REVIEWS

Metforminmode of action and clinical

implications for diabetes and cancer
Ida Pernicova and Mrta Korbonits
Abstract | Metformin has been the mainstay of therapy for diabetes mellitus for many years; however, the
mechanistic aspects of metformin action remained ill-defined. Recent advances revealed that this drug, in
addition to its glucose-lowering action, might be promising for specifically targeting metabolic differences
between normal and abnormal metabolic signalling. The knowledge gained from dissecting the principal
mechanisms by which metformin works can help us to develop novel treatments. The centre of metformins
mechanism of action is the alteration of the energy metabolism of the cell. Metformin exerts its prevailing,
glucose-lowering effect by inhibiting hepatic gluconeogenesis and opposing the action of glucagon. The
inhibition of mitochondrial complex I results in defective cAMP and protein kinase A signalling in response to
glucagon. Stimulation of 5'-AMP-activated protein kinase, although dispensable for the glucose-lowering effect
of metformin, confers insulin sensitivity, mainly by modulating lipid metabolism. Metformin might influence
tumourigenesis, both indirectly, through the systemic reduction of insulin levels, and directly, via the induction
of energetic stress; however, these effects require further investigation. Here, we discuss theupdated
understanding of the antigluconeogenic action of metformin in the liver and the implications of the discoveries
of metformin targets for the treatment of diabetes mellitus and cancer.
Pernicova, I. & Korbonits, M. Nat. Rev. Endocrinol. 10, 143156 (2014); published online 7 January 2014; doi:10.1038/nrendo.2013.256

Introduction
The biguanide metformin has been used for its glucose-
lowering effect since 1957 in Europe and since 1995
in the USA. Yet despite being the most frequently pre-
scribed antidiabetic treatment worldwide, its mechanism
of action remains largely elusive. Metformin and the
related phenformin are derivatives of guanidine (Table1).
Discovered in the 1920s in extracts of the plant Galega
officinalis (French lilac), isoamylene guanidine, also called
galegine, had been used for centuries for the treatment
of diabetes mellitus.1 Phenformin has been withdrawn
for use in humans as a result of safety concerns in most
parts of the world, whereas the clinical indications for
metformin therapy have expanded from type2 diabetes
mellitus (where it offers undisputable benefits) to gesta-
tional diabetes mellitus, polycystic ovary syndrome, the
metabolic syndrome and diabetesprevention.2
Metformin lowers glucose levels and improves insulin
sensitivity.3 In addition, metformin has gained atten-
Department of
Endocrinology, William
tion for its pleiotropic effects. It has been shown to
Harvey Research decrease food intake (Box1)4 and body weight3 in some
Institute, Barts and studies. Moreover, this drug can positively influence
TheLondon School of
Medicine and Dentistry, multiple cardiovascular risk markers (Box2), includ-
Queen Mary University ing lipid profile3,5 and fatty liver,6 modulate inflamma-
of London,
Charterhouse Square,
tory markers7,8 (Box2) and possibly reduce cancer risk.9
London EC1A 6BQ, UK However, many promising results are not without dispute.
(I. Pernicova, For example, whether metformin treatment can improve
M.Korbonits).
Correspondence to:
M. Korbonits Competing interests
m.korbonits@ The authors declare an association with the following company:
qmul.ac.uk Merck. See the article online for full details of the relationships.
cardiovascular morbidity and mortality remains con-
troversial. Although the UK Prospective Diabetes Study
and several systematic reviews found an overall reduced
cardiovascular mortality and morbidity risk with met-
formin treatment,10 two meta-analyses have failed to
determine any benefit of metformin therapy on all-cause
or cardiovascular-related mortality or on diabetes-related
macrovascular complications, pointing to methodological
weaknesses of previous studies and insufficient data.11,12
Here, we discuss the updated understanding of the
molecular mechanisms through which metformin acts
on metabolism, mainly focussing on liver gluconeo
genesis, and on tumourigenesis. In addition, we will
review the potential implications of new discoveries
about metformin molecular targets for the development
of antidiabetic and anticancer therapies.
Metformin and diabetes mellitus
Antidiabetic actions of metformin
The antihyperglycaemic action of biguanides is mainly a
consequence of reduced glucose output owing to inhibi-
tion of liver gluconeogenesis13 and, possibly to a lesser
extent, increased insulin-mediated glucose uptake in the
skeletal muscle.14 Metformin has little effect on glucose
absorption through the gastrointestinal tract but slightly
delays the absorption process.15,16 The pharmacokinetics
and response to metformin reveal a wide interindividual
variability. The polarity of metformin makes it depend-
ent on membrane transporters for cellular uptake and
secretion. The main metformin transporters are solute

