diabetes research and clinical practice 160 (2020) 108025

Contents available at ScienceDirect

Diabetes Research
and Clinical Practice
journal homepage: www.elsevier.com/locat e/dia bre s

Metformin: An old drug against old age and

associated morbidities

Teresa Salvatore a, Pia Clara Pafundi b, Floriana Morgillo c, Raimondo Di Liello c,

Raffaele Galiero b, Riccardo Nevola b, Raffaele Marfella b, Lucio Monaco b, Luca Rinaldi b,
Luigi Elio Adinolfi b, Ferdinando Carlo Sasso b,*
a
Unit of Internal Medicine, Department of Precision Medicine, University of Campania ‘‘Luigi Vanvitelli”, Via Pansini, 5, 80131 Naples, Italy
b
Unit of Internal Medicine, Department of Advanced Medical and Surgical Sciences, University of Campania ‘‘Luigi Vanvitelli”, Piazza
Miraglia, 2, 80138 Naples, Italy
c
Division of Medical Oncology, Department of Precision Medicine, University of Campania ‘‘Luigi Vanvitelli”, Via Pansini, 5, 80131 Naples,
Italy

A R T I C L E I N F O A B S T R A C T

Article history: Metformin represents a striking example of a ‘‘historical nemesis” of a drug. About 40 years
Received 9 December 2019 after its marketing in Europe, once demonstrated its efficacy and safety, metformin was
Received in revised form registered also in the U.S. A few years later, it has become a mainstay in T2DM treatment,
2 January 2020 according to all international Scientific Societies guidelines.
Accepted 13 January 2020 Today, despite the advent of new innovative drugs, metformin still persists as a first-
Available online 16 January 2020 choice drug in T2DM.
This success is largely justified. In fact, over the years, also positive effects on health
increased. In particular, evidence has been accumulated on a beneficial impact against
Keywords:
many other aging-related morbidities (obesity, metabolic syndrome, cardiovascular dis-
Metformin
ease, cancer, cognitive decline and mortality). This literature review describes preclinical
Aging
and clinical evidence favoring the ‘‘anti-aging” therapeutic potential of metformin outside
Obesity
of T2DM. The rationale to the use of metformin as part of a combined therapy in a variety of
Mortality
clinical settings, allowing for a reduction of the chemotherapy dose in cancer patients, has
Cardiovascular diseases
also been discussed. In particular, the focus was on metformin action on RAS/RAF/MAPK
Cancer
pathway.
In the end, the real challenge for metformin could be to fully demonstrate beneficial
effects on health even in non-diabetic subjects.
Ó 2020 Elsevier B.V. All rights reserved.

* Corresponding authors at: University of Campania ‘‘Luigi Vanvitelli”, Department of Advanced Medical and Surgical Sciences, Italy.
E-mail addresses: teresa.salvatore@unicampania.it (T. Salvatore), piaclara.pafundi@unicampania.it (P.C. Pafundi), floriana.morgil-
lo@unicampania.it (F. Morgillo), diliello90@gmail.com (R. Di Liello), raffaele_ga@outlook.it (R. Galiero), riccardo.nevola@unicampania.it
(R. Nevola), raffaele.marfella@unicampania.it (R. Marfella), monacolucio@live.it (L. Monaco), luca.rinaldi@unicampania.it (L. Rinaldi),
luigielio.adinolfi@unicampania.it (L.E. Adinolfi), ferdinando.sasso@unicampania.it (F.C. Sasso).
https://doi.org/10.1016/j.diabres.2020.108025
0168-8227/Ó 2020 Elsevier B.V. All rights reserved.
2 diabetes research and clinical practice 160 (2020) 108025

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2. Benefits of metformin on Age-Related morbidities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2.1. Obesity and associated conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2.2. Cardiovascular disease and mortality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
3. Anticancer properties. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
4. Cognitive decline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
5. Anti-Aging potential of metformin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
7. Authors’ contributions statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Declaration of Competing Interest. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Appendix A. Supplementary material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

1. Introduction

Metformin is one of the oldest and most widely prescribed
anti-hyperglycemic drug worldwide. Its history begins in the
1920s, when compounds related to the active agents in the
Galega officinalis, the French lilac, were accidentally noted
to have anti-hyperglycemic properties. Firstly introduced in
Europe in 1957, the drug was registered in U.S. much later,
in 1995, after its efficacy and safety were confirmed and pub-
lished. A few years after its introduction into the American
market, metformin has become a mainstay in the treatment
of type 2 diabetes mellitus (T2DM) thanks to the support of
UKPDS results [1]. Still today, the guidelines on diabetes man-
agement indicate that the drug should be continued as long
as tolerated and not contraindicated, in monotherapy as well
as in combination with other oral and subcutaneous antihy-
perglycemic drugs.
