Metformin for Cardiovascular Protection, Inflammatory Bowel Disease, Osteoporosis, Periodontitis, Polycystic Ovarian Syndrome, Neurodegeneration, Cancer, Inflammation and Senescence: What Is Next?

Abstract

Diabetes is accompanied by several complications. Higher prevalence of cancers, cardiovascular diseases, chronic kidney disease (CKD), obesity, osteoporosis, and neurodegenerative diseases has been reported among patients with diabetes. Metformin is the oldest oral antidiabetic drug and can improve coexisting complications of diabetes. Clinical trials and observational studies uncovered that metformin can remarkably prevent or alleviate cardiovascular diseases, obesity, polycystic ovarian syndrome (PCOS), osteoporosis, cancer, periodontitis, neuronal damage and neurodegenerative diseases, inflammation, inflammatory bowel disease (IBD), tuberculosis, and COVID-19. In addition, metformin has been proposed as an antiaging agent. Numerous mechanisms were shown to be involved in the protective effects of metformin. Metformin activates the LKB1/AMPK pathway to interact with several intracellular signaling pathways and molecular mechanisms. The drug modifies the biologic function of NF-κB, PI3K/AKT/mTOR, SIRT1/PGC-1α, NLRP3, ERK, P38 MAPK, Wnt/β-catenin, Nrf2, JNK, and other major molecules in the intracellular signaling network. It also regulates the expression of noncoding RNAs. Thereby, metformin can regulate metabolism, growth, proliferation, inflammation, tumorigenesis, and senescence. Additionally, metformin modulates immune response, autophagy, mitophagy, endoplasmic reticulum (ER) stress, and apoptosis and exerts epigenetic effects. Furthermore, metformin protects against oxidative stress and genomic instability, preserves telomere length, and prevents stem cell exhaustion. In this review, the protective effects of metformin on each disease will be discussed using the results of recent meta-analyses, clinical trials, and observational studies. Thereafter, it will be meticulously explained how metformin reprograms intracellular signaling pathways and alters molecular and cellular interactions to modify the clinical presentations of several diseases.

It is estimated that type 2 diabetes affects more than 462 million individuals or 6.28% of the world’s population.¹ Type 2 diabetes is the major endocrine driver for global burden of diseases and has been the ninth leading cause of death during recent years.² The prevalence of diabetes abruptly increased during the last decades, and it was observed that diabetes directly caused 1.3 million deaths worldwide in 2010. In addition, the death number doubled from 1990 to 2010.³ Diabetic patients usually have complications with several coexisting conditions such as obesity, frailty, CKD, hypertension and other cardiovascular diseases, neuropathy, retinopathy, and increased prevalence of osteoporosis, neurodegenerative diseases, and cancers.⁴⁻⁷ These complications, particularly, cardiovascular and renal diseases, are the leading causes of mortality among diabetic patients.⁸

Metformin is an old, inexpensive, and first-line medication for type 2 diabetes.⁹ It is a biguanide derivative reducing hepatic production of glucose and is safe in a wide range of ages. Despite its contra-indications such as high-grade heart failure, advanced CKD, lactic acidosis, and advanced chronic liver disease (CLD), its prescription increased during recent years.^10,11 Additionally, Its contra-indication for CKD patients has been limited during recent years thanks to its newly observed effects.⁹ Numerous studies showed that metformin can improve several coexisting complications of diabetes.¹²⁻¹⁵ Even it was observed that metformin can improve these diseases in nondiabetic patients.^16,17

Metformin enhances the function of liver kinase B1 (LKB-1), a serine–threonine kinase. Subsequently, it enhances the LKB-1/AMP-activated protein kinase (AMPK) pathway.¹⁸ The LKB-1/AMPK pathway is heavily involved in the regulation of metabolism, cellular energy expenditure, cell proliferation, and growth and widely interacts with other signaling pathways.¹⁹ Metformin, through AMPK, can favorably alter the course of several diseases.²⁰

In this review, the beneficial effects of metformin on several diseases is discussed, separately. In addition, the underlying molecular mechanisms leading to these effects are precisely explained. In this review, diabetes is deemed type 2 diabetes, unless otherwise mentioned.

Effect of Metformin on Inflammation and Immune System

There is an immense amount of evidence from cell culture to human studies showing that metformin can vigorously modulate inflammation that is a major propellant of metabolic syndrome and the mainstay of many chronic diseases.²¹⁻²³ Long-term (12 months) administration of metformin led to a mild reduction in C-reactive protein (CRP) in both men and women with impaired glucose tolerance.²⁴ Consistently, it was observed that use of metformin is associated with lower mortality rate and organ failure among sepsis patients.^25,26

Metformin modifies inflammation via AMPK-dependent and independent inhibition of nuclear factor kappa B (NF-κB).^27,28 P65 phosphorylation and nuclear translocation of NF-κB lead to the expression of many inflammatory mediators and have been implicated in the pathogenesis of cancer and inflammatory and autoimmune diseases.²⁹ Negative regulation of NF-κB can effectively improve several inflammatory diseases.²⁹ In addition, metformin potentiates the antioxidant defense system.^25,30

Metformin could decrease T helper 17 (Th17) differentiation in patients with rheumatoid arthritis (RA) and change T regulatory (T Reg)/Th17 balance in favor of T Reg cells. Suppression of mechanistic target of rapamycin (mTOR), signal transducer and activator of transcription 3 (STAT3), and hypoxia-inducible factor 1 (HIF-1) through AMPK has been implicated in these effects.³¹ Indeed, AMPK activates tuberous sclerosis complex 2 (TSC2), thereby inhibiting mTOR.³² Inhibition of mTOR/STAT3 by metformin could also modulate B cell response and mitigate autoimmune diseases.³³ mTOR/STAT3 signaling participates in the differentiation and maturation of effector T cells and B cells.³⁴⁻³⁶ Metformin increased T Reg cell population in the airway of asthmatic mice and confined the production of inflammatory cytokines such as tumor necrosis factor α (TNF-α).³⁷ Metformin modulates T cell response to alleviate several medical conditions. The drug promotes T Reg response and confines cytotoxic activity of T cells in autoimmune diseases and inflammation. However, it enhances cytotoxic activity of T cells and decreases tumor cell immune-evasion.^38,39 Metformin strongly promotes immune cells to identify and invade tumor cells.⁴⁰ Paradoxical behavior in different conditions makes metformin a unique drug that can improve a wide variety of medical conditions.

In order to restrict inflammatory response, metformin shifts the balance between M1 and M2 subtypes of macrophages in favor of M2 subtype. This shift in macrophages polarization decreases the release of inflammatory cytokines such as TNF-α, interleukin (IL) 1β, and IL6 and augments the anti-inflammatory response.⁴¹ Metformin also attenuates the pattern recognition receptor (PRR)-mediated inflammatory response.⁴²⁻⁴⁴ PRR signaling pathways such as those mediated by nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain containing 3 (NLRP3) and Toll-like receptor 4 (TLR4) signaling pathways have been implicated in the pathogenesis of several inflammatory and autoimmune diseases such as inflammatory bowel disease (IBD), gout, asthma, rheumatoid arthritis, psoriasis, multiple sclerosis, and atherosclerosis.^45,46 Metformin can modify innate immune response and inflammation through inhibition of TLR4, NLRP3, and other PRRs.^43,44

Moreover, metformin modulates inflammatory cell infiltration by decreasing cell adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1).⁴⁷ Treatment with metformin attenuates neutrophil-associated inflammation and suppresses the release of numerous destructive enzymes.^48,49 Metformin can decrease neutrophil extracellular traps (NET) and oxidative stress to suppress neutrophil-associated inflammatory response.¹⁷

Anti-inflammatory properties of metformin can be helpful in a wide variety of diseases. Even meta-analysis of several studies demonstrated that preadmission use of metformin is associated with significantly lower mortality rate among hospitalized COVID-19 patients (95% CI, odds ratio (OR) 0.64 (0.43–0.97)).¹² Uncontrolled activation of the immune system, dysregulated inflammatory response, and cytokine storm are the main causes of multiorgan failure, cardiovascular and respiratory collapse, and death in severe COVID-19.⁵⁰ The release of immense amounts of inflammatory cytokines such as IL1β, IL6, IL18, TNF-α, and interferon γ (INF-γ) from macrophages, dendritic cells, and T cells impairs the function of different organs such as the heart, lungs, kidneys, and liver.⁵⁰ The protective effects of metformin against inflammation and its interaction with immune cells can explain the lower mortality rate observed in COVID-19 patients who used metformin.

