Dataset Information

Cellular signaling underlying GSIS in pancreatic β-cells

ABSTRACT: In the present study, we investigated signaling pathways associated with GSIS in freshly isolated rat Islets of Langerhans. Specifically, our aim was to reveal which phosphosites and N-linked sialylated glycosylation sites that were reversible regulated upon brief glucose stimulation and link these to cellular signaling pathways using bioinformatics tools.

INSTRUMENT(S): Orbitrap Fusion, Q Exactive

ORGANISM(S): Rattus Norvegicus (rat) Mus Musculus (mouse)

TISSUE(S): Pancreatic Islet, Primary Cell, Islet Of Langerhans

SUBMITTER: Taewook Kang

LAB HEAD: Martin R. Larsen

PROVIDER: PXD007689 | Pride | 2017-11-14

REPOSITORIES: Pride

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Characterization of the Molecular Mechanisms Underlying Glucose Stimulated Insulin Secretion from Isolated Pancreatic β-cells Using Post-translational Modification Specific Proteomics (PTMomics).

Kang Taewook T Jensen Pia P Huang Honggang H Lund Christensen Gitte G Billestrup Nils N Larsen Martin R MR

Molecular & cellular proteomics : MCP 20171107 1

Normal pancreatic islet β-cells (PBCs) abundantly secrete insulin in response to elevated blood glucose levels, in order to maintain an adequate control of energy balance and glucose homeostasis. However, the molecular mechanisms underlying the insulin secretion are unclear. Improving our understanding of glucose-stimulated insulin secretion (GSIS) mechanisms under normal conditions is a prerequisite for developing better interventions against diabetes. Here, we aimed at identifying novel signal ...[more]

PMID: 29113996

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Project description:The homeodomain transcription factor Nkx6.1 plays an important role in pancreatic islet Î²-cell development, but its effects on adult Î²-cell function, survival, and proliferation are not well understood. In the present study, we demonstrated that treatment of primary rat pancreatic islets with a cytomegalovirus promoter-driven recombinant adenovirus containing the Nkx6.1 cDNA (AdCMV-Nkx6.1) causes dramatic increases in [methyl-3H] thymidine and 5-bromo-2'-deoxyuridine (BrdU) incorporation and in the number of cells per islet relative to islets treated with a control adenovirus (AdCMV-Î²GAL), whereas suppression of Nkx6.1 expression reduces thymidine incorporation. Immunocytochemical studies reveal that >80% of BrdU-positive cells in AdCMV-Nkx6.1-treated islets are Î² cells. Microarray, real-time PCR, and immunoblot analyses reveal that overexpression of Nkx6.1 in rat islets causes concerted upregulation of a cadre of cell cycle control genes, including those encoding cyclins A, B, and E, and several regulatory kinases. Cyclin E is upregulated earlier than the other cyclins, and adenovirus-mediated overexpression of cyclin E is shown to be sufficient to activate islet cell proliferation. Moreover, chromatin immunoprecipitation assays demonstrate direct interaction of Nkx6.1 with the cyclin A2 and B1 genes. Overexpression of Nkx6.1 in rat islets caused a clear enhancement of glucose-stimulated insulin secretion (GSIS), whereas overexpression of Nkx6.1 in human islets caused an increase in the level of [3H]thymidine incorporation that was twice the control level, along with complete retention of GSIS. We conclude that Nkx6.1 is among the very rare factors capable of stimulating Î²-cell replication with retention or enhancement of function, properties that may be exploitable for expansion of Î²-cell mass in treatment of both major forms of diabetes. Keywords: Insulin secretion, islet biology, transcription factor, cell cycle regulation, diabetes We utilized a âsample x referenceâ experimental design strategy in which RNA extracted from rat pancreatic islets was hybridized to the microarray slide in the presence of labeled rat reference RNA (RRR, Stratagene, LaJolla, CA). Cultures were treated with adenoviruses expressing either the hamster form of Nkx6.1 or the beta-galactosidase enzyme. 5 biological replicates each representing independent islet isolations were used for microarray analysis. Briefly, five hundred nanograms of total RNA were used for gene expression profiling following reverse transcription and T-7 polymerase-mediated amplification/labeling with Cyanine-5. Labeled subject cRNA was co-hybridized to Operon rat 27K oligonucleotide arrays with equimolar amounts of Cyanine-3 labeled RRR. Slides were hybridized, washed, and scanned on a Gene Pix 5000 microarray scanner.

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