The chronic effects of metformin on liver gluconeogenesis involve repression of the G6pc gene, which is regulated by the Carbohydrate response element binding protein through raised cellular intermediates of glucose metabolism. In this study we determined the candidate mechanisms by which metformin lowers glucose 6-phosphate (G6P) in mouse and rat hepatocytes challenged with high glucose or gluconeogenic precursors. Cell metformin loads in the therapeutic range lowered cell G6P but not ATP and decreased G6pc mRNA at high glucose. The G6P lowering by metformin was mimicked by a Complex 1 inhibitor (rotenone), an uncoupler (dinitrophenol) and by overexpression of mGPDH, which lowers glycerol 3-phosphate and G6P and also mimics the G6pc repression by metformin. In contrast, direct allosteric activators of AMPK (A-769662, 991, C13) had opposite effects from metformin on glycolysis, gluconeogenesis and cell G6P. The G6P lowering by metformin which also occurs in hepatocytes from AMPK-knock-out mice, is best explained by allosteric regulation of phosphofructokinase-1 (PFK-1) and/or fructose bisphosphatase-1, as supported by: increased metabolism of [3-3H]glucose relative to [2-3H]glucose; an increase in lactate m2/m1 isotopologue ratio from [1,2-13C2]glucose, by lowering of glycerol 3-phosphate an allosteric inhibitor of phosphofructokinase-1 and by marked G6P elevation by selective inhibition of phosphofructokinase-1 but not by a more reduced cytoplasmic NADH/NAD redox state. We conclude that therapeutically relevant doses of metformin lower G6P in hepatocytes challenged with high glucose by stimulation of glycolysis by an AMPK-independent mechanism through changes in allosteric effectors of PFK-1 and fructose bisphosphatase-1, including AMP, inorganic phosphate and glycerol 3-phosphate.

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