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Regulatable production of insulin from primary-cultured hepatocytes: insulin production is up-regulated by glucagon and cAMP and down-regulated by insulin

Regulatable production of insulin from primary-cultured hepatocytes: insulin production is... To utilize hepatocytes for insulin-producing surrogate cells, we devised a regulatory secretion system by placing proinsulin DNA under the regulatable promoter for phosphoenolpyruvate carboxykinase (PEPCK). The expression of PEPCK is down-regulated by insulin, and up-regulated by cAMP and glucagon. To express insulin in hepatocytes, we constructed an adenoviral insulin expression system. After infection, the hepatocytes secreted immunoreactive insulin (IRI) at an increasing rate. IRI secretion increased over four-fold upon stimulation with 300 μM cAMP and 500 μM of the cAMP-dependent phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). This increase was also observed with glucagon and IBMX. Production was augmented two-fold by the addition of wortmannin, a phosphatidylinositol (PI)-3-kinase inhibitor, suggesting that inhibitory insulin signaling to the PEPCK promoter may be mediated through PI-3-kinase. Addition of exogenous insulin to the culture decreased insulin mRNA expression remarkably on Northern blot. Thus, by using a PEPCK promoter for insulin expression, we were able to up-regulate insulin production from hepatocytes with cAMP and glucagon, and down-regulate with insulin itself. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Gene Therapy Springer Journals

Regulatable production of insulin from primary-cultured hepatocytes: insulin production is up-regulated by glucagon and cAMP and down-regulated by insulin

Gene Therapy , Volume 5 (7) – Jul 10, 1998

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References (48)

Publisher
Springer Journals
Copyright
Copyright © 1998 by Macmillan Publishers Limited
Subject
Biomedicine; Biomedicine, general; Human Genetics; Cell Biology; Nanotechnology; Gene Therapy; Gene Expression
eISSN
1476-5462
DOI
10.1038/sj.gt.3300677
Publisher site
See Article on Publisher Site

Abstract

To utilize hepatocytes for insulin-producing surrogate cells, we devised a regulatory secretion system by placing proinsulin DNA under the regulatable promoter for phosphoenolpyruvate carboxykinase (PEPCK). The expression of PEPCK is down-regulated by insulin, and up-regulated by cAMP and glucagon. To express insulin in hepatocytes, we constructed an adenoviral insulin expression system. After infection, the hepatocytes secreted immunoreactive insulin (IRI) at an increasing rate. IRI secretion increased over four-fold upon stimulation with 300 μM cAMP and 500 μM of the cAMP-dependent phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). This increase was also observed with glucagon and IBMX. Production was augmented two-fold by the addition of wortmannin, a phosphatidylinositol (PI)-3-kinase inhibitor, suggesting that inhibitory insulin signaling to the PEPCK promoter may be mediated through PI-3-kinase. Addition of exogenous insulin to the culture decreased insulin mRNA expression remarkably on Northern blot. Thus, by using a PEPCK promoter for insulin expression, we were able to up-regulate insulin production from hepatocytes with cAMP and glucagon, and down-regulate with insulin itself.

Journal

Gene TherapySpringer Journals

Published: Jul 10, 1998

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