Marta Patrycja Wróbel M.D., Ph.D., Department of Internal Medicine, Diabetology and Cardiometabolic Disorders, Silesian Centre for Heart Diseases, Medical University of Silesia, Curie-Skłodowskiej St. 9, 41–800 Zabrze, Poland, tel.: +48 32 373 38 23, e-mail: mwrobel@sum.edu.pl

Introduction

Since the late 1990s metformin is the gold standard in the treatment of type 2 diabetes. According to recommendations of the most important diabetes associations, this is the first-choice drug for use as monotherapy in patients with newly diagnosed type 2 diabetes. Metformin is also recommended in combined treatment when monotherapy is no longer effective. It is then combined with a sulfonylurea, an incretin, flozin, or insulin, irrespective of the number of insulin injections per day [1, 2]. Metformin is the only commonly economically available product that, besides the glucose lowering effect, has a favourable effect on the body weight. This is important because the diabetic population are usually overweight. Besides its properties used in the treatment of diabetes, metformin has been treated for some time as a drug of a socalled pleiotropic activity, as each year brings new reports about its favourable effect in different conditions [3, 4]. At present, the scope of reimbursed indications of this drug has been expanded to include prediabetes, insulin resistance syndromes, and polycystic ovary syndrome.

Metformin’s mechanism of action

Metformin does not stimulate insulin secretion by the beta cells of the pancreas, and thus it is a drug that does not cause hypoglycaemia. The blood glucose-lowering effect of the drug is a consequence of hepatic glucose production inhibition and of peripheral tissue (muscle tissue, fatty tissue) sensitisation to the effect of insulin, of both endogenous and exogenous origin. The exact mechanism of metformin action at the cellular level remained unknown for a long time. Studies performed in recent years provided much information that enables better understanding of the mechanism of action of the drug as well as the clinical effects resulting from its use. The current knowledge in this field is presented below.

• Metformin interferes with cellular respiration by inhibition of mitochondrial respiratory chain complex I (mitochondrial stress), which leads to a decrease of intracellular ATP, which imitates a cell starvation condition. As a result, the AMP pool increases and this stimulates the AMP-activated protein kinase (AMPK) that is a so-called cellular energy sensor. AMPK is activated even at a minor ATP deficit. This leads to “downregulation” of all metabolic pathways that consume energy – proliferation, and protein and lipid synthesis [5]. ATP production increases, in turn, through an increased uptake of glucose from the blood, increased glycolysis (and thus increased glucose utilisation and a decrease of its blood level), and fatty acid beta-oxidation. AMP molecules activate AMPK directly through binding to the gamma-subunit of the enzyme or indirectly through inhibition of its dephosphorylation. Stimulation of the AMPK increases also translocation of glucose transporters – GLUT – towards the cell membrane, and intensifies glucose transport into the cell (increased glucose utilisation) (Fig. 1, Table I) [6];

• Based on the results of recent studies, metformin appears to act also independently from AMPK activation. By inhibiting mitochondrial respiratory chain complex I, metformin leads to production of reactive oxygen species (ROS) and generates a so-called integrated stress response (ISR) via activation of transcription factor 4 (ATF-4), which activates fibroblast growth factor-21 (FGF-21) [7, 8]. This pathway is completely independent of AMPK, which was confirmed in studies conducted on a culture of fibroblasts from rodents deprived of the AMPK gene. Until recently, FGF-21 produced by hepatic cells was considered to be a factor involved only in glucose and lipid metabolism, but it appears to have also a role in the mechanism of action of metformin. FGF-21 level was observed to change in people treated with metformin [9];

• Metformin is widely known to inhibit hepatic gluconeogenesis [10]. However, the mechanism of action of this drug in this respect has been understood only recently. There are data suggesting that metformin inhibits mitochondrial glycerophosphate dehydrogenase (mGDP) – a key enzyme linking metabolic pathways of carbohydrates and lipids (without affecting the cytosolic glycerophosphate dehydrogenase; cGDP) [11]. In normal conditions, this pathway allows for entrance of the reduced form of NAD (NADH+), being the product of cytoplasmic glycolysis, into the mitochondria, where ATP production and cytoplasmic NAD+ regeneration occurs. Inhibition of mGDP hampers conversion of glycerol-3-phosphate to dihydroxyacetone phosphate (DHAP), thus inhibiting gluconeogenesis from glycerol. As a result of decreased NAD+ level (oxidised form of NAD) lactate cumulates as its transformation to pyruvate (a reaction catalysed by lactate dehydrogenase) is impaired in such conditions, and additionally hepatic gluconeogenesis is inhibited [10] (Fig. 1). Lactate accumulation at the cellular level is a typical phenomenon during metformin therapy; however, its amount is harmless if the drug is used after consideration of contraindications (Table III);