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REVIEWS
Key points
The glucose-lowering, insulin-sensitizing agent metformin works mainly by
reducing gluconeogenesis and opposing glucagon-mediated signalling in the
liver and, to a lesser extent, by increasing glucose uptake in skeletal muscle
The primary site of metformin action is the mitochondrion
The antihyperglycaemic effect of metformin is probably owing to defective
protein kinase A signalling
Metformin affects lipid metabolism primarily via 5'-AMP-activated protein kinase
(AMPK) activation
Antitumourigenic effects of metformin, which require further study, might
be partially due to systemic metabolic alterations, including the reduced
availability of insulin
In cancer cells, metformin acts as an inducer of energetic stress; AMPK-driven
inhibition of mTOR seems to be required for much of its antimitotic activity
carrier family 22 members (SLC22A) 1 and 4 (also
known as OCT1 and OCTN1, respectively),17 multi
drug and toxin extrusion protein (MATE) 1 and 2, and
the plasma membrane monoamine transporter hENT4
(also known as PMAT). The function of these metformin
transporters is summarized in Table2.
Inhibition of gluconeogenesis in the liver
The liver, which expresses high levels of SLC22A1, is
considered to be the main site of action of metformin
(Table2). In addition, metformin concentration is higher
in the portal circulation than elsewhere in the body, which
might contribute to metformin accumulation in the liver.18
In this organ, metformin is suggested to have an effect
on the regulation of glucose uptake, gluconeogenesis,
glycolysis and glycogen synthesis (Figure1).
Metformin increases the activity of the insulin receptor
and of insulin receptor substrate 2 (IRS2) and enhances
glucose uptake via increased translocation of glucose
transporters, such as GLUT1 (also known as SLC2A1),
to the plasma membrane. 19 As a result, metformin
enhances the insulin-mediated suppression of gluconeo-
genesis.20 Furthermore, and possibly of greater impor-
tance, metformin opposes the gluconeogenic action of
the peptide hormone glucagon.2022 The net effect of the
interactions is that metformin inhibits gluconeogenic
enzymes and stimulates glycolysis by altering the activ-
ity of multiple enzymes in these pathways (Figure1).23
Gluconeogenesis accounts for 2897% of overall hepatic
glucose output depending on the feeding status in non-
diabetic individuals, the rate being higher in patients with
advanced type2 diabetes mellitus.24 In this patient popu-
lation, metformin was reported to reduce hepatic glucose
output by up to 75%.25
The uptake of gluconeogenic substrates, such as alanine
and lactate,26 is reduced in the presence of metformin,
possibly owing to depolarization of the hepatocyte mem-
brane through metformin-stimulated Cl efflux.26 The
effects of metformin on hepatic glycogen metabolism
are not well-established; however, invitro treatment of
hepatocytes decreased glycogen synthesis.27
Increased glucose uptake in skeletal muscle
Metformin improves insulin sensitivity and insulin-
mediated glucose uptake in skeletal muscle. This effect
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2014 Macmillan Publishers Limited. All rights reserved
is mediated through an increase in the tyrosine kinase
activity of the insulin receptor 19 and through enhanced
activity and translocation of glucose transporters, such
as GLUT4 (also known as SLC2A4), to the plasma
membrane.28 Increased insulin receptor expression19,29
and an enhanced ability to restore enzymatic pathways
involved in insulin signalling 30 have also been attributed
to metformin.
Altered endocrine function in the pancreas
The antidiabetic action of metformin is not the result of
increased insulin levels31,32 but rather has been associated
with reduced insulin concentrations in the circulation.33,34
Consequently, metformin monotherapy is associated with
only a minimal risk of hypoglycaemia.16 Glucose clamp
studies in nondiabetic individuals treated with metformin
suggest that a reduction in glucose levels lowers insulin
secretion and increases glucagon secretion.35 However,
this mechanism might be less effective in patients with
type2 diabetes mellitus, which might account for rare
cases of hypoglycaemia among patients with diabetes
mellitus receiving metforminmonotherapy.25,35
Importantly, metformin seems to interact with the
incretin axis, as an enhancer and sensitizer for the actions
of glucagon-like peptide 1 (GLP1).36 GLP1 increases
secretion of insulin and reduces secretion of glucagon in
response to glucose and has widespread tissue-specific
metabolic effects.37 Metformin stimulates expression
of GLP1 receptor in the pancreas and increases plasma
GLP1 levels.37,38 Moreover, circulating levels of dipep-
tidyl peptidase 4 (DDP4), which is known to degrade
incretins, were reported to be lower in patients treated
with metformin than in untreated individuals.39 However,
metformin did not directly inhibit DDP4 activity
invitro.38,40 Combining DDP4 inhibitors with metformin
improved glycaemic control in patients with type2 dia-
betes mellitus.41 The full clinical relevance of the effect of
metformin on GLP1 is yet to be established. Metformin
did not preserve cell function in outcomestudies.42
To summarize, the ability of metformin to reduce cir-
culating glucose levels in patients with type2 diabetes
mellitus can be explained by multiple mechanisms, such
as increased glucose uptake in liver and muscle, reduced
gluconeogenesis, improved GLP1 and reduced gluca-
gon functions. Nevertheless, the molecular principles of
metformin action remain debated.
Molecular targets of antidiabetic effects
Mitochondriathe primary site of action
The primary target of metformin within the cell is the
mitochondrion, where metformin transiently inhib-
its complex I of the mitochondrial electron transport
chain, which induces a drop in energy charge, 4346 a
measure of the energetic state of the cell (defined as
[ATP]+0.5[ADP])/([ATP]+[ADP]+[AMP]). 46 This
inhibition has been demonstrated more markedly invitro
than invivo.45 The resulting decrease in ATP production
and increase in AMP levels probably drive two major
pathways: inhibition of glucagon-induced cAMP syn
thesis, as demonstrated in the liver,21 and the activation
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Table 1 | Comparison of phenformin and metformin

Characteristic Phenformin Metformin
Chemical structure Biguanide Biguanide
Two guanidine groups linked together with the loss of an Two methyl groups on a guanide side-chain (Dimethylbiguanide)
ammonia group
Contains a phenyl-ethyl ring on a guanidine side-chain
(Phenethylbiguanide)
Physical properties Less polar and more lipid soluble than metformin127 Polar and unusually hydrophilic
and cellular Phenformin exhibits higher affinity and transport activity More reliant on active transcellular transport129*
transport with marked differences in uptake kinetics compared Lower affinity for mitochondrial membranes than phenformin
withmetformin128*
High affinity for mitochondrial membranes60
Effect on Powerful inhibitory effect on functioning of the Less powerful inhibitor of the mitochondrial respiratory chain (probably
mitochondrial mitochondrial respiratory chain the main reason for decreased risk of lactic acidosis)45
respiratory chain Inhibits lactate oxidation/increases lactate concentration Metformin increases lactate oxidation25
in plasma25 Not altering the release of lactate from muscle25
Increased release of lactate from muscle130
Drug metabolism Metabolized by CYP2D6 in the liver131 Not metabolized131
Phenformin metabolism can differ depending on
CYP2D6polymorphisms
Half-life Half-life of 715h132,133 Half-life of 1.56.5h20,132134
Elimination through the kidneys Food decreases and slightly delays absorption of metformin; metformin
is excreted by kidneys
Renal clearance is reduced by other factors, such as concomitant use
ofcimetidine
Clinical use Withdrawn from the markets in most parts of the world Widespread use and excellent safety profile
in the 1970s for safety reasons135 Many nephrologists propose that metformin is under-prescribed in
chronic kidney disease and guidelines should be revisited136
(metforminis deemed safe unless eGFR levels fall <30ml/min/1.73m2)
Adverse effects
Risk of lactic Increased risk of lactic acidosis, with a mortality rate Incidence of lactic acidosis is 0.03 cases per 1,000 patient-years
acidosis of3050% (1020times lower than with phenformin).131 The reported incidence of
0.40.64 cases per 1,000 patient-years131 lactic acidosis in diabetic patients on metformin is similar to the one in
diabetic patients not taking metformin137
Metformin has a wider therapeutic window and requires higher blood
levels than phenformin to cause lactic acidosis;132 metformin is generally
believed not to cause accumulation of lactate unless metabolism of
lactate is impaired for another reason
Cardiovascular risk Increased cardiovascular risk11 Reported cardiovascular benefits138
Gastrointestinal Gastrointestinal disturbance Gastrointestinal disturbance (especially at initiation of therapy), reported in
disturbances Reduced gastrointestinal glucose absorption15,16 ~30% of patients,139 may be one of the reasons for compliance issues
(reported in up to one third of patients140). The mechanisms of gastro-
intestinal side-effects are unknown but an increased mucosal serotonin
production139 and possibly rise in GLP1 may be partially responsible.
Little effect on intestinal glucose absorption15,16
B12 malabsorption and increased, homocystein levels with long-term use
Other clinical In cancer, invitro and invivo animal models show greater Following promising results of invitro and invivo animal models, the
effects antineoplastic activity with phenformin than metformin129,141 anti-tumour effects of metformin are being intensively investigated in
and phenformin was linked to an improved immunologic clinical trials. To date most available data is in colon and breast cancer,
status in breast cancer patients.142 However, the reporting surrogate markers143
anti-tumour effect of phenformin has not been formally
investigated in clinical trials
*Active transport is predominant in both phenformin and metformin.128 Abbreviations: CYP2D6, cytochrome P450 2D6; eGFR, estimated glomelural filtration rate; GLP1, glucagon-like peptide 1.

