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  • 51. Olofsson, Charlotta S
    et al.
    Göpel, Sven O
    Barg, Sebastian
    Galvanovskis, Juris
    Ma, Xiaosong
    Salehi, Albert
    Rorsman, Patrik
    Eliasson, Lena
    Fast insulin secretion reflects exocytosis of docked granules in mouse pancreatic B-cells.2002In: Pflügers Archiv: European Journal of Physiology, ISSN 0031-6768, E-ISSN 1432-2013, Vol. 444, no 1-2, p. 43-51Article in journal (Refereed)
    Abstract [en]

    A readily releasable pool (RRP) of granules has been proposed to underlie the first phase of insulin secretion. In the present study we combined electron microscopy, insulin secretion measurements and recordings of cell capacitance in an attempt to define this pool ultrastructurally. Mouse pancreatic B-cells contain approximately 9,000 granules, of which 7% are docked below the plasma membrane. The number of docked granules was reduced by 30% (200 granules) during 10 min stimulation with high K+. This stimulus depolarized the cell to -10 mV, elevated cytosolic [Ca2+] ([Ca2+](i)) from a basal concentration of 130 nM to a peak of 1.3 microM and released 0.5 ng insulin/islet, corresponding to 200-300 granules/cell. The Ca2+ transient decayed towards the prestimulatory concentration within approximately 200 s, presumably reflecting Ca2+ channel inactivation. Renewed stimulation with high K+ failed to stimulate insulin secretion when applied in the absence of glucose. The size of the RRP, derived from the insulin measurements, is similar to that estimated from the increase in cell capacitance elicited by photolytic release of caged Ca2+. We propose that the RRP represents a subset of the docked pool of granules and that replenishment of RRP can be accounted for largely by chemical modification of granules already in place or situated close to the plasma membrane.

  • 52.
    Omar-Hmeadi, Muhmmad
    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.
    Plasma membrane PI(4,5)P-2 is critical for secretory granule exocytosis2017In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 28, no 26, p. 3727-3727Article in journal (Other academic)
  • 53.
    Omar-Hmeadi, Muhmmad
    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.
    PtdIns(4,5)P2 is not required for secretory granule docking2018In: Traffic: the International Journal of Intracellular Transport, ISSN 1398-9219, E-ISSN 1600-0854, Vol. 19, no 6, p. 436-445Article in journal (Refereed)
    Abstract [en]

    Phosphoinositides (PtdIns) play important roles in exocytosis and are thought to regulate secretory granule docking by co-clustering with the SNARE protein syntaxin to form a docking receptor in the plasma membrane. Here we tested this idea by high-resolution total internal reflection imaging of EGFP-labeled PtdIns markers or syntaxin-1 at secretory granule release sites in live insulin-secreting cells. In intact cells, PtdIns markers distributed evenly across the plasma membrane with no preference for granule docking sites. In contrast, syntaxin-1 was found clustered in the plasma membrane, mostly beneath docked granules. We also observed rapid accumulation of syntaxin-1 at sites where granules arrived to dock. Acute depletion of plasma membrane phosphatidylinositol (4,5) bisphosphate (PtdIns(4,5)P-2) by recruitment of a 5-phosphatase strongly inhibited Ca2+-dependent exocytosis, but had no effect on docked granules or the distribution and clustering of syntaxin-1. Cell permeabilization by -toxin or formaldehyde-fixation caused PtdIns marker to slowly cluster, in part near docked granules. In summary, our data indicate that PtdIns(4,5)P-2 accelerates granule priming, but challenge a role of PtdIns in secretory granule docking or clustering of syntaxin-1 at the release site.

  • 54.
    Omar-Hmeadi, Muhmmad
    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.
    Barg, Sebastian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Fusion pore regulation by transient local generation of PI(4,5)P2 in pancreatic in β-cellsManuscript (preprint) (Other academic)
  • 55.
    Omar-Hmeadi, Muhmmad
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Lund, Per-Eric
    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.
    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.
    Paracrine control of α-cell glucagon exocytosis is compromised in human type-2 diabetesManuscript (preprint) (Other academic)
  • 56. Renström, Erik
    et al.
    Barg, Sebastian
    Thévenod, Frank
    Rorsman, Patrik
    Sulfonylurea-mediated stimulation of insulin exocytosis via an ATP-sensitive K+ channel-independent action.2002In: Diabetes, ISSN 0012-1797, E-ISSN 1939-327X, Vol. 51 Suppl 1, p. S33-6Article in journal (Refereed)
    Abstract [en]

