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Loss of metabolic plasticity underlies metformin toxicity in aged Caenorhabditis elegans

Nature Metabolism volume 2, pages 1316–1331 (2020)Cite this article

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Abstract

Current clinical trials are testing the life-extending benefits of the diabetes drug metformin in healthy individuals without diabetes. However, the metabolic response of a non-diabetic cohort to metformin treatment has not been studied. Here, we show in C. elegans and human primary cells that metformin shortens lifespan when provided in late life, contrary to its positive effects in young organisms. We find that metformin exacerbates ageing-associated mitochondrial dysfunction, causing respiratory failure. Age-related failure to induce glycolysis and activate the dietary-restriction-like mobilization of lipid reserves in response to metformin result in lethal ATP exhaustion in metformin-treated aged worms and late-passage human cells, which can be rescued by ectopic stabilization of cellular ATP content. Metformin toxicity is alleviated in worms harbouring disruptions in insulin-receptor signalling, which show enhanced resilience to mitochondrial distortions at old age. Together, our data show that metformin induces deleterious changes of conserved metabolic pathways in late life, which could bring into question its benefits for older individuals without diabetes.

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Fig. 1: Late-life metformin treatment reduces survival independently of AMPK and microbiome.
Fig. 2: Metformin toxicity is caused by mitochondrial impairments.
Fig. 3: Metformin toxicity associates with ATP exhaustion.
Fig. 4: Insulin-receptor deficiency confers metformin resilience.
Fig. 5: Metformin activates longevity-assurance pathways in young, but not old, animals.
Fig. 6: Ageing abrogates lipid mobilization by metformin.
Fig. 7: Rapamycin cotreatment alleviates metformin toxicity.
Fig. 8: Mitochondrial dysfunction abrogates longevity benefits of metformin in ageing.

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Data availability

The mass-spectrometry proteomics data, including the exact UniProt information, have been deposited to the ProteomeXchange under accession code PXD011579. Maximum-size microscopy images have been deposited on figshare: https://doi.org/10.6084/m9.figshare.c.5124524. Remaining data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank K. L. Rudolph and H. Morrison for critical discussions that were very helpful and important throughout the study and during manuscript preparation. We also thank the Proteomics Core Facility at FLI for supporting this study. The FLI is a member of the Leibniz Association and is financially supported by the Federal Government of Germany and the State of Thuringia. L.E. is supported by the EU-ESF Thüringer Aufbaubank funding (2019 FBR 0082); A.D. was, and A.M. is, supported by the German Research Council (Deutsche Forschungsgemeinschaft, DFG) via the Research Training Group Adaptive Stress Responses (GRK 1715); T.P. is supported by the German Academic Exchange Services (Deutsche Akademische Austauschdienst, DAAD).

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Author notes

  1. These authors contributed equally: Lilia Espada, Alexander Dakhovnik, Prerana Chaudhari.

Authors and Affiliations

  1. Leibniz Institute on Aging – Fritz Lipmann Institute (FLI), Jena, Germany

    Lilia Espada, Alexander Dakhovnik, Prerana Chaudhari, Asya Martirosyan, Tetiana Poliezhaieva, Yvonne Schaub, Ashish Nair, Nadia Döring, Norman Rahnis, Alessandro Ori & Maria A. Ermolaeva

  2. Institute of Pharmacy, Friedrich Schiller University Jena, Jena, Germany

    Laura Miek, Oliver Werz & Andreas Koeberle

  3. Michael Popp Research Institute, University of Innsbruck, Innsbruck, Austria

    Andreas Koeberle

  4. Francis Crick Institute, London, UK

    Joanna Kirkpatrick

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Contributions

M.A.E. conceptualized and designed the study; L.E., A.D., P.C., A.M., T.P., A.N., Y.S. performed experiments; M.A.E., L.E., A.D., P.C. analysed the data; N.D., N.R. performed sample curation for proteomics analysis; J.K., A.O. developed proteomics-analysis protocols and analysed proteomics data; A.K. designed the lipidomics study; L.M. and A.K. performed the lipidomics analysis; A.K., O.W. and M.A.E interpreted the lipidomics data; L.E., A.D., P.C. and A.O. prepared figures and performed statistical analysis; M.A.E. wrote the manuscript; L.E., A.D., P.C., J.K., A.K., A.O. reviewed the manuscript.

