title: Proteomics analysis of cytokine-induced dysfunction and death in insulin-producing INS-1E cells: new insights into the pathways involved.
abstract: Cytokines released by islet-infiltrating immune cells play a crucial role in beta-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 beta-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 < or = 0.01) by a combination of interleukin-1beta and interferon-gamma, 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 beta-cell dysfunction/apoptotic beta-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 beta-cell dysfunction and apoptosis. The present findings may open new avenues for the understanding and prevention of beta-cell loss in type 1 diabetes.
authors.affiliation: Laboratory for Experimental Medicine and Endocrinology, University Hospital Gasthuisberg, Catholic University of Leuven, Herestraat 49, box 902, B-3000 Leuven, Belgium.
authors.forename: Wannes
authors.initials: W
authors.lastname: D'Hertog
authors.forename: Lut
authors.initials: L
authors.lastname: Overbergh
authors.forename: Kasper
authors.initials: K
authors.lastname: Lage
authors.forename: Gabriela Bonfim
authors.initials: GB
authors.lastname: Ferreira
authors.forename: Michael
authors.initials: M
authors.lastname: Maris
authors.forename: Conny
authors.initials: C
authors.lastname: Gysemans
authors.forename: Daisy
authors.initials: D
authors.lastname: Flamez
authors.forename: Alessandra Kupper
authors.initials: AK
authors.lastname: Cardozo
authors.forename: Gert
authors.initials: G
authors.lastname: Van den Bergh
authors.forename: Liliane
authors.initials: L
authors.lastname: Schoofs
authors.forename: Lut
authors.initials: L
authors.lastname: Arckens
authors.forename: Yves
authors.initials: Y
authors.lastname: Moreau
authors.forename: Daniel Aaen
authors.initials: DA
authors.lastname: Hansen
authors.forename: Decio Laks
authors.initials: DL
authors.lastname: Eizirik
authors.forename: Ettienne
authors.initials: E
authors.lastname: Waelkens
authors.forename: Chantal
authors.initials: C
authors.lastname: Mathieu
mesh_terms: D000595:Amino Acid Sequence; D002460:Cell Line; D016207:Cytokines / Q000502:physiology*; D006801:Humans; D007328:Insulin / Q000096:biosynthesis*; D040901:Proteomics; D053719:Tandem Mass Spectrometry
chemical_list: D016207:Cytokines; D007328:Insulin