Mechanisms of toxicity by proinflammatory cytokines in a novel human pancreatic beta cell line, 1.1B4.

Srividya Vasu, Neville McClenaghan, Janie McCluskey, Peter Flatt

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

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-1β, IFN-γ, TNF-α.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 Ca2+, GLP-1 or forskolin. Glucokinase enzyme activity, regulation of intracellular Ca2+ 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.
LanguageEnglish
Pages136-145
Number of pages10
JournalBIOCHIMICA ET BIOPHYSICA ACTA
Volume1840
Issue number1
Early online date1 Sep 2013
DOIs
Publication statusPublished - 31 Jan 2014

Fingerprint

Insulin-Secreting Cells
Cytokines
Cell Line
Glucokinase
Insulin
Nitrites
Proto-Oncogene Proteins c-bcl-2
Oxidative Stress
Caspase 7
Glucagon-Like Peptide 1
Comet Assay
DNA Fragmentation
Colforsin
Caspases
Caspase 3
Alanine
Reverse Transcription
Radioimmunoassay
Culture Media
Tail

Cite this

Vasu, Srividya ; McClenaghan, Neville ; McCluskey, Janie ; Flatt, Peter. / Mechanisms of toxicity by proinflammatory cytokines in a novel human pancreatic beta cell line, 1.1B4. 2014 ; Vol. 1840, No. 1. pp. 136-145.
@article{35daf0dc07834e468e78ad8a98d1647c,
title = "Mechanisms of toxicity by proinflammatory cytokines in a novel human pancreatic beta cell line, 1.1B4.",
abstract = "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-1β, IFN-γ, TNF-α.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 Ca2+, GLP-1 or forskolin. Glucokinase enzyme activity, regulation of intracellular Ca2+ 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.",
author = "Srividya Vasu and Neville McClenaghan and Janie McCluskey and Peter Flatt",
year = "2014",
month = "1",
day = "31",
doi = "10.1016/j.bbagen.2013.08.022",
language = "English",
volume = "1840",
pages = "136--145",
number = "1",

}

Mechanisms of toxicity by proinflammatory cytokines in a novel human pancreatic beta cell line, 1.1B4. / Vasu, Srividya; McClenaghan, Neville; McCluskey, Janie; Flatt, Peter.

Vol. 1840, No. 1, 31.01.2014, p. 136-145.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Mechanisms of toxicity by proinflammatory cytokines in a novel human pancreatic beta cell line, 1.1B4.

AU - Vasu, Srividya

AU - McClenaghan, Neville

AU - McCluskey, Janie

AU - Flatt, Peter

PY - 2014/1/31

Y1 - 2014/1/31

N2 - 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-1β, IFN-γ, TNF-α.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 Ca2+, GLP-1 or forskolin. Glucokinase enzyme activity, regulation of intracellular Ca2+ 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.

AB - 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-1β, IFN-γ, TNF-α.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 Ca2+, GLP-1 or forskolin. Glucokinase enzyme activity, regulation of intracellular Ca2+ 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.

U2 - 10.1016/j.bbagen.2013.08.022

DO - 10.1016/j.bbagen.2013.08.022

M3 - Article

VL - 1840

SP - 136

EP - 145

IS - 1

ER -