on line editionMetformin: from mechanisms of action to advanced clinical use Professional article id 1 Department of Vascular Diseases, Division of Internal Medicine, University Medical Centre Ljubljana 2 Department of Endocrinology, Diabetes and Metabolic Diseases, Division of Internal Medicine, University Medical Centre Ljubljana Correspondence: Miodrag Janić, e: miodrag.janic@kclj.si Key words: metformin; main effects; pleiotropic effects; lactacidosis; iodine contrast imaging Cite as: Zdrav Vestn. 2017; 86: 138–57. received: 12. 3. 2016 accepted: 24. 2. 2017 Metabolic and hormonal disordersProfessional article Zdrav Vestn | March – april 2017 | Volume 86 Metformin: from mechanisms of action to advanced clinical use Miodrag Janić,1 Špela Volčanšek,2 Mojca lunder,2 andrej Janež2 Abstract Metformin represents the-first line treatment and the most widely prescribed anti-hyperglycaemic drug for patients with type 2 diabetes. It can be used as monotherapy or in combination with other oral anti-hyperglycaemic drugs or insulin. Additionally, it is prescribed in type 1 diabetes; it proved to be effective in prediabetes and to provide beneficial effects in other insulin resistant states, such as polycystic ovary syndrome. However, the exact molecular mechanism of its action remains un- known. It was shown that it inhibits liver gluconeogenesis, facilitates glucose uptake into peripheral tissues, such as striated muscle and acts in the gut. In addition to anti-hyperglycaemic effects, met- formin was shown to exert several beneficial, protective pleiotropic effects, particularly on the car- diovascular system, and that it is protective against cancer. Metformin has only a few side effects, the most serious one being metformin-associated lactic acidosis. The latter appears in rare clinical cases of pre-existent chronic kidney disease or advanced heart failure with tissue hypo-perfusion, which represent relative contraindications to metformin use. In the past the treatment with metformin was usually discontinued before iodinated contrast-enhanced imaging, but recently there is evidence of its safety even in patients with higher stages of chronic kidney disease. All in all, metformin is the drug with a long tradition and a promising future. Cite as: Zdrav Vestn. 2017; 86: 138–57. 1 Introduction Metformin (1.1-dimethyl-biguanide) is the most widely used drug for the treatment of hyperglycaemia in type 2 diabetes. It is prescribed to more than 100 million patients worldwide yearly (1- 4). Although it has been in clinical use for more than half a century, the exact mechanisms of its action have not yet been fully explained. The beginnings of clinical use of metformin date back to 1957, when it was first prescribed to treat diabetes in humans  (5); while the effectiveness of guanidine hydrochloride in an animal model was demonstrated by Wattanabe as early as 1918 (6). Despite the fact that metformin had been present on the UK market since 1958, it was approved by the US Food and Drug Administration (FDA) as late as 1995  (7). These reser- vations could be partially attributed to the fact that in 1970 another biguanide, phenformin, was withdrawn from the market because of the high incidence of lactic acidosis and cardiovascular com- plications (8). on line edition Zdrav Vestn | March – april 2017 | Volume 86 Metabolic and horMonal disorders 2 The evidence-based options for metformin use In international and Slovenian nation- al guidelines, metformin is recommend- ed as the first-choice drug to be used after failure of non-pharmacological measures in type 2 diabetes mellitus (1-4). It is also used as an add-on-drug to insulin in pa- tients with type 1 diabetes mellitus (9,10) and as monotherapy in some other insu- lin-resistant conditions (e.g. polycystic ovary syndrome) (11). Metformin became the gold standard therapy in the treat- ment of type 2 diabetes mellitus because of its efficiency, its ability to be used in combination with other anti-hypergly- caemic agents, its safety profile, low price and favourable metabolic and cardiovas- cular effects  (12). In the UKPDS multi- center prospective study (UK Prospec- tive Diabetes Study)the subset of patients with newly diagnosed diabetes treated with metformin (UKPDS34) had a 32 % reduced risk for all outcomes associated with diabetes, a 42 % reduced risk of dia- betes-related death and a 36 % reduction in the total mortality (13). The first results of this research were published in 1998 and after a 10-year observation period, a 21 % reduction in risk for any diabetes-re- lated outcome, a 27 % reduction in overall mortality, and a 33 % lower incidence of myocardial infarction in the subgroup of obese patients treated with metfor- min (UKPDS80) were recorded (all sta- tistically significant)  (14). The results of some of the subsequent meta-analyses of randomized controlled trials, which included a small number of studies, did not support the findings of the UKPDS trial (15). A meta-analysis of more than 30 studies confirmed the reduced mortality in patients treated with metformin com- pared to those receiving placebo or any other diabetes drug, which was consistent with the results of the UKPDS trial (16). 