• Another explanation of metformin’s inhibitory effect on hepatic gluconeogenesis was published a few years ago in “Nature”. Glucagon is also responsible for hepatic gluconeogenesis. Excessive glucagon secretion by pancreatic alpha cells in type 2 diabetic patients is one of the elements of complex pathogenesis of this type of disease. Glucagon, by binding to a receptor on the hepatocyte activates the adenylate cyclase and thus promotes cyclic AMP (cAMP) formation, which stimulates protein kinase A (PKA), leading to hepatic glucose production. The authors of this work state that metformin weakens the effect of glucagon after binding to the receptor, through reduction of cAMP production in liver cells. This is because metformin, which, by inhibiting mitochondrial respiratory chain complex I, leads to AMP accumulation, which in turn inhibits adenylate cyclase, and thus cAMP production. A decreased amount of cAMP inhibits the activity of PKA and thus diminishes hepatic gluconeogenesis [12] (Fig. 1).
• Additionally, metformin induces secretion of glucagon-like peptide-1 (GLP-1) by L-cells of the small intestine and sensitises beta cells of the pancreas to its action, among others by affecting the expression of the GLP-1 receptor on these cells [13, 14]. GLP-1 is an incretin hormone, whose deficiency or impaired activity plays an important role in the pathogenesis of type 2 diabetes. In physiological conditions, GLP-1 is responsible for increased insulin secretion by the beta cells of the pancreas in response to food ingestion. GLP-1, by binding to the relevant receptor on the beta cell (other than sulfonylurea receptor), stimulates an insulin secretion adjusted to the blood glucose level, thus not generating hypoglycaemia. Due to treatment safety (a minimal risk of hypoglycaemia), among others, the drugs improving the incretin effect that is impaired in diabetes (so-called incretins) are more and more widely used in the treatment of type 2 diabetes. They include GLP-1 analogues (agonists of the GLP-1 receptor on the beta cells of the pancreas) and inhibitors of dipeptidyl-peptidase 4 – the enzyme responsible for GLP-1 breakdown. A combination of an incretin and metformin is therefore fully justified, taking into consideration the confirmed synergism of action of these drugs;
• Metformin is also active within the intestines, where it increases glucose uptake and utilisation. It was found that the metformin level in the intestinal mucosa reaches the highest values, as compared to other tissues, which may explain the occurrence of gastrointestinal adverse effects with the use of this drug in some patients [15, 16]. Additionally, metformin accumulation in the intestinal mucosa may interfere with FDG (18F-deoxyglucose) PETCT image assessment (see below). Furthermore, metformin use is associated with a change in intestinal flora composition, and in particular with and increased amount of a Gram-negative anaerobic bacteria – Akkermansia muciniphila (belonging to Verrucomicrobia), residing in the intestine, within the mucous membrane. These bacteria are specialised in the degradation of mucin (a glycoprotein found in the mucus). According to the most recent data, the Akkermansia bacteria not only improve the function of the intestinal barrier but they also increase the number of regulatory T cells (Treg) and reduce mild inflammation present in the fatty tissue of obese experimental animals [17, 18];
• Metformin, via activation of AMPK, followed by phosphorylation of the Unc-51-like kinase 1 (ULK-1) and beclin 1, also initiates autophagy [19]. Autophagy is a katabolic process during which macromolecular components of the cytoplasm are destroyed. Initially, part of the cytoplasm is surrounded by a double membrane that finally forms a follicle – autophagosome. The autophagosome unites with a lysosome and is digested. Autophagy is necessary for the maintenance of the intracellular balance, thus enabling survival of the cells under stress conditions. It seems that metformin-induced increased autophagic activity may result in an improvement of the metabolic profile in stress conditions, and this may lead, among others, to reduction of the mild inflammation that accompanies obesity;
• Some data suggest that this drug prolongs the survival of Caenorhabditis elegans (a type of nematode), which probably is the result of metabolism of folates and methionine changed by metformin (reduced methionine availability in the intestine). Similar observations were made with experimental rodents [20, 21].

Clinical effects during metformin use in diabetic patients

From the clinical point of view, it is important that metformin reduces the demand for insulin (including the exogenous forms) by improving sensitivity of peripheral tissues to this hormone and by reducing hepatic glucose production. Metformin does not stimulate hypoglycaemia, so it is a safe drug in this respect. This is particularly important in patients with cardiovascular risk, in whom stress reaction of the body in response to glucose level decreased below the recommended range may trigger myocardial infarction or dangerous arrhythmias. Metformin is a completely safe drug if it is used in patients without contraindications to this treatment (Table III).

