Metformin Modulates the Mechanisms of Ageing

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 oldestablished, 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.


Introduction
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. Galega officinalis, also termed as French lilac, Italian fitch, or Spanish sainfoin, the herb metformin derives from, has been known as a traditional medicine since the seventeenth century and was recommended for the treatment of thirst and frequent urination (symptoms of diabetes) by John Hill in 1772. The identification of guanidine and of its related compounds within Galega officinalis, which proved to be able to reduce blood glucose in animals, led to the synthesis of metformin (dimethylbiguanide) in 1922. However, it was only in the 1950s that more information on metformin's properties was published and when the name of Glucophage, meaning glucose eater, was suggested by Jean Sterne. Metformin was introduced as a treatment for T2DM in 1958 in the UK and in other European countries, whereas in the USA it was approved only in 1994 and started to be used beginning in 1995 [5]. A milestone multicentre trial, the United Kingdom Prospective Diabetes Study (UKPDS) in 1998, showed that the newly T2DM diagnosed patients receiving metformin for more than a decade displayed significant reduction of the cardiovascular events and of diabetesrelated death and highlighted that these effects were independent of the glucoselowering efficacy. Moreover, the potentially beneficial effects of metformin on the macro-and microvasculature have also been revealed [5][6][7][8]. Finally, in a 10-year posttrial analysis, metformin continues to offer cardiovascular benefits [9]. Based on these evidence data, in 2009, the American Diabetes Association (ADA) and the European Association for the Study of Diabetes (EASD) indicated metformin as the first-line therapy for T2DM [10]. Furthermore, metformin holds a significant role in the delay/prevention of T2DM onset, as shown by the randomised trial conducted in the USA, i.e. the Diabetes Prevention Program (DPP). The study highlighted that metformin reduces the incidence of T2DM by 31% compared to placebo in adults at high risk for T2DM (obese and with impaired glucose tolerance) [11][12][13][14]. Hence, metformin is also recommended as a pharmacologic tool for the prevention of T2DM in subjects with prediabetes, mainly for those with a BMI ≥ 35 kg/m [2], those aged <60 years, and in women with prior gestational diabetes mellitus [15][16][17].
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 [18]. 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 [19]. 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 [24]. 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 [27]. As such, metformin has been proven useful in overweight and obesity [28,29], hypertension [30], atherosclerosis [31], coronary artery disease [32], dementia [33] and cancer [34]. Moreover, in terms of mortality [35], 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 [27]. 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 [37]. DOI: http://dx.doi.org/10.5772/intechopen.89431 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.

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 [23]. 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. [23] 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 [23]. 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. [23] 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 [23].
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 [42].