3 Pharmacokinetics of metformin Metformin is ingested orally in a daily dose of 500 mg to a maximum of 3000 mg, or 35 mg/kg of body weight per day. Absorption is slow and takes place in the proximal part of the small intes- tine, i.e. in the duodenum and jejunum. Higher doses of metformin tend to slow down its absorption and reduce its bio- availability. Slow absorption and sub- sequent accumulation in the digestive tract may lead to adverse effects (17). On the Slovenian market metformin is for- mulated as immediate release (IR) tab- lets usually taken 2 to 3 times daily. The advantages of metformin extended re- lease formulation (XR), which will soon be available on the Slovenian market, are similar pharmacokinetic properties with once-daily dosing and a lower incidence of gastrointestinal tract side-effects (18). The use of a new formulation of metfor- min with delayed release (DR), revealed that most of its anti-hyperglycaemic ef- fects were caused by metformin action in the gastrointestinal tract and were not dependent on its bioavailability. The results of studies using DR metformin promise greater safety, since with lower bioavailability most adverse events and contraindications could be avoided (19). Metformin is a biguanide that is a strong base with a pKa of 12.4 and exists mainly as a cation in compartments at physiological pH. Metformin is a hydro- philic base and is unable to cross plasma membranes by passive diffusion (20). The membrane transport therefore needs to be facilitated by organic cationic trans- porters (OCT). OCT1 is expressed in the endothelium of the digestive tract, liver and red blood cells, a subset of OCT2 is expressed in kidney cells, and OCT3 is present in skeletal muscle, brain cells, pla- centa and other cells (17,21). Plasma con- on line editionMetformin: from mechanisms of action to advanced clinical use Professional article centrations of metformin normally reach values between 1 and 50 μM, with the highest concentration detected in the por- tal system and liver  (22). Metformin re- mains mostly non-metabolized and 90 % of the drug is excreted in urine by tubular secretion via OCT2 in the kidneys (17,23). Because of metformin's inability to pas- sively diffuse into intracellular space, therapeutic effectiveness of the drug may depend on the expression and various ge- netic forms of the membrane transporter OCT (1/2/3) (24,25). Metformin's absorp- tion takes place slower than its excretion, therefore the former represents a rate limiting step in its metabolism. The half- life of metformin in plasma is usually 2 to 6 hours, but it may be extended up to 14 hours due to possible accumulation in the digestive tract and erythrocytes (17). 4 Pharmacodynamics of metformin Metformin's lowers blood glucose lev- els by reducing hepatic glucose produc- tion (inhibition of gluconeogenesis) (26) and by lowering liver and skeletal muscle insulin resistance. Yet, some of its almost forgotten mechanisms of action that take place in the gastrointestinal tract before absorption into the blood flow should not to be neglected (27). Metformin mechanisms of action on the molecular level are complex and not yet fully understood; a schematic sum- mary of its action is shown in Figure 1. It is known that metformin acts through the AMP-activated protein kinase route (AMPK pathway) or by mechanism of action independent of AMPK. 4.1 Effects in the liver 4.1.1 AMPK-dependent effects Upon entering the liver cell metfor- min inhibits gluconeogenesis primarily through its action in the mitochondria, where it inhibits complex 1 in mitochon- drial respiratory chain. This prevents the formation of energy-rich ATP (ad- enosine-5’-triphosphate). As a result, the concentrations of both, ADP (adenosine diphosphate) and AMP (adenosine mo- nophosphate) increase. Increased levels of ADP and AMP in comparison with ATP lead to a lack of energy needed for energy-consuming enzymatic processes of gluconeogenesis. Increased levels of ADP and AMP in the signaling pathway of metformin have two effects: the acti- vation of (1) AMPK, and (2) liver kinase B1 (LKB1), which regulates AMPK (28). AMPK is the most important enzyme in the regulation of the cellular energy balance. It is activated by the binding of ADP or AMP molecule on its subunit (γ subunit). LKB1 further increases activa- tion of AMPK through the mechanism of positive feedback  (29,30). Activated AMPK inhibits gluconeogenesis through actions targeting several key proteins in- volved in gluconeogenesis  (31). Firstly, it inhibits core protein CRTC2 (CREB- regulated transcription coactivator 2), which has a key role in regulating the expression of enzymes of gluconeo- genesis. The inhibited CRTC2 exits the cell nucleus. Further, AMPK indirectly stimulates an increase in liver sirtuin 1, which also inhibits CRTC2 and leads to its degradation. The inhibited and dis- located CRTC2 disables expression of enzymes involved in gluconeogenesis, which is further facilitated by AMPK- induced dissociation of the transcription complex CREB (CAMP response element binding protein)-CBP (CREB binding protein)-CRTC2 (29) (Figure 1). The activation of cytoplasmic AMPK inhibits the action of acetyl-CoA car- boxylase, thereby reducing the produc- tion of malonyl-CoA. The latter is an important precursor in the process of on line edition Zdrav Vestn | March – april 2017 | Volume 86 Metabolic and horMonal disorders lipogenesis, and inhibits beta-oxidation of fatty acids. AMPK further inhibits the expression of several enzymes involved in lipogenesis indirectly by inhibiting the transcriptional activity of SREBP-1 (ste- rol regulatory element binding protein-1) and ChREBP (carbohydrate-responsive element binding protein) (29). 4.1.2 AMPK-independent effects AMPK-independent effects of met- formin are generated through several different pathways as follows: 1. Metformin increases the activity of the insulin receptor and its substrate, which increases the uptake of glucose in the liver cell (32). Figure 1: a schematic representation of metformin molecular mechanisms of action. (c)aMP – (cyclic) adenin monophosphate, ac – adenylate cyclase, aKt – protein kinase b, aMPK – 5-adenosine monophosphate- activated protein kinase, atP – adenosine triphosphate, chrebP – carbohydrate-responsive element- binding protein, creb – caMP response element binding protein, crtc2 – creb-regulated transcription coactivator 2, enos – endothelial nitric oxide synthase, GlUt 4 – glucose transporter type 4, irs – insulin receptor substrate, lKb1 - liver kinase b1, m/cGPd(h) – guanosine diphoshate, mPtP – mitochondrial permeability transition pore, mtor – mechanistic target of rapamycin, nad(h) – nicotinamide adenine dinucleotide, no – nitric oxide, oct 1 – organic cation transporter 1, Pi3K – phosphatidylinositol-4.5- bisphosphate 3-kinase, PKa – protein kinase a, ros – reactive oxygen species, sirt-1 – sirtuin 1, srebP – sterol regulatory element-binding protein, tsc2 – tuberous sclerosis complex 2. ChREBP ↓ INSULIN RESISTANCE skeletal muscle ⊖ ⊖⊖ ⊖ ⊖ ⊖ ⊖ ⊖ ⊖ ↑ Ca2 + ⊖ ⊖ ⊕ ↓cAMPATP ⊖ Glucagon receptor mkATP MPTP nucleus NADH + H+ NAD+ Glycerol 3-P mitochondrion Complex 1 ↓ ATP production ↑AMP METFORMIN OCT1 mGPDH cGPDH ↓ GLUCONEOGENSIS AMPK LKB1 SIRT-1 SREBP CREBP ↓ expression of lipogenesis genes ↓ LIPOGENESIS ↓ expression of gluconeogenesis genes ↓ GLUCONEOGENESIS Insulin receptor PI3K Akt eNOS NO ROS ANTIOXIDATIVE EFFECT IRS ↑ENDOTHELIAL FUNCTION mTOR TSC2 GLUT-4 translocation ↑GLUCOSE UPTAKE ↓ cell growth ↓ autophagy ↓ protein synthesis ↓ proliferation ANTI-CANCER EFFECT - ↓ apoptosis - ↓ cellular ageing - ↓ inflammation cell membrane PKA ⊕ ⊕ ⊕ ⊕ ⊕ ⊕ p53 AC ↓ adenylate cyclase activity ⊖ ⊕ CRTC2 ⊕ on line editionMetformin: from mechanisms of action to advanced clinical use Professional article 2. Metformin opposes the effect of glu- cagon, thereby lowering fasting gly- caemia. Indirectly, through increased formation of AMP, which binds di- rectly to adenylate cyclase enzyme and inhibits its function, metformin also inhibits glucagon-mediated formation of cAMP, which inhibits glycogenolysis  (33). Treatment with metformin does not give rise to hy- poglycaemia which would be expect- ed after the suppression of glucagon effects. It is believed that metformin- mediated inhibition of glucagon in humans is incomplete, or that com- pensatory mechanisms that prevent hypoglycaemia are simultaneously activated (32). 3. Metformin inhibits mitochondrial glycerophosphate dehydrogenase (mGDP), to which increasing impor- tance has been attributed recently. Inhibition of mGDP prevents glyc- erol from entering into the process of gluconeogenesis, and changes the oxido-reduction state of the cell. This reduces the conversion of lactate into pyruvate and thus the entry of lactate into the gluconeogenesis (34). 4.2 Effects on skeletal muscle Metformin increases the uptake and utilization of glucose in peripheral tis- sues (skeletal muscle), thereby reducing insulin resistance. It increases glucose uptake in skeletal muscle via increased translocation of GLUT4 (glucose trans- porter type 4) transporters to the plasma membrane, which may be carried out via two different signaling pathways: (1) via stimulation of signaling pathways of protein kinase C (PKC), also influenced by insulin action  (35), and (2) through activation of AMPK pathway (Figure 1) (36). 4.3 Effects in the gastrointestinal tract In the gastrointestinal tract, metfor- min slows the absorption of glucose. It also increases the production of intes- tinal hormones and peptides. It plays a particularly important role in the in- cretin axis: it increases the action of glucagon-like peptide 1 (GLP-1), which stimulates glucose-dependent insulin secretion and inhibits glucagon secre- tion  (37). Metformin-treated patients showed lower activity of the enzyme dipeptidyl peptidase-4 (DDP-4), which otherwise degrades incretins. (38). Met- formin is also assumed to have a favour- able effect on the composition of intesti- nal microbiota (39). 5 The role of metformin in the treatment of hyperglycaemia 5.1 Type 2 diabetes mellitus Metformin monotherapy as the first- line treatment in patients with type 2 diabetes mellitus is started after the non- pharmacological treatment, including lifestyle change, regular exercise, healthy diet and weight reduction, has failed to achieve appropriate glycaemic control. Metformin is introduced into the ther- apy if there are no contraindications for its use (as described further on) (4,40). It is usually started at a low dose (500 mg once or twice daily), and gradually in- creased (individually) over a few weeks to 850–1000 mg 2 to 3 times per day. In a patient with newly diagnosed type 2 diabetes it can be expected that after 2 months of metformin therapy (total dose of 2000 mg daily) the values of gly- cated haemoglobin (HbA1c) and fasting glucose will decrease on average by 1.4 % and 2.9 mmol/l, respectively (41). on line edition Zdrav Vestn | March – april 2017 | Volume 86 Metabolic and horMonal disorders Metformin can be used in combina- tion with all other anti-hyperglycaemic drugs. The drug to be combined with metformin is selected individually for each patient, based on the risk of hypo- glycaemia, weight gain and other side effects and potential contraindications. Metformin is most often used in combi- nation with sulfonylureas or with insu- lin. In the latter case, a 15–25 % reduction in insulin consumption, less weight gain and a lower incidence of hypoglycaemia than in insulin monotherapy can be ex- pected (12). No significant differences in total or cardiovascular mortality were found between patients treated with the combination of metformin and insulin and patients treated with insulin alone. It is important to note that previous studies lasted for too short time period (approx. 6 months on average) to provide suf- ficient data to prove that the treatment would result in decreased mortality (42). Metformin can also be used in combi- nation with DDP-4 inhibitors. This com- bination improves glycaemic control in patients with type 2 diabetes, and is a promising option for providing cardio- vascular protection, mainly due to the si- multaneous and synchronous protective action of both drugs (43). 5.2 Type 1 diabetes mellitus Adding metformin to insulin treat- ment in patients with type 1 diabetes mellitus reduces insulin consumption, however the incidence of hypoglycaemia with combined treatment varies (44,45). According to the meta-analysis by Vella and co-workers a combination of insu- lin and metformin decreases daily insu- lin consumption by 5.7–10.1 units/day, HbA1c by 0.6–0.9 % and total cholester- ol levels by 0.3–0.41 mmol/l. This meta- analysis suggests that metformin reduces insulin consumption and decreases body weight in patients with type 1 diabetes, but that its effect on glycaemic control is relatively small. There is no evidence yet that the use of metformin in patients with type 1 diabetes improves survival or reduces the incidence of chronic diabet- ic complications  (45). Guidelines of the American Diabetes Association (ADA) and the Canadian Diabetes Association (CDA) suggest an off-label indication for treatment with metformin in over- weight or obese patients with type 1 dia- betes mellitus (40,46). 