of 5'-AMP-activated protein kinase (AMPK).47 AMPK is
an energy sensor and a master coordinator of an integra
ted signalling network that comprises metabolic and
growth pathways acting in synchrony to restore cellu-
lar energy balance. When activated, AMPK switches on
catabolic pathways that generate ATP and switches off
anabolic, ATP-consumingpathways.
The specific molecular mechanisms involved in the
inhibition of mitochondrial complex I remain unclear.
An interaction between biguanides and mitochondrial
copper ions has been observed and reported to be crucial
for the metabolic effects of metformin.48 At normal pH,
metformin is positively charged; therefore, its intra
cellular transport depends on cationic transporters
(Table2). The mitochondrial membrane potential might
drive accumulation of the positively charged metformin
in the mitochondrial matrix.
The inhibition of complex I by metformin reduces
NADH oxidation, lowering the proton gradient across
the inner mitochondrial membrane and the proton-
driven synthesis of ATP. As a result, the ATP:ADP:AMP
equilibrium is shifted towards increased AMP synthesis

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Box 1 | The role of metformin on feeding behaviour
In some studies, metformin suppresses food intake,4 probably by increasing
the levels of glucagon-like peptide 1 (GLP-1)37 and by interacting with signalling
upstream or downstream of other hormones or cytokines (such as ghrelin,
leptin and insulin)
Metformin has been detected in the cerebrospinal fluid after oral administration158
Metformin has been shown to reduce appetite by counteracting ghrelin-induced
5'-AMP-activated kinase (AMPK) signalling and inhibition of mammalian target
of rapamycin (mTOR) in the hypothalamus;159 metformin has also been reported
to inhibit dexamethasone-induced hypothalamic AMPK activity invitro157
AMPK has been proposed to mediate feeding behaviour in response to different
signalling pathways (including ghrelin, leptin and cannabinoid signalling) 160163
In the hypothalamus, metformin inhibits hypoglycaemia-induced AMPK activity
and the expression of the orexigenic neuropeptide Y,164 although the inhibition
of neuropeptide Y and agouti-related peptide in other scenarios was mediated
by signal transducer and activator of transcription 3 (STAT3) signalling rather
than AMPK activity158,165
Box 2 | Metabolic* and anti-inflammatory benefits associated with metformin
Moderate reduction of BMI or weight-neutral effects (as opposed to the effects
of many other antidiabetic treatments)3
Weight loss preferentially involving adipose tissue25
Reduction in blood pressure that is independent of weight change;33 however,
no change in blood pressure could be detected in other studies166,167
Beneficial effect on lipid levels in some studies,3,5 but not others166,167
Reduced resistin levels168 and increased adiponectin levels169
Reduced fatty liver in rodents6
Reduced fibrinogen170 and upregulated fibrinolytic system171
Reduced levels of PAI1172
Inhibition of platelet aggregation171
Improved endothelium-dependent vasodilatation173
Reduced progression of carotid intima thickening was identified in patients
with type2 diabetes mellitus174 treated with metformin; however, no difference
in carotid intima progression was noted in nondiabetic patients with proven
coronary artery disease175
Reduced levels of Creactive protein167
Reduced levels of proinflammatory cytokines;7,8 for example, metformin
inhibited production of TNF by human monocytes invitro176 and lowered TNF
levels in patients with diabetes mellitus7
Reduced expression of the adhesion molecules ICAM1 and VCAM1;177 for
example, metformin exerted an antiatherogenic effect in vascular endothelial
cells through inhibition of TNFNF-B-driven expression of proinflammatory
and cell adhesion molecules, in an AMPK-dependent manner178 and through
blockade of the PI3KAKT pathway179
Metformin has been reported to enhance CD8+ Tcell memory by altering fatty
acid metabolism,180 thereby linking metabolism and immunomodulation
Metformin has been shown to reduce endotoxin-induced hepatic injury in mice181
Metformin inhibited osteoclasts and bone resorption through immune
mechanisms182
Metformin favourably modulated immune responses in animal models of
multiple sclerosis,8 uveitis,183 autoimmune arthritis,184 and chronic asthma185
*The metabolic benefits of metformin administration listed here are not consistent in all
studies. Abbreviations: AMPK, 5'-AMP-activated kinase; NFB, nuclear factor B; PI3K,
phosphoinositide 3kinase; PAI1, plasminogen activator inhibitor 1; TNF, tumor necrosis factor.
by adenylate kinase,45 thereby reducing the energy charge
of the cell. Direct inhibition of AMP deaminase, an
enzyme that degrades AMP, by metformin might also
increase AMP levels.49 Raised AMP levels inhibit ade-
nylate cyclase (see below), a membrane-bound enzyme
that catalyses the conversion of ATP to cAMP. Hence,
metformin action ultimately reduces cAMP levels. A
link has been made between reduced cAMP synthesis
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and glucagon signalling, and the antidiabetic effect
ofmetformin.21
Interestingly, although mitochondria seem to be the
primary targets of metformin action, metformin can also
influence erythrocytes, which lack mitochondria, possi-
bly by affecting membrane fluidity.50 Further exploration
of this mechanism is awaited.
Glucagon signalling and metformin
Glucagon concentrations are abnormally elevated in
the circulation of individuals with diabetes mellitus,
as hyperglycaemia blunts suppression of the glucagon-
secreting pancreatic cells. Glucagon promotes gluco-
neogenesis, glycogenolysis and ketogenesis, and reduces
glycogenesis and glycolysis (Figure1). These actions
lead to increased hepatic glucose production during
fasting and an attenuated decline in hepatic glucose
synthesispostprandially.51
Glucagon performs its main effect via activation
of adenylate cyclase. The adenylate-cyclase-derived
cAMP activates protein kinase A (PKA), which then
phosphorylates downstream targets, such as the
bifunctional enzyme 6phosphofructo2-kinase/
fructose2,6-bisphosphatase (one of the isoforms being
PFK/FBPase1, encoded by PFKFB1). PFK/FBPase1
phosphorylation inhibits PFK activity and stimulates
FBPase1 activity, thereby lowering the intracellular
levels of fructose2,6-bisphosphate (Figure2). This com-
pound is an allosteric inhibitor of glycolysis and activa-
tor of gluconeogenesis, as it alters the activity of several
enzymes in these pathways (Figures1 and 2). Activation
of PKA also leads to changes in gene expression, but this
effect is somewhat slower than the one on enzymatic
activity. For example, phosphorylation of the cAMP-
responsive element-binding protein (CREB1) induces
binding of this protein to cAMP response element sites
within the promoter of gluconeogenic genes such as
PEPCK and G6PC (Figure3).52,53 During fasting, PKA
also phosphorylates inositol 1,4,5-triphosphate receptors
(I3PRs), thereby inducing an increase in intracellular
Ca2+ levels.54 This increase, in turn, activates CREB-
regulated transcription coactivator 2 (CRTC2), enab
ling interaction between CRTC2 and CREB1 to activate
gluconeogenic gene expression (Figure3).55 This process
is reduced during feeding owing to an increase in insulin
signalling induced by the inactivation of I3PR via AKT
(also known as PKB).55
Following on from earlier reports,22 Miller etal.
showed in a series of invitro and invivo experiments
that biguanides exert their glucose-lowering effect mainly
by opposing glucagon signalling (Figure3).21 Biguanides
were shown to rapidly inhibit the glucagon-stimulatory
effect on cAMP levels in hepatocytes, leading to dis-
rupted PKA activity and repressed phosphorylation of
PFK/FBPase 1, I3PR and CREB1.21 So, it was proposed
that, at therapeutic concentrations, metformin leads to
inhibition of glucagon signalling, possibly by increas-
ing AMP levels through inhibition of mitochondrial
complexI (Figure3). AMP then binds to the regulatory
intracellular inhibitory Psite (a purine moietysite)
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Table 2 | Membrane transporters involved in metformin pharmacokinetics

