The development and benefits of metformin in various diseases

Abstract

Metformin has been used for the treatment of type II diabetes mellitus for decades due to its safety, low cost, and outstanding hypoglycemic effect clinically. The mechanisms underlying these benefits are complex and still not fully understood. Inhibition of mitochondrial respiratory-chain complex I is the most described downstream mechanism of metformin, leading to reduced ATP production and activation of AMP-activated protein kinase (AMPK). Meanwhile, many novel targets of metformin have been gradually discovered. In recent years, multiple pre-clinical and clinical studies are committed to extend the indications of metformin in addition to diabetes. Herein, we summarized the benefits of metformin in four types of diseases, including metabolic associated diseases, cancer, aging and age-related diseases, neurological disorders. We comprehensively discussed the pharmacokinetic properties and the mechanisms of action, treatment strategies, the clinical application, the potential risk of metformin in various diseases. This review provides a brief summary of the benefits and concerns of metformin, aiming to interest scientists to consider and explore the common and specific mechanisms and guiding for the further research. Although there have been countless studies of metformin, longitudinal research in each field is still much warranted.

Keywords

metformin / metabolism / cancer / aging / neurological disorder

1 Introduction

Metformin, a first line oral therapy, is the most widely prescribed clinical drug for the treatment of type 2 diabetes (T2D) [1]. Metformin inhibits glucose production in liver by increasing insulin sensitivity in the peripheral tissues, resulting in elevated glucose uptake and consumption by skeletal muscle and adipose tissues [2]. It is accepted that the central anti-diabetes mechanism of metformin is to inhibit the mitochondrial respiratory-chain complex I, resulting in the decrease of ATP and the increase of AMP. The transient decrease in cellular energy promotes the activation of AMPK, a cellular energy sensor and the target of metformin, to further impair the overall synthesis of glucose, lipid, and protein, but increases fatty acids usage [3,4].

Metformin originates from Galega officinalis, which has been served as a traditional medicine for treatment of worms, epilepsy, fever and pestilence in medieval Europe, and the first-known literature was published in Culpeper’s Complete Herbal in the 18th century, around that time G. officinalis was described for reducing thirst and frequent urination [5–7]. In the 20th century, isolation and chemical analysis of the plant identified guanidine and related compounds, and guanidine was later reported to reduce blood glucose in rabbits and dogs, but initial optimism was overshadowed by disappointment because of toxicity profile in clinical use [5,8–11]. In the early 20th century, diguanide (such as synthalin A and synthalin B) and biguanides (such as phenformin, buformin and metformin) with less toxicity, lower blood glucose and more effective were synthesized for diabetes treatment [12–16]. The use of diguanides declined in the 1930s and had been forgotten for decades with their toxicity and the availability of insulin [17–21]. Despite that, there is always a way out, and renewed attention was paid to metformin for diabetes treatment because of accidental reports to lower blood glucose in malaria and influenza treatment. In the 1940s, a guanidine derivative, proguanil, was first introduced for malaria treatment, as well metformin was reported to be helpful for influenza outbreak in Philippines, thus interest was sparked for metformin to reduce blood glucose in some of the influenza patients [22,23]. Breakthrough for metformin occurred in the 1950s that Jean Sterne, a French physician-scientist, conducted the first human experiments of metformin and coined the compound “glucophage” (meaning glucose eater), igniting the further research and promoting the clinical translation of metformin. Sterne published his finding that is now widely known as a landmark paper of metformin for diabetes treatment [24,25]. Biguanides became the most prescribed glucose-lowering drugs worldwide. Although approved in the UK in 1958 and in Canada in 1972, metformin was initially of little clinical interest and subjected to doubts because phenformin and buformin, more effective biguanides in clinical use, were rediscovered, published and approved around the same time, but withdrawn from market in 1978 with inducing fatal lactic acidosis [26–29]. To prove the hypoglycemic effects and low toxicity of metformin, the famous UKPDS study (UK Diabetes Prospective Study), a prospective randomized trial of 5100 T2D patients, was conducted to note the long-term benefits of glucose-lowering and cardiovascular protection of metformin. The study was an epoch-making milestone in the history of diabetes treatment, started in 1977 and ended in 1997; median follow-up was 10 years, which brought metformin to the first line for type 2 diabetes treatment [30–33]. Thus, metformin was approved by FDA in 1994 and introduced in 1995 in the USA. In 2013, metformin was accepted by WHO Model List of Essential Medicines (Tab.1). Besides hypoglycemic effects, metformin also has several other non-FDA-approved indications, including gestational diabetes, antipsychotic-induced weight gain, polycystic ovary syndrome (PCOS), Alzheimer’s disease, and various types of cancer [34–39].

In this review, we present the pharmacokinetics and pharmacology of metformin, discuss the relation between metformin with systemic metabolism, cancer and the combination strategy, aging, and neurological disorders, to direct the potential clinical programs of metformin in the future.

