Marc Foretz

Bruno Guigas

Luc Bertrand

Michael Pollak

Benoit Viollet
email: benoit.viollet@inserm.fr

Metformin: From Mechanisms of Action to Therapies

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Introduction

Metformin has been an important drug for treatment of type 2 diabetes (T2D) for decades. It isthe most widely used oral anti-hyperglycemic agent, andis currently recommended as first line therapy for all newly diagnosedT2D patients(American Diabetes Association, 2014). Metformin(N, N-dimethylbiguanide) belongs to the biguanide class of anti-diabetic drugs (containing two linked guanidine rings) originally derived fromgalegine (isoamylene guanidine), a guanidine derivative found in the French lilac Galegaofficinalis.Among the threebiguanidesdeveloped for diabetes therapy, metformin has a superior safety profile and it is well tolerated. Two other biguanides, phenformin and buformin, were withdrawn in the early 1970's due to risk of lactic acidosis and increased cardiac mortality. The incidence of lactic acidosis with metformin at therapeutic doses is rare (less than 3 cases per 100.000 patient-years) and not greater than with non-metformin therapies. Metformin has been used widely in the treatment of T2Dfor over 50 years and has been found to be safe and efficacious both as monotherapy and in combination with other oral anti-diabetic agents and insulin. It offers the major clinical advantage of not inducing hypoglycemia or weight gain and ameliorates hyperglycemia with remarkable cardiovascular safety. Besides its use in T2D, there is interest in the use of metformin for the treatment of polycystic ovary disease,diabetic nephropathy and gestational diabetes [START_REF] Viollet | Cellular and molecular mechanisms of metformin: an overview[END_REF]. The drug also has the advantage of counteracting the cardiovascular complications associated with diabetesas reportedin a large cohort of individuals fromthe United Kingdom Prospective Diabetic Study (UKPDS)(1998).Another possible benefit for metformin use is the association with decreased cancer risk and improved cancer prognosis (Pollak, 2012b;[START_REF] Viollet | Cellular and molecular mechanisms of metformin: an overview[END_REF]. Although metformin has been used in Europe for treatment of hyperglycemiasince 1957 (and inthe USA sinceFDA approval in 1994), the exact molecular mechanisms of itstherapeutic action remain obscure. In this review, we summarize what is currently known about these molecular mechanismsin the context of classic use for T2D and also for possible novel areas of therapeutic application.

Pharmacokinetics and pharmacogenomics

Pharmacokinetics of metformin

The optimal oral metformin dose for many diabetic patients is ~2g/day. After a single oral dose, metformin is rapidly distributed tomany tissues following partial absorption by the small intestine, but the luminal concentration in the gastrointestinal tract remains high. The peak plasma concentration occurs in 3 h (increasing from 1.0 to 1.6 µg/ml [about 6 to 10 µM]after a 0.5 g dose and to ~3 µg/ml [about18 µM]after a 1.5 g dose) with a mean plasma half-life of about 20 hours [START_REF] Tucker | Metformin kinetics in healthy subjects and in patients with diabetes mellitus[END_REF].When the human metformin dose of 20 mg/kg/day orally is translated to the mouse equivalent dose of 250 mg/kg/day, according to the normalization to body surface area, murine plasma levels of metformin of up to 1.7 µg/ml(about 10 µM) are achieved [START_REF] Memmott | Metformin prevents tobacco carcinogen--induced lung tumorigenesis[END_REF]. This is in the range achieved when conventional anti-diabetic doses are used in humans [START_REF] Tucker | Metformin kinetics in healthy subjects and in patients with diabetes mellitus[END_REF]. Biodistribution studies in mice using 14 C-labelled metformin showed accumulation mainly in the gastrointestinal tract, kidney and liver [START_REF] Wilcock | Accumulation of metformin by tissues of the normal and diabetic mouse[END_REF]. It is important to notethat being supplied directly by blood coming from the portal vein, the liver may contain a concentration of orally administered metformin substantially higher than in the general circulation and other organs [START_REF] Wilcock | Accumulation of metformin by tissues of the normal and diabetic mouse[END_REF]. Metformin liver concentrations of greater than 180 µmol/kgwet weight and 250 µmol/kgwet weight in normal and diabetic rodents, respectively, can be achieved after a single dose of 50 mg/kg [START_REF] Wilcock | Accumulation of metformin by tissues of the normal and diabetic mouse[END_REF].

Cellular uptake of metformin

Metformin is an unusually hydrophilic drug that mostly exists in a positively charged protonated form under physiological conditions. These physicochemical properties make rapid and passive diffusion through cell membranes unlikely. Indeed, transport of metformin involves an active uptake process viasolute carrier organic transporters. The intestinal absorption of metformin is primarily mediated by the plasma membrane monoamine transporter (PMAT, SLC29A4 gene), which is localized on the luminal side of enterocytes. Organic cation transporter1 (Oct1, SLC22A1 gene) is expressed on the basolateral membrane of enterocytes and may be responsible for the transport of metformin into the interstitial fluid [START_REF] Gong | Metformin pathways: pharmacokinetics and pharmacodynamics[END_REF]. The primary mediator of hepatic metformin uptake is OCT1 and possibly OCT3 (SLC22A3 gene), expressed at the basolateral membrane of hepatocytes [START_REF] Gong | Metformin pathways: pharmacokinetics and pharmacodynamics[END_REF]. The clearance of metformin is dependent on renal elimination as metformin does not undergo relevant biotransformation in the liver or biliary excretion. In the kidney, metformin is taken up into renal epithelial cells by OCT2 (SLC22A2 gene), expressed on the basolateral membrane, and excreted into the urine via multidrug and toxin extrusion 1 and 2 (MATE1 geneSLC47A1 andMATE2 SLC47A2 genes).

Pharmacogenomics of metformin

Considerable inter-individual heterogeneity in clinical efficacy and the pharmacokinetic disposition of metforminhas been reported in the treatment of diabetic patients. This may be explained by variability in genetic polymorphisms of cation transporters. It was first reported that individuals carrying polymorphisms of the OCT1 gene SLC22A1 display an impaired effect of metformin in lowering blood glucose levels, consistent with the great reduction of hepatic metformin uptake observed in OCT1 -/-mice [START_REF] Shu | Effect of genetic variation in the organic cation transporter 1 (OCT1) on metformin action[END_REF].However, these results have not been confirmed in the long-term follow-up of a large observational cohort of patients treated with metformin [START_REF] Zhou | Reduced-function SLC22A1 polymorphisms encoding organic cation transporter 1 and glycemic response to metformin: a GoDARTS study[END_REF]. Conversely, variants in the MATE1 gene SLC47A1 enhance the effect of metformin on glycated hemoglobin (HbA1c) and glucose tolerance in T2D patients [START_REF] Becker | Genetic variation in the multidrug and toxin extrusion 1 transporter protein influences the glucose-lowering effect of metformin in patients with diabetes: a preliminary study[END_REF]. In MATE 1 -/-mice, urinary excretion of metformin is significantly decreased,suggesting that MATE1 is essential for renal clearance of the drug [START_REF] Tsuda | Targeted disruption of the multidrug and toxin extrusion 1 (mate1) gene in mice reduces renal secretion of metformin[END_REF]. Among new candidate genetic determinants of metformin response, single nucleotide polymorphisms have been identified in the AMPK subunit genes, PRKAA1, PRKAA2 and PRKAB2 and the LKB1 gene, STK11 [START_REF] Jablonski | Common variants in 40 genes assessed for diabetes incidence and response to metformin and lifestyle intervention in the diabetes prevention program[END_REF]. In addition, a recent genome-wide association study showed association between a large locus on chromosome 11, encompassing several genes, and glycemic variability in response to metformin therapy [START_REF] Zhou | Common variants near ATM are associated with glycemic response to metformin in type 2 diabetes[END_REF]. This locus includes the ataxia telangiectasia mutated (ATM) geneand it was suggested as the most likely candidate given its association with insulin resistance and T2D. However, additional studies are needed to clearly delineate genetic influences on the clinical response to metformin.