Metformin transporter Encoded by Function
SLC22A1 (also known SLC22A1 Main transporter accountable for metformin uptake18
as OCT1) Expressed in liver and kidney
Some SNPs shown to associate with reduced metformin uptake, increased metformin
elimination as a result of reduced renal tubular reabsorption and lower therapeutic
response owing to diminished action of metformin in the liver144
Wide disparity in frequency distribution of SNPs among ethnic groups18
SLC22A2 (also known SLC22A2 Mediates metformin secretion (kidney)
as OCT2) Accountable for 80% of the total metformin clearance85
SLC22A3 (also known SLC22A3 Expressed in multiple tissues (including liver, kidney, heart, skeletal muscle, brain, placenta)
as OCT3) May be important in the uptake of metformin in muscle145
SLC22A4 (also known SLC22A4 Involved in the gastrointestinal absorption of metformin146
as OCTN1) Role in the mitochondrial uptake of phenformin17
MATE1 SLC47A1 Mediates metformin secretion (kidney; liverexcretion into bile)
Rs2289669 polymorphism was associated with an amplified glucose-lowering effect of
metformin in diabetic patients147
MATE2 SLC47A2 Mediates metformin secretion (kidney)
hENT4 (also known SLC29A4 Mediates renal and intestinal metformin uptake148
as PMAT)
Abbreviations: hENT4, equilibrative nucleotide transporter 4; MATE1, multidrug and toxin extrusion transporter 1; PMAT, plasma membrane monoamine transporter;
SLC22A1, solute carrier family 22 member 1; SNP, single nucleotide polymorphism.

of adenylate cyclase, 56 thereby inhibiting glucagon-
mediated activation of adenylate cyclase and reducing
endogenous synthesis of cAMP and PKA activity. This
action culminates in a glucose-lowering effect (Figure3).
It was also demonstrated that AMPKalthough acti-
vated by metforminis dispensable for the above action
and that AKT phosphorylation is unaltered shortly after
metformin administration,21 which suggests that met-
formin does not affect insulin responsiveness acutely.
However, how metformin works at higher concentrations
remains unclear, asin addition to inhibiting glucagon
signallingit reduces glucose output in response to a
membrane-p ermeable analogue of cAMP, bypassing
adenyl ate cyclase. 21 Furthermore, whereas glucagon
receptor knockout mice show hypoglycaemia,57 this
outcome is unusual in patients receiving metformin
monotherapy, which suggests that either metformin inhi
bits glucagon signalling incompletely in humans or that
other compensatory mechanisms are present.
The role of AMPK in hepatic gluconeogenesis
Biguanides are recognized as indirect activators of
AMPK.47 For about a decade, AMPK was the assumed
prime mediator of metformin action. As mentioned
above, metformin can activate AMPK by promot-
ing AMP accumulation. Moreover, the drug has been
shown to activate AMPK without inducing any detect-
able changes in AMP, ADP and ATP,58,59 whereas phen-
formin has been more consistent in modifying adenine
nucleotide ratio.60
The link between metformin and AMPK activa-
tion was supported by experiments showing that the
effects of metformin on reduced glucose output and
lipogenesis were mitigated by treatment with the
AMPK inhibitor compound C47 (although it was later
deemed nonselective), or under adenoviral-mediated
expression of dominant negative AMPK.61 Moreover,
gluconeogenesis was shown to be inhibited by the AMPK
activator AICAR,62 and was also reduced in a mutant
rodent model expressing a constitutively active form of
AMPK.63 In the liver, deletion of liver kinase B1 (LKB1;
an upstream kinase that phosphorylates the catalytic
domain of AMPK) prevented activation of AMPK and
negated the antidiabetic effect of metformin in mice fed
a high-fat diet.13 Multiple studies reported involvement
of AMPK in the deactivation of CRTC2,13,6466 one of the
key regulators of gluconeogenic gene expression.
However, reservations regarding the hypothesis of
AMPK being the main driving force behind reduced
hepatic gluconeogenesis have been accumulating
over years, primarily owing to a lack of correlation
between gluconeogenic gene expression and hepatic
glucose output. 67,68 The AMPK model was seriously
challenged when metformin lowered glucose produc-
tion in the liver of transgenic mice that lacked AMPK
or its upstream activator LKB1.69 The investigators of
this breakthrough study69 hypothesized that the dis
cordance between their findings and the previous
model, which suggested that the antidiabetic action of
metformin requires LKB1,13 reflected possible benefits
of the LKB1AMPK axis for lipotoxicity in animals fed
a high-fat diet rather than a direct effect on gluconeo
genesis.69 Unlike PKA, AMPK does not control PFK
activity in the liver.70 Foretz etal. demonstrated that
the ability of metformin to lower glucose output was
preserved under a forced overe xpression of pe roxi
some p roliferator-activated receptor- coactivator-1
(PGC-1) and an increase in protein levels of gluconeo-
genic enzymes, such as phosphoenolpyruvate carboxy
kinase (PEPCK) and glucose-6-phosphatase (G6Pase),
concluding that metformin disrupts the activity of the
gluconeogenic enzymes rather than the expression of the
genes encoding them (PEPCK and G6PC, respectively).69
The proposed role of metformin in glucagon signalling

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oxidation71 (by regulating the activity of acetyl coen-

Glucose
zyme A carboxylase47 and the expression of multiple
GLUT-2 Plasma membrane enzymes involved in lipogenesis47,63 and oxidation23).
This mechanism is consistent with the observations of
altered fatty acid metabolism and improved hepatic stea-
+ Gluconeogenesis Glycolysis +
Glucose Glycogen tosis in mice receiving metformin.6 Free fatty acids can
G6Pase + GCK GYS PYGL
reduce glucose transport by hampering insulin signal-
+ +
ling 72 and have been implicated as the main culprit of
Glucose-6-phosphate Glycogen-1-phosphate
impaired insulin secretion by pancreatic cells.73 In addi-
Glucogenesis Glucogenolysis +
tion to free fatty acids, acetyl coenzymeA and citrate
Fructose-2,6-bisphosphate byproducts of oxidationinhibit key enzymes of the
Fructose-6-phosphate AMP glycolytic pathway (Figure1).70 Therefore, metformin
+ +
might improve insulin secretion and sensitivity by low-
FBP1 PFKL Fructose-2,6-bisphosphate ering the levels of glucose and free fatty acids, and by
+
preventing lipid deposition in insulin-sensitivetissues.
AMP Fructose-1,6-bisphosphate ATP, citrate, fatty acids

