Benefits of Metformin in Attenuating the Hallmarks of Aging

Biological aging involves an interplay of conserved and targetable molecular mechanisms, summarized as the hallmarks of aging. Metformin, a biguanide that combats age-related disorders and improves health span, is the first drug to be tested for its age-targeting effects in the large clinical trial—TAME (targeting aging by metformin). This review focuses on metformin’s mechanisms in attenuating hallmarks of aging and their interconnectivity, by improving nutrient sensing, enhancing autophagy and intercellular communication, protecting against macromolecular damage, delaying stem cell aging, modulating mitochondrial function, regulating transcription, and lowering telomere attrition and senescence. These characteristics make metformin an attractive gerotherapeutic to translate to human trials. Biological aging involves an interplay of conserved and targetable molecular mechanisms, summarized as the hallmarks of aging. Metformin, a biguanide that combats age-related disorders and improves health span, is the first drug to be tested for its age-targeting effects in the large clinical trial—TAME (targeting aging by metformin). This review focuses on metformin’s mechanisms in attenuating hallmarks of aging and their interconnectivity, by improving nutrient sensing, enhancing autophagy and intercellular communication, protecting against macromolecular damage, delaying stem cell aging, modulating mitochondrial function, regulating transcription, and lowering telomere attrition and senescence. These characteristics make metformin an attractive gerotherapeutic to translate to human trials. Aging is characterized by a progressive loss of physiological function, which drives the development of chronic morbidities including metabolic, cardiovascular, neoplastic, and neurodegenerative disorders as well as geriatric symptoms, such as frailty and immobility. Aging is accompanied by an inherent biological mechanism that is malleable and can be targeted using therapeutic interventions. Indeed, over the past few decades, scientists have achieved remarkable progress in extending health span and lifespan of model organisms using several genetic, dietary, and pharmacological interventions. These advancements have urged the geroscience research community to initiate clinical trials to investigate the efficacy of interventions in targeting human aging, starting with the TAME (targeting aging with metformin) study (Barzilai, 2017Barzilai N.R. Targeting aging WITH metformin (TAME).Innov. Aging. 2017; 1: 743Crossref Google Scholar, Campisi et al., 2019Campisi J. Kapahi P. Lithgow G.J. Melov S. Newman J.C. Verdin E. From discoveries in ageing research to therapeutics for healthy ageing.Nature. 2019; 571: 183-192Crossref PubMed Scopus (52) Google Scholar). The TAME study, soon to be launched in the near future, aims to prove the concept that human aging can be targeted while simultaneously preventing a multitude of major age-related outcomes. Furthermore, TAME is a potential tool to facilitate the FDA to approve “aging” as a target for drug discovery and development. Thus, TAME will pave the way for the development of novel interventions that could target and delay the aging process and improve human health span, by modulating the conserved mechanistic pathways involved in aging. To systematically dissect the biological aging process, Lopez-Otin et al. characterized nine major hallmarks of aging, widely accepted by the geroscience research community, namely (1) genomic instability, (2) epigenetic alterations, (3) loss of proteostasis, (4) deregulated nutrient sensing, (5) mitochondrial dysfunction, (6) cellular senescence, (7) stem cell