Regulated Production of Mature Insulin in Rat Hepatoma Cells:Insulin Production is Up-regulated by Dexamethasone and Down-regulated by Insulin

摘要

We engineered an artificial β cell line that produces an up-regulation of insulin in response to dexamethasone, and a down-regulation in response to insulin. A regulatory secretion system was devised by placing proinsulin cDNA containing genetically engineered furin endoprotease cleavage sites and a regulatory promoter for phosphoenolpyruvate carboxykinase (PEPCK), and an insulin expressing retrovirus vector (pN-PEPCK-mINS) was constructed and transfected into Hepa1-6 cells. The levels of insulin in culture medium and expression of insulin gene was estimated by radioimmunoassay and reverse transcriptionpolymerase chain reaction (RT-PCR), respectively. The clone (Hepa1-6/INS21), which secreted the highest level of insulin (10.79 μIU/106 cells per day), was selected for the regulation experiment. Compared with the non-treated Hepa1-6/INS21 cells, insulin production was augmented 3.6-fold by the addition of 10-7 M of dexamethasone. Addition of exogenous insulin to the culture medium decreased insulin mRNA expression remarkably on RT-PCR results, while dexamethasone increased insulin gene expression at the transcriptional level. The data indicated that genetically engineered Hepa1-6 cells could synthesize process and secrete insulin in a physiological manner.