Dietetics, Nutrition and Biological Sciences
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Item Dietary sources of vitamin D in school children in Northern Ireland(The Nutrition Society, 2021-08-17) Benson, H.; Glatt, Dominique; Beggan, L.; McSorley, E. M.; Pourshahidi, L. K.; McCluskey, Jane T.; Revuelta-Iniesta, Raquel; Gleeson, Nigel; Magee, P. J.Item 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.Item 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.Item Comparison of Insulin Release From MIN6 Pseudoislets and Pancreatic Islets of Langerhans Reveals Importance of Homotypic Cell Interactions(Wolters Luwer, 2010-10) Kelly, Catriona; Guo, Hong; McCluskey, Jane T.; Flatt, Peter R.; McClenaghan, Neville H.OBJECTIVES: Cellular communication is required for normal patterns of insulin secretion from _ cells. Experiments using isolated islets of Langerhans are hampered by lack of supply and the consuming isolation process. Pseudoislets comprising clonal cells have emerged as an alternative to study islet-cell interactions and insulin secretion. The current study compared MIN6 pseudoislets and freshly isolated mouse islets. METHODS: Insulin content and release were measured by insulin radioimmunoassay. Reverse transcription polymerase chain reaction and Western blot analysis of adhesion molecule expression were performed on MIN6 monolayers and pseudoislets. MIN6 cellular proliferation and viability were measured by 5-bromo-2-deoxyuridine (BrdU) enzyme-linked immunosorbent assay, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and lactate dehydrogenase assays. RESULTS: Mouse islets were found to have greater insulin content than pseudoislets. However, insulin release was comparable between the 2 groups. With the use of MIN6 monolayers as a control, the expression of the adhesion molecule E-cadherin and connexin 36 were found to be enhanced in cells cultured as pseudoislets. Moreover, connexin 43 was shown to be absent from MIN6 cells irrespective of configuration. Finally, MIN6 pseudoislets seem able to manage their rate of proliferation with apoptosis resulting in a static size in the culture for extended periods. CONCLUSIONS: The current study found that MIN6 pseudoislets share many important functional and molecular features with islets of Langerhans.Item Daily administration of the GIP-R antagonist (Pro3)GIP in streptozotocin-induced diabetes suggests that insulin-dependent mechanisms are critical to anti-obesity-diabetes actions of (Pro3)GIP(Wiley, 2008-04) McClean, P. L.; Gault, V. A.; Irwin, N.; McCluskey, Jane T.; Flatt, P. R.AIM: Glucose-dependent insulinotropic polypeptide-receptor (GIP-R) antagonism using (Pro3)GIP improves glucose tolerance and ameliorates insulin resistance and abnormalities of islet structure and function in a commonly used model of obesity-diabetes, namely ob/ob mice. The effect of GIP-R antagonism in a streptozotocin (STZ)-induced model of insulin deficiency has not been evaluated. The present study has investigated the effects of daily administration of (Pro(3))GIP to STZ-treated mice. METHODS: Swiss TO mice received once-daily injection of (Pro3)GIP (25 nmol/kg body weight) or saline 4 days prior to and 16 days after injection of STZ, and effects on metabolic parameters and islet architecture were assessed. RESULTS: (Pro3)GIP treatment had no significant effect on hyperphagia or body weight loss. However, hyperglycaemia and glycated haemoglobin were worsened, glucose tolerance further decreased and insulin sensitivity was impaired by (Pro3)GIP. These effects were observed on an STZ-induced background characterized by severe reductions of circulating insulin, beta-cell mass and pancreatic insulin stores. CONCLUSIONS: These data indicate that the beneficial actions of the GIP-R antagonist, (Pro3)GIP, in obesity-diabetes appear to be largely mediated through insulin-dependent mechanisms that merit further investigation.Item The role of glucagon- and somatostatin-secreting cells in the regulation of insulin release and beta-cell function in heterotypic pseudoislets(Wiley, 2010-08-18) Kelly, Catriona; Parke, Hong Guo; McCluskey, Jane T.; Flatt, Peter R.; McClenaghan, Neville H.BACKGROUND: Pseudoislet studies have concentrated on single beta-cell lines or a combination of insulin and glucagon-secreting cells, overlooking the potential role of somatostatin in insulin release. This study sought to evaluate a heterotypic pseudoislet model containing insulin- (MIN6), glucagon- (TC1.9) and somatostatin (TGP52)-secreting cells of mouse origin and to compare these pseudoislets with traditional monolayer preparations. METHODS: Cellular viability (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and lactate dehydrogenase assays), proliferation (5-bromo-2-deoxyuridine ELISA), hormone content and functional insulin release in response to a variety of stimuli were measured. Differential expression of E-cadherin, connexin 36 and connexin 43 was assessed by reverse transcriptase-polymerase chain reaction and Western blot to determine a possible role for adherens in insulin release from these pseudoislets. RESULTS: All pseudoislet cells displayed reduced proliferation coupled with an increase in cell death which may contribute to their static size in culture. While MIN6 and TGP52 cells expressed E-cadherin and showed sustained or improved hormone content when configured as pseudoislets, TC1.9 lacked E-cadherin and contained less glucagon following pseudoislet formation. MIN6 and TC1.9 cells expressed connexin 36, but not connexin 43 and TGP52 cells expressed connexin 43 only. In the presence of Alanine, Arginine and glucagon-like peptide-1, heterotypic pseudoislet cultures secreted levels of insulin that were comparable to that of MIN6 pseudoislets. In addition, pseudoislets comprising all three cell lines released more insulin into the surrounding culture medium than