Browsing by Person "Vasu, Srividya"

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Cellular responses of novel human pancreatic _-cell line, 1.1B4 to hyperglycemia
(Taylor & Francis, 2013-08-28) Vasu, Srividya; McClenaghan, Neville H.; McCluskey, Jane T.; Flatt, Peter R.
The novel human-derived pancreatic _-cell line, 1.1B4 exhibits insulin secretion and _-cell enriched gene expression. Recent investigations of the cellular responses of this novel cell line to lipotoxicity and cytokine toxicity revealed similarities to primary human _ cells. The current study has investigated the responses of 1.1B4 cells to chronic 48 and 72 h exposure to hyperglycemia to probe mechanisms of human _-cell dysfunction and cell death. Exposure to 25 mM glucose significantly reduced insulin content (p < 0.05) and glucokinase activity (p < 0.01) after 72 h. Basal insulin release was unaffected but acute secretory response to 16.7 mM glucose was impaired (p < 0.05). Insulin release stimulated by alanine, GLP-1, KCl, elevated Ca2+ and forskolin was also markedly reduced after exposure to hyperglycemia (p < 0.001). In addition, PDX1 protein expression was reduced by 58% by high glucose (p < 0.05). Effects of hyperglycemia on secretory function were accompanied by decreased mRNA expression of INS, GCK, PCSK1, PCSK2, PPP3CB, GJA1, ABCC8, and KCNJ11. In contrast, exposure to hyperglycemia upregulated the transcription of GPX1, an antioxidant enzyme involved in detoxification of hydrogen peroxide and HSPA4, a molecular chaperone involved in ER stress response. Hyperglycemia-induced DNA damage was demonstrated by increased % tail DNA and olive tail moment, assessed by comet assay. Hyperglycemia-induced apoptosis was evident from increased activity of caspase 3/7 and decreased BCL2 protein. These observations reveal significant changes in cellular responses and gene expression in novel human pancreatic 1.1B4 _ cells exposed to hyperglycemia, illustrating the usefulness of this novel human-derived cell line for studying human _-cell biology and diabetes.
Effects of lipotoxicity on a novel insulin-secreting human pancreatic _-cell line, 1.1B4
(De Gruyter, 2013-07-01) Vasu, Srividya; McClenaghan, Neville H.; McCluskey, Jane T.; Flatt, Peter R.
The novel insulin-secreting human pancreatic _-cell line, 1.1B4, demonstrates stability in culture and many of the secretory functional attributes of human pancreatic _-cells. This study investigated the cellular responses of 1.1B4 cells to lipotoxicity. Chronic 18-h exposure of 1.1B4 cells to 0.5 mm palmitate resulted in decreased cell viability and insulin content. Secretory responses to classical insulinotropic agents and cellular Ca2+ handling were also impaired. Palmitate decreased glucokinase activity and mRNA expression of genes involved in secretory function but up-regulated mRNA expression of HSPA5, EIF2A, and EIF2AK3, implicating activation of the endoplasmic reticulum stress response. Palmitate also induced DNA damage and apoptosis of 1.1B4 cells. These responses were accompanied by increased gene expression of the antioxidant enzymes SOD1, SOD2, CAT and GPX1. This study details molecular mechanisms underlying lipotoxicity in 1.1B4 cells and indicates the potential value of the novel _-cell line for future research.
Mechanisms of toxicity by proinflammatory cytokines in a novel human pancreatic beta cell line, 1.1B4.
(Elsevier, 2014-01-01) Vasu, Srividya; McClenaghan, Neville H.; McCluskey, Jane T.; Flatt, Peter R.
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.