exhaustion, (8) altered intercellular communication, and (9) telomere attrition (López-Otín et al., 2013López-Otín C. Blasco M.A. Partridge L. Serrano M. Kroemer G. The hallmarks of aging.Cell. 2013; 153: 1194-1217Abstract Full Text Full Text PDF PubMed Scopus (4120) Google Scholar). These are divided as primary, antagonistic, and integrative hallmarks depending on their functional characteristics as causes of damage, responses to damage, and end results of the first two categories, respectively (López-Otín et al., 2013López-Otín C. Blasco M.A. Partridge L. Serrano M. Kroemer G. The hallmarks of aging.Cell. 2013; 153: 1194-1217Abstract Full Text Full Text PDF PubMed Scopus (4120) Google Scholar). The hallmarks and their interconnectivity can serve as an evaluation tool to assess and prioritize interventions that can be deemed effective in targeting aging. Although the contribution of each of these hallmarks toward the progression of biological aging is not yet fully elucidated, interventions that can modulate several of these hallmarks, at least in part, need to be studied extensively to provide newer insights into the druggable targets of biological aging. In humans, metformin has been in clinical use for over 60 years, studied extensively, has a high safety profile, and is uniquely positioned to intervene several crucial pathways responsible for aging and age-related diseases (Barzilai et al., 2016Barzilai N. Crandall J.P. Kritchevsky S.B. Espeland M.A. Metformin as a tool to target aging.Cell Metab. 2016; 23: 1060-1065Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar). As recommended by the American Diabetes Association, due to its glucose-lowering effects, metformin monotherapy is the preferred first-line pharmacological action against type 2 diabetes (American Diabetes Association, 2019American Diabetes AssociationPharmacologic approaches to glycemic treatment: standards of medical care in diabetes-2019.Diabetes Care. 2019; 42: S90-S102Crossref PubMed Scopus (0) Google Scholar). Epidemiological studies have revealed metformin’s gerotherapeutic effect in lowering the incidence of multiple age-related diseases as well as all-cause mortality, in both diabetics and non-diabetics (Campbell et al., 2017Campbell J.M. Bellman S.M. Stephenson M.D. Lisy K. Metformin reduces all-cause mortality and diseases of ageing independent of its effect on diabetes control: A systematic review and meta-analysis.Ageing Res. Rev. 2017; 40: 31-44Crossref PubMed Scopus (78) Google Scholar, Valencia et al., 2017Valencia W.M. Palacio A. Tamariz L. Florez H. Metformin and ageing: improving ageing outcomes beyond glycaemic control.Diabetologia. 2017; 60: 1630-1638Crossref PubMed Scopus (0) Google Scholar). Clinical studies including the diabetes prevention program (DPP) in non-diabetics and UK prospective diabetes study (UKPDS) in diabetics, support metformin’s role as an effective intervention against diabetes and cardiovascular disease. Association studies suggest a decrease in the incidence of most age-related cancers, Alzheimer’s disease while clinical studies support metformin’s role in a decrease in cognitive decline and reduced mortality in diabetics taking metformin compared with non-diabetics (Barzilai et al., 2016Barzilai N. Crandall J.P. Kritchevsky S.B. Espeland M.A. Metformin as a tool to target aging.Cell Metab. 2016; 23: 1060-1065Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar). Studies in multiple model organisms and human cell lines have elucidated metformin’s role in targeting multiple mechanisms of aging. In mice and C. elegans, metformin extends lifespan and improves several indicators of health span (Anisimov et al., 2008Anisimov V.N. Berstein L.M. Egormin P.A. Piskunova T.S. Popovich I.G. Zabezhinski M.A. Tyndyk M.L. Yurova M.V. Kovalenko I.G. Poroshina T.E. Semenchenko A.V. Metformin slows down aging and extends life span of female SHR mice.Cell Cycle. 