2 Pharmacokinetics of metformin

Since its publication in 1957, metformin has become one of the most widely used oral drugs for treatment T2D in the world, and increasing attempts to use metformin for treatment of various diseases other than diabetes have been mentioned. It is necessary and meaningful to clarify the pharmacokinetics and pharmacology of metformin in humans and provide insights and ideas for further understanding of metformin. It is well known that metformin is transported across cellular membranes by numerous transporters because of its hydrophilic base chemically. Transporters play an important role in the absorption, distribution, and elimination of metformin, and serve as determinants for its bioavailability, clearance, and pharmacological effects. Here, we summarize the pharmacokinetics of metformin including its absorption, distribution, metabolism, and elimination.

2.1 Absorption of metformin

Metformin is slowly and incompletely absorbed. After an oral administration of 500 mg or 850 mg metformin hydrochloride tablets (immediate-release formulation), the absolute oral bioavailability (F) is approximately 50% to 60% under fasting conditions while rapidly and largely absorbed from the small intestine [47,48]. Following a single oral administration of immediate-release formulation, peak plasma concentrations (C_max) occur approximately at 3 h, and C_max ranges from 1.0 to 1.6 mg/L after a 500 mg dose. After a single oral administration of metformin hydrochloride extended-release tablet (extended-release formulation), C_max is reached at 7 h, and the bioavailability is very similar to that of immediate-release formulation [47,49,50]. The C_max is about 30% higher for taking extended-release formulation 2000 mg once daily than for taking immediate-release formulation 1000 mg twice daily, while the C_max of extended-release formulation is lower than the same dose of immediate-release formulation, but the extent of absorption (as measured by plasma concentration-time curve, AUC) is similar for both formulations [51,52]. Under the fed condition, although the extent of absorption is slightly reduced and delayed, and the absorption is increased by about 50% compared to the fasting condition, there is no effect on C_max and T_max of metformin [49,53].

For the absorption of metformin, the plasma membrane monoamine transporter (PMAT, SLC29A4) expressed in the apical membranes of polarized enterocyte has an apparent affinity K_m for metformin of 1.32 mmol/L and acts as a major role in the intestinal absorption [54]. The other important transporters, organic cation transporter 1 (OCT1, SLC22A1) and organic cation transporter 3 (OCT3, SLC22A3) in the SLC22 family, have apparent affinity K_m for metformin of 1.47 mmol/L and 1.10 mmol/L, respectively. OCT1 is expressed in the basolateral membrane of the enterocyte and hepatocytes and is assumed to play a major role in metformin transportation into the hepatocytes or the portal vein [55,56]. But the steady-state pharmacokinetics of metformin is independent of the OCT1 genotype in healthy volunteers [57]. OCT3 is expressed in the brush border membrane of enterocytes and is responsible for metformin absorption in vivo [58–61]. Carnitine/organic cation transporter (OCTN1, SLC22A4) localized to the apical membrane of enterocytes have also been identified to participate in the intestinal absorption of metformin [62]. Organic cation transporter 2 (OCT2, SLC22A2) with a K_m of 0.99 mmol/L for metformin is predominantly expressed in the basolateral membrane of renal tubules and facilitates to absorb metformin from circulation into renal epithelial cells [58,63]. The pharmacokinetics of metformin is significantly affected by genetic variants of OCT2-808 G>T and OCTN1-917C>T polymorphisms, and this may influence the clinical response to metformin therapy [64]. Other transporters such as serotonin reuptake transporter (SERT, SLC6A4), thiamine transporter 2 (THTR2, SLC19A3) may also contribute to the intestinal absorption of metformin [65,66]. In addition to active transport, passive transport has been recently reported to be responsible for metformin distribution dynamics between plasma and red blood cells in humans [67].

2.2 Distribution of metformin

Metformin is widely distributed into body tissues, including the intestine, liver and kidney, with negligible binding to plasma proteins [47,48,68]. The volume of distribution (V_d) after intravenous administration has been reported to range from 63 to 276 L [68,69], and the apparent volume of distribution (V_d/F) after oral administration of immediate-release formulation of 850 mg metformin averages at 654 ± 358 L, and the actual V_d after multiple doses of metformin is about 300 L [47]. Following a single oral administration, concentrations in the kidneys, adrenal glands, pancreas and liver are up to seven times higher than in blood, and concentrations in the lung, muscle and spleen are lower. In addition, the concentration of metformin in the liver is higher than that in the portal vein, as well as in general circulation and other organs [3,47]. The higher concentration in the kidney may be due to the higher concentration of metformin in the urinary tract rather than absorption in renal tissue [47]. Since the glucose-lowering effect of metformin is mainly to inhibit hepatic glucose output and the concentration of metformin in the liver is much higher than that in other organs, it is considered that the liver is the primary site of metformin function [70]. Steady-state plasma concentrations (C_ss) of metformin are usually reached within 1 to 2 days and are generally less than 1 μg/mL [47,52].

For the distribution of metformin, OCT3 is an important transporter expressed in various tissues including lung, prostate, skeletal muscle, and adipose tissue. Furthermore, OCT3 has also been detected in the blood-brain barrier, placenta, and salivary glands, and studies have shown that OCT3 is responsible for the accumulation and secretion of metformin [58,71]. In addition, multidrug and toxin extrusion transporter 1 (MATE1, SLC47A1), which is mainly expressed in the kidney and liver, may contribute to the transport and excretion of metformin [72].

