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Functional expression of the human insulin gene in a human hepatoma cell line (HEP G2).

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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Gene therapy Pubmed

Functional expression of the human insulin gene in a human hepatoma cell line (HEP G2).

Gene therapy , Volume 2 (3): 9 – Aug 24, 1995

Functional expression of the human insulin gene in a human hepatoma cell line (HEP G2).


Abstract

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.

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ISSN
0969-7128
pmid
7614254

Abstract

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.

Journal

Gene therapyPubmed

Published: Aug 24, 1995

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