNAP-25 over syntaxin-1. Fifty to seventy copies each of syntaxin-1 and SNAP-25 molecules were associated with a single docked granule, many more than have been reported to be required for fusion.

  • 43.
    Korol, Sergiy V
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Physiology.
    Jin, Zhe
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Physiology.
    Jin, Yang
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Physiology.
    Bhandage, Amol K.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Physiology.
    Tengholm, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Gandasi, Nikhil R.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Physiology.
    Barg, Sebastian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Espes, Daniel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Carlsson, Per-Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Laver, Derek
    University of Newcastle, Callaghan, Australia.
    Birnir, Bryndis
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Physiology.
    Functional Characterization of Native, High-Affinity GABAA Receptors in Human Pancreatic β Cells2018In: EBioMedicine, ISSN 0360-0637, E-ISSN 2352-3964, Vol. 30Article in journal (Refereed)
    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.

  • 44. Krus, Ulrika
    et al.
    King, Ben C
    Nagaraj, Vini
    Gandasi, Nikhil R
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Sjölander, Jonatan
    Buda, Pawel
    Garcia-Vaz, Eliana
    Gomez, Maria F
    Ottosson-Laakso, Emilia
    Storm, Petter
    Fex, Malin
    Vikman, Petter
    Zhang, Enming
    Barg, Sebastian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Blom, Anna M
    Renström, Erik
    The Complement Inhibitor CD59 Regulates Insulin Secretion by Modulating Exocytotic Events2014In: Cell Metabolism, ISSN 1550-4131, E-ISSN 1932-7420, Vol. 19, no 5, p. 883-890Article in journal (Refereed)
    Abstract [en]

    Type 2 diabetes is triggered by reduced insulin production, caused by genetic and environmental factors such as inflammation originating from the innate immune system. Complement proteins are a component of innate immunity and kill non-self cells by perforating the plasma membrane, a reaction prevented by CD59. Human pancreatic islets express CD59 at very high levels. CD59 is primarily known as a plasma membrane protein in membrane rafts, but most CD59 protein in pancreatic β cells is intracellular. Removing extracellular CD59 disrupts membrane rafts and moderately stimulates insulin secretion, whereas silencing intracellular CD59 markedly suppresses regulated secretion by exocytosis, as demonstrated by TIRF imaging. CD59 interacts with the exocytotic proteins VAMP2 and Syntaxin-1. CD59 expression is reduced by glucose and in rodent diabetes models but upregulated in human diabetic islets, potentially reflecting compensatory reactions. This unconventional action of CD59 broadens the established view of innate immunity in type 2 diabetes.

  • 45. Krus, Ulrika
    et al.
    King, Benjamin C.
    Nagaraj, Vini
    Gandasi, Nikhil
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Sjolander, Jonatan
    Buda, Pawel
    Garcia-Vaz, Eliana
    Gomez, Maria
    Ottosson-Laakso, Emilia
    Storm, Petter
    Fex, Malin
    Vikman, Petter
    Zhang, Enming
    Barg, Sebastian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Blom, Anna M.
    Renstrom, Erik
    CD59 is required for pancreatic beta cell insulin exocytosis2014In: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 61, no 2, p. 279-279Article in journal (Other academic)
  • 46. Lin, Chia-Wei
    et al.
    Yan, Feifei
    Shimamura, Satoko
    Barg, Sebastian
    Shyng, Show-Ling
    Membrane phosphoinositides control insulin secretion through their effects on ATP-sensitive K+ channel activity.2005In: Diabetes, ISSN 0012-1797, E-ISSN 1939-327X, Vol. 54, no 10, p. 2852-8Article in journal (Refereed)
    Abstract [en]

    ATP-sensitive K(+) channels (K(ATP) channels) of pancreatic beta-cells play key roles in glucose-stimulated insulin secretion by linking metabolic signals to cell excitability. Membrane phosphoinositides, in particular phosphatidylinositol 4,5-bisphosphates (PIP(2)), stimulate K(ATP) channels and decrease channel sensitivity to ATP inhibition; as such, they have been postulated as critical regulators of K(ATP) channels and hence of insulin secretion in beta-cells. Here, we tested this hypothesis by manipulating the interactions between K(ATP) channels and membrane phospholipids in a beta-cell line, INS-1, and assessing how the manipulations affect membrane excitability and insulin secretion. We demonstrate that disruption of channel interactions with PIP(2) by overexpressing PIP(2)-insensitive channel subunits leads to membrane depolarization and elevated basal level insulin secretion at low glucose concentrations. By contrast, facilitation of channel interactions with PIP(2) by upregulating PIP(2) levels via overexpression of a lipid kinase, phosphatidylinositol 4-phosphate 5 kinase, decreases the ATP sensitivity of endogenous K(ATP) channels by approximately 26-fold and renders INS-1 cells hyperpolarized, unable to secrete insulin properly in the face of high glucose. Our results establish an important role of the interaction between membrane phosphoinositides and K(ATP) channels in regulating insulin secretion.

