Living in a time when population is continuously ageing, the challenge and demand for assessing the age-related pathways, potential diseases and longevity have become of major interest. The pharmaceutical industry possesses huge resources in this field, mainly due to the recent discoveries of novel mechanisms of action of old-established, classical drugs. Here we find metformin, a well-established antidiabetic medicine but with new potential benefits, as the most recent reports quote. We present the main pathways of the possible implications of metformin in the modulation of ageing processes, evolution and diseases, focussing on its ageing counteraction, based on the latest scientifically based biochemical reports.
- type 2 diabetes
- mechanisms of ageing
At present, metformin is the preferred first-line drug used for the treatment of type 2 diabetes mellitus (T2DM) [1, 2, 3, 4]. However, the journey of metformin (1,1-dimethylbiguanide hydrochloride) has not been a simple one.
Ageing continues to be an intruding topic and an area of great interest, constantly addressed by researchers worldwide. It encompasses a plethora of complex processes that have urged scientists to decipher its underlying mechanisms and to find the possible avenues to postpone its onset and that of its associated diseases . Data from the literature have demonstrated a sustained ageing of the world’s population, estimating a total of around 21.8% of subjects over 60 years old in 2050 and 32.2% in 2100 . Installed as a result of the interaction between genetic, epigenetic, environmental and stochastic factors, ageing involves a progressive decline of the body functions as a consequence of the gradual cellular impairment due to a failure of the repair mechanisms [20, 21, 22, 23]. Age is a major risk factor for the onset of metabolic, cardiovascular, neurodegenerative, immune and malignant diseases . Ageing has been reported to be conditioned by the genetic factor in a proportion of 25–30%, while the remaining 70–75% is ruled by the environmental factor, making it a possible target for therapeutic tools among which metformin has been found [25, 26].
Beyond its blood glucose-lowering effect, metformin has been described as a drug used for preventing or delaying several conditions associated with ageing . As such, metformin has been proven useful in overweight and obesity [28, 29], hypertension , atherosclerosis , coronary artery disease , dementia  and cancer . Moreover, in terms of mortality , it has been shown that patients with T2DM under metformin monotherapy had a longer survival than the matched, nondiabetic controls. However, the precise beneficial mechanisms by which metformin performs its non-glycaemic work are yet to be analysed. Hence, given the complex mechanisms of action of metformin, there is a growing interest in approaching and studying the potential anti-ageing effect of this drug. With regard to this interest, some large randomised clinical trials have been recently set up in order to evaluate the potential role of metformin in reducing the burden of age-related diseases. The Investigation of Metformin in Pre-Diabetes on Atherosclerotic Cardiovascular outcomes (VA-IMPACT) trial is a placebo-controlled study started in February 2019 and aimed at shedding light on the potential role of metformin in reducing mortality and cardiovascular morbidity in patients with prediabetes and established atherosclerotic cardiovascular disease. More precisely, the primary outcomes include the time to death from any cause, nonfatal myocardial infarction, stroke, hospitalisation for unstable angina, or symptom-driven coronary revascularisation . The other clinical trial, also a placebo-controlled trial, i.e. Targeting Ageing with Metformin (TAME), investigates subjects who have been diagnosed with one single age-associated disease and will provide insight on the ability of metformin to postpone and/or prevent the installation of a second pathology, such as cancer, CVD and dementia [13, 36]. Finally, more information is needed for a better understanding of the mechanistic targets and therapeutic implications of certain drugs (such as metformin) that might delay/alleviate the development of age-related diseases .
Herein, we revisit the mechanisms involved in ageing and the mechanistic target of metformin as a potential anti-ageing drug, and we review the available data on the clinical and experimental results showing the ability of metformin to promote healthspan and longevity.
