Metformin in Non-Diabetic Conditions: An Overview

Abstract

Metformin has been proven to be one of the most safe and effective antihyperglycemic agent. Jean Sterne in 1957 first used metformin for treatment of diabetes mellitus type II. The main effect of this drug from the biguanide family is to acutely decrease hepatic glucose production, mostly through a mild and transient inhibition of the mitochondrial respiratory chain complex I. The drug is an insulin sensitizer, leading to reduction in insulin resistance and significant plasma fasting insulin levels. Additionally, the resulting decrease in hepatic energy status activates AMPK (AMP-activated protein kinase), a cellular metabolic sensor, having action on hepatic gluconeogenesis. It depicted marvelous non-glycemic related effects. The drug because of positive charge, can only partially cross the plasma membrane by passive diffusion. Its intracellular pathways are mediated by different isomers of organic cation transporters (OCT 1 for liver tissues and OCT 2 in the kidneys). These effects include modulation of different points of cancer timeline, weight reduction, cardiovascular health, thyroid diseases, polycystic ovaries disease and many other medical conditions. The aim of this review is to familiarize the effects of metformin in non-diabetes related medical disorders, advances in our understanding of this drug and its pathways in health and diseases.

1. Introduction

History of metformin is linked to Galega officinalis (also known as goat’s rue), a traditional herbal medicine in Europe, found to be rich in guanidine, which, in 1918, was shown to lower blood glucose. Guanidine derivatives, including metformin, were synthesized and some (not metformin) were used to treat diabetes in the 1920s and 1930s but were discontinued due to toxicity. Metformin was rediscovered in the search for antimalarial agents in 1940s. It proved to be useful in clinical cases to treat influenza when it had also shown to lower blood glucose. This property was pursued by the French physician Jean Sterne, who first reported the use of metformin to treat diabetes in 1957.

Metformin is under use over 65 years worldwide. The data has shown beneficial effects of the drug on obesity, PCOS, fatty liver disease, cardiovascular dysfunction as anti-oxidant and on inflammation, besides its use in pre diabetic and diabetic subjects. Moreover, drug has ardent usefulness in various malignant conditions, Alzheimer’s disease and dementias. The literature is also rich on use of metformin in diabetic and non-diabetic related effects on endocrinal disorders, especially on thyroid and different malignant conditions. The present review is focused on these alternate non-diabetic uses of metformin.

2. Metformin structure and pharmacology

Metformin is a plant based medicinal product a synthetic guanidine with two coupled molecules with loss of ammonia, hence named biguanide with additional substitution. Metformin structure is shown below:

Metformin is conjugate base, a member of the class of guanidines that is biguanide the carrying two methyl substituents at position 1. It has a role as a hypoglycemic agent, a xenobiotic, an environmental contaminant and a gero protector. It derives from a biguanide.

Galega or guanidine is extracted from herbal plant G. officinalis [1]. Attention was paid initially on guanidine itself. The drug proved too toxic for clinical use. The drug is mainly absorbed from upper intestine with bioavailability of around 50%. The plasma half-life ranges from 0.9 to 2.6 h varying with different formulations. The transportation into the cell is via organic transporter-3 (OCT 3 & OCT 1). The drug is excreted unchanged in urine. This oral medicine is mainly concentrated in liver, kidney and jejunum site [2]. Because of short half-life and low oral bioavailability GI tract upsets are often noticed with the therapy. This limitations have overcome by designing delivery system and development of novel formulations. The drug delivery system have been investigated to decrease the side effects, frequency of dosage and enhancing the effect of oral anti-diabetic drug. The interest with novel formulations with nanoparticles is expected to improve drug bioavailability, dosing, frequency and GI tract side effects. The process of nano particles in the optimized concentration and surface characteristics has generated a great potential offer for treatment of type 2 diabetic mellitus [3]. The rational development of nano skilled delivery system has been described in literature by many workers [4, 5]. Their objectives were to achieve specific advantages using apt nano size and surface modifications of the particles to improve the target delivery of the drug to any compartment of the body at cellular and subcellular levels. Improving therapeutic potentials are the main goals of this drug delivery system in their researches [5].

