Metformin promotes lifespan through mitohormesis via the peroxiredoxin PRDX-2

Abstract

The antiglycemic drug metformin, widely prescribed as first-line treatment of type II diabetes mellitus, has lifespan-extending properties. Precisely how this is achieved remains unclear. Via a quantitative proteomics approach using the model organism Caenorhabditis elegans, we gained molecular understanding of the physiological changes elicited by metformin exposure, including changes in branched-chain amino acid catabolism and cuticle maintenance. We show that metformin extends lifespan through the process of mitohormesis and propose a signaling cascade in which metformin-induced production of reactive oxygen species increases overall life expectancy. We further address an important issue in aging research, wherein so far, the key molecular link that translates the reactive oxygen species signal into a prolongevity cue remained elusive. We show that this beneficial signal of the mitohormetic pathway is propagated by the peroxiredoxin PRDX-2. Because of its evolutionary conservation, peroxiredoxin signaling might underlie a general principle of prolongevity signaling.

Fig. 1.

Metformin treatment does not induce the mitochondria-specific unfolded protein response (UPR^mt). Day-1 adult metformin-treated hsp-6::GFP worms show no difference in fluorescence compared with untreated control worms. hsp-6::GFP worms were exposed to cco-1 RNAi as a positive control. cco-1 encodes a cytochrome c oxidase subunit, an integral part of the mitochondrial electron transport chain (24).

Fig. 2.

Metformin increases lifespan according to the principle of mitohormesis, for which it requires PRDX-2. (A) Metformin treatment increases metabolic heat production (**P < 0.01; n = 3 for untreated and n = 4 for treated worms) and respiration (*P < 0.05; n = 3). Bars represent mean ± SEM. (B) Metformin induced a significant increase in H₂O₂ release in day-1 adult worms after both continuous exposure during development (*P < 0.05; n = 7) and after 24 h of exposure, starting from the young adult stage (*P < 0.05; n = 7 for untreated and n = 5 for treated worms). Exposing the worms for 4 h before measurement did not result in a significant increase (^n.s.P > 0.05; n = 7). Bars represent mean ± SEM. (C) The antioxidant NAC abolishes the lifespan-extending effect of metformin (***P < 0.001; n ≥ 169 for each curve; Table S1). (D) prdx-2 is required for metformin-induced lifespan extension. Metformin treatment significantly reduces lifespan of prdx-2 mutants (***P < 0.001; n ≥ 127 for each curve; Table S1). (E) Metformin treatment promotes the formation of PRDX-2 disulfide dimers (**P < 0.01), implied to function in signal transduction. Bars represent mean ± SEM (n = 4). (F) PRDX-2 is required for metformin-induced phosphorylation of the p38 MAP kinase PMK-1. Metformin treatment of wild-type worms induced phosphorylation of PMK-1, inferred from a larger band observed on the Western blot. This metformin-mediated induction of PMK-1 phosphorylation is absent in prdx-2 knockout worms. Histone H3 levels were used as a loading control.

Fig. 3.

Metformin inhibits complex I of the ETC in a way distinct from rotenone. (A) Metformin inhibits complex I- but not complex II-based respiration. Higher values for O₂ flux indicate higher respiration, negative values indicate a small influx of O₂, usually due to the injection of a compound. The effect of metformin on mitochondrial respiration was measured by sequentially adding compounds to stimulate different parts of the ETC. Green arrows indicate compounds that were added in both the control and metformin-treated cells; red arrows indicate compounds that were only added in the treated cell. After adding an equal volume of mitochondria (↓start), metformin was added to one of the cells (↓metformin). Subsequently, the complex I substrates pyruvate and malate (↓P+M) were added, followed by the addition of ADP (↓ADP), initiating electron transport from complex I. Metformin-treated mitochondria clearly fail to initiate complex I-based respiration, thus indicating that metformin inhibits electron transport from complex I in vitro. Finally, the complex II substrate succinate (↓S) was added to the metformin-treated cell, which led to a marked increase in mitochondrial respiration, indicating that metformin does not block electron transport from complex II. Two variations of this experiment were executed to also use the potent complex I inhibitor rotenone as a positive control (Fig. S4A) and to add succinate to the negative control condition as well (Fig. S4B). (B) Mitochondria treated with metformin produce H₂O₂ at a higher rate than untreated mitochondria (***P < 0.001; n = 3). Treatment with the complex I inhibitor rotenone had the opposite effect (***P < 0.001; n = 3).

Fig. 4.

Metformin induces the BCAA degradation and β-oxidation pathways but the β-oxidation enzyme ACDH-1 is not required for metformin-mediated longevity. (A) Metformin treatment stimulates the BCAA degradation pathway (Fig. S5) and in turn reduces the concentration of free BCAAs in C. elegans (**P < 0.01). Bars represent mean ± SEM (n = 6). (B) Metformin-treated worms show reduced fat storage (***P < 0.001; n = 30 for untreated and n = 33 for treated worms), possibly indicating increased flux through the β-oxidation pathway. Bars represent mean ± SEM. (C) Deletion of β-oxidation enzyme acdh-1 results in a proportionally larger effect of metformin on longevity (***P < 0.001; n ≥ 54 for each curve; Table S1).

Fig. 5.

Metformin attenuates the morphological decline with aging in C. elegans. (A) Metformin-treated worms retain a stable volume, whereas control worms older than 6 d start shrinking. (B) Electron micrograph of the cuticle of a day-9 adult nontreated wild-type worm. Some deformations of the cuticle (seen as “wrinkling,” marked with an arrow) are starting to manifest. (C) No structural abnormalities can be seen in a metformin-treated day-9 wild-type adult.

Fig. 6.

The mitohormetic signaling cascade as induced by metformin. Metformin induces an increase in activity in several catabolic pathways (Fig. S6), including the TCA cycle and β-oxidation, with β-oxidation producing a relatively higher amount of FADH₂ per cycle. This increase in substrate allows for an increase in mitochondrial respiration, which in turn leads to an increase in ROS production, possibly through metformin-mediated perturbation of electron transport (marked in red). These ROS oxidize PRDX-2 peroxiredoxins, which subsequently dimerize and enter their active state. Active PRDX-2 will activate a conserved MAPK cascade containing the p38 MAPK PMK-1, likely leading to the activation of SKN-1 (28) and a concomitant increase in longevity and stress protection (dashed arrows: literature-based evidence; Fig. S6 provides more information on the pathways induced by metformin treatment).