Metformin has also a number of proven favourable effects other than carbohydrate metabolism, which is very important in the case of diabetic patients, who often have macroangiopathic complications (atheromatosis and its consequences).

Additional effects of metformin:

• neutral effect on the body weight or its reduction, primarily through reduction of the visceral fatty tissue [22];
• cardioprotective: in the studies of UKPDS and UKPDS 10 years later, metformin use was associated with a 30% reduction of the risk of myocardial infarction. This drug has a favourable effect on the vascular wall through activation of the endothelial NO synthase. It has also a favourable influence on clotting system parameters: plasminogen activator inhibitor 1 (PAI-1) and von Willebrand factor levels are decreased, and the activity of the tissue activator of plasminogen is increased [23, 24].
• improvement of lipidogram: decreased LDL cholesterol and TG levels, increased HDL cholesterol level [4];
• probable anti-cancer effect: AMPK activated with participation of metformin inhibits the mTOR (a threonine-serine protein kinase), which leads to inhibition of proliferation of tumour cells and thus inhibits its growth [25]. Ongoing clinical studies (about 100) will evaluate the effect of metformin on the survival of cancer patients, both with local and with metastatic disease. There are works suggesting a benefit from use of metformin as an adjuvant therapy during chemotherapy [26]. The use of metformin in diabetic patients, irrespective of whether the purpose of drug administration is only diabetes treatment or the drug is used as an add-on therapy in cancer patients, may affect interpretation of the PET scan (positron emission tomography) and thus the staging of the neoplastic disease (see below).

The effect of metformin on PET imaging

Positron emission tomography (PET) is at present a basic tool in molecular imaging because it provides information about metabolic processes and organ function. PET in combination with computed tomography (PET-CT) allows imaging of both anatomy (computed tomography assures a high spatial resolution) and physiology (functional imaging) owing to fusion of PET and CT images with use of the appropriate software. The capabilities of the positron emission tomography are currently used mainly in oncology, but also in neurology (Alzheimer’s disease, epilepsy), cardiology (diagnosis of coronary artery disease), and in imaging of infections and inflammatory conditions (fever of unknown background, immunocompromised patient). PET imaging uses various radiopharmaceuticals that are metabolic molecules and are labelled with radioactive isotopes emitting positrons during their decay (particles of the same weight as electrons but positively charged). Fluor 18 (F-18) is currently most commonly used for labelling. A PET gamma camera detects photons of gamma radiation created during annihilation of positrons and electrons, and the picture is reconstructed based on this phenomenon. Glucose labelled with radioactive fluorine is a metabolic tracer widely used in the assessment of neoplastic processes with PET (synonyms: FDG, 18F-FDG, 2 [fluorine-18] fluoro-2-deoxy-d-glukose). It is a glucose analogue with a tendency for higher accumulation in the tissues with increased metabolic activity, e.g. cancer cells (enhanced mitosis, increased activity of glucose transport mechanisms). FDG-glucose is taken up by GLUT transporters in the cell membrane (competing with glucose) and after it gets into the cells and gets phosphorylated (to 6-phosphate FDG) during the first reaction of the Krebs cycle, and is captured in the cells because it is not further metabolised as “normal” glucose. Owing to this fact, FDG concentration in some tumours is higher than in a healthy tissue. FDG is an unspecific tracer, so it accumulates in the region of the highest metabolic activity (benign and malignant tumours, inflammatory foci) [27]. Therefore, differentiation of whether increased tracer uptake is related to a neoplastic process or not remains a problem in PET image interpretation. Furthermore, FDG distribution in the body is affected, among others, by metabolic activity of the tissues – their blood supply and glycolysis rate (brain accumulates more FDG), blood glucose and insulin levels, or muscle activity. As presented above, metformin use is associated with AMPK activation, and some metabolic processes are down-regulated and others are activated. This drug also affects the insulin level. Based on previous data, it is known that changes in glucose metabolism and proliferative activity of the cells may affect FDG uptake. There are not many publications concerning the effects of glucose metabolism modulators, including metformin, on PET-CT imaging in cancer patients. One of these works showed the presence of a reversible increase of intestinal FDG uptake during metformin therapy, which significantly affected interpretation of the image. According to the authors of this work, the diagnostic effectiveness of PET-CT scans is particularly limited by intense diffuse 18F-FDG uptake within the colon, and to a lesser extent – within the small intestine. This may mask the presence of a neoplastic process within the gastrointestinal tract and the actual response of the body to chemotherapy [28]. A recent study, in which human and murine colorectal cancer cells incubated with metformin were assessed, has shown that the effect of this drug on 18F-FDG uptake by the tumour varies from the effect of other, typical chemotherapeutic agents. Under the influence of metformin, cellular uptake of 18F-FDG increases (the patient may be considered a non-responder), and then, due to the death of tumour cells (the result of metformin/chemotherapy administration), 18F-FDG uptake decreases. Such a two-phase response may interfere with the assessment of the efficacy of anticancer therapy. On the other hand, one should bear in mind that even if the volume of the tumour decreases, increased 18F-FDG uptake (due to chronic activation of AMPK) may impede accurate assessment. Use of a proliferation marker, 18F-tymidine (18F-FLT), instead of a cell metabolism marker, such as 18F-FDG, seems to be a better solution for the assessment of treatment efficacy in patients using glucose modulators, such as metformin. The uptake of this tracer correlates well with tumour response to metformin – decreased tumour proliferation = decreased 18F-FLT accumulation [29]. This tracer is not commonly used. In the face of the fact that currently FDG is usually used in PET scans, and metformin is the most commonly prescribed anti-diabetic drug, it should be discontinued before the examination so that it does not interfere with image interpretation. There is no unequivocal opinion among researchers as to a specific wash-out period; however, some authors suggest that metformin should be discontinued in oncological patients at least 48 hours before the scan [28].