2008; 7: 2769-2773Crossref PubMed Google Scholar, Martin-Montalvo et al., 2013Martin-Montalvo A. Mercken E.M. Mitchell S.J. Palacios H.H. Mote P.L. Scheibye-Knudsen M. Gomes A.P. Ward T.M. Minor R.K. Blouin M.J. et al.Metformin improves healthspan and lifespan in mice.Nat. Commun. 2013; 4: 2192Crossref PubMed Scopus (542) Google Scholar, De Haes et al., 2014De Haes W. Frooninckx L. Van Assche R. Smolders A. Depuydt G. Billen J. Braeckman B.P. Schoofs L. Temmerman L. Metformin promotes lifespan through mitohormesis via the peroxiredoxin PRDX-2.Proc. Natl. Acad. Sci. USA. 2014; 111: E2501-E2509Crossref PubMed Scopus (151) Google Scholar, Chen et al., 2017aChen S.C. Brooks R. Houskeeper J. Bremner S.K. Dunlop J. Viollet B. Logan P.J. Salt I.P. Ahmed S.F. Yarwood S.J. Metformin suppresses adipogenesis through both AMP-activated protein kinase (AMPK)-dependent and AMPK-independent mechanisms.Mol. Cell. Endocrinol. 2017; 440: 57-68Crossref PubMed Scopus (43) Google Scholar, Chen et al., 2017bChen J. Ou Y. Li Y. Hu S. Shao L.W. Liu Y. Metformin extends C. elegans lifespan through lysosomal pathway.eLife. 2017; 6: e31268Crossref PubMed Scopus (0) Google Scholar). Intermittent metformin treatment, when initiated in mice, even later in life and administered every other week, has also shown to provide health benefits, such as a reduction in hepatic steatosis through regulation of the liver transcriptome and metabolome (Alfaras et al., 2017Alfaras I. Mitchell S.J. Mora H. Lugo D.R. Warren A. Navas-Enamorado I. Hoffmann V. Hine C. Mitchell J.R. Le Couteur D.G. et al.Health benefits of late-onset metformin treatment every other week in mice.npj Aging Mech. Dis. 2017; 3: 16Crossref PubMed Scopus (11) Google Scholar). Recently, metformin’s extraordinary ability as a gerotherapeutic is established through several experimental, clinical, and observational evidence (Novelle et al., 2016Novelle M.G. Ali A. Diéguez C. Bernier M. de Cabo R. Metformin: a hopeful promise in aging research.Cold Spring Harb. Perspect. Med. 2016; 6: a025932Crossref PubMed Scopus (42) Google Scholar, Piskovatska et al., 2019Piskovatska V. Stefanyshyn N. Storey K.B. Vaiserman A.M. Lushchak O. Metformin as a geroprotector: experimental and clinical evidence.Biogerontology. 2019; 20: 33-48Crossref PubMed Scopus (16) Google Scholar, Glossmann and Lutz, 2019Glossmann H.H. Lutz O.M.D. Metformin and aging: a review.Gerontology. 2019; 65: 581-590Crossref PubMed Scopus (7) Google Scholar, Soukas et al., 2019Soukas A.A. Hao H. Wu L. Metformin as anti-aging therapy: is it for everyone?.Trends Endocrinol. Metab. 2019; 30: 745-755Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). Moreover, in older human adults, we recently demonstrated metformin’s efficacy in targeting multiple age-associated metabolic and non-metabolic pathways and reverting derangements, using cardiometabolic and transcriptomic outcomes (Kulkarni et al., 2018Kulkarni A.S. Brutsaert E.F. Anghel V. Zhang K. Bloomgarden N. Pollak M. Mar J.C. Hawkins M. Crandall J.P. Barzilai N. Metformin regulates metabolic and nonmetabolic pathways in skeletal muscle and subcutaneous adipose tissues of older adults.Aging Cell. 2018; 17 (https://onlinelibrary.wiley.com/action/showCitFormats?doi=10.1111%2Facel.12723)Crossref PubMed Scopus (19) Google Scholar). In this review, we aim to determine the influence of metformin’s mechanisms of action on the hallmarks of biological aging. We demonstrate that each hallmark of aging, as well as their interconnectivity is attenuated by metformin’s direct and/or downstream effects (Figure 1). Metformin was introduced to the world in 1957, as an antihyperglycemic agent by the French physician Jean Sterne, and today, it has become one of the most commonly used pharmaceutical interventions and the most prescribed glucose-lowering medication worldwide (Bailey, 2017Bailey C.J. Metformin: historical overview.Diabetologia. 