Around 120 million people worldwide are using the drug as antihyperglycemic agent, without much having overt hypoglycemic episodes. In human beings, the drug molecules works at 2 levels: at liver level and at peripheral tissues.

The process is active through AMPK (adenosine monophosphate protein kinase) as cell regulatory mechanism. In humans, AMPK is essential for metabolism of glucose and fatty acids by downsizing glucose output from the liver, reducing gluconeogenesis and fatty acid synthesis in the liver. AMPK is essential for enhancing glucose uptake and fatty acid oxidation in peripheral muscles. The beneficial effects are seen on multiple organ system. Mitochondrial metabolism is also found altered by metformin which helps in reduction in gluconeogenesis by down regulation. The drug mechanism has tumor modulation as prerogative role as adjunct therapy in cancer [6]. Metformin also inhibits GI uptake of carbohydrates, reduces leptin levels and results in augmentation of glucose like peptide on gut cells. At molecular level, metformin modulates Adenosine A1 receptors (ADORA 1) which are essential in the cellular energy cycle. The human colorectal cancer cells may remain energy deprived when ADORA 1 are downregulated with resultant apoptosis [6].

The certain authorities have commented that drug probably does not directly activate AMPK and LKB1gene. LKB1 gene is tumor suppressor gene {Serine/threonine kinase 11 (STK11) also known as liver kinase B1 (LKB1)}. The drug does not influence the phosphorylation of AMPK by LKB1 in a cell free assay [7]. Metformin activates AMPK through its effect on mitochondria. Growth inhibition include mTOR activity resulting in inhibition of protein synthesis and cell growth. AMPK activation also enhances p53 and p21 along with inhibition of cyclin D1. These small molecules exhibit diverse biological activities with inhibition of cell cycle. Inhibition of lipid and sterol biosynthesis pathway wide inhibition of sterol regulatory element – binding protein-1c (SREBP 1) and down regulation of fatty acid synthase. Inhibition of CO-A carboxylase (ECC) influences tumor suppression. The main mechanism of action of metformin and cancer is summarized in Figure 1. (Adopted from Chow Elaine and colleagues) [8].

Figure 1.

Mechanism of action of metformin. Metformin activates AMPK through its effect on mitochondria. Growth inhibition include mTOR activity resulting in inhibition of protein synthesis and cell growth. AMPK activation also enhances p53 and p21 along with inhibition of cyclin D1. These small molecules exhibit divers’ biological activities with inhibition of cell cycle. Inhibition of lipid and sterol biosynthesis pathway wide inhibition of sterol regulatory element – binding protein-1c (SREBP 1) and downregulation of fatty acid synthase. Inhibition of CoA carboxylase (ECC) influences tumor suppression. Source: Pharmaceuticals 2022, 15, 442. DOI: 10.3390/ph15040442.

3. Multifaceted impact of metformin

3.1 Antioxidant activity

The exact privileged mechanism is yet unclear. The antioxidant effect includes decreased gluconeogenesis and activation of AMPK system, upregulation of protein to fat and beta cell oxidation that lead to increase in the fatty acid metabolism and beta oxidation in fatty tissue [9]. A healthy heart obtains 60–90% energy for oxidative phosphorylation from fatty acid oxidation, whereas a failing heart has a balance for increase the glucose uptake and utilization [10]. The utilization of fatty acids use more oxygen per unit of ATP than the generated glucose which may improve ventricular performance. The drug also normalized non-esterified fatty acids, suggesting metabolic adaptation with use of the drug, as the drug modifies cardiac lipid/glucose oxidation ratio. Further, metformin action on cardiomyocytes attenuates the production of pro-apoptotic proteins, increase in anti-apoptotic proteins that reduce the percentage of apoptotic cardiomyocytes [11]. The studies with support of AMPK on endothelial functions revealed beneficial effects on it, suppressing oxidative stress in endothelial cell [12]. Thus, findings related to oxidative stress in endothelial cells, cardiomyocytes functionality and production of radical oxidative substrates (ROS) are inhibited by metformin which helps in preventing endothelial dysfunction, atherogenesis and improves myocardial dysfunction. Metformin is avoided in patient with heart failure because of risk of lactic acidosis. Currently, this contraindication weighs insignificance in literature [13]. Eurich and coworkers in 2011, indeed suggested that metformin alone or in combination with sulphonylurea reduce both the mortality and morbidity of type 2 diabetic patients with heart failure in comparison with Sulphonylurea as monotherapy [14].