Metformin and mitochondrial oxidative phosphorylation

It is generally accepted that actions of metformin (and other biguanides) on mitochondria underlie most of the pleiotropic effects of the drug. This emerging consensus originates from two seminal papers published in 2000 reporting that metformin decreases cellular respiration by a mild and specific inhibition of the respiratory-chain complex 1 (NADH:ubiquinoneoxidoreductase) without affecting any other steps of the mitochondrial machinery [START_REF] El-Mir | Dimethylbiguanide inhibits cell respiration via an indirect effect targeted on the respiratory chain complex I[END_REF][START_REF] Owen | Evidence that metformin exerts its anti-diabetic effects through inhibition of complex 1 of the mitochondrial respiratory chain[END_REF]. However, reviewing early mechanistic studies, the first observation reporting effects of biguanide derivatives on mitochondrial oxidative phosphorylation (OXPHOS) comes from the pioneering work of Hollunger in the 50's that linked the increase in glycolysis induced by this class of molecules to the inhibition of cellular respiration [START_REF] Hollunger | Guanidines and oxidative phosphorylations[END_REF]. Later, high concentrations of various biguanide derivatives were found to specifically reduce NADH but not succinate oxidation in submitochondrial particles, assigning the inhibitory effect of these compounds on OXPHOS to the complex 1 of the mitochondrial electron transport chain (ETC). Thus, the concept that members of the biguanide family, including phenformin and metformin, exert many of their actions though modulation of mitochondrial energetics is not a recent proposal. During the last decade, the specific inhibition of the mitochondrial respiratory-chain complex 1 by metformin was confirmed in many cellular models, including rat, mouse and human primary hepatocytes [START_REF] El-Mir | Dimethylbiguanide inhibits cell respiration via an indirect effect targeted on the respiratory chain complex I[END_REF][START_REF] Owen | Evidence that metformin exerts its anti-diabetic effects through inhibition of complex 1 of the mitochondrial respiratory chain[END_REF][START_REF] Stephenne | Metformin activates AMP-activated protein kinase in primary human hepatocytes by decreasing cellular energy status[END_REF]hepatoma and adrenocortical carcinoma immortalized cell lines [START_REF] Guigas | Metformin inhibits mitochondrial permeability transition and cell death: a pharmacological in vitro study[END_REF][START_REF] Hirsch | Metformin inhibits human androgen production by regulating steroidogenic enzymes HSD3B2 and CYP17A1 and complex I activity of the respiratory chain[END_REF][START_REF] Kim | Metformin-induced inhibition of the mitochondrial respiratory chain increases FGF21 expression via ATF4 activation[END_REF], skeletal muscle homogenates [START_REF] Brunmair | Thiazolidinediones, like metformin, inhibit respiratory complex I: a common mechanism contributing to their antidiabetic actions?[END_REF], endothelial cells [START_REF] Detaille | Metformin prevents high-glucose-induced endothelial cell death through a mitochondrial permeability transition-dependent process[END_REF], pancreatic beta cells [START_REF] Hinke | Methyl succinate antagonises biguanide-induced AMPK-activation and death of pancreatic beta-cells through restoration of mitochondrial electron transfer[END_REF], neurons [START_REF] El-Mir | Neuroprotective role of antidiabetic drug metformin against apoptotic cell death in primary cortical neurons[END_REF], peripheral blood mononuclear cells and platelets [START_REF] Piel | Metformin induces lactate production in peripheral blood mononuclear cells and platelets through specific mitochondrial complex I inhibition[END_REF]and more recently in cancer cells [START_REF] Bridges | Effects of metformin and other biguanides on oxidative phosphorylation in mitochondria[END_REF][START_REF] Janzer | Metformin and phenformin deplete tricarboxylic acid cycle and glycolytic intermediates during cell transformation and NTPs in cancer stem cells[END_REF][START_REF] Scotland | Mitochondrial energetic and AKT status mediate metabolic effects and apoptosis of metformin in human leukemic cells[END_REF][START_REF] Wheaton | Metformin inhibits mitochondrial complex I of cancer cells to reduce tumorigenesis[END_REF]. It has been reported that thistransient inhibition of complex 1 induces a drop in cellular energy charge, a measure of the energetic state of the cell defined as ( [START_REF] Foretz | Metformin inhibits hepatic gluconeogenesis in mice independently of the LKB1/AMPK pathway via a decrease in hepatic energy state[END_REF][START_REF] Stephenne | Metformin activates AMP-activated protein kinase in primary human hepatocytes by decreasing cellular energy status[END_REF]. The resulting fall in cellular ATP concentration and an increase in both ADP/ATP andAMP/ATP ratiosactivates the AMPactivated protein kinase (AMPK),acritical energy sensor of cellular energy homeostasiswhich integrates multiple signaling networks to coordinate a wide array of compensatory, protective, and energy-sparing responses [START_REF] Viollet | Cellular and molecular mechanisms of metformin: an overview[END_REF].

[ATP] + 0.5[ADP])/([ATP] + [ADP] + [AMP])
Although the exact mechanism(s) by which metformin inhibits complex 1 remains unknown, some studies were unable to detect a direct effect on isolated mitochondria except at very high concentrations, suggesting that the mitochondrial action of the drug requires intact cells [START_REF] El-Mir | Dimethylbiguanide inhibits cell respiration via an indirect effect targeted on the respiratory chain complex I[END_REF][START_REF] Guigas | Metformin inhibits mitochondrial permeability transition and cell death: a pharmacological in vitro study[END_REF]. On the other hand, it was recently reported that metformin (and other biguanides) can directly inhibit function of purified respiratory-chain complex 1, as well as in isolated mitochondria and submitochondrial particles from bovine heart{Andrzejewski, 2014 #286} [START_REF] Bridges | Effects of metformin and other biguanides on oxidative phosphorylation in mitochondria[END_REF], in line with previous data reporting similar properties using very high concentrations (20-100 mM) of the drug [START_REF] Dykens | Biguanide-induced mitochondrial dysfunction yields increased lactate production and cytotoxicity of aerobically-poised HepG2 cells and human hepatocytes in vitro[END_REF][START_REF] Owen | Evidence that metformin exerts its anti-diabetic effects through inhibition of complex 1 of the mitochondrial respiratory chain[END_REF]. However, it remains to be demonstrated if these mechanisms operate in vivo.

It was also shown that the inhibitory effect of metformin on complex 1 was not prevented by nitric oxide (NO) synthase inhibitors or reactive oxygen species (ROS) scavengers [START_REF] El-Mir | Dimethylbiguanide inhibits cell respiration via an indirect effect targeted on the respiratory chain complex I[END_REF], and was independent of AMPK, at least in primary mouse hepatocytes [START_REF] Stephenne | Metformin activates AMP-activated protein kinase in primary human hepatocytes by decreasing cellular energy status[END_REF]. Among the possible underlying mechanisms, it was proposed that the positive charge of metformin might account for its accumulation within the matrix of energized mitochondria, driven by the membrane potential [START_REF] Owen | Evidence that metformin exerts its anti-diabetic effects through inhibition of complex 1 of the mitochondrial respiratory chain[END_REF]. In addition, its apolar hydrocarbon side-chain would also promote its binding to hydrophobic structures, such as the constitutive phospholipids of mitochondrial membranes. Importantly, the lipophilicity of metformin, which is mostly due to its dimethyl-substituted terminal amino group, is much less than those of phenformin, which is more evenly distributed along its biguanide backbone. These peculiar physicochemical characteristics explain why two structurally-related biguanides affect differently the mitochondrial machinery, metformin being a weak but specific inhibitor of complex 1 whereas phenformin exerting a more potent but less specific action on the mitochondrial ETC [START_REF] Drahota | Biguanides inhibit complex I, II and IV of rat liver mitochondria and modify their functional properties[END_REF]. It is also worth mentioning that the inhibition of complex 1 activity by metformin is rather mild when compared to the reference inhibitor rotenone [START_REF] El-Mir | Dimethylbiguanide inhibits cell respiration via an indirect effect targeted on the respiratory chain complex I[END_REF].Furthermore, metformin was shown to significantly reduce mitochondrial ROS production by selective inhibition of the reverse electron flow through the respiratory-chain complex 1, whereas rotenone triggers ROS production by increasing forward electron flow [START_REF] Batandier | The ROS production induced by a reverse-electron flux at respiratorychain complex 1 is hampered by metformin[END_REF]. Taken together, this suggests that their respective site of action on one or several of the subunits constituting the respiratory-chain complex 1 differ. Although recent studies have led to significant improvements in the understanding of its structurefunction relationships [START_REF] Bridges | Effects of metformin and other biguanides on oxidative phosphorylation in mitochondria[END_REF],many aspects of the regulation of mitochondrial complex 1, such as the so-called active/deactive transition, remains incompletely understood. Further investigations are therefore still required to clarify the mechanism(s) by which metformin modulates the respiratory-chain complex 1 in such a unique way. Interestingly, it has been reported that direct binding of metformin to mitochondrial copper ions might be crucial for the metabolic effects of the drug [START_REF] Logie | Cellular responses to the metal-binding properties of metformin[END_REF]. These findings point out again the crucial involvement of mitochondria in the molecular mechanism of action of metformin. Of note, not all the effects of biguanides are mediated by mitochondria since the glucose metabolism of erythrocytes, which are devoid of this organelle, was shown to be significantly affected secondary to metformin-induced change in cell membrane fluidity induced by the drug [START_REF] Muller | Action of metformin on erythrocyte membrane fluidity in vitro and in vivo[END_REF].

