Regulatable production of mature insulin from a hepatocyte cell line: Insulin production is up-regulated by cAMP and glucocorticoids, and down-regulated by insulin

We engineered a hepatoma cell line that produces an up-regulation of insulin in response to cAMP, dexamethasone, and retinoic acid, and a down-regulation in response to insulin. We devised a regulatory secretion system by placing proinsulin DNA under the regulatable promoter for phosphoenolpyruvate carboxykinase (PEPCK). To assess the ability to regulate insulin secretion, we used the rat hepatoma cell line, H4IIE. The H4IIE cells secreted immunoreactive insulin (IRI) constantly at a level of 1-3 fmol/10(6) cells/h. IRI increased approximately two-fold upon stimulation with 0.5 mM cAMP and five-fold with the addition of the cAMP-dependent phosphodiesterase inhibitor IBMX, as compared to baseline IRI secretion. IRI increased 18-fold by 1-500 nM dexamethasone together with cAMP and IBMX. Addition of exogenous insulin to the culture medium significantly decreased insulin mRNA expression on Northern blot.