Direct modulation through energy charge
Phosphoenolpyruvate
The data presented by Miller etal. suggest a good corre
PEPCK + lation between metformin-induced elevation of AMP and
Pyruvate kinase + ATP the drop in cAMP and glucose levels in mice.21 In addition
Oxaloacetate Pyruvate Lactate to fructose2,6-bisphosphate, which regulates phospho
fructokinase1 (also called 6phosphofructokinase, liver
type; PFKL) and fructose1,6-bisphosphatase1 (FBP1)
Malate Acetyl-CoA with the greatest affinity, various other factors modulate
Oxaloacetate
gluconeogenic and glycolytic enzymes independently
Citric of glucagon and AMPK signalling, including metabo-
acid cycle Ketogenesis +
lites (such as citrate), the AMP:ATP ratio46,70,74 and the
Malate Citrate NADH:NAD+ status46 (Figure1). These findings suggest
-oxidation +
that metformin-induced changes in energy charge have
Mitochondrion contributory effects on gluconeogenesis, independent of
Glucagon effect cAMP and AMPK signalling.
Biguanide effect The molecular targets of metformin include the mito-
Figure 1 | The effects of glucagon and biguanides on gluconeogenic and glycolytic chondrial complex I and other enzymes modulated by
fluxes. The role of glucagon signalling on the expression and activity of various the altered energy charge, notably adenylate cyclase
enzymes and its opposition by biguanides is illustrated in a simplified scheme of (inhibited by increased AMP levels), affecting gluca-
hepatic glucose metabolism. Glycolysis is a pathway that converts glucose into gon signalling, and AMPK (stimulated by the increased
pyruvate whilst generating ATP; gluconeogenesis is an energy-consuming process AMP:ATPratio).
of glucose synthesis from non-carbohydrate precursors such as lactate or pyruvate.
Many of the metabolic steps of gluconeogenesis are the reverse of the glycolytic
Metformin and cancer
pathway. Both glucagon and biguanides can regulate these pathways. A rise in
fructose2,6-bisphosphate, induced by metformin, inhibits FBP1 and activates
In 2005, a report associated metformin use with a
PFKL. Biguanides abrogate glucagons effect on the gluconeogenic flux and reduced incidence of cancer,75 putting the drug into the
influence fatty acid metabolism. AMP and ATP have modulatory effects on several cancer research spotlight. As of October 2013, 173 clini-
metabolic steps. The rate of glycolysis and gluconeogenesis is also determined by cal trials on metformin and cancer have been registered.76
the concentration of glucose and lactate (and other precursors of glucose). The Diabetes mellitus has been associated with a 1.22.0-fold
stimulatory (+) and inhibitory () effects are highlighted in green for glucagon and in increase in cancer incidence.9 A report in 2010 suggested
red for biguanide signalling. Abbreviations: FBP1, fructose1,6-bisphosphatase 1; that metformin reduces this risk by approximately 40%
G6Pase, glucose6-phosphatase; GCK, glucokinase; GLUT2, glucose transporter 2;
compared with any other antidiabetic treatment.9 Several
GYS, glycogen synthase; PEPCK, phosphoenolpyruvate carboxykinase; PFKL,
6phosphofructokinase, liver type; PYGL, glycogen phosphorylase. studies have claimed a notable reduction in the risk of
all-cause and cancer-specific mortality, and with attenu-
ated cancer progression. 77,78 However, a population-
(increased AMP reduces cAMP levels)21 has supported based analysis has failed to show an association between
the AMPKindependent antihyperglycaemic action improved survival and metformin use in diabetic patients
ofmetformin. with breast cancer aged >65 years,79 and these findings
Nevertheless, a number of metformin effects are were in line with results of several other studies.80,81
still attributed to AMPK. AMPK phosphorylates and Little is known about the cancer-related effect of met-
increases the activity of insulin receptor and IRS2 formin in nondiabetic patients. A human study showed
and enhances translocation of glucose transporters that short-term treatment with metformin suppressed
(Figure3).19 AMPK is also responsible for the effect of formation of colorectal aberrant crypt foci;82 a trial of
metformin on fatty acid metabolism (Figure3).47 Acti longer duration is currently underway. Metformin treat-
vated AMPK inhibits fatty acid synthesis and enhances ment also reduced the cellular marker of proliferation,

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 REVIEWS

a Glucagon b Biguanides

cAMP cAMP

PKA PKA
Phosphorylation Lack of phosphorylation
promotes FBPase 1 prevents FBPase 1
P PFK/FBPase 1 activity and activity and promotes PFK/FBPase 1
inactivates PFK PFK activity

FBPase 1 FBPase 1
F2,6BP F6P + P F2,6BP F6P + P
PFK PFK
Allosteric Allosteric Allosteric Allosteric
inhibition activation inhibition activation
by F2,6BP by F2,6BP by F2,6BP by F2,6BP
FBP1 PFKL FBP1 PFKL

Gluconeogenesis Glycolysis Gluconeogenesis Glycolysis

Figure 2 | The effect of glucagon and biguanide signalling on fructose2,6-bisphosphate. a | Glucagon binds to glucagon
receptor coupled to Gs-protein (not shown). Gs activates adenylate cyclase which generates cAMP from ATP (not shown).
cAMP stimulates protein kinase A (PKA) that phosphorylates 6phosphofructo2-kinase/fructose2,6-bisphosphatase 1
(PFK/FBPase 1). PFK/FBPase 1 is a bifunctional enzyme that regulates the levels of fructose2,6-biphosphate (F2,6BP) by
catalysing both synthesis and hydrolysis of F2,6BP to and from fructose6-phosphate (F6P), respectively. F2,6BP is an
allosteric activator of phosphofructokinase1 (PFKL), therefore it controls the rate of conversion of fructose6-phosphate to
fructose1,6-bisphosphate in the glycolytic pathway. At the same time, F2,6BP is also an allosteric inhibitor of fructose1,6-
bisphosphatase 1 (FBP1), thereby regulating the rate of gluconeogenesis. PFK/FBPase 1 phosphorylation leads to
conformational changes that favour FBPase 1 activity, therefore lowering F2,6BP levels. A drop in F2,6BP levels promotes
gluconeogenesis and inhibits glycolysis. b | Biguanides abrogate glucagon signalling. cAMP production is inhibited resulting
in decreased PKA activity, increased PFK activity leading to increased F2,6BP levels. A rise in F2,6BP levels suppresses
gluconeogenesis and stimulates glycolysis. Abbreviation: F2,6BP, fructose2,6-bisphosphate.

