Metformin improves healthspan and lifespan in mice

Abstract

Metformin is a drug commonly prescribed to treat patients with type 2 diabetes. Here we show that long-term treatment with metformin (0.1% w/w in diet) starting at middle age extends healthspan and lifespan in male mice, while a higher dose (1% w/w) was toxic. Treatment with metformin mimics some of the benefits of calorie restriction, such as improved physical performance, increased insulin sensitivity, and reduced low-density lipoprotein and cholesterol levels without a decrease in caloric intake. At a molecular level, metformin increases AMP-activated protein kinase activity and increases antioxidant protection, resulting in reductions in both oxidative damage accumulation and chronic inflammation. Our results indicate that these actions may contribute to the beneficial effects of metformin on healthspan and lifespan. These findings are in agreement with current epidemiological data and raise the possibility of metformin-based interventions to promote healthy aging.

Figure 1. Metformin increases survival and improves physical performance

(a, b) Kaplan–Meier survival curve for mice treated either with 0.1% or 1% metformin. n = 64 for metformin 0.1% group and n = 83 for their untreated counterparts; n = 90 for metformin 1% group and n = 88 for their untreated counterparts. The arrows at 54 weeks indicate the age at which metformin treatment was initiated. (c) Body weights. (d) Food consumption. (e, f) In vivo metabolic response to 0.1% metformin treatment. n = 9 per group. (e) Energy expenditure. (f) Respiratory exchange ratio. (g) Time to fall from an accelerating rotarod. n = 16 per group. (h) Distance ran on treadmill performance. n = 9 per group. (i) Average speed of animals in the open-field test. n = 15–16 per group. (j) Metformin treatment delayed the onset of age-related cataracts. n = 93–124 eyes per group. (k) Plasma levels of glucose after oral glucose load (OGTT). n = 8 per group. (l) Area under OGTT curve. (m) Plasma levels of glucose after intraperitoneal insulin injection (ITT). n = 9 per group. (n) Area under ITT curve. Metformin, Met. Unless otherwise stated n = all live animals in the study. Data are represented as the mean ± SEM. * p < 0.05 compared to standard diet (SD)-fed mice (t-test two tailed).

Figure 2. Metformin shifts expression patterns of mice towards those on calorie restriction

(a) Principal component analysis (PCA) was performed on differentially expressed genes from the liver and muscle tissue of mice maintained on SD and 0.1% metformin. Each data point corresponds to the PCA analysis of each subject. (b) Gene expression profile comparing genes significantly up- (red) and down-regulated (blue) by either calorie restriction (CR) or metformin compared to SD mice (z-ratio). The percentage of significant gene expression changes shifted in the same direction in CR and metformin treatments compared to SD mice is presented in brackets. (c) Comparison of gene sets significantly altered by CR and metformin treatment compared to SD expression (z-score); up- (red) and down-regulated (blue) gene sets. The percentage of significant gene sets changes shifted in the same direction in CR and metformin treatments compared to SD mice is presented in brackets. (d) Effect of metformin on mitochondria (Mito), glycolysis, lipid metabolism (Lipid Met) and stress response (Stress Resp) related gene sets. The list of all the significantly modified gene sets can be found in Supplementary table S6. Metformin, Met; Calorie restriction, CR.

Figure 3. Metformin activates AMPK without altering in vivo electron transport chain activities

(a) Activation of AMPK by metformin in MEFs. AMPK and acetyl-coA carboxylase (ACC) phosphorylation by metformin. n = 3 per group. (b) Activation of AMPK and ACC phosphorylation in the liver of 0.1% metformin-treated mice. n = 4–6 per group. (c) Oxygen consumption in MEFs treated with 1 mM metformin. n = 3 per group. (d, e), Mitochondrial content in MEFs treated with metformin was determined by tetramethyl rhodamine methyl ester (TMRM) (d) and MitoTracker green (e) staining, M.F.I., mean fluorescence intensity. n = 3 per group. (f) Mitochondrial DNA content analyzed by quantitative PCR in the liver. n = 5–8 per group. (g) Mitochondrial protein levels in MEFs treated with metformin. n = 3 per group. (h) Mitochondrial protein levels in the liver from 0.1% metformin-treated mice. n = 4–6 per group. (i, j) Effect of metformin on mitochondrial enzymatic activities. (i) MEFs treated with 1 mM metformin (n = 3 per group) and (j) Liver lysates from 0.1% metformin-treated mice (n = 5–6 per group). Metformin, Met: Calorie restriction, CR; Ut, untreated. Data are represented as the mean ± SEM. * p < 0.05 versus untreated controls or standard diet (SD)-fed mice (t-test two tailed).

Figure 4. Metformin enhances antioxidant defenses and inhibits inflammation

(a) Rate of electrons derived to superoxide generation in mitochondrial complexes I and II to III in the liver of 0.1% metformin-treated mice. n = 5–6 per group. (b) Oxidative damage in proteins determined by lysine-4-hydroxinonenal levels in the liver of 0.1% metformin-treated mice. (n = 4–6 per group) (c) Oxidative damage in lipids determined by 8-iso-PGF_2α levels in the liver of 0.1% metformin-treated mice. (n = 4–6 per group) (d) Nrf2-ARE assay determining Nrf2-ARE-dependent expression in metformin-treated HepG2 cells. tBHQ was added as positive control for NRF2-ARE induction. (n = 3 per group) (e) Antioxidant and stress response protein levels in the liver of 0.1% metformin-treated mice. (n = 4–6 per group) (f) Activation of pro-inflammatory signaling pathways in the liver of 0.1% metformin-treated mice. (n = 4–6 per group) (g) Expression of multiple inflammatory-related genes in the liver of 0.1% metformin-treated mice. (n = 5 per group) Metformin, Met; Calorie restriction, CR. Data are represented as the mean ± SEM. * p < 0.05 versus standard diet (SD)-fed mice (t-test two tailed).