2.3 Elimination or excretion of metformin

Metformin undergoes renal clearance rather than goes through hepatic metabolism, biliary excretion or gastrointestinal excretion in humans, with a half-life of 4.0 to 8.7 h [48,69]. Since renal clearance is approximately 4–5 times higher than glomerular filtration rate (GFR), tubular secretion is responsible for the elimination of metformin [73,74]. Following oral administration, about 90% of the absorbed drug is excreted in the kidney within the first 24 h, while a population mean of renal clearance (CL_r) is 510 ± 130 mL/min and apparent total clearance (CL/F) is 1140 ± 330 mL/min [47,68,75]. Furthermore, metformin is little or undetectable in feces after intravenous administration, and 20%–30% of metformin may recover from feces after oral administration [76]. So, it is clinically recommended to take it with food to minimize gastrointestinal side effects of metformin, such as bloating, flatulence, and diarrhea. The rapid elimination of metformin via kidney was reported to be associated with a lower number of episodes of lactic acidosis than phenformin [77]. While another study also showed that most of the absorbed dose of metformin is excreted very quickly, a small fraction is removed much more slowly. Thus in patients with moderate renal impairment, creatinine clearance is not suggested as a reliable indicator of potential metformin accumulation [78].

For the elimination of metformin, transporters in the kidney, such as MATE1 (SLC47A1) and MATE2 (SLC47A2), facilitate the clearance of metformin. MATE1 with K_m of 0.23 mmol/L is localized in the bile canaliculus or kidney and is a carrier of metformin, thus possibly transporting metformin into bile or transporting metformin from renal tubule into urine, whereas MATE2 with K_m of 1.05 mmol/L is expressed in the apical membrane of the renal proximal tubule cells and plays a key role in renal excretion of metformin [47,75,79,80].

2.4 Conclusions

Metformin is eliminated intactly by renal excretion with high clearance rates. The maximum approved total daily dose of metformin for diabetes treatment is 2.5 g (35 mg/kg body weight). After treatment, the portal vein plasma concentration of animals was between 40 and 70 μmol/L, and the concentration would reduce to 10–40 μmol/L after hepatic uptake [80]. However, very high concentration of metformin in vitro (5 mmol/L) has been applied in multiple studies, which may lead to a non-specific effect of the drug or toxic effect. Therefore, given its pharmacokinetic characteristics, the concentration of metformin should be carefully used.

3 Metformin and systemic metabolism

Metformin has been the first-line drug treatment for type 2 diabetes for several decades. Besides its glucose-lowering effect, metformin also has the potential relevance on systemic metabolism, including regulating gut homeostasis, thermogenesis, appetite, and so on. However, the underlying mechanism of its action remains elusive. Some convincing data have documented that metformin-induced activation of the energy-sensor AMPK is its core mechanism [81,82], but may not account for all effects. Here, we summarize current knowledge for the action of metformin and its potential mechanisms (Fig.1).

3.1 Metformin and adipose tissue

White adipose tissue (WAT) and brown adipose tissue (BAT) compose the adipose organ that constitutes almost a quarter of body weight even in a lean state, playing pivot roles in energy homeostasis in mammals through multiple mechanisms [83,84]. Compared with other metabolic organs such as the liver, kidney, and intestine, it is impossible that the relatively lower concentration of metformin in adipose tissue can exert the effect of reduction in body fat mass only through the glucose-lowering effect [85]. Metformin has a positive regulation of the lipid metabolism of adipose tissue. It was found that metformin increases the uptake and utilization of fatty acids in adipose tissue, which is related to the decrease of VLDL-TG and adipose tissue mass in HFD-feeding mice and patients [86,87]. Moreover, metformin can reduce visceral fat mass through increased oxidation of fatty acids by adaptive thermogenesis. After the treatment of metformin, the baseline respiratory quotient decreased and the postprandial respiratory quotient increased significantly in healthy subjects and patients with T2D [88], and metformin treatment reduces the content of visceral fat mass accompanied by the upregulation of UCP-1 in the brown adipose tissue [89]. Metformin can also prevent the loss of BAT caused by olanzapine treatment and gene expression analysis found a great change in the expression of multiple key genes controlling energy expenditure, which confirms that the regulation of energy expenditure is a primary effect of metformin [90]. Except for the regulation of the metabolism of adipose tissue, metformin also influences the balance of remodeling of adipose tissue. Metformin can induce macrophages into an anti-inflammatory phenotype and the reduction of inflammation in adipose tissue caused by metformin has been found to improve obesity-related metabolic disorders [91,92]. Adipose tissue fibrosis is another target of metformin. Extracellular matrix (ECM) remodeling, especially adipose tissue fibrosis induced by obesity, can lead to a dysfunctional process of adipose tissue homeostasis [93]. Metformin treatment can activate AMPK and suppress transforming growth factor-β1 (TGF-β1)/Smad3 signaling, therefore decreasing collagen deposition and the expression of fibrotic genes in adipose tissue to inhibit ECM deposition in obese mice, leading to improved systemic insulin sensitivity [94]. Except as the classical metabolic organ to maintain the balance of lipid dynamics, adipose tissue also can act as an endocrine organ to orchestrate metabolic homeostasis. Up to now, more than 100 adipokines are involved in the regulation of local or systematic tissue homeostasis [95]. Two kinds of miRNA secreted by adipose tissue may be the target of metformin. First, metformin can increase the insulin sensitivity of adipose tissue in patients with diabetes by reducing the expression of miR-223 and activating the IRS/Akt/GLUT4 signaling pathways [96]. Moreover, exposure to metformin can decrease the accumulation of intracellular lipid of adipose-derived stem cells that have the potency to differentiate to mature adipocytes by regulating the expression of miR-145 [97]. Collectively, as an active organ with complex properties, adipose tissue is an important target of metformin.