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Correspondence to Maria A. Ermolaeva.

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Peer review information Primary handling editor: George Caputa. Nature Metabolism thanks Michael Ristow, Nektarios Tavernarakis and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Life extension by metformin deteriorates with age.

a, b, Wild type (WT, N2 Bristol strain) worms were treated with indicated doses of metformin (Met) from day 4 (a) and day 8 (b) of adulthood (AD4 and AD8 respectively), survival was scored daily. c, % of total deaths following metformin exposure on AD10 is shown for the first 24 h and 48 h of exposure, depicting the highest number of metformin-induced deaths during the 1st 24 h; the graph is based on the survival data from Fig. 1b; the stars describe the difference between metformin exposed and control animals for each time point. Results are representative of at least 3 independent tests. d, Wild type worms were treated with 50 mM metformin on adulthood day 1 (young) and 10 (old) for 24 h and 48 h. Heatmaps of selected AMPK target proteins are shown (metformin treated versus age- and time point matched untreated control); log2 fold changes are color coded as indicated. Absolute log2 fold changes above 0.5 with Q value below 0.25 were considered significant, only proteins with significance in at least one age/treatment combination are depicted. 3 independent populations with n=500 were measured for each condition; the expression levels and significance for individual proteins are reported in Supplementary Table 3. For a and b significance was determined by Mantel-Cox test and two-tailed p values were computed, all n numbers and statistical values are presented in Supplementary Table 1. For c, mean and SEM are presented, two-tailed unpaired t-test was used for the statistical analysis, the statistical values are presented in Supplementary Table 2; * p<0.05; ** p<0.01; **** p<0.0001.

Source data

Extended Data Fig. 2 Mitochondrial impairments pre-dispose late passage cells to metformin toxicity.

a, WT animals (left panel) and ubl-5(ok3389) mutants (right panel) were treated with 50 mM metformin on days 1 and 10 of adulthood (AD1 and AD10 respectively), survival was scored daily. b,c, Early passage (population doubling, PD38) human primary fibroblasts were co-treated with FCCP and metformin for 24 h; cell death (b, LDH assay) and mitochondrial membrane potential (c, JC-1 assay) were measured. DMSO was used as a vehicle control for FCCP, and DMEM media - as a control for metformin. All values are relative to the respective untreated control (no metformin, no FCCP). A proof of concept decline of MMP is seen at 5μM of FCCP (no metformin, grey bar). Stars depict differences with the respective vehicle control for each dose of metformin (b) or FCCP (c). For a significance was calculated by Mantel-Cox test and two-tailed p values are shown, all n numbers and statistical values are presented in Supplementary Table 1; for b and c, n=3, mean and SEM are presented; two-tailed unpaired t-test was used for the statistical analysis, statistical values are shown in Supplementary Table 2; ** p<0.01; *** p<0.001; **** p<0.0001. Results are representative of at least 3 independent tests.

Source data

Extended Data Fig. 3 Aging and mitochondrial dysfunction impair metabolic adaptation to metformin.

