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BETA
Dyachok, Oleg
Alternative names
Publications (10 of 12) Show all publications
Kononenko, O., Bazov, I., Watanabe, H., Gerashchenko, G., Dyachok, O., Verbeek, D. S., . . . Bakalkin, G. (2017). Opioid precursor protein isoform is targeted to the cell nuclei in the human brain. Biochimica et Biophysica Acta, 1861(2), 246-255
Open this publication in new window or tab >>Opioid precursor protein isoform is targeted to the cell nuclei in the human brain
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2017 (English)In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1861, no 2, p. 246-255Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: Neuropeptide precursors are traditionally viewed as proteins giving rise to small neuropeptide molecules. Prodynorphin (PDYN) is the precursor protein to dynorphins, endogenous ligands for the κ-opioid receptor. Alternative mRNA splicing of neuropeptide genes may regulate cell- and tissue-specific neuropeptide expression and produce novel protein isoforms. We here searched for novel PDYN mRNA and their protein product in the human brain.

METHODS: Novel PDYN transcripts were identified using nested PCR amplification of oligo(dT) selected full-length capped mRNA. Gene expression was analyzed by qRT-PCR, PDYN protein by western blotting and confocal imaging, dynorphin peptides by radioimmunoassay. Neuronal nuclei were isolated using fluorescence-activated nuclei sorting (FANS) from postmortem human striatal tissue. Immunofluorescence staining and confocal microscopy was performed for human caudate nucleus.

RESULTS: Two novel human PDYN mRNA splicing variants were identified. Expression of one of them was confined to the striatum where its levels constituted up to 30% of total PDYN mRNA. This transcript may be translated into ∆SP-PDYN protein lacking 13 N-terminal amino acids, a fragment of signal peptide (SP). ∆SP-PDYN was not processed to mature dynorphins and surprisingly, was targeted to the cell nuclei in a model cellular system. The endogenous PDYN protein was identified in the cell nuclei in human striatum by western blotting of isolated neuronal nuclei, and by confocal imaging.

CONCLUSIONS AND GENERAL SIGNIFICANCE: High levels of alternatively spliced ∆SP-PDYN mRNA and nuclear localization of PDYN protein suggests a nuclear function for this isoform of the opioid peptide precursor in human striatum.

Keywords
Alternative splicing, Human brain, Neuropeptide precursor protein, Nuclear localization, Prodynorphin
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-316836 (URN)10.1016/j.bbagen.2016.11.002 (DOI)000392680200023 ()27838394 (PubMedID)
Funder
Swedish Research Council, K2014-62X-12190-19-5Swedish Research Council Formas, 2009-1709 259-2012-23
Note

Shared first authorship for Kononenko O., Bazov I.

Available from: 2017-03-07 Created: 2017-03-07 Last updated: 2017-11-29
Abels, M., Riva, M., Poon, W., Bennet, H., Nagaraj, V., Dyachok, O., . . . Wierup, N. (2015). CART is a novel glucose-dependent peptide with antidiabetic actions in humans. Paper presented at 51st Annual Meeting of the European-Association-for-the-Study-of-Diabetes (EASD), SEP 14-18, 2015, Stockholm, SWEDEN. Diabetologia, 58(Suppl. 1), S279-S280
Open this publication in new window or tab >>CART is a novel glucose-dependent peptide with antidiabetic actions in humans
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2015 (English)In: Diabetologia, ISSN 0012-186X, E-ISSN 1432-0428, Vol. 58, no Suppl. 1, p. S279-S280Article in journal, Meeting abstract (Other academic) Published
National Category
Endocrinology and Diabetes
Identifiers
urn:nbn:se:uu:diva-264886 (URN)000359820901266 ()
Conference
51st Annual Meeting of the European-Association-for-the-Study-of-Diabetes (EASD), SEP 14-18, 2015, Stockholm, SWEDEN
Note

Meeting Abstract: 576

Available from: 2015-11-05 Created: 2015-10-19 Last updated: 2017-12-01Bibliographically approved
Alenkvist, I., Dyachok, O., Tian, G., Li, J., Mehrabanfar, S., Jin, Y., . . . Welsh, M. (2014). Absence of Shb impairs insulin secretion by elevated FAK activity in pancreatic islets. Journal of Endocrinology, 223(3), 267-275
Open this publication in new window or tab >>Absence of Shb impairs insulin secretion by elevated FAK activity in pancreatic islets
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2014 (English)In: Journal of Endocrinology, ISSN 0022-0795, E-ISSN 1479-6805, Vol. 223, no 3, p. 267-275Article in journal (Refereed) Published
Abstract [en]

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

National Category
Endocrinology and Diabetes
Identifiers
urn:nbn:se:uu:diva-236195 (URN)10.1530/JOE-14-0531 (DOI)000345292500007 ()25274988 (PubMedID)
Available from: 2014-11-14 Created: 2014-11-14 Last updated: 2017-12-05Bibliographically approved
Chowdhury, A. I., Dyachok, O., Tengholm, A., Sandler, S. & Bergsten, P. (2013). Functional differences between aggregated and dispersed insulin-producing cells. Diabetologia, 56(7), 1557-1568
Open this publication in new window or tab >>Functional differences between aggregated and dispersed insulin-producing cells
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2013 (English)In: Diabetologia, ISSN 0012-186X, E-ISSN 1432-0428, Vol. 56, no 7, p. 1557-1568Article in journal (Refereed) Published
Abstract [en]