Ki67, in biopsy samples obtained from nondiabetic
women with breast cancer treated with metformin.83
Mechanisms by which metformin attenuates tumouri-
genesis and has chemoprotective properties are not well-
defined. An important limitation of many experimental
studies is that metformin inhibits cell proliferation invitro
at supraphysiological concentrations,84 which are gener-
ally thought to be unachievable in patients. Moreover,
many factors influence availability and response to met-
formin in tissue.85 For example, the tissue expression
of transporters that mediate metformin uptake varies
between normal and tumour cells, and can be influenced
by various drugs such as antibiotics and proton pump
inhibitors.86 Poor uptake into target cells might limit
the therapeutic potential of metformin in cancer; on the
other hand, this fact could play a crucial part in the drugs
excellent safety profile. Overall, the interplay between the
patients metabolic make-up and the molecular character-
istics of the tumour adds to the complexity of the effects
of metformin on tumourigenesis, which are probably
both systemic (indirect) and local (direct) (Figure4).
Systemic metformin effects on tumourigenesis
Hyperinsulinaemia has been associated with adverse
prognosis in several cancers (including breast, colon and
prostate cancer).84 In states of insulin resistance, met-
formin action in the liver lowers systemic glucose levels
and improves secondary hyperinsulinaemia, preventing
the latters effects on tumour growth and progression.
This action means that metformin can affect insulin-
sensitive neoplastic tissues indirectly, without the need
to accumulate in cancer cells. Our understanding of this
principle remains limited. Although exogenous insulin
used in the treatment of diabetes mellitus does not pro
mote cancer,84 there is some controversy regarding the
risk for patients treated with the long-acting insulin
analogueglargine.87
Moreover, it is possible that other hormones, cytokines
and metabolic intermediates that influence tumourigenesis
are also affected by metformin. A systemic effect related
to reduced glucagon signalling in glucagon-responsive
cells21 warrants exploration. More studies are needed to
clarify this principle in patients withnormoglycaemia.
Direct effects of metformin in cancer
The antitumourigenic effects of metformin can be inde-
pendent of insulinaemia.88 Several findings support the
notion of a direct action of metformin in cancer cells. In
this context, AMPK-dependent and AMPK-independent
mechanisms have been described, which are likely to
coexist and interact.89,90
AMPK-dependent metfomin effects in cancer
LKB1-dependent and AMPK-dependent suppression of
the mammalian target of rapamycin (mTOR) pathway
is possibly the most potent antineoplastic effect of met-
formin (Figure4). mTOR inhibition disturbs protein
synthesis and, thereby, tumour cell proliferation. Loss
of LKB1 is frequent in cancer, and a germline mutation
in LKB1 is responsible for PeutzJeghers syndrome, a
cancer-predisposing condition.91,92
mTOR is a catalytic subunit of two multiprotein com-
plexes, mTORC1 and mTORC2. These complexes are
central in the regulation of cellular growth and integrate

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REVIEWS

to a complex tumour syndrome, which suggests that

Metformin Glucagon
Adenylate cyclase
induction of DICER1 expression by metformin could
Plasma
membrane SLC22A1 activity be an additional antineoplastic mechanism (Figure4).98
Metformin also inhibits the proto-oncogene cMYC
Mitochondrion
P and hypoxia-inducible factor 1 (HIF1) via AMPK
Complex I Binding to (Figure4).98 Importantly, AMPK and HIF1 have a key
P-site
Pyruvate kinase ATP cAMP levels role in metabolic transformation in cancer.
PFKL activity ATP production Rapidly dividing cells, such as tumour or inflamma-
tory cells, show altered metabolism that favours glyco
PFKL activity AMP PKA activity
FBP1 activity lysis to oxidative phosphorylation as energy source, even
AMPK activation
under aerobic conditions, a process called the Warburg
effect.99,100 This metabolic transformation promotes fatty
I3PR CREB-1/PGC-1 PFK/FBPase 1
acid synthesis and ensures availability of intermediates as
Improved Improved Reduced
insulin glucose fatty building blocks for proliferating cells. Probably one of the
receptor transport acid
function synthesis key mediators of this reprogramming is the mTORC1-
Ca+ activated HIF1,101 a transcription factor promoting
PEPCK
and G6Pase Glycolytic Gluconeogenic expression of glycolytic enzymes, GLUT1 and mono-
Improved insulin sensitivity CRTC2 expression pathways pathways carboxylate transporter 4 (MCT4), involved in lactic
Figure 3 | Model of metformin action in the hepatocyte. Metformin enters the cell
acidtransport.
through transporters such as SLC22A1 and inhibits mitochondrial complex I. A defect in the LKB1AMPK pathway potentiates the
Complex I inhibition results in reduced ATP levels and an accumulation of AMP. risk of metabolic transformation of pre-neoplastic cells;
AMP binds to the so called P-site at the adenylate cyclase enzyme and inhibits its hence, many tumours have inactive LKB1.102 AMPK has
activity, leading to reduced generation of cAMP upon stimulation of the glucagon been shown to negatively regulate the Warburg effect
receptor. As a result, PKA activation and its downstream pathways are inhibited. and tumour growth invivo.103 Metformin decreased
Gluconeogenesis is suppressed as a result of reduced activity of enzymes involved HIF1 mRNA and protein levels in a breast cancer
in the gluconeogenic flux (for example, owing to lack of phosphorylation of PFK/
model,98 exhibiting an antiproliferative, anti-Warburg
FBPase 1) and reduced gene expression (owing to decreased phosphorylation of the
transcription factor CREB1). Metformin-induced change in energy charge also potential, probably via AMPK. Metformin also acted
activates AMPK, which suppresses fat metabolism and possibly also contributes to antimitotically by inhibiting expression of fatty acid
the reduced gluconeogenic gene expression. In addition, AMP and ATP have an synthase104 and favouring fatty acid oxidation in cancer
independent modulatory role on these pathways. Abbreviations: AMPK, 5'-AMP- cells of the colon.105
activated protein kinase; CREB1, cAMP response element-binding protein; CRTC2, The effect of metformin on cell metabolism and
CREB regulated transcription coactivator 2; G6Pase, glucose6-phosphatase; I3PR, tumour growth is controversial. In pre-neoplastic cells
inositol 1,4,5-triphosphate receptors; PEPCK, phosphoenolpyruvate carboxykinase;
with an intact AMPK axis, metformin can counter-
PFK/FBPase 1, 6phosphofructo2-kinase/fructose2,6-bisphosphatase 1; PGC1,
peroxisome proliferator-activated receptor coactivator1; PKA, protein kinase A;
act the Warburg effect (that is, inhibit oxidative glyco
SLC22A1, solute carrier family 22 member 1. lysis).106 However, in established tumours, as opposed to
pre-neoplastic cells, the presence of LKB1 and AMPK
might confer a survival advantage to tumour cells by
input from various hormonal signalling and energy- protecting them against energetic stress.107 It can even
sensing pathways, including the insulin, insulin-like growth cause increased glycolytic flux under certain micro
factor 1 (IGF1), IGF2 and AMPK pathways (Figure4).93 environments, such as acidic pH;108 therefore, AMPK
AMPK-dependent downregulation of mTORC1 results activators could potentially be harmful. Conversely,
from activation of the tumour suppressor genes tuber- absence of LKB1 or AMPK in established tumours makes
ous sclerosis complex 1 (TSC1) and TSC2, which form the cancer cells selectively more vulnerable to depleted
an mTOR-inhibiting complex. Moreover, AMPK directly ATP incurred by metformin, as their ability to restore
inhibits RAPTOR, a positive regulator of mTOR.94 energy balance is impaired.109
Metformin can inhibit IGF1insulin signalling
through AMPK-dependent phosphorylation of IRS1, AMPK-independent metformin effects in cancer
which transmits signals from the insulin receptor and Whilst growth factors regulate mTORC1 through the
IGF1 receptor to the PI3KAKT pathway (Figure4). PI3KAKTTSC1TSC2 axis, amino acids can activate
This activity also has the potential to downregulate mTORC1 signalling through the RAG family of GTPases
mTOR signalling.95 However, multiple regulatory feed- (also called the Ras-related GTPases), independently of
back loops possibly counteract the antitumour effect of AMPK.110,111 Following its activation by the Ragulatory
this cascade during chronic metformin exposure.96 complex, the RAG GTPases recruit mTORC1 to the lyso-
The tumour suppressor protein p53 activates various somal surface, where it is activated by RHEB. Metformin
genes that can inhibit the AKT and mTORC1 pathways.97 can inhibit mTORC1 signalling by inactiv ating the
p53 is one of the targets of AMPK (Figure4), but the role Ragulatory complex, thereby mimicking the effect of
of metformin in p53 activation remains controversial.97 amino-acid withdrawal.89 This mechanism of action,
Furthermore, metformin induces expression of which is responsive to perturbations in energy status, may
DICER1, an enzyme that is involved in microRNA prove important in certain types of cancer 110 and, parallel
synthesis. Loss of function mutations in DICER1 lead to dietary energy restriction, in cancerprevention.112

