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PRM_control_10 minutes.sky.zip (8 MB)2017-09-211213262862
Cellular signaling underlying GSIS in pancreatic β-cells

  • Organism: Rat
  • Instrument: Thermo QE-HF
  • SpikeIn: No
Abstract
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 signaling pathways involved in the initial release of insulin from PBCs after glucose stimulation using quantitative strategies for the assessment of phosphorylated proteins and sialylated N-linked (SA) glycoproteins. Islets of Langerhans derived from newborn rats with a subsequent 9-10 days of maturation in vitro were stimulated with 20 mM glucose for 0 min (control), 5 min, 10 min and 15 min. The isolated islets were subjected to time-resolved quantitative phosphoproteomics and sialiomics using iTRAQ-labeling combined with enrichment of phosphorylated peptides and formerly SA glycopeptides and high-accuracy LC-MS/MS. Using bioinformatics we analyzed the functional signaling pathways during GSIS, including well-known insulin secretion pathways. Furthermore, we identified six novel activated signaling pathways (e.g., agrin interactions and prolactin signaling) at 15 min GSIS, which may increase our understanding of the molecular mechanism underlying GSIS. Moreover, we validated some of the regulated phosphosites by parallel reaction monitoring, which resulted in the validation of eleven new phosphosites significantly regulated upon GSIS. Besides protein phosphorylation, alteration in SA glycosylation was observed on a number of surface proteins upon brief GSIS. Interestingly, proteins important for cell-cell interaction, cell movement, cell-ECM interaction and Focal Adhesion (e.g., integrins, semaphorins and plexins) were found regulated at the level of sialylation, but not in protein expression. Collectively, we believe that this comprehensive Proteomics and PTMomics survey of signaling pathways taking place during brief GSIS of primary PBCs is contributing to understanding the complex signaling underlying GSIS.
Created on 9/21/17, 9:09 AM