2017; 60: 1566-1576Crossref PubMed Scopus (133) Google Scholar). Indeed, it is important to note that metformin does not cause hypoglycemia per se but rather reduces hepatic glucose production through improved hepatic insulin sensitivity, which results in the reduction of fasting plasma glucose levels (Jackson et al., 1987Jackson R.A. Hawa M.I. Jaspan J.B. Sim B.M. Disilvio L. Featherbe D. Kurtz A.B. Mechanism of metformin action in non-insulin-dependent diabetes.Diabetes. 1987; 36: 632-640Crossref PubMed Google Scholar, DeFronzo et al., 1991DeFronzo R.A. Barzilai N. Simonson D.C. Mechanism of metformin action in obese and lean noninsulin-dependent diabetic subjects.J. Clin. Endocrinol. Metab. 1991; 73: 1294-1301Crossref PubMed Google Scholar). Furthermore, with the epidemiological, preclinical, and clinical evidence of metformin exhibiting beneficial effects beyond glycemic control in diabetics, it has been suggested to be repurposed against cancer and its recurrence (Heckman-Stoddard et al., 2017Heckman-Stoddard B.M. DeCensi A. Sahasrabuddhe V.V. Ford L.G. Repurposing metformin for the prevention of cancer and cancer recurrence.Diabetologia. 2017; 60: 1639-1647Crossref PubMed Scopus (69) Google Scholar), cardiovascular disease (Rena and Lang, 2018Rena G. Lang C.C. Repurposing metformin for cardiovascular disease.Circulation. 2018; 137: 422-424Crossref PubMed Scopus (20) Google Scholar), neurodegenerative diseases (Rotermund et al., 2018Rotermund C. Machetanz G. Fitzgerald J.C. The therapeutic potential of metformin in neurodegenerative diseases.Front. Endocrinol. 2018; 9: 400Crossref PubMed Scopus (34) Google Scholar), autoimmune diseases (Ursini et al., 2018Ursini F. Russo E. Pellino G. D'Angelo S. Chiaravalloti A. De Sarro G. Manfredini R. De Giorgio R. Metformin and autoimmunity: a "new deal" of an old drug.Front. Immunol. 2018; 9: 1236Crossref PubMed Scopus (0) Google Scholar), and most recently, systemic aging as a whole (Barzilai et al., 2016Barzilai N. Crandall J.P. Kritchevsky S.B. Espeland M.A. Metformin as a tool to target aging.Cell Metab. 2016; 23: 1060-1065Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, Barzilai, 2017Barzilai N.R. Targeting aging WITH metformin (TAME).Innov. Aging. 2017; 1: 743Crossref Google Scholar). Despite such widespread use and efficacy, the mechanisms by which metformin regulates fundamental pathways in aging and diseases are not fully elucidated. Metformin’s antihyperglycemic role is attributed to its action on glucose metabolism, specifically as a suppressor of hepatic gluconeogenesis by inhibiting mitochondrial glycerophosphate dehydrogenase, thereby modifying the hepatocellular redox state to reduce glucose formation from lactate and glycerol (Madiraju et al., 2014Madiraju A.K. Erion D.M. Rahimi Y. Zhang X.M. Braddock D.T. Albright R.A. Prigaro B.J. Wood J.L. Bhanot S. MacDonald M.J. et al.Metformin suppresses gluconeogenesis by inhibiting mitochondrial glycerophosphate dehydrogenase.Nature. 