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 REVIEWS

Indirect effects Direct effects

Metformin Metformin Insulin Metformin p53

IGF-1 and insulin receptors

IRS-1
P ROS ATM
Body PI3K
weight Ragulator

Other upstream AKT

Inflammation kinases RAG GTPases
P
P
Insulin resistance LKB1 TSC2TSC1 RHEB
Glucose levels P P
Insulin levels P
CaMKK2 AMPK mTORC1
P P

P
P P P HIF-1 Protein synthesis
Fatty acid Cell growth
synthesis cMYC DICER p53 NF-B IL-6 Cell viability

Figure 4 | Proposed actions of metformin in cancer. The antitumour effect of metformin is probably a combination of indirect
(systemic) and direct effects. Systemic influence is secondary to the effects of metformin on metabolism in insulin-sensitive
target tissues. Metformin lowers systemic glucose and insulin levels, which decreases insulin-mediated tumour growth and
progression. Possible anti-inflammatory effects may also reduce cancer risk. Metformin might inhibit mTORC1 through
regulation at multiple levels. Low energy charge in metformin-treated cancer cells activates AMPK, which can restrain cell
growth and proliferation. AMPK activates the tumour suppressor gene TSC2, resulting in inhibition of the mTORC1 activator
RHEB. AMPK can directly phosphorylate and inhibit RAPTOR, a member of the mTORC1 complex. Metformin might also
decrease signalling downstream of IGF1insulin receptors by lowering insulin levels and through AMPK-dependent
phosphorylation of IRS1. This action inhibits AKT and mTORC1 signalling. Moreover, AMPK has been suggested to hinder
cancer cell growth through numerous other mechanisms, including activation of HIF1, p53, cMYC and DICER1, and
suppression of fatty acid synthesis. AMPK may also be a mediator of an attenuated inflammatory feedback loop (NF-B/IL-6
pathway), restraining malignant transformation. Metformin can inhibit mTORC1 also in an AMPK-independent manner via direct
inactivation of the Ragulator complex, which results in inhibition of RAG GTPases and dissociation of mTORC1 from its activator
RHEB. Other AMPK-independent effects include inhibition of the serine-protein kinase ATM and reduction of ROS levels, which
are produced by mitochondrial complex I and confer mutagenesis risk. Abbreviations: AMPK, 5'-AMP-activated protein kinase;
CaMKK2, Calcium/calmodulin-dependent protein kinase kinase 2; IGF1, insulin-like growth factor 1; IRS1, insulin receptor
substrate 1; IL-6, interleukin 6; LKB1,liver kinase B1; mTORC1, mammalian target of rapamycin complex 1; NF-B, nuclear
factor B, PI3K, phosphatidylinositide 3kinase; ROS, reactive oxidative species; TSC2, tuberous sclerosis complex 2.

Through inhibition of mitochondrial complex I, met-
formin reduces production of reactive oxygen species,
oxidative stress and DNA damage,113 therefore reduc
ing the risk of mutagenesis. Variation in the glycaemic
response to metformin in patients with type2 dia
betes mellitus has been associated with the presence of
common genetic variants near the ataxia telangiectasia
mutated (ATM) gene locus.114 This observation suggests
that metformin-dependent signalling involves ATM
activation. ATM encodes a tumour-suppressor protein,
a crucial component of the DNA-damage response
network system that is required for DNA repair and
cell-cycle control. ATM is mutated in ataxia telangi
ectasia, a neurodegenerative condition associated with
a predisposition to cancer, insulin resistance and type2
diabetes mellitus. Both AMPK-dependent and AMPK-
independent mechanisms are probably involved in the
ATM-mediated reparatory effect. Metformin, as a cellu
lar stressor, activates reparatory processes, even in the
absence of DNA damage, which might be protective
against premalignant to malignant transformation.115
Metformin combined with cytotoxic therapy
Multiple studies have looked into the antineoplastic
potential of metformin when used in combination with
cytotoxic therapies.116 Metformin has been reported to
improve cancer responses to radiation therapy, probably
via downregulation of the hyperactive PI3KAKTmTOR
pathway. 117 Moreover, metformin enhanced apopto-
sis induced by paclitaxel and cisplatin in endometrial
cancer by repressing glyoxalase I expression through an
undefined mechanism.118 However, metformin antago-
nized cisplatin-induced cytotoxicity in glioma, neuro
blastoma, fibrosarcoma and leukaemia cell lines through
an AMPK-independent activation of AKT.119 Thus, use
of metformin in combination with chemotherapy drugs
in cancer warrants a degree ofcaution.119
Tyrosine kinase inhibitors reduce insulinIGF1
receptor signalling, therefore metformin is frequently
used in combination with these agents to counter
iatrogenic hyperglycaemia. The contribution of met-
formin to the antineoplastic success of this combination
remainsunclear.84
In mouse xenograft cancer models, metformin selec-
tively kills cancer stem cells, even at low concentrations,
and prolongs tumour remission when combined with
chemotherapy agents,120,121 probably through the regu-
lation of inflammatory pathways (Box2). Metformin is
thought to inhibit cancerous transformation by attenuat-
ing the inflammatory feedback loop, a signalling cascade

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REVIEWS

Table 3 | Potential therapeutic targetslessons learned from metformin-related metabolic studies

