The mechanisms by which metformin (dimethylbiguanide) inhibits hepatic gluconeogenesis at concentrations relevant for type 2 diabetes therapy remain debated. Two proposed mechanisms are: inhibition of mitochondrial Complex 1 with consequent compromised ATP and AMP homeostasis; or inhibition of mitochondrial glycerophosphate dehydrogenase (mGPDH) and thereby attenuated transfer of reducing equivalents from the cytoplasm to mitochondria resulting in a raised lactate/pyruvate ratio and redox-dependent inhibition of gluconeogenesis from reduced but not oxidised substrates. Here we show that metformin has a biphasic effect on the mitochondrial NADH/NAD redox state in mouse hepatocytes. A low cell dose of metformin (therapeutic equivalent: < 2nmol / mg) caused a more oxidized mitochondrial NADH/NAD state and an increase in lactate / pyruvate ratio, whereas a higher metformin dose (³5nmol/mg) caused a more reduced mitochondrial NADH/NAD state similar to Complex 1 inhibition by rotenone. The low metformin dose inhibited gluconeogenesis from both oxidized (dihydroxyacetone) and reduced (xylitol) substrates by preferential partitioning of substrate towards glycolysis by a redox-independent mechanism that is best explained by allosteric regulation at phosphor-fructokinase-1 (PFK1) and/or fructose bisphosphatase-1 (FBP-1) in association with a decrease in cell glycerol 3-P, an inhibitor of PFK1 rather than by inhibition of transfer of reducing equivalents. We conclude that at a low pharmacological load, the metformin effects on the lactate / pyruvate ratio and glucose production are explained by attenuation of transmitochondrial electrogenic transport mechanisms with consequent compromised malate-aspartate shuttle and changes in allosteric effectors of PFK1 and FBP1. Read more »