Use of metformin has also been associated with lower active tuberculosis (TB) incidence (95% CI, relative risk (RR) 0.51 (0.38–0.69)) and mortality (95% CI, RR 0.34 (0.20–0.57)) among diabetic patients.⁵¹ Interestingly, the drug accelerated sputum culture conversion in patients with diabetes and cavitary pulmonary tuberculosis.⁵² In response to Mycobacterium tuberculosis, metformin enhanced phagocytosis and oxidative stress, while in the absence of the stimulus, metformin suppressed oxidative stress and inflammatory cytokines such as IL1β, INF-γ, and TNF-α.⁵³ Although inflammation, infection, and tumorigenesis are interacting with the same molecular mechanisms, metformin selectively regulates signaling pathways to provide the utmost benefit.

The major effects of metformin on inflammation were briefly discussed in this section. However, metformin recruits various mechanisms to halt inflammation. The detailed interactions of metformin with numerous signaling pathways will be separately discussed for each disease.

Metformin for Cardiovascular and Renal Protection

It was observed that metformin can improve moderate CKD, congestive heart failure (CHF), and chronic liver disease (CLD) in patients with diabetes. Metformin has been associated with decreased all-cause mortality, reduced incidence of heart failure, and decreased readmission because of CKD or CHF among patients with diabetes.⁵⁴⁻⁵⁶ Additionally, metformin could decrease all-cause mortality in myocardial infarction and heart failure among patients with diabetes.^57,58 Metformin led to 21% decrease in mortality because of myocardial infarction and 16% decrease in mortality because of heart failure.⁵⁷ Recent meta-analysis showed that metformin has been associated with decreased cardiovascular mortality (95% CI, OR 0.44 (0.34–0.57)) or incidence of cardiovascular diseases (95% CI, OR 0.73 (0.59–0.90)) among patients with diabetes.⁵⁹ Similarly, metformin has been associated with lower risk of all-cause mortality (95% CI, RR 0.71 (0.61–0.84)) and cardiovascular events (95% CI, RR 0.76 (0.60–0.97)) among diabetic patients with CKD at stage G1-3.¹³ Long-term administration of metformin in patients with diabetic nephropathy has been associated with lower all-cause mortality (95% CI, hazard ratio (HR) 0.65 (0.57–0.73)) and prevented progression to end-stage renal disease (ESRD) (95% CI, HR 0.67 (0.58–0.77)).⁶⁰

The drug can protect against arterial calcification, ameliorates endothelial dysfunction, and preserves the integrity of vasculature in patients with diabetes.^61,62 Metformin accelerates endothelial nitric oxide (NO) production to improve cardiovascular outcome.⁶³ Augmentation of AMPK/endothelial NO synthase (eNOS)/NO was shown to protect against aortic smooth muscle cell calcification in rats.⁶⁴ NO, in a cGMP-dependent manner, inhibits the differentiation of vascular smooth muscle cells (VSMCs) into osteoblastic cells by decreasing transforming growth factor β (TGF-β) and attenuating TGF-β signaling. NO can also downregulate TGF-β receptor and TGF-β-induced Smad2/3 phosphorylation.⁶⁵ Consistent with its vascular protection, it was observed that diabetic patients who used metformin had a lower below-the-knee arterial calcification score.⁶¹ Activation of the AMPK/eNOS pathway by metformin improves blood flow and angiogenesis and was protective in several animal models of ischemia/reperfusion.⁶⁶⁻⁶⁸ Augmentation of the NO system after administration of metformin was associated with cardioprotection in the mice model of myocardial infarction.⁶⁹ Moreover, metformin recruits several other mechanisms to decrease the tonicity of VSMCs, which results in vasodilatation and prevents cardiovascular events.⁷⁰ Long-term (>12 months) administration of metformin for patients has been associated with a significant decrease in carotid intima-media thickness.⁷¹ Consistently, mortality and adverse cardiovascular events were less common among patients with peripheral artery disease who used metformin.⁷²

It was shown that metformin improves mitochondrial function to prevent atherosclerosis. The effect was exerted through AMPK-dependent downregulation of dynamin-related protein (Drp1).⁷³ Drp1-mediated mitochondrial fission has been implicated in cardiomyocyte injury and myocardial hypertrophy.^74,75 Furthermore, it was shown that inhibition of Drp1 can protect against myocardial infarction and prevent apoptosis.⁷⁵ Moreover, metformin through AMPK/silent mating-type information regulation 2 homologue-1 (SIRT1) signaling activates peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α).⁷⁶ PGC-1α is the chief regulator of mitochondrial biogenesis, protects the integrity of mitochondrial membrane, and improves mitochondrial respiration and myocardial energy metabolism in heart failure.⁷⁶

Metformin activates AMPK to attenuate oxidative stress and augment autophagy.^77,78 The drug attenuates mTOR signaling to promote autophagy, prevent the development and progression of atherosclerosis, and provide cardiovascular protection.⁷⁹ Autophagy is crucial for cardiovascular health. Attenuation of autophagy has been associated with disorganization of heart sarcomeres, age-related cardiomyopathy, heart failure, and shorter lifespan.^80,81 Meanwhile, metformin activates the AMPK/peroxisome proliferator-activated receptor δ (PPARδ) pathway to suppress endoplasmic reticulum (ER) stress and improve cardiac injury and endothelial dysfunction.^82,83 Uncontrolled activation of ER stress upregulates CCAAT-enhancer-binding protein homologous protein (CHOP), thereby activating apoptotic cell death.⁸⁴ AMPK/PPARδ-mediated attenuation of ER stress and oxidative stress is markedly involved in maintaining vascular health.⁸⁵ Attenuation of ER stress by metformin can protect against vascular damage in hypertension.⁸⁶

Metformin, via AMPK, could also downregulate the TLR4/NF-κB signaling pathway in a rat’s heart and prevent the release of several inflammatory cytokines such as TNF-α and IL6.⁸⁷ Sustained activation of NF-κB and subsequent production of several inflammatory cytokines can lead to endothelial and myocardial injury and heart failure in the long term.⁸⁸ Metformin, in an AMPK-dependent manner, could improve diabetic cardiomyopathy by suppressing the mTOR/NLRP3 signaling pathway.⁴² Downregulation of mTOR/NLRP3 signaling helps M2 macrophage polarization, suppresses inflammation, and promotes autophagy^43,89 (Figure 1).

Figure 1.

Protective effect of metformin on cardiovascular diseases at the molecular level. Metformin activates the LKB-1/AMPK pathway to modulate other signaling pathways. AMPK activation can enhance the eNOS/NO pathway, which leads to vasodilation and protects against fibrosis and vascular calcification. AMPK-mediated activation of SIRT1/PGC-1α and inhibition of Drp1 improves mitochondrial biogenesis and protects against mitochondrial fission. Inhibition of mTOR signaling by AMPK/TSC2 can enhance autophagy. Moreover, metformin inhibits major molecules involved in the inflammatory response such as TLR4, NLRP3, and NF-κB. Activation and nuclear translocation of NF-κB leads to the expression of several inflammatory mediators. Furthermore, metformin can attenuate ER stress through PPARδ and prevent ER-stress-mediated apoptosis.

Furthermore, metformin can increase glucagon-like peptide 1 (GLP-1) secretion.^11,90 GLP-1 receptor agonists have been associated with considerably better cardiovascular and renal outcome among patients with diabetes.⁹¹ Recent meta-analysis uncovered that GLP-1 receptor agonists could decrease cardiovascular mortality (95% CI, RR 0.88 (0.80–0.97)), major adverse cardiac events (95% CI, RR 0.88 (0.81 to 0.97)), and nonfatal stroke (95% CI, RR 0.87 (0.76 to 0.99)) among diabetic patients.⁹²

Consistent with its renoprotective effects in diabetic patients, metformin could protect against renal fibrosis in animal models. Furthermore, it could attenuate the deleterious effects of angiotensin II, aldosterone, TGF-β, and high glucose concentration on the progression of renal fibrosis. It could also hinder ER stress in the kidneys. Metformin activated AMPK to exert these effects, which were abolished in the presence of an AMPK inhibitor.⁹³ Metformin decreased the production of inflammatory and fibrogenic mediators such as MCP-1, ICAM-1, TGF-β, fibronectin, and type IV collagen in the kidney. In addition, it inhibited TGF-β/Smad3, extracellular signal-regulated kinase (ERK), and p38 mitogen-activated protein kinase (P38 MAPK) signaling pathways in the kidneys of mice.⁹⁴ Previously, it was revealed that ERK and TGF-β/Smad are involved in the development of renal fibrosis.⁹⁵ ERK and P38 MAPK signaling can aggravate tubular epithelial cell loss in damaged kidneys.⁹⁶

Metformin enhances AMPK/SIRT1-mediated inhibition of the forkhead box protein O1 (FOXO1) pathway to increase autophagy in diabetic nephropathy.⁹⁷ SIRT1 and FOXO1 are markedly involved in the pathogenesis of diabetic nephropathy. It was observed that particular polymorphisms of these molecules can increase the susceptibility to diabetic nephropathy.⁹⁸ Knockdown of SIRT1 results in podocyte dysfunction and leads to age-related changes in the kidneys. SIRT1 knockdown has been associated with increased activity of NF-κB, FOXO3, and FOXO4 in the kidneys that led to inflammation and apoptosis.⁹⁹ Additionally, SIRT1 attenuates hypoxia-inducible factor 1-alpha (HIF1α) signaling to restrict mitochondrial damage, oxidative stress, apoptosis, and fibrogenesis in the kidneys.¹⁰⁰ Potentiation of SIRT1 signaling by metformin can vigorously maintain the structure and function of the kidneys.¹⁰¹

Metformin can improve cardiovascular diseases through different mechanisms (Figure 1). Similarly, metformin modifies several signaling pathways to ameliorate renal injury and prevent apoptosis of tubular cells and substitution of fibrotic tissue. These protective effects can mitigate the annual decline in renal function. Here, we have addressed how metformin can ameliorate the leading causes of mortality among diabetic patients. Future studies should answer the question of whether metformin can replicate the same results in nondiabetic subjects or not.

