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 on GSIS. Besides protein phosphorylation, alteration in SA glycosylation was observed on several surface proteins on 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.
Highlights
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, 5 min, 10 min, and 15 min
Temporal Quantification of the PBC Proteome, Phosphoproteome, and Sialiome—To characterize the initial mechanisms underlying glucose stimulated insulin secretion (GSIS) in PBCs, we applied a modification of our TiSH quantitative proteomics and PTMomics approach [17, 19, 36] with iTRAQ labeling using newborn rat derived islets (ϳ1,500 islets per condition), which had been matured for 9 –10 days in vitro (DIV)
By ELISA we confirmed significantly increased insulin release in response to high glucose stimulation for 10 and 15 min compared with controls (Fig. 2)
Summary
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. Cellular Signaling Underlying GSIS in Pancreatic -cells among the most important endocrine cells, constituting around ϳ80% of the cell type percentage within the rodent islets of Langerhans. Substantial studies have showed epidemic and physiological evidence for the glucose-stimulated insulin secretion (GSIS) process in PBCs, the molecular mechanism underlying GSIS and hyperglycemia regulated insulin action is still unclear. The common GSIS model of action [9], involving glucose metabolism, closure of ATP sensitive potassium channels leading to depolarization of the plasma membrane and subsequent opening of Calcium (Ca2ϩ) channels, Ca2ϩ influx and activation of exocytotic pathways leading to emptying of insulin granules, have been challenged as incomplete [10].
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