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

Keywords
Beta cell, Ca2+, Insulin secretion, IRS-1, Islets, Mitochondrial metabolism, PI3-kinase
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-203520 (URN)10.1007/s00125-013-2903-3 (DOI)000319881300013 ()
Available from: 2013-07-16 Created: 2013-07-15 Last updated: 2017-12-06Bibliographically approved
Dezaki, K., Damdindorj, B., Sone, H., Dyachok, O., Tengholm, A., Gylfe, E., . . . Yada, T. (2011). Ghrelin Attenuates cAMP-PKA Signaling to Evoke Insulinostatic Cascade in Islet beta-Cells. Diabetes, 60(9), 2315-2324
Open this publication in new window or tab >>Ghrelin Attenuates cAMP-PKA Signaling to Evoke Insulinostatic Cascade in Islet beta-Cells
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2011 (English)In: Diabetes, ISSN 0012-1797, E-ISSN 1939-327X, Vol. 60, no 9, p. 2315-2324Article in journal (Refereed) Published
Abstract [en]

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

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-159241 (URN)10.2337/db11-0368 (DOI)000294699600014 ()
Available from: 2011-09-27 Created: 2011-09-26 Last updated: 2017-12-08Bibliographically approved
Dyachok, O., Idevall-Hagren, O., Sågetorp, J., Tian, G., Wuttke, A., Arrieumerlou, C., . . . Tengholm, A. (2008). Glucose-induced cyclic AMP oscillations regulate pulsatile insulin secretion. Cell Metabolism, 8(1), 26-37
Open this publication in new window or tab >>Glucose-induced cyclic AMP oscillations regulate pulsatile insulin secretion
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2008 (English)In: Cell Metabolism, ISSN 1550-4131, E-ISSN 1932-7420, Vol. 8, no 1, p. 26-37Article in journal (Refereed) Published
Abstract [en]

Cyclic AMP (cAMP) and Ca2+ are key regulators of exocytosis in many cells, including insulin-secreting β-cells. Glucose-stimulated insulin secretion from β cells is pulsatile and involves oscillations of the cytoplasmic Ca2+ concentration ([Ca2+]i), but little is known about the detailed kinetics of cAMP signalling. Using evanescent-wave fluorescence imaging we found that glucose induces pronounced oscillations of cAMP in the sub-membrane space of single MIN6-cells and primary mouse β-cells. These oscillations were preceded and enhanced by elevations of [Ca2+]i. However, conditions raising cytoplasmic ATP could trigger cAMP elevations without accompanying [Ca2+]i rise, indicating that adenylyl cyclase activity may be controlled also by the substrate concentration. The cAMP oscillations correlated with pulsatile insulin release. Whereas elevation of cAMP enhanced secretion, inhibition of adenylyl cyclases suppressed both cAMP oscillations and pulsatile insulin release. We conclude that cell metabolism directly controls cAMP, and that glucose-induced cAMP oscillations regulate the magnitude and kinetics of insulin exocytosis.

Place, publisher, year, edition, pages
Cell Press, 2008
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-86694 (URN)10.1016/j.cmet.2008.06.003 (DOI)000257339600007 ()
Available from: 2008-12-03 Created: 2008-11-27 Last updated: 2017-12-14Bibliographically approved
Dyachok, O., Sagetorp, J., Isakov, Y. & Tengholm, A. (2006). cAMP oscillations restrict protein kinase A redistribution in insulin-secreting cells.. Biochem Soc Trans, 34(Pt 4), 498-501
Open this publication in new window or tab >>cAMP oscillations restrict protein kinase A redistribution in insulin-secreting cells.
2006 (English)In: Biochem Soc Trans, ISSN 0300-5127, Vol. 34, no Pt 4, p. 498-501Article in journal (Refereed) Published
Keywords
Active Transport; Cell Nucleus, Animals, Calcium/metabolism, Catalytic Domain, Cell Line; Tumor, Cyclic AMP/*metabolism, Cyclic AMP-Dependent Protein Kinases/*metabolism, Insulin-Secreting Cells/*metabolism, Rats
Identifiers
urn:nbn:se:uu:diva-22882 (URN)16856843 (PubMedID)
Available from: 2007-01-23 Created: 2007-01-23 Last updated: 2011-01-11
Dyachok, O., Isakov, Y., Sågetorp, J. & Tengholm, A. (2006). Oscillations of cyclic AMP in hormone-stimulated insulin-secreting beta-cells.. Nature, 439(7074), 349-52
Open this publication in new window or tab >>Oscillations of cyclic AMP in hormone-stimulated insulin-secreting beta-cells.
2006 (English)In: Nature, ISSN 1476-4687, Vol. 439, no 7074, p. 349-52Article in journal (Refereed) Published
Keywords
Animals, Calcium/metabolism, Calcium Signaling/drug effects, Cell Line, Cell Nucleus/drug effects/metabolism, Cyclic AMP/*metabolism, Cyclic AMP-Dependent Protein Kinases/metabolism, Cytoplasm/drug effects/metabolism, Glucagon/*pharmacology, Glucagon-Like Peptide 1/*pharmacology, Insulin/*secretion, Islets of Langerhans/*drug effects/*metabolism/secretion, Protein Subunits/metabolism, Protein Transport/drug effects, Rats
Identifiers
urn:nbn:se:uu:diva-22916 (URN)16421574 (PubMedID)
Available from: 2007-01-23 Created: 2007-01-23 Last updated: 2011-01-11
Thore, S., Dyachok, O., Gylfe, E. & Tengholm, A. (2005). Feedback activation of phospholipase C via intracellular mobilization and store-operated influx of Ca2+ in insulin-secreting β-cells. Journal of Cell Science, 118(Pt 19), 4463-4471
Open this publication in new window or tab >>Feedback activation of phospholipase C via intracellular mobilization and store-operated influx of Ca2+ in insulin-secreting β-cells
2005 (English)In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 118, no Pt 19, p. 4463-4471Article in journal (Refereed) Published
Abstract [en]

