Abstract
BackgroundGlucose is the main secretagogue of pancreatic beta-cells. Uptake and metabolism of the nutrient stimulates the beta-cell to release the blood glucose lowering hormone insulin. This metabolic activation is associated with a pronounced increase in mitochondrial respiration. Glucose stimulation also initiates a number of signal transduction pathways for the coordinated regulation of multiple biological processes required for insulin secretion.MethodsShotgun proteomics including TiO2 enrichment of phosphorylated peptides followed by liquid chromatography tandem mass spectrometry on lysates from glucose-stimulated INS-1E cells was used to identify glucose regulated phosphorylated proteins and signal transduction pathways. Kinase substrate enrichment analysis (KSEA) was applied to identify key regulated kinases and phosphatases. Glucose-induced oxygen consumption was measured using a XF96 Seahorse instrument to reveal cross talk between glucose-regulated kinases and mitochondrial activation.ResultsOur kinetic analysis of substrate phosphorylation reveal the molecular mechanism leading to rapid activation of insulin biogenesis, vesicle trafficking, insulin granule exocytosis and cytoskeleton remodeling. Kinase-substrate enrichment identified upstream kinases and phosphatases and time-dependent activity changes during glucose stimulation. Activity trajectories of well-known glucose-regulated kinases and phosphatases are described. In addition, we predict activity changes in a number of kinases including NUAK1, not or only poorly studied in the context of the pancreatic beta-cell. Furthermore, we pharmacologically tested whether signaling pathways predicted by kinase-substrate enrichment analysis affected glucose-dependent acceleration of mitochondrial respiration. We find that phosphoinositide 3-kinase, Ca2+/calmodulin dependent protein kinase and protein kinase C contribute to short-term regulation of energy metabolism.ConclusionsOur results provide a global view into the regulation of kinases and phosphatases in insulin secreting cells and suggest cross talk between glucose-induced signal transduction and mitochondrial activation.
Highlights
Glucose is the main secretagogue of pancreatic beta-cells
As these metabolic changes occur, glucose stimulation initiates a number of signal transduction pathways, which are important for the regulation of insulin secretion in pancreatic beta-cells [19]
We have analyzed the kinetic changes of protein phosphorylation during glucose stimulation of INS-1E cells in order to improve our understanding of glucose-induced signal transduction and its possible link to mitochondrial activation
Summary
Uptake and metabolism of the nutrient stimulates the beta-cell to release the blood glucose lowering hormone insulin. This metabolic activation is associated with a pronounced increase in mitochondrial respiration. Glucose stimulation initiates a number of signal transduction pathways for the coordinated regulation of multiple biological processes required for insulin secretion. Pancreatic beta-cells secrete the blood glucose lowering hormone insulin. Glucose rises in the blood and acts as the primary secretagogue for insulin secretion [1]. This can take up to several hours when glucose remains elevated [3]. During continued glucose stimulation beta-cells are able to adjust insulin biosynthesis and release to restore glucose homeostasis
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