Properties and functions of a human islet cell line
A major goal for the treatment of diabetes is the generation of a replenishable source of human β-cells for transplantation. A study of PHHI identified a β-like cell line that could grow and divided in culture and expressed detectable levels of insulin. Firstly this study has characterised the properties and functions of the Nes2Y(P) cell in terms of insulin expression and β-cell phenotype, by RT-PCR and immunocytochemistry. Nes2Y(P) cells could proliferate both rapidly and indefinitely in culture. Major β-cell phenotypic markers including GLUT2, NeuroD and Nkx6.1 were also detected. Insulin expression however, was dramatically reduced at the level of mRNA, insulin release and intracellular stores. This led to the conclusion that in their native state, Nes2Y(P) cells lost their differentiated function in continuous sub-culture and were of limited use as an insulin secreting cell line for therapeutic purposes. It also highlighted the importance of a detailed understanding of those factors that regulate β-cell differentiation, of which the homeodomain transcription factor PDX1 plays a particularly important role. PDX1 is involved in pancreas development, growth and differentiation of islet cells and in regulation of insulin gene expression. This study used immunocytochemistry to investigate the nuclear translocation of PDX1 in response to extracellular stimuli. PDX1 was located in the cytoplasm, predominantly associated with the nuclear membrane in non-stimulatory conditions. Rapid nuclear translocation could be induced through stimulation with glucose, insulin and stress agents. Moreover, PDX1 shuttled between the nucleus and cytoplasm in response to fluctuating glucose conditions. Specific kinase inhibitors were used to characterise the signalling pathway that links PDX1 activation and nuclear translocation. Nuclear import was inhabitable by wortmannin and SB203580, suggesting a role for PtdIns 3-kinase and SAPK2. Nuclear export of PDX1 was inhabitable by phosphatase inhibitors, indicating the action of a nuclear phosphatase. In summary, the results presented in this study have important implications for a greater knowledge of the mechanisms through which nutrients regulate insulin gene expression in β-cells.