Metformin and Obesity

Meta-analysis of six randomized clinical trials disclosed that metformin led to 2.23 kg weight loss (95% CI, WMD −2.23 kg (−2.84 to −1.62)) and simultaneously improved total cholesterol (−0.184 mmol/L, p < 0.001) and low-density lipoprotein (LDL) levels (−0.182 mmol/L, p < 0.001) in elderly (>60 years of age) diabetic patients.¹⁰² Another meta-analysis of clinical trials by Pu et al. revealed that metformin could significantly lower body mass index (BMI) (95% CI, WMD −0.98 kg/m² (−1.25 to −0.72)), particularly among nondiabetic patients.¹⁰³ Similarly, Sadeghi et al. revealed that use of metformin has been associated with significant decrease in BMI (95% CI, WMD −1.07 kg/m² (−1.43 to −0.72)), body weight (95% CI, WMD −2.51 kg (−3.14 to −1.89)), and waist circumference (95% CI, WMD −1.93 cm (−2.69 to −1.16)) in children and adolescents and significantly reduced body fat mass in overweight or obese patients (95% CI, WMD −1.90% (−3.25 to −0.56)).¹⁰⁴ Interestingly, it was shown that metformin led to modest weight loss in patients who used olanzapine or clozapine, thus metformin can prevent antipsychotic-induced weight gain.^14,105 Likewise, Ellul et al. showed that metformin led to 0.98, 1.83, and 3.23 kg weight loss after 4, 12, and 16 weeks of treatment in children and adolescents who used second-generation antipsychotics.¹⁰⁶ Furthermore, the drug could prevent the development of metabolic syndrome in large trials.^107,108

Administration of metformin for 6 months to nondiabetic but obese children decreased their BMI and increased their insulin sensitivity and adiponectin/leptin ratio (Figure 2).¹⁰⁹ Metformin improved leptin sensitivity in the rat model of obesity.¹¹⁰ Leptin resistance impairs endogenous mechanisms of satiety, increases food intake, and leads to weight gain.¹¹¹ Leptin decreases intracellular lipid accumulation and improves insulin sensitivity, hence leptin resistance contributes to the development of diabetes and metabolic syndrome.¹¹² Metformin increases the bioavailability of adiponectin that can maintain body composition and prevent excessive fat accumulation (Figure 2).^113,114

Figure 2.

Effect of metformin on hormonal changes and obesity. Metformin can lead to a modest weight loss or prevent weight gain. It increases insulin sensitivity, leptin sensitivity and adiponectin/leptin ratio, and reduces insulin requirement. Furthermore, it can suppress appetite by increasing GLP-1 and GDF-15 secretion and decreasing ghrelin.

As mentioned previously, the drug increases the plasma levels of GLP-1 in both diabetic and nondiabetic patients (Figure 2).^115,116 GLP-1 receptor agonists could lead to pronounced weight loss in previous clinical trials. Even, once-weekly 2.4 mg of semaglutide caused 15.3 kg weight loss after 68 weeks of use.¹¹⁷ Enterochromaffin cells release GLP-1, within a few minutes of ingestion. GLP-1 postpones gastric emptying, decreases appetite, and limits food intake. It targets several sites on the central nervous system (CNS) to exert these effects.¹¹⁸

Moreover, metformin increases growth differentiation factor 15 (GDF-15), which can decrease appetite and is associated with weight loss and cardioprotection.^119,120 GDF-15 stimulates glial cell-derived neurotrophic factor (GDNF) family receptor α-like (GFRAL) in the hind brain that leads to anorexia, nausea, and emesis.¹²¹ Lack of GDF-15 in null mice attenuated the weight lowering effect of metformin. Furthermore, GDF-15 plasma level is positively correlated with weight loss in diabetic patients.¹²⁰ GDF-15 is distinctively involved in the pathophysiology of cachexia, and cachectic cancer patients have higher levels of GDF-15.^122,123 Similarly, attenuation of GDF-15 signaling by specific antibodies ameliorated cancer-associated cachexia.¹²⁴ Metformin suppresses mitochondrial function to enhance GDF-15 release from intestinal cells.¹²⁵ It can also prolong the postprandial decrease in plasma ghrelin. Ghrelin is a positive regulator of appetite, and its absence may decrease snack eating between the main meals (Figure 2).¹²⁶

Metformin can act through different mechanisms to cause a modest weight loss (Figure 2). It modulates the cross-talk between gastrointestinal tract and the brain through GDF-15, ghrelin, and GLP-1. Furthermore, it improves leptin sensitivity and increases adiponectin. These effects can be considered as additional advantages of metformin for obese or overweight patients who are using metformin for diabetes or other reasons.

Metformin for Neuronal Damage, Neurodegenerative Diseases, and Cognitive Disorder

Diabetes and long-lasting hyperglycemia damage nerve fibers and are characterized by neuropathy and neurodegeneration.¹²⁷ Metformin (500 mg of TDS for 1 year) could improve cognitive function in patients with impaired glucose metabolism and nondementia vascular cognitive impairment.¹²⁸ Guo et al. reported that metformin not only improved cognitive performance but also alleviated depressive symptoms in patients with diabetes.¹²⁹ Metformin enhanced the antidepressant effects of fluoxetine in nondiabetic patients with major depressive disorder.¹⁶ Similarly, Shi et al. indicated that long-term use of metformin is associated with a lower risk of neurodegenerative diseases among elderly adults with type 2 diabetes (11.48 per 1000 person-years among metformin users, compared with 25.45 per 1000 person-years for nonusers).¹³⁰ Particularly, more than 6 years of treatment with metformin vigorously (95% CI, OR 0.30 (0.11–0.80)) protected against cognitive impairment among diabetic patients. Likewise, a positive correlation was observed between the duration of use and the protective effects of metformin.¹³¹ A pilot study revealed that 8 week administration of metformin brought improvement in executive functioning, learning, memory, and attention of nondiabetic patients with mild cognitive impairment or mild dementia because of Alzheimer’s disease.¹³² Primary results of another pilot study showed that use of metformin is associated with better performance than placebo on tests of declarative and working memory of survivors of pediatric brain tumors.¹³³

Metformin promotes the AMPK signaling pathway and enhances autophagy, which can vigorously improve neurological diseases and increase nerve repair.¹³⁴ There are several preclinical studies reporting that metformin can improve spinal cord injury. It accelerates autophagy via inhibition of mTOR. The drug can downregulate NF-κB signaling in the damaged foci to alleviate neuronal inflammation. Furthermore, metformin decreases the expression of caspases in the damaged segment of the spinal cord and promotes the expression of antiapoptotic proteins such as bcl-2.¹³⁵ The drug increases the expression of nuclear factor erythroid-related factor 2 (Nrf2). Nrf2 binds to antioxidant response element to promote the expression of endogenous antioxidants.¹³⁶ It was shown that metformin also activates phosphoinositide 3-kinases (PI3K)/protein kinase B (AKT) and Wnt/β-catenin signaling pathways in spinal cord injury. These signaling pathways can accelerate axon regeneration and nerve repair after spinal cord injury.^135−140 The drug also impedes microglial activation after spinal cord injury. Microglial activation strongly exacerbates neuroinflammation and causes secondary damage after spinal cord injury.¹⁴¹