3.2 Endothelial function and anti-inflammatory agent

The mechanism of metformin on endothelial function is unclear. However, endothelial function modulation was suggested in acquiring insulin resistance improvement. Endothelial dysfunctions are chiefly observed in diabetic and patients of ischemic heart disease [15]. The results of metformin as anti-inflammatory agent is controversial [16]. Metformin inhibits nuclear factor kappa B (NF-kB) by down regulating inflammatory responses. The factor NF-kB, a protein transcription factor is also a regulator of innate immunity.

3.3 Obesity and hormonal imbalances

It is argued by many researchers that weight reduction by metformin per say is owing to diabetic related medications rather the drug itself. The weight changes are evidently observed with impaired glucose tolerance unlike diabetics without obesity where in the drug failed to reduce the weight significantly [17]. Others elaborated the reasons for reduction in weight with metformin which are (1) reduction in leptin levels, (2) augmentation of glucagon like peptide one effects on fatty tissues, (3) reduction in carbohydrates uptake by the gut [18]. Metformin induced reduction in hepatic lipid content is consistent with increase in fatty acid oxidation and inhibition of lipogenesis mediated through AMPK activation [19].

3.4 PCOS (polycystic ovarian syndrome)

It is now recognized that insulin resistance, is a common feature of PCOS. The disorder affects at least 5–15% of reproductive age women. Pharmacological option in PCOS and insulin resistance with insulin sensitizer are being proposed. Metformin medications increased ovulation, reduce serum androgen levels [20].

The beneficial effects of metformin are based on production of excess insulin or ovarian effects. Insulin directly stimulates enzymes in ovary such as cytochrome P₄₅₀, 3beta SHD (3 beta hydroxysteroids) by improving insulin sensitivity by CYP 17 (cytochrome P450) activity, inhibits androstenedione effects on theta cells, reduces factors such as endometrial androgen receptor expression - factors with high risk of abortion. The observations have clinical significance that the use of metformin in an overweight PCOS patient would be fruitful. Metformin has been shown to reduce risks of abortion in PCOS in pregnancy.

3.5 Thyroid gland function

Metformin is considered a cornerstone of type 2 diabetes medication umbrella. Many prospective and retrospective studies have shown that serum TSH levels in hypothyroid patients decreased in response to metformin therapy and increased again when metformin was withdrawn [21]. Relevant changes were not observed in serum thyroxine (T4), triiodothyronine (T3) levels. In a study, by Cappali et al. [22] in 2009 that diabetic patient on metformin, had significant decline levels of TSH, but T4, T3 remained unchanged for 1 year. The changes were also noticed in group with already existing hypothyroidism on medication irrespective of metformin therapy. Following this initial findings a number of studies that were performed elucidated various mechanisms of the drug on TSH level. Metformin was shown to have a lowering effect on TSH level. Metformin likely affects thyroid function through peripheral conversion of thyroxine to triiodothyronine [23] when TSH value was higher than 2.98 mU/L, whereas the opposite effect was seen on individual having serum TSH level lower than 2.98 mU/L. The thyrotropin effect was not observed in hyperthyroid subjects with type 2 diabetes [24].

Use of metformin is hypothesized to change the affinity of thyroid hormones to increase the dopaminergic tone or induce activation of TSH receptors [21].

Metformin is also found to modulate hypothalamic pituitary thyroid axis at the level of peripheral tissues. The observations indicated that metformin treatment could have an impact on thyrotropin function in hypothyroid patient in apart associated with alterations in dopaminergic regulation of thyrotropin secretion. Duntas et al. [25] suggested that metformin is proved to have inhibitory effect on AMPK activity in hypothalamus. Clinical studies under taken by Al-Alu-Si 2015 [26], concluded that levothyroxine absorption remains unchanged by concomitant metformin. TSH suppression effect of metformin may be useful in clinical hyperthyroidism. However, complexity exist for monitoring thyroid function status in diabetic patient on metformin. The depressed TSH level may provide false reassurance in decreasing T4 (levothyroxine) dosage with low TSH level. The proper interpretation of thyroid functions is mandatory in patients receiving metformin therapy in diabetes.