Metformin and treatment of type 2 diabetes

Metformin exerts its glucose-lowering effect primarily by decreasing hepatic glucose production through suppression of gluconeogenesis and enhancing insulin suppression of endogenous glucose productionand to a lesser extent, by reducing intestinal glucose absorption and possibly improving glucose uptake and utilization by peripheral tissues, such as skeletal muscle and adipose tissue [START_REF] Natali | Effects of metformin and thiazolidinediones on suppression of hepatic glucose production and stimulation of glucose uptake in type 2 diabetes: a systematic review[END_REF]. Of note, it has been reported that metformin does not improve peripheral insulin sensitivity [START_REF] Natali | Effects of metformin and thiazolidinediones on suppression of hepatic glucose production and stimulation of glucose uptake in type 2 diabetes: a systematic review[END_REF] and improvements in insulin sensitivity in muscle may be related to the use of higher doses of metformin than clinically relevant.Additionally, metformin may also improve glucose homeostasis by interacting with the incretin axis through the action of glucagon-like peptide 1 (GLP-1) [START_REF] Maida | Metformin regulates the incretin receptor axis via a pathway dependent on peroxisome proliferator-activated receptor-alpha in mice[END_REF][START_REF] Mulherin | Mechanisms underlying metformin-induced secretion of glucagon-like peptide-1 from the intestinal L cell[END_REF].A recent study has found evidence that metformin and phenformin antagonize the action of the counter-regulatory hormone glucagon to suppress hepatic glucose production [START_REF] Miller | Biguanides suppress hepatic glucagon signalling by decreasing production of cyclic AMP[END_REF]. Furthermore, Fullerton and colleagues recently showed that metformin-induced improvements in insulin action operatethrough alterations in hepatic lipid homeostasisvia theinhibitory phosphorylation of acetyl CoA carboxylase (ACC) by AMPK [START_REF] Fullerton | Single phosphorylation sites in Acc1 and Acc2 regulate lipid homeostasis and the insulinsensitizing effects of metformin[END_REF].

Inhibition of hepatic gluconeogenesis

An important breakthrough in the understanding of the molecular mechanism underlying metformin action was the demonstration that metformin-induced AMPK activation is associated with the inhibition of glucose production in primary hepatocytes [START_REF] Zhou | Role of AMP-activated protein kinase in mechanism of metformin action[END_REF].The role for AMPK in mediating the action of metformin was initially supported by the reduction in metformin's effect on glucose production in primary hepatocytes treated with compound C [START_REF] Zhou | Role of AMP-activated protein kinase in mechanism of metformin action[END_REF], an AMPK inhibitor whichis now recognized to be non-selective. Thereafter, it was reported that, ablation of liver kinase B1 (LKB1, the upstream kinase that phosphorylates and activates AMPK)in the liver prevented the anti-hyperglycemic effects of metformin in high-fat fedmice [START_REF] Shaw | The kinase LKB1 mediates glucose homeostasis in liver and therapeutic effects of metformin[END_REF], also supporting the involvement of the kinase in the inhibition of glucose production by the drug. In this study, it was shownthat LKB1/AMPK signaling controls the phosphorylation and nuclear exclusion of the transcriptional coactivatorcAMPresponse element-binding protein (CREB)-regulated transcription coactivator 2 (CRTC2, also known as TORC2) [START_REF] Shaw | The kinase LKB1 mediates glucose homeostasis in liver and therapeutic effects of metformin[END_REF], a pivotal regulator of gluconeogenic gene transcription in response to fasting.In addition, AMPK activation by metformin has also been reported to be involved in the transcriptional regulation of hepatic gluconeogenic enzyme genes by different mechanisms: i) dissociation of the CREB-CBP (CREB-binding protein)-TORC2 transcription complex, through the phosphorylation of the transcriptional coactivator CBP via atypical protein kinase C /λ [START_REF] He | Metformin and insulin suppress hepatic gluconeogenesis through phosphorylation of CREB binding protein[END_REF], ii) increased expression of the orphan nuclear receptor small heterodimer partner [START_REF] Lee | AMPK-dependent repression of hepatic gluconeogenesis via disruption of CREB.CRTC2 complex by orphan nuclear receptor small heterodimer partner[END_REF], and iii) induction of SIRT1-mediated CRTC2 deacetylation [START_REF] Caton | Metformin suppresses hepatic gluconeogenesis through induction of SIRT1 and GCN5[END_REF].However, the impact of reduction ingluconeogenic gene expression in metformin action has been recently disputed. Forced increase in gluconeogenic enzymes expression did not counteract the metformin-induced reduction in glucose output [START_REF] Foretz | Metformin inhibits hepatic gluconeogenesis in mice independently of the LKB1/AMPK pathway via a decrease in hepatic energy state[END_REF], this being in line with the emerging concept that transcriptional expression of PEPCK and G6Pase only weakly influences hepatic glucose output in patients with T2D [START_REF] Samuel | Fasting hyperglycemia is not associated with increased expression of PEPCK or G6Pc in patients with Type 2 Diabetes[END_REF].

Over the last years, controversy has arisen concerning the involvement of AMPK in the therapeutic effects of metformin on hepatic glucose production. Indeed, although metformin activates AMPK, this may not explain all of the therapeutic effects of the drug.

Recent work in liver and primary hepatocytes from knockout models for both AMPK1/2 catalytic subunits and the upstream activating kinase LKB1 reveal that neither AMPK nor LKB1 are essential for metformin inhibition of hepatic glucose production [START_REF] Foretz | Metformin inhibits hepatic gluconeogenesis in mice independently of the LKB1/AMPK pathway via a decrease in hepatic energy state[END_REF].However, a new report challenges these results and now shows that low concentrations of metformin suppress glucose production via AMPK activation independently of an increase in the AMP/ATP ratio [START_REF] Cao | Low Concentrations of Metformin Suppress Glucose Production in Hepatocytes through AMP-activated Protein Kinase (AMPK)[END_REF]. A central question raised by this work is how does metformin activate AMPK without affecting energy charge.As described above, there is a consensus that metformin activates AMPK indirectly, secondary to the inhibition of the mitochondrial respiratory-chain complex 1, leading to ATP depletion and increase in AMP levels [START_REF] Hawley | Use of cells expressing gamma subunit variants to identify diverse mechanisms of AMPK activation[END_REF]. In addition, it has been suggested that perturbation of intracellular ATP levels, but not AMPK activationper se or inhibition of gluconeogenic gene expression, constitutes the critical factor underlying the effects of metformin on hepatic glucose output [START_REF] Foretz | Metformin inhibits hepatic gluconeogenesis in mice independently of the LKB1/AMPK pathway via a decrease in hepatic energy state[END_REF].

Gluconeogenesis is an energetically costly anabolic process, requiring 6 ATP equivalents per molecule of glucose synthesized, and it seems likely that the metformin-induced increase in AMP exerts a major role in the flux control of hepatic gluconeogenesis by the drug. Indeed, AMP is a potent allosteric inhibitor of fructose 1,6-bisphosphatase, a key enzyme in gluconeogenesis. Additionally, high AMP levels inhibit adenylatecyclase, thereby reducing cyclic AMP (cAMP) formation in response to glucagon and thus, fasting glucose levels [START_REF] Miller | Biguanides suppress hepatic glucagon signalling by decreasing production of cyclic AMP[END_REF].Taken together, a growing body of data indicates that multiple AMPK-independent regulatory points exist for direct AMP-and ATP-mediated effects of metformin on gluconeogenesis (Figure 1). In addition, it was very recently reported that the reduction in hepatic gluconeogenesis by metformin might result from a direct inhibition of the mitochondrial glycerophosphate dehydrogenase (mGPD), identifying another putative mitochondrial target of the drug [START_REF] Madiraju | Metformin suppresses gluconeogenesis by inhibiting mitochondrial glycerophosphate dehydrogenase[END_REF].Inhibition of mGPDhalts the glycerophosphate shuttle, blockinggluconeogenesis from glycerol and raising cytosolic NADH that feeds back on lactate dehydrogenase and also impairsincorporation of lactate into glucose.