Metformin is a multifaceted drug, with multiple sites of
action and molecular mechanisms [2]. Its anti-
hyperglycemic effects are mainly produced by the either
direct or indirect reduction of hepatic glucose production
and, according to several studies, by an increase in insulin
sensitivity of peripheral tissues. Moreover, for time gut has
been recognized as a target organ for metformin where the
drug increases glucose consumption, stimulates GLP-1 pro-
duction and modifies the microbiome.
The complex I of the mitochondrial respiratory chain is
the ‘‘classic” molecular target of metformin. Its inhibition
prevents mitochondrial ATP production and increases cyto-
plasmic ADP:ATP and AMP:ATP ratios, thus leading to activa-
tion of AMP-activated protein kinase (AMPK). AMPK is an
important energy sensing enzyme which regulates cellular
energy status by enhancing insulin sensitivity (through
effects on fat metabolism) and lowering cAMP, thus reducing
the expression of gluconeogenic enzymes. AMPK-
independent mechanisms of action may include inhibition
of fructose-1,6-bisphosphatase by AMP.
Over the years, metformin has been shown to exert an
increasingly wide range of positive pleiotropic effects on
health, in addition to those associated with an improvement
in glycemia. In particular, data has accumulated on a benefi-
cial impact against many aging-related morbidities other
than diabetes, including obesity, metabolic syndrome, cardio-
vascular disease, cancer, cognitive decline and mortality.
Some of these benefits have been known since long time
(e.g., metabolic disorders and atherosclerotic burden) [3,4].
Others have emerged later, i.e. anti-cancer [5] and anti-
neurodegeneration properties [6]. As a result, metformin
appears like a gerontoprotector agent that may substantially
extend lifespan and lifetime free from diseases in diabetic
subjects taking the drug, even compared with non-diabetic
people [7].
In this review we provide a brief picture of current preclin-
ical and clinical evidences about the anti-aging therapeutic
potential of metformin outside of T2DM, with a short discus-
sion on the underlying hypothesized pathophysiological
mechanisms.
2. Benefits of metformin on Age-Related
morbidities
2.1. Obesity and associated conditions
The excess of body fat must be considered to right reason an
age-related disease as overweight and obesity have been
demonstrated to markedly increase worldwide as individuals
age [8]. It is widely accepted that obesity increases the risk of
cardiovascular diseases (e.g., atherosclerotic disease, chronic
heart failure, and arterial hypertension), type 2 diabetes, var-
ious types of cancers, and sleep apnea/sleep-disordered
breathing. However, whereas weight excess is associated with
a higher mortality risk in young and middle-aged individuals,
a similar relationship is not observed in adults aged 60 to
75 years [9]. This phenomenon, known as the ‘‘obesity sur-
vival paradox”, represents an unresolved matter of debate,
which needs further clarifications, particularly a more accu-
rate risk status stratification based on comorbidities, sex,
body fat distribution, preserved amount of lean mass, nutri-
tional status, cardiorespiratory fitness, socioeconomic envi-
ronment, etc. [10].
The belief in a protective role of excess adiposity from
health disorders in the elderly may be a simplistic way of
 diabetes research and clinical practice
dealing with the issue if considering that a high risk of dis-
ability and falls, as well as a low quality of life, may result
from reduced physical function. In addition, individuals over
80 years old are characterized by a high prevalence of ‘‘sar-
copenic obesity” (8.4% in women and 13.5% in men) [11]. This
clinical condition combining sarcopenia and obesity, and not
easily identifiable by anthropometric measures, is worse than
the other forms of obesity associated with an increased all-
cause mortality [12].
Metformin is one of the few oral agents preventing weight
gain or even favoring weight loss in T2DM patients. Due to the
progressive nature of disease, many diabetic subjects will
need insulin treatment, a regimen that may induce weight
gain in various ways (anabolic effect of hormone, correction
of glycosuria, and increase in dietary intake to prevent/cor-
rect episodes of hypoglycemia). In this setting, the HOME trial
has indicated that continuing metformin beyond the initia-
tion of insulin therapy may prevent insulin-induced weight
gain [13].
It is usually thought that weight loss associated to met-
formin therapy is determined by a reduction in systemic insu-
lin requirement and gastrointestinal side effects such as
nausea, diarrhea, and dysgeusia. Currently, emerging evi-
dence suggests multiple mechanisms underlying this effect
mediated by modification of gut microbiome and modulation
of hypothalamic appetite regulatory centers, both directly or
indirectly, i.e. through the gut-brain axis [14].