Numerous experimental studies revealed that metformin is able to alleviate neuropathy through activation of AMPK. AMPK activation attenuates oxidative stress and prevents intradermal nerve fibers degeneration.^142−144 Metformin hinders the atrophy of myelinated axons and decreases the expression of inducible nitric oxide synthase (iNOS) and inflammatory cytokines such as IL1β. Interestingly, metformin augments the expression of neurotrophic factors such as myelin basic protein and nerve growth factor and increases anti-inflammatory cytokines such as IL10 and inhibitor kappa B-alpha (IκBα).¹⁴⁵

Metformin improved mechanical and cold hypersensitivity induced by oxaliplatin in rats.¹⁴⁶ Use of metformin has been significantly associated with decreased lumbar radiculopathy pain among patients.¹⁴⁷ Lin et al. reported that metformin can decrease the increased number of myelinated fibers in spinothalamic tract to reduce pain.¹⁴⁸ Moreover, it was indicated that peripheral diabetic neuropathy is associated with numerical increase of synapses in the spinal dorsal horn, and metformin can hamper this numerical increase of synapses to mitigate diabetic neuropathy.¹⁴⁹ Meanwhile, metformin partially activates the opioid system to silence neuropathic pain.¹⁵⁰ Zhang et al. revealed that metformin can decrease diabetic neuropathy and the increased levels of IL33.¹⁵¹ IL33 and its receptor play a pivotal role in the conduction of neuropathic pain.¹⁵² The drug could also ameliorate diabetic retinopathy and optic neuropathy in diabetic rats.¹⁵³ Similarly, metformin attenuated cisplatin-induced ototoxicity in mice and zebrafish.¹⁵⁴ Likewise, Das et al. observed that metformin can improve complex regional pain syndrome subsequent to tibial fracture in mice. The effect was mediated through upregulation of AMPK and downregulation of mTOR and eukaryotic initiation factor 2α (eIF-2α).¹⁵⁵ EIF-2α is a regulatory target for translation whose overactivation is an ER stressor, involved in apoptosis of neurons and neurodegeneration.^156,157 Interestingly, a recent cross-sectional study in the U.K. reported that diabetic patients using metformin were less likely to complain of back, knee, neck, shoulder, and multisite musculoskeletal pain than diabetic patients with no prior use of metformin.¹⁵⁸ Consistently, recent clinical trial performed by El-Fatatry et al. indicated that metformin can markedly alleviate oxaliplatin-induced peripheral neuropathy in patients with stage III colorectal cancer.¹⁵⁹ Taken together, metformin can enhance autophagy and ameliorate oxidative stress, ER stress, inflammation, and apoptosis in the nervous system. The neuroprotective effects of metformin can help to better the management of neuropathy, spinal cord injury, and several neurodegerative diseases that are currently without an effective treatment.

Use of metformin may be associated with increased risk of B12 deficiency and its subsequent clinical neuropathy.^160,161 B12 deficiency, which is evident in long-term use of metformin for diabetic patients, strongly correlates with diabetic neuropathy.¹⁶² Hashem et al. indicated that the dose of metformin and duration of administration are positively correlated with the severity of vitamin B12 deficiency and predict the severity of diabetic neuropathy. Moreover, metformin increases homocysteine and methylmalonic acid in patients with diabetes that can accentuate diabetic neuropathy, as well.^163,164 Metformin leads to B12 malabsorption that will be reversed by stoppage of treatment or increase in calcium intake.¹⁶⁵ B12 deficiency should be strongly considered when using metformin long-term to improve the neuroprotective effects of metformin.^166,167

Metformin and Cancer

Diabetes is associated with increased risk of several cancers such as colorectal cancer, endometrial cancer, prostate cancer, lung cancer, and hepatocellular carcinoma.^168−172 Metformin may have a dose-dependent effect on cancer prevention. Use of metformin has been associated with a 31% decrease in overall cancer risk, compared with other antidiabetic medications and insulin. The effect was statistically significant for hepatocellular carcinoma, pancreatic cancer, lung cancer, and colorectal cancer.^{15,58,173−175} Use of metformin has been associated with 23% decrease in the incidence of colorectal adenoma, 39% decrease in the incidence of advanced adenoma, and 24% decrease in the incidence of colorectal cancer among diabetic patients. Metformin similarly improved overall survival (95% CI, HR 0.6 (0.53–0.67)) and colorectal cancer-specific survival (95% CI, HR 0.66 (0.59–0.74)) among patients with colorectal cancer and concurrent diabetes.¹⁷⁶ Patients with acromegaly are at higher risk of colorectal polyps and cancers. Albertelli et al. performed a cross-sectional study to measure the association between metformin and new formation of colorectal polyps in 153 patients with acromegaly. The study uncovered that use of metformin is significantly associated with lower risk of colonic polyps (95% CI, OR 0.22 (0.06–0.77) P = 0.01) in patients with acromegaly.¹⁷⁷

Metformin could also significantly improve the incidence of lung cancer (95% CI, HR 0.78 (0.70–0.86)), the incidence of hepatocellular carcinoma (95% CI, OR 0.468 (0.275–0.799)), the incidence of pancreatic cancer (95% CI, RR 0.63 (0.46–0.86)), survival of lung cancer (95% CI, HR 0.65 (0.55–0.77)), overall survival of hepatocellular carcinoma (95% CI, OR 3.31 (2.39–4.59)), and survival of pancreatic cancer (95% CI, HR 0.83 (0.74–0.91)) among diabetic patients.^178−182 However, some systematic reviews and meta-analyses revealed that metformin may not decrease the incidence of other cancers such as breast cancer, endometrial cancer, and prostate cancer; instead, they showed that metformin can improve prognosis, overall survival, and recurrence-free survival of these cancers.^183−185 Metformin can prevent the incidence of cancers, improve response to chemotherapy and radiotherapy, hinder the relapse of cancers, and hamper their progression to malignancy.^175,186 In addition, use of metformin is associated with reduced all-cause mortality, compared to insulin and other antidiabetic drugs.⁵⁸

Although the exact antitumor mechanisms have not been disclosed yet, activation of AMPK and inhibition of proliferative signaling pathways have been implicated in this regard. Metformin inhibits the PI3K/AKT/mTOR signaling pathway, which is a major stimulator of proliferation in cancer cells.¹⁸⁷ Likewise, metformin acts through AMPK to suppress the dishevelled segment polarity protein 3 (DVL3)/Wnt/β-catenin signaling pathway.¹⁸⁸ Furthermore, some studies reported that metformin can also inhibit the Raf/mitogen-activated extracellular signal-regulated kinase (MEK)/ERK pathway.¹⁸⁹ The Raf/MEK/ERK pathway is a major proliferative pathway in tumorigenesis and interacts with growth factor receptors such as epidermal growth factor receptor (EGFR) and proto-oncogenes such as KRAS.^190,191

Uncontrolled activation of Wnt/β-catenin, PI3K/AKT/mTOR, and Raf/MEK/ERK signaling pathways is the major cause of neoplasia and leads to the development of several cancers.^{190,192−194} These signaling pathways interact with numerous growth factors and are affected by several oncogenes. Indeed, they are the effector parts of oncogenic networks and play pivotal roles in the pathogenesis of cancers, hence, their blockade may inhibit a chain of neoplastic alterations, tumor cell proliferation, migration, invasion, and metastasis.^195−202 As mentioned previously, metformin activates AMPK/TSC2 to directly inhibit mTOR. mTOR inhibition results in the autophagy of tumor cells, cell cycle arrest, and inhibition of tumor cell proliferation.³²

Metformin could augment the effect of rapamycin and cisplatin on human gastric cancer cells. It activated AMPK and subsequently inhibited the phosphorylation of mTOR and also its downstream molecules such as S6, eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1), and P70S6K.²⁰³ Metformin similarly attenuated mTOR and its downstream molecules in other cancers such as endometrial cancer to stop tumor cell proliferation.²⁰⁴ Increased phosphorylation of S6, 4E-BP1, and P70S6K following mTOR activation was observed in cancers. This signaling pathway enhances ribosomal translation of proteins and has been implicated in cancer cell growth.^205,206 These proteins are needed for cancer cell homeostasis, cell cycle progression, and proliferation of cancer cells.²⁰⁷

