Low metformin causes a more oxidized mitochondrial NADH/NAD redox state in hepatocytes and inhibits gluconeogenesis by a redox-independent mechanism

Abstract

The mechanisms by which metformin (dimethylbiguanide) inhibits hepatic gluconeogenesis at concentrations relevant for type 2 diabetes therapy remain debated. Two proposed mechanisms are 1) inhibition of mitochondrial Complex 1 with consequent compromised ATP and AMP homeostasis or 2) 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 oxidized 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: <2 nmol/mg) caused a more oxidized 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) substrates by preferential partitioning of substrate toward glycolysis by a redoxindependent 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 malate-aspartate shuttle and changes in allosteric effectors of PFK1 and FBP1.

Mitochondrial targets of metformin—Are they physiologically relevant?

“…Finally, it was shown that low concentrations of metformin (≤2 nmol/mg of protein), which does not inhibit complex I, may attenuate gluconeogenesis by a redox-independent mechanism-allosteric regulation of phosphofructokinase-1 (PFK1) and/or fructose bisphosphatase-1 (FBP-1). 13 This study is also in direct disagreement with observations of Madiraju et al, 17,37 which proposed redox dependent mechanism and ascribed the shift in NADH/NAD + ratio to inhibition of mGPDH (see above).…”

“…Metformin action on mGPDH has also been addressed using isolated mouse hepatocytes, where biphasic effect of metformin on mitochondrial NADH/NAD + redox state was observed. 13 Low doses of the drug (<2 nmol/mg) resulted in more oxidized mitochondrial NADH/NAD + state and increased lactate to pyruvate ratio, whereas higher doses (>5 nmol/mg) induced more reduced NADH/NAD + expected for complex I inhibition. However, the authors implied that the effect of low doses of metformin cannot be explained by complex I inhibition or AMPK activation and they suggested inhibition of gluconeogenesis via redox independent mechanismallosteric regulation of phosphofructokinase-1 and/or fructose bisphosphatase-1 in conjunction with decrease of glycerol-3-phosphate and increase in lactate to pyruvate ratio.…”

“…Metformin action on mGPDH has also been addressed using isolated mouse hepatocytes, where biphasic effect of metformin on mitochondrial NADH/NAD + redox state was observed . Low doses of the drug (<2 nmol/mg) resulted in more oxidized mitochondrial NADH/NAD + state and increased lactate to pyruvate ratio, whereas higher doses (>5 nmol/mg) induced more reduced NADH/NAD + expected for complex I inhibition.…”

“…Similarly, at the molecular level, several mechanisms of metformin action have been suggested. These include inhibition of adenylate cyclase, alterations of mitochondrial fission, or changes of allosteric effectors of phosphofructokinase‐1 or fructose bisphosphatase‐1 . Nevertheless, the most frequently associated target is AMPK.…”

“…These include inhibition of adenylate cyclase, 11 alterations of mitochondrial fission, 12 or changes of allosteric effectors of phosphofructokinase-1 or fructose bisphosphatase-1. 13 Nevertheless, the most frequently associated target is AMPK. Metformin was proposed to activate AMPK directly, 4 through its upstream regulator LKB1 14 or act as an inhibition of mitochondrial oxidative phosphorylation and activate AMPK indirectly as a consequence of associated decrease in energy charge.…”