5.2 Gestational diabetes According to the current Slovenian guidelines, insulin represents the first- line therapy for gestational diabetes. Even though previous studies have not confirmed any risk of metformin for pregnant women or foetus, metformin can be prescribed for the treatment of gestational diabetes only in rare cases when treatment with insulin is not pos- sible (4). Metformin crosses the placenta, but its effects on the foetus have not yet been well studied, thus its use in pregnancy is limited  (47). Meta-analyses of observa- tional studies did not provide evidence of increased incidence of foetal malfor- mations or neonatal mortality with the use of metformin in the first trimester of pregnancy. The Metformin in Gestation- al Diabetes (MiG) study did not show any differences in the incidence of foe- tal hypoglycaemia, respiratory distress, need for phototherapy, birth injuries, Apgar score value or prematurity rates between the group of pregnant women treated with metformin and the group receiving insulin. Women with gesta- tional diabetes treated with metformin had significantly higher rates of pre- term births, but the difference between the groups was not clinically important. on line editionMetformin: from mechanisms of action to advanced clinical use Professional article There is a hypothesis that treatment with metformin during pregnancy may have beneficial effects on the metabolic pro- file of offspring, but larger studies are needed to confirm this assertion (48). 5.4 Conditions associated with increased risk for the development of type 2 diabetes mellitus Impaired glucose tolerance (IGT) and impaired fasting glycaemia (IFG) carry an increased risk for the develop- ment of type 2 diabetes mellitus. While IGT is statistically significantly associat- ed with an increased risk for cardiovas- cular events, this was not confirmed for IFG (49). The most convincing evidence that metformin prevents progression of previously described conditions to type 2 diabetes mellitus was provided by the Diabetes Prevention Program (DPP) study, which enrolled 3,234 subjects. Treatment with metformin reduced the incidence of type 2 diabetes mellitus by 31 % compared to placebo; this risk was additionally halved by lifestyle changes. Maximum effect of metformin was seen in the subgroup of individuals under 60 years of age and in those with body mass index over 35 kg/m2 (50). A slightly lesser effect (risk reduction of 18 %) was seen 10 years after the end of the inter- vention. Later meta-analyses of random- ized trials revealed beneficial effects of metformin on slowing the progression of conditions associated with increased risk for the development of type 2 diabe- tes mellitus to a clinically overt disease, even when using metformin at low doses (250 mg twice daily) (51). 6 The role of metformin in decreasing insulin resistance It was shown that metformin de- creases insulin resistance in the liver and skeletal muscles. This beneficial effect of Figure 2: a schematic review of basic anti-hyperglicaemic and additional beneficial (pleiotropic) effects of metformin. Basic effects Decreased insulin resistance Decreased glucose absorption in the gastrointestinal system Increased glucose uptake in skeletal muscles Decreased effect of glucagon Decreased gluconeogenesis Stimulation of GLP-1-mediated insulin secretion Lipogenesis inhibition Pleiotropic effects Antiinflammatory action Antioxidative action Beneficial effects on cardiovascular system Anti-cancer effects Protective effect on central nervous system? Increased lifespan? METFORMIN on line edition Zdrav Vestn | March – april 2017 | Volume 86 Metabolic and horMonal disorders metformin is also important in the treat- ment of non-diabetic patients. 6.2 Polycystic ovary syndrome Insulin resistance is present in poly- cystic ovary syndrome (PCOS). In Slo- venia, metformin is used to treat PCOS although it has not yet been officially approved for the treatment of this con- dition  (52). A meta-analysis of 31 clini- cal trials showed that treatment with metformin in overweight women with PCOS increases the probability of ovula- tion, improves the regularity of menstru- al cycles and reduces the level of serum androgens, but there was no convinc- ing evidence that it increased live birth rates  (53). Its effects are due to the de- creased influence of insulin excess on the ovarian cells, direct effects of metformin on theca and granulosa ovarian cells, diminished oxidation of free fatty ac- ids and reduced secretion of androgens from ovaries and adrenal glands (11). 7 Pleiotropic effects of metformin In addition to its basic anti-hypergly- caemic action, metformin has also ad- ditional pleiotropic effects, that are not mediated directly through the influence on the glucose metabolism (12,29,54,55). These include benefits for cardiovascular system and other organs, as well as an- ti-cancer effects and positive effects on non-alcoholic fatty liver disease. Basic and pleiotropic effects of metformin are summarized in Figure 2. 