a, Relative expression of glycolytic enzymes in old (adulthood day 10, AD10) versus young (adulthood day 1, AD1) wild type (N2, Bristol strain) nematodes is presented. b, The expression of glycolysis enzymes in young and old WT animals treated with 50 mM metformin for indicated times is depicted. For a and b, individual proteins are shown as blue dots, the median fold change of the proteins belonging to each plot is shown as a bold line, the upper and lower limits of the boxplot indicate the first and third quartile, respectively, and whiskers extend 1.5 times the interquartile range from the limits of the box. n=500 for each condition and 3 independent populations were analyzed. The statistics was assessed by Wilcoxon rank-sum test presenting two-tailed p values; the complete list of proteins used for the boxplot analysis along with individual fold changes and Q values is reported in Supplementary Table 3. The stars refer to log2 fold changes of the entire protein group in old versus young animals (a) and in metformin treated versus age- and time point matched control animals (b), these values are reported in Supplementary Table 2. c, Young (AD1) and Old (AD10) wild type worms were washed and incubated for 30 minutes with unbuffered EPA water before loading on Seahorse XFe 96 well plate; the analysis of glycolysis was performed by measuring extracellular acidification rate (ECAR) following injection of FCCP (200 µM) and antimycin-A (5µM) plus rotenone (5µM), at indicated times. The stars depict the difference between young and old worms at indicated time points. d, ATP levels were measured in young (AD1) atfs-1(gk3094) mutants or wild type worms treated with 25mM FCCP following 24h of exposure to 50mM metformin; wild type (N2, Bristol strain) exposed to metformin (no FCCP) were used as control; the stars depict the difference between metformin treated and control animals for each genotype. In (c) n≥209 and in (d) n=100 for each condition, mean and SEM are presented, two-tailed unpaired t-test was used for the statistical analysis, all values are presented in Supplementary Table 2; * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001. Results are representative of at least 3 independent tests.

Source data

Extended Data Fig. 4 Ectopic ATP supplementation alleviates metformin toxicity in human fibroblasts.

Pre-senescent (PD44) primary human skin fibroblasts were treated with indicated doses of metformin in presence or absence of indicated concentrations of ATP for 24h hours; cell death (a, LDH assay), cell survival (b, MTT assay), ATP content (c) and mitochondrial membrane potential (d, JC-1 assay) were measured; the data are complementary to Fig. 3i, j; values are relative to the respective untreated control (no ATP, no metformin) for each assay; the stars depict differences between ATP supplemented and non-supplemented cells for each metformin dose. n=3, mean and SEM are presented, two-tailed unpaired t-test was used for the statistical analysis, statistical values are shown in Supplementary Table 2; * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001. Results are representative of at least 3 independent tests.

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Extended Data Fig. 5 Metformin resilience of daf-2(e1370) mutants is mediated by DAF-16/FOXO.

a, daf-2(e1370) mutants were treated with 50mM metformin (Met) on adulthood day 21 (AD21) along with AD10 wild type (N2 Bristol strain) control animals, survival was scored daily. b, daf-2(e1370);daf-16(mu86) mutants and age-matched wild type controls were treated with 50mM metformin on adulthood day 10 (AD10), survival was scored daily. c, ATP levels were measured in wild type, daf-2(e1370) and daf-2(e1370);daf-16(mu86) worms after 36h of treatment with 50mM metformin initiated on AD1 or AD10. The data complements Fig. 4c, all presented ATP measurements were performed in parallel to ensure comparability; the stars show differences between metformin treated and untreated animals for each age and genotype. d, Boxplots showing the expression of selected mitochondrial proteins in young (AD1) daf-2(e1370) and daf-2(e1370);daf-16(mu86) mutant nematodes relative to age-matched wild type (N2, Bristol strain) control are presented. Individual proteins are shown as blued dots, the median fold change of the proteins belonging to each group is shown as a bold line, the upper and lower limits of the boxplot indicate the first and third quartile, respectively, and whiskers extend 1.5 times the interquartile range from the limits of the box. For each condition n=700 and at least 3 independent populations were measured; statistics was calculated by Wilcoxon rank-sum test, two-tailed p values are presented in Supplementary Table 2. The list and data of individual proteins are reported in Supplementary Table 3. For a, b significance was measured by Mantel-Cox test and two-tailed p values were computed; n numbers and statistics are presented in Supplementary Table 1. For c n≥50, mean and SEM are presented, two-tailed unpaired t-test was used for the statistical analysis, all values are presented in Supplementary Table 2, * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001. Results are representative of at least 3 independent tests.