2. Epidemiological data on the anti-ageing effect of metformin
A large body of evidence has demonstrated that metformin could be considered a geroprotective agent in humans . As explained, the protective role of metformin in survival has been largely demonstrated by the UKPDS multicentre trial in terms of cardiac and all-cause mortality, as compared with usual care [8, 9]. However, given its main role, that is to reduce hyperglycaemia, and knowing that a good control of diabetes correlates with an extended lifespan, the question arises whether metformin could be accounted as a tool to prolong longevity in patients that do not display T2DM. In keeping with this question, a recent systematic review by Campbell et al.  summarised the studies in which the effects of metformin on all-cause mortality or diseases of ageing have been compared to the nondiabetic or general population or to diabetics controlling the disease through other means. Overall, the meta-analysis revealed that subjects with T2DM under metformin treatment have a lower rate of all-cause mortality and longer survival than people free of T2DM not using metformin and the general population, suggesting that this drug could be an effective instrument to extend the lifespan of those not affected by T2DM [23, 35, 38, 39, 40]. Moreover, the meta-analysis revealed that subjects with T2DM taking metformin had lower rates of all-cause mortality than those following other therapies, such as insulin or sulphonylurea . Given these results, it may be argued that the outcome is attained by the geroprotective role of metformin resulting in delaying or preventing diseases of ageing, such as cancer or cardiovascular disturbances, which are the two most encountered ageing-related diseases [23, 41]. Firstly, in terms of malignancies, Campbell et al.  showed that people with T2DM taking metformin had a lower rate of developing any cancer compared with the general population. Moreover, the risk of developing colorectal, breast or lung cancer in individuals with T2DM on metformin treatment, as compared to those using other therapies, was lower. Secondly, subjects with T2DM following metformin therapy displayed a lower rate of any form of cardiovascular disease with respect to those managing their T2DM through any non-metformin therapy. In addition, although the incidence of stroke was also lower with metformin, for myocardial infarction the effect of the drug seems to be non-significant .
Finally, apart from the cardiovascular diseases and cancer, there are also other age-related pathologies that could be targeted by metformin, such as cognitive dysfunction. However, the evidence in patients with T2DM is conflicting with some studies showing a protective role of metformin against cognitive decline, whereas others are arguing that metformin treatment could induce neurodegeneration as well as Parkinson’s and Alzheimer’s disease. Nevertheless, the interpretation of the data is difficult given the possible presence of other concomitant conditions that may contribute to this cognitive decline .
3. Mechanisms involved in ageing
Ageing is a complex process that occurs at the molecular, cellular, organ and organismal level that everyone faces in time . It involves the loss of the body’s ability to overcome and respond to stress (homeostenosis) by repair and regeneration, thus leading to various disturbances within the human body . Overall, the ageing processes are of a heterogeneous and heterochronic nature. As a heterogeneous process, ageing can evolve at different rates in diverse organisms, while the heterochronic feature implies that cells and tissues within a single organism can age in an asynchronic manner, finally making chronological age different as compared to biological age [24, 43]. Growing body of evidence has shown that ageing involves multiple mechanisms that inter-relate with and modulate each other. In this respect, two elegant reviews have described nine hallmarks of ageing, which have been classified into primary hallmarks (genomic instability, telomere attrition, epigenetic alterations, and loss of proteostasis) as the main culprit of molecular damage, antagonistic hallmarks (deregulated nutrient sensing, mitochondrial dysfunction, and cellular senescence) with beneficial effects when at low levels, by protecting the human organism against damage, but with deleterious effects when at high levels, and finally, the integrative hallmarks (stem cell exhaustion and altered intercellular communication) that arise when the accumulating damage cannot be balanced by homeostatic mechanisms, thus ultimately inducing ageing [22, 36].
The “insulin and/IGF1-signalling” pathway (IIS) operates on the forkhead box proteins or FOXO family of transcription factors and on the mTOR complexes and has been reported to be the most conserved ageing-controlling pathway. Indeed, mutations that reduce the functions of insulin and IGF-1 receptor or downregulate the intracellular effectors, i.e. AKT, mTOR and FOXO, result in increased lifespan [22, 69, 71].
The mTOR kinase is part of two complex proteins and is sensitive to high levels of amino acids controlling a wide range of cellular functions, mostly anabolic metabolism . It is noteworthy that mTOR is a target of rapamycin (an mTOR inhibitor), an antibiotic that exerts anti-proliferative effects by acting through this specific pathway. Several studies have shown that mTOR manipulation by inducing downregulation is involved in extending longevity [22, 43].
Finally, the AMPK pathway and sirtuins that sense changes in energy levels, i.e. low levels of ATP, act in the opposite direction as compared to IIS and mTOR, their activation leading to increased energy production and decreased ATP utilisation [22, 43]. In fact, caloric restriction seems to activate the AMPK pathway . Finally, upregulation of both AMPK and sirtuins favours healthy ageing .
Apart from the ROS theory, accumulating data have revealed that impaired mitochondrial function may contribute to ageing through other mechanisms, such as the increase of permeability in response to stress that triggers inflammatory reactions, the damaged interface between the outer mitochondrial membrane and the endoplasmic reticulum as well as reduced biogenesis of mitochondria . Furthermore, it seems that both endurance training and alternate-day fasting have the ability to improve healthspan through mitochondrial degeneration avoidance [82, 83].