Mechanisms involved in ageing
Ageing is a complex process that occurs at the molecular, cellular, organ and organismal level that everyone faces in time [43]. 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 [24]. 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].
Genomic instability has been revealed to be a major stochastic mechanism of ageing [44,45]. Broadly, deoxyribonucleic acid (DNA) damage can be induced by both exogenous genotoxic factors, such as ionising radiation and ultraviolet irradiation as well as endogenous genotoxic agents, i.e. products of normal metabolism that lead to the formation of reactive oxygen species (ROS) and subsequently to oxidative stress, that may finally result in deleterious effects on the cell. DNA lesions can cause mutations, block transcription and replication but can also trigger DNA damage response (DDR), which implies mechanisms that intervene and arrest cell cycle progression, resulting in the repair of almost all the alterations that occur within the genome. However, when DNA damage is extensive and prevails over repair, DDR effectors trigger cell death (apoptosis) or cell senescence, contributing to ageing and age-related diseases [46,47]. In fact, in ageing, DNA damage overtakes DNA repair, leading to genomic instability, a fact sustained by studies showing accumulation of DNA alterations in old tissues [48]. On the other hand, genomic instability has been reported to be a driver of accelerated ageing, widely demonstrated by the presence of hypersensitivity to genotoxins and defects in genome maintenance in progeroid syndromes termed as diseases of accelerated ageing. Collectively, DNA damage as a culprit in ageing is highlighted by the accrual of sources of damage, i.e. oxidative stress (the oxidative stress theory of ageing) associated with the mitochondrial theory of ageing, as mitochondria is the primary source of ROS, increased activation of the DDR, mutations and presence of senescent cells along with a decreased capacity for DNA repair [47]. Among these factors oxidative stress is a well-known pathogenic mechanism and seems to be the most important one [49]. The overproduction of ROS along with a reduced antioxidant defence, i.e. oxidative stress, leads to DNA, protein and lipid damage [50,51]. Also, ROS lead to age-related DNA lesions acting via nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) which controls cytokine and chemokine expression and regulates adhesion molecules [45,52,53].
Telomeres are chromosomal end structures that play important roles in the protection of DNA from degradation [54]. In each cell division, 20-200 base pairs are lost within the telomeres, and telomerase is in charge of repairing telomeres after cell division. However, when they reach a certain critical length, i.e. shortening or attrition, the cells stop replicating and die [43]. The shortening process, as the telomerase fails to replicate completely the terminal ends of the DNA molecules, has been reported in ageing [55,56]. Moreover, in humans, damaged telomerase can cause degenerative defects associated with ageing [57,58]. DOI: http://dx.doi.org/10.5772/intechopen.89431 Epigenetics meaning "above the genes" is termed as the inheritance of changes in gene function with no modifications in the nucleotide sequence of DNA [43,59]. Epigenetic changes that comprise alterations in DNA itself as DNA methylation and modifications of histones (acetylation and methylation) as well as of other chromatin-associated proteins and chromatin remodelling can also be involved in ageing [22]. Sirtuins, a family of NAD-dependent deacetylases that act on Lys16 of histone H4, are emerging as a link between cellular transformation and lifespan [59]. Of note, epigenetic alterations seem to be reversible, underpinning the anti-ageing interventions [60]. Moreover, Greer et al. [61] showed transgenerational epigenetic inheritance of longevity in Caenorhabditis elegans suggesting that manipulation of specific chromatin modifiers in parents can induce an epigenetic memory of longevity in descendants.
Proteostasis or protein stability is an important feature of the cells and involves a complex network that coordinates protein synthesis with polypeptide folding, conservation of protein conformation and protein degradation [62,63]. When damaged, as a consequence of various external and endogenous stress factors, it leads to the accumulation of protein aggregates holding proteotoxic effects and becomes a contributor to ageing and to age-related diseases [63][64][65]. In fact, it has been demonstrated that with age, proteostasis becomes compromised, leading to proteotoxicity [43,62,66]. More precisely, intracellular damaged protein deposition has been described in age-related diseases such as Alzheimer's and Parkinson's [62,63,67]. Finally, evidence data have revealed a double-sense link between DNA damage and proteostasis, which jointly induce an increased cellular lesion [63].
Deregulated nutrient sensing represents another important hallmark of ageing [22,68]. Nutrient sensing is mediated by specific molecular pathways, such as insulin and insulin-like growth factor 1 (IGF-1 informs the cells about the presence of glucose and has the same intracellular signalling pathway as insulin), termed as "insulin and/IGF1-signalling" pathway (IIS) as well as the mechanistic target of rapamycin (mTOR) that senses nutrients, whereas AMP-activated protein kinase (AMPK) and sirtuins detect the energy levels [22,43]. All these systems named as "nutrient sensing" pathways regulate metabolism and influence ageing [43]. More precisely, current data show that anabolic signalling induces accelerated ageing, while decreased nutrient signalling (attained through caloric restriction) promotes a healthy span and extends longevity [69,70].
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 [72]. 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 [73]. Finally, upregulation of both AMPK and sirtuins favours healthy ageing [74].
Mitochondrial dysfunction is a feature of ageing that refers to reduced respiratory chain efficiency, resulting in electron leak and diminished ATP production [75]. The consequence of mitochondrial dysfunction, installed across ageing, is the formation of ROS, and the theory of free radicals as a mechanism inducing ageing has been widely discussed [76]. However, this theory has been re-analysed and reconsidered as emerging data show that oxidative stress up to a specific threshold has, in fact, a beneficial effect in prolonging lifespan [77,78]. More specifically, it seems that ROS in a certain amount may play a role as a trigger of compensatory homeostatic reactions as a response to the ongoing and increasing stress factors that come along with ageing, resulting in facing damage and maintaining survival [79]. Still, when over the specific threshold, ROS change their purpose and induce deleterious age-related effects [77,80,81].
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 [22]. 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 [63]. Hence, although severe mitochondrial dysfunction is deleterious, mild respiratory damage may increase lifespan, possibly subsequently to a hormetic response [84]. 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 [85]. In this respect, Anisimov et al. [74] showed that when administrated early in life, metformin treatment increases life span in mice.
Senescence is an age hallmark that stands out as a response triggered by genomic instability and telomere attrition resulting in growth arrest, thus limiting the proliferation of aged and damaged cells [22,46,47,86]. A second important feature of senescent cells is the development of a peculiar secretome, termed as the senescence-associated secretory phenotype (SASP), which encompasses cytokines, chemokines and proteases, resulting in a pro-inflammatory state [87,88]. Under normal conditions the SASP is involved in the recruitment of macrophages, neutrophils and natural killer (NK) cells, thus holding a beneficial effect in eliminating the senescent cells. However, across the ageing process, the senescence cells accumulate resulting in increased cytokine production and recruitment of more immune cells, which jointly contribute to the onset of the inflammageing state, a true driver of ageing [36,87]. Moreover, a declined activity of the immune system, termed as immunosenescence, is installed in aged people, thus impairing the clearance of senescent cells and, in turn, increasing even more the chronic inflammation state. Collectively, senescence, inflammageing and immunosenescence promote ageing and operate together, rendering aged people more susceptible to age-related diseases [87,89]. Finally, interestingly, mitochondrial dysfunction can also trigger cellular senescence, a process termed as "mitochondrial dysfunction-associated senescence" (MiDAS). MiDAS support the existence of a strong inter-relation between cellular senescence and metabolic dysfunction, highlighting that targeting metabolism may be a proper way to extend lifespan in humans [36].
Stem cell exhaustion, i.e. the progressive decline in the regenerative potential of the stem cells needed for tissue repair, is another characteristic of ageing. As explained, ageing is accompanied by immunosenescence, a condition that results from reduced haematopoiesis and that has several deleterious consequences [22]. DOI: http://dx.doi.org/10.5772/intechopen.89431 Finally, apart from cellular damage, ageing also implies altered intercellular communication. Inflammation is an ageing-associated damage in intercellular communication termed as "inflammageing," as previously described. Inflammageing may result from multiple causes, such as the accumulation of tissue damage, the reduced ability of the immune system to remove pathogens, the increase of senescent cells that produce pro-inflammatory cytokines, immunosenescence that fails to remove the senescent cells, the activation of the NFkB transcription factor, as well as the onset of a dysfunctional autophagic response [22]. Noteworthy, that inflammation is involved in the pathogenesis of obesity and T2DM, diseases that contribute to the onset of ageing [71]. Apart from inflammation, the intercellular communication has been revealed by the bystander effect referring to senescent cells inducing senescence in neighbouring cells via gap-junction-mediated cell-cell cross talk [90].