    Several reports indicate that hypoglycemic sulfonylureas augment Ca(2+)-dependent insulin secretion via mechanisms other than inhibition of the ATP-sensitive K(+) channel. The effect involves a 65-kd protein in the granule membrane and culminates in intragranular acidification. Lowering of granule pH is necessary for the insulin granule to gain release competence. Proton pumping into the granule is driven by a v-type H(+)-ATPase, but requires simultaneous Cl(-) uptake into the granule via metabolically regulated ClC-3 Cl(-) channels to maintain electroneutrality. Here we discuss the possibility that modulation of granule ClC-3 channels represents the mechanism whereby sulfonylureas directly potentiate the beta-cell exocytotic machinery.

  • 57. Rorsman, Patrik
    et al.
    Eliasson, Lena
    Renström, Erik
    Gromada, Jesper
    Barg, Sebastian
    Göpel, Sven
    The Cell Physiology of Biphasic Insulin Secretion.2000In: News in Physiological Sciences - NIPS, ISSN 0886-1714, E-ISSN 1522-161X, Vol. 15, p. 72-77Article in journal (Refereed)
    Abstract [en]

    Glucose-stimulated insulin secretion consists of a transient first phase followed by a sustained second phase. Diabetes (type II) is associated with abnormalities in this release pattern. Here we review the evidence that biphasic insulin secretion reflects exocytosis of two functional subsets of secretory granules and the implications for diabetes.

  • 58.
    Salunkhe, V. A.
    et al.
    Lund Univ, Ctr Diabet, Clin Res Ctr Malmo, Islet Cell Exocytosis, Malmo, Sweden..
    Ofori, J.
    Lund Univ, Ctr Diabet, Clin Res Ctr Malmo, Islet Cell Exocytosis, Malmo, Sweden..
    Gandasi, Nikhil R.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Salo, S. A.
    Lund Univ, Ctr Diabet, Clin Res Ctr Malmo, Islet Cell Exocytosis, Malmo, Sweden.;Roskilde Univ, Dept Sci Syst & Models, Roskilde, Denmark..
    Hansson, S.
    Lund Univ, Ctr Diabet, Clin Res Ctr Malmo, Islet Cell Exocytosis, Malmo, Sweden..
    Andersson, M. E.
    Lund Univ, Ctr Diabet, Clin Res Ctr Malmo, Islet Cell Exocytosis, Malmo, Sweden..
    Wendt, A.
    Lund Univ, Ctr Diabet, Clin Res Ctr Malmo, Islet Cell Exocytosis, Malmo, Sweden..
    Barg, Sebastian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Esguerra, J. L. S.
    Lund Univ, Ctr Diabet, Clin Res Ctr Malmo, Islet Cell Exocytosis, Malmo, Sweden..
    Eliasson, L.
    Lund Univ, Ctr Diabet, Clin Res Ctr Malmo, Islet Cell Exocytosis, Malmo, Sweden..
    MiR-335 regulates exocytotic proteins and affects glucose-stimulated insulin secretion through decreased Ca2+-dependent exocytosis in beta cells2015In: Diabetologia, ISSN 0012-186X, E-ISSN 1432-0428, Vol. 58, no Suppl. 1, p. S128-S128Article in journal (Other academic)
  • 59.
    Salunkhe, Vishal A.
    et al.
    Lund Univ, Lund Univ Diabet Ctr, Dept Clin Sci Malmo, Islet Cell Exocytosis, CRC 91-11,SUS Malmo,Box 50332, S-20213 Malmo, Sweden..
    Ofori, Jones K.
    Lund Univ, Lund Univ Diabet Ctr, Dept Clin Sci Malmo, Islet Cell Exocytosis, CRC 91-11,SUS Malmo,Box 50332, S-20213 Malmo, Sweden..
    Gandasi, Nikhil R
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Salo, Sofia A.
    Lund Univ, Lund Univ Diabet Ctr, Dept Clin Sci Malmo, Islet Cell Exocytosis, CRC 91-11,SUS Malmo,Box 50332, S-20213 Malmo, Sweden..
    Hansson, Sofia
    Lund Univ, Lund Univ Diabet Ctr, Dept Clin Sci Malmo, Islet Cell Exocytosis, CRC 91-11,SUS Malmo,Box 50332, S-20213 Malmo, Sweden..
    Andersson, Markus E.
    Lund Univ, Lund Univ Diabet Ctr, Dept Clin Sci Malmo, Islet Cell Exocytosis, CRC 91-11,SUS Malmo,Box 50332, S-20213 Malmo, Sweden..
    Wendt, Anna
    Lund Univ, Lund Univ Diabet Ctr, Dept Clin Sci Malmo, Islet Cell Exocytosis, CRC 91-11,SUS Malmo,Box 50332, S-20213 Malmo, Sweden..
    Barg, Sebastian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Esguerra, Jonathan L. S.
    Lund Univ, Lund Univ Diabet Ctr, Dept Clin Sci Malmo, Islet Cell Exocytosis, CRC 91-11,SUS Malmo,Box 50332, S-20213 Malmo, Sweden..
    Eliasson, Lena
    Lund Univ, Lund Univ Diabet Ctr, Dept Clin Sci Malmo, Islet Cell Exocytosis, CRC 91-11,SUS Malmo,Box 50332, S-20213 Malmo, Sweden..
    MiR-335 overexpression impairs insulin secretion through defective priming of insulin vesicles2017In: Physiological Reports, E-ISSN 2051-817X, Vol. 5, no 21, article id e13493Article in journal (Refereed)
    Abstract [en]