3.2 Metformin and microbiome

Although metformin is generally thought to mediate its antihyperglycemic effects by inhibiting hepatic glucose output by activating AMPK-dependent [98–100] and AMPK-independent pathways in the liver, there is increasing evidence that it may also act through the intestinal pathway. Recent evidence suggests that the gut microbiome is a site of action of metformin. Transfer of fecal samples from metformin-treated donors into germ-free mice showed improved glucose tolerance in mice receiving metformin-treated altered microbiota. When administered intravenously, metformin does not lower blood glucose [101]. In addition, a delayed-release formulation of metformin, which delivers the drug to the lower plasma-exposed intestinal segments, exerts the same glucose-lowering effect comparing to the standard immediate release formulation, providing further evidence for a gut-mediated mechanism of metformin [102]. Recent studies in rodents [103–105] and humans [106,107] have suggested that changes in the gut microbiome may contribute to the antidiabetic effects of metformin. In recent years, studies have reported that the clinical benefits of metformin are related to the composition and function of intestinal microbes [108]. The gut-mediated health effects of metformin may originate specifically from alterations in the gut microbiota. A seminal study by Cabreiro et al. [109] showed that metformin prolongs the lifespan of Caenorhabditis elegans (C. elegans) by altering the production of folate and methionine by Escherichia coli, providing evidence that metformin affects the composition of the gut microbiota. In a randomized trial, overweight/obese adults had significantly altered microbiota composition after metformin treatment, with the relative abundance of E. coli increasing in the metformin group, while that of Intestinibacter bartlettii decreased. Circulating SCFAs were altered by metformin. Metformin increases butyrate, acetate, and valerate, and the increase in acetate is associated with a decrease in fasting insulin. Whole-genome metagenomic sequencing revealed that metformin altered 62 metagenomic functional pathways, including one acetate production pathway and three glucose metabolism pathways. Bauer et al. identified glucose-sensing pathways in the upper small intestine of rodents that reduce glucose production. The high-fat diet altered the microbiota in the upper small intestine and impaired glucose sensing, whereas metformin treatment in the upper small intestine counteracted the microbiota changes and restored glucose sensing. The high-fat diet reduced glucose-SGLT1 sensing and metformin restored glucose-SGLT1 sensing, whereas increased metformin-treated microbial transplantation restored glucose-SGLT1 sensing [110]. Metformin treatment in the upper small intestine restored SGLT1 expression and glucose sensing while partially altering the upper small intestinal microbiota by increasing Lactobacillus abundance. Transplantation of the upper small intestine microbiota from metformin-treated HFD rats into the upper small intestine of untreated HFD rats also increased Lactobacillus abundance and glucose sensing in the upper small intestine by upregulating SGLT1 expression. Thus, we demonstrate that metformin alters the upper small intestine microbiota and affects the glucose-SGLT1-sensing glucose regulation pathway. Some studies have identified a bacterial signaling pathway that integrates metformin and nutrition to alter the microbial metabolites, and the changes produced by the metabolites in turn affect the host fatty acid metabolism. How do gut microbes interact with metformin to affect the host? It has been reported that microbes integrate cues from metformin and diet to alter microbial metabolites through the transcriptional regulator CRP-mediated phosphotransferase signaling pathway. The microbiota of metformin-treated patients increased the capacity of agmatine production. Agmatine is a regulator of host lipid metabolism and lifespan, which in turn affects host fatty acid metabolism [111].

