Regulation of Glucose-6-phosphatase Gene Expression by Protein Kinase Bα and the Forkhead Transcription Factor FKHR

Abstract

Glucose-6-phosphatase plays an important role in the regulation of hepatic glucose production, and insulin suppresses glucose-6-phosphatase gene expression. Recent studies indicate that protein kinase B and Forkhead proteins contribute to insulin-regulated gene expression in the liver. Here, we examined the role of protein kinase B and Forkhead proteins in mediating effects of insulin on glucose-6-phosphatase promoter activity. Transient transfection studies with reporter gene constructs demonstrate that insulin suppresses both basal and dexamethasone/cAMP-induced activity of the glucose-6-phosphatase promoter in H4IIE hepatoma cells. Both effects are partially mimicked by coexpression of protein kinase B␣. Coexpression of the Forkhead transcription factor FKHR stimulates the glucose-6-phosphatase promoter activity via interaction with an insulin response unit (IRU), and this activation is suppressed by protein kinase B. Coexpression of a mutated form of FKHR that cannot be phosphorylated by protein kinase B abolishes the regulation of the glucose-6-phosphatase promoter by protein kinase B and disrupts the ability of insulin to regulate the glucose-6-phosphatase promoter via the IRU. Mutation of the insulin response unit of the glucose-6-phosphatase promoter also prevents the regulation of promoter activity by FKHR and protein kinase B but only partially impairs the ability of insulin to suppress both basal and dexamethasone/ cAMP-stimulated promoter function. Taken together, these results indicate that signaling by protein kinase B to Forkhead proteins can account for the ability of insulin to regulate glucose-6-phosphatase promoter activity via the IRU and that other mechanisms that are independent of the IRU, protein kinase B, and Forkhead proteins also are important in mediating effects of in insulin on glucose-6-phosphatase gene expression.Glucose-6-phosphatase (Glc-6-Pase) 1 catalyzes the hydrolysis of glucose 6-phosphate to glucose, which is the terminal step of both hepatic gluconeogenesis and glycogen breakdown. Glc-6-Pase is induced in starved and diabetic animals (1, 2). In vitro models have shown that glucocorticoids and cAMP induce Glc-6-Pase gene expression. This effect is opposed by insulin, which also is able to reduce basal expression of the Glc-6-Pase gene (3-6). Identification of the signaling events that connect the insulin receptor to the Glc-6-Pase promoter, leading to the subsequent repression of gene transcription, is of particular interest because Glc-6-Pase plays a key role in the regulation of hepatic glucose production and blood glucose homeostasis.In H4IIE hepatoma cells, activation of class 1a phosphoinositide 3-kinase (PI 3-kinase), but not of the Ras/Raf/MAP kinase pathway, is necessary for the suppression of Glc-6-Pase promoter activity by insulin (6). The formation of PtdIns(3,4,5)P 3 catalyzed by PI 3-kinase has been shown to increase the activity of 3-phosphoinositide-dependent protein kinase-1 (PDK1) and to result in a conformational change in PKB which renders it susceptible...

The Three Insulin Response Sequences in the Glucose-6-phosphatase Catalytic Subunit Gene Promoter Are Functionally Distinct

“…IRSs Does Not-Results from a number of studies suggest that, when overexpressed, FKHR can mediate insulin repression of IGFBP-1 and G6Pase gene transcription through their PEPCK-like IRS motifs (32,34,38,60). In addition, we have previously shown that recombinant FKHR can bind an oligonucleotide representing the wild-type G6Pase region B sequence, but not an oligonucleotide containing point mutations in each of the three region B IRS motifs ( Fig.…”

“…Subsequently, it was shown that FKHR can bind the PEPCK, IGFBP-1, and G6Pase IRSs in vitro (16,(32)(33)(34). In addition, FKHR was shown to stimulate PEPCK, IGFBP-1, and G6Pase fusion gene expression (32,34,35).…”

“…Subsequently, it was shown that FKHR can bind the PEPCK, IGFBP-1, and G6Pase IRSs in vitro (16,(32)(33)(34). In addition, FKHR was shown to stimulate PEPCK, IGFBP-1, and G6Pase fusion gene expression (32,34,35). It has been proposed that insulin inhibits FKHR-, FKHRL1-, and AFX-mediated transcriptional activation through the phosphatidylinositol 3-kinase-dependent activation of PKB, which leads to the phosphorylation and nuclear exclusion of these factors (33, 36 -42).…”

Convergence of Peroxisome Proliferator-activated Receptor γ and Foxo1 Signaling Pathways

“…Recent evidence suggests that the phosphorylation status of Foxo1 might also regulate transcriptional activation properties in addition to regulating cytolocalization (24). Insulin-induced inactivation of Foxo1 appears to be one of the mechanisms through which insulin suppresses gluconeogenic gene expression as phosphoenolpyruvate carboxykinase and glucose-6-phosphatase, two enzymes involved in gluconeogenesis, are encoded by Foxo1 target genes (25,26). While regulation of phosphoenolpyruvate carboxykinase via this mechanism is controversial, recent evidence from Foxo1Ϯ mice supports such a mechanism for regulation of glucose-6-phosphatase (27).…”

Regulation of hepatic glucose production and AMPK by AICAR but not by metformin depends on drug uptake through the equilibrative nucleoside transporter 1 (ENT1)

“…The cAMP analogue dibutyryl cAMP (bucladesine, db‐cAMP), a cell‐permeable stabilized cAMP mimic that also inhibits phosphodiesterase (PDE) activity, is commonly used 6, 7. Treatment of cells with db‐cAMP causes a significant stimulation of PEPCK and G6‐Pase expression, which is inhibited by the addition of insulin in a dose‐dependent manner 8, 9, 10. 8‐(4‐chlorophenylthio)cAMP (8CPT‐cAMP), like db‐cAMP, is a membrane‐permeable cAMP analogue that stimulates PEPCK and G‐6‐Pase 4, 11.…”