2014; 510: 542-546Crossref PubMed Scopus (565) Google Scholar). On the other hand, metformin’s suppression of hepatic glucose production is shown to be the result of AMP-induced inhibition of fructose-1,6-bisphosphatase-1, a rate-controlling enzyme in gluconeogenesis (Hunter et al., 2018Hunter R.W. Hughey C.C. Lantier L. Sundelin E.I. Peggie M. Zeqiraj E. Sicheri F. Jessen N. Wasserman D.H. Sakamoto K. Metformin reduces liver glucose production by inhibition of fructose-1-6-bisphosphatase.Nat. Med. 2018; 24: 1395-1406Crossref PubMed Scopus (46) Google Scholar). In addition to these mechanisms, metformin’s metabolic action includes a reduction in glucose absorption in the intestine (Wu et al., 2017Wu T. Xie C. Wu H. Jones K.L. Horowitz M. Rayner C.K. Metformin reduces the rate of small intestinal glucose absorption in type 2 diabetes.Diabetes Obes. Metab. 2017; 19: 290-293Crossref PubMed Scopus (18) Google Scholar), restoring insulin secretion in pancreatic beta cells (Patanè et al., 2000Patanè G. Piro S. Rabuazzo A.M. Anello M. Vigneri R. Purrello F. Metformin restores insulin secretion altered by chronic exposure to free fatty acids or high glucose: a direct metformin effect on pancreatic beta-cells.Diabetes. 2000; 49: 735-740Crossref PubMed Google Scholar) and to a lesser extent increasing insulin-mediated glucose uptake in the peripheral tissues of muscle and adipose (Galuska et al., 1991Galuska D. Zierath J. Thörne A. Sonnenfeld T. Wallberg-Henriksson H. Metformin increases insulin-stimulated glucose transport in insulin-resistant human skeletal muscle.Diabete Metab. 1991; 17: 159-163PubMed Google Scholar). Being a hydrophilic compound charged positively at physiological pH, metformin enters and leaves the cells mainly via organic cationic transporters (OCTs) and multidrug and toxin extrusion transporters (MATEs) (Gong et al., 2012Gong L. Goswami S. Giacomini K.M. Altman R.B. Klein T.E. Metformin pathways: pharmacokinetics and pharmacodynamics.Pharmacogenet. Genomics. 2012; 22: 820-827Crossref PubMed Scopus (200) Google Scholar). The hepatic, intestinal, and adipocytic uptake of metformin is primarily mediated through the OCT1 (Wang et al., 2002Wang D.S. Jonker J.W. Kato Y. Kusuhara H. Schinkel A.H. Sugiyama Y. Involvement of organic cation transporter 1 in hepatic and intestinal distribution of metformin.J. Pharmacol. Exp. Ther. 2002; 302: 510-515Crossref PubMed Scopus (325) Google Scholar, Moreno-Navarrete et al., 2011Moreno-Navarrete J.M. Ortega F.J. Rodríguez-Hermosa J.I. Sabater M. Pardo G. Ricart W. Fernández-Real J.M. OCT1 expression in adipocytes could contribute to increased metformin action in obese subjects.Diabetes. 2011; 60: 168-176Crossref PubMed Scopus (0) Google Scholar) (Figure 1). Furthermore, it has been shown to accumulate in mitochondria due to the membrane potential across the mitochondrial inner membrane (Owen et al., 2000Owen M.R. Doran E. Halestrap A.P. Evidence that metformin exerts its anti-diabetic effects through inhibition of complex 1 of the mitochondrial respiratory chain.Biochem. J. 2000; 348: 607-614Crossref PubMed Scopus (1293) Google Scholar). One of the main actions of metformin is the inhibition of mitochondrial complex I (NADH:ubiquinone oxidoreductase) that leads to multiple downstream effects both on metabolic and non-metabolic pathways responsible in the aging process (El-Mir et al., 2000El-Mir M.Y. Nogueira V. Fontaine E. Avéret N. Rigoulet M. Leverve X. Dimethylbiguanide inhibits cell respiration via an indirect effect targeted on the respiratory chain complex I.J. Biol. Chem. 2000; 275: 223-228Crossref PubMed Scopus (850) Google Scholar, Foretz et al., 2014Foretz M. Guigas B. Bertrand L. Pollak M. Viollet B. Metformin: from mechanisms of action to therapies.Cell Metab. 2014; 20: 953-966Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar, Barzilai et al., 2016Barzilai N. Crandall J.P. Kritchevsky S.B. Espeland M.A. Metformin as a tool to target aging.Cell Metab. 