As well as the effect of metformin on AMPK and thus on tumour proliferation, it seems that this drug may show anti-cancer activity also through its effect on insulin levels. In a study using murine colorectal cancer cells, exposition to metformin had no effect on the insulin level or FDG uptake by tumour cells in normoinsulinaemic mice, but a decrease of insulin levels and FDG uptake in mice with baseline hyperinsulinaemia was found. The authors suggest that the above effect of metformin use should be taken into consideration in the evaluation of its anticancer activity because the response may depend on whether we have a patient with hyperinsulinaemia and an insulin-sensitive tumour or not [30]. Further improvement of understanding of the effect of metformin (as a drug used in chronic treatment of diabetes or as adjuvant therapy in clinical studies in oncological patients) on FDG uptake and PET image interpretation is necessary. Furthermore, based on the information presented above, a question arises whether FDG is an appropriate marker in the assessment of anticancer activity of metformin [31].

Established clinical applications of metformin other than type 2 diabetes

Prediabetes

Before overt diabetes appears, frequently for many years we have to do with a minor elevation of glycaemia values resulting from growing resistance to insulin and concomitant compensatory hyperinsulinaemia. This is so-called prediabetes. Prediabetes is defined as: impaired fasting glucose (IFG) (glucose levels: 100–125 mg/dL) or impaired glucose tolerance (IGT) diagnosed based on the result of the oral glucose loading test 75 g (glucose levels at two hours: ≥ 140 and ≤ 199, according to the WHO), or both these abnormalities at the same time. Impaired fasting glucose and impaired glucose tolerance are not benign conditions. Patients with these abnormalities have a high risk of both type 2 diabetes development and cardiovascular complications. According to recent data, prediabetes affects 344 million people all over the world, and this number is steadily increasing [32].

Benefits from metformin use in people with prediabetes were shown in a three-year study: the Diabetes Prevention Program (DPP). The use of metformin reduced the risk of type 2 diabetes occurrence by 31%, and change of the lifestyle alone (without medication) by as much as 58%. Body weight reduction was also highest in the behavioural therapy group (–5.6 kg), and patients using metformin (at the dose of 2 × 850 mg/24 h) reduced their body weight by 2 kg, as compared to a reduction by 0.1 kg in the placebo group [33]. By inhibiting hepatic glucose production, which takes place mainly during the night hours, metformin improves fasting glycaemia, which translates into a reduction of cardiovascular complications. There is also data suggesting that use of metformin in patients with IGT is associated with reduction of blood pressure and of hs-CRP levels [34]. According to the guidelines of the Polish Diabetes Association, people diagnosed with prediabetes (IFG or IGT) should be instructed to reduce their body weight and to increase physical activity. Additionally, the start of metformin should be considered as a prevention of type 2 diabetes occurrence in people with high risk of its development, in particular in patients with cardiac risk factors unable (unwilling) to modify their lifestyle.

Insulin resistance and metabolic syndrome

Insulin resistance means the absence of appropriate response of the body to insulin. Insulin resistance may precede development of type 2 diabetes by many years. This is a condition where both endogenous and exogenous insulin do not have an appropriate effect on glucose uptake and utilisation, as they would in healthy people.

