Regulated production of mature insulin in rat hepatoma cells: insulin production is up-regulated by dexamethasone and down-regulated by insulin.
We engineered an artificial beta 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 microIU/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.
Qin
Xin-Yu
XY
Department of General Surgery, Zhongshan Hospital, Fudan University, Shanghai 200032, China. xyqin@zshospital.com
Shen
Kun-Tang
KT
Song
Lu-Jun
LJ
Zhang
Xin
X
Han
Ze-Guang
ZG
D000818:Animals; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular / Q000473:pathology; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D003907:Dexamethasone / Q000494:pharmacology*; D015536:Down-Regulation; D045683:Furin / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects*; D005822:Genetic Vectors / Q000235:genetics; D007328:Insulin / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D011863:Radioimmunoassay; D051381:Rats; D012190:Retroviridae / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000187:drug effects* / Q000378:metabolism; D015854:Up-Regulation
D018076:DNA, Complementary; D007328:Insulin; D012333:RNA, Messenger; D003907:Dexamethasone; D045683:Furin; D010729:Phosphoenolpyruvate Carboxykinase (GTP)