    MicroRNAs contribute to the maintenance of optimal cellular functions by fine-tuning protein expression levels. In the pancreatic beta-cells, imbalances in the exocytotic machinery components lead to impaired insulin secretion and type 2 diabetes (T2D). We hypothesize that dysregulated miRNA expression exacerbates beta-cell dysfunction, and have earlier shown that islets from the diabetic GK-rat model have increased expression of miRNAs, including miR-335-5p (miR-335). Here, we aim to determine the specific role of miR-335 during development of T2D, and the influence of this miRNA on glucose-stimulated insulin secretion and Ca2+-dependent exocytosis. We found that the expression of miR-335 negatively correlated with secretion index in human islets of individuals with prediabetes. Overexpression of miR-335 in human EndoC-beta H1 and in rat INS-1 832/13 cells (OE335) resulted in decreased glucose-stimulated insulin secretion, and OE335 cells showed concomitant reduction in three exocytotic proteins: SNAP25, Syntaxin-binding protein 1 (STXBP1), and synaptotagmin 11 (SYT11). Single-cell capacitance measurements, complemented with TIRF microscopy of the granule marker NPY-mEGFP demonstrated a significant reduction in exocytosis in OE335 cells. The reduction was not associated with defective docking or decreased Ca2+ current. More likely, it is a direct consequence of impaired priming of already docked granules. Earlier reports have proposed reduced granular priming as the cause of reduced first-phase insulin secretion during prediabetes. Here, we show a specific role of miR-335 in regulating insulin secretion during this transition period. Moreover, we can conclude that miR-335 has the capacity to modulate insulin secretion and Ca2+-dependent exocytosis through effects on granular priming.

  • 60. Somanath, Sangeeta
    et al.
    Barg, Sebastian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Marshall, Catriona
    Silwood, Christopher J
    Turner, Mark D
    High extracellular glucose inhibits exocytosis through disruption of syntaxin 1A-containing lipid rafts2009In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 389, no 2, p. 241-246Article in journal (Refereed)
    Abstract [en]

    Diabetes is characterized by high blood glucose which eventually impairs the secretion of insulin. Glucose directly affects cholesterol biosynthesis and may in turn affect cellular structures that depend on the sterol, including lipid rafts that help organize the secretory apparatus. Here, we investigated the long-term effects of glucose upon lipid rafts and secretory granule dynamics in pancreatic beta-cells. Raft fractions, identified by the presence of GM1 and flotillin, contained characteristically high levels of cholesterol and syntaxin 1A, the t-SNARE which tethers granules to the plasma membrane. Seventy-two hours exposure to 28mM glucose resulted in approximately 30% reduction in membrane cholesterol, with consequent redistribution of raft markers and syntaxin 1A throughout the plasma membrane. Live cell imaging indicated loss of syntaxin 1A from granule docking sites, and fewer docked granules. In conclusion, glucose-mediated inhibition of cholesterol biosynthesis perturbs lipid raft stability, resulting in a loss of syntaxin 1A from granule docking sites and inhibition of insulin secretion.