Insulin resistance is promoted, among others, by disturbances of expression of proteins involved in the insulin action pathway and visceral obesity. Adipocytes of the visceral fatty tissue are large and have a higher metabolic activity. They are also less sensitive to insulin [35]. The most important feature of insulin resistance of the fatty tissue is reduced inhibition of lipolysis by insulin, which results in increased levels of free fatty acids. The excess of free fatty acids reduces muscle glucose uptake and leads to disturbances in insulin signalling. Sensitivity to insulin decreases also naturally along with advancing age, probably as a result of the increase in the amount of the visceral fatty tissue and of the levels of proinflammatory factors as well as triacylglycerol deposition in cells [4]. Insulin resistance may lead to development of prediabetes, type 2 diabetes, metabolic syndrome, and PCOS. Insulin resistance/overt type 2 diabetes is one of the components of metabolic syndrome [36, 37]. Based on data regarding the pleiotropic activity of metformin, its use in the above conditions is highly justified. By reducing insulin resistance and thus reducing compensatory hyperinsulinaemia, metformin is the chance to delay type 2 diabetes onset.

Insulin resistance may also be of secondary nature and it may occur in the course of such conditions as: liver cirrhosis, cancers, autoaggressive diseases, uraemia, and endocrinopathies: Cushing syndrome, acromegaly, or pheochromocytoma [4].

Not only an excess but also a deficit of fatty tissue leads to insulin resistance, which is observable in lipodystrophy (lipoatrophy) syndromes. In these conditions metformin is also used [38, 39].

Polycystic ovary syndrome

A compensatory hyperinsulinaemia, secondary to insulin resistance, occurs usually in PCOS, which seems to be important for hyperandrogenism development because it leads to stimulation of the cells of the follicles that express receptors for IGF-1 and insulin. Insulin may modify LH receptors on the thecal cells of the follicles, thus contributing to increased androgenesis. Consequently, resistance of peripheral tissues to insulin and increased sensitivity of the ovary to insulin occur concurrently. Besides, hyperinsulinaemia decreases the levels of the IGF binding protein (IGFBP-1), which results in increased levels of available IGF-1, which enhances LH stimulation of the ovary to androgen production [40]. Increased levels of insulin are also associated with decreased levels of sex hormone binding globulin (SHGB), which results in an excess of active testosterone. It was shown that insulin resistance affects not only obese but also slim women with PCOS [41, 42]. Patients with polycystic ovary syndrome have an increased risk of development of metabolic syndrome and its consequences, in the form of cardiovascular complications, prediabetes, and type 2 diabetes. Metformin has had its place in the treatment of polycystic ovary syndrome for many years. This drug, by reducing insulin resistance in the PCOS patients, has a favourable effect on fertility, regulates menstrual periods, decreases hirsutism, and may lead to body weight reduction [42]. Metformin is also related with significant decreases in proinsulin and androstenedione levels in obese or overweight PCOS patients [43]. Additionally, it improves lipid metabolism parameters and lowers CRP levels. Combined treatment with clomiphene citrate and metformin improves ovulation frequency and thus increases the number of pregnancies. So this drug is used both in the treatment of PCOS symptoms and in the treatment of infertility [44]. Decreased incidence of spontaneous abortions, obstetric complications, and of congenital defects in the foetus were found in PCOS patients using metformin before and during pregnancy, as well as a lower risk of gestational diabetes mellitus (GDM) development [45, 46]. It should be stressed that metformin use does not release PCOS patients from the necessity to modify their lifestyle (appropriate diet and physical activity).

Conclusions

Metformin is the most frequently prescribed oral antidiabetic drug in the world. Its position in the treatment of type 2 diabetes has been established for over two decades. For many years this drug has also been used in the treatment of prediabetes, of the polycystic ovary syndrome, and of insulin resistance conditions. Additional, favourable effects of the drug on the cardiovascular system as well as its frequently suggested anti-cancer activity further justify the presence of metformin at each level of the algorithm of type 2 diabetes treatment in national and global recommendations of diabetes associations. Images obtained with PET scans using FDG as the radiotracer represent a tangible effect of drug interference with glucose and cancer metabolism. One should, however, bear in mind the fact that chronic intake of metformin by patients with type 2 diabetes, as well as by oncological patients (as adjuvant therapy), significantly affects interpretation of the obtained image. Therefore, the drug should be discontinued for an adequate time period before the scan. Numerous studies are currently ongoing that will certainly enlarge our knowledge about the mechanisms of action of metformin and will give us the final answer to the question of whether the drug has a real anti-cancer effect in humans and whether prolonged survival of treated nematodes and rodents may translate into a similar effect in primates.