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 label free shot-gun proteomics. Methods: Aliquots of 40 µg of human islets protein extracts (n=3), with and without cytokines treatment, 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. 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. 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 changes. Further studies are necessary to clarify the correlation between these deregulation and type 1 diabetes features. Keywords: cytokines, human pancreatic islets, type-1 diabetes.
SHOTGUN PROTEOMICS OF HUMAN PANCREATIC ISLETS: EFFECT OF CYTOKINES EXPOSURE
Laura GiustiUltimo
2018-01-01
Abstract
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 label free shot-gun proteomics. Methods: Aliquots of 40 µg of human islets protein extracts (n=3), with and without cytokines treatment, 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. 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. 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 changes. Further studies are necessary to clarify the correlation between these deregulation and type 1 diabetes features. Keywords: cytokines, human pancreatic islets, type-1 diabetes.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