  • 61. Timerman, A P
    et al.
    Onoue, H
    Xin, H B
    Barg, S
    Copello, J
    Wiederrecht, G
    Fleischer, S
    Selective binding of FKBP12.6 by the cardiac ryanodine receptor.1996In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 271, no 34, p. 20385-91Article in journal (Refereed)
    Abstract [en]

    The calcium release channels (CRC)/ryanodine receptors of skeletal (Sk) and cardiac (C) muscle sarcoplasmic reticulum (SR) are hetero-oligomeric complexes with the structural formulas (ryanodine recepter (RyR)1 protomer)4(FKBP12)4 and (RyR2 protomer)4(FKBP12.6)4, respectively, where FKBP12 and FKBP12.6 are isoforms of the 12-kDa receptor for the immunosuppressant drug FK506. The sequence similarity between the RyR protomers and FKBP12 isoforms is 63 and 85%, respectively. Using 35S-labeled FKBP12 and 35S-labeled FKBP12.6 as probes to study the interaction with CRC, we find that: 1) analogous to its action in skeletal muscle sarcoplasmic reticulum (SkMSR), FK506 (or analog FK590) dissociates FKBP12.6 from CSR; 2) both FKBP isoforms bind to FKBP-stripped SkMSR and exchange with endogenously bound FKBP12 of SkMSR; and 3) by contrast, only FKBP12. 6 exchanges with endogenously bound FKBP12.6 or rebinds to FKBP-stripped CSR. This selective binding appears to explain why the cardiac CRC is isolated as a complex with FKBP12.6, whereas the skeletal muscle CRC is isolated as a complex with FKBP12, although only FKBP12 is detectable in the myoplasm of both muscle types. Also, in contrast to the activation of the channel by removal of FKBP from skeletal muscle, no activation is detected in CRC activity in FKBP-stripped CSR. This differential action of FKBP may reflect a fundamental difference in the modulation of excitation-contraction coupling in heart versus skeletal muscle.

  • 62. Wiser, O
    et al.
    Trus, M
    Hernández, A
    Renström, E
    Barg, S
    Rorsman, P
    Atlas, D
    The voltage sensitive Lc-type Ca2+ channel is functionally coupled to the exocytotic machinery.1999In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 96, no 1, p. 248-53Article in journal (Refereed)
    Abstract [en]

    Although N- and P-type Ca2+ channels predominant in fast-secreting systems, Lc-type Ca2+ channels (C-class) can play a similar role in certain secretory cells and synapses. For example, in retinal bipolar cells, Ca2+ entry through the Lc channels triggers ultrafast exocytosis, and in pancreatic beta-cells, evoked secretion is highly sensitive to Ca2+. These findings suggest that a rapidly release pool of vesicles colocalizes with the Ca2+ channels to allow high Ca2+ concentration and a tight coupling of the Lc channels at the release site. In binding studies, we show that the Lc channel is physically associated with synaptotagmin (p65) and the soluble N-ethylmaleimide-sensitive attachment proteins receptors: syntaxin and synaptosomal-associated protein of 25 kDa. Soluble N-ethylmaleimide-sensitive attachent proteins receptors coexpressed in Xenopus oocytes along with the Lc channel modify the kinetic properties of the channel. The modulatory action of syntaxin can be overcome by coexpressing p65, where at a certain ratio of p65/syntaxin, the channel regains its unaltered kinetic parameters. The cytosolic region of the channel, Lc753-893, separating repeats II-III of its alpha1C subunit, interacts with p65 and "pulls" down native p65 from rat brain membranes. Lc753-893 injected into single insulin-secreting beta-cell, inhibits secretion in response to channel opening, but not in response to photolysis of caged Ca2+, nor does it affect Ca2+ current. These results suggest that Lc753-893 competes with the endogenous channel for the synaptic proteins and disrupts the spatial coupling with the secretory apparatus. The molecular organization of the Lc channel and the secretory machinery into a multiprotein complex (named excitosome) appears to be essential for an effective depolarization evoked exocytosis.

  • 63.
    Xie, Beichen
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Nguyen, Phuoc My
    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.
    Thonig, Antje
    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.
    Idevall-Hagren, Olof
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Plasma Membrane Phosphatidylinositol 4,5-Bisphosphate Regulates Ca2+-Influx and Insulin Secretion from Pancreatic beta Cells2016In: CELL CHEMICAL BIOLOGY, ISSN 2451-9448, Vol. 23, no 7, p. 816-826Article in journal (Refereed)
    Abstract [en]

    Insulin secretion from pancreatic beta cells is regulated by the blood glucose concentration and occurs through Ca2+-triggered exocytosis. The activities of multiple ion channels in the beta cell plasma membrane are required to fine-tune insulin secretion in order to maintain normoglycemia. Phosphoinositide lipids in the plasma membrane often gate ion channels, and variations in the concentration of these lipids affect ion-channel open probability and conductance. Using light-regulated synthesis or depletion of plasma membrane phosphatidylinositol 4,5-bisphosphate (PI[4,5]P-2), we found that this lipid positively regulated both depolarization- and glucose-triggered Ca2+ influx in a dose-dependent manner. Small reductions of PI(4,5)P-2 caused by brief illumination resulted in partial suppression of Ca2+ influx that followed the kinetics of the lipid, whereas depletion resulted in marked inhibition of both Ca2+ influx and insulin secretion.