Some studies only reported in abstract form associate
metformin treatment with a redistribution of fat from central
to subcutaneous depots and with a reduction in the thickness
of epicardial adipose tissue [15].
The role of metformin as a primary treatment for obesity
is still equivocal and its current use as weight loss agent out-
side of T2DM remains off-label. However, the American Dia-
betes Association (ADA) suggests taking into consideration
the prescription of metformin also to prediabetic patients
with a BMI > 35 kg/m2 and/or other additional risk factors
for diabetes. This recommendation mainly arises from the
impressive findings of the Diabetes Prevention Program
(DPP) [16], a study involving overweight patients with
impaired glucose tolerance (IGT) and fasting plasma glucose
95–125 mg/dL. During the 2-year double-blind period, subjects
assigned to the metformin arm experienced significant body
weight and waist circumference reductions, in addition to a
31% reduction in T2DM incidence compared to placebo.
Patients initially randomized to the drug maintained these
benefits, both for weight loss and diabetes incidence, also
after a follow-up >15 years [17].
As widely reviewed by Hostalek et al. [18], a strong evi-
dence from a number of clinical trials other than DPP, as well
as the clinical experience, support both efficacy and safety
profiles of metformin for diabetes prevention. To this concern,
it should be remarked that aging is associated with major
changes in glucose metabolism, with a progressive decline
in glucose tolerance from the third through the ninth decade
of life [19] and a rise of blood glucose levels per decade after
50 years by 0.06 mmol/L at fasting and by 0.05 mmol/L 2-
hours after an oral glucose tolerance test [20].
Data from the literature support a positive effect of met-
formin even on metabolic syndrome components other than
160 (2020) 108025 3
obesity and prediabetes. Metformin monotherapy may signif-
icantly improve dyslipidemia on statin-naı̈ve people with
newly diagnosed T2DM [21] and a meta-analysis suggests that
biguanide could effectively lower systolic blood pressure in
nondiabetic patients, especially in those with IGT or obesity
[22].
2.2. Cardiovascular disease and mortality
Metformin is also one of the few anti-hyperglycemic drugs
with significant results in cardiovascular events reduction
and diabetes-related mortality. These benefits are mostly sup-
ported by the seminal trial UK Prospective Diabetes Study
(UKPDS 34), in which a small subgroup of overweight patients
randomized to metformin displayed a risk reduction of 39%
for nonfatal myocardial infarct, 42% for diabetes-related
death and 36% for all-cause mortality, compared to conven-
tional dietary measures [1]. Remarkably, beneficial effects
were still appreciable throughout the 10-year post-trial mon-
itoring, despite similarities in glycated hemoglobin levels [23].
Besides UKPDS, only two small randomized, placebo-
controlled trial were published. The first conducted on
insulin-treated diabetic patients showed a 40% reduction of
macrovascular events with the addition of metformin vs pla-
cebo [13]. In the other, T2DM patients with coronary artery
disease (CAD) obtained a 46% reduction of primary cardiovas-
cular composite endpoint when treated with metformin vs
glipizide [24].
Solid evidence of cardiovascular protection also derives
from large observational studies in people with early- and
late-stage diabetes, showing improved outcomes with met-
formin as compared with sulfonylurea monotherapy, both
for cardiovascular events and all-cause mortality. This advan-
tage over sulfonylureas is confirmed by a recently published
updated meta-analysis including over one million patients
with CAD, in which a higher reduction in incidence rate of
cardiovascular events and all-cause and cardiovascular mor-
tality was shown with metformin rather than with sulfony-
lureas [25].
An interesting finding is reported by a retrospective obser-
vational study on a population of about 80,000 T2DM subjects,
showing that metformin therapy determined not only a
longer survival than in diabetic patients not treated with met-
formin, as well as in their matched non‐diabetic controls,
despite the higher BMI and greater comorbidities at baseline
[26].
Clinical literature is rich in data suggesting a substantial
benefit from metformin in chronic heart failure (CHF) popula-
tion, even on patients with CHF in III and IV NYHA class. A
retrospective cohort study on 16,417 Medicare beneficiaries
with diabetes hospitalized for CHF showed that patients dis-
charged on metformin experimented a reduced mortality
(13%) and readmission rates (8%) [27]. In another retrospective
study on a large British database, metformin therapy in out-
patients with newly diagnosed T2DM and CHF significantly
reduced mortality rates with respect to diet and lifestyle
changes [28]. In a systematic review of observational studies
involving 34,000 patients with diabetes and CHF, a 20% lower
mortality in metformin users was recorded, mostly compared
with sulfonylureas [29].
