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Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease
Published on: September 7, 2012
Glucose-induced changes of multiple mouse islet proteins analysed by two-dimensional gel electrophoresis and mass
1Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden.
Aims/Hypothesis:
The aim of this study was to investigate molecular mechanisms of glucose-induced changes in islets of Langerhans by analysing global changes in protein patterns of islets exposed to elevated glucose concentrations.
Methods:
Islets were isolated from C57BL/6J mice and used either directly or after exposure to 11 mmol/l glucose for 24 h. Islet protein profiles were obtained by two-dimensional gel electrophoresis, and protein spots were identified by peptide mass fingerprinting using mass spectrometry.
Results:
Two-dimensional gels of freshly isolated islets and islets exposed to 11 mmol/l glucose contained 1,074 and 1,254 spots, respectively. The number of differentially expressed spots was 379, with 20 spots appearing as new proteins in islets exposed to 11 mmol/l glucose. We identified 124 spots corresponding to 77 protein entries and generated a reference map from freshly isolated islets. Actin, alpha enolase, cytokeratin 8, endoplasmin, glucose-regulated proteins, heat shock proteins, peroxiredoxins, prohormone convertase 2, protein disulphide isomerase, superoxide dismutase, tubulin, and V-type H+ -ATPase (V1 subunit A) were upregulated in islets exposed to 11 mmol/l glucose. In contrast, exocrine proteins and secretagogin were downregulated in these islets compared with in freshly isolated islets.
Conclusions/Interpretation:
The islet proteome approach revealed simultaneous changes in protein patterns of islets exposed to elevated glucose concentrations, indicating enhanced insulin synthesis, granular mobilisation and maturation, and increased stress response. The changes may be of relevance for the understanding of altered islet function in the hyperglycaemic state. It is expected that the islet reference map will become an important tool for dissecting multifactorial islet processes.