7.1 Mechanisms of pleiotropic effects Similarly to the basic anti-hypergly- caemic action of metformin, its pleio- tropic effects are AMPK-dependent and -independent. Through the above de- scribed pathways, metformin activates AMPK, which not only controls or in- hibits gluconeogenesis and lipogenesis, but also affects the enzymes and pro- teins involved in cell cycle and protein metabolism. The AMPK-dependent pathway plays an important role in cell proliferation and differentiation  (56). Signalisation via this pathway is impor- tant for the basic anti-hyperglicaemic effects of metformin; it causes phos- phorylation of endothelial nitric oxide synthase (eNOS), which leads to an in- crease in nitric oxide (NO) synthesis. Metformin also phosphorylates eNOS independently of AMPK, via increased activity of the insulin receptor and con- sequent activation of phosphatidylino- sitol-4.5-bisphosphate 3-kinase/pro- tein kinase B (PI3K/Akt) pathway. The increased NO synthesis prevents the opening of the mitochondrial perme- ability transition pore (mPTP) on the mitochondrial membrane and the re- lease of reactive oxygen species (ROS) into the cell – anti-oxidative activ- ity (54). Additionally, metformin inhibits the mechanistic target of rapamycin (mTOR), which is involved in the pro- cess of cell growth via two pathways: (1) directly through AMPK, or (2) through the activation of the PI3K/Akt signal- ing pathway, which is AMPK-indepen- dent. The signaling pathway PI3K/Akt activates the tumour suppressor com- plex of the tuberous sclerosis complex 2 (TSC2), which inhibits the mTOR (Fig- ure 1) (55,56). 7.2 Pleiotropic cardiovascular effects Since cardiovascular diseases re- main the leading cause of morbidity and mortality in patients with diabetes mel- on line editionMetformin: from mechanisms of action to advanced clinical use Professional article litus (52 %), the beneficial cardioprotec- tive activity of metformin represents one of its most important pleiotropic effects  (29,54). The UKPDS study and some other studies have shown that met- formin reduces the risk of cardiovascu- lar disease and total mortality (13,29,57). This is probably not only due to the di- rect effect of metformin on glycaemia, but also to its beneficial pleiotropic ac- tivity. Pleiotropic effects of metformin on the cardiovascular system are mediated through the following mechanisms: 1. Improvement of the endothelial func- tion: metformin improves endothe- lium-dependent vasorelaxation  (58) by increasing eNOS and NO and decreasing sVCAM-1 and E-selec- tin  (59). Metformin also decreases the endothelial and vascular smooth muscle cell death through reduced expression of angiotensin II type 1 re- ceptor gene (60). 2. Effects on the lipid profile: metformin has beneficial effects on serum lipid concentration, since it decreases the concentration of free fatty acids, tri- glycerides, total cholesterol and LDL cholesterol. Some studies also found that metformin could increase HDL cholesterol levels (61). 3. Effects on haemostats: metformin in- hibits coagulation and stimulates fi- brinolysis. In patients with type 2 dia- betes metformin decreased the levels of several coagulation factors, such as von Willebrand’s factor (vWF) and factor VII. In addition it reduced the levels of plasminogen-activator-in- hibitor-1 (PAI-1), which inhibits fibri- nolysis (62). Also, metformin directly affects fibrin structure and function by decreasing factor XIII activity and changing fibrin structure  (12). Met- formin also inhibits thrombocyte ag- gregation (29). 4. Anti-inflammatory activity: metfor- min may inhibit pro-inflammatory responses through direct inhibition of the nuclear factor κB (NF-κB) pathway  (63). Its anti-inflammatory activity may be mediated through sirtuin 1, a protein important in in- tracellular pathways associated with metabolism, stress response, cell cycle and ageing (12). Metformin acts anti- inflammatory by inhibiting tumour necrosis factor α (TNFα) in human monocytes and by decreasing C-reac- tive protein (CRP) (64). 5. Anti-oxidative activity: metformin decreases the production of reactive oxygen species (ROS) in mitochon- dria, inhibits the production of ad- vanced glycosylation end products (AGE) in arterial wall (12,56,65), and decreases the concentration of re- duced glutathione (12). 6. Blood pressure decrease: the influ- ence of metformin on blood pres- sure has not yet been fully explored. In the BIGPRO1 trial, carried out in overweight subjects, blood pressure decreased in the group treated with metformin. Other studies found no- significant effect of metformin on blood pressure. Metformin probably influences blood pressure through its action on vascular smooth muscle cells (12,66). 7. Decreased myocardial ischaemic- reperfusion injury: metformin de- creases the extent of myocardial isch- aemic-reperfusion injury through activation of protein kinase B (Akt) signaling pathway or through AMPK activation. The latter activates eNOS, which prevents the mPTP pore open- ing on the mitochondrial membrane, thus acting in a cardioprotective way (29,54). 8. Effects on the cardiac function: met- formin decreases the incidence of on line edition Zdrav Vestn | March – april 2017 | Volume 86 Metabolic and horMonal disorders chronic heart failure (mostly through AMPK activation and myocardial fibrosis inhibition). Therefore, it is suggested that chronic heart failure should not be an absolute contraindi- cation for metformin treatment (see further text) (67). 7.3 Pleiotropic effects on cancer It was found that patients with type 2 diabetes mellitus may have an in- creased risk of various types of cancer, particularly of liver, pancreas, endome- trium, colon, breast and bladder can- cer  (12,29,55,68), but a decreased risk for prostate cancer  (69). Compared to the general population, patients with diabetes mellitus have increased mor- tality due to malignant diseases  (12). Treatment with metformin may lower mortality due to malignant diseases and decrease cancer incidence (by 25–30 %), this effect being more pronounced with higher metformin dosages (55,68,70). In vivo studies showed greater cytotoxicity with the combination of metformin and different cytostatic agents. Radiotherapy was more effective in patients treated with metformin (56). Increased incidence of malignant diseases in diabetic patients is probably the consequence of increased insulin concentration, increased insulin resis- tance, hyperglycaemia and increased concentration of IGF-1 (and indirect activation of mTOR via PI3K/Akt path- way) (12,71). The proposed mechanisms of metformin anti-cancer properties are not fully understood. They are mainly mediated through the AMPK-depen- dent and AMPK-independent