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

Abstract

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 l-3 fmoll106 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 1% fold by l-500 nM dexamethasone together with CAMP and IBMX. Addition of exogenous insulin to the culture medium significantly decreased insulin mRNA expression on Northern blot.

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

“…9,10,15 As expected, the hepatocytes infected with the adenoviral insulin expression vector produced mature insulin as a major fraction and proinsulin as a minor fraction (Figure 7a). Since we did not make an adenoviral insulin expression vector carrying native insulin gene, we could not compare the processing between native and mutated proinsulin in hepatocytes.…”

“…We also used a mutant proinsulin DNA carrying furin-cleavable processing sites, 9-11 expression of which resulted in the production of biologically active mature insulin in hepatocytes ( Figure 7). By using these insulin expression vectors, we were successful in achieving regulatable production of insulin from H4IIE cells, 15 however, we could not obtain enhanced production of insulin from the H4IIE using glucagon, which is known to increase the cAMP level in hepatocytes. In severe diabetes, a high level of plasma glucagon is invariably observed, and this level is decreased by insulin therapy, 43 therefore, regulation of insulin production by glucagon is preferable.…”

“…By a 50 MOI, IRI was highly produced, but the production plateaued after 48 h. To examine the effect of dbcAMP and IBMX, we used the optimal amounts previously reported for the H4IIE, which were 300 m dbcAMP and 500 m IBMX. 15 The addition of these doses to the culture medium enhanced IRI production at all time-points for hepatocytes infected with all three MOI numbers of AdP 3 ␤Ins (Figure 2a). By 2 and 50 MOI, the slope of increase became steeper after 24 h. By 50 MOI, the increase became smaller toward 72 h. In contrast, the increase was not observed in hepatocytes infected with any MOI of AdP 0 ␤Ins (Figure 2b).…”

“…[9][10][11] For regulating insulin production from non-neuroendocrine cells, we utilized a phosphoenolpyruvate carboxykinase (PEPCK) promoter. 15 Because these cells do not carry secretory granules but secrete proteins and peptides through a constitutive pathway without being retained in the cytoplasm, the regulation of insulin production is limited to the gene transcription level. Production of a bioactive peptide from its gene activation requires more time than does exocytosis in neuroendocrine cells.…”

“…We previously examined insulin production by expressing a PEPCK promoter plus insulin DNA construct in the rat hepatoma cell line H4IIE. 15 H4IIE cells secreted immunoreactive insulin (IRI), which we increased five-fold using cAMP and the cAMP-dependent phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), and 18-fold by the further addition of dexamethasone. The addition of exogenous insulin to the culture medium significantly decreased insulin mRNA expression in H4IIE cells.…”