MIN6 pseudoislets when studied over a 1-week period. CONCLUSIONS: The current model may prove useful in studying the role of islet cell interactions in the release of insulin from pancreatic islets.Item 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.Item Beneficial effects of parenteral GLP-1 delivery by cell therapy in insulin-deficient streptozotocin diabetic mice(Springer Nature, 2013-11) Vasu, S.; Moffett, R. C.; McCluskey, Jane T.; Hamid, M. H.; Irwin, N.; Flatt, P. R.Parenteral delivery of long-acting glucagon-like peptide-1 (GLP-1) mimetics has received much attention as a therapeutic option for diabetes. However, cell therapy-based GLP-1 treatments may provide a more physiological regulation of blood glucose. The present study assessed the effects of chronic GLP-1 delivery by cell therapy, using the GLP-1-secreting GLUTag cell line, in normoglycemic and streptozotocin-induced diabetic mice. GLUTag cell aggregates were transplanted into the subscapular region of mice. Over 30 days, cellular transplantation gave rise to encapsulated and well-vascularized growths, which contained immunoreactive GLP-1. Cell implantation was well tolerated and had no appreciable metabolic effects in normal mice. However, transplantation significantly (P<0.001) countered excessive food and fluid intake in diabetic mice and maintained normal body weight. Circulating glucose (P<0.01) and glucagon (P<0.05) were significantly reduced and plasma insulin and GLP-1 dramatically increased. This was associated with significantly (P<0.01) improved glucose tolerance in diabetic mice. Histological examination of the pancreata of these mice revealed elevations (P<0.001) in islet and _-cell area, with reduced (P<0.001) -cell area. Increased _-cell mass reflected the enhanced proliferation relative to apoptosis. These studies emphasize the potential of chronic GLP-1 delivery by cell therapy as a potential therapeutic option for diabetes.Item Development and Functional Characterization of Insulin-releasing Human Pancreatic Beta Cell Lines Produced by Electrofusion(2011) McCluskey, Jane T.; Hamid, M.; Guo-Parke, H.; McClenaghan, N. H.; Gomis, R.; Flatt, P. R.Three novel human insulin-releasing cell lines designated 1.1B4, 1.4E7, and 1.1E7 were generated by electrofusion of freshly isolated of human pancreatic beta cells and the immortal human PANC-1 epithelial cell line. Functional studies demonstrated glucose sensitivity and responsiveness to known modulators of insulin secretion. Western blot, RT-PCR, and immunohistochemistry showed expression of the major genes involved in proinsulin processing and the pancreatic beta cell stimulus-secretion pathway including PC1/3, PC2, GLUT-1, glucokinase, and K-ATP channel complex (Sur1 and Kir6.2) and the voltage-dependent L-type Ca(2+) channel. The cells stained positively for insulin, and 1.1B4 cells were used to demonstrate specific staining for insulin, C-peptide, and proinsulin together with insulin secretory granules by electron microscopy. Analysis of metabolic function indicated intact mechanisms for glucose uptake, oxidation/utilization, and phosphorylation by glucokinase. Glucose, alanine, and depolarizing concentrations of K(+) were all able to increase [Ca(2+)](i) in at least two of the cell lines tested. Insulin secretion was also modulated by other nutrients, hormones, and drugs acting as stimulators or inhibitors in normal beta cells. Subscapular implantation of the 1.1B4 cell line improved hyperglycemia and resulted in glucose lowering in streptozotocin-diabetic SCID mice. These novel human electrofusion-derived beta cell lines therefore exhibit stable characteristics reminiscent of normal pancreatic beta cells, thereby providing an unlimited source of human insulin-producing cells for basic biochemical studies and pharmacological drug testing plus proof of concept for cellular insulin replacement therapy.Item Configuration of electrofusion-derived human insulin-secreting cell line as pseudoislets enhances functionality and therapeutic utility(2012-09-01) Guo-Parke, H.; McCluskey, Jane T.; Kelly, C.; Hamid, M.; McClenaghan, N. H.; Flatt, P. R.Formation of pseudoislets from rodent cell lines has provided a particularly useful model to study homotypic islet cell interactions and insulin secretion. This study aimed to extend this research to generate and characterize, for the first time, functional human pseudoislets comprising the recently described electrofusion-derived insulin-secreting 1.1B4 human _-cell line. Structural pseudoislets formed readily over 3-7 days in culture using ultra-low-attachment plastic, attaining a static size of 100-200 _m in diameter, corresponding to ~6000 _ cells. This was achieved by decreases in cell proliferation and integrity as assessed by BrdU ELISA, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, and lactate dehydrogenase assays. Insulin content was comparable between monolayers and pseudoislets. However, pseudoislet formation enhanced insulin secretion by 17- to 125-fold in response to acute stimulation with glucose, amino acids, incretin hormones, or drugs compared with equivalent cell monolayers. Western blot and RT-PCR showed expression of key genes involved in cell communication and the stimulus-secretion pathway. Expression of E-Cadherin and connexin 36 and 43 was greatly enhanced in pseudoislets with no appreciable connexin 43 protein expression in monolayers. Comparable levels of insulin, glucokinase, and GLUT1 were found in both cell populations. The improved secretory function of human 1.1B4 cell pseudoislets over monolayers results from improved cellular interactions mediated through gap junction communication. Pseudoislets comprising engineered electrofusion-derived human _ cells provide an attractive model for islet research and drug testing as well as offering novel therapeutic application through transplantation.