3.3 Metformin and pancreas

Though there is extensive research focusing on the pivotal roles of metformin in regulating insulin sensitivity and glucose homeostasis in insulin-targeted organs, the exact function of metformin in pancreatic β cells is still controversial. In 2005, a study showed that metformin can increase insulin secretion slightly, in addition to the known function of improvement of insulin sensitivity [112]. In brief, metformin exerts a beneficial influence on β cell function directly in many aspects such as insulin release, transcriptional regulation, and islet cell viability, and these effects are dependent on the presence of glucose [113]. After chronic exposure to free fatty acids or high glucose, pancreatic β cell show decreased insulin secretion which can be restored by metformin treatment. However, the roles of metformin are not observed in normal conditions. Furthermore, this phenomenon can be observed in both human islets and cell lines accompanied with reduced oxidative stress [114,115]. It is known that glucotoxicity-induced oxidative and endoplasmic reticulum (ER) stress are essential in the development of β cell dysfunction. Human islets cultured with high glucose revealed reduced glucose-stimulated insulin secretion. However, these effects can be reversed with metformin treatment via inhibiting the activity of mitochondrial complex I [114]. Research shows that hyperglycemia induces oxidative stress and inflammation which eventually lead to impaired insulin secretion and increased apoptosis in β cells, which can be improved by the treatment of metformin. Human islet cells in culture with palmitate for seven days show reduced expression of p-AMPK and a significantly increased expression of phosphorylated eukaryotic initiation factor-2 (p-EIF2α), C/EBP homologous protein, and cleaved caspase 3, but their expressions were back to a normal state in the presence of metformin at a dose of 25 µmol/L [116]. This suggests that metformin can promote pancreatic β cell function following chronic fatty acid exposure. Also, metformin is able to protect against glucotoxicity-induced reactive oxygen species production and inhibits the cluster determinant 36-mediated free fatty acid influx in pancreatic β cells [117]. In the aspect of inflammation, researchers observed that the pancreatic mRNA expressions of inflammation factors including TLR4, NF-κB, JNK, IL-6, TNF-α along with p-NF-κB p65 protein levels in diabetic KKAy mice were downregulated by metformin, while expressions of genes participating in the insulin secretion including SERCA2 and Kir6.2 are upregulated. These observations indicate that metformin alleviates pancreatic inflammation, and elevates β cell function in diabetic KKAy mice [118]. On the other hand, metformin was also reported to play roles in pancreatic β cell survival under different conditions. Under normal growth condition, metformin suppresses MIN6 β cell proliferation and promotes apoptosis via an AMPK-dependent and autophagy-mediated mechanism. While metformin protects MIN6 and INS-1 cells against palmitic acid (PA)-induced apoptosis, suggesting a dual role of metformin in regulating MIN6 pancreatic β cell survival [119]. Under glucolipotoxicity conditions, low dose metformin was reported to prevent Ca²⁺-induced PTP opening in permeabilized and intact INS-1 cells to preserve β cell viability [120]. And metformin can inhibit the ER stress-induced apoptosis in NIT-1 cells or pancreatic islets, via the regulation of AMPK-PI3 kinase-JNK pathway in lipotoxicity [121]. In conclusion, metformin exerts beneficial effects on β cell functions such as increased insulin secretion and β cell viability depending on the presence of glucose. Although accumulated evidence shed light on metformin activities, the precise mechanisms of metformin in pancreatic β cells remain unclear, needing further investigations in the future.

3.4 Metformin and anorexia effect

The contribution of metformin administration on weight loss is attributed to food intake rather than energy consumption, according to the number of results both in human and experimental animals [122–124]. Although recent studies attempted to assess the role of metformin in weight loss induced by food intake reduction, the precise mechanism of its anorexigenic effect has not been elucidated. There are proven studies that metformin acts directly on the central nervous system and thus leads to the suppression of appetite. The increasing neuropeptide Y (NPY, a known orexigenic neuropeptide) gene expression resulted from the phosphorylation of AMPK was detected in low-glucose medium cultured primary rat hypothalamic neurons, which was blocked by the addition of metformin [125]. It is implied that metformin inhibits AMPK activity and then regulates the expression of the orexigenic peptide NPY. Other research also demonstrates that metformin inhibits the increased food intake caused by acute ghrelin application [126]. Also, metformin administration restored the impaired response to leptin in HFD-induced obese mice, which suggests that metformin could improve leptin sensitivity [127]. Moreover, in an acute intracerebroventricular leptin injection study, metformin has been identified to enhance the anorexigenic effect mediated by leptin, accompanied by increased phosphorylation of stat3 [128]. In addition to direct action on the neural center, metformin may orchestrate the secretion of some endocrine factor communicating with the central nervous system indirectly. Growth differentiation factor 15 (GDF15), a member of the TGF-β superfamily, has been classified as responsible for the weight loss of metformin. GDF15 binds to the GDNF family receptor α-like (GFRAL) in the hindbrain, thus inhibiting food intake [129–131]. Coll et al. present that the circulating GDF15 level significantly increases upon metformin administration in a short-term human study and a series of animal experiments, and the lowering of weight is dependent on the GDF15-GFRAL pathway which has no impact on the hypoglycemic and insulin-sensitizing effects [132]. In addition, they found that GDF15 induced by metformin was predominantly expressed in the small intestine and colon. Hence, its further mechanism remains to be determined. Unexpectedly, Klein et al. definitely verify the causal correlation between circulating GDF15 and metformin, while there was no difference in the weight loss and food intake between GDF15 or GFRAL-knockout mice and their littermates, suggesting GDF15 dispensable for the effects of metformin [133]. In fact, it is still controversial whether the weight loss induced by metformin depends on the GDF15-GFRAL pathway. Therefore, whether GDF15 contributes to the anorexigenic effect of metformin requires additional studies. Moreover, Day et al. conduct experiments in primary mouse hepatocytes and indicate that the expression of activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP; also known as DDIT3) might play an important role in the secretion of GDF15 caused by metformin [134]. Taken together, the molecular mechanism underlying metformin-induced weight loss has remained unclear, making it a big hurdle for the clinical application of metformin. It is essential to illustrate the identified targets or metabolic pathways, which perhaps provide promising strategies for obesity.