Regulation of lipid metabolism

Another effect of metformin is to improve lipid metabolism by reducing hepatic steatosisas demonstrated in rodent liver [START_REF] Lin | Metformin reverses fatty liver disease in obese, leptin-deficient mice[END_REF][START_REF] Woo | Metformin ameliorates hepatic steatosis and inflammation without altering adipose phenotype in diet-induced obesity[END_REF]and also reported in a clinical study [START_REF] Marchesini | Metformin in non-alcoholic steatohepatitis[END_REF]. It was also recently reported that metformin exerts a beneficial effect on circulating lipids by lowering plasma triglycerides, through a selective increase in VLDL-triglyceride uptake and fatty acid oxidation in brown adipose tissue [START_REF] Geerling | Metformin lowers plasma triglycerides by promoting VLDL-triglyceride clearance by brown adipose tissue in mice[END_REF]. The metformin-induced reduction in tissue lipid storage is consistent with an increase in both fatty acid oxidation and inhibition of lipogenesis, presumably mediated by AMPK activation [START_REF] Geerling | Metformin lowers plasma triglycerides by promoting VLDL-triglyceride clearance by brown adipose tissue in mice[END_REF][START_REF] Zang | AMP-activated protein kinase is required for the lipidlowering effect of metformin in insulin-resistant human HepG2 cells[END_REF][START_REF] Zhou | Role of AMP-activated protein kinase in mechanism of metformin action[END_REF]. Further support for a role of AMPK in the mechanisms of metformin action on lipid metabolism was recently provided inknock-in mouse models in which ACC1 and ACC2 were rendered insensitive to AMPK phosphorylation [START_REF] Fullerton | Single phosphorylation sites in Acc1 and Acc2 regulate lipid homeostasis and the insulinsensitizing effects of metformin[END_REF]. These mice are refractory to the lipidlowering and insulin-sensitizing effects of metformin, showing that metformin-induced reduction in blood glucose levels depends on its ability to lower cellular fatty acid levels through the AMPK-dependent phosphorylation of ACC. Thus, the inhibition of hepatic glucose production by metformin may be, at least in certain conditions, secondary to the effects of the drugon ACC. These observations offer a potential explanation for the lack of metformin action on blood glucose levels in liver-specific LKB1-knockout mice fed on a high-fat diet [START_REF] Shaw | The kinase LKB1 mediates glucose homeostasis in liver and therapeutic effects of metformin[END_REF]. Indeed, impaired metformin-induced AMPK phosphorylation in the absence of LKB1 would prevent ACC phosphorylation and the ability of metformin to improve insulin sensitivity and lower blood glucose.Therefore, metformin can acutely suppress hepatic glucose output by acting on distinct metabolic pathways via AMPK-independent and AMPK-dependent mechanisms in the context of insulin resistance (Figure 1).

Metformin action in cardiovascular system

Metformin andcardioprotection

Cardiovascular diseases are undoubtedly associated with T2D and ischemic heart disease is the main cause of death in type 2 diabetic population [START_REF] Grundy | Diabetes and cardiovascular disease: a statement for healthcare professionals from the American Heart Association[END_REF]. The cardiovascular dysfunctions associated with T2D are macro-and micro-vascular abnormalities including atherosclerosis of large arteries and coronary atherosclerosis which contributes to not only diabetes-related mortality and morbidity, but also include diabetic cardiomyopathy, a specific heart muscle dysfunction that occurs independently of coronary artery disease [START_REF] Bugger | Molecular mechanisms of diabetic cardiomyopathy[END_REF]. Although metformin is a first-line glucose-lowering pharmacological agent, its use was historically contraindicated in patients with heart failure due to concerns regarding increased risk of lactic acidosis.

However, numerous studies revealed that metformin-associated lactic acidosis is minimaland that metformin treatment clearly reduces mortality and morbidity of type 2 diabetic patients with cardiovascular diseases such as stable coronary artery disease, acute coronary syndrome and myocardial infarction [START_REF] Eurich | Comparative safety and effectiveness of metformin in patients with diabetes mellitus and heart failure: systematic review of observational studies involving 34,000 patients[END_REF][START_REF] Masoudi | Thiazolidinediones, metformin, and outcomes in older patients with diabetes and heart failure: an observational study[END_REF]. Currently, clinical practice guidelines recommend using metformin as first-line therapy in diabetic patients with heart failure(American Diabetes Association, 2014).

The UKPDS trial nicely demonstrated that metformin was more effective than sulphonylurea or insulin in patients allocated to intensive blood-glucose control (1998).

In this study, metformin significantly reduced all-cause mortality and diabetes-related death by 36% and 42%, respectively with a significant reduction in myocardial infarction events, which persisted after a 10-year follow-up [START_REF] Holman | 10-year follow-up of intensive glucose control in type 2 diabetes[END_REF][START_REF]Effect of intensive blood-glucose control with metformin on complications in overweight patients with type 2 diabetes (UKPDS 34)[END_REF].

Numerous recent studies have confirmed the UKPDS conclusions. In these studies, metformin use was associated with better short-and/or long-term prognosis than other antidiabetic treatments in diabetic patients with acute coronary syndrome [START_REF] Hong | Effects of metformin versus glipizide on cardiovascular outcomes in patients with type 2 diabetes and coronary artery disease[END_REF] or chronic heart failure [START_REF] Eurich | Comparative safety and effectiveness of metformin in patients with diabetes mellitus and heart failure: systematic review of observational studies involving 34,000 patients[END_REF]. However, it has to be noted that the benefit obtained with metformin in comparison to the other glucose-lowering agents is generally quite modest. Importantly, the use of metformin in non-diabetic patients suffering from coronary heart disease was not associated with any beneficial effects [START_REF] Lexis | Effect of metformin on left ventricular function after acute myocardial infarction in patients without diabetes: the GIPS-III randomized clinical trial[END_REF][START_REF] Preiss | Metformin for non-diabetic patients with coronary heart disease (the CAMERA study): a randomised controlled trial[END_REF]. Several meta-analyses have been recently performed [START_REF] Boussageon | Reappraisal of metformin efficacy in the treatment of type 2 diabetes: a meta-analysis of randomised controlled trials[END_REF][START_REF] Eurich | Comparative safety and effectiveness of metformin in patients with diabetes mellitus and heart failure: systematic review of observational studies involving 34,000 patients[END_REF][START_REF] Lamanna | Effect of metformin on cardiovascular events and mortality: a meta-analysis of randomized clinical trials[END_REF]. Their inclusion criteria (only population at low risk of mortality; including or not patients with heart failure; including or not non-diabetic patients) and their conclusions slightly differ.

The first concludes that metformin monotherapy improves survival whereas concomitant utilization with sulphonylurea was associated with reduced survival [START_REF] Lamanna | Effect of metformin on cardiovascular events and mortality: a meta-analysis of randomized clinical trials[END_REF]. In the second, the effectiveness of metformin to avoid death or cardiovascular events is not established by existing studies for the second meta-analysis [START_REF] Boussageon | Reappraisal of metformin efficacy in the treatment of type 2 diabetes: a meta-analysis of randomised controlled trials[END_REF]. Finally, the more recent meta-analysis indicates that metformin treatment is associated with reduced mortality compared with controls and is at least as safe as other glucose-lowering agents in diabetic patients with heart failure [START_REF] Eurich | Comparative safety and effectiveness of metformin in patients with diabetes mellitus and heart failure: systematic review of observational studies involving 34,000 patients[END_REF]. Future clinical trials are clearly necessary to definitively conclude about the safety and cardioprotective effects of metformin treatment.

Metformin in hypertension and atherogenesis

The mechanisms involved in the beneficial cardiovascular effects of metformin in diabetic patients are not fully understood. A plausible explanation is the systemic antihyperglycemic effect of the drug,whereby lowered gluconeogenesis decreases glucose levels and secondarily reduces insulin levels. However, several other molecular mechanisms directly targeting the cardiovascular system have been suggested by animal studies to participate in the benefits of metformin (Figure 2). A large part of these effects appears to be mediated by AMPK. The activation of AMPK by metformin-induced energy stressis relevant to the vascular system, which is known to be alteredin diabetes by endothelial dysfunction and atherogenesis. Atherogenesisis accompanied by an impairment of endothelium-dependent relaxation, increased reactive oxygen species (ROS) production and reduced nitric oxide (NO) bioavailability, mediating proinflammatory and pro-thrombotic mechanisms including platelet aggregation and leukocyte adhesion to the wall. Several cellular and animal studies have evaluated the potential anti-atherogenic action of metformin and AMPK activation. Metformin has been shown to inhibit high glucose-dependent ROS overproduction in aortic endothelial cells and its consequent endothelial dysfunction [START_REF] Detaille | Metformin prevents high-glucose-induced endothelial cell death through a mitochondrial permeability transition-dependent process[END_REF][START_REF] Ouslimani | Metformin decreases intracellular production of reactive oxygen species in aortic endothelial cells[END_REF]. The decrease in ROS production is mediated by a double mechanism involving a reduction in NADPH oxidase activity and an inhibition of the respiratory-chain complex 1. Two more recent studies proposed that AMPK mediates this metformin-induced NADPH oxidase inactivation reducing cytoplasmic ROS production [START_REF] Batchuluun | Metformin and liraglutide ameliorate high glucose-induced oxidative stress via inhibition of PKC-NAD(P)H oxidase pathway in human aortic endothelial cells[END_REF][START_REF] Bhatt | C-peptide activates AMPKalpha and prevents ROS-mediated mitochondrial fission and endothelial apoptosis in diabetes[END_REF]. In parallel, Kukidome and colleagues showed that AMPK activation reduces the hyperglycemia-mediated mitochondrial ROS overproduction via the induction of manganese superoxide dismutase and the promotion of peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1α)-dependent mitochondrial biogenesis in human umbilical vein endothelial cells [START_REF] Kukidome | Activation of AMP-activated protein kinase reduces hyperglycemia-induced mitochondrial reactive oxygen species production and promotes mitochondrial biogenesis in human umbilical vein endothelial cells[END_REF]. AMPK activation is also known to induce endothelial NO synthase (eNOS) activation and NO-dependent vasodilation. In line with this action, it has been recently

