The signaling pathways of metformin (MF) in cells are still not fully understood, they seem to be dependent on species and cell type, as well as doses and routes of administration, along with metabolic and hormonal status of subjects [22,24,25,26,27].

The molecule of MF, a small hydrophilic cation, is transported from the extracellular space to the cytoplasm of the target cell through organic cation transporters-1 and -2 (OCT1, OCT2), multidrug and toxin extrusion transporters (MATE), and ATM (ataxia telangiectasia mutated) transporter, and OCT1 and OCT2 are considered as the main functional units of MF transmembrane transport [28]. The transfer of MF across the placental barrier during pregnancy is largely dependent on the transporter OCT3 [29]. The ultimate intracellular target for MF is the 5′-adenosine monophosphate-activated protein kinase (AMPK), the key energy sensor of the cell, although MF does not interact directly with the enzyme [22,30,31,32]. In pathological conditions, like type 2 diabetes mellitus (T2DM) and metabolic syndrome (MetS), the activity of AMPK is reduced. MF’s action increases the activity of AMPK, and consequently normalizes the energy metabolism of the target cell. The AMPK consists of a catalytic α-subunit and the regulatory β- and γ-subunits that form a functionally active αβγ-heterotrimeric complex, and is widely distributed in all subcellular compartments (cytoplasmic, lysosomal, mitochondrial, and nuclear). AMPK is activated by increasing levels of AMP, a positive allosteric regulator of the enzyme [31,33,34]. The interaction of AMP with the adenine nucleotides-binding sites located in the γ subunit leads to stabilization of the αβγ heterotrimeric complex and enables phosphorylation of the α-subunit by liver kinase B1 (LKB1), which leads to the increase in AMPK activity [31,32,35] (Figure 1). Activating phosphorylation of AMPK may be also mediated by Ca²⁺-calmodulin-dependent protein kinase kinase 2 (CaMKK2) [36,37] and transforming growth factor β activated kinase-1 (TAK1) [38,39,40], but LKB1 is most important for AMPK activation [31,34,41,42,43]. Allosteric binding of AMP and ADP to γ-subunit of AMPK increases the ability of LKB1 and CaMKK2 to phosphorylate AMPK α-subunit at the Thr¹⁷² [44,45,46]. In the lysosomes, the “non-canonical” pathway of LKB1-mediated AMPK activation is carried out through dissociation of fructose 1,6-bisphosphate from aldolase. At the lysosomal surface, free aldolase promotes the formation of a multiprotein complex, including the vacuolar H⁺-ATPase and the scaffold protein AXIN, and this complex ensures the effective binding between AMPK and LKB1, thereby activating AMPK [47,48]. A negative regulator of AMPK is the protein phosphatase 2C (PP2C), which dephosphorylates and inactivates the α-subunit of AMPK, causing the dissociation of the αβγ-heterotrimeric complex. Elevated levels of AMP lead to an inhibition of PP2C activity, which allows AMPK to remain stable in the active Thr¹⁷²-phosphorylated state [49,50].

 

MF penetrates into the mitochondria through intracellular space and accumulates in them. While in the mitochondria, MF inhibits the mitochondrial ETC complex I, which leads to decrease in ATP production and increase in the [AMP]_i/[ATP]_i and [ADP]_i/[ATP]_i ratios [51,52,53,54]. Moreover, MF decreases the activity of the enzyme AMP-deaminase (AMPD), which converts AMP to inosine monophosphate, inducing the accumulation of AMP within the cell [55]. The MF-induced increase in the intracellular AMP level leads to the activation of AMPK as described above [41,56]. The MF effect on AMPK activity is observed at drug concentrations below 80 μM, which are achieved with oral administration of therapeutic doses of MF [57]. The MF-induced activation of AMPK results in the stimulation of energy-producing catabolic pathways that mediate the increased glucose uptake by cells, the increased expression and activity of the membrane glucose transporters, the activated metabolic processes such as glycolysis and oxidative phosphorylation, and the normalization of mitochondrial biogenesis [20,24,58,59]. The MF-induced AMPK stimulation leads to phosphorylation of types 1 and 2 acetyl-CoA carboxylases (ACC1 and ACC2), inducing an inhibition of lipogenesis and stimulation of the β-oxidation of free fatty acids [60,61,62] (Figure 1). The ultimate results of this metabolic cascade is the decrease of T2DM- and MetS-produced dyslipidemia, and the normalization of lipid metabolism. In addition, the AMPK activation induces a plethora of cellular events, including regulation of autophagy and apoptotic processes, a decrease in the activity of inflammatory factors, including nuclear factor κB (NF-κB) and interleukin 1β, an inhibition of the ROS production, a decrease in the ER stress, as well as a decrease in insulin/IGF-1-induced activation of the mTORC1/2 complexes and a decrease in the protein synthesis [60,62,63,64,65,66].