  • 47. Lotharius, Julie
    et al.
    Barg, Sebastian
    Wiekop, Pia
    Lundberg, Cecilia
    Raymon, Heather K
    Brundin, Patrik
    Effect of mutant alpha-synuclein on dopamine homeostasis in a new human mesencephalic cell line.2002In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 277, no 41, p. 38884-94Article in journal (Refereed)
    Abstract [en]

    Mutations in alpha-synuclein have been linked to rare, autosomal dominant forms of Parkinson's disease. Despite its ubiquitous expression, mutant alpha-synuclein primarily leads to the loss of dopamine-producing neurons in the substantia nigra. alpha-Synuclein is a presynaptic nerve terminal protein of unknown function, although several studies suggest it is important for synaptic plasticity and maintenance. The present study utilized a new human mesencephalic cell line, MESC2.10, to study the effect of A53T mutant alpha-synuclein on dopamine homeostasis. In addition to expressing markers of mature dopamine neurons, differentiated MESC2.10 cells are electrically active, produce dopamine, and express wild-type human alpha-synuclein. Lentivirus-induced overexpression of A53T mutant alpha-synuclein in differentiated MESC2.10 cells resulted in down-regulation of the vesicular dopamine transporter (VMAT2), decreased potassium-induced and increased amphetamine-induced dopamine release, enhanced cytoplasmic dopamine immunofluorescence, and increased intracellular levels of superoxide. These results suggest that mutant alpha-synuclein leads to an impairment in vesicular dopamine storage and consequent accumulation of dopamine in the cytosol, a pathogenic mechanism that underlies the toxicity of the psychostimulant amphetamine and the parkinsonian neurotoxin 1-methyl-4-phenylpyridinium. Interestingly, cells expressing A53T mutant alpha-synuclein were resistant to amphetamine-induced toxicity. Because extravesicular, cytoplasmic dopamine can be easily oxidized into reactive oxygen species and other toxic metabolites, mutations in alpha-synuclein might lead to Parkinson's disease by triggering protracted, low grade dopamine toxicity resulting in terminal degeneration and ultimately cell death.

  • 48.
    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 University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Yang, Mingyu
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    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 University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Tengholm, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    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 Handling2019In: Diabetes, ISSN 0012-1797, E-ISSN 1939-327X, Vol. 68, no 4, p. 747-760Article in journal (Refereed)
    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.

  • 49. Obermüller, Stefanie
    et al.
    Calegari, Federico
    King, Angus
    Lindqvist, Anders
    Lundquist, Ingmar
    Salehi, Albert
    Francolini, Maura
    Rosa, Patrizia
    Rorsman, Patrik
    Huttner, Wieland B
    Barg, Sebastian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Defective secretion of islet hormones in chromogranin-B deficient mice2010In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 5, no 1, p. e8936-Article in journal (Refereed)
    Abstract [en]

    Granins are major constituents of dense-core secretory granules in neuroendocrine cells, but their function is still a matter of debate. Work in cell lines has suggested that the most abundant and ubiquitously expressed granins, chromogranin A and B (CgA and CgB), are involved in granulogenesis and protein sorting. Here we report the generation and characterization of mice lacking chromogranin B (CgB-ko), which were viable and fertile. Unlike neuroendocrine tissues, pancreatic islets of these animals lacked compensatory changes in other granins and were therefore analyzed in detail. Stimulated secretion of insulin, glucagon and somatostatin was reduced in CgB-ko islets, in parallel with somewhat impaired glucose clearance and reduced insulin release, but normal insulin sensitivity in vivo. CgB-ko islets lacked specifically the rapid initial phase of stimulated secretion, had elevated basal insulin release, and stored and released twice as much proinsulin as wildtype (wt) islets. Stimulated release of glucagon and somatostatin was reduced as well. Surprisingly, biogenesis, morphology and function of insulin granules were normal, and no differences were found with regard to beta-cell stimulus-secretion coupling. We conclude that CgB is not required for normal insulin granule biogenesis or maintenance in vivo, but is essential for adequate secretion of islet hormones. Consequentially CgB-ko animals display some, but not all, hallmarks of human type-2 diabetes. However, the molecular mechanisms underlying this defect remain to be determined.

  • 50.
    Obermüller, Stefanie
    et al.
    Department of Experimental Medicinal Sciences, Lund University, Lund.
    Lindqvist, Anders
    Department of Experimental Medicinal Sciences, Lund University, Lund.
    Karanauskaite, Jovita
    Department of Experimental Medicinal Sciences, Lund University, Lund.
    Galvanovskis, Juris
    OCDEM, Nuffield Department of Clinical Medicine, University of Oxford, Churchill Hospital, Oxford.
    Rorsman, Patrik
    Department of Experimental Medicinal Sciences, Lund University, Lund.
    Barg, Sebastian
    Department of Experimental Medicinal Sciences, Lund University, Lund.
    Selective nucleotide-release from dense-core granules in insulin-secreting cells2005In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 118, no Pt 18, p. 4271-82Article in journal (Refereed)
    Abstract [en]

    Secretory granules of insulin-secreting cells are used to store and release peptide hormones as well as low-molecular-weight compounds such as nucleotides. Here we have compared the rate of exocytosis with the time courses of nucleotide and peptide release by a combination of capacitance measurements, electrophysiological detection of ATP release and single-granule imaging. We demonstrate that the release of nucleotides and peptides is delayed by approximately 0.1 and approximately 2 seconds with respect to membrane fusion, respectively. We further show that in up to 70% of the cases exocytosis does not result in significant release of the peptide cargo, likely because of a mechanism that leads to premature closure of the fusion pore. Release of nucleotides and protons occurred regardless of whether peptides were secreted or not. These observations suggest that insulin-secreting cells are able to use the same secretory vesicles to release small molecules either alone or together with the peptide hormone.

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