Mechanisms of toxicity by proinflammatory cytokines in a novel human pancreatic beta cell line, 1.1B4.
McClenaghan, Neville H.
McCluskey, Jane T.
Flatt, Peter R.
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Vasu, S., McClenaghan, N., McCluskey, J. & Flatt, P. (2014) Mechanisms of toxicity by proinflammatory cytokines in a novel human pancreatic beta cell line, 1.1B4., Biochimica et biophysica acta, vol. 1840, , pp. 136-45,
BACKGROUND Molecular mechanisms of toxicity and cell damage were investigated in the novel human beta cell line, 1.1B4, after exposure to proinflammatory cytokines - IL-1_, IFN-_, TNF-. METHODS MTT assay, insulin radioimmunoassay, glucokinase assay, real time reverse transcription PCR, western blotting, nitrite assay, caspase assay and comet assay were used to investigate mechanisms of cytokine toxicity. RESULTS Viability of 1.1B4 cells decreased after 18h cytokine exposure. Cytokines significantly reduced cellular insulin content and impaired insulin secretion induced by glucose, alanine, KCl, elevated Ca(2+), GLP-1 or forskolin. Glucokinase enzyme activity, regulation of intracellular Ca(2+) and PDX1 protein expression were significantly reduced by cytokines. mRNA expression of genes involved in secretory function - INS, GCK, PCSK2 and GJA1 was downregulated in cytokine treated 1.1B4 cells. Upregulation of transcription of genes involved in antioxidant defence - SOD2 and GPX1 was observed, suggesting involvement of oxidative stress. Cytokines also upregulated transcriptions of NFKB1 and STAT1, which was accompanied by a significant increase in NOS2 transcription and accumulation of nitrite in culture medium, implicating nitrosative stress. Oxidative and nitrosative stresses induced apoptosis was evident from increased % tail DNA, DNA fragmentation, caspase 3/7 activity, apoptotic cells and lower BCL2 protein expression. CONCLUSIONS This study delineates molecular mechanisms of cytokine toxicity in 1.1B4 cells, which agree with earlier observations using human islets and rodent beta cells. GENERAL SIGNIFICANCE This study emphasizes the potential usefulness of this cell line as a human beta cell model for research investigating autoimmune destruction of pancreatic beta cells.