The MF is a functional antagonist of cAMP-dependent signaling cascades, which are stimulated by hormones, glucagon in particular, through the G_s protein-coupled receptors and the membrane-bound forms of adenylyl cyclase (AC) [67,68]. The stimulation of AC results in an increase in the intracellular cAMP level and the activation of the protein kinase A (PKA) and the cAMP-activated transcription factor CREB (cAMP response element-binding protein). The MF-induced activation of AMPK promotes phosphorylation and activation of cAMP-specific 3′,5′-cyclic phosphodiesterase 4B (PDE4B), thereby reducing the intracellular level of cAMP [68]. Moreover, MF causes an increase in the intracellular level of AMP, a negative regulator of the catalytic site of AC, which leads to inhibition of AC activity and a decrease in cAMP production. An increase in the level of AMP can be the result of both inhibition of the mitochondrial ETC complex I, and suppression of the activity of AMP deaminase [55,69] (Figure 1). A decrease in the activity of cAMP-dependent pathways in the liver, like activation of AMPK, leads to the inhibition of glucose synthesis in hepatocytes. Furthermore, MF-induced AMPK activation induces the protein kinase ι/λ-mediated phosphorylation of cyclic AMP response element binding (CREB)-binding protein (CBP or CREBBP) at the Ser⁴³⁶, which leads to the inability of the phospho-CBP to form a functionally active complex with the factor CREB and thereby inhibits the cAMP-dependent gene transcription [70].

Along with AMPK-dependent, there are also AMPK-independent pathways of MF action on the intracellular effector systems and gene expression. High-dose MF inhibits the activity of the mitochondrial glycerol-3-phosphate dehydrogenase (mG3PDH) [71]. The inhibition of mG3PDH leads to an increase in NADH levels and decreases NAD⁺ levels, and this causes a deficiency in NAD⁺, which is involved in the conversion of lactate to pyruvate (Figure 1). Since a decrease in mG3PDH activity inhibits the conversion of lactate to glucose, the result of impaired gluconeogenesis in hepatocytes is an accumulation of lactate, which can cause lactic acidosis in the conditions of high-dose MF treatment [71,72]. Another target of MF is the enzyme H3K27me3-demethylase KDM6A/UTX, which is responsible for the transcriptional activity of a large number of genes [73].

The antidiabetic effects of MF may be due to the changes in the gut microbiota, due to stimulation of the growth of bacteria that produce short-chain fatty acids [74]. By modulating the composition of the microbiota in rodents with T2DM and MetS, MF reduces the levels of bacterial lipopolysaccharides in the blood [75], and activates AMPK-dependent pathways in the mucosal layer of the intestine, reducing glucose absorption [76].

The most important mechanism of action of MF on target cells is the enhancement of the insulin signaling pathways and the decrease in insulin resistance (IR). This may be due to inhibition of hyperactivated nuclear factor κB (NF-κB), a transcription factor that provokes the development of IR, as well as a decrease in the expression of the phosphatase and tensin homolog (PTEN), which dephosphorylates phosphatidylinositol-3,4,5-triphosphate and thereby prevents insulin-induced stimulation of Akt kinase, a key effector component in the 3-phosphoinositide signaling pathway. The inhibitory effect of MF on the activity of NF-κB-dependent signaling pathways is carried out mainly through the stimulation of AMPK [25,77,78]. Since NF-κB plays a key role in inflammatory reactions, its inhibition by MF promotes the weakening of inflammation and increases the cell survival, and these effects of MF are prevented by AMPK inhibitors [25,79,80].

