Functional expression of the human insulin gene in a human hepatoma cell line (HEP G2).
To develop a model somatic gene therapy system for diabetes, a human hepatoma cell line (HEP G2) was transfected with a mammalian expression vector carrying the full-length human insulin cDNA. More proinsulin than insulin was released daily by the stably transformed cell line (HEP G2ins). However, on acute stimulation with 5mM 8-Br-cAMP and 10mM theophylline the HEP G2ins cells released predominantly insulin into the medium. The cells did not secrete insulin in response to glucose. Examination of acid-ethanol extracts confirmed insulin was preferentially being stored. Immunohistochemical analysis of the cells also showed (pro)insulin was being stored. Electron microscopy revealed large membrane-bound vacuoles, containing electron-dense material, which were not seen in control cells. Glucokinase activity and albumin secretion of the transfectants were unaltered from the controls. Five-minute pulse-chase labelling of the HEP G2ins cells with 3H-leucine confirmed insulin synthesis in the presence of 20mM glucose and 5mM 8-Br-cAMP. A dose-response curve for insulin synthesis was also generated to increasing concentrations of glucose with a half Vmax of 4.9mM. Our results show that the introduction of insulin cDNA into a human hepatoma cell line results in synthesis, storage and acute regulated insulin release and lend credence to the possibility of engineering a liver cell to secrete insulin acutely in response to physiological stimuli.
Simpson
A M
AM
Department of Endocrinology, Prince of Wales Hospital, Randwick, NSW, Australia.
Tuch
B E
BE
Swan
M A
MA
Tu
J
J
Marshall
G M
GM
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D006528:Carcinoma, Hepatocellular; D002467:Cell Nucleus / Q000648:ultrastructure; D003593:Cytoplasm / Q000648:ultrastructure; D018076:DNA, Complementary / Q000235:genetics; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D005941:Glucokinase / Q000378:metabolism; D005947:Glucose / Q000494:pharmacology; D006593:Hexokinase / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D000078790:Insulin Secretion; D007700:Kinetics; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D011384:Proinsulin / Q000096:biosynthesis / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D013806:Theophylline / Q000494:pharmacology; D014162:Transfection; D014407:Tumor Cells, Cultured; D014617:Vacuoles
D018076:DNA, Complementary; D007328:Insulin; D012333:RNA, Messenger; D015124:8-Bromo Cyclic Adenosine Monophosphate; D011384:Proinsulin; D013806:Theophylline; D006593:Hexokinase; D005941:Glucokinase; D005947:Glucose