Proteomics analysis of cytokine-induced dysfunction and death in insulin-producing INS-1E cells: new insights into the pathways involved.
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.
D'Hertog
Wannes
W
Laboratory for Experimental Medicine and Endocrinology, University Hospital Gasthuisberg, Catholic University of Leuven, Herestraat 49, box 902, B-3000 Leuven, Belgium.
Overbergh
Lut
L
Lage
Kasper
K
Ferreira
Gabriela Bonfim
GB
Maris
Michael
M
Gysemans
Conny
C
Flamez
Daisy
D
Cardozo
Alessandra Kupper
AK
Van den Bergh
Gert
G
Schoofs
Liliane
L
Arckens
Lut
L
Moreau
Yves
Y
Hansen
Daniel Aaen
DA
Eizirik
Decio Laks
DL
Waelkens
Ettienne
E
Mathieu
Chantal
C
D000595:Amino Acid Sequence; D002460:Cell Line; D016207:Cytokines / Q000502:physiology*; D006801:Humans; D007328:Insulin / Q000096:biosynthesis*; D040901:Proteomics; D053719:Tandem Mass Spectrometry
D016207:Cytokines; D007328:Insulin