Over the past years, the focus has been on the validity of the use of MF for the treatment of GDM and the prevention of GDM-associated preeclampsia, which is of great importance for a normal pregnancy and the birth of healthy offspring [309,310,311,312,313,314,315,316,317,318]. GDM is recognized on average in 10–20% of pregnant women, and the incidence is largely depends on diagnostic criteria and genetic, environmental and ethnic factors [319,320,321]. The dietary and exercise interventions are often used to prevent and treat GDM and improve pregnancy [317,322,323], but such interventions are effective only when used concurrently, and with pharmacological intervention [322,324]. Combining diet and exercise with vitamin D and myo-inositol supplement has been shown to be effective [324,325,326,327]. The most widely used pharmacological approach for GDM correction is insulin therapy, which is currently considered the “gold standard” of care in GDM [317,328,329]. Notably, insulin treatment leads to a number of side effects, primarily hypoglycemic episodes, dangerous for the health of pregnant women and fetus. As a result, the development of alternative pharmacological approaches is now underway, among which the use of MF and other oral hypoglycemic agents is of the greatest interest [317,330,331,332].

In 2007–2013, the first series of randomized clinical trials was carried out to assess the efficacy and safety of MF therapy in GDM, including the comparison with insulin therapy [333,334,335,336,337,338]. It was concluded that MF, to a greater extent than insulin, reduces body weight gain in pregnant women, and also reduces the frequency and severity of gestational hypertension. Unlike insulin, MF did not induce hypoglycemic episodes in the mother and fetus. At the same time, MF therapy had a little or no effect on the incidence of small for gestational age (SGA) or large for gestational-age (LGA) neonates, and had a little effect on the incidence of preeclampsia.

Subsequently, there were the additional evidences of the effectiveness of MF as a drug that reduces maternal body weight and prevents gestational hypertension [314,339,340]. Based on a large clinical data [337,341,342,343,344,345,346,347,348,349], it was concluded that MF in GDM and T2DM, to a greater extent than insulin, prevents neonatal hypoglycemia, and significantly reduces the incidence of fetuses with LGA, thereby reducing the number of newborns who require intensive care after birth [321,339,350]. It is important to note that MF therapy does not increase the rate of preterm delivery, Cesarean section, and SGA neonates [321,339,340], despite the fact that earlier studies showed the risks of MF therapy for a normal pregnancy in GDM [334,351]. Polish scientists found some increase in triglyceride levels and atherogenic index in the third trimester of pregnancy in MF-treated women with GDM. At the same time, they indicated that short-term treatment with MF and insulin had a similar impact on lipid markers of MetS in women with GDM [352].

Several advantages of MF therapy in GDM treatment were demonstrated when comparing MF efficiency with that of other oral hypoglycemic agents, including glibenclamide and its analogues [330,347,353,354,355]. The use of glyburide, which, like MF, provides adequate glycemic control in GDM, is associated with an increased risk of neonatal hypoglycemia, high neonatal birth weight and macrosomia [330,353,355].

As noted above, there is evidence that MF reduces preeclampsia in GDM, and this is based on the ability of MF to prevent endothelial dysfunctions via normalizing the levels of the anti- and pro-angiogenic factors and improving the mitochondrial energy and biogenesis [313,356,357]. MF decreases the production of anti-angiogenic factors, including soluble forms of receptors of angiogenic factors. Among them, there are the placenta-produced soluble vascular endothelial growth factor receptor-1 (VEGFR-1), also called soluble fms-like tyrosine kinase-1 (sFlt-1), which specifically binds to vascular endothelial growth factor-A (VEGF-A) and placental growth factor (PlGF), and the soluble endoglin, a soluble isomer of endoglin, which inhibits the specific binding of TGF-β1 to its receptor [313,356,357,358]. Moreover, MF and its combination with other drugs (esomeprazole, sulfasalazine) lead to an increase in the expression of VEGF-A and placental growth factor (PlGF), both powerful activators of angiogenesis, and to a decrease in TNFα-induced expression of endothelin-1, a potent vasoconstrictor [356,357]. In pregnant women, the increased activity of anti-angiogenic factors induces the systemic endothelial dysfunctions and vasospasm and provokes preeclampsia [358]. MF treatment of mice with a preeclampsia-like model induced by a six-week high-fat diet (HFD) before pregnancy reduces the blood pressure, prevents the proteinuria, normalizes the fetal and maternal weight, and leads to an improvement of the placental labyrinth and fetal vascular development, impaired in the conditions of preeclampsia [359]. This is based on MF-induced normalization of placental production of MMP-2 and VEGF in preeclamptic mice, which, in turn, improves the pregnancy outcomes [359].