Previous studies illuminated that chronic inflammation is a main driver for neoplastic reactions and is associated with a higher incidence of cancers.^208,209 As mentioned, metformin possesses numerous anti-inflammatory characteristics that can inhibit cancer development and progression.^210,211 For instance, it was uncovered that metformin attenuates the NF-κB/IL6 axis in fibroblasts to inactivate fibroblast-mediated ovarian cancer progression.²¹² The drug could also hinder the cyclooxygenase 2 (COX-2)/prostaglandin E₂ (PGE2)/STAT3 axis to inhibit epithelial–mesenchymal transition (EMT) in prostate cancer.²¹³ Similarly, metformin inhibited COX-2 to decrease the migration and invasion of breast cancer cells.²¹⁴ STAT3 is a transcription factor that can expedite EMT, invasion, and metastasis of cancer cells.²¹⁵ Metformin ameliorated colitis and colitis-associated colorectal cancer in mice.²¹⁶ It also prevented progression from NAFLD to hepatocellular carcinoma by modulating inflammation in animal models.²¹⁷ Interestingly, metformin could improve chemoresistance in cisplatin- and paclitaxel-resistant tumor cells. Further measurements uncovered that attenuation of NF-κB-mediated inflammatory response in tumor cells is involved in such an effect.²¹⁸ These findings are just some examples of anti-inflammatory properties of metformin which could potentiate its anticancer effects. Hence, the anti-inflammatory property of metformin can be an advantage to counterattack cancer cells.²¹⁹

Importantly, metformin could decrease the expression of HIF-1α through AMPK, thereby downregulating pyruvate kinase M2 (PKM2) and vascular endothelial growth factor (VEGF) in cancer cells.^220,221 VEGF stimulates angiogenesis, and PKM2 increases tumor cell aerobic glycolysis, known as the Warburg effect. Angiogenesis and aerobic glycolysis expedite tumor cell proliferation and accelerate their metastasis.^220,221 Further, metformin downregulates c-Myc, which is a transcription factor for glycolytic enzymes and a major enhancer of Warburg effect. c-Myc potentiates tumor cell biosynthesis and provides the substrates for proliferation in cancer cells.²²²

Song et al. revealed that metformin downregulates the expression of Snail through activation of LKB-1/AMPK.²²³ Snail decreases E-cadherin, which is a major cell adhesion molecule, and its absence accelerates EMT, tumor cell invasion, and metastasis.²²⁴ In addition to Snail, metformin downregulates Slug and zinc finger E-box-binding homeobox 1 (ZEB1), other transcription factors, and promoters of EMT.²²⁵ Hence, metformin can downregulate the major transcription factors in cancer cells such as c-Myc, HIF-1α, STAT3, Slug, Snail, and ZEB1, thereby impairing cancer cell metabolism, proliferation, and metastasis.

Addition of metformin to conventional chemotherapy regimens could improve the efficacy of treatment with several anticancer drugs and overcome cancer cell chemoresistance. Metformin improved the efficacy of cisplatin and paclitaxel for ovarian cancer cells. It reduced chemoresistance and increased differentiation among cancer cells.²²⁶ Also, ovarian cancer cells from patients who were treated with metformin had better in vitro sensitivity to chemotherapy agents.²²⁷ Likewise, metformin enhanced sensitivity to 5-fluorouracil in rectal cancer cells. It increased tumor cell apoptosis and prevented their proliferation, migration, and invasion.²²⁸ It was found that metformin accelerates Nrf2 degradation and suppresses Nrf2-mediated expression of antioxidants in cancer cells. These effects were shown to be involved in the attenuation of chemoresistance of non-small cell lung cancer and hepatocellular carcinoma.^189,229 Nrf2 is a major transcription factor for numerous endogenous antioxidants; therefore, Nrf2 downregulation can paralyze tumor cells’ self-defense system and makes them vulnerable to apoptosis.^189,229

DNA repair contributes to the integrity of the cancer cell genome and increases cancer cell survival. Cancer cells possess high potential of DNA repair and utilize it against chemotherapy.²³⁰ Inhibitors of DNA repair can increase the efficacy of cancer chemotherapy and decrease chemoresistance.²³¹ Metformin significantly reduces the expression of DNA-repairing proteins in cancer cells.²³² Inhibition of DNA repair by metformin can enhance the effect of radiotherapy and chemotherapeutic drugs.^232,233

It was uncovered that metformin can upregulate PTEN and P21 in gastric and lung cancer cells.^203,234 P21 is downstream of P53 and a potent cyclin-dependent kinase (CDK) inhibitor whose upregulation leads to cell cycle arrest on the G1/S phase. CDKs are critical regulators of the cell cycle. Their overactivity is involved in cancer pathogenesis, and their inhibition can effectively contribute to the treatment of cancer.²³⁵ PTEN is an important tumor suppressor which effectively attenuates proliferative signaling of the PI3K/AKT pathway. Loss of PTEN function due to its methylation, genetic mutation, and deletion correlates with a higher risk of cancers and aggressive and metastatic phenotypes of cancer cells.²³⁶ Metformin upregulates PTEN through AMPK activation.²³⁷ Interestingly, metformin decreases the secretion of IGF-1 and the expression of growth factor receptors such as the IGF-1 receptor. Stimulation of the IGF-1 receptor results in the activation of the PI3K/AKT/mTOR pathway. Metformin targets this growth and proliferation signal from different points.^238,239 Therefore, metformin can partly inhibit cell cycle progression and collaborate with oncosuppressors to attenuate the proliferation of tumor cells and decrease their invasiveness.

Interestingly, it was uncovered that metformin affects the bioavailability of noncoding RNAs, which are heavily involved in the pathogenesis of cancer. For instance, the inhibitory effects of metformin on breast cancer cell viability was associated with increased expression of miR-26a. miR-26a overexpression was associated with decreased viability of breast cancer cells, and miR-26a deletion partly attenuated the inhibitory effect of metformin on breast cancer cell viability.²⁴⁰ Metformin also upregulated miR-483-3p to increase the expression of p21.²⁴¹ Metformin via downregulation of miR-222 could increase the protein abundance of p27, p57, and PTEN in human lung cancer cells and prevent cancer cell proliferation.²³⁴ P27 is a CDK inhibitor encoded by CDKN1B. Lack of P27 is a common finding in most human epithelial cancer cells, and augmentation of its function improves the outcome of cancer chemotherapy.²⁴² Similar to P21 and P27, P57 is a CKD inhibitor and a tumor suppressor that negatively regulates cell cycle progression, intratumor angiogenesis, and metastasis in cancer.²⁴³ P57 also promotes tumor cell differentiation and apoptosis.²⁴³ Long noncoding RNA H19 was shown to be involved in the inhibitory effects of metformin on the invasive behavior of human gastric cancer cells. Additionally, after deletion of long noncoding RNA H19, metformin could no longer activate AMPK or decrease matrix metalloproteinase 9 (MMP-9).²⁴⁴ It seems that metformin recruits many noncoding RNAs to modulate several signaling pathways.

Metformin can potentiate the immune system to attack immune cells. It decreases the differentiation of T Reg cells and reduces their population in the tumor microenvironment. Diminished population of T Reg cells accelerates T-cell-mediated cytotoxicity and removal of tumor cells.²⁴⁵ Interestingly, metformin enhanced the antitumor effects of chimeric antigen receptor (CAR) T cells via AMPK.³⁹ Metformin, in an AMPK-dependent manner, decreases the expression of program cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) to prolong the CD8+ T cell lifespan, thereby increasing their antitumor efficacy.^39,246 Metformin also improves the antitumor activity of PD-1 inhibitors.²⁴⁷ Consistently, using metformin as an additive treatment for head and neck squamous cell carcinoma resulted in a significant increase in CD8+ population and decrease in T Reg cell population in the tumor.²⁴⁸

Metformin is a safe drug, with wide variety of interactions with numerous oncogenic and tumor-suppressing pathways which make it a good choice for cancer prevention and treatment (Figure 3).

Figure 3.

Underlying mechanisms involved in the protective effects of metformin against cancers. Metformin hinders the development of cancers and inhibits cancer cells proliferation, invasion, and metastasis through different mechanisms. The drug attenuates cancer cell proliferation through inhibition of major proliferative pathways such as PI3K/AKT/mTOR, DVL3/Wnt/β-catenin, and Raf/MEK/ERK. Simultaneously, metformin increases the expression of tumor suppressors such as PTEN, P21, P27, and P57. These tumor suppressors inhibit CKD that results in cell cycle arrest. Furthermore, metformin downregulates Snail, Slug, and ZEB1 to maintain E-cadherin expression and prevent cancer cell metastasis. Interestingly, metformin decreases PD-1 and PD-L1 and impairs tumor cell immune-evasion. Metformin also attenuates the growth signals mediated by EGFR and IGF1 receptors. The drug downregulates Nrf2, thereby impairing tumor cells’ antioxidant defense and improving chemoresistance. Furthermore, metformin can reduce the expression of HIF-1α and c-Myc. HIF-1α and c-Myc can promote the expression of glycolytic enzymes, enhance intratumor angiogenesis, and increase tumor cell invasion and metastasis.

