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Molecular & Cellular Proteomics 8:393-408, 2009.
© 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Rapid Changes of mRNA-binding Protein Levels following Glucose and 3-Isobutyl-1-methylxanthine Stimulation of Insulinoma INS-1 Cells ^*,S

Christin Süss, Cornelia Czupalla^,¶, Christof Winter^¶,||, Theresia Pursche^,¶, Klaus-Peter Knoch, Michael Schroeder^¶,||,**, Bernard Hoflack^,¶,** and Michele Solimena^,**,,,¶¶

From the Experimental Diabetology, Departments of Proteomics, ^|| Bioinformatics, and Medicine III, ^¶ Biotechnology Center, ^** Center for Regenerative Therapies, Dresden University of Technology, and Max Planck Institute of Molecular Cell Biology and Genetics, Dresden 01307, Germany

Glucose and cAMP-inducing agents such as 3-isobutyl-1-methylxanthine (IBMX) rapidly change the expression profile of insulin-producing pancreatic β-cells mostly through post-transcriptional mechanisms. A thorough analysis of these changes, however, has not yet been performed. By combining two-dimensional differential gel electrophoresis and mass spectrometry, we identified 165 spots, corresponding to 78 proteins, whose levels significantly change after stimulation of the β-cell model INS-1 cells with 25 mM glucose + 1 mM IBMX for 2 h. Changes in the expression of selected proteins were verified by one- and two-dimensional immunoblotting. Most of the identified proteins are novel targets of rapid regulation in β-cells. The transcription inhibitor actinomycin D failed to block changes in two-thirds of the spots, supporting their post-transcriptional regulation. More spots changed in response to IBMX than to glucose alone conceivably because of phosphorylation. Fourteen mRNA- binding proteins responded to stimulation, thus representing the most prominent class of rapidly regulated proteins. Bioinformatics analysis indicated that the mRNA 5'- and 3'-untranslated regions of 22 regulated proteins contain potential binding sites for polypyrimidine tract-binding protein 1, which promotes mRNA stability and translation in stimulated β-cells. Overall our findings support the idea that mRNA-binding proteins play a major role in rapid adaptive changes in insulin-producing cells following their stimulation with glucose and cAMP-elevating agents.

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