In GDM, MF-induced normalization of angiogenesis and blood circulation prevents hypoperfusion of the placenta. In this regard, it should be noted that hypoperfusion leads to placental hypoxia and ischemia and oxidative stress at the maternal-fetal interface, and then the inflammatory factors and the oxidized and degraded biomolecules are transferred into the mother’s bloodstream, causing preeclamptic symptoms [360,361]. Another mechanism for the prevention of preeclampsia may be a MF-induced decrease in IR and normalization of insulin levels in women with GDM, since there is an evidence that a decrease in insulin sensitivity is an important factor provoking preeclampsia [362,363].

It is also assumed that in GDM the protective effect of MF on preeclampsia may be due to its effect on the sensitivity of the placenta to human chorionic gonadotropin (hCG) during the early pregnancy. hCG is the most important regulator of placental development and angiogenesis and is able to influence the ratio of PlGF and sFlt-1 receptors in the blood and, thereby, control the activity of pro- and anti-angiogenic factors [364]. During early pregnancy, high levels of hCG are associated with the elevated sFlt-1/PlGF ratio and the increased risk of preeclampsia [364]. The pregnant women with GDM are more likely to deliver by Cesarean section. The success of Cesarean section is significantly increased with the combined use of MF and insulin and is higher than with the use of insulin or MF alone [365]. The combination MF plus insulin had an improving effect on the delivery of LGA infant as compared to insulin monotherapy. These data indicate the need for the combined use of MF and insulin in treating GDM women with high-risk pregnancy and recommendation for Cesarean section [365].

When using MF to treat GDM, it is important to take into account the ability of this drug to easily cross the placenta barrier to affect the embryo and developing fetus [366]. At the same time, the available data do not support the teratogenic effect of MF when used during pregnancy [315,345,346,351,353,367,368,369,370,371]. Moreover, a number of positive effects of prenatally administered MF on fetal development and functional indices of newborns in GDM were demonstrated [366,372,373]. In GDM, the side effects of MF, as in the treatment of PCOS and T2DM, are mainly limited to gastrointestinal dysfunctions [287,334,374,375].

The fact that, in GDM, MF therapy in about half of cases has little or no effect [334,376], may be due to the genetic and ethnically defined environmental ethnic features, as well as the severity and pattern of hormonal and metabolic disorders in women with GDM. In this regard, the situation is similar to that in PCOS women, where the factors that determine the effectiveness of MF therapy are hyperinsulinemia, HA, functional activity of the gonadotropin and insulin/IGF-1 signaling systems in the ovaries, lipids spectrum, expression and activity of inflammatory factors and antioxidant enzymes, and also organic cation transporter-dependent transfer of MF into the target cells (see Figure 3).

To fully assess the response of pregnant women with GDM to MF, the effect of this drug on glucose homeostasis should be investigated. The glucose level one hour after the start of the oral glucose tolerance test above 212 mg/dL and the mean fasting glucose level during the first week of MF therapy above 95 mg/dL are both reliable indices for supplementing MF therapy with insulin [376]. The differences in responsiveness to MF therapy are believed to explain the results for a number of recent studies and meta-analyzes that did not show a significant effect of MF on pregnancy and its outcomes in women with GDM and on the development of preeclampsia. Based on this, a number of authors have expressed the opinion that insulin is preferable to MF in GDM treatment [377,378,379].