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Extended Data Fig. 6 Increased mitochondrial content confers resilience to metformin toxicity.

Boxplots showing relative expression of selected mitochondrial proteins (a) and ETC complex I (b), complex II (c), complex III (d), complex IV (e) and complex V (f) components in old (AD10) versus young (AD1) wild type (N2, Bristol strain) and daf-2 mutant nematodes are presented. Individual proteins are shown as blue dots, the median fold change of the proteins belonging to each group is shown as a bold line, the upper and lower limits of the boxplot indicate the first and third quartile, respectively, and whiskers extend 1.5 times the interquartile range from the limits of the box. 3 independent populations with n=500 were measured for each condition; Wilcoxon rank-sum test was used for the statistical analysis, two-tailed p values are presented. The stars above the plots refer to log2 fold changes of the entire protein group in old versus young animals for each genotype; additional bars compare log2 fold change distributions between WT and mutant animals (a and b). The lists of all depicted proteins with individual expression and Q values are reported in Supplementary Table 3, and the grouped statistics is shown in Supplementary Table 2. (g) Wild type and pink-1(tm1779) mutant animals were treated with 50mM metformin from AD1 and AD10, survival was scored daily. Significance was measured by Mantel-Cox test and two-tailed p values are shown; all n numbers and statistical values are presented in Supplementary Table 1, results are representative of 3 independent tests; * p<0.05; ** p<0.01; **** p<0.0001.

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Extended Data Fig. 7 Aging blunts the adaptive responses to metformin.

WT worms were treated with 50mM metformin on adulthood day 1 (young) and 10 (old). a, Venn diagram of significantly altered proteins after 48h of early and late life metformin treatment is shown. In circles all proteins with absolute log2 fold change above 0.5 and Q value below 0.25 are shown; the number in the outside box indicates non-regulated proteins; fold changes were calculated against age- and time point matched untreated controls. Three independent pools of n=500 worms were analyzed for each sample group. b, Scatter plot comparing individual log2 fold changes (shown as dots) after 48h of metformin treatment at young and old age is shown. Proteins significantly regulated at both ages are colored: the green-highlighted proteins are consistently regulated at both ages while black-highlighted ones show opposite regulation between young and old age. The correlation coefficient (Rho) between overall young and old responses is shown. Boxplots showing fold changes of selected ribosomal proteins (c) and proteins involved in general autophagy (d) are presented. The median fold change of the proteins belonging to each group is shown as a bold line, the upper and lower limits of the boxplot indicate the first and third quartile, respectively, and whiskers extend 1.5 times the interquartile range from the limits of the box. e, Heatmaps of selected dehydrogenases are shown; only fold changes with significance in at least one age/treatment combination are depicted, color bar depicts log2 fold changes. Box plots depicting log2 fold changes of selected peroxisomal proteins (f) and vitellogenins (g) in old versus young WT animals are presented. Wilcoxon rank-sum test was used for the statistical analysis, two-tailed p values are shown. Stars refer to log2 fold changes of the entire protein group in metformin treated versus age- and time point matched control (c and d), and in old versus young animals (f and g); in c the bar compares log2 fold change distributions between young and old animals treated with metformin for 24h. The lists, expression data and Q values for individual proteins are reported in Supplementary Table 3, and whole group statistics is shown in Supplementary Table 2. * p<0.05; *** p<0.001; **** p<0.0001.