  • 64.
    Yin, Peng
    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.
    Arora, Swati
    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.
    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.
    Syntaxin clusters at secretory granules in a munc18-bound conformation2018In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 29, no 22, p. 2700-2708Article in journal (Refereed)
    Abstract [en]

    Syntaxin (stx)-1 is an integral plasma membrane protein that is crucial for two distinct steps of regulated exocytosis, docking of secretory granules at the plasma membrane and membrane fusion. During docking, stx1 clusters at the granule docking site, together with the S/M protein munc18. Here we determined features of stx1 that contribute to its clustering at granules. In live insulin-secreting cells, stx1 and stx3 (but not stx4 or stx11) accumulated at docked granules, and stx1 (but not stx4) rescued docking in cells expressing botulinum neurotoxin-C. Using a series of stx1 deletion mutants and stx1/4 chimeras, we found that all four helical domains (Ha, Hb, Hc, SNARE) and the short N-terminal peptide contribute to recruitment to granules. However, only the Hc domain confers specificity, and it must be derived from stx1 for recruitment to occur. Point mutations in the Hc or the N-terminal peptide designed to interfere with binding to munc18-1 prevent stx1 from clustering at granules, and a mutant munc18 deficient in binding to stx1 does not cluster at granules. We conclude that stx1 is recruited to the docking site in a munc18-1-bound conformation, providing a rationale for the requirement for both proteins for granule docking.

  • 65.
    Zang, Guangxiang
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Christoffersson, Gustaf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology, Integrative Physiology.
    Tian, Geng
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Harun-Or-Rashid, Mohammad
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Neuroscience.
    Vågesjö, Evelina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology, Integrative Physiology.
    Phillipson, Mia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology, Integrative Physiology.
    Barg, Sebastian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Tengholm, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Welsh, Michael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Aberrant association between vascular endothelial growth factor receptor-2 and VE-cadherin in response to vascular endothelial growth factor-a in Shb-deficient lung endothelial cells2013In: Cellular Signalling, ISSN 0898-6568, E-ISSN 1873-3913, Vol. 25, no 1, p. 85-92Article in journal (Refereed)
    Abstract [en]

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

  • 66.
    Åkerblom, Björn
    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.
    Calounova, Gabriela
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Mokhtari, Dariush
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Jansson, Leif
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Welsh, Michael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Impaired glucose homeostasis in Shb-/- mice2009In: Journal of Endocrinology, ISSN 0022-0795, E-ISSN 1479-6805, Vol. 203, no 2, p. 271-279Article in journal (Refereed)
    Abstract [en]

    Src homology 2 domain-containing protein B (SHB) is an adapter protein involved in the regulation of beta-cell and endothelial cell function. We have recently obtained the Shb knockout mouse, and consequently, the aim of this study was to assess the effect of Shb deletion upon beta-cell function and blood glucose homeostasis. Shb-/- mice display an elevated basal blood glucose concentration, and this increase is maintained during insulin challenge in insulin sensitivity tests. To assess glucose-induced insulin secretion, pancreata were perfused, and it was observed that Shb-/- first phase insulin secretion was blunted during glucose stimulation. Gene expression of Shb-/- islets shortly after isolation was altered, with increased pancreatic and duodenal homeobox gene-1 (Pdx1) gene expression and reduced expression of Vegf-A. Islet culture normalized Pdx1 gene expression. The microvascular density of the Shb-/- islets was reduced, and islet capillary endothelial cell morphology was changed suggesting an altered microvascular function as a contributing cause to the impaired secretory activity. Capacitance measurements of depolarization-induced exocytosis indicate a direct effect on the exocytotic machinery, in particular a dramatic reduction in readily releasable granules, as responsible for the insulin-secretory defect operating in Shb-/- islets. Shb-/- mice exhibited no alteration of islet volume or beta-cell area. In conclusion, loss of Shb impairs insulin secretion, alters islet microvascular morphology, and increases the basal blood glucose concentration. The impaired insulin secretory response is a plausible underlying cause of the metabolic impairment observed in this mutant mouse.

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Output format
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  • asciidoc
  • rtf