MF, the main oral hypoglycemic agent for the treatment of T2DM, is also widely used to treat women with T2DM before pregnancy. At the onset of pregnancy, for a significant proportion of diabetic women, on average from 11.4% to 62.5%, MF therapy is replaced by insulin therapy, which is still considered more acceptable for glycemic control in pregnancy [380,381]. However, with the accumulation of clinical data, the validity increases for the use of MF to treat women with T2DM during pregnancy, mainly due to the optimization of strategy of MF use and strong evidence of its prenatal safety [341,343,346,349,370,375,381,382,383,384,385,386,387].

As in the case of PCOS and GDM, the effectiveness of MF in the treatment of pregnant women with T2DM depends on a number of factors, including individual susceptibility to this drug. Reduced response to MF requires the use of insulin supplements to improve glycemic control. The number of pregnant T2DM women receiving MF treatment, which require supplementary insulin therapy varied significantly and ranged widely from 4% [385] to 43% [341] and even up to 84% [346]. Supplementary insulin therapy usually begins on average after the 25th week of gestation [346]. The need to add insulin is due to the ineffectiveness of glycemic control using MF alone, even with a significant increase in the daily doses of this drug. Egyptian scientists have shown that in 37% and 39% of pregnant women with T2DM, MF in the daily doses of 1500 and 2000 mg, respectively, provided a good glycemic control, while in 24% of pregnant diabetic women the use of a daily dose of 2000 mg did not secure reliable glycemic control, thus these patients required supplementary insulin treatment [343].

In contrast to women with T2DM, the use of MF in obese pregnant women who do not have severe IR, hyperglycemia and dyslipidemia is not effective. Most clinical studies indicate that MF does not significantly affect the pregnancy and the health of newborns in obese pregnant women [318,388,389,390]. The use of MF in pregnant women with overweight or obesity at the end of the first and beginning of the second trimesters does not significantly improve the outcomes of pregnancy and childbirth [390]. Although some meta-analyzes show that MF treatment of obese women can reduce maternal gestational weight gain and prevent preeclampsia, without negatively affecting the fetus, including stillbirth and congenital abnormalities [314,388], there is currently no reliable reason to recommend MF for the prevention of adverse pregnancy outcomes in obese or overweight women without pronounced signs of T2DM [388,390].

Women with type 1 diabetes mellitus (T1DM) are also characterized by an increased risk of developing preeclampsia and adverse pregnancy outcomes, and these risks are significantly higher than those in T2DM [391,392,393,394,395]. In this regard, it becomes necessary to reduce these risks, for which, traditionally, insulin therapy is used, both in the period before and during pregnancy. Adequate insulin therapy for women with T1DM before pregnancy reduces the risk of hypertensive disorder of pregnancy [396].

However, a long-term therapy with increasing doses of insulin leads to IR and hypoglycemic episodes, and also increases the mother’s body weight, thereby worsening the outcome of pregnancy [397,398,399]. In the third trimester of pregnancy, the daily insulin dose can increase to 1.0 IU/kg, and the increase in this dose can occur rapidly [400]. Weight gain in pregnant women with T1DM, which is more pronounced than among non-diabetic patients, and persistent hyperglycemia lead to a higher risk of fetal overgrowth, resulting in LGA and macrosomia [401,402].

Optimization of insulin therapy using automated insulin delivery is one of the approaches to prevent IR and normalize the body weight and glucose homeostasis [399,403,404]. Another, alternative, approach is the use of MF therapy to correct hyperglycemia in pregnant women with T1DM, especially since MF has been successfully used for a long period of time to correct hyperglycemia in non-pregnant patients with T1DM, which makes it possible to increase insulin sensitivity and reduce the insulin dose [405,406,407,408,409]. The data on MF treatment of pregnant women with T1DM are encouraging, demonstrating improved glycemic control without the need to increase the insulin dose and reducing maternal weight gain and the risk of neonatal hypoglycemia [410].