shown that metformin restores endothelial function through the inhibition of endoplasmicreticulum (ER) stress and oxidative stress and via the increase in NO bioavailability in obese diabetic mice, these effects being mediated by the AMPK/peroxisome proliferator-activated receptor  pathway [START_REF] Cheang | Metformin protects endothelial function in diet-induced obese mice by inhibition of endoplasmic reticulum stress through 5' adenosine monophosphateactivated protein kinase-peroxisome proliferator-activated receptor delta pathway[END_REF]. In addition, metformin targets advanced glycationend-products (AGEs), which are significant contributors of complications linked to diabetes. Indeed, metformin, independently of its anti-hyperglycemic property, is able to reduce AGEs synthesis and the expression of their specific cell receptor called RAGE in endothelial cells [START_REF] Ouslimani | Metformin reduces endothelial cell expression of both the receptor for advanced glycation end products and lectin-like oxidized receptor 1[END_REF]. Finally, metformin treatment of patient with T2D was associated with a decrease in the level of the soluble intercellular adhesion molecule-1 (ICAM-1) and the soluble vascular cell-adhesion molecule-1 (VCAM-1), both being directly correlated to increase in cardiovascular events in such population [START_REF] Jager | Effects of short-term treatment with metformin on markers of endothelial function and inflammatory activity in type 2 diabetes mellitus: a randomized, placebo-controlled trial[END_REF].

Interestingly, as for AGEs, this decrease in ICAM-1 and VCAM-1 level was independent of the anti-hyperglycemic action of the drug.

Metformin in myocardial injury

The protective action of metformin also occurs at the myocardium and cardiomyocyte levels. Several ex vivo experiments using perfused heart protocols revealed a protective action of metformin during an ischemic episode. In a workingheart perfusion model where metformin is administered before a mild ischemic episode, the anti-diabetic drug improved rat cardiac functional post-ischemic recovery [START_REF] Legtenberg | Metformin improves cardiac functional recovery after ischemia in rats[END_REF]. It has been also shown that metformin given at the time of reperfusion reduced myocardial infarct size in both non-diabetic and diabetic hearts [START_REF] Bhamra | Metformin protects the ischemic heart by the Aktmediated inhibition of mitochondrial permeability transition pore opening[END_REF][START_REF] Paiva | Metformin prevents myocardial reperfusion injury by activating the adenosine receptor[END_REF]. In these two last studies, the protective action of metformin was associated with a PI3K-mediated inhibition of the mitochondrial permeability transition pore opening and with increased intracellular formation of adenosine. However, the same group also showed that chronic metformin treatment of diabetic rats augments their myocardial resistance to ischemia-reperfusion injury via a pathway involving AMPK activation and PGC-1 [START_REF] Whittington | Chronic metformin associated cardioprotection against infarction: not just a glucose lowering phenomenon[END_REF]. Similar results have been obtained using in vivo models of myocardial infarction. In mice subjected to permanent left coronary artery occlusion or to one hour left coronary artery occlusion followed by reperfusion, 4 weeks metformin treatment improved survival and preserved left ventricular dimensions and left ventricular ejection fraction [START_REF] Gundewar | Activation of AMP-activated protein kinase by metformin improves left ventricular function and survival in heart failure[END_REF]. These effects were concomitant to AMPK and eNOS activation, to increase in PGC-1 expression and disappeared in mice lacking AMPK or eNOS. In a similar study, Yin and colleagues demonstrated that metformin treatment increases AMPK activity, improves cardiac function and reduces infarct size after a myocardial infarction in rats [START_REF] Yin | Metformin improves cardiac function in a nondiabetic rat model of post-MI heart failure[END_REF]. This protective action of metformin was also established in a dog model of heart failure [START_REF] Sasaki | Metformin prevents progression of heart failure in dogs: role of AMP-activated protein kinase[END_REF]. The authors nicely showed that other AMPK activators, such as 5-aminoimidazole-4-carboxamide 1--Dribofuranoside (AICAr), have the same effect than metformin, suggesting a role of this protein kinase. In cardiomyocytes, metformin reduced apoptosis by increasing antiapoptotic proteins and attenuating the production of pro-apoptotic proteins [START_REF] Yeh | AMP-activated protein kinase activation during cardioplegia-induced hypoxia/reoxygenation injury attenuates cardiomyocytic apoptosis via reduction of endoplasmic reticulum stress[END_REF]. These effects correlate with AMPK activation and can be reproduced by AICAr [START_REF] Yeh | AMP-activated protein kinase activation during cardioplegia-induced hypoxia/reoxygenation injury attenuates cardiomyocytic apoptosis via reduction of endoplasmic reticulum stress[END_REF]. Metformin and AMPK also influence autophagy, which is known to be dysregulated in diabetic cardiomyopathy and heart failure. Indeed, metformin is able to restore impaired autophagy via the dissociation of Bcl-2 from Beclin1 in diabetic wild-type mice but not in cardiac-specific AMPK-dominant-negative transgenic diabetic mice [START_REF] He | Dissociation of Bcl-2-Beclin1 complex by activated AMPK enhances cardiac autophagy and protects against cardiomyocyte apoptosis in diabetes[END_REF][START_REF] Xie | Improvement of cardiac functions by chronic metformin treatment is associated with enhanced cardiac autophagy in diabetic OVE26 mice[END_REF].

Metformin in cardiac hypertrophy

Left ventricular hypertrophy is common among patients with T2D, who commonly have chronic hypertension. AMPK is a known inhibitor of cardiac hypertrophy via the negative regulation of protein synthesis (via mTOR inhibition) and of gene transcription including mitogen-activated protein kinase and calcineurinnuclear factor of activated T cells pathways [START_REF] Horman | AMP-activated protein kinase in the control of cardiac metabolism and remodeling[END_REF]. In agreement with the anti-hypertrophic action of AMPK, metformin inhibits cardiac hypertrophy in a rat model of pressure overload (transverse aortic constriction) [START_REF] Zhang | Metformin attenuates ventricular hypertrophy by activating the AMP-activated protein kinase-endothelial nitric oxide synthase pathway in rats[END_REF]. Avery recent publication has reported that metformin protects against transverse aortic constriction-mediated cardiac hypertrophy independently of AMPK [START_REF] Xu | Metformin protects against systolic overload-induced heart failure independent of AMPactivated protein kinase alpha2[END_REF].

