Analysis of gene expression and insulin secretion by monolayer-forming adult porcine pancreatic endocrine cells.

Abstract

We recently established a method of isolation and primary monolayer culture of porcine pancreatic endocrine cells that involves the use of nicotinamide. To obtain genetic information on cultured porcine endocrine cells and to examine cell function in relation to insulin secretion during long-term culture. Gene expression of insulin and several transcription factors, including PDX-1, Beta2/NeuroD, Pax6, and Nkx6.1, was assessed by reverse transcription-polymerase chain reaction analysis, and the insulin protein level was estimated by immunohistochemistry and enzyme assay during a 12-week period. During the culture period, insulin accumulation in the medium at 5 weeks had decreased by almost half the level of accumulation in the first week. In contrast to the alteration of secretory function, insulin gene expression was maintained for at least 12 weeks, and regulatory transcription factors were expressed at the same levels until 9 weeks. These observations suggest that gene expression is not involved in the cause of decreased baseline insulin secretion. Moreover, although the insulin response to high glucose and potassium loading was maintained, the magnitude of the responses to both stimuli was attenuated in the late period of culture. Insulin secretion tended to decrease in our culture system, and the secretory response to pharmacological stimulation was attenuated despite maintenance of messenger RNA expression of insulin and other islet-specific genes for at least 9 weeks in vitro. These findings indicate that cell integrity is maintained and that the alteration in insulin secretion must be explained by another mechanism.