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Extended Data Fig. 8 Late life metformin toxicity is not mediated by autophagy.

a, Representative images of baseline diffused mCherry::LGG-1 expression (left panel) and autophagy puncta (right panel) are shown, scale bar is 100µm. Young (adulthood day 1, AD1) and old (AD10) transgenic animals were treated with 50mM metformin (Met), the number of puncta per animal was quantified; b and c show autophagy fold induction relative to time point matched untreated control in each case; d and e show absolute numbers of puncta per animal used for the calculation of values shown in b-c. The baseline elevation of autophagy over time is likely due to fresh plate transfer in all cases. (f) Transgenic animals were exposed to bec-1 or control RNAi from the L4 larval stage; number of puncta was quantified over time. g, Wild type animals were grown on HT115 E. coli and exposed to bec-1 RNAi from AD1 or AD4, followed by metformin treatment on AD10, survival was scored daily; empty vector RNAi was used as control. For b-f n=10, mean and SEM are presented, two-tailed unpaired t-test was used for the statistical analysis, all statistical values are presented in Supplementary Table 2; for g statistics was assessed by Mantel-Cox test, two-tailed p values and n numbers are shown in Supplementary Table 1; **** p<0.0001. Results are representative of at least 3 independent tests.

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Extended Data Fig. 9 Late life metformin toxicity is not mediated by oxidative stress.

a, Wild-type nematodes were treated with 50mM metformin at old age (AD10) with and without 5mM NAC co-supplementation (provided from AD8), survival was scored daily. Significance was measured by Mantel-Cox test and two-tailed p values are shown; all n numbers and statistical values are presented in Supplementary Table 1. b, d, Young (PD35) and old (PD60) cells were treated with indicated concentrations of metformin for 20h and assayed for ROS production (b) and cell death (d, LDH assay); all values are relative to the untreated control of a given age and stars describe the same comparison. c, Young (PD35) cells were exposed to FCCP 5μM and H2O2 250 5μM to induce ROS production as a proof of concept, and NAC 4mM was used as a control for ROS scavenging; the data is complementary to the experiment shown in b; values are relative to the untreated control of PD35 (presented in b), stars depict the difference between NAC exposed and unexposed cells. For b-d, n=3, mean and SEM are presented, two-tailed unpaired t-test was used for the statistical analysis, all statistical values are presented in Supplementary Table 2; * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001. Results are representative of at least 3 independent tests.

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Extended Data Fig. 10 Lipid mobilization by metformin is blunted at old age.

Wild type animals were treated with 50mM metformin for 24h on AD1 and AD10; lipids were isolated and analyzed by UPLC-MS/MS. Absolute intensities for phosphatidylethanolamines (PEs) (c) and phosphatidylinositols (PIs) (d) are shown for treated and untreated animals, and absolute intensities of PEs, PIs, free fatty acids (FFAs) and phosphatidylcholines (PC) are shown for untreated young and old animals (a). (b) Relative intensities of lyso-phosphatidylcholines (LPCs) and lyso-phosphatidylethanolamines (LPEs) as well as of polyunsaturated fatty acid (PUFA) containing LPCs and LPEs are shown for untreated young and old animals. e, Relative intensities of triglycerides with PUFAs containing more than 3 double bonds (PUFA n>3 TAGs) are shown for young and old treated and untreated animals. All values are normalized to the AD10 untreated control. Definitions of absolute and relative intensities are provided in the Methods section. f, g, Representative images of the Oil Red O whole body lipid staining are shown for Fig. 6a, e, respectively; scale bar is 100µm. For a-e n=700, all individual lipid values are presented in Supplementary Table 4, mean and SEM are depicted, two-tailed unpaired t-test was used for the statistical analysis, all statistical values are presented in Supplementary Table 2. In a and b the stars depict the differences in the abundance of each lipid type between young and old animals, in c and d the difference between age-matched metformin treated and untreated animals is highlighted, and in e the difference between young and old metformin exposed nematodes is shown; * p<0.05; ** p<0.01. Results are representative of at least 3 independent tests.

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Supplementary information

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Espada, L., Dakhovnik, A., Chaudhari, P. et al. Loss of metabolic plasticity underlies metformin toxicity in aged Caenorhabditis elegans. Nat Metab 2, 1316–1331 (2020). https://doi.org/10.1038/s42255-020-00307-1

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