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

Abstract

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

Adult rat liver cells transdifferentiated with lentiviral IPF1 vectors reverse diabetes in mice: an ex vivo gene therapy approach

“…This has stimulated efforts to expand existing pancreatic islets or grow new ones. As beta cells have an extremely low replication rate [1], transplantation of non-beta cells that produce insulin has become a reasonable alternative [2][3][4][5][6][7][8][9][10][11][12].…”

Glucose-stimulated and self-limiting insulin production by glucose 6-phosphatase promoter driven insulin expression in hepatoma cells

“…The promoter of PEPCK (phosphoenolpyruvate carboxykinase) is insulin suppressible and has been used to direct insulin expression in order to achieve the feedback control of insulin production. 25,26 However, its activity is inhibited by glucose, 27 the opposite of what is required to direct insulin expression. The glucose inducible promoter of L-PK (liver-type pyruvate kinase) has been used to achieve insulin production in response to glucose.…”

“…The insulin inhibitory effect on insulin driving promoters has been assessed either by measuring insulin production with or without phosphatidylinositide 3-kinase inhibitors (eg Wortmannin), which block the insulin receptor-mediated signaling pathway, 34 or by measuring insulin mRNA levels in insulin-expressing cells cultured with/without exogenous insulin. 25,26 However, blockage of the insulin-signaling pathway may not be complete or sufficient for the repression of G6Pase expression by insulin and may result in unexpected metabolic changes, which may not reflect conditions observed in the absence of insulin. Exogenous insulin added to the culture could not be maintained at the original concentration over time since hepatocytes actively degrade insulin.…”

Glucose regulates secretion of exogenously expressed insulin from HepG2 cells in vitro and in a mouse model of diabetes mellitus in vivo

“…Previous attempts to mimic pancreatic function in regulating insulin have used nonislet cells, including hepatocytes, to express and secrete insulin (Lu et al 1998, Chen et al 2005, Wilson et al 2005, Hsu et al 2008. The general approach involved use of a glucose-responsive promoter or motif to control insulin gene transcription , Chen et al 2001, Alam & Sollinger 2002, Hsu et al 2008).…”