4 diabetes research and clinical practice
Finally, data collected on T2DM patients with established
atherothrombosis participating to the REACH (Reduction of
Atherothrombosis for Continued Health) Registry [30] deserve
to be mentioned. In this study, metformin used in secondary
prevention resulted associated with a 24% lower mortality
rate. Such a finding was consistent among subsets of patients
in whom biguanide was not recommended, noticeably those
with a history of congestive heart failure (31%), >65 years
(23%), and with an estimated creatinine clearance of 30 to
60 mL/min/1.73 m2 (36%). The REACH registry results have
thus helped to reconsider the traditional contraindications
to the use of metformin and the concerns about the risk of
lactic acidosis, especially in the elderly [31,32].
Though the extensive literature, as well as the positive
results recently reported in landmark large-scale randomized
CV outcome trials (CVOT) with glucagon-like peptide-1 recep-
tor agonists (GLP-1 RAs) and sodium-glucose cotransporter
type 2 inhibitors (SGLT2), the most recent 2019 ESC guidelines
on diabetes, prediabetes and CVD [33] recommend these
glucose-lowering agents in drug-naı̈ve patients at high/very
high CV risk instead of metformin.
Metformin would be reserved as first choice only to over-
weight T2DM patients without CVD and at moderate CV risk,
or added in second line only if HbA1c is not at target. Scheen
suggests several reasons to challenge this downgrading of
metformin use, first of all the lack of a meta-analysis on
CVOTs of both SGLT2i and GLP-1RAs separating T2DM
patients treated (about three quarters of the enrolled popula-
tion) or not with metformin as background therapy [34].
Moreover, we cannot ignore the huge amount of data on both
animal and human studies, proving the cardiovascular pro-
tection exerted by metformin.
As recently reviewed by Zilov et al. [15], this beneficial
effect derives from an improvement either of glycemic con-
trol and of other traditional risk factors (i.e., body fat and lipid
profile), but also from the correction of a series of disorders
such as haemostasis, inflammation and oxidative stress, as
well as from the direct inhibition of atherosclerotic plaque
development. In particular, as shown by the major part of
controlled trials on T2DM patients, metformin seems to exert
a protective effect against endothelial dysfunction which rep-
resents an early step both in the initiation and progression of
atherosclerosis. A recently published trial, the CODICE multi-
center prospective study, has assessed in prediabetic patients
with stable angina and nonobstructive coronary stenosis the
effect of metformin therapy on coronary endothelial function
and major adverse cardiac events (MACEs) at 24 months of
follow-up [35]. The findings demonstrated a reduction of car-
diovascular events of 40% and a significantly lower percent-
age of endothelial dysfunction (left anterior descending
coronary diameter at baseline and after acetylcholine infu-
sion) in prediabetics treated with metformin 850 mg twice a
day as compared with untreated prediabetics.
This topic is as well the aim of an ongoing randomized
clinical trial exploring primary prevention of cardiovascular
events by metformin in people with hyperglycemia below
the threshold diagnosis for diabetes [36]. Moreover, the Inves-
tigation of Metformin in Pre-Diabetes on Atherosclerotic Car-
diovascular OuTcomes (VA-IMPACT; https://clinicaltrials.gov/
ct2/show/NCT02915198) aims to provide insights into met-
160 (2020) 108025
formin efficacy in reducing both mortality and cardiovascular
morbidity in patients with pre-diabetes and established car-
diovascular disease.
3. Anticancer properties
Pharmaco-epidemiology has provided several lines of evi-
dence suggesting a protective role of metformin on malignan-
cies development, which impacts differently based on the
type of cancer, with promising results with some and mini-
mal effects on others [37]. Nevertheless, these considerations
arise from observational retrospective studies, most of which
deprived of important covariates (e.g., BMI and glycemic con-
trol), and with several methodological limitations. These lat-
ter, along with an insufficient follow-up in most of cases,
render the relationship between metformin and cancer inci-
dence still object of debate [38].
Few groups have studied the antiproliferative effects of
metformin as single agent in different cancer settings. Anti-
cancer effects of metformin are associated with both direct
(insulin-independent) and indirect (insulin-dependent)
actions of the drug. Two main mechanisms have been pro-
posed: (i) the correction of hyperinsulinemia and (ii) the
reduced energy consumption in neoplastic cells [37]
(Fig. 1). Both mechanisms involve the activation of AMPK,
which inhibits the mammalian target of rapamycin (mTOR)
signaling pathway, whose role in tumorigenesis is well-
known [39].
