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The Carbohydrate response element binding protein, ChREBP encoded by the MLXIPL gene, is a transcription factor that is expressed at high levels in the liver and has a prominent function during consumption of high-carbohydrate diets. ChREBP is activated by raised cellular levels of phosphate ester intermediates of glycolysis, gluconeogenesis and the pentose phosphate pathway. Its target genes include a wide range of enzymes and regulatory proteins, including G6pc, Gckr, Pklr, Prkaa1,2, and enzymes of lipogenesis. ChREBP activation cumulatively promotes increased disposal of phosphate ester intermediates to glucose, via glucose 6-phosphatase or to pyruvate via glycolysis with further metabolism by lipogenesis. Dietary fructose is metabolized in both the intestine and the liver and is more lipogenic than glucose. It also induces greater elevation in phosphate ester intermediates than glucose, and at high concentrations causes transient depletion of inorganic phosphate, compromised ATP homeostasis and degradation of adenine nucleotides to uric acid. ChREBP deficiency predisposes to fructose intolerance and compromised cellular phosphate ester and ATP homeostasis and thereby markedly aggravates the changes in metabolite levels caused by dietary fructose. The recent evidence that high fructose intake causes more severe hepatocyte damage in ChREBP-deficient models confirms the crucial protective role for ChREBP in maintaining intracellular phosphate homeostasis. The improved ATP homeostasis in hepatocytes isolated from mice after chronic activation of ChREBP with a glucokinase activator supports the role of ChREBP in the control of intracellular homeostasis. It is hypothesized that drugs that activate ChREBP confer a protective role in the liver particularly in compromised metabolic states.

Aim To test the hypothesis that glucokinase activators (GKAs) induce hepatic adaptations that alter intra-hepatocyte metabolite homeostasis. Methods C57BL/6 mice on a standard rodent diet were treated with a GKA (AZD1656) acutely or chronically. Hepatocytes were isolated from the mice after 4 or 8 weeks of treatment for analysis of cellular metabolites and gene expression in response to substrate challenge. Results Acute exposure of mice to AZD1656 or a liver-selective GKA (PF-04991532), before a glucose tolerance test, or challenge of mouse hepatocytes with GKAs ex vivo induced various Carbohydrate response element binding protein (ChREBP) target genes, including Carbohydrate response element binding protein beta isoform (ChREBP-β), Gckr and G6pc. Both glucokinase activation and ChREBP target gene induction by PF-04991532 were dependent on the chirality of the molecule, confirming a mechanism linked to glucokinase activation. Hepatocytes from mice treated with AZD1656 for 4 or 8 weeks had lower basal glucose 6-phosphate levels and improved ATP homeostasis during high substrate challenge. They also had raised basal ChREBP-β mRNA and AMPK-α mRNA (Prkaa1, Prkaa2) and progressively attenuated substrate induction of some ChREBP target genes and Prkaa1 and Prkaa2. Conclusions Chronic GKA treatment of C57BL/6 mice for 8 weeks activates liver ChREBP and improves the resilience of hepatocytes to compromised ATP homeostasis during high-substrate challenge. These changes are associated with raised mRNA levels of ChREBP-β and both catalytic subunits of AMP-activated protein kinase.

International audience; The chronic effects of metformin on liver gluconeogenesisinvolve repression of theG6pcgene, which is regulated by thecarbohydrate-response element–binding protein throughraised cellular intermediates of glucose metabolism. In thisstudy we determined the candidate mechanisms by which met-formin lowers glucose 6-phosphate (G6P) in mouse and rathepatocytes challenged with high glucose or gluconeogenic pre-cursors. Cell metformin loads in the therapeutic range loweredcell G6P but not ATP and decreasedG6pcmRNA at high glu-cose. The G6P lowering by metformin was mimicked by a com-plex 1 inhibitor (rotenone) and an uncoupler (dinitrophenol)and by overexpression of mGPDH, which lowers glycerol3-phosphate and G6P and also mimics theG6pcrepression bymetformin. In contrast, direct allosteric activators of AMPK(A-769662, 991, and C-13) had opposite effects from metforminon glycolysis, gluconeogenesis, and cell G6P. The G6P loweringby metformin, which also occurs in hepatocytes from AMPKknockout mice, is best explained by allosteric regulation ofphosphofructokinase-1 and/or fructose bisphosphatase-1, assupported by increased metabolism of [3-3H]glucose relative to[2-3H]glucose; by an increase in the lactate m2/m1 isotopologratio from [1,2-13C2]glucose; by lowering of glycerol 3-phos-phate an allosteric inhibitor of phosphofructokinase-1; and bymarked G6P elevation by selective inhibition of phosphofruc-tokinase-1; but not by a more reduced cytoplasmic NADH/NADredox state. We conclude that therapeutically relevant doses of metformin lower G6P in hepatocytes challenged with high glu-cose by stimulation of glycolysis by an AMP-activated proteinkinase–independent mechanism through changes in allostericeffectors of phosphofructokinase-1 and fructose bisphospha-tase-1, including AMP, Pi, and glycerol 3-phosphate

Metformin therapy lowers blood glucose in type 2 diabetes by targeting various pathways including hepatic gluconeogenesis. Despite widespread clinical use of metformin the molecular mechanisms by which it inhibits gluconeogenesis either acutely through allosteric and covalent mechanisms or chronically through changes in gene expression remain debated. Proposed mechanisms include: inhibition of Complex 1; activation of AMPK; and mechanisms independent of both Complex 1 inhibition and AMPK. The activation of AMPK by metformin could be consequent to Complex 1 inhibition and raised AMP through the canonical adenine nucleotide pathway or alternatively by activation of the lysosomal AMPK pool by other mechanisms involving the aldolase substrate fructose 1,6-bisphosphate or perturbations in the lysosomal membrane. Here we review current interpretations of the effects of metformin on hepatic intermediates of the gluconeogenic and glycolytic pathway and the candidate mechanistic links to regulation of gluconeogenesis. In conditions of either glucose excess or gluconeogenic substrate excess, metformin lowers hexose monophosphates by mechanisms that are independent of AMPK-activation and most likely mediated by allosteric activation of phosphofructokinase-1 and/or inhibition of fructose bisphosphatase-1. The metabolite changes caused by metformin may also have a prominent role in counteracting G6pc gene regulation in conditions of compromised intracellular homeostasis.