3.5 Metformin and muscle

Metformin is recommended as the most prescribed antidiabetic in the world taking its safety and efficacy into account, which has been considered the first-line drug of type 2 diabetes mellitus [135]. As a metabolically highly active tissue, skeletal muscle whose metabolic disturbances are associated with metabolism syndrome plays an important role in blood glucose regulation, which uses both circulating plasma glucose as well as stored glycogen as fuel sources, and insulin action, dysregulation of lipid metabolism, reduced mitochondrial oxidation. Metformin is suggested to have an effect on skeletal muscle energy metabolism and is beneficial to glucose homeostasis. Studies have shown that metformin can stimulate skeletal muscle glucose uptake and oxidation [136], and can lower intramuscular triglyceride content and bioactive acyl-chain bioactive lipids by partially increasing fat oxidation [137,138]. On the one hand, metformin can induce the transport of glucose transporter 4 to the cell plasma membrane, thereby increasing glucose uptake and improving insulin resistance [139]. Moreover, skeletal muscle is responsible for 70%–80% of whole-body insulin-stimulated glucose uptake and plays an important role in lipid metabolism, where insulin resistance results from bioactive lipid accumulation such as intramuscular long-chain acyl-CoA (LCACoA), diacylglycerols (DAG), and ceramide (Cer) [138]. And AMPK also can be activated by metformin in skeletal muscle, which can promote increased fatty acid oxidation because of the activation of acetyl-CoA carboxylase 2 and the reduction of malonyl-CoA [140]. Additionally, metformin also can enhance mitochondrial biogenesis and mitochondrial oxidase activity in skeletal muscle by activating PGC-1α through AMPK phosphorylation. In summary, these results illustrate that metformin is of importance in skeletal muscle energy metabolism which will improve glucose homeostasis, lipid metabolism, and insulin resistance.

3.6 Metformin and liver

Metformin exerts its glucose-lowering effect primarily by decreasing hepatic glucose production through suppression of gluconeogenesis and enhancing insulin suppression of endogenous glucose production [141]. The role of AMPK in mediating the action of metformin in primary hepatocytes was initially supported by Zhou and colleagues [142]. It was reported that ablation of liver kinase B1 (LKB1) in the liver prevented the antihyperglycemic effects of metformin in high-fat-fed mice [99]. AMPK activation by metformin has also been reported to be involved in the transcriptional regulation of hepatic gluconeogenic enzyme genes by different mechanisms: (1) dissociation of the CREB-CBP (CREB binding protein)-TORC2 transcription complex, through the phosphorylation of the transcriptional coactivator CBP via atypical protein kinase C i/l [143], (2) increased expression of the orphan nuclear receptor small heterodimer partner [144], and (3) induction of SIRT1-mediated CRTC2 deacetylation [145]. In addition, it was reported that the reduction in hepatic gluconeogenesis by metformin might result from direct inhibition of the mitochondrial glycerophosphate dehydrogenase (mGPD), identifying another putative mitochondrial target of the drug [146]. Another effect of metformin is to improve lipid metabolism by reducing hepatic steatosis as demonstrated in rodent liver [147,148]. Fullerton and colleagues recently showed that metformin-induced improvements in insulin action operate through alterations in hepatic lipid homeostasis via the inhibitory phosphorylation of acetyl CoA carboxylase (ACC) by AMPK, the role of AMPK in the mechanisms of metformin action on lipid metabolism was provided in knock-in mouse models in which ACC1 and ACC2 were rendered insensitive to AMPK phosphorylation [100]. ACC is a rate-determining enzyme for the synthesis of malonyl-CoA, both a critical substrate for fatty acid biosynthesis and a potent inhibitor of fatty acid oxidation. It is generally accepted that the actions of metformin on mitochondria underlie most of the pleiotropic effects of the drug. Two seminal papers published in 2000 reported that metformin decreases cellular respiration by a mild and specific inhibition of the respiratory-chain complex I (NADH: ubiquinone oxidoreductase) [149,150]. The specific inhibition of the mitochondrial respiratory-chain complex I by metformin was confirmed in many cellular models, including rat, mouse, and human primary hepatocytes [149–151]. It was shown that the inhibitory effect of metformin on complex I was not prevented by nitric oxide (NO) synthase inhibitors or reactive oxygen species (ROS) scavengers [149] and was independent of AMPK, at least in primary mouse hepatocytes [151]. To investigate the mechanisms by which metformin affects the cellular energy state, together with the putative involvement of AMPK in this process, they used hepatocytes from wild-type and liver-specific AMPKα1/2^−/− mice for permeabilisation of the plasma membrane by digitonin, allowing the mitochondrial OXPHOS pathway to be investigated in situ [151]. It is also worth mentioning that the inhibition of complex I activity by metformin is rather mild when compared to the reference inhibitor rotenone [149]. Furthermore, metformin was shown to significantly reduce mitochondrial ROS production by selective inhibition of the reverse electron flow through the respiratory-chain complex I, whereas rotenone triggers ROS production by increasing forward electron flow [152]. However, it is still unclear concerning the mechanism by which metformin inhibits complex I despite its use for 60 years. Some researchers proposed a direct effect of metformin on complex I involving an accumulation of metformin inside the mitochondria while others proposed an indirect effect (the drug no longer having to diffuse into the mitochondria), which is in urgent clarification [153].

