Proteome Analysis of Interleukin-1β–Induced Changes in Protein Expression in Rat Islets of Langerhans

Abstract

The intracellular molecular events involved in the ␤-cell death process are complex but poorly understood. Cytokines, e.g., interleukin (IL)-1␤, may play a crucial role in inducing this process. Protein synthesis is necessary for the deleterious effect of IL-1, and induction of both protective and deleterious proteins has been described. To characterize the rather complex pattern of islet protein expression in rat islets in response to IL-1, we have attempted to identify proteins of altered expression level after IL-1 exposure by 2D gel electrophoresis and mass spectrometry. Of 105 significantly changed (i.e., up-or downregulated or de novo-induced) protein spots, we obtained positive protein identification for 60 protein spots. The 60 identifications corresponded to 57 different proteins. Of these, 10 proteins were present in two to four spots, suggesting that posttranslatory modifications had occurred. In addition, 11 spots contained more than one protein. The proteins could be classified according to their function into the following groups: 1) energy transduction; 2) glycolytic pathway; 3) protein synthesis, chaperones, and protein folding; and 4) signal transduction, regulation, differentiation, and apoptosis. In conclusion, valuable information about the molecular mechanisms involved in cytokine-mediated ␤-cell destruction was obtained by this approach. Protein synthesis inhibitors (e.g., cycloheximide) effectively protect IL-1-exposed islets from destruction (12). Hence, protein synthesis is necessary for the deleterious effect of IL-1. Previous studies have shown that IL-1 induces the synthesis of members of the heat shock protein (HSP) family, like heme oxygenase (13) and HSPs 70 and 90 (14,15), and hyperexpression of HSPs in islets is partially protective against cytokine-induced ␤-cell destruction (16). Furthermore, it has been reported that IL-1 may induce the synthesis of unknown proteins with molecular weights of 45,50, 75, 85, 95, and 120 kDa (17). It has previously been shown that rat islets exposed to IL-1 release NO into the culture media (18). iNOS has been cloned from islets (19) in which it has been shown to be inducible in ␤-cells only (20). We have further shown that IL-1 also upregulates IL-1 converting enzyme mRNA transcription (21) in rat islets. Also, SOD is shown to be upregulated in islets by cytokines (15,22).Based on ␤-cell-selective toxic effects, we hypothesized that IL-1 induces a rather complex pattern of both protective and deleterious events and mechanisms in islets cells, and that in ␤-cells the deleterious events seem to prevail (23). We further suggested that this might be reflected at the level of islet protein expression (24). To examine this hypothesis, we used 2D gel electrophoresis to produce a database of rat islet proteins containing about 2,200 protein spots characterized by molecular weight and isoelectric point (pI). The data presented here provide the first global assessment of the IL-1-mediated ␤-cell-damaging processes at the protein level. We could demonstra...

Characterization of a nuclear-factor-kappa B (NFκB) genetic marker in type 1 diabetes (T1DM) families

“…7 Several of these genes are putative targets for NFkB. 7,8 The action of NFkB can be of pro-or anti-apoptotic character, 9 but in beta-cells the action is predominantly pro-apoptotic. 3,7 A highly polymorphic CA-dinucleotide repeat microsatellite marker has been identified close to the NFKB1 gene, located at chromosome 4q24.…”

IL-1β induced protein changes in diabetes prone BB rat islets of Langerhans identified by proteome analysis

“…haeme oxygenases and manganese superoxide dismutase (MnSOD)] [5,6]. Many IL-1β effects are mediated by inducing the inducible nitric oxide syntase (iNOS) and its product, nitric oxide [7]; however, NO-independent mechanisms also occur [8,9,10]. We hypothesize that the beta cell, when exposed to IL-1β initiates a protective response in competition with a series of deleterious events, and that in beta cells the deleterious events prevail [3].…”

“…The majority of these proteins have been identified by mass spectrometry [8,10] and identified proteins were classified into the following groups on the basis of their known and perceived function: (i) energy transduction and redox potential; (ii) glycolytic enzymes; (iii) protein synthesis chaperones and protein folding; (iv) signal tranduction, regulation, differentiation and apoptosis suggesting a broad variety of pathways involved in IL-1β mediated islet toxicity [10].…”

“…Proteome analysis applied to WF rat islets exposed to IL-1β has provided a detailed picture of beta-cell destruction at the protein level [9,10]. Comparison of IL-1β induced pertubation of protein patterns (PIPP), i.e.…”

“…The aim of this study was to identify the proteins in these 82 spots by mass spectrometry and to compare the findings with protein identifications from IL-1β exposed WF islets [10] and to describe briefly their known functions with respect to the pathogenesis of Type I diabetes for some of the proteins.…”