Browsing by Person "Kelly, Catriona"

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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.
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.