In our previous experience, treatment with metformin
resulted in a dose-dependent inhibition of growth in selected
non-small cell lung cancer (NSCLC) cell lines, which also
showed a significant reduction in the anchorage-
independent colony-forming ability, with a nonsignificant
antiproliferative effect in other NSCLC cell lines [40–42]. The
reason for the different response to metformin among the
NSCLC cell lines examined may depend on LKB1 gene inacti-
vation, a common event in NSCLC, especially in lung adeno-
carcinoma cells [43]. In this sense, loss of function of LKB1
may reduce the sensitivity to metformin. However, several
preclinical studies suggest the existence of an AMPK-
independent mechanism of action of metformin [2].
Several studies have assessed the risk of cancer in diabetic
patients, as well as any protective role of metformin on can-
cer development, whilst there is less evidence about how
antidiabetic drugs act on the outcome after anticancer ther-
apy. One study by Tan BX et al. on 99 patients with NSCLC
assessed both metformin and insulin influence on
chemotherapy response rates and survival [44]. In particular,
chemotherapy with metformin produced higher results com-
pared to insulin and other compounds, both in terms of pro-
gression, free-survival and overall survival.
Evidence of increased antitumor effects by the concomi-
tant treatment with metformin and chemotherapeutics (e.g.,
cisplatin, paclitaxel or doxorubicin) arises from in vivo studies
on breast, prostate and lung cancer models [45–48].
The reason for such a synergism is supported by several
hypotheses. Metformin has been demonstrated to decrease cel-
lular thymidine phosphorylase (TP) and excision repair cross-
complementation 1 (ERCC1) protein and mRNA levels by down
regulating phosphorylated MEK1-ERK1/2 protein levels, in both
 diabetes research and clinical practice
Fig. 1 – Direct cardioprotective effects of metformin. AMPK, adenosine monophosphate activated protein kinase; eNOS,
endothelial nitric oxide synthase; ER, endoplasmic reticulum; HIF-1, hypoxia-inducible factor-1; LKB1, liver kinase B1; mTOR,
mammalian target of rapamycin; NADPH, nicotinamide adenine dinucleotide phosphate; NFkB, nuclear factor-kappa B; ROS,
reactive oxygen species; TGF-b1 transforming growth factor-b1.
a dose- and time-dependent manner [45]. These two enzymes
are involved in protection from apoptosis and DNA damage
recognition and repair, respectively. Both are considered nega-
tive prognostic factors and negatively correlate with the efficacy
of chemotherapy. Similarly, metformin has been demonstrated
to enhance paclitaxel cytotoxicity in human lung cancer cells
(H1650 and H1703) by inhibiting the paclitaxel-induced p38
MAPK-mediated ERCC1 expression [46,47].
These observations provide an experimental rationale to
the use of metformin as part of a combined therapy in a vari-
ety of clinical settings, and allows for a reduction of the
chemotherapy dose in cancer patients. The evidence of a sig-
nificant synergism of metformin with chemotherapy in lung
cancer models led to examine the effects of a combined treat-
ment of metformin with gefitinib, a selective EGFR tyrosine
kinase inhibitor (EGFR-TKI) on NSCLC cell lines. To this end,
a panel of human NSCLC cell lines with a defined spectrum
of sensitivity to EGFR inhibition by gefitinib was used, includ-
ing an in vitro model of acquired resistance to gefitinib devel-
oped from the sensitive human CALU-3 lung cancer cells [40].
Combination of metformin with gefitinib strongly reduced
proliferation and induced apoptosis of NSCLC cell lines which
had harbored wild-type LKB1 gene. Such effects were also
shown in NSCLC cell lines resistant to the EGFR TKI, suggest-
ing a potential metformin-induced reversal of resistance to
gefitinib in some cancer cell lines. The effects were associated
with a pronounced decrease in the levels of MAPK and Akt
proteins phosphorylation, key intracellular mediators of
growth factor-activated cell survival and proliferation signals.
The combined treatment also affected mTOR signaling, as
suggested by the sustained inhibition of S6 and p70S6K
phosphorylation.
