Abstract
Glucose-6-phosphatase catalyzes the terminal step in the gluconeogenic and glycogenolytic pathways. In HepG2 cells, the maximum repression of basal glucose-6-phosphatase catalytic subunit (G6Pase) gene transcription by insulin requires two distinct promoter regions, designated A (located between -231 and -199) and B (located between -198 and -159), that together form an insulin response unit. Region A binds hepatocyte nuclear factor-1, which acts as an accessory factor to enhance the effect of insulin, mediated through region B, on G6Pase gene transcription. We have previously shown that region B binds the transcriptional activator FKHR (FOXO1a) in vitro. Chromatin immunoprecipitation assays demonstrate that FKHR also binds the G6Pase promoter in situ and that insulin inhibits this binding. Region B contains three insulin response sequences (IRSs), designated IRS 1, 2, and 3, that share the core sequence T(G/A)TTTT. However, detailed analyses reveal that these three G6Pase IRSs are functionally distinct. Thus, FKHR binds IRS 1 with high affinity and IRS 2 with low affinity but it does not bind IRS 3. Moreover, in the context of the G6Pase promoter, IRS 1 and 2, but not IRS 3, are required for the insulin response. Surprisingly, IRS 3, as well as IRS 1 and IRS 2, can each confer an inhibitory effect of insulin on the expression of a heterologous fusion gene, indicating that, in this context, a transcription factor other than FKHR, or its orthologs, can also mediate an insulin response through the T(G/A)TTTT motif.
Highlights
Glucose-6-phosphatase catalyzes the final step in the glycogenolytic and gluconeogenic pathways, the hydrolysis of glucose-6-phosphate (G6P)1 to glucose and inorganic phosphate
One of these inhibitory insulin response sequences (IRSs) was identified through studies on the glucagon promoter [18, 19], whereas the other was first identified through studies on the phosphoenolpyruvate carboxykinase insulin response factor; CAT, chloramphenicol acetyltransferase; TK, thymidine kinase; GST, glutathione S-transferase; HGP, hepatic glucose production; IPTG, isopropyl-1-thio--D-galactopyranoside; WT, wild-type; TM, triple mutant; PKB, protein kinase B; SDM, site-directed mutation
We have previously shown that insulin has little effect on reporter gene expression directed by the native TK promoter, whereas ligation of an oligonucleotide representing the wildtype (WT) glucose6-phosphatase catalytic subunit (G6Pase) region B sequence between Ϫ197 and Ϫ159 into the TK promoter confers an insulin-dependent inhibition of fusion gene expression (Fig. 1B; Ref. 14)
Summary
G6P, glucose 6-phosphate; G6Pase, glucose-6-phosphatase catalytic subunit; HNF, hepatocyte nuclear factor; IRS, insulin response sequence; ChIP, chromatin immunoprecipitation; ER, endoplasmic reticulum; PEPCK, phosphoenolpyruvate carboxykinase; IGFBP-1, insulin-like growth factor binding protein-1; IRF,. (PEPCK) promoter [20, 21] This PEPCK IRS has the sequence TGTTTTG [21, 22]; similar elements were subsequently identified that mediate inhibitory effects of insulin on transcription of the genes encoding insulin-like growth factor-binding protein-1 (IGFBP-1) [23], tyrosine aminotransferase [24], and G6Pase [14]. 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) These studies support the hypothesis that FKHR and its orthologs are the insulin response factors that bind the PEPCK-like IRS motif and mediate the inhibitory effect of insulin on gene transcription through this element, other results are not consistent with this model.
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