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Research

Proteomics Analysis of Cytokine-induced Dysfunction and Death in Insulin-producing INS-1E Cells

New Insights into the Pathways Involved^*,S

From the ^a Laboratory for Experimental Medicine and Endocrinology, University Hospital Gasthuisberg, Catholic University of Leuven, Herestraat 49, box 902, B-3000 Leuven, Belgium, ^d Centre for Biological Sequence Analysis, BioCentrum-DTU, Technical University of Denmark, Building 208, DK-2800 Lyngby, Denmark, ^e Laboratory for Experimental Medicine, Université Libre de Bruxelles, route de Lennik 808, box CP618, B-1070 Brussels, Belgium, ^g Laboratory of Neuroplasticity and Neuroproteomics and ^h Functional Genomics and Proteomics Research Unit, Catholic University of Leuven, Naamsestraat 59, B-3000 Leuven, Belgium, ⁱ ProMeta and ^k Laboratory of Biochemistry, University Hospital Gasthuisberg, Catholic University of Leuven, Herestraat 49, box 901, B-3000 Leuven, Belgium, and ^j ESAT-SDC, Department of Electrical Engineering, Catholic University of Leuven, Kasteelpark Arenberg 10, box 2446, B-3001 Heverlee, Belgium

Cytokines released by islet-infiltrating immune cells play a crucial role in β-cell dysfunction and apoptotic cell death in the pathogenesis of type 1 diabetes and after islet transplantation. RNA studies revealed complex pathways of genes being activated or suppressed during this β-cell attack. The aim of the present study was to analyze protein changes in insulin-producing INS-1E cells exposed to inflammatory cytokines in vitro using two-dimensional DIGE. Within two different pH ranges we observed 2214 ± 164 (pH 4–7) and 1641 ± 73 (pH 6–9) spots. Analysis at three different time points (1, 4, and 24 h of cytokine exposure) revealed that the major changes were taking place only after 24 h. At this time point 158 proteins were altered in expression (4.1%, n = 4, p 0.01) by a combination of interleukin-1β and interferon-, whereas only 42 and 23 proteins were altered by either of the cytokines alone, giving rise to 199 distinct differentially expressed spots. Identification of 141 of these by MALDI-TOF/TOF revealed proteins playing a role in insulin secretion, cytoskeleton organization, and protein and RNA metabolism as well as proteins associated with endoplasmic reticulum and oxidative stress/defense. We investigated the interactions of these proteins and discovered a significant interaction network (p < 1.27e–05) containing 42 of the identified proteins. This network analysis suggests that proteins of different pathways act coordinately in a β-cell dysfunction/apoptotic β-cell death interactome. In addition the data suggest a central role for chaperones and proteins playing a role in RNA metabolism. As many of these identified proteins are regulated at the protein level or undergo post-translational modifications, a proteomics approach, as performed in this study, is required to provide adequate insight into the mechanisms leading to β-cell dysfunction and apoptosis. The present findings may open new avenues for the understanding and prevention of β-cell loss in type 1 diabetes.