To note, single-agent metformin treatment caused an
unexpected paradoxical increase in activated phosphory-
lated MAPK (P-MAPK) levels, as a result of an increased B-
RAF and C-RAF association. This observation could be thera-
peutically relevant, since it has been shown that, while
exerting antiproliferative and proapoptotic effects, single
160 (2020) 108025 5
agent metformin treatment could enhance proliferating sig-
nals through the RAS/RAF/MAPK pathway. Such action could
in turn, induce cell proliferation in those cell lines with con-
stitutively activating Ras mutations. In this scenario, met-
formin enhances MEK-Is antitumor activity in human
LKB1-wild-type NSCLC cell lines by downregulating GLI1
and reducing the NF-kB (p65)-mediated transcription of
MMP-2 and MMP-9 [49]. Similarly, a recent work by Martin
et al. showed that melanoma cells driven by oncogenic BRAF
are resistant to the growth-inhibitory effects of metformin,
which in turn increases ERK activity and upregulates the
VEGF-A protein. Likewise to the synergism identified
between gefitinib and metformin, Martin et al. also identified
another unexpected ‘synthetic lethality’ whereby metformin
and VEGF inhibitors cooperate to suppress BRAF mutant
tumor growth [50].
Actually, it is mandatory to use caution to extrapolate
from in vitro to in vivo effects, due to a potential difference
between high pharmacological concentrations in vitro com-
pared to lower physiological concentrations used in clinical
setting.
Moreover, future studies in cancer patients, alongside with
extensive preclinical investigations, are thus required to bet-
ter investigate metformin effect on the RAS/RAF/MAPK path-
way and the best setting in which to use metformin combined
with molecularly targeted agents.
4. Cognitive decline
In the last decade, emerging evidence has accumulated also
suggesting an exciting metformin property of counteracting
neurodegenerative diseases.
It is reported that higher glucose levels may represent a
risk factor for dementia, even among persons without dia-
betes [51]. On the contrary, individuals affected by Alzhei-
mer’s disease (AD), the major neurocognitive disorder, are
characterized by an increased likelihood of T2DM develop-
ment [52]. This epidemiological link, along with several bio-
6 diabetes research and clinical practice
logical and clinical data, have provided convincing evidence
that the pathogenesis of these two disorders may share a ser-
ies of common mechanisms (e.g., impaired insulin signaling,
altered glucose metabolism, inflammation, increased oxida-
tive stress, and premature senescence), which may represent
the rationale for anti-hyperglycemic drugs (e.g., metformin)
use in dementia [53].
Laboratory research studies on spontaneously obese or
high fat fed murine models with insulin resistance report a
reduction by metformin in cognitive deficits [54].
In humans, although several publications favor an appre-
ciable metformin benefit, epidemiological data are still
ambiguous. In a large observational study on about 70.000
subjects aged 65 or over, non-dement and non-diabetic at
baseline, new-onset T2DM was associated with an increased
risk of dementia, which resulted weaker in patients treated
with metformin rather than other antidiabetic medications
[55]. A best neuroprotection offered by metformin vs sulfony-
lureas is supported by a retrospective study on 28,640 older
veterans, new users of metformin or sulfonylureas, showing
a little (11%) though significant lower risk of dementia in
patients taking biguanide and aged <75 years with no differ-
ence in patients aged 75 years [56]. In the population-
based Singapore Longitudinal Aging Study, metformin admin-
istration in a very low sample size (365 T2DM patients)
resulted associated with a very impressive lower risk of cogni-
tive decline (51%; until 73% for treatment duration >6 years),
even after adjusting for vascular and non-vascular risk factors
[57]. The matter of treatment duration has emerged even in a
recently published study on few thousand of elderly veterans
with T2DM in which only a metformin exposure longer than
2 years showed a significant beneficial influence [58].
As suggested by a pilot trial on 80 subjects with amnestic
mild cognitive impairment followed for 12 months, met-
formin seems also to improve cognition in either overweight
or obese individuals without treated diabetes, though more
likely affected by prediabetes (HbA1c: 6.1% + 0.8 in 40 met-
formin subjects vs. 6.1 + 0.5 in 40 placebo subjects) [59]. On
the other hand, a recent study on a large population free of
dementia and anti-hyperglycemic therapy for 2 years before
Fig. 2 – Potential protective mechanisms of metformin against Alzheimer’s disease.
160 (2020) 108025
enrollment, provided a robust evidence that metformin use
for about 6 years is associated with an only modestly lower
(8%) risk of incident dementia than sulfonylurea, but no effect
after 75 years [60]. Other publications report no association
with metformin therapy or rather an opposite effect of
increasing dementia. In a cohort study from Taiwan’s
National Health Insurance Research Database, metformin
users showed, during a 12-yrs follow-up, not only an
increased risk for all-cause dementia, but also a higher inci-
dence of Parkinson’s disease [61].
Further large-scale long-term controlled trials are needed
to finally establish an eventual beneficial role of metformin
in neurodegenerative disorders development.