Polycystic Ovarian Syndrome (PCOS) and Metformin

Adolescent PCOS is associated with greater risk of metabolic syndrome particularly among overweight/obese patients.^249,250 Use of metformin (1700 mg/day for 6 months) for adolescent girls with PCOS improved menstrual cycles and made them ovulatory. In addition, metformin ameliorated patients’ BMI and their hyperandrogenic symptoms such as hirsutism and acne and reduce their total testosterone, androstenedione, and free testosterone levels.^251,252 The modulatory effects of metformin on the hormonal and metabolic status of patients with PCOS have been associated with clinical improvement in ovulation, pregnancy rate, and androgenic symptoms.²⁵³ Metformin increased the clinical pregnancy rate among patients with PCOS and BMI ≥26 who underwent in vitro fertilization.²⁵⁴ Three months of pretreatment with metformin effectively increased pregnancy rate and live birth rate in both obese and non-obese PCOS patients who had non-ovulatory menstrual cycles.²⁵⁵ Metformin prevented early pregnancy loss, preterm labor, and pregnancy complications such as gestational diabetes mellitus (GDM) and pregnancy-induced hypertension (PIH) and preeclampsia in pregnant women with PCOS.^256−259 Moreover, metformin lowered the chance of neonatal hypoglycemia and being large for gestational age (LGA)²⁶⁰ (Figure 4). Contrary to insulin, metformin can freely pass through the placenta, hence, the fetus will be exposed to the concentrations of metformin comparable to its therapeutic concentrations.^261,262 According to available evidence, metformin is safe during gestation and currently, further studies are measuring its safety during pregnancy.^256,257 Although, a follow-up study reported that use of metformin for patients with PCOS has been associated with a higher risk of being overweight among 4 year old children, which should be considered and further investigated.²⁶³

Figure 4.

Effect of metformin on different dimensions of PCOS. The effects of metformin on PCOS is not restricted to its beneficial effects on metabolic syndrome. Metformin can mitigate infertility and pregnancy complications. The drug improves anovulation, pregnancy loss, PIH, preeclampsia, LGA, neonatal hypoglycemia, and preterm labor. In addition, it was observed that metformin can decrease androgens and hyperandrogenism symptoms such as hirsutism and acne.

Activation of the ovarian AMPK/SIRT1 signaling pathway has been implicated in the beneficial effects of metformin on ovulation and hormonal changes.²⁶⁴ Impaired function of the AMPK/SIRT1 signaling pathway in granulosa cells is associated with insulin resistance and hyperandrogenism. Likewise, activation of the AMPK/SIRT1 pathway modulates insulin resistance and modifies hormonal status.^265,266 AMPK activation by metformin also led to suppression of mTOR signaling and decreased the expression of MMP-2 and MMP-9 in the animal model of PCOS.²⁶⁷ Previously, it was reported that patients with PCOS have considerably higher levels of MMP-2 and MMP-9, which may be involved in menstrual irregularity or higher risk of cardiovascular events.^268,269 Furthermore, metformin upregulates the expression of implantation-associated genes.²⁷⁰ It was observed that metformin interacts with noncoding RNAs to regulate the expression of these genes.²⁷¹

It was uncovered that higher expression of endometrial progesterone receptor is associated with anovulation in PCOS patients.²⁷² Indeed, higher expression of endometrial progesterone receptor implies an underlying progesterone resistance in PCOS patients that can progress to endometrial hyperplasia.²⁷³ Metformin can improve progesterone resistance in PCOS and decrease the expression of the endometrial progesterone receptor.^270,273,274 Metformin also attenuates androgen-mediated endometrial inflammation. It inhibits endometrial TLR4 and NF-κB signaling and prevents the release of inflammatory cytokines such as INF-γ and TNF-α in the endometrium.⁴⁴

Metformin markedly ameliorates PCOS and its complications. It is relatively safe during pregnancy, increases the chance of a successful pregnancy, and minimizes maternal and fetal complications.

Metformin and Osteoporosis

Interestingly, metformin has been associated with decreased risk of fracture (95% CI, RR 0.82 (0.72, 0.93)) among diabetic patients, indicated by meta-analyses of several observational studies.^275,276 Eighteen months use of metformin could significantly increase bone mineral content and bone mineral densitometry (BMD) in overweight patients with type 2 diabetes.²⁷⁷ Tseng et al. showed that long-term use of metformin, particularly for more than 2 years, not only prevents osteoporosis but also protects against vertebral fracture.²⁷⁸ Blumel et al. assessed the association between metformin and osteoporosis among Latin American adult women aged 40 or more who did not use antiosteoporotic drugs. The study uncovered that metformin use has been associated with a lower risk of osteoporosis, regardless of the presence of diabetes, obesity, and other factors.²⁷⁹ Interestingly, it was observed that metformin reduces the incidence of osteoporosis among patients with carcinoma in situ.²⁸⁰ It seems that metformin has several benefits for cancer patients including decreasing cancer incidence and facilitating cancer treatment, improving chemotherapy-induced neuropathy, and preventing osteoporosis among patients.

Metformin ameliorated glucocorticoid-associated osteoporosis in rats.²⁸¹ Use of metformin was associated with increased levels of osteocalcin, collagen type I, and OSTERIX in the rat model of osteoporosis. These proteins are markers of osteoblastic activity and bone formation. Similarly, higher BMDs were found in metformin-treated rats.^281,282 In addition, metformin increased osteoblast proliferation and decreased their apoptosis in cell culture.²⁸³ Metformin increases the expression of runt-related transcription factor 2 (RUNX-2) through AMPK. RUNX-2 promotes osteoblast differentiation.²⁸⁴ RUNX-2 is a key factor to induce a group of genes involved in the differentiation of osteoblast progenitor cells into osteoblasts.²⁸⁵ AMPK-mediated expression of bone morphogenetic protein 2 (BMP-2) and eNOS was also shown to be involved in osteoblastic differentiation after administration of metformin.²⁸⁶ BMP-2 is an upstream of RUNX-2 and promotes osteoblastogenesis. Activation of the BMP-2/RUNX-2 pathway leads to the expression of osteoblastogenesis-related proteins such as osteocalcin and OSTERIX.²⁸⁷ Moreover, Mai et al. uncovered that metformin can enhance the expression of osteoprotegerin (OPG), suppress the expression of receptor activator of nuclear factor kappa-B ligand (RANKL), and decrease osteoclast differentiation in cell culture and ovariectomized rats. Despite OPG, RANKL is a positive regulator of osteoclastogenesis. Increased OPG/RANKL ratio reduces osteoclast differentiation, inhibits bone resorption, and prevents osteoporosis.^288,289 It was revealed that SIRT6 plays a critical role in antiosteoporosis effects of metformin. It was shown that knockout of SIRT6 reverses the beneficial effects of metformin on OPG, RANKL, and RUNX-2.^282,290 SIRT6 is a histone deacetylase and regulates gene expression and glucose metabolism.²⁹¹ Previously, it was shown that SIRT6 promotes osteogenic differentiation of bone marrow mesenchymal stem cells via inhibition of the NF-κB signaling pathway.²⁹² Consistently, SIRT6 deficiency has been associated with osteopenia in the animal model.²⁹³ Metformin protects bone mesenchymal stem cells against oxidative stress and promotes their autophagy. These cells are the precursors of osteoblasts and needed for osteoblastogenesis.¹⁴⁸ Similarly, it was shown that metformin can prevent hyperglycemia-induced apoptosis of osteoblasts through attenuation of the TLR4/MyD88/NF-κB signaling pathway.²⁹⁴ In addition to protecting mesenchymal stem cells, metformin promotes their differentiation to osteoblasts and simultaneously hinders their differentiation into adipocytes.²⁹⁵ Hence, metformin can prevent osteoporosis by protecting osteoblasts, improving their differentiation, and decreasing osteoclast function (Figure 5).

Figure 5.

Underlying molecular mechanisms involved in the protective effects of metformin on osteoporosis. Metformin protects against MSC damage. The drug accelerates osteoblastogenesis through promoting the AMPK/SIRT6/BMP-2/RUNX-2 pathway. Moreover, metformin promotes the expression of OPG and downregulates RANKL/RANK/NF-κB pathway to decrease osteoclastogenesis.

