Flux through the MA-shuttle involves an electrogenic transporter for aspartate and is highly dependent on mitochondrial membrane potential (45,C48). mitochondrial NADH/NAD state and an increase in lactate/pyruvate ratio, whereas a higher metformin dose (5 nmol/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) Rabbit Polyclonal to Cytochrome P450 19A1 substrates by preferential partitioning of substrate toward glycolysis by a redox-independent mechanism that is best explained by allosteric regulation at phosphofructokinase-1 (PFK1) and/or fructose 1,6-bisphosphatase (FBP1) in association with a decrease in cell glycerol 3-phosphate, 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 malateCaspartate shuttle and changes in allosteric effectors of PFK1 and FBP1. (19, 20) but not in isolated hepatocytes. The aims of this study were, first, to test whether metformin has a dose-dependent effect on the mitochondrial NADH/NAD ratio in hepatocytes Lexibulin dihydrochloride and, second, to explore the mechanism(s) by which a low metformin dose that is within the therapeutic range, affects gluconeogenesis and compare this with inhibition and/or stimulation of transfer of NADH-reducing equivalents from the cytoplasm to the mitochondria by the GP-shuttle or the malateCaspartate shuttle (MA-shuttle). We report that clinically relevant doses of metformin cause a more oxidized mitochondrial NADH/NAD redox state and a more reduced cytoplasmic redox state but inhibit gluconeogenesis from oxidized substrates. This is best explained by a redox-independent mechanism involving allosteric regulation at the level of PFK1 and/or FBP1 that is in part explained by a decrease in cell glycerol 3-phosphate, an inhibitor of PFK1. Results Biphasic effect of metformin on the mitochondrial redox state: more oxidized at low metformin Studies showed that metformin causes either a more reduced (10) or a more oxidized (19, 20) mitochondrial NADH/NAD redox state in liver based on the ratio of 3-hydroxybutyrate/acetoacetate that correlates with the mitochondrial NADH/NAD ratio through the hydroxybutyrate dehydrogenase equilibrium (22). Our first aim was to determine whether metformin (100C500 m) has a dose-dependent effect on the mitochondrial NADH/NAD redox state in hepatocytes incubated with octanoate. This medium-chain fatty acid enters the mitochondria as the free acid by a mechanism independent of regulation by malonyl-CoA and thereby AMPK activity and is metabolized predominantly to acetoacetate and 3-hydroxybutyrate. We used 100 m as the lowest metformin concentration because in hepatocytes incubated with Lexibulin dihydrochloride 100 m metformin for 2C4 h, metformin accumulates in the cells to 1C2 nmol/mg protein (23). This is within the range observed in mouse liver after an oral dose of 50 mg metformin/kg body weight (24). At the highest concentration (500 m), metformin accumulates to 5C10 nmol/mg (23) in rat and mouse hepatocytes. In both mouse and rat hepatocytes, high metformin (500 m) increased the ratio of 3-hydroxybutyrate/acetoacetate as did rotenone, the Complex I inhibitor (Fig. 1, and and and and and and and and = 8C14 hepatocyte preparations; *, 0.05 relative to control. and and and = 5C7. *, 0.05 relative to respective control; $, metformin or DNP effect. = 4 mouse hepatocyte preparations. *, 0.05 relative to respective control; $, 0.05 octanoate effect. Metformin causes greater inhibition of glucose production from dihydroxyacetone than from glycerol Having confirmed that low metformin (100 m) causes a more oxidized mitochondrial NADH/NAD redox state without inhibiting ketone body production, we next determined the effects of 100 m metformin on glucose production from oxidized (dihydroxyacetone (DHA)) and reduced (glycerol and xylitol) substrates. Glucose production was significantly higher from DHA than from glycerol (Fig. 2). Metformin inhibited glucose production from both oxidized (DHA) and reduced (xylitol and 0.25 mm glycerol) substrates, and it increased the production of lactate and pyruvate with both DHA and xylitol (Fig. 2, and 0.05 relative to DHA; $, metformin effect. Low metformin, but not inhibitors of the NADH shuttles, favors metabolism of DHA and xylitol to glycolysis relative to glucose To test whether inhibition of glucose production by low metformin can be explained by inhibition of NADH shuttles, we compared 100 Lexibulin dihydrochloride m metformin with aminooxyacetate (AOA), an inhibitor of the MA-shuttle (27), or GPi (STK017597, GPI), a recently.
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