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 Caenorhabditis elegans [105,106] and mice [107], but not in Drosophila melanogaster [108,109]. In this respect, metformin supplementation was shown to increase mean lifespan and to prolong the healthspan of nematode Caenorhabditis elegans (an experimental model often used to study ageing and anti-ageing therapies) via AMPK [106]. Moreover, other authors have shown that metformin has the ability to retard ageing in Caenorhabditis elegans by metabolic alteration of its trophic microbial partner, E. coli. In brief, metformin disrupts the bacterial folate cycle, which reduces the levels of methionine in the worm. Finally, this results in postponing ageing by triggering a metabolic dietary restriction phenomenon and AMPK activation [105,110]. Based on these results, we might argue another important role of metformin, that of modulating human microbiota, i.e. an increased abundance of E. coli, resulting in an increased production of short-chain fatty acids, such as butyrate and propionate, by which metformin might induce significant positive results in T2DM and might interfere with longevity [36,111,112].
In a very recent study, Song et al. [113] 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 [113].
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. [116] 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 [74]. Besides the increase of lifespan in mice, Martin-Montalvo et al. [107] pointed out that metformin seems to mimic some of the benefits of calorie restriction and leads to improved glucosetolerance 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 [107].
Finally, as previously mentioned, not all experimental models confirm the antiageing role of metformin. It is the case of Drosophila fruit fly, another animal model where the authors showed that metformin induced a robust activation of AMPK and reduced lipid stores, but did not increase lifespan. Moreover, they found that when administered in high concentrations, metformin is toxic to flies. Finally, it seems that metformin appears to have evolutionarily conserved effects on metabolism but not on fecundity or lifespan [108].

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 [119]. Although other organs have been discussed as a target for metformin, such as the gut [120], liver remains the main ground of action, as reduced hepatic uptake of metformin prevents the lowering blood glucose effect [91]. There are several mechanisms by which metformin downregulates gluconeogenesis. Firstly, metformin induces alterations in cellular energetics [117], 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 [42]. Noteworthy, AMPK is one of the molecular pathways that can modify the rate of ageing [43]. The importance of the activation of AMPK in obtaining the reduction in hepatic glucose production was investigated by Hawley et al. [85] who showed that an AMPK mutant does not respond to metformin treatment. On the other hand, Foretz et al. [124] 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 [126] as physiological/low concentration of metformin, which cannot induce AMP/ATP change, can still activate AMPK [125].
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 [127]. Furthermore, activated AMPK DOI: http://dx.doi.org/10.5772/intechopen.89431 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 [128]. Moreover, the decreased PKA activity promotes glucose consumption and inhibits glucose output [129]. Finally, metformin inhibits hepatic gluconeogenesis through AMPK-dependent regulation of the orphan nuclear receptor small heterodimer partner (SHP) [130].
Secondly, AMPK-independent mechanisms by which metformin inhibits hepatic gluconeogenesis have been reported [117]. In this respect, Miller et al. [131] 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 [131]. 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 [132].
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 [117]. 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 [72]. Metformin has been demonstrated to downregulate mTOR in both a AMPK-dependent and AMPKindependent 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 [42].
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 [45]. Metformin has been demonstrated to inhibit NF-kB resulting in suppressing the inflammatory response via AMPK-dependent and independent pathways [138]. Also, metformin seems to hold the ability to reduce the endogenous ROS production [93] by acting at a mitochondrial level through blockage of the reverse electron flow at the respiratory chain complex 1 [139].
Finally, a very recent pathway has been described by Chen et al. [140]. The authors showed through genetic manipulation that metformin extends the Caenorhabditis elegans lifespan and attenuates age-related fitness decline via a mechanism that requires v-ATPase-Ragulator-AXIN/LKB1 of the lysosomal pathway [140].

Conclusions
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.