Effect of Stimulators such as GLP-1, PACAP, and Nicotinamide on Glucose-stimulated Insulin Secretion from Porcine Pancreatic Endocrine Cells in Long-term Culture.

摘要

INTRODUCTION: Transplantation of glucose-responsive insulin-secreting cells has the potential to result in a cure for diabetes. AIM: To report the development of a model of adult porcine pancreatic endocrine cells (PE cells) exhibiting glucose-stimulated insulin secretion during a long-term culture period, in vitro. METHODOLOGY: The PE cells were prepared by non-enzymatic digestion and purified by modifying a technique developed in our laboratory. The cells were first cultured for 7 days in RPMI-1640 medium containing 10% fetal bovine serum and 10 mM nicotinamide. On adhesion to the culture flasks, cells were collected by trypsinization, and then cultured in tissue culture dishes in medium with or without stimulators such as glucagon-like peptide-1 (GLP-1), pituitary adenylate cyclase-activating polypeptide (PACAP), and nicotinamide. The ability of the cells to respond to glucose-stimulated insulin secretion was also observed with and without stimulators. The immunocytochemical studies demonstrated pancreatic islets with well-preserved insulin and glucagon-containing cells. The morphologic integrity of cultured porcine cells was observed for up to 5-6 weeks after the purification. RESULTS: At a concentration of 3.3 mM glucose, PACAP and nicotinamide did not affect glucose-dependent insulin secretion, whereas 10 nM GLP-1 stimulated insulin secretion significantly. However, when glucose concentration was increased to 20 mM, 10 nM GLP-1 had no effect on insulin secretion. We also demonstrated that GLP-1 and PACAP could maintain insulin secretion better than control in the culture up to 5 weeks. Also GLP-1 and PACAP increased the number of insulin-secreting cells in culture. CONCLUSION: These results demonstrate that GLP-1 and PACAP increased the number of pancreatic beta-cells in culture.