Finally, the mitochondrial dysfunction seems to be related to the hormesis which is deemed as an adaptive response of the organism to low doses of a toxic agent or physical condition, such as ROS, that induces the ability of the organism to tolerate higher doses of the same toxic agent . Hence, although severe mitochondrial dysfunction is deleterious, mild respiratory damage may increase lifespan, possibly subsequently to a hormetic response . In fact, data from the literature have shown that metformin could be considered a mild mitochondrial “toxic agent” as it induces a low energy state and activates AMPK . In this respect, Anisimov et al.  showed that when administrated early in life, metformin treatment increases life span in mice.
Finally, apart from cellular damage, ageing also implies
Given the aforementioned complex hallmarks of ageing, researchers worldwide have searched for proper tools to obtain the delay of ageing and the avoidance of age-related diseases. Here we find metformin, a drug that has been reported to be useful in modulating some of the age-related features. In fact, in cellular and animal models, metformin has been shown to influence and to hold beneficial effects on the following age related hallmarks : (1) genomic instability [92, 93], (2) telomere attrition , (3) epigenetic changes , (4) proteostasis [96, 97], (5) nutrient-sensing pathways [98, 99], (6) mitochondrial function , (7) cellular senescence [101, 102], (8) stem cell function , and (9) low-grade inflammation .
4. Experimental evidence on the anti-ageing effect of metformin
Evidence-based data have revealed that metformin holds an important role in extending survival and delaying the onset of age-related diseases in nematode
In a very recent study, Song et al.  used the silkworm, a popular experimental model, to investigate the impact of metformin on lifespan and the underlying molecular pathways. They found that metformin prolonged lifespan without reducing body weight, which suggests that it can increase lifespan by remodelling the animal’s energy distribution strategy. Also, metformin increased fasting tolerance and levels of the antioxidant glutathione and activated APMK. Finally, these results suggest that activity in this pathway may contribute to metformin-induced lifespan extension in silkworm by increasing stress resistance and anti-oxidative capacity, while reducing energy output for silk product .
Studies on ageing and lifespan have also been performed on mice, highlighting the potential anti-ageing effect of metformin, resulting in an extended lifespan [114, 115, 116]. Anisimov et al.  demonstrated that chronic treatment of female mice with metformin significantly increased mean and maximum lifespan, even without cancer prevention in that model. In a further study, the authors showed that in female mice, metformin increased lifespan and postponed tumours when started at young and middle, but not at the old age . Besides the increase of lifespan in mice, Martin-Montalvo et al.  pointed out that metformin seems to mimic some of the benefits of calorie restriction and leads to improved glucose-tolerance test, increased insulin sensitivity and reduced low-density lipoprotein and cholesterol levels without a decrease in the caloric intake. With respect to the mechanisms of action, metformin seems to increase the antioxidant activity, resulting in reductions in both oxidative stress and chronic inflammation .
Finally, as previously mentioned, not all experimental models confirm the anti-ageing role of metformin. It is the case of
5. Mechanisms of metformin action: A focus on molecular pathways that modulate ageing
The main universally accepted role of metformin is to alleviate hyperglycaemia. This outcome is obtained through the inhibition of hepatic gluconeogenesis [117, 118]. Metformin holds an insulin-sensitising action and insulin-induced suppression of endogenous glucose production . Although other organs have been discussed as a target for metformin, such as the gut , liver remains the main ground of action, as reduced hepatic uptake of metformin prevents the lowering blood glucose effect . There are several mechanisms by which metformin downregulates gluconeogenesis. Firstly, metformin induces alterations in cellular energetics , i.e. by decreasing cellular respiration through inhibition of the complex I mitochondrial respiratory chain [121, 122]. The result of this inhibition is the increase of the ADP:ATP and AMP:ATP ratios, which subsequently activate the cellular energy state sensor AMP-activated protein kinase (AMPK) [91, 110, 123], the key player of metformin. Once activated, AMPK leads to an increase in ATP production and a decrease in ATP consumption . Noteworthy, AMPK is one of the molecular pathways that can modify the rate of ageing . The importance of the activation of AMPK in obtaining the reduction in hepatic glucose production was investigated by Hawley et al.  who showed that an AMPK mutant does not respond to metformin treatment. On the other hand, Foretz et al.  showed that in AMPK knockout mice, the inhibition of gluconeogenesis is still present and associated with a reduction in energy state, but this happens in response to higher concentrations of metformin as compared to standard treatment. With regard to therapeutic concentrations of metformin, it seems that AMPK activation is mandatory for the suppression of gluconeogenesis [117, 125]. Finally, we have to mention that the activation of AMPK via inhibition of the complex I mitochondrial respiratory chain has been recently debated  as physiological/low concentration of metformin, which cannot induce AMP/ATP change, can still activate AMPK .