3.7 Summary and future perspectives

In recent decades, the lack of effective treatment contradicts the rapidly increasing incident rate of metabolic diseases for the reason of the complexity of pathogenesis. The heterogeneity of different metabolic organs makes it a great challenge to find a key common target to improve metabolism-related disorders. However, metformin brings the hope that various metabolic diseases may be improved only by one compound. It is a miracle that metformin exerts its beneficial effects in almost all the metabolic organs including direct metabolic organs, the liver, adipose tissue, and muscle, and indirect regulatory organs, the pancreas, and the brain. This extensive therapeutic effect of metformin may attribute to its simple carbon structure, which makes metformin bind with various proteins to involve in the regulation of key signal transduction or functions. Up to now, metformin is expected to become the panacea in improving metabolic diseases, even though its potential target is still under exploration.

4 Metformin and cancer

Metformin is a well-tolerated antidiabetic drug, with potential as an anticancer agent [154,155]. It has been reported to reduce the risk of cancer in diabetic patients [156,157]. In 2005, Evans et al. suggested that taking metformin may be associated with reduced risk of cancer in patients with type 2 diabetes applied in a cohort study and the potential link with the tumor suppressor role of LKB1, which enhanced great interest in metformin as an anti-cancer drug since then [156]. Metformin possesses beneficial clinical efficacy, including synergistically inhibiting cancer cell or cancer stem cell growth, reducing recurrence, cardiotoxicity, and mortality, as well as increasing the efficacy of other agents in various types of cancer models [156,158,159]. It is used in monotherapy or in combination with other various chemotherapeutic agents [160–162].

The anti-tumor effect of metformin probably attributes to a combination of indirect and direct mechanisms (Fig.2). Metformin may indirectly contribute to anti-tumor effects by lowering the systemic glucose and insulin levels, and reducing cancer risk [80,163,164]. In the other direct context, AMPK-dependent and AMPK-independent mechanisms have been described previously, which are likely to coexist and interact together to suppress tumor development [154]. LKB1, a tumor suppressor protein, serves as an activator of AMPK in response to cellular stresses and previous reports have demonstrated that LKB1 is essential for the inhibition of cancer cell proliferation [165]. Activation of AMPK can affect multiple signaling in cells, such as mTOR, p53 and NF-κB pathway, to regulate cell proliferation and survival. mTOR, the mammalian target of rapamycin, plays a vital role in tumor proliferation and inhibition of cell death [166,167]. Metformin activates AMPK via activation of LKB1, subsequently inhibits mTOR activity resulting in inhibition of protein synthesis, induction of cell cycle arrest, apoptosis, or autophagy [168–172]. p53, a critical tumor suppressor, regulates cell growth, survival, and development. AMPK activated by metformin induces p53 activity leading to cell cycle arrest and autophagy [173,174]. NF-κB, the nuclear factor kappa B, is a protein complex and is involved in cell migration and proliferation. Metformin inhibits NF-κB pathway dependently on AMPK, reforming chemotherapy resistance and inducing cancer cell pyroptosis, metastasis, and invasion [175–177]. Our team recently found that metformin activates AMPK-PHF2 axis to downregulate H3K9me2 and inhibits lung cancer metastasis [178]. Furthermore, studies also reported that metformin inhibits activation of NF-κB via AMPK activation to inhibit release of cytokines and exert anti-inflammatory effects [179–181]. Among these, LKB1-AMPK-mTOR signaling pathway may be one of the main mechanisms of metformin to exert anticancer effects [169,182]. In addition, metformin also inhibits the occurrence and development of tumor cells through AMPK mediated Warburg effect, fatty acid synthesis, cell cycle regulation, and its regulatory role in tumor metastasis and angiogenesis [183–185]. In AMPK independent manner, metformin, acts as an insulin-sensitizing agent, inhibits insulin and insulin-like growth factor 1 (IGF-1), key regulators of metabolism and growth, to control tumor growth [186–189]. Decreased insulin after administration of metformin also inhibits phosphoinositide 3-kinase (PI3K)-protein kinase B (AKT) pathway, leading to inhibit mTOR [190–192]. And, metformin inhibits mTOR independently on AMPK via activation of REDD1 to exert anticancer effects [193,194]. Besides, mTORC1 activated in the amino acid metabolic signaling pathway could also be directly inhibited by metformin, thereby controlling the energy metabolism of tumor cells [195]. And, metformin directly interferes NF-κB signaling pathway, inducing cancer cell pyroptosis, metastasis, and invasion [196]. In addition, metformin inhibits mitochondrial complex I and reduce the ATP/AMP ratio leading to activation of AMPK and reduce tumorigenesis [197]. Therefore, based on the complex regulatory network of metformin, it is important to further study the anticancer mechanism of metformin.

Though metformin with the favorable safety profile exerts the anti-tumor effects, the drug administrated alone may not achieve the desired results in certain scenes; meanwhile, pleiotropic effects of metformin on cancers also make it a unique adjuvant to combination with other effective anti-cancer drugs [159,161,198–203]. Instead, most of the preclinical and clinical data are exploring and supporting its role as an adjuvant drug in the treatment of cancer (Tab.2–Tab.4), although the action mechanisms are not fully elucidated. Herein, we focus on the combination strategy of metformin with chemotherapy, targeted molecular drugs, and immunotherapy to promote clinical application of metformin in cancer therapy.