At same time, we also need further investigations into the
molecular mechanisms possibly mediating this protective
effect, for which the activation of AMPK-dependent pathways
with resulting inhibition of mTor and PI3K-Akt signaling, has
been hypothesized as main actor [6] (Fig. 2). As shown by
postmortem brain examination, the AD neuropathological
hallmark is the presence of: (1) extracellular amyloid plaques
resulting from the aggregation of amyloid-b (Ab) peptides; (2)
intracellular neurofibrillary tangles (NFT) made by hyper-
phosphorylated tau protein and (3) neuronal loss [55]. Met-
formin may affect all these pathological conditions through
mechanisms related to its anti-hyperglycemic actions, i.e.
correction of insulin-resistance (and therefore of hyperinsu-
linemia) and hyperglycemia, as described below.
Since the insulin-degrading enzyme acts on both insulin
and Ab, but more selective for insulin, high levels of insulin
in the brain may deprive Ab of its main clearance mechanism
[62]. Again, insulin seems to increase tau phosphorylation
and cleavage, thus leading to NFT formation and accumula-
tion, as shown by both in vitro [63] and in vivo experiments
[64].
Chronic hyperglycemia is mainly associated with
advanced glycation end-products (AGEs) accumulation, which
induces Ab and tau proteins glycation and lead to Ab aggrega-
tion and NFT formation [65]. Hyperglycaemia also increases
the production of reactive oxygen and nitrogen species, thus
determining oxidative stress. This event negatively reflects on
 diabetes research and clinical practice
neuronal cells, given the brain high susceptibility to an oxida-
tive imbalance due to its high-energy demand and oxygen
consumption and abundance of easily peroxidable polyunsat-
urated fatty acids [66].
Neuronal damage in AD also derives from mitochondrial
structural and functional abnormalities, neuroinflammation,
and senescence [67]. Even these processes may be relieved
by metformin, as the drug inhibits NF-kB signaling and proin-
flammatory cytokines [68] and regulates mitochondrial bio-
genesis and autophagy by mTOR inhibition [54]. As reported
in the literature, various others mechanisms may contribute.
As an example, metformin has been found to remarkably
decrease, via AMPK, the expression and activity of Beta-
secretase 1 protein involved in the cleavages of the amyloid
precursor protein and generation of Ab [69], as well as to
reduce tau phosphorylation via mTOR/PP2A [70].
5. Anti-Aging potential of metformin
Growing experimental and clinical data have recently turned
the spotlight on metformin as a potential therapeutic agent to
slow ageing.
The life-extending capability of the drug has been revealed
in a number of recent experimental studies on various non-
diabetic animal models such as rodents and invertebrates,
i.e. Drosophila melanogaster and mostly Caenorhabditis elegans.
In humans, the anti-aging effect is reported in a population
of diabetic subjects taking metformin who exhibits a signifi-
cantly lower (7%) all-cause mortality than non-diabetics [7].
The ongoing clinical trial TAME (Targeting Aging with Met-
formin) [71] is testing metformin as a tool to target ageing in
non-diabetic people. To this purpose, healthy subjects aged
Fig. 3 – Potential mechanisms of metformin anti-aging properties.
160 (2020) 108025 7
65–79 yrs. were enrolled to evaluate the time until appearance
of any ageing-related morbidity by taking metformin.
The recent publication of the results of Metformin in Long-
evity Study (MILES; https://clinicaltrials.gov/ct2/show/
NCT02915198) provided the first direct evidence that met-
formin exerts tissue-specific effects on the expression of
metabolic and non-metabolic human genes implicated in
ageing [72]. Besides this interesting suggestion, the met-
formin anti-aging potential currently remains a highly
debated topic, as it is unclear whether the ability of drug to
target multiple pathways of aging reflects downstream conse-
quences of a primary action on a single mechanism or
whether it involves direct effects on multiple aging regulators
(Fig. 3).
Some of the nine alterations indicated by López-Otı́n et al.
as the hallmarks of the aging process (genomic instability,
telomere attrition, epigenetic alterations, loss of proteostasis,
deregulated nutrient sensing, mitochondrial dysfunction, cel-
lular senescence, stem cell exhaustion, and altered intercellu-
lar communication) may be modulated by metformin [73]. An
example may be represented by cellular senescence, an irre-
versible cellular growth arrest resistant to apoptosis and asso-
ciated to secretion of a variety of bioactive factors inducing, in
turn, senescence. Metformin has been described to control
this process through limitation of ceramide harmful effects
[74] or inhibition of genes coding for multiple inflammatory
cytokines [75]. Even if it is only an untested hypothesis, met-
formin may exert some of its health-promoting effects
through epigenomic alterations, i.e. by modulation of micro-
biota, as it has been reported in C. elegans [76] and in many
human age-related disorders (e.g., T2DM, obesity, and cancer)
[77].