3.6 Central nervous system

Metformin effect on nervous system is also puzzling. In Alzheimer’s disease, there is remarkable progressive insulin resistance of the brain cells leading to formation of amyloid cells. The disease is unofficially considered as type-3 diabetes. The brain cells here are dependent on glucose for survival and have low antioxidant enzyme contents as a result of oxidative injuries. Activation of AMPK pathway play a vital role in reducing insulin resistance and oxidative stress. The drug metformin is known to activate AMPK thus, partially could have a protective role in Alzheimer’s disease and others oxidative distress related neurological disorders. Metformin also plays a pivotal role in breaking the cycle of permeability transition protein release in the mitochondria. By virtue of drug effect, metformin in the mitochondria, plays a role in this cascade with resultant delay in the program of cell death [27]. The drug metformin influences and reduces neuro inflammatory status along with glucose metabolism leading to protein dephosphorylation, acts as an anti-oxidant and helps reducing neuro inflammation and degeneration (Figure 2).

Figure 2.

Metformin effects in neuron. It shows metformin counteraction protein hyperphosphorylation, oxidative stress and neuro inflammation, neuro fibrillary tangles, processes known to drive neural loss. The drug metformin influence neuro inflammatory status along with glucose metabolism leading to protein dephosphorylation, as an anti-oxidant and reducing neuro inflammation. Source: (https://www.researchgate.net/figure/Metformins-potential-as-a-neuroprotective-agent-Metformin-can-counteract-protein).

3.7 Anti-retroviral agents

HIV treatments lead to metabolic consequences as insulin resistance, dysregulation of glucose metabolism, dyslipidemia, and lipodystrophy in 80% of subjects as side effects of medication. Metformin as an adjunct therapy, diet and exercise effectively prevent these consequences by improving visceral fat distribution, reduces risk of insulin insensitivity, dyslipidemia, weight gain, hyperglycemia and endothelial dysfunctions.

3.8 Renal cancer

AMPK which suppresses the cell proliferation. AMPK pathway subsequently lead to inhibition of mTOR that is responsible for cell growth. mTOR the mammalian/mechanistic target of rapamycin is a kinase that in humans is encoded of MTOR gene. mTOR pathway is involved in number of important physiological functions including cell growth, cell proliferation, metabolism and protein synthesis. Inhibition of mTOR vide AMPK pathway is responsible for anti-proliferative effect. Metformin is also known to inhibit mTOR independently of AMPK pathway, making the outcome more effective in controlling the proliferation of cell growth. Metformin not only prevents phosphorylation of mTORc1 complex component but also inhibits phosphorylation of AKT, a mTORc2 substrate which is beneficial in the treatment of cancer.

3.9 COVID19

Metformin blocks the viruses from binding the host receptors ACE2. Other effects of metformin are AKT inhibition, AMPK activation, inhibition of mTOR activity there by suppressing virus – host protein interaction. The drug also inhibits mitochondrial generation of ROS which leads to rising intracellular calcium and subsequent release of pro-inflammatory cytokines (IL1, IL6, TNF alpha and IL beta). In COVID 19 infection, the drug metformin increases insulin sensitivity that modify endosomal pH and reduces viral replications and maturation. ACE2 downregulation by SARS Co-2 is prevented by metformin. This drug mediated increase levels of ACE2 and phosphorylation of ACE2 subsequently regulates RAAS and offers cardiopulmonary protection.

4. Therapeutic applications of metformin

4.1 Cancer

The large number of cohort studies have confirmed the significance of associated diabetes mellitus with increased risk of cancer affecting pancreas, kidney, prostate, colon and breast. This increased risk is attributed to persistently elevated plasma glucose and plasma insulin levels. Insulin resistance, hyperinsulinemia that might promote carcinogenesis, directly or indirectly increasing levels of insulin-like growth factor (IGF) (formerly called somatomedin) that functions primarily to stimulate growth. However, that also possess some ability to decrease blood glucose levels and inflammatory process [28].