Another effect mediated by AMPK activation by metformin refers to the inhibitory phosphorylation of acetyl-CoA carboxylase (ACC), which leads to increased fatty acid uptake and β-oxidation and hence to improved lipid metabolism and subsequently to improved insulin sensitivity . Furthermore, activated AMPK decreases glucagon-stimulated cyclic AMP (cAMP) accumulation, cAMP-dependent protein kinase (PKA) activity and downstream PKA target phosphorylation and increases cyclic nucleotide phosphodiesterase 4B (PDE4B). The authors provided a new mechanism by which AMPK antagonises hepatic glucagon signalling via phosphorylation-induced PDE4B activation . Moreover, the decreased PKA activity promotes glucose consumption and inhibits glucose output . Finally, metformin inhibits hepatic gluconeogenesis through AMPK-dependent regulation of the orphan nuclear receptor small heterodimer partner (SHP) .
Secondly, AMPK-independent mechanisms by which metformin inhibits hepatic gluconeogenesis have been reported . In this respect, Miller et al.  point towards the ability of the drug to inhibit adenylate cyclase, reduce levels of cAMP and PKA activity, abrogate phosphorylation of critical protein targets of PKA, and block glucagon-dependent glucose output from hepatocytes through accumulation of AMP and related nucleotides independently of AMPK . In addition, metformin inhibits the mitochondrial glycerophosphate dehydrogenase, resulting in an altered hepatocellular redox state, reduced conversion of lactate and glycerol to glucose and hence decreased hepatic gluconeogenesis .
Taken together, given the important role of metformin in inhibiting hepatic gluconeogenesis and therefore in reducing hyperglycaemia and subsequently hyperinsulinemia, jointly, important accelerators of ageing, several studies regard metformin as a potential anti-ageing drug [42, 117]. Metformin works through complex mechanisms that have been demonstrated to be similar to those associated with caloric restriction, a well-known model that underpins extended lifespan and healthspan. More precisely, it seems that both metformin and caloric restriction induce the same gene expression profile [107, 117, 133].
Another important target involved in changing the rate of ageing is mTOR . TOR responds to insulin, amino acids and hormones and is involved in controlling a wide range of cellular functions, such as glucose metabolism, lipid and protein synthesis, inflammation and mitochondrial function . Metformin has been demonstrated to downregulate mTOR in both a AMPK-dependent and AMPK-independent manner [98, 134, 135, 136]. Through stimulation of AMPK, metformin induces suppression of ATP consumption by inhibiting energy needing processes, such as protein synthesis via mTOR [42, 137]. In addition, through downregulation of mTOR signalling and of insulin-like growth factor 1 (IGF-1), metformin influences cell growth, proliferation and autophagy .
NF-kB pathway is another key mediator of ageing. As previously described, it is activated by genotoxic, oxidative and inflammatory stress and regulates the expression of cytokines, inflammation, growth factors and genes that regulate apoptosis . Metformin has been demonstrated to inhibit NF-kB resulting in suppressing the inflammatory response via AMPK-dependent and independent pathways . Also, metformin seems to hold the ability to reduce the endogenous ROS production  by acting at a mitochondrial level through blockage of the reverse electron flow at the respiratory chain complex 1 .
Finally, a very recent pathway has been described by Chen et al. . The authors showed through genetic manipulation that metformin extends the
In toto, the possible molecular mechanisms by which metformin exerts anti-ageing effects are [13, 91]: (1) inhibition of mitochondrial complex 1 in the electron transport chain and decrease of ROS production [139, 141], (2) activation of AMPK [106, 124, 140, 142, 143, 144], (3) inhibition of mTOR [106, 134, 135, 140], (4) NF-ĸB inhibition , and (5) reduced IGF-1 signalling .
Ageing encompasses a cluster of processes that induce a gradual decline of the human body functions, a condition that everyone faces in time. Also, ageing is a risk factor for a gamut of disturbances such as cancer, T2DM and cardiovascular and neurodegenerative diseases. Therefore, researchers worldwide strive to find the adequate tools in order to delay/avoid the onset of age-related diseases and hence promote healthspan. In keeping with this aim, metformin emerges as a drug that, beyond its main role to reduce hyperglycaemia, has antitumor effects and works as a protector against cardiovascular and neurodegenerative diseases making it a potential anti-ageing medicine. Importantly, metformin seems to possess positive effects even in nondiabetic subjects. However, the exact mechanisms of action and the molecular pathways involved in ageing that are modulated by metformin are not fully explained, and further studies are warranted for a better understanding of the beneficial effects of this drug.
Mr. Manfredi Rizzo serves as Director, Clinical Medical & Regulatory Affairs, Novo Nordisk Europe East and South. The publication was supported by funds from the National Research Development Projects to finance excellence (PFE)-37/2018–2020 granted by the Romanian Ministry of Research and Innovation.