2016; 23: 1060-1065Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar). The mechanisms by which metformin inhibits mitochondrial complex I remain unsolved. Although, very high concentrations of metformin are needed to directly inhibit complex I activity in isolated mitochondria, micromolar concentrations of the drug are effective in achieving a dose- and time-dependent weak, reversible, and selective complex I inhibition (Vial et al., 2019Vial G. Detaille D. Guigas B. Role of mitochondria in the mechanism(s) of action of metformin.Front. Endocrinol. (Lausanne). 2019; 10: 294Crossref PubMed Scopus (10) Google Scholar). As a result, the effects of metformin diverge on metabolic and oxidative pathways and their downstream targets. However, some actions of metformin, including its antiproliferative effect can be demonstrated regardless of its effect on mitochondria, especially in Rho0 cells deficient in mitochondrial DNA (Liu et al., 2014Liu X. Chhipa R.R. Pooya S. Wortman M. Yachyshin S. Chow L.M.L. Kumar A. Zhou X. Sun Y. Quinn B. et al.Discrete mechanisms of mTOR and cell cycle regulation by AMPK agonists independent of AMPK.Proc. Natl. Acad. Sci. USA. 2014; 111: E435-E444Crossref PubMed Scopus (143) Google Scholar). In parallel to complex I inhibition and its action on the mitochondrial electron transport chain (ETC), metformin’s mechanism of action is attributed both to its 5′ adenosine monophosphate-activated protein kinase (AMPK)-dependent and AMPK-independent roles (Figure 1). The downstream effect of mitochondrial complex I inhibition is directly evident from the increase in cytoplasmic AMP:ATP and ADP:ATP ratios, which in turn leads to phosphorylation and activation of AMPK (Foretz et al., 2014Foretz M. Guigas B. Bertrand L. Pollak M. Viollet B. Metformin: from mechanisms of action to therapies.Cell Metab. 2014; 20: 953-966Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar). The direct activation of AMPK and inhibition of mTORC1 is a result of metformin’s action on the lysosomal pathway, requiring v-ATP-ase-AXIN-LKB1, proposed to be a mechanism to increase the lifespan of C. elegans (Zhang et al., 2016Zhang C.S. Li M. Ma T. Zong Y. Cui J. Feng J.W. Wu Y.Q. Lin S.Y. Lin S.C. Metformin activates AMPK through the lysosomal pathway.Cell Metab. 2016; 24: 521-522Abstract Full Text Full Text PDF PubMed Google Scholar, Chen et al., 2017aChen S.C. Brooks R. Houskeeper J. Bremner S.K. Dunlop J. Viollet B. Logan P.J. Salt I.P. Ahmed S.F. Yarwood S.J. Metformin suppresses adipogenesis through both AMP-activated protein kinase (AMPK)-dependent and AMPK-independent mechanisms.Mol. Cell. Endocrinol. 2017; 440: 57-68Crossref PubMed Scopus (43) Google Scholar, Chen et al., 2017bChen J. Ou Y. Li Y. Hu S. Shao L.W. Liu Y. Metformin extends C. elegans lifespan through lysosomal pathway.eLife. 2017; 6: e31268Crossref PubMed Scopus (0) Google Scholar). Phosphorylation and activation of AMPK leads to further inhibition of mTORC1, activation of peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC1-α) and mitochondrial biogenesis, activation of SIRT1 and other nutrient-sensing pathways, inhibition of advanced-glycation end products partly by inhibiting nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and pro-inflammatory cytokines, activation of Ulk1 and regulation of autophagy, among others (Kim et al., 2011Kim J. Kundu M. Viollet B. Guan K.L. AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1.Nat. Cell Biol. 2011; 13: 132-141Crossref PubMed Scopus (2948) Google Scholar, Salminen and Kaarniranta, 2012Salminen A. Kaarniranta K. AMP-activated protein kinase (AMPK) controls the aging process via an integrated signaling network.Ageing Res. Rev. 2012; 11: 230-241Crossref PubMed Scopus (309) Google Scholar, Barzilai et al., 2016Barzilai N. Crandall J.P. Kritchevsky S.B. Espeland M.A. Metformin as a tool to target aging.Cell Metab. 