Metformin in diabetic cardiomyopathy

A healthy human heart produces around 5-6 kg of ATP each day to sustain its function, representing 20 times its own weight [START_REF] Horman | AMP-activated protein kinase in the control of cardiac metabolism and remodeling[END_REF]. This ATP is generated via the mitochondrial oxidation of fatty acids (for 70 %) and glucose/pyruvate (for 30 %). Fatty acids are preferred substrates because their oxidation inhibits glucose catabolism via the Randle cycle. However, in the postprandial state, insulin favors glucose utilization by promoting glucose uptake and glycolysis and inhibiting fatty acid oxidation. Currently, it is commonly accepted that this metabolic flexibility is essential for the maintenance of heart function. On the other hand, the metabolic inflexibility of diabetic hearts, which are insulin resistant and almost exclusively use fatty acids as source of energy, participates in the development of the diabetic cardiomyopathy [START_REF] Bugger | Molecular mechanisms of diabetic cardiomyopathy[END_REF]. Thus, therapies that promote glucose metabolism and normalize insulin sensitivity may reduce cardiac complications linked to diabetes. Metformin and AMPK are potential therapeutic candidates. Indeed, AMPK activation by metformin or other activators is able to stimulate cardiac glucose uptake and glycolysis independently of insulin, bypassing insulin resistance in insulin-resistant cardiomyocytes [START_REF] Bertrand | AMPK activation restores the stimulation of glucose uptake in an in vitro model of insulin-resistant cardiomyocytes via the activation of protein kinase B[END_REF][START_REF] Ginion | Inhibition of the mTOR/p70S6K pathway is not involved in the insulin-sensitizing effect of AMPK on cardiac glucose uptake[END_REF]. Even more interestingly, increase in AMPK activity by the same activators restored insulin sensitivity of insulin-resistant cardiomyocytes by a mechanism that still needs to be identified [START_REF] Bertrand | AMPK activation restores the stimulation of glucose uptake in an in vitro model of insulin-resistant cardiomyocytes via the activation of protein kinase B[END_REF][START_REF] Ginion | Inhibition of the mTOR/p70S6K pathway is not involved in the insulin-sensitizing effect of AMPK on cardiac glucose uptake[END_REF]. The concentration of metformin used in these acute (few hours) studies was however higher than those used in diabetic patients. We can postulate that lower concentrations of metformin could play similar role when utilized during longer time of exposure. In relation to the problematic of the effective dose of metformin in the heart, a new specific activator of AMPK (A-769662) has been recently

shown to increase metformin sensitivity independently of the AMP/ATP ratio [START_REF] Timmermans | A-769662 potentiates the effect of other AMP-activated protein kinase activators on cardiac glucose uptake[END_REF]. Such compound might be useful clinically to increase metformin cardiac sensitivity.

On the other hand, similarly to the situation found in endothelial cells [START_REF] Detaille | Metformin prevents high-glucose-induced endothelial cell death through a mitochondrial permeability transition-dependent process[END_REF][START_REF] Ouslimani | Metformin decreases intracellular production of reactive oxygen species in aortic endothelial cells[END_REF], hyperglycemia induced NADPH oxidase-mediated ROS production and cell death in cardiomyocytes [START_REF] Balteau | NADPH oxidase activation by hyperglycaemia in cardiomyocytes is independent of glucose metabolism but requires SGLT1[END_REF]. It is tempting to speculate that AMPK activation by metformin would be able to reduce this ROS production and to increase cardiomyocyte survival under hyperglycemic condition as demonstrated in the endothelium [START_REF] Batchuluun | Metformin and liraglutide ameliorate high glucose-induced oxidative stress via inhibition of PKC-NAD(P)H oxidase pathway in human aortic endothelial cells[END_REF][START_REF] Bhatt | C-peptide activates AMPKalpha and prevents ROS-mediated mitochondrial fission and endothelial apoptosis in diabetes[END_REF]. In agreement with this hypothesis, it has been recently shown that AMPK activation by A-769662, the metformin analog phenformin or the new anti-diabetic drug glucagon-like peptide 1 limited glucotoxicity in adult cardiomyocytes [START_REF] Balteau | AMPK activation by glucagon-like peptide-1 prevents NADPH oxidase activation induced by hyperglycemia in adult cardiomyocytes[END_REF]. The mechanism proposed in this study is the AMPK-dependent suppression of the hyperglycemia-mediated ROS production via the inhibition of the NADPH oxidase NOX2.

Metformin in cardiac fibrosis

Cardiac fibrosis is another element of diabetic cardiomyopathy and more generally heart failure. Interestingly, metformin is able to attenuate fibrosis in a canine model of heart failure,presumably via AMPK activation and its inhibitory action on transforminggrowthfactor- (TGF-) expression [START_REF] Sasaki | Metformin prevents progression of heart failure in dogs: role of AMP-activated protein kinase[END_REF]. Similar results were obtained in mice subjected to left ventricular pressure overload by transverse aortic constriction even if, in this case, AMPK did not seem to be involved [START_REF] Xiao | Metformin attenuates cardiac fibrosis by inhibiting the TGFbeta1-Smad3 signalling pathway[END_REF]. In other studies, it has been established that metformin inhibits myofibroblast differentiation by suppressing ROS generation via the inhibition of the NADPH oxidase pathway [START_REF] Bai | Metformin inhibits angiotensin II-induced differentiation of cardiac fibroblasts into myofibroblasts[END_REF], AMPK probably mediating this effect. Of note, a genetic link between AMPK and cardiac fibrosis has been recently demonstrated in a myocardial infarction mouse model [START_REF] Noppe | Reduced scar maturation and contractility lead to exaggerated left ventricular dilation after myocardial infarction in mice lacking AMPKalpha1[END_REF].

Anti-neoplastic actions of metformin

Pharmaco-epidemiology

Pharmaco-epidemiologic evidence has played a major role in generating the hypothesis that metformin has utility in cancer prevention and/or treatment. A seminal report published in 2005 [START_REF] Evans | Metformin and reduced risk of cancer in diabetic patients[END_REF] presented evidence that diabetics treated with metformin had a substantially lower cancer burden than diabetics treated with other agents, and many other studies reached similar conclusions [START_REF] Gandini | Metformin and Cancer Risk and Mortality: A Systematic Review and Meta-Analysis taking into account Biases and Confounders[END_REF]. However, while results of certain studies are encouraging, there are contradictions in the available data and the hypothesis remains controversial [START_REF] Tsilidis | Metformin Does Not Affect Cancer Risk: A Cohort Study in the U.K. Clinical Practice Research Datalink Analyzed Like an Intention-to-Treat Trial[END_REF].

Obviously, the populations under study were type 2 diabetic patients, and the conclusions may not be applicable to non-diabetic subjects, even if the conclusions were validated for diabetics. More importantly, all these studies are based on retrospective reviews of medical records, and are subject to a variety of potential biases [START_REF] Suissa | Metformin and cancer: mounting evidence against an association[END_REF]. On the other hand, some recent reports are consistent with the earlier pharmaco-epidemiologic data that provided evidence for reduced cancer burden among users of metformin. Examples include a study of multiple myeloma outcomes [START_REF] Wu | The association of diabetes and anti-diabetic medications with clinical outcomes in multiple myeloma[END_REF], one of prostate cancer risk based on the Danish cancer registry [START_REF] Preston | Metformin Use and Prostate Cancer Risk[END_REF], and one regarding prostate cancer prognosis [START_REF] Margel | Metformin use and all-cause and prostate cancer-specific mortality among men with diabetes[END_REF]. Thus, pharmaco-epidemiologic data have played an important role in generating the hypothesis that metformin may be useful in cancer prevention or treatment, but have not been consistent in supporting this concept.

Indirect effects of metformin on cancer

The proposed mechanisms of action of metformin relevant to oncology can be divided into two broad, non-mutually exclusive categories: effects on the host that indirectly influence the cancer, and direct effects on cancer cells (Pollak, 2012b) (Figure 3). As noted earlier in this review, direct actions of metformin on the liver inhibit hepatic glucose production, resulting in systemic metabolic and endocrine effects that may influence cancer biology. The most obvious candidate change of oncologic relevance is the reduction of hyperinsulinemia, given prior evidence that high insulin levels can stimulate proliferation of a subset of common cancers (Pollak, 2012a). Importantly, however, the magnitude of metformin-induced decline in insulin levels is greater in type 2 diabetics than in metabolically normal subjects and it is not clear if metformin-induced changes in plasma insulin levels, particularly in non-diabetics, are sufficient to perturb tumor biology. Nevertheless, there is evidence from a murine model that metformin administration is more effective with respect to tumor growth inhibition when dietinduced obesity and hyperinsulinemia are present, and is associated with decreased activation of insulin receptors of cancer xenografts [START_REF] Algire | Diet and tumor LKB1 expression interact to determine sensitivity to anti-neoplastic effects of metformin in vivo[END_REF]. Levels of variousadipokines relevant to cancer biology are also influenced by metformin in models, but further clinical data in this area are required. Also, a few studies have suggested immunological or anti-inflammatory actions of metformin [START_REF] Moiseeva | Metformin inhibits the senescence-associated secretory phenotype by interfering with IKK/NF-kappaB activation[END_REF][START_REF] Pearce | Enhancing CD8 T-cell memory by modulating fatty acid metabolism[END_REF] relevant to oncology, but there are to date no clinical data to support or refute these observations. Thus, the role of "indirect" actions of metformin remains an active research topic.