Metformin for Periodontitis

Recently, meta-analysis of several studies has shown that locally delivered metformin particularly with a gel concentration of 1% can markedly improve the management of chronic periodontitis and significantly increase treatment response.^296,297 It was observed that metformin 1% gel can improve probing depth (95% CI, WMD 2.12 mm (1.83–2.42)) and clinical attachment level (95% CI, WMD 2.29 mm (1.72–2.86)). These findings show the efficacy of metformin 1% gels to enhance the effect of mechanical periodontal therapy for periodontitis.²⁹⁷

Metformin could increase the decreased levels of AMPK and attenuate NF-κB and NIMA-related kinase 7 (Nek7)/NLRP3 signaling pathways in the animal models of periodontitis. Thereby, metformin suppressed the release of several inflammatory cytokines such as IL-1β, IL18, and TNF-α and prevented apoptosis and inflammasome-associated pyroptosis of periodontal cells.^298−301 Moreover, metformin promoted autophagy and suppressed oxidative stress in periodontal ligament cells.^148,302 Intracanal use of metformin inhibited the upregulated iNOS/NO system and prevented monocyte infiltration in periodontitis.³⁰³ iNOS overactivity is a strong activator of osteoclasts and facilitates alveolar bone loss.³⁰⁴ Interestingly, metformin protects against dental stem cell damage and preserves the regenerative capacity of periodontal tissue.³⁰⁵ Metformin enhances Nrf2 function during periodontitis to protect periodontal stem cells against oxidative stress.³⁰⁶ Nrf2 signaling has been implicated in the attenuation of oxidative stress and osteogenic differentiation of periodontal ligament stem cells.^307,308

Additionally, metformin downregulates the RANK/RANKL signaling pathway and increases OPG to decrease bone resorption.³⁰⁰ As mentioned, decreased RANKL/OPG ratio can hinder osteoclast differentiation and maintain bone structure.³⁰⁹ The NLRP3 inflammasome promotes osteoclast differentiation, augments alveolar bone resorption and exacerbates periodontitis.³¹⁰ Therefore, the inhibitory effect of metformin on the NLRP3 inflammasome not only alleviates inflammation but also prevents osteoclast differentiation.²⁹⁸ The drug also promotes the osteogenic differentiation and facilitates the proliferation and migration of osteocytes in the periodontal ligament, which may contribute to regeneration of periodontal tissue.³¹¹ Furthermore, use of metformin has been associated with decreased expression of MMP-9.³⁰⁰ MMPs play a critical role in the degeneration of periodontal soft and hard tissues, leading to the progression of periodontitis.³¹² Salivary MMPs, particularly MMP9, have been proposed as markers of periodontal inflammation and periodontitis.³¹³

Metformin 1% gel has been strongly proposed as a protective agent in the management of chronic periodontitis. Preclinical studies showed that metformin can mechanistically improve periodontitis, as well. These findings support the use of metformin for periodontitis.

Metformin for IBD and Colitis

Recently, it was reported that patients with type 2 diabetes who received metformin were at lower risk of IBD (95% CI, HR 0.55 (0.51–0.60)), compared with those whom never received this drug. Interestingly, longer duration of use has been associated with lower risk of IBD. Use durations of <26.0 months, 26.0–58.3 months, and >58.3 months were associated with HR 1.00 (0.93–1.09), HR 0.57 (0.52–0.62), and HR 0.24 (0.22–0.26) risk of IBD, respectively. Furthermore, metformin decreased the risk of IBD in combination with insulin and other antidiabetic medications.³¹⁴ Recently, a study with 30 patients with ulcerative colitis and 10 healthy subjects revealed that metformin (850 mg, BID, for 3 months) significantly improved patients’ symptoms such as diarrhea, bloody stool, and abdominal pain. Further, metformin markedly decreased colon endoscopic scores, histological disease scores, erythrocyte sedimentation rate, tissue malondialdehyde, tissue myeloperoxidase, tissue TNF-α, and increased catalase.³¹⁵

Metformin similarly alleviated colitis in animal models.³¹⁶ Metformin upregulated the TFG-β signaling pathway and increased CD4+ Foxp3+ T Reg cell abundance in the animal models of colitis. The drug also reduced Th17 population and downregulated IL17 expression.^316,317 Aberrant function or overactivity of T cells, particularly Th17 cells, is the mainstay of inflammatory response in IBD. Th17 cells respond to extracellular bacteria and provoke inflammatory response.³¹⁸ Inflamed mucosa is also recognized by decreased abundance of T Reg cells.³¹⁸

The ameliorative effects of metformin on colitis and colitis-associated colorectal cancer has been associated with inhibition of NF-κB, as one of the main drivers of inflammation. Metformin improved mitochondrial function, as well.²¹⁶ Metformin could decrease the severity of colitis even in IL10^–/– mice via activation of AMPK and inhibition of NF-κB.³¹⁹ In addition to NF-κB, metformin attenuates intestinal inflammatory response through inactivation of mTOR, TLR4, and NLRP3. The drug also negatively regulates heat shock protein 90 (HSP90) in intestinal inflammation.³²⁰ Each one of these molecules has been implicated in the initiation or exacerbation of colitis, and their overactivation endangers mucosal integrity. Interestingly, metformin modulates all of them to alleviate intestinal inflammation. Moreover, metformin enhances the expression of Beclin-1 in colitis, which positively regulates autophagy.³²⁰

JNK and p38 MAPK signaling pathways are the other inflammatory pathways in IBD and colitis that can be attenuated by metformin.^321,322 JNK is a major promoter of inflammatory response and leads to apoptosis and disruption of the intestinal epithelial barrier. Likewise, JNK inhibitors were shown to be protective against colitis.³²³ P38 MAPK is markedly overexpressed in the intestinal mucosal cells of patients with IBD, and pharmacological attenuation of P38 MAPK could bring remission for patients IBD in previous studies.³²⁴ Pandey et al. indicated that metformin decreases COX-2 and iNOS expression to improve colitis and maintain mucosal integrity.³²⁵ Previously, it was shown that inhibition of P38 MAPK decreases COX-2 and iNOS expression and modulates experimental colitis.³²⁶ Moreover, COX-2 plays a crucial role in the pathogenesis of colorectal cancer, and COX-2 inhibitors can modify the risk of colorectal cancer.^327,328 Similarly, it was observed that iNOS is overexpressed in colitis and colorectal cancer.³²⁹

Metformin activates the LKB-1/AMPK axis, thereby reprogramming a wide variety of intracellular signaling pathways.²¹⁶ Patients with IBD have lower amounts of activated AMPK in their mucosal cells, and treatment with metformin can increase it.³²² Consistently, it was revealed that phosphorylation of AMPK is negatively correlated with the severity of colitis in the dextran sulfate sodium colitis model.³³⁰ AMPK activation by metformin restores the integrity of intestinal epithelial barrier through upregulation of tight junction proteins.³³⁰ Metformin promotes the expression of tight junction proteins such as ZO-1, occludin, claudin-1, claudin-3, and E-cadherin to prevent colitis or alleviate it.^330−332

Treatment with metformin led to lower expression of caspase-1, caspase-3, and MCP-1 in the in rat model of colitis. Decreased expression of caspases prevents intestinal mucosal cells apoptosis, and diminished expression of MCP-1 hinders immune cell infiltration into inflamed foci.^331,333 Furthermore, the drug decreased the release of IL-1β, IL6, IL18, TNF-α, and IFN-γ and reduced M1 macrophage abundance.^331,333 Metformin upregulated glutathione peroxidase (GPx) 4 activity and reduced myeloperoxidase activity and lipid peroxidation in the colon.³²⁰ The drug leads to activation and nuclear translocation of Nrf2 in colitis, thereby promoting the expression of endogenous antioxidants and suppressing oxidative stress.³³² Interestingly, metformin changed gut microbial composition. These alterations in gut microbial composition improved body weight, glycemic control, and decreased intestinal inflammation.^334,335

Furthermore, the drug could decrease the increased activity of sphingosine kinase 1 (SPHK1) and the concentration of sphingosine 1 phosphate (S1P) in intestinal inflammation.³³³ SPHK1 produces S1P. SPHK1 overexpression and activation of SPHK1/S1P/S1P receptor have been implicated in the pathophysiology of colitis and colorectal cancer. Consistently, SPHK1 inhibitors have been developed and showed anti-inflammatory and anticancer effects.³³⁶ S1P is involved in the activation of NF-κB and STAT3.³³⁷ STAT3 has mutual interactions with NF-κB, which results in gastrointestinal tract inflammation and carcinogenicity.³³⁸ Interestingly, it was also revealed that metformin inhibits STAT3 to mitigate colitis.³¹⁷

Metformin can modify immune response, suppress inflammation, prevent intestinal lining cells apoptosis, and improve the integrity of mucosal barriers in IBD. These characteristics makes it a good additive therapeutic option for IBD and colitis.

