Glucose metabolism in the liver activates the transcription of various genes encoding enzymes of glycolysis and lipogenesis and also G6pc (glucose-6-phosphatase). Allosteric mechanisms involving glucose 6-phosphate or xylulose 5-phosphate and covalent modification of ChREBP (carbohydrate-response element-binding protein) have been implicated in this mechanism. However, evidence supporting an essential role for a specific metabolite or pathway in hepatocytes remains equivocal. By using diverse substrates and inhibitors and a kinase-deficient bisphosphatase-active variant of the bifunctional enzyme PFK2/FBP2 (6-phosphofructo-2-kinase-fructose-2,6-bisphosphatase), we demonstrate an essential role for fructose 2,6-bisphosphate in the induction of G6pc and other ChREBP target genes by glucose. Selective depletion of fructose 2,6-bisphosphate inhibits glucose-induced recruitment of ChREBP to the G6pc promoter and also induction of G6pc by xylitol and gluconeogenic precursors. The requirement for fructose 2,6-bisphosphate for ChREBP recruitment to the promoter does not exclude the involvement of additional metabolites acting either co-ordinately or at downstream sites. Glucose raises fructose 2,6-bisphosphate levels in hepatocytes by reversing the phosphorylation of PFK2/FBP2 at Ser32, but also independently of Ser32 dephosphorylation. This supports a role for the bifunctional enzyme as the phosphometabolite sensor and for its product, fructose 2,6-bisphosphate, as the metabolic signal for substrate-regulated ChREBP-mediated expression of G6pc and other ChREBP target genes.

OBJECTIVEThe induction of hepatic glucose 6-phosphatase (G6pc) by glucose presents a paradox of glucose-induced glucose intolerance. We tested whether glucose regulation of liver gene expression is geared toward intracellular homeostasis.RESEARCH DESIGN AND METHODSThe effect of glucose-induced accumulation of phosphorylated intermediates on expression of glucokinase (Gck) and its regulator Gckr was determined in hepatocytes. Cell ATP and uric acid production were measured as indices of cell phosphate homeostasis.RESULTSAccumulation of phosphorylated intermediates in hepatocytes incubated at elevated glucose induced rapid and inverse changes in Gck (repression) and Gckr (induction) mRNA concomitantly with induction of G6pc, but had slower effects on the Gckr-to-Gck protein ratio. Dynamic metabolic labeling in mice and liver proteome analysis confirmed that Gckr and Gck are low-turnover proteins. Involvement of Max-like protein X in glucose-mediated Gck-repression was confirmed by chromatin immunoprecipitation analysis. Elevation of the Gck-to-Gckr ratio in hepatocytes was associated with glucose-dependent ATP depletion and elevated urate production confirming compromised phosphate homeostasis.CONCLUSIONSThe lowering by glucose of the Gck-to-Gckr ratio provides a potential explanation for the impaired hepatic glucose uptake in diabetes. Elevated uric acid production at an elevated Gck-to-Gckr ratio supports a role for glucose regulation of gene expression in hepatic phosphate homeostasis.

dIn the liver, a high glucose concentration activates transcription of genes encoding glucose 6-phosphatase and enzymes for glycolysis and lipogenesis by elevation in phosphorylated intermediates and recruitment of the transcription factor ChREBP (carbohydrate response element binding protein) and its partner, Mlx, to gene promoters. A proposed function for this mechanism is intracellular phosphate homeostasis. In extrahepatic tissues, MondoA, the paralog of ChREBP, partners with Mlx in transcriptional induction by glucose. We tested for glucose induction of regulatory proteins of the glycogenic pathway in hepatocytes and identified the glycogen-targeting proteins, G L and PTG (protein targeting to glycogen), as being encoded by Mlx-dependent glucose-inducible genes. PTG induction by glucose was MondoA dependent but ChREBP independent and was enhanced by forced elevation of fructose 2,6-bisphosphate and by additional xylitol-derived metabolites. It was counteracted by selective depletion of fructose 2,6-bisphosphate with a bisphosphatase-active kinase-deficient variant of phosphofructokinase 2/fructosebisphosphatase 2, which prevented translocation of MondoA to the nucleus and recruitment to the PTG promoter. We identify a novel role for MondoA in the liver and demonstrate that elevated fructose 2,6-bisphosphate is essential for recruitment of MondoA to the PTG promoter. Phosphometabolite activation of MondoA and ChREBP and their recruitment to target genes is consistent with a mechanism for gene regulation to maintain intracellular phosphate homeostasis.