Metformin,phenformin, and galegine inhibit complex IV activity and reduce glycerol-derived gluconeogenesis

Metformin exerts its plasma glucose-lowering therapeutic effect primarily through inhibition of hepatic gluconeogenesis. However, the precise molecular mechanism by which metformin inhibits hepatic gluconeogenesis is still unclear. Although inhibition of mitochondrial complex I is frequently invoked as metformin’s primary mechanism of action, the metabolic effects of complex I inhibition have not been thoroughly evaluated in vivo. Here, we show that acute portal infusion of piericidin A, a potent and specific complex I inhibitor, does not reduce hepatic gluconeogenesis in vivo. In contrast, we show that metformin, phenformin, and galegine selectively inhibit hepatic gluconeogenesis from glycerol. Specifically, we show that guanides/biguanides interact with complex IV to reduce its enzymatic activity, leading to indirect inhibition of glycerol-3-phosphate (G3P) dehydrogenase (GPD2), increased cytosolic redox, and reduced glycerol-derived gluconeogenesis. We report that inhibition of complex IV with potassium cyanide replicates the effects of the guanides/biguanides in vitro by selectively reducing glycerol-derived gluconeogenesis via increased cytosolic redox. Finally, we show that complex IV inhibition is sufficient to inhibit G3P-mediated respiration and gluconeogenesis from glycerol. Taken together, we propose a mechanism of metformin action in which complex IV–mediated inhibition of GPD2 reduces glycerol-derived hepatic gluconeogenesis.

Metformin (1,1-dimethylbiguanide) is the standard first-line pharmaceutical intervention for type 2 diabetes mellitus (T2D) and is one of the most widely prescribed drugs worldwide (1, 2). Metformin and other more potent synthetic guanide/biguanide derivatives, such as phenformin (N-phenethylbiguanide), have glucose-lowering effects in patients with T2D. Following oral administration, metformin accumulates to a high degree within the liver due to first-pass uptake in the portal vein following absorption from the gut, and the presence of the organic cation transporter 1 (OCT1) in the sinusoidal endothelial cells of the liver (3–7). This is in contrast to skeletal and cardiac muscle, where OCT1 is not highly expressed. The observed glucose-lowering effects in individuals with poorly controlled T2D can mostly be attributed to inhibition of hepatic gluconeogenesis, as opposed to altering insulin sensitivity or secretion (8–13); however, despite the extensive literature spanning several decades examining metformin’s effects in vivo and in vitro, a consensus on metformin’s precise mechanism of action still does not exist.The most well-studied mechanism is complex I inhibition, which is central to several frequently invoked mechanisms of metformin action, including adenosine monophosphate (AMP)-activated protein kinase activation, decreased energy charge ([adenosine triphosphate {ATP}]:[adenosine diphosphate {ADP}] and [ATP]:[AMP] ratios), and AMP inhibition of fructose 1,6-bisphosphatase, among others (14–18). Yet, complex I inhibition is only observed at suprapharmacological concentrations (>1 mM) of metformin, which is severalfold higher than concentrations achieved in vivo (3, 7, 19, 20). Furthermore, no study to date has convincingly demonstrated that complex I inhibition can, in fact, replicate metformin’s glucose-lowering effects in vivo. To address this question, we sought to specifically inhibit complex I activity in vivo to determine whether the metabolic effects of impaired complex I activity resemble those observed with metformin.We and others have previously proposed an alternative mechanism of metformin action, in which alterations in hepatic redox state and inhibition of glycerol-3-phosphate dehydrogenase (GPD2) potentiate metformin’s glucose-lowering effects (20–22). GPD2 is central to the α-glycerophosphate shuttle, one of two redox shuttles, which transfers reducing equivalents from the cytosol to the mitochondrial matrix. Specifically, GPD2 transfers electrons to mitochondrial ubiquinone, generating ubiquinol that is reoxidized by complex III (ubiquinol cytochrome c reductase) of the electron transport chain (ETC) (23). Thus, excess mitochondrial ubiquinol decreases GPD2 activity and increases reducing equivalents in the cytosol, which can be experimentally represented by an increased [lactate]:[pyruvate] ratio (referred to here as increased cytosolic redox state). Increased cytosolic redox is predicted to selectively reduce gluconeogenesis from reduced substrates (e.g., lactate and glycerol), while gluconeogenesis from nonreduced substrates (e.g., alanine, dihydroxyacetone phosphate [DHAP], and pyruvate) is unaffected (24), which is in contrast to a complex I-dependent mechanism of metformin action. This is consistent with metformin’s effects in both humans and rodents (12, 20, 25).Clinical studies have shown that the glucose-lowering effects of metformin in patients with fasting hyperglycemia, due to poorly controlled T2D, can mostly be attributed to reductions in hepatic glucose production (HGP); however, these effects are not consistently observed in normoglycemic individuals (8, 26, 27). These paradoxical effects provide insights into potential mechanisms of metformin action in humans: Individuals with poorly controlled T2D have dysregulated white adipose tissue (WAT) lipolysis, leading to increased flux of fatty acids and glycerol delivery to the liver, the latter of which increases hepatic gluconeogenesis through a substrate push mechanism (28–33). Accordingly, selective inhibition of glycerol-derived gluconeogenesis, due to increased cytosolic redox, may explain metformin’s paradoxical effects, but this has not yet been demonstrated in vivo.Here, we examined whether targeted inhibition of complex I using piericidin A, a specific and irreversible inhibitor of mitochondrial nicotinamide adenine dinucleotide reduced (NADH)–ubiquinone oxidoreductase (complex I), is sufficient to mediate metformin’s glucose-lowering effects in vitro and in vivo. We also examine the effects of metformin, two more potent guanides/biguanides (phenformin and galegine [isoamylene guanidine]), and piericidin A on glycerol-derived gluconeogenesis and cytosolic redox state in liver slices, as well as their effects on [¹³C₃]glycerol incorporation into [¹³C₃]glucose in awake rats with indwelling intraportal catheters. Finally, we investigated a mechanism of metformin/phenformin/galegine action through cytochrome c oxidase (complex IV)-mediated inhibition of GPD2 activity, which, in turn, can explain guanide/biguanide effects to increase the hepatic cytosolic redox state and reduce glycerol-derived hepatic gluconeogenesis and glycerol-3-phosphate (G3P)-dependent mitochondrial oxidation.