Metformin lowers glucose 6-phosphate in hepatocytes by activation of glycolysis downstream of glucose phosphorylation

Tabassum Moonira,Shruti S Chachra,Brian E Ford,Silvia Marin,Ahmed Alshawi,Natasha S Adam-Primus,Catherine Arden,Ziad H Al-Oanzi,Marc Foretz,Benoit Viollet,Marta Cascante,Loranne Agius

doi:10.1074/jbc.ra120.012533

Abstract

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) and 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, and C-13) had opposite effects from metformin on glycolysis, gluconeogenesis, and cell G6P. The G6P lowering by metformin, which also occurs in hepatocytes from AMPK knockout mice, is best explained by allosteric regulation of phosphofructokinase-1 and/or fructose bisphosphatase-1, as supported by increased metabolism of [3-3H]glucose relative to [2-3H]glucose; by an increase in the lactate m2/m1 isotopolog 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 AMP-activated protein kinase–independent mechanism through changes in allosteric effectors of phosphofructokinase-1 and fructose bisphosphatase-1, including AMP, Pi, and glycerol 3-phosphate.

Highlights

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
The G6pc gene is of particular interest because it was identified as a component of the metformin mechanism in both animal diabetes and in man by nontargeted approaches [11,12,13] and because G6pc is regulated by the transcription factor ChREBP [14], which is activated by raised cellular phosphorylated intermediates of glucose metabolism in conditions of raised blood glucose or compromised intracellular homeostasis, resulting in raised glucose 6-phosphate, G6P4 (14 –17)
We show in this study that metformin lowers G6P in hepatocytes challenged with gluconeogenic precursors or high glucose by increased flux through glycolysis, downstream of G6P

Summary

Introduction

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. Metformin lower G6P in hepatocytes challenged with high glucose by stimulation of glycolysis by an AMP-activated protein kinase–independent mechanism through changes in allosteric effectors of phosphofructokinase-1 and fructose bisphosphatase-1, including AMP, Pi, and glycerol 3-phosphate. Its therapeutic effect involves inhibition of glucose absorption by the gut and inhibition of glucose production by the liver [1] Evidence for the latter mechanism in man is largely derived from chronic studies demonstrating efficacy after 2–26 weeks therapy [2]. Suppression of hepatic glucose production during chronic therapy may involve either acute inhibition of gluconeogenic flux or chronic changes in gene expression [3,4,5], with various arguments in support of the latter One such argument is a lack of acute effect of intravenously administered metformin on hepatic glucose production in man (6 –8). Various sets of evidence support lowering of G6P by increased glycolysis via allosteric effectors of phosphofructokinase-1

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