2016; 23: 1060-1065Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, Zhou et al., 2016Zhou Z.E. Tang Y. Jin X. Chen C. Lu Y. Liu L. Shen C. Metformin inhibits advanced glycation end products-induced inflammatory response in murine macrophages partly through AMPK activation and RAGE/NFκB pathway suppression.J. Diabetes Res. 2016; 2016: 4847812Crossref PubMed Scopus (8) Google Scholar, Herzig and Shaw, 2018Herzig S. Shaw R.J. AMPK: guardian of metabolism and mitochondrial homeostasis.Nat. Rev. Mol. Cell Biol. 2018; 19: 121-135Crossref PubMed Scopus (367) Google Scholar) (Figure 1). Metformin’s AMPK-independent mechanisms also contribute to the direct activation of SIRT1, direct inhibition of mTORC1 via Rag-GTPases, suppression of adipogenesis through inhibition of p70S6K pathway, activation of DNA-damage-like response via the activation of ATM-Chk2 pathway, and activation of nuclear factor erythroid 2-related factor 2 (Nrf2), all of which result in downregulation of inflammatory responses (Kalender et al., 2010Kalender A. Selvaraj A. Kim S.Y. Gulati P. Brûlé S. Viollet B. Kemp B.E. Bardeesy N. Dennis P. Schlager J.J. et al.Metformin, independent of AMPK, inhibits mTORC1 in a rag GTPase-dependent manner.Cell Metab. 2010; 11: 390-401Abstract Full Text Full Text PDF PubMed Scopus (501) Google Scholar, Vazquez-Martin et al., 2011Vazquez-Martin A. Oliveras-Ferraros C. Cufí S. Martin-Castillo B. Menendez J.A. Metformin activates an ataxia telangiectasia mutated (ATM)/Chk2-regulated DNA damage-like response.Cell Cycle. 2011; 10: 1499-1501Crossref PubMed Scopus (63) Google Scholar, Chen et al., 2017aChen S.C. Brooks R. Houskeeper J. Bremner S.K. Dunlop J. Viollet B. Logan P.J. Salt I.P. Ahmed S.F. Yarwood S.J. Metformin suppresses adipogenesis through both AMP-activated protein kinase (AMPK)-dependent and AMPK-independent mechanisms.Mol. Cell. Endocrinol. 2017; 440: 57-68Crossref PubMed Scopus (43) Google Scholar, Chen et al., 2017bChen J. Ou Y. Li Y. Hu S. Shao L.W. Liu Y. Metformin extends C. elegans lifespan through lysosomal pathway.eLife. 2017; 6: e31268Crossref PubMed Scopus (0) Google Scholar, Prasad et al., 2017Prasad S. Sajja R.K. Kaisar M.A. Park J.H. Villalba H. Liles T. Abbruscato T. Cucullo L. Role of Nrf2 and protective effects of metformin against tobacco smoke-induced cerebrovascular toxicity.Redox Biol. 2017; 12: 58-69Crossref PubMed Scopus (42) Google Scholar, Cuyàs et al., 2018aCuyàs E. Fernández-Arroyo S. Verdura S. García R.Á.-F. Stursa J. Werner L. Blanco-González E. Montes-Bayón M. Joven J. Viollet B. et al.Metformin regulates global DNA methylation via mitochondrial one-carbon metabolism.Oncogene. 2018; 37: 963-970Crossref PubMed Scopus (0) Google Scholar, Cuyàs et al., 2018bCuyàs E. Verdura S. Llorach-Parés L. Fernández-Arroyo S. Joven J. Martin-Castillo B. Bosch-Barrera J. Brunet J. Nonell-Canals A. Sanchez-Martinez M. Menendez J.A. Metformin is a direct SIRT1-activating compound: computational modeling and experimental validation.Front. Endocrinol. (Lausanne). 2018; 9: 657Crossref PubMed Google Scholar) (Figure 1). A novel role of metformin was elucidated in modulating the gut microbiota and the availability of branched-chain amino acids to the gut microbiota, which is directly attributable to its effect on nutrient sensing and aging. This function is further discussed below in metformin’s capability to improve intercellular signaling and attenuate inflammation. More recently, metformin’s beneficial effects on maintaining energy balance and body weight were shown to be regulated via growth differentiation factor 15 (GDF15), which further warrants more understanding of metformin’s GDF15-mediated targeting of biological aging (Coll et al., 2020Coll A.P. Chen M. Taskar P. Rimmington D. Patel S. Tadross J.A. Cimino I. Yang M. Welsh P. Virtue S. et al.GDF15 mediates the effects of metformin on body weight and energy balance.Nature. 