path- ways  (12,29,55,56). Through the AMPK pathway, metformin inhibits the synthe- sis of fatty acids, cholesterol, mTOR ac- tivity, stimulates the tumour suppressor gene p53 activity and decreases the con- centration of c-myc oncogene, which is one of the crucial factors for the growth of malignant cells. Independently of AMPK, metformin also inhibits mTOR, chronic inflammation and the forma- tion of oxygen free radicals that would otherwise stimulate the development of malignant cells  (29,55). Some stud- ies have shown that the anti-cancer ef- fect of metformin is mediated through microRNAs (miRNA), key regulators of many biological processes, such as cell proliferation, differentiation, apoptosis, stress response and angiogenesis  (56). Modulation of miRNAs may play an important role in the above mentioned processes. MiRNAs often behave as tu- mour suppressors or oncogenes, thereby either promoting or inhibiting the pro- gression of malignant cells. Anti-cancer activity of metformin could be mediated through the stimulation or inhibition of different miRNA subtypes  (56,72). The body of available data is large, so we would refer only to the following find- ings: metformin stimulated the expres- sion of miRNA-33a in breast cancer and therefore inhibited cell proliferation through the inhibition of c-myc onco- gene  (73); metformin increased the ex- pression of miRNA-192 and miRNA-26a, which resulted in the apoptosis of malig- nant cells in pancreatic tumour (74); by inhibiting the miRNA-222 expression in lung cancer, metformin increased the ac- tivity of p27 and p57 tumour suppressor genes (75). Some evidence exists to sup- port anti-cancer effects of metformin via miRNAs in other types of cancer (56,72), but a detailed description would go be- yond the scope of this manuscript. Several studies have shown that met- formin decreases the incidence of various types of cancer (76-80). In patients with type 2 diabetes treatment with metfor- min reduced the incidence and mortality of colorectal carcinoma (79,81). In female on line editionMetformin: from mechanisms of action to advanced clinical use Professional article patients with type 2 diabetes metformin lowered the incidence of breast cancer. In women with breast cancer metfor- min decreased the incidence of metas- tases  (77,78). In addition, women with breast cancer who received metformin plus neoadjuvant chemotherapy had a higher disease remission rate than women not treated with metformin (82). Metfor- min extended the survival rate in patients with pancreatic cancer  (83). Metformin inhibited the growth of pancreatic cancer cells by its direct influence on fatty acid synthesis (84). It also decreased the pro- gression of renal cell carcinoma (85,86). Treatment with metformin reduced the incidence and progression of prostate cancer,  (87) primarily by reducing c-myc protein  (88) levels and through IGF-1 reduced the formation of androgens. It may act synergistically with anti-andro- gen drugs commonly prescribed for the treatment of metastatic prostate carci- noma (56). Despite extensive evidence of metformin effectiveness in diabetic pa- tients with malignant diseases, the use of metformin for this population has not yet been established in clinical practice. 7.4 Pleiotropic effects in non- alcoholic fatty liver disease Currently, the best treatment op- tions for non-alcoholic fatty liver dis- ease (NAFLD) include weight reduction, regular physical activity and healthy diet (89). The effectiveness of metformin in decreasing the content of liver fat was shown in a mice model with NAFLD, while clinical findings are opposing. One of the possible reasons is the small num- ber of patients included in the research. However, even in the larger TONIC clinical trial, the efficacy of metformin in patients with NAFLD without con- comitant diabetes mellitus was not con- firmed (90). 7.5 Pleiotropic effects on other organs Metformin was shown to decrease se- rum levels of thyrotropin (TSH) (91). It influences redistribution and decrease of body fat (12). Non-clinical studies on nematodes and rodents have confirmed that met- formin extended their life span  (92,93). There are no clinical studies that would confirm the direct effect of metformin on ageing or on increasing life span. The UKPDS study showed that treat- ment with metformin reduced the risk of death from diabetes by 42 %, and de- creased total mortality by 36 % (13). Moreover, in vitro studies showed that metformin could increase neuro- genesis, improve neuron activity and decrease their degradation  (94). Fur- ther clinical research in this field is re- quired. 8 Metformin adverse reactions 8.1 Gastrointestinal adverse reactions The most common adverse effects of metformin occur in the gastrointestinal system, and include flatulence, cramps, diarrhoea, nausea and vomiting. Rarely, it causes a metallic taste in the mouth. Gastrointestinal adverse reactions are usually caused by fast titration of the drug or initial high dose regimens. Usu- ally, these effects disappear with time. They usually disappear with dose reduc- tion or after changing drug formulation to XR or DR option (12). 8.2 Vitamin B12-associated adverse reactions In addition to causing gastrointesti- nal adverse reactions, metformin may on line edition Zdrav Vestn | March – april 2017 | Volume 86 Metabolic and horMonal disorders diminish vitamin B12 absorption in the gut. Consequently, patients with type 2 diabetes treated with metformin may have diminished levels of vitamin B12. Vitamin B12 levels reduction is de- pendent on the cumulative metformin dose  (95). Clinical significance of these findings is still a matter of debate as only several studies in this field showed asso- ciation with the development of mega- loblastic anaemia (96), cognitive decline and advancement of peripheral diabetic neuropathy (97). Because of the high vi- tamin B12 availability in the organism, B12 deficiency usually becomes appar- ent only after several years of metformin use. Monitoring vitamin B12 levels and substituting B12 in case of diminished availability are recommended. Preven- tive substitution is not recommended at the moment (95). 