Metformin therapy in these malignant cases were associated with relative reduce risk of cancer and cancer mortality in diabetic patient [29]. These observations are consistently seen with in vitro and invivo studies revealing anti-proliferative action of metformin on various cancer lines [30].

4.1.1 Breast cancer

As mentioned previously, the action of metformin is having direct effect on AMPK that when occurs, lowers the ATP ratio in cells resulting in stimulating AMPK pathway for regularizing energy homeostasis. Excess energy, in turn will signal the need to decrease the energy consumption. Thus, resulting cytotoxic effects are met with inhibition of cell growth and proliferation of breast tissue. Further, Metformin is known to exert an indirect effect on cells by lowering insulin levels and decreasing PI3k pathway, inhibiting cell growth and proliferation. PI3k signaling pathway stimulates the cells for proliferation and growth, simultaneously inhibiting cell apoptosis. It is an enzyme that transmit signal in cells that helps to control the cell growth. Some tumors have higher level than normal of PI3k (PI3 kinase). By inhibiting this enzyme, PI3k inhibitors cause cell death and inhibit the proliferation of malignant cell. Iliopoulos and coworkers [31] in 2011, suggested the need of higher dosage of metformin (1.5–2.5 g) to attain anti-neoplastic effect [31].

4.1.2 Liver cancer

Lack of well-designed trials on the subject, reserves the outcome of metformin use in liver cancer. However, in metaanalysis compiled by zhang and colleagues concluded that metformin appears playing a role in reducing liver cancer risk in type 2 DM [32]. More well designed trials would be needed to evaluate this point.

4.1.3 Pancreatic cancer

Many trials have assessed possible link between type 2 diabetic and pancreatic cancer. The study by Huxley et al. [33] 2005, noticed in their study a higher risk of pancreatic cancer development in diabetic subjects who are having short duration of illness. Yet another study revealed to significantly lower risk of mortality in patients with pancreatic cancer who used metformin [34]. More further studies needed to consolidate this conclusion.

4.1.4 Endometrial cancer

A large numbers of studies had explained the improvement of overall survival in patient with advanced endometrial cancer taking metformin. Studied patients were either diabetic or non-diabetic [35, 36]. Metformin’s role in recurrence of endometrial cancer as remained unclear. Prospective study under taken by Soliman et al. in 2016 in newly diagnosed endometrial cancer, demonstrated the reduction in serum molecular markers of cancer who were using metformin in a week’s period [36]. It is also described that mechanism of metformin in tumor cell is vide FOXO 1 pathway involvement. FOXO family is a subclass of fork head transcription factors. The pathway is responsible for regulating the expression of gene in the cellular physiological events including apoptosis, cell cycling control, glucose metabolism, oxidative stress resistance etc. It was also revealed that providing a AMPK inhibition in the FOXO 1 pathway by injecting silencing RNA for FOXO 1 in endometrial cell, subsequently eradicated anti-proliferative effects of metformin. The observation bears strong therapeutic implication [37, 38]. The proposed scheme for the antiproliferative mechanism of metformin in estrogen-dependent endometrial cancer (EC) cells is shown in Figure 3 (Source: [39]).

Figure 3.

Proposed scheme for the antiproliferative mechanism of metformin in estrogen-dependent endometrial cancer (EC) cells. Metformin activates AMPK/human forkhead FOXO1 –P in the cytoplasm thus decreasing phosphorylation (P) of FOXO1 protein. There by organizing RE localisation of FOXO1 protein from cytoplasm to nucleus with enhance FOXO1 activity. FOXO1 function contributes to increased efficacy of metformin therapy against EC-novel mechanism of metformin as anti-neoplastic. Findings are supportive for prostate cervical cancer.