2020; 578: 444-448Crossref PubMed Scopus (10) Google Scholar). Although the mechanisms of metformin in targeting fundamental pathways in biological aging are far from completely understood, here, we attempt to link its mode of action by highlighting its role on individual hallmarks of biological aging as evidenced in cell lines and model organisms (Table 1; Figure 1).Table 1Summary of Key Targets and Pathways Impacted by Metformin as Evidenced by Their Modulation in Cell Lines, C. elegans, Drosophila, and RodentsAttenuation of Hallmarks of AgingEffects of Metformin on key targets and pathways involved in regulating each hallmark of agingImproved Nutrient Signaling•AMPK ↑ (Hawley et al., 2002Hawley S.A. Gadalla A.E. Olsen G.S. Hardie D.G. The antidiabetic drug metformin activates the AMP-activated protein kinase cascade via an adenine nucleotide-independent mechanism.Diabetes. 2002; 51: 2420-2425Crossref PubMed Google Scholar)•SIRT1 ↑ in low NAD+ concentrations (Cuyàs et al., 2018bCuyàs E. Verdura S. Llorach-Parés L. Fernández-Arroyo S. Joven J. Martin-Castillo B. Bosch-Barrera J. Brunet J. Nonell-Canals A. Sanchez-Martinez M. Menendez J.A. Metformin is a direct SIRT1-activating compound: computational modeling and experimental validation.Front. Endocrinol. (Lausanne). 2018; 9: 657Crossref PubMed Google Scholar)•Insulin/IGF-1 signaling ↓ (Sarfstein et al., 2013Sarfstein R. Friedman Y. Attias-Geva Z. Fishman A. Bruchim I. Werner H. Metformin downregulates the insulin/IGF-I signaling pathway and inhibits different uterine serous carcinoma (USC) cells proliferation and migration in p53-dependent or -independent manners.PLoS One. 2013; 8: e61537Crossref PubMed Scopus (0) Google Scholar)•AGEs ↓ (Chung et al., 2017Chung M.M. Nicol C.J. Cheng Y.C. Lin K.H. Chen Y.L. Pei D. Lin C.H. Shih Y.N. Yen C.H. Chen S.J. et al.Metformin activation of AMPK suppresses AGE-induced inflammatory response in hNSCs.Exp. Cell Res. 2017; 352: 75-83Crossref PubMed Scopus (13) Google Scholar)•mTORC1 ↓ via Rag-GTPase ↓ (Kalender et al., 2010Kalender A. Selvaraj A. Kim S.Y. Gulati P. Brûlé S. Viollet B. Kemp B.E. Bardeesy N. Dennis P. Schlager J.J. et al.Metformin, independent of AMPK, inhibits mTORC1 in a rag GTPase-dependent manner.Cell Metab. 2010; 11: 390-401Abstract Full Text Full Text PDF PubMed Scopus (501) Google Scholar), via TSC2 ↑ (Dowling et al., 2007Dowling R.J. Zakikhani M. Fantus I.G. Pollak M. Sonenberg N. Metformin inhibits mammalian target of rapamycin-dependent translation initiation in breast cancer cells.Cancer Res. 2007; 67: 10804-10812Crossref PubMed Scopus (656) Google Scholar) & via REDD1 ↑ (Ben Sahra et al., 2011Ben Sahra I. Regazzetti C. Robert G. Laurent K. Le Marchand-Brustel Y. Auberger P. Tanti J.F. Giorgetti-Peraldi S. Bost F. Metformin, independent of AMPK, induces mTOR inhibition and cell-cycle arrest through REDD1.Cancer Res. 2011; 71: 4366-4372Crossref PubMed Scopus (382) Google Scholar)•AMPK ↑ (Onken and Driscoll, 2010Onken B. Driscoll M. Metformin induces a dietary restriction–like state and the oxidative stress response to extend C. elegans healthspan via AMPK, LKB1, and SKN-1.PLoS One. 2010; 5: e8758Crossref PubMed Scopus (0) Google Scholar, Cabreiro et al., 2013Cabreiro F. Au C. Leung K.Y. Vergara-Irigaray N. Cochemé H.M. Noori T. Weinkove D. Schuster E. Greene N.D. Gems D. Metformin retards aging in C. elegans by altering microbial folate and methionine metabolism.Cell. 2013; 153: 228-239Abstract Full Text Full Text PDF PubMed Sc