8.3 Metformin-associated lactic acidosis Metformin-associated lactic acidosis (MALA) is a rare, but potentially fatal adverse effect associated with metformin treatment. Its incidence is estimated to be around 1/23,000–30,000 patient-years. Interestingly, the incidence of lactic aci- dosis in diabetic patients treated with other oral anti-hyperglycaemics with- out metformin, is estimated at 1/18,000– 21,000 patient-years, and is significantly higher than with metformin (98). Metformin-associated lactic acidosis is called high anion gap acidosis with lactate levels > 5 mmol/l and pH ≤ 7.35. Severe acidosis is associated with multi- organ failure, characterized by neuro- logic signs (stupor, coma, convulsions) and cardiovascular manifestations (hy- Table 1: recommended metformin dose adjustments according to the stage of chronic kidney disease (4,104,105). Chronic kidney disease stage eGFR (ml/min/1,73 m2) Maximum total daily metformin dose (mg) Other recommendations 1 ≥90 3000 2 60–89 3000 3a 45–59 2000–3000 • safe use at full dose, unless worsening of kidney function suspected • frequent follow-up of kidney function advised 3b 30–44 1000 • safe use in adjusted dose • continue already initiated drug use • cessation of metformin use, if worsening of kidney function suspected • Very frequent follow-up of kidney function recommended 4 metformin contraindicated 5 metformin contraindicated eGFR – estimated glomerular filtration rate on line editionMetformin: from mechanisms of action to advanced clinical use Professional article potension, heart rhythm disturbances). Mortality is very high, and is estimated at 30–50 % (99). Pathophysiologically, there are two types of lactic acidosis. Type A lactic aci- dosis is due to overproduction of lactate, occurring to restore cellular energy lev- els (in the form of ATP) under anaerobic conditions (without oxygen). This type of lactic acidosis can be seen in states of circulatory collapse, such as severe heart failure, sepsis, and other states of shock. Type B lactic acidosis, on the other hand, is a consequence of underutilisation of lactate due to its abnormal removal through oxidation and gluconeogenesis under aerobic conditions. Type B lactic acidosis is seen in liver failure, diabetes mellitus, cancer, and intoxications with alcohol or metformin. A combination of both type A and B lactic acidosis also ex- ists (99,100). The exact mechanism of MALA is not clear yet. It can usually be seen in patients with pre-existent chronic kid- ney disease, advanced heart failure, or other chronic conditions, that could lead to the lactate acid forming state. In mitochondria, lactate is oxidised into carbon monoxide and water, resulting in cellular energy generation. Addition- ally, lactate can be metabolised back to glucose through gluconeogenesis in the liver and, to a lower extent, in the kidneys. Metformin inhibits the mito- chondrial respiratory chain in the liver and muscles, responsible for lactate re- moval. Therefore, this inhibition leads to accelerated lactate formation and, on the other hand, diminishes its remov- al (99,100). MALA is primarily treated with haemodialysis that allows excess drug removal and acid-base balance regula- tion (99,100). 9 Risk factors for the development of metformin- associated lactic acidosis and the use of metformin in states predisposing to increased lactic acid production 9.1 Chronic kidney disease Ninety percent of metformin is ex- creted unchanged by the kidneys. There- fore in chronic kidney disease (CKD) it can start accumulating in the body, which leads to unacceptable over-therapeutic levels in plasma. This causes increased inhibition of gluconeogenesis and mito- chondrial respiratory chain, leading to a higher risk of lactic acidosis. Therefore, CKD stage 3 or higher (eGFR ≤ 60 ml/ min/1.73 m2) represents a relative con- traindication for metformin use. Nev- ertheless, evidence is accumulating that metformin treatment in CKD patients is safe (101). The Cochrane analysis of 347 controlled trials with more than 70,490 patient-years of metformin use reported no MALA or significant increase in lac- tate levels. In 43 % of those trials, CKD was not a contraindication for metfor- min treatment  (102). Another analysis of the Swedish registry of patients with type 2 diabetes including more than 50,000 patients showed that metformin treatment was safe even in patients with eGFR as low as 30 ml/min/1.73 m2 (103). Table 1 presents the recommended met- formin dose adjustments based on these data (4,104,105). 9.2 Heart failure In view of the accumulating evidence on a very low incidence of lactic acido- sis in patients with both type 2 diabetes on line edition Zdrav Vestn | March – april 2017 | Volume 86 Metabolic and horMonal disorders and heart failure treated with metfor- min  (102), FDA dismissed heart failure as an absolute contraindication for met- formin treatment (106). At least five ran- domized trials showed favourable out- come in heart failure in diabetic patients treated with metformin (107-109). Con- sequently, metformin treatment is rec- ommended in patients with stable heart failure regardless of its stage (NYHA I-IV), as long as their kidney function is stable or they do not have additional risk factors for acute deterioration of heart failure or lactic acidosis (110). 9.3 Stable coronary artery disease and acute coronary syndrome Treatment with metformin may have beneficial cardiovascular effects in diabetic patients with stable coro- nary disease, as revealed by several tri- als  (13,29,57,111). Similar findings were reported for diabetic patients with acute coronary syndrome. In the latter, treat- ment with metformin was associated with better short- and long-term prog- nosis, as compared to patients treated with other anti-hyperglycaemic drugs. Consequently, treatment with metfor- min is not contraindicated in diabetic patients with either stable coronary dis- ease or acute coronary syndrome, as long as they are at low risk for cardiogenic shock in case of an acute event (112). 