8 diabetes research and clinical practice
A series of other pathological mechanisms implied in the
aging process seem to be modulated by metformin. Inflam-
mation may be suppressed by inhibition of NF-jB transcrip-
tion factor via AMPK-dependent and AMPK-independent
pathways [68]. In addition, also autophagy, a form of pro-
grammed cell death highly implicated in shortened lifespan
and development of aging-associated diseases, may be
enhanced [78]. Metformin may even correct the imbalance
in the amount of endogenous reactive oxygen species pro-
duced by mitochondria to antioxidant defense [79], with a
resulting decrease of oxidant stress responsible of proteins,
lipids and nucleotide damage. Expression of SIRT1, a member
of the class III (NAD+-dependent) histone deacetylases con-
tributing to enhanced healthy aging, is induced by metformin
as a downstream consequence of AMPK mediated NAD+ ele-
vation [80].
To sum up, many of the above described actions could be
traced to the metformin direct inhibitory action on mitochon-
drial respiratory complex I and elevation of cellular AMP
levels that allosterically primes AMPK activation, a principal
energy sensor that switches on catabolic pathways breaking
down complex molecules to produce more ATP [2]. This is
what most likely happens in Caenorhabditis elegans, where
metformin-mediated lifespan extension seems to be geneti-
cally dependent upon AMPK and its upstream effects [81].
The inhibition of mTOR complex, another hub for energy
and nutrient sensing that switches on anabolism and
increases cell growth when activated, has been shown to
extend lifespan across multiple model systems [82]. Met-
formin suppresses mTOR function, both through AMPK-
independent [83] or AMPK-dependent mechanisms [84]. Wu
et al. recently observed that the drug inhibits mTOR by acting
on the transport through the nuclear pore complex [85],
which has been shown as increased in aging [86].
An interesting unifying molecular mechanism has been
suggested by Chen et al. [87]. Based on biochemical and
genetic experiments involving Caenorhabditis elegans and
human cells, the authors found that metformin regulates
AMPK activation and mTORC1 suppression, two metabolic
pathways that both converge on lysosomes, membrane-
bound cellular organelles involved in nutrient sensing and
recycling.
6. Conclusions
Since the latest version of the International Classification of
Diseases (ICD-11, code ‘Ageing-related’ XT9T) has formally
recognized ageing as a disease, now therapeutic strategies
targeting this condition should be developed.
To this aim, metformin may represent an interesting
option, since this drug has long been approved for human
use, with an excellent safety record for over six decades and
low costs. Moreover, in patients with T2DM, metformin can
be combined with any other anti-hyperglycemic agent, both
orally and parenterally administered, and therefore contin-
ued for life, unless contraindications.
The experimental and clinical data collected till now are
very encouraging. However, it remains still unknown whether
non-diabetic individuals taking metformin will live longer
160 (2020) 108025
and healthier without manifesting negative pleiotropic
actions as well as patients with or at risk for T2DM. Moreover,
we do not even have reliable biomarkers which could objec-
tively monitor the healthy aging process, and we do not know
the optimal anti-aging dosage of the drug.
Given these ambiguities, further findings from laboratory
research and new clinical trials such as TAME [71] could pro-
vide definitive answers to the ongoing debate on metformin
role as gerontoprotector. In the meantime, it is worthwhile
to point out that: (1) metformin should be the preferred treat-
ment option for T2DM, even in older patients; (2) the drug
may represent a therapeutic chance even for subjects with
prediabetes and (3) an excessive caution due to the fear of
side effects (i.e. lactic acidosis) may deprive a substantial
number of diabetic individuals from its wide potential bene-
fits [32].
7. Authors’ contributions statement
Conception and design: TS, FCS. Development of methodol-
ogy: TS, FCS, PCP. Acquisition of data: RG, LM, RDL, RN. Anal-
ysis and interpretation of data: PCP. Writing, review, and/or
revision of the manuscript: TS, PCP, FM, RDL, RG, RM, LR,
LM, LEA, RN, FCS. Study supervision: TS, FCS, PCP, FM. All
authors read and approved the final draft of the manuscript.
Funding
The authors received no funding from an external source.
Declaration of Competing Interest
The authors declared that there is no conflict of interest.
Appendix A. Supplementary material
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.diabres.2020.108025.
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