4.1.5 Cervical cancer

Limited data is available on this subject. Xiao et al. 2012 [40] examined metformin dynamics in these cancer cells with focus on LKB1. Cell lines were either responsive to metformin or remained non-responsive to metformin. The cell lines which are responsive to metformin stimulate AMPK via LKB1 and prevents mTOR. Nonresponsive cell to metformin were void of LKB1. LKB1 gene also known as STK 1 gene, provides instruction for making enzyme called serine/threonine kinase 1. This enzyme is a tumor suppresser that helps the cells in growing and its division rapidly. Metformin also suppress other cell lines of cervical cancer cell viz. C33A, ME180. Metformin may, Therefore, have an adjuvant role especially in such cervical cancer having presence of LKB1 cell line. C33A is an epithelial cell line isolated from cervix of uterine cancer patient. Herein, arginine to cystine substitution takes place at codon 273.

4.1.6 Lung cancer

Poor association are shown between use of metformin and lung cancer [41]. A retrospective cohort study by wink et al. [42] had concluded that patients of diabetics on metformin and with locally advanced non-small cell (NSCL) cancer where having less chances of disease progression and metastasis, than those who were not kept on metformin [42].

4.1.7 Colorectal cancer

Diabetic patients are shown to have lower risk of colorectal carcinoma when treated with metformin. Besides metformin is having the ability to lower the risk of colorectal cancer, improvement and survival in such patients [43, 44, 45].

4.1.8 Renal cancer

Metformin is a common therapeutic agent with anti-tumor activity in various cancer types. However, its use remains controversial in renal cell carcinoma. Adjunct metformin treatment in RCC (renal cell carcinoma) lead to activation of. In RCC additional, anti-proliferative effects are also achieved by suppression of cyclin D gene responsible for cell proliferation and cell growth [46]. Metformin is also able to prevent renal cell carcinoma by incorporating the regulation of gene miR-26a, which will inhibit cyclin D1 expression- a factor incumbent for the cell growth. mTORc1and c2 promote the cell growth by inducing and inhibiting anabolic and catabolic processes respectively and drives the cell cycle progression. In clinical practice, metformin has shown insignificant association in preventing the recurrences of RCC after its resection [47].

4.2 COVID19

After the emergence in late December 2019, it was observed by many workers, that diabetic individuals were at high risk of COVID 19 infection and its associated complications. Interestingly, diabetic COVID 19 patients had more adverse outcome while on insulin when compared to those who were on metformin. Metformin treatment correlated with significant reduction in disease severity and mortality in such cases. Excellent review on the subject is published by Varghese E and colleagues in 2021 [48]. The drug has its role as anti-oxidant, anti-inflammatory, immunomodulator, and protective effects on vasculature and endothelial functions, already discussed earlier in the text. It is noteworthy to mention that ACE2 receptor is expressed in various organs viz. brain, heart, lung, intestine, kidney, liver, vasculature and adipose tissues making them targets for COVID infections. Diabetes has increased expression of ACE2 in various tissues hence leading to increase in viral load Metformin provides protection metabolically and reduces complications related to immune response and thrombotic events. Metformin in COVID 19 be judiciously used with conditions such as renal failure, diabetic ketoacidosis and severely ill patients.

5. Conclusion

Despite newer advances made for oral hypoglycemic agents metformin remains as main agent under diabetic umbrella. The drug has effectively demonstrated to possess anti-tumor activity of value in varied cancer subjects. The main effect of drug is to decrease hepatic glucose production by mitochondrial respiratory chain complex resulting in transient decline in cellular energy status that promotes the activation of AMPK, a well-known cellular sensor. There are multiple molecular mechanisms proposed about metformin with protective properties in diabetic and non-diabetic users. Morewider researches with their clinical implications still are awaiting for metformin use as an adjunct therapy in various cancer subjects.

6. Future perspective

Well-designed human and animal researches are needed to confirm the benefits of drugs in as anti-inflammatory agent, anti-proliferative agent in various inflammatory and malignant disorders. Nanoparicles/microbubbles generated drug for treatment of diabetes mellitus has great potential but needs further researches with specific attention to optimized concentration. Extensive physiologic and biochemical studies are still required to identify indirect targets influenced by relatively limited number of direct targets.

7. Recommendations

Metformin is currently recommend as first line glucose lowering drug in type 2 diabetes mellitus. Molecular studies though highlight the knowledge gap in areas of unearth needs, However, neo clinical and mechanistic studies on metformin and its usefulness on central nervous system, infections and cancers are warranted with new insight into its therapeutic value beyond boundaries of diabetes.