Direct effects of metformin on cancer

Dozens of in vivo and in vitro studies have shown direct anti-neoplastic activity of biguanides in model systems, but most of these have not provided mechanistic details nor considered dose-response issues relevant to clinical applications (Pollak, 2012b;[START_REF] Viollet | Cellular and molecular mechanisms of metformin: an overview[END_REF]. One of these [START_REF] Huang | Important role of the LKB1-AMPK pathway in suppressing tumorigenesis in PTEN-deficient mice[END_REF] provided evidence that AMPK activation is important in the action of biguanides by showing that the direct AMPK activator A-769662 (which does not inhibit OXPHOS) has in vivo anti-neoplastic activity.

Other observations suggest the relevance of inhibition of respiratory-chain complex 1 within tumors underlies the therapeutic effect of metforminin vivo under conditions where dosing is adequate:a keyfinding was that tumor growth inhibition by metformin occurs under control conditions, but not when the tumor model is engineered to express the yeast metformin-resistant Saccharomyces cerevisiae NADH dehydrogenase NDI1 protein [START_REF] Birsoy | Metabolic determinants of cancer cell sensitivity to glucose limitation and biguanides[END_REF][START_REF] Wheaton | Metformin inhibits mitochondrial complex I of cancer cells to reduce tumorigenesis[END_REF].

Alterations in cellular metabolism in a manner that is influenced by mutations in exposed cancer cells are important consequences of metformin-inducedreduction of oxidative phosphorylation [START_REF] Buzzai | Systemic treatment with the antidiabetic drug metformin selectively impairs p53-deficient tumor cell growth[END_REF]. This suggests the possibility of rational drug combinations [START_REF] Ben Sahra | Targeting cancer cell metabolism: the combination of metformin and 2-deoxyglucose induces p53-dependent apoptosis in prostate cancer cells[END_REF].Particularly interesting work has provided a metabolic rationale for combining biguanides with small molecule drugs that inhibit oncogenic kinases that drive glycolysis [START_REF] Pollak | Targeting oxidative phosphorylation: why, when, and how[END_REF]. These studies support the possibility that cancer cells have a requirement to increase OXPHOS, at least transiently, to compensate for the decreased glycolysis that arises as a consequence of oncogenic kinase inhibition. In the presence of biguanides, this compensatory increase is attenuated, resulting in enhanced antineoplastic activity of the kinase inhibitor [START_REF] Haq | Oncogenic BRAF regulates oxidative metabolism via PGC1alpha and MITF[END_REF]. Mutation of isocitrate dehydrogenase-1 (IDH-1) is an important topic in cancer metabolism, and there is preclinical evidence justifying study of the combination of a biguanide and inhibitors of mutated IDH-1 [START_REF] Grassian | IDH1 mutations alter citric acid cycle metabolism and increase dependence on oxidative mitochondrial metabolism[END_REF]. There is also evidence of a benefit of combining biguanides with conventional chemo-and radio-and hormonal therapies (Pollak, 2012b).

An early finding supportinga direct action of metformin on cancer cells was the observation that the drug was growth inhibitory in vitro in a manner that was associated withAMPK activation and mTOR inhibition, as a consequence of metformin-induced energetic stress [START_REF] Zakikhani | Metformin is an AMP kinase-dependent growth inhibitor for breast cancer cells[END_REF]. Further work in this direction showed that genetic ablation of the α1 catalytic subunit of AMPK accelerates Myc-induced lymphomagenesis, consistent with the observation that AMPK activation not only downregulatesmTOR, but also suppresses the excess aerobic glycolysis (Warburg effect) characteristic of most transformed cells [START_REF] Faubert | AMPK is a negative regulator of the Warburg effect and suppresses tumor growth in vivo[END_REF]. This line of investigation suggests a tumor suppressor role for AMPK. However, it is now recognized that AMPK activation under conditions of energetic stress can improve cell survival [START_REF] Jeon | AMPK regulates NADPH homeostasis to promote tumour cell survival during energy stress[END_REF] by tuning cellular energy metabolism to reduce energy consumption, in keeping with the evolutionary role of AMPK in adjusting metabolism to cope with low nutrient supply. Thus, it remains to be determined if the anti-proliferative but pro-survival consequences of AMPK activation in cancer cells, either due to the inhibition of oxidative phosphorylation by metformin or the direct activation by specific pharmacological activators,will be of clinical benefit. Consideration of the consequences of biguanide-induced energetic stress in cancer cells defective in AMPK activation is of particular interest. Xenograft [START_REF] Algire | Diet and tumor LKB1 expression interact to determine sensitivity to anti-neoplastic effects of metformin in vivo[END_REF] and transgenic [START_REF] Shackelford | LKB1 inactivation dictates therapeutic response of non-small cell lung cancer to the metabolism drug phenformin[END_REF] models have shown that cancers with loss of function of LKB1are hypersensitive to biguanides. This observation is consistent with the notion that activation of AMPK, while associated with reduction in proliferation, actually mitigates energetic stress induced by metformin by reducing energy consumption, thereby favoring cell survival. Cancer cells functionally deficient in AMPK, in contrast, will be less likely to reduce energy consumption in face of biguanideinduced reduction in ATP generation, and therefore more likely to experience a lethal energetic crisis. This scenario is attractive because it implies a favorable therapeutic index (a greater effect on the cancer than the normal host tissues), as AMPK-defective cancers would be more sensitive to biguanides than normal tissues that retain a functional AMPK signaling system. This situation may be relevant to a sizable proportion of human cancers: for example, more than a third of non-small cell lung cancers are reported to be LKB1-defective. Cancers with mutations in genes encoding respiratorychaincomplex 1 components have also been shown to be hypersensitive to biguanides [START_REF] Birsoy | Metabolic determinants of cancer cell sensitivity to glucose limitation and biguanides[END_REF].

Collectively, pre-clinical studies raise enthusiasm for clinical trials of metformin, in a manner that is less controversial than the pharmaco-epidemiologic rationale. However, it is clear that precise mechanisms have not been defined, and it is important to point out that in order for metformin to have a direct anti-neoplastic effect, an adequate drug concentration must be achieved in neoplastic tissue. This concentration will be determined not only by the plasma level, which is related to the administered dose and to pharmacokinetic variables, but also to cellular uptake in cancer cells which may vary with respect to expression of OCT1 and other relevant transporters. The serum levels of metformin achieved in diabetic patients and in many in vivo models (as reviewed above) are in the micromolar range, while growth inhibition in vitro is usually observed at millimolar concentrations. Thus, an overarching research question is what metformin concentrations are achieved in neoplastic tissue of patients receiving conventional anti-diabetic doses of metformin. Even before consideration of tumor biology, there may well be heterogeneity between cancer patients with respect to direct actions of metformin that arise as a consequence of whole organism and cellular pharmacokinetic factors. Imprecision regarding achieved drug levels in target tissues in preclinical models that demonstrate antineoplastic activity makes extrapolation to the clinic hazardous: it remains uncertain if the laboratory data should be used to support clinical trials of conventional anti-diabetic doses of metformin or clinical trials of more aggressive and novel methods of biguanide administration designed to maximize tumor drug concentration.

Clinicaladvances and outlook

While many clinical trials are ongoing, few actual clinical outcome results have been reported to date. However, there have been many publications of pilot studies that refer to surrogate endpoints such as changes in tumor proliferation rate between serial tumor biopsies obtained from patients prior to and during metformin exposure. Many of these have shown encouraging decreases in proliferation in breast, prostate, and endometrial cancers (Pollak, 2012b;[START_REF] Viollet | Cellular and molecular mechanisms of metformin: an overview[END_REF], but overall results have been difficult to interpret because the magnitude of declines is in many cases smaller than those associated with currently approved treatments, and because in some studies declines are confined to subsets of patients. It should be noted that the design of ongoing randomized clinical trials may not address some of the specific contexts suggested to be of high interest by pre-clinical work performed only after the trials were designed, such as strategic combinations of biguanides with tyrosine kinase inhibitors [START_REF] Pollak | Targeting oxidative phosphorylation: why, when, and how[END_REF]or selective use of biguanides for tumors with respiratory-chaincomplex 1mutations [START_REF] Birsoy | Metabolic determinants of cancer cell sensitivity to glucose limitation and biguanides[END_REF].

Anecdotal clinical evidence of stimulation of glucose uptake by intestinal mucosa by metformin has been presented in the nuclear medicine literature [START_REF] Gontier | High and typical 18F-FDG bowel uptake in patients treated with metformin[END_REF]. While from a radiologic perspective this was reported as a bothersome artifact, from a physiologic perspective, the data may represent pharmacodynamic evidence of biguanide-induced inhibition of OXPHOS in intestinal mucosa, leading to energetic stress and a compensatory increase in glycolysis, resulting in the observed increase in glucose uptake. This may take place preferentially in bowel as compared to other tissues because of the high luminal concentration of metformin following oral administration, emphasizing the importance of pharmacokinetic factors in determining tissues where direct biguanide actions are likely to occur. These data are correlated with greater reductions of proliferation rate by metformin in colon (Pollak, 2013a) than in most other tissues examined, and reduced aberrant crypt foci in patients [START_REF] Hosono | Metformin suppresses colorectal aberrant crypt foci in a short-term clinical trial[END_REF], thus raising the possibility of specific applications for metformin in colorectal cancer prevention.

