Shotgun proteomic analysis and protein lysine acetylation in cytokine exposed human pancreatic islets

Background: The effect of cytokines on β-cell function and survival was studied in β-cells as INS1-E and a different role was suggested for INF-γ and IL-1 which act on activation of cellular defense mechanism and cell functions, respectively. In addition, pro-inflammatory cytokines also showed to induce β-cell apoptosis and endoplasmic reticulum stress. In order to investigate the effect of cytokines on human islets, the complete proteome before and after exposure to cytokines was analyzed using two complementary approaches the 2DE and label free shot-gun proteomics. In addition we investigated the presence of lysine-acetylation deregulation. Methods: We started to assay the effect of cytokines by 2DE before moving to shotgun approach. For 2DE we used 250 µg of human islets protein extracts (n=5), with and without cytokines treatment as previously described (reference). For shotgun aliquots of 40 µg of human islets protein extracts (n=3) were loaded onto 12% acrylamide resolving gel. After separation, gel pieces (13 pieces for lane) were excised from the gel and the proteins were identified by Shotgun methodology after an in-gel trypsin digestion. Mass spectrometry data were acquired according to the novel label free quantitation workflow developed by Bruker Daltonik. For purpose of lysine-acetylation, 2DE of human islets coupled with Western blot has been performed using specific anti-acetylated lysine antibody. Results: Around 3000 proteins were identified and out of 307 differentially expressed proteins, 184 resulted increased (among these chemokines, oxidative stress related proteins and immunoproteasome proteins) and 123 reduced (i.e cathepsines, antioxidant proteins, Krebs enzymes) after treatment with cytokines. Ingenuity pathways analysis highlighted the activation of upstream regulators such as STAT1 and 2, NFKb, JAK1, and inhibition of MAPK1, atypical chemokine receptor 2, transcription intermediary factor 1-alpha and small ubiquitin-related modifier 3. Concerning lysine-acetylation pattern we detected 151 spots. Overall, after exposure to cytokines, a general reduction of acetylation pattern come to light. In particular, 6 spots showed a statistical significant alteration of acetylation immunoreactivity. Mass spectrometry analysis allowed to identify these spots as: glutamate dehydrogenase, cathepsin D, ACAD9 and EIF4A1 showed a decrease in acetylation, while peroxiredoxin 3 and superoxide dismutase 1 where the only spots whose acetylation level was increased. Finally, the treatment with cytokines induced a significant decrease (-28±10%) of insulin secretion with respect to control, together with an increase of apoptotic beta cells and a reduction of volume density of grain of insulin. Conclusions: Overall our results show how the detrimental effects of cytokines treatment in human pancreatic islets could be associated to proteome and post translational modification changes. Further studies are necessary to clarify the correlation between this deregulation and type 1 diabetes features. Keywords: cytokines, human pancreatic islets, type-1 diabetes; lysine-acetylation; post-traslational modification Reference: Ciregia F, Giusti L, Ronci M, Bugliani M, Piga I, Pieroni L, Rossi C,Marchetti P, Urbani A, Lucacchini A. Glucagon-like peptide 1 protects INS-1E mitochondria against palmitate-mediated beta-cell dysfunction: a proteomic study. Mol Biosyst. 2015 Jun;11(6):1696-707.