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

Keywords
Animals, Boron Compounds/metabolism, Ca(2+)-Transporting ATPase/antagonists & inhibitors/metabolism, Calcium/*metabolism, Calcium Channel Blockers/metabolism, Calcium Channels/metabolism, Cells; Cultured, Diazoxide/metabolism, Enzyme Activation, Feedback; Biochemical, Green Fluorescent Proteins/genetics/metabolism, Inositol 1;4;5-Trisphosphate/metabolism, Insulin/*metabolism, Insulin-Secreting Cells/cytology/*metabolism, Isoenzymes/genetics/*metabolism, Lanthanum/metabolism, Mice, Microscopy; Fluorescence/methods, Phospholipase C/genetics/*metabolism, Recombinant Fusion Proteins/genetics/metabolism, Research Support; Non-U.S. Gov't, Signal Transduction/*physiology
National Category
Cell and Molecular Biology
Research subject
Medical Cell Biology
Identifiers
urn:nbn:se:uu:diva-79778 (URN)10.1242/jcs.02577 (DOI)16159958 (PubMedID)
Available from: 2009-01-29 Created: 2008-11-21 Last updated: 2018-01-13Bibliographically approved
Dyachok, O., Tufveson, G. & Gylfe, E. (2004). Ca2+-induced Ca2+ release by activation of inositol 1,4,5-trisphosphate receptors in primary pancreatic β-cells. Cell Calcium, 36(1), 1-9
Open this publication in new window or tab >>Ca2+-induced Ca2+ release by activation of inositol 1,4,5-trisphosphate receptors in primary pancreatic β-cells
2004 (English)In: Cell Calcium, ISSN 0143-4160, E-ISSN 1532-1991, Vol. 36, no 1, p. 1-9Article in journal (Refereed) Published
Abstract [en]

The effect of sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) inhibition on the cytoplasmic Ca2+ concentration ([Ca2+]i) was studied in primary insulin-releasing pancreatic β-cells isolated from mice, rats and human subjects as well as in clonal rat insulinoma INS-1 cells. In Ca2+-deficient medium the individual primary β-cells reacted to the SERCA inhibitor cyclopiazonic acid (CPA) with a slow rise of [Ca2+]i followed by an explosive transient elevation. The [Ca2+]i transients were preferentially observed at low intracellular concentrations of the Ca2+ indicator fura-2 and were unaffected by pre-treatment with 100 μM ryanodine. Whereas 20 mM caffeine had no effect on basal [Ca2+]i or the slow rise in response to CPA, it completely prevented the CPA-induced [Ca2+]i transients as well as inositol 1,4,5-trisphosphate-mediated [Ca2+]i transients in response to carbachol. In striking contrast to the primary β-cells, caffeine readily mobilized intracellular Ca2+ in INS-1 cells under identical conditions, and such mobilization was prevented by ryanodine pre-treatment. The results indicate that leakage of Ca2+ from the endoplasmic reticulum after SERCA inhibition is feedback-accelerated by Ca2+-induced Ca2+ release (CICR). In primary pancreatic β-cells this CICR is due to activation of inositol 1,4,5-trisphosphate receptors. CICR by ryanodine receptor activation may be restricted to clonal β-cells.

Keywords
Ca2+-induced Ca2+ release, IP3 receptors, Ryanodine receptors, Insulin secretion, Endoplasmic reticulum, Calcium, Signaling
National Category
Cell and Molecular Biology Physiology Endocrinology and Diabetes
Research subject
Medical Cell Biology
Identifiers
urn:nbn:se:uu:diva-72702 (URN)10.1016/j.ceca.2003.11.004 (DOI)
Available from: 2009-02-02 Created: 2008-11-21 Last updated: 2018-01-14Bibliographically approved
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