Metformin and Senescence

Aging is accompanied by remarkable changes in cellular and molecular levels. These changes results in reprograming of many signaling pathways and shifts the equilibrium between restorative and destructive mechanisms, in favor of destructive mechanisms. The manifestation of these alterations in molecular and cellular levels is cancer, chronic inflammatory and noninflammatory diseases, decreased functional capacity, and frailty.³³⁹ Cellular senescence is associated with increased production of inflammatory mediators, oxidative stress, mitochondrial dysfunction, ER stress, imbalance between apoptosis and autophagy, telomere shortening, DNA damage, genomic instability, stem cell exhaustion, and neurodegeneration.^340−343

Because of its beneficial effects on several dimensions of age-related diseases, metformin has been proposed as an antiaging drug.³⁴⁴ This review mentioned how metformin can ameliorate chronic inflammation and protect against age-related comorbidities such as neuronal damage and neurodegenerative disorders, osteoporosis, periodontitis, cancer, cardiovascular diseases, and CKD. These medical conditions account for a great proportion of age-related complications and morbidities. Moreover, metformin accelerates self-renewal mechanisms such as autophagy and prevents several pathologic and destructive changes.³⁴⁴

Autophagy provides the opportunity to degrade aggregated and misfolded proteins and remove damaged or dysfunctional organelles. Dysregulation of autophagy is accompanied by neurodegenerative diseases, cardiovascular diseases, and age-related cellular damage in different organs.^345,346 Autophagy is negatively regulated by mTOR.³⁴⁷ The AMPK/TSC2 pathway activation by metformin effectively attenuates mTOR signaling and promotes autophagy.¹³⁸

Metformin enhances mitophagy in diabetic patients.³⁴⁸ Mitophagy is the selective removal of damaged and dysfunctional mitochondria. Mitophagy is involved in several physiological and pathological processes and can protect against aging, diabetes, cancers, and neurodegenerative diseases.³⁴⁹ Metformin, in an SIRT3/PTEN-induced kinase-1 (PINK1)-dependent manner, can enhance Parkin-dependent mitophagy.³⁵⁰ In response to mitochondrial damage, the mitochondrial kinase, PINK1, phosphorylates Parkin. Parkin translocation onto the mitochondrial membrane and subsequent ubiquitin chain formation leads to lysosomal degradation of damaged mitochondria.³⁵¹ Consistently, Parkin overexpression has been proposed as a promoter of lifespan prolongation and protector against neurodegenerative diseases.³⁵² Metformin also activates AMPK/PGC-1α pathway, which is necessary for competent mitochondrial biogenesis.³⁵³ Improved mitochondrial biogenesis can heavily ameliorate metabolic syndrome, cardiovascular diseases and neurodegenerative diseases.^354,355

It was demonstrated that age-related mutation in mitochondrial DNA markedly accelerates apoptosis. Similarly, it was shown that apoptosis is associated with aging, and apoptotic markers are increased in older age.³⁵⁶ Metformin, in a AMPK-dependent manner, suppresses mitochondrial ROS production and inhibits JNK signaling to prevent apoptosis.^357,358 In response to the stress signal, JNK leads to apoptosis and pharmacological inhibition of JNK prevents apoptosis.³⁵⁹ Moreover, metformin can attenuate ER stress to prevent organ damage.^86,360 ER stress vigorously activates JNK signaling, leading to apoptosis.³⁶⁰

Metformin modulates gene expression by exerting epigenetic changes. Indeed, metformin via AMPK and SIRT1 selectively alters gene expression.³⁶¹ Metformin can activate SIRT1 signaling, which strongly inhibits aging-related pathways.^97,101,362 SIRT1 inactivation accelerates the aging process.³⁶³ SIRT1 promotes autophagy and inhibits FOXO1 signaling to attenuate oxidative stress. Furthermore, SIRT1 prevents mitochondrial damage and suppresses NF-κB signaling, thereby decreasing the release of inflammatory cytokines and abrogating apoptosis.³⁶⁴ Regulation of SIRT1 enables metformin to modify several pathological processes. For instance, metformin augments SIRT1 signaling to modulate p53/p21-mediated apoptosis.³⁶³ Metformin promotes AMPK/SIRT1 signaling to enhance PGC-1α expression. Subsequently, AMPK/SIRT1/PGC-1α pathway improves mitochondrial function and autophagy and hampers apoptosis.³⁶⁵ The SIRT1/PGC-1α pathway is a major regulator and promoter of autophagy and mitophagy.³⁶⁶

Aging is associated with mitochondrial and nuclear DNA damage. Accumulation of oxidative damage in DNA, particularly in mitochondrial DNA, shortens cellular lifespan and accelerates cellular aging.³⁶⁷ Metformin attenuates oxidative stress to protect against DNA damage and genomic instability.³⁶⁸ Metformin, in an Nrf2-dependent manner, promotes the expression of several antioxidants such as GPx7.³⁶⁹ Potentiation of Nrf2/GPx7 axis by metformin was associated with enhanced antioxidant capacity and longevity of human diploid fibroblasts and human mesenchymal stem cells.³⁶⁹ In addition, it was observed that metformin activates an intracellular ataxia telangiectasia mutated (ATM)/checkpoint kinase 2 (CHK2) checkpoint to promote DNA-damage response (DDR).³⁷⁰ Enhancement of ATM-mediated DDR by metformin not only improves DNA repair in senescence but also improves cancer treatment. ATM activation decreases tumor progression and facilitates cancer cell apoptosis.³⁷¹ ATM is a member of kinases involved in DNA repair. These kinases phosphorylate several proteins to repair the damage when possible; otherwise, apoptosis happens. Furthermore, ATM is needed for preventing telomere shortening and genomic instability in stem cells.³⁷² Consistently, it was observed that metformin could partly prevent placental telomere shortening in GDM patients.³⁷³ Similarly, it was shown that metformin can prevent telomere shortening in diabetic patients particularly in patients with mild age-related diabetes.³⁷⁴ After 18 months of treatment, the drug could also improve telomere length in adolescent girls with hyperinsulinemic androgen excess.³⁷⁵

Interestingly, it was demonstrated that metformin or fasting can prevent stem cell exhaustion. They preserve the capacity of stem cells to respond to pro-differentiation signals.³⁷⁶ For instance, metformin could potentiate the capability of oligodendrocyte progenitor cells to produce mature oligodendrocytes and remyelinate the CNS.³⁷⁶ Pavlidou et al. indicated that inactivation of mTOR/ribosomal protein S6 (RPS6) propels satellite cells in a quiescent period.³⁷⁷ Satellite cells are responsible for muscle fiber regeneration, and metformin may help to preserve their regenerative capacity and prevent their exhaustion during repeated regeneration cycles.³⁷⁷ Previously, we have mentioned that metformin activates LKB1 and subsequently activates its downstream AMPK. It was shown that LKB1 deletion in adult mice resulted in exhaustion of hematopoietic stem cells, severe pancytopenia, and death.³⁷⁸ Indeed, LKB1/AMPK pathways are crucially involved in stem cell quiescence, and their deletion leads to stem cell depletion.³⁷⁸

Older age is associated with fibrotic changes in different organs such as the kidneys, liver, and heart.³⁷⁹ Similarly, aging is associated with fibrogenic conversion of stem cells at the cellular level. Aged fibroblasts and extracellular matrix provoke these fibrogenic alterations in stem cells.³⁸⁰ Metformin effectively prevented TGF-β-induced fibrosis of endothelial stem cells, epithelial progenitor cells, and mesenchymal stem cells.^379,381,382 This protective effect of metformin can prolong the lifespan and functional capacity of stem cells (Figure 6).

Figure 6.

Protective effects of metformin on cellular senescence, other than its protective effects on inflammation, cancer, cardiovascular, renal and neuronal diseases. Metformin reprograms intracellular signaling pathways to exert its antiaging effects. Metformin activates several mechanisms to prevent ER stress, oxidative stress, apoptosis, and stem cell exhaustion and improves mitochondrial biogenesis and autophagy. Additionally, metformin activates the SIRT3/PINK1/Parkin signaling pathway to enhance mitophagy. Meanwhile, it activates DDR through ATM/CHK2. DRR can prevent tumorigenesis, genomic instability, and telomere shortening.

However, metformin protects normal cells against apoptosis and DNA damage, and it can oppositely enhance the apoptosis of tumor cells or prevent their DNA repair. Metformin also promotes excessive autophagy of tumor cells to destroy them.^232,233,383 These paradoxical behaviors of metformin in different conditions are interesting and make it unique.

Conclusion and Future Direction

Numerous cell culture studies, animal studies, and clinical trials revealed that metformin can improve inflammation, obesity, cardiovascular and renal diseases, IBD, cancers, PCOS, osteoporosis, and periodontitis. Moreover, various molecular interactions were shown to be involved in such effects. Future studies should determine the effect of metformin in nondiabetic patients who are suffering from these diseases. Finally, it is needed to assess the effect of metformin on the prevention or treatment of similar medical conditions such as autoimmune diseases.

Author Contributions

Moein Ala had the idea for this article. Moein Ala and Mahan Ala performed literature search and wrote the article. Moein Ala critically edited the article.

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

The authors declare no competing financial interest.