Open AccessRegulated Production of Mature Insulin in Rat Hepatoma Cells: Insulin Production is Up‐regulated by Dexamethasone and Down‐regulated by Insulin
Author(s) -
QIN XinYu,
SHEN KunTang,
SONG LuJun,
ZHANG Xin,
HAN ZeGuang
Publication year - 2006
Publication title -
acta biochimica et biophysica sinica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.771
H-Index - 57
eISSN - 1745-7270
pISSN - 1672-9145
DOI - 10.1111/j.1745-7270.2006.00138.x
Subject(s) - insulin , proinsulin , medicine , endocrinology , transfection , cell culture , phosphoenolpyruvate carboxykinase , biology , downregulation and upregulation , reverse transcription polymerase chain reaction , gene expression , chemistry , gene , biochemistry , genetics
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 transcription‐polymerase chain reaction (RT‐PCR), respectively. The clone (Hepa1–6/INS21), which secreted the highest level of insulin (10.79 μIU/10 6 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. Edited by
Ming‐Hua XU