Ongoing research comprises two broad areas. One examines the hypothesis that metformin, when administered in the manner used for diabetes therapy, has utility for cancer prevention or treatment. There has been relatively little effort to address this possibility in a quantitative fashion in preclinical model systems because it is difficult to accurately simulate human pharmacokinetics following oral dosing in rodents, and because there has been enthusiasm among physicians to proceed directly to human studies [START_REF] Pollak | Overcoming Drug Development Bottlenecks With Repurposing: Repurposing biguanides to target energy metabolism for cancer treatment[END_REF]. Thus, there are more than 200 Oncology clinical trials involving metformin in progress. Another research area, which has not yet led to clinical trials, involves optimization of biguanide pharmacokinetics to maximize exposure of neoplastic tissue. This work is based on one hand on the attractive laboratory data related to the 'direct' mechanism of action, and on the other hand on concern that conventional anti-diabetic dosing may be inadequate for the desired effects. Relative to metformin, phenformin or novel biguanides [START_REF] Narise | Optimization of biguanide derivatives as selective antitumor agents blocking adaptive stress responses in the tumor microenvironment[END_REF] may have reduced requirements for cell surface transporters to enter cancer cells or other desirable pharmacokinetic properties.This has been clearly evidenced in rat hepatoma cells H4IIE, where quinidine, a competitive OCT1 inhibitor, completely blocked AMPK activation by metformin, while AMPK activation byphenforminandgalegine was notaffected [START_REF] Hawley | Use of cells expressing gamma subunit variants to identify diverse mechanisms of AMPK activation[END_REF]. Metformin may have very different effects if given parentally rather than orally. However, while such approaches may have advantages, they also may be associated with unacceptable toxicity, so that conventional preclinical work leading to phase I safety and dose finding clinical trials would be required as a first step.

Conclusionand therapeutic perspectives

After more than 50 years of clinical experience, the utility of metformin in the control of hyperglycemia in T2D has been well established. The safety of metformindifferentiated it fromphenformin and buformin,which were withdrawn in most countries due to a higher risk incidence of lactic acidosis. The favorable risk/benefit profile of metformin hasmade it one of the most widely prescribed drugs in the world.Clinically, metformin exerts its anti-hyperglycemic effect mostly through the inhibition of hepatic gluconeogenesis. Its primary site of action is the mitochondria via its mild and specific inhibition of the respiratory-chain complex 1, thereby lowering energy charge and ultimately leading to a reduction in hepatic glucose output. Although the energy sensor AMPK is activated by a decrease in the cellular energy charge, it appears to be dispensable for the direct inhibitory effect of metformin on hepatic gluconeogenesis, but still may indirectly inhibit by restoring hepatic insulin sensitivity. This is consistent with the role of AMPK in the lipid-lowering effects and improvements in insulin sensitivity by metformin in the liver. Recent advances revealed a new molecular target in the mitochondria with the direct inhibition of mGPD, resulting in altered cellular redox state and limitinglactate and glycerol contribution to hepatic gluconeogenesis.

The evidence presented in this review suggests that metformin may have clinical value in the treatment of cardiovascular complications associated with T2D by exerting a variety of cellular actions in different tissues and cell types. Chronic metformin treatment of patients leads to a basal state of cardioprotection, thereby potentially limiting the occurrence of myocardial infarction, heart failure, diabetic cardiomyopathy and cardiac hypertrophy. Multiple molecular mechanisms were proposed including reduction of ER stress, oxidative stress, apoptosis, protein synthesis and insulin resistance in endothelial cells, cardiomyocytes and cardiac fibroblasts through AMPKdependent and AMPK-independent pathways. Some studies have raised the possibility that metformin may also be effective in providing protection against cancer.The concept of 'repurposing' metformin for cancer prevention or treatment is appealing [START_REF] Pollak | Overcoming Drug Development Bottlenecks With Repurposing: Repurposing biguanides to target energy metabolism for cancer treatment[END_REF], as the drug is inexpensive and well tolerated relative to commonly used anti-neoplastic agents. The original and most optimistic hypothesis, that this drug at conventional anti-diabetic doses is useful for a wide variety of indications in oncology, was based in part on pharmaco-epidemiologic data that are now considered controversial. As most experimental evidence for antineoplastic activity of metformin involves drug exposure levels considerably higher than those in serum of metformin-treated diabetics, the use of novel metformin dosing strategies, including intravenous rather than oral administration, as well as the use of phenformin or novel biguanides designed to have pharmacokinetic characteristics optimized for oncologic indications may be worthwhile. Furthermore, pre-clinical studies have identified not only rational drug combinations involving biguanides that deserve evaluation but also specific tumor characteristics, such as mutations of STK11/LKB1, that may be associated with biguanide sensitivity. Therefore, even if the first generation of metformin trials for cancer treatment are disappointing, there will be interesting questions to address in subsequent studies. On the other hand, demonstration of clinical benefit of any of the ongoing trials would represent an important example of productive 'repurposing' research, and lead to efforts to build on that success.

Among clinicians, metformin is regarded as a widely used and "mature" generic drug with a well-established and important role in treatment of T2D, for which there is a limited research agenda remaining. However, as reviewed here, from a mechanistic perspective, there are many important unanswered questions (Table 1), and a recognition that biguanides have unique therapeutic properties arising from their effects on cellular energy metabolism. It is possible that further understanding of the mechanistic aspects of biguanide pharmacology will result in advances in treatment not only of diabetes, but also in cardiovascular and neoplastic diseases.

Table 1: Examples of challenges in biguanide research

Elucidate the precise mechanism(s) by which metformin interacts with and inhibits the mitochondrial respiratory-chain complex 1

Clarify the effect of metformin on tissue-specific regulation of mitochondrial biogenesis and its subsequent impact on cellular energy homeostasis Clarify if metformin actually acts as an "insulin sensitizer" in vivo, or if the reduction of insulin level associated with use of the drug is completely accounted for by decrease in glucose secondary to decrease gluconeogenesis Define mechanisms that limit duration of efficacy of metformin in treatment of type 2 diabetes Clarify why cardiac benefits of metformin are greater in diabetics than non-diabetics Provide clinical evidence for or against clinical anti-neoplastic action of metformin in diabetic and non-diabetic cancer patients Define optimum dosing of biguanides for cancer treatment in diabetic and non-diabetic patients and establish if phenformin or novel biguanides offer advantages for cancer treatment Delineate the role of AMPK in metformin action in different tissues and different disease states Design and evaluate rational combinations of metformin with other pharmacological agents for various indications Understand the genetic influences on efficacy of metformin for various indications and move towards personalized medicine to optimize therapies Are recently described actions of metformin on the intestinal microbiome [START_REF] Cabreiro | Metformin retards aging in C. elegans by altering microbial folate and methionine metabolism[END_REF] relevant to any of the clinical effects of the drug ? influence not only the extent to which biguanides accumulate, but also its ability to deal with energetic stress, and thus the degree of therapeutic benefit. Abbreviations: GI, gastrointestinal; LKB1, liver kinase B1; OXPHOS, oxidative phosphorylation.

Figure3:

Francophone

  du Diabète, Région Ile de France and Association pour la Recherchesur le Diabète. The work of Guigas' group is supported by Société Francophone du Diabète and European Federation for the Study of Diabetes. The work of Bertrand's group is supported by the Fonds National de la RechercheScientifiqueetMédicale (Belgium), the Actions de RechercheConcertées (Belgium) and Société Francophone du Diabète. LB is Research Associates of the Fonds National de la RechercheScientifique, Belgium. MP is supported by FRSQ, CIHR and CCSRI. The authors have no conflict of interest related to this work.

Figure 1 :

 1 Figure legends:

Figure 2 :

 2 Figure 2: Main putative molecular mechanisms involved in the cardioprotective

Figure 3 :

 3 Figure 3: Proposed mechanisms by which biguanides may influence cancer

Acknowledgments

The authorswant to apologize for the omission of references to many important contributions due to space and citation number limitations. The work ofViollet's groupis performed within the DépartementHospitalo-Universitaire (DHU) AUToimmune and HORmonaldiseaseS and supported by grants from INSERM, CNRS, Université Paris Descartes, AgenceNationale de la Recherche, Société