The data presented in the review convincingly prove that MF has an improving effect on reproductive functions in women with PCOS, GDM and T2DM. At the same time, the effectiveness of MF therapy is due to a large number of different factors that must be taken into account when choosing this therapy and also when developing a strategy for using MF. Firstly, it is necessary to assess the efficiency of MF transport into the cell, which depends on the functional activity of the organic cation transporters and can be disrupted by inactivating mutations in their genes. The presence of certain mutations leads to a loss of responsiveness to MF and makes the use of MF therapy meaningless. Secondly, MF therapy is more effective in the severely overweight and obese patients with IR, compensatory hyperinsulinemia, impaired glucose tolerance, as well as with dyslipidemia, which is due to a decrease in the blood levels of HDL-C. This is not surprising, since the clinical effect of MF therapy is due to an improvement in insulin sensitivity, a decrease in the adipose tissue mass, and restoration of the glucose and lipid metabolism.

In addition, as demonstrated in a number of clinical studies in PCOS women, the effectiveness of MF is largely determined by the hormonal status of the ovaries and the functioning of the HPG axis. The MF therapy may be most effective in PCOS women who have: (1) severe HA, which may be due to hyperactivation of ovarian insulin/IGF-1-regulated signaling pathways that stimulate androgen synthesis, as well as an increase in LH levels and the LH/FSH ratio and a decrease in the blood levels of IGFBP-1 and SHBG; (2) an increase in the AMH production; and (3) an increased aromatase expression and FSH-induced estrogen synthesis in the ovaries. There is reason to believe that various combinations of these factors, including those with IR and metabolic disorders, may become reliable indications for prescribing MF alone and in combinations with other drugs, diet or exercises to correct the reproductive functions in PCOS. This can be helpful when using MF to treat the pregnant women with GDM and T2DM.

Since some of MF targets overlap well with those of leptin, the assessment of leptin status in patients with reproductive disorders may also be important. As a result, leptin resistance, both systemic and in the ovaries, as well as the changes in the hypothalamic leptin signaling pathways, negatively affecting the production of GnRH, can become factors that will determine the effectiveness of MF therapy. In this regard, it should be noted that the central mechanisms of action of MF, which easily penetrates the CNS and improves the metabolism of the neuronal and glial cells, still remain underestimated. By acting on the CNS, MF restores the signaling networks of the hypothalamus and the other brain regions that are involved in the control of reproductive functions and undergo significant compensatory and pathological changes in metabolic and endocrine disorders, including PCOS, GDM, T2DM, and MetS.

A unique feature of MF is the multiplicity of molecular mechanisms of its action on target cells, which include direct or indirect regulation of the AMPK-, calcium- and cAMP-dependent signaling pathways, as well as the MAPK cascade and the IRS/PI 3-K/Akt pathway. As a result, MF controls not only energy and metabolic processes in the cell, but also the processes of growth, differentiation, apoptosis, inflammation, and ER stress. At the same time, most of the regulatory effects of MF are based largely on its modulating and normalizing influence on intracellular signaling cascades than on their prolonged stimulation or suppression. Depending on the functional state of the IRS/PI 3-K/Akt pathway, MF can either prevent its hyperactivation, which is especially important for its antitumor effect, or, on the contrary, restore its reduced activity, improving the survival of target cells and their sensitivity to insulin and leptin. As expected, MF therapy affects the responsiveness of hypothalamic neurons, pituitary gonadotrophs, and ovarian cells to the hormones, growth factors, adipokines and cytokines, but more studies are required for complete elucidation of all regulatory mechanisms involved.

The use of MF in combination with the other drugs has great potential. This is supported by the encouraging results of clinical trials of the combined therapy with MF and insulin in pregnant women with GDM and T2DM. In metabolic and endocrine disorders, the combined therapy not only allows to increase the efficiency and pattern of the effects of MF on the HPG axis, but also to reduce the pharmacological doses of drugs, including MF, thus avoiding possible side effects of high-dose drug administration, including the undesirable effect of MF on the functioning of the gastrointestinal tract.

The presented results indicate a significant and not yet fully understood potential of MF therapy for the correction of reproductive dysfunctions in women. Significantly, of great importance are the absence of a teratogenic effect of MF and the low risks of MF therapy on the health of the mother and child. It should be taken into account that the unjustified use of MF for the treatment of patients lacking profoundly manifesting metabolic and endocrine disorders can lead to energy and metabolic imbalance and further deterioration of the functional state of their reproductive system.