Regulation by glucagon (cAMP) and insulin of the promoter of the human phosphoenolpyruvate carboxykinase gene (cytosolic) in cultured rat hepatocytes and in human hepatoblastoma cells

Abstract

A promoter fragment (-457 to +65) of the human cytosolic phosphoenolpyruvate carboxykinase gene, which by analogy to the rat promoter contains regulatory regions conferring glucagon (cAMP) and insulin responsiveness to the phosphoenolpyruvate carboxykinase gene, was cloned into a luciferase expression vector and transfected into cultured rat hepatocytes and human hepatoblastoma cells (HepG2) to study the regulation of the transgene by glucagon (cAMP) and insulin. A reporter gene that contained the rat promoter sequence from -493 to +33 was used for comparison. In cultured rat hepatocytes glucagon and its second messenger cAMP increased luciferase expression 4-6-fold over basal levels. Insulin reduced this effect by 40-70%. Luciferase expression was also stimulated by the combination of dexamethasone and cAMP in HepG2 cells and this effect was inhibited by insulin. The phosphoinositide 3-kinase (PI 3-kinase) inhibitor, wortmannin, abolished this action of insulin in cultured rat hepatocytes. The results show that the promoter of the human phosphoenolpyruvate carboxykinase gene mediates the stimulatory action of glucagon and its second messenger cAMP. The inhibitory action of insulin was exerted through the PI 3-kinase pathway in cultured rat hepatocytes.

Identification of a novel human glucagon receptor promoter: Regulation by cAMP and PGC-1α

“…2, there is some E-box like elements in the proximal promoter of the human glucagon receptor. Although others have shown HepG2 cells to be glucose responsive (Carriere et al, 2005;Elsas et al, 2002), in our hands this cell line was non-responsive to glucose when tested with a PEPCK promoter construct previously shown to be glucose responsive in primary hepatocytes (Cournarie et al, 1999;Rucktaschel et al, 2000) (data not shown). Thus, we were unable to examine the glucose regulation of the human glucagon receptor promoters in the HepG2 cells.…”