9.4 Liver failure Advanced decompensated liver cir- rhosis and severe liver hypo-perfusion represent absolute contraindications for metformin treatment in diabetic patients, even though there has been no research in this field (112). Yet in 100 patients with type 2 diabetes and liver cirrhosis due to hepatitis C followed up for 5.7 years, a statistically significant reduction in the incidence of hepatocellular carcinoma and mortality from liver disease was found  (113). These results suggest that metformin can be used in diabetic pa- tients with liver cirrhosis. Additionally, treatment with metformin is possible in patients with non-alcoholic fatty liver disease and non-alcoholic steatohepati- tis, although no significant reduction of liver fat content can be expected in these patient groups (112). 9.5 Chronic respiratory failure Advanced respiratory failure may lead to hypoxic states and consequent accel- erated glycolysis and lactic acid forma- tion. In this field, there have been no tri- als that would identify the risk of MALA development in patients with diabetes. Consequently, metformin treatment is contraindicated in patients with diabetes mellitus and advanced chronic respira- tory failure  (112). Metformin treatment is not started in this patient population, but patients already on chronic metfor- min therapy can continue receiving this medication (110). 9.6 Elderly patients The use of metformin in diabetic pa- tients of advanced age has proved effec- tive and well tolerated by the patients. Moreover, elderly patients treated with metformin had no hypoglycaemic epi- sodes. Yet caution with the use of met- formin is advised in frail diabetic pa- tients with low body/muscle mass or with other relative contraindications for treatment with this drug (114). on line editionMetformin: from mechanisms of action to advanced clinical use Professional article 10 Use of metformin in patients undergoing intravascular iodinated contrast –enhanced imaging investigations Exposure of patients to intravas- cular iodinated contrast agents used for imaging investigations may lead to acute kidney injury, i.e. the so-called contrast-induced nephropathy. Its inci- dence is dependent of the stage of the pre-existent CKD and the amount of the iodinated contrast used. Also, the latter can accumulate in case of acute kidney injury occurring in patients treated with metformin. Since MALA is a relatively rare phenomenon, the previously strict guidelines on interruption of metfor- min therapy before intravascular ad- ministration of iodinated contrast agent were softened. The European guidelines issued by the European Society of Uro- genital Radiology in 2013 recommend that in metformin-treated diabetic pa- tients with CKD the following protocol should be followed before performing intravascular iodinated contrast-en- hanced imaging (115): • Patients with eGFR ≥ 60 ml/min/1.73 m2 (CKD stages 1 and 2) can con- tinue taking metformin, i.e. stopping the metformin treatment before and after the investigation is not neces- sary. In patients with eGFR 30–59 ml/ min/1.73 m2 (CKD stage 3), decision on metformin use is based on the fol- lowing: (a) with intravenous adminis- tration of iodinated contrast and if the patients have eGFR ≥ 45 ml/min/1.73 m2, metformin treatment discontinu- ation is not necessary; (b) with intra- arterial administration of iodinated contrast or if the patients have eGFR 30–44 ml/min/1.73 m2, metformin is stopped 48 hours before the investi- gation. In these patients, metformin should be restarted at least 48 hours after the investigation, if the kidney function remains stable. • In patients with eGFR ≤ 30 ml/ min/1.73 m2 (CKD stages 4 and 5) or with a disease that may lead to acute liver failure or a hypoxic state, met- formin treatment is contraindicated. In emergency situations, metformin treatment is discontinued before in- travascular iodinated contrast ad- ministration; after the investigation, the patient is monitored for possible MALA development. In case of sta- ble kidney function and absence of MALA, metformin can be reintro- duced 48 hours after the investigation. With intravascular use of gadolini- um contrast agent metformin discon- tinuation is not necessary at any CKD stage (115). In addition to the above described guidelines, all additional measures used in the prevention of contrast-induced nephropathy should be followed, in par- ticular good hydration of patients with CKD. 11 Conclusion Metformin has been on the market for almost 60 years, and thanks to its efficacy and safety it remains the gold standard and the first-choice therapy for patients with type 2 diabetes melli- tus. There is a plethora of evidence for its anti-hyperglycaemic action and its ben- eficial effects in other diabetes mellitus- associated and insulin-resistant states. Metformin provides additional, benefi- cial pleiotropic effects, the best described being cardioprotective and anti-cancer effects. Additionally, it shows promise in treating degenerative diseases of the central nervous system and, perhaps, has a role in slowing the ageing. Metformin on line edition Zdrav Vestn | March – april 2017 | Volume 86 Metabolic and horMonal disorders has a relatively low incidence of adverse reactions, the most common being gas- trointestinal side effects. A relatively rare, but most dangerous complication is the development of metformin-as- sociated lactic acidosis associated with pre-existent chronic kidney disease or advanced heart failure with tissue hy- po-perfusion. Recently, there has been evidence that metformin can safely be used in patients undergoing intravascu- lar iodinated contrast-enhanced imag- ing studies even in those with advanced chronic kidney disease. Thanks to the above described anti-hyperglycaemic and other beneficial effects, along with its proven efficacy and safety, metformin has a bright future. References 1. 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