Insulin and Epidermal Growth Factor Suppress Basal Glucose-6- Phosphatase Catalytic Subunit Gene Transcription Through Overlapping but Distinct Mechanisms

In liver, glucose-6-phosphatase catalyses the final step in the gluconeogenic and glycogenolytic pathways, the hydrolysis of glucose-6-phosphate (G6P)1 to glucose and inorganic phosphate [1]. Glucose-6-phosphatase is located in the endoplasmic reticulum (ER) membrane and is thought to exist as a multi-component enzyme system in which a glucose-6-phosphatase catalytic subunit (G6Pase) has its catalytic site directed towards the lumen of the ER with a G6P transporter delivering substrate from the cytosol to the active site of the catalytic subunit and transporters for inorganic phosphate and glucose returning the reaction products back to the cytosol [1]. The glucose transporter remains to be identified but recent data suggest that a single transporter transports both G6P and inorganic phosphate [2]. G6Pase gene transcription is regulated by multiple factors, including some that stimulate gene expression (glucose, glucocorticoids, glucagon, fatty acids) and some that repress (insulin, TNFα and phorbol esters). In human HepG2 hepatoma cells, the inhibitory effect of insulin on basal mouse and human G6Pase gene transcription is mediated through a multi-component insulin response unit that is composed of two regions, designated Regions A and B [3]. In the mouse G6Pase promoter, Region A is located between −231 and −199, while Region B is located between −198 and −158, relative to the transcription start site. Region A binds hepatocyte nuclear factor-1 (HNF-1), which acts as an accessory factor to enhance the effect of insulin that is mediated through Region B [3]. Region B contains three insulin response sequences (IRSs), designated IRS 1, 2 & 3 [3] but only IRS 1 and 2 are required for the suppression of basal G6Pase gene expression by insulin [4]. Detailed mutagenesis experiments and chromatin immunoprecipitation (ChIP) assays have shown that FOXO1 is the transcription factor binding IRS 1 and 2 and mediating the effect of insulin [4, 5]. Insulin inhibits FOXO1-mediated transcriptional activation through the PI 3-kinase dependent activation of PKB, which leads to the phosphorylation and nuclear exclusion of this factor [6]. Overexpression of G6Pase is thought to contribute to the increased hepatic glucose production (HGP) characteristic of both type 1 and type 2 diabetes [7]. Indeed, overexpression of G6Pase in liver [8] is sufficient to stimulate HGP. Understanding how hormones/growth factors repress G6Pase gene expression may therefore be helpful in the design of strategies to treat patients with diabetes. With this goal in mind we have investigated the effect of epidermal growth factor (EGF) on G6Pase gene expression since it inhibits the expression of another gluconeogenic enzyme encoding gene, namely phosphoenolpyruvate carboxykinase (PEPCK) [9]. EGF signaling is extremely complex in that four homologous transmembrane receptors have been identified, designated ErbB1-4. These receptor tyrosine kinases have differing ligand specificities and are able to form both homo- and heterodimers [10, 11]. Adding to the complexity, ErbB2 lacks a ligand-binding domain whereas ErbB3 lacks an active intracellular tyrosine kinase domain [10, 11]. Finally, EGF is but one of a family of ligands for these receptors that also includes epiregulin, β-cellulin, amphiregulin, transforming growth factor-α, heparin binding EGF-like growth factor, and neuregulins; each ligand can bind different combinations of ErbB receptors [10, 11]. Our results show that EGF, like insulin, inhibits G6Pase fusion gene expression in HepG2 cells. However, EGF not only works through FOXO1 but also but through an additional promoter element that binds both FOXA2 and the glucocorticoid receptor. EGF also inhibits hepatic G6Pase gene expression in vivo but in cultured hepatocytes EGF has the opposite effect stimulating G6Pase gene expression, an observation that may be explained by a switch in ErbB receptor sub-type expression following hepatocyte isolation.