Theoretical targets or compounds under investigation Insulinresistance Oncology
and/or diabetes
mellitus
Potential renaissance of phenformin use (better absorbed and a probably more potent mitochondrial
inhibitor than metformin; its adverse effect profile is better than that of most cytotoxic drugs129)
Short courses of high-dose metformin84 (mechanism of action of high doses of metformin is unknown)
Novel biguanides
New molecules that are better tolerated and have less adverse effects than current biguanides
Molecules less dependent on active transport than metformin and with more suitable
pharmacokinetics (better absorbed in the gut, better absorbed in the target cells)
Molecules affecting mitochondrial respiratory chain
Tissue-selective (e.g. liver)
Different degree of energy stress
Different inhibitory effect on reactive oxygen species production
Molecules targeting copper ion transport
Metabolic inhibitors and targeted therapies based on tumour metabolic profiles (for example,
DICER, cMYC,98 HIF1, MCT4, and MCT1,149 GLUT1, HKII, phosphoglycerate dehydrogenase,
LDHA,150 6phosphofructo2-kinase (PFKFB3 isoform),151 fatty acid synthase,152 CPT1C153)
P-site of adenylate cyclase21
Other targets specific to glucagon signalling (e.g. glucagon receptor, cells)154
New GLP1 receptor agonists and DPP4 inhibitors (to reduce plasma glucagon levels)
Targeting abnormal metabolism linked to different Gprotein coupled receptors (e.g. hybrid peptides
acting through GIP, glucagon and GLP1 receptors at the same time,155 Gproteincoupled receptor
40 agonist156)
Other targets of insulin signalling (e.g. anti-IGF-1R antibodies or small molecule IGF-1R compounds96)
Other AMPK activators and tissue specific AMPK activators
AMPK inhibitors (for cases where AMPK potentiates tumour metabolism)
Other mTOR inhibitors
Agents targeting appetite and obesity
Anti-inflammatory molecules
New chronotherapeutics (that is, modulators of insulin release to mimic cyclicity; targets against
dysregulated circadian rhythms which may underlie insulin resistance, obesity and can have
association with cancer; for example, CLOCK, BMAL1, PER, CRY)
Targeting complications of other treatments (e.g. steroids157)
Combination therapies (for example, chemotherapeutic agents, other antidiabetics)

mediated by the transcription factor NFB and its down-
stream cytokine interleukin 6 (IL6).122 Interestingly, the
inhibitory effect is more pronounced in cancer stem cells
than noncancer stem cells, as the sensitivity of trans-
formed cell lines to metformin is determined by the
degree of immune-cell-mediated tumour inflammation,
which is reflected by IL6 levels.122 How metformin inhib-
its NFB is unclear, but AMPK activation in the pres-
ence of a metabolic difference between cancer stem cells
and noncancer stem cells may play a part.123 The main
systemic effect of metformin on cancer is via its bene
ficial effect on glucose metabolism, whereas the main
direct effect is via LKB1 and AMPK. More research is
needed to dissect the intersection between dysregulated
metabolism and cancer and whether there is a benefit
of using metformin as a single agent or incombination.
Future therapeutic directions
The AMP-binding Psite on adenylate cyclase has been
proposed as a new therapeutic target in insulin resistance
and type2 diabetes mellitus.21 Molecules that abrogate
cAMP and PKA signalling would bypass the mito
chondrial action of metformin and thus confer a pro-
glycolytic effect without changes in energy charge and
the resulting AMPK activation. The efficacy and safety of
such molecules would need to be established. What about
the risk of hypoglycaemia or pancreatic cell hyper
plasia, as seen in glucagon knockout mice?57 Could there
be a risk of an undesirable pro-Warburg effect? Could
such molecules affect different enzymatic isoforms of
adenylate cyclase or be tissue-specific? How will speci-
ficity be established? Glucagon receptor is expressed in
various tissues, and the cAMPPKA pathway is involved
in a plethora of signalling pathways, highlighting the
need for a specific targeting strategy. Investigating this
newly proposed pathway beyond hepatoc ytes is of
interest, given that an upregulated, rather than down-
regulated, cAMPPKA pathway, as mediated by GLP1,
is deemed protective of myocardial infarct size. 124
Metformin-induced activation of AMPK also confers

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 REVIEWS

cardioprotection;124 however, other frequently prescribed
drugs such as statins can activate PKA125 and AMPK126 in
the heart. Interaction of various pathways and drugs and
their contribution to the pleiotropic effects attributed to
metformin will probably remain a topic of debate in the
future. The knowledge gained from the molecular mecha-
nisms of metformin action could lead to the development
of novel therapies across multiple fields in medicine with
more suitable pharmacokinetic and pharmacodynamic
properties (Table3).
Conclusions
Metformin acts as a metabolic inhibitor and alters both
whole-body and cellular energy metabolism. It is pri
marily used in patients with type2 diabetes mellitus,
and its main mechanism of action in this disease setting
is inhibition of hepatic gluconeogenesis. Metformin
interacts with complex I in the mitochondrial electron
transport chain, thereby lowering cellular ATP levels and
causing AMP accumulation. AMP binds to the Psite of
adenylate cyclase and inhibits its action in response to
glucagon, thereby disrupting downstream cAMPPKA
signalling. As a result, the activity of enzymes of the
gluconeogenic pathway is inhibited in favour of glyco
lysis. This mechanism is probably the main mode through
which metformin lowers hepatic glucose output.
In addition, the reduction in energy charge leads to
AMPK activation and downregulation of gluconeogenic
gene expression. Although AMPK activation is dis-
pensable for the glucose-lowering effect of metformin,
it probably triggers important insulin-sensitizing and
lipid-modulating mechanisms. Much of the regulatory
components of this model of metformin action, as well as
parameters that determine its kinetics (such as the rate of
dissociation of AMP from the Psite of adenylate cyclase)
and specificity remain undefined.
Laboratory evidence of the antimitotic action of met-
formin is promising, although results from epidemio-
logical studies remain controversial.80 The combination
of tumour genetics, patient metabolic profile and the cel-
lular microenvironment determine the antitumour effect
of metformin treatment. Focussed use of metformin to
specifically target metabolic differences between normal
and abnormal signalling offers a vast, though challenging,
therapeutic potential. The metabolic similarities between
activated proinflammatory cells and cancer cells suggest
that metabolic inhibitors may modulate immune cells.
The immune system could thus be a mediator of some
of the pleiotropic benefits of metformin. Many details of
metformin action remain to be discovered, and the
risk of harm must be considered when designing new
metformin-based therapies. Hopefully, the knowledge
gained from dissecting the pathways that metformin acts
on will propel the development of multiple noveltherapies.
Review criteria
A PubMed database search was performed using the
following terms: metformin, gluconeogenesis,
glycogenolysis, glucagon, inflammation, cancer,
AMPK, cAMP, mitochondrium, GLP1, liver,
phenformin, and hypoglycaemia. No restriction was
placed on the year the paper was published. However,
due to the sheer volume of publications relating to this
topic, spreading over decades, more weight was given to
original articles, meta-analysis and reviews published in
the last 5years. The references given in selected papers
were scrutinised. Manuscripts written in English, French,
Spanish and Czech were considered.

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Acknowledgements
We would like to thank Professor A. B. Grossman,
Oxford Centre for Diabetes, Endocrinology and
Metabolism, for his expert review of this manuscript.
I.Pernicova is supported by a Project Grant from the
Barts Charity.
Author contributions
The authors contributed equally to all aspects
ofthemanuscript.