4.1 Combination of metformin with chemotherapeutic agents

4.1.1 In combination with antimetabolite drugs

Metabolic reprogramming in cancer cell is described as deregulating cellular metabolism and it is considered one of “hallmarks of cancer” [204]. It is involved in tumor development and proliferation. Cancer cells survive in a state of aerobic glycolysis which produces ATP for emergency, intermediate metabolites for biosynthesis, low pH, and hypoxic microenvironment. Antimetabolite drugs, also known as nucleoside analogs, have been used as primary and effective cancer treatment with the effect to disturb the biosynthesis of DNA and block cancer cell division and proliferation [205]. Antimetabolite agents are widely conducted in a clinical setting with poor pharmacokinetics and common side effects such as poor bioavailability, low penetration in blood-brain barrier, bone marrow suppression, and gastrointestinal toxicity.

4.1.1.1 Methotrexate

Methotrexate (MTX), a folate analog that interrupts one-carbon transfer reactions and inhibits de novo nucleotide synthesis, has been used as a traditional chemotherapeutic agent for treatment of many cancers [206–208]. Unfortunately, hepatorenal toxicity is the underlying adverse effect and has been reported in many cases of high doses or long-term treatments of MTX [209–212]. Previous studies reported that metformin shows a protective effect in MTX-induced hepatorenal toxicity due to its anti-oxidant, anti-inflammatory, and anti-apoptotic properties in rats [212]. Subsequently, combining metformin with MTX has shown to decrease nucleotide metabolism, inhibit cell proliferation, increase sensitivity, and overcome resistance of hepatocarcinoma cell to MTX by transcriptionally inhibiting dihydrofolate reductase (DHFR) [213].

4.1.1.2 (5-FU)

5-FU is an analog of uracil, that can be converted to 5F-dUMP and 5F-UTP to affect biosynthesis of DNA or disturb the biosynthesis of proteins [214]. It is widely used to treat a range of cancers in clinical settings. Metformin combined with 5-FU synergistically and significantly increases chemosensitivity, inhibits cell proliferation, induces cell cycle arrest and apoptosis in cancer cells both in vitro and in vivo. Present studies showed that the mechanisms of metformin in combination with 5-FU are complex and unclear in different cancer types. The relevant signaling pathways involved are PI3K/mTOR, YAP, AMPK/mTOR/HIF1α/P-gp, etc. [215–217].

There are some controversial cases that should not be neglected. A study reported that metformin combined with 5-FU did not show anticancer activity in vitro and in vivo for colorectal cancer [199]. In a clinical trial for refractory metastatic colorectal cancer, metformin had a longer median survival rate when used alone, but it did not show significant difference overall. When combined with 5-FU, metformin showed a modest boost in survival rate compared with either drug used alone [218]. Thus, metformin might show variable effect in different stages of multiple cancers.

4.1.1.3 Other antimetabolite drugs

Cytarabine (Ara-C) has been used for the first-line treatment of acute myeloid leukemia (AML). Metformin combined with Ara-C significantly enhances chemosensitivity of AML cells through reducing the mitochondrial transfer and oxidative phosphorylation (OXPHOS) [219].

Hydroxycarbamide has been used for treatment of melanoma, resistant chronic myelocytic leukemia, head and neck cancer as well as recurrent or metastatic ovary carcinoma. Interestingly, metformin combines with hydroxyurea to induce fetal hemoglobin (HbF) and reverse the arrest in erythroid maturation caused by hydroxyurea treatment alone [220].

Gemcitabine is a first-line drug used for the treatment of pancreatic cancer. Studies reported that heat shock protein 27 (HSP27) is phosphorylated in gemcitabine-resistant pancreatic cancer cells and metformin reverses chemoresistance through blocking the phosphorylation of HSP27 [221,222], indicating a potential and synergistic effect in pancreatic cancer. Further studies showed that metformin combined with gemcitabine increases chemosensitivity by reducing proportion of CD133⁺ cells and suppressing ERK/p70S6K signaling pathway [223].

4.1.2 Metformin combines with drugs interfering with microtubules synthesis

4.1.2.1 Vincristine and its derivatives

Vincristine and its derivatives are alkaloids isolated from Vinca rosa Linn., which have been used for the treatment of leukemia due to cell cycle specific cytotoxicity. The drugs bind specifically to tubulin, leading to microtubule depolymerization, cell cycle arrest, and cell apoptosis. Metformin combined with vincristine sensitizes leukemia cells through activation of AMPK [224]. A clinical trial showed that metformin in combination with VPLD (vincristine/dexamethasone/doxorubicin/PEG-asparaginase) can treat relapsed childhood acute lymphoblastic leukemia (ALL) (NCT01324180) with better outcome than patients who received VPLD alone by inducing ER stress, activating AMPK, and inhibiting the unfolded protein response (UPR) [225]. In another study, metformin was used in combination with R-CHOP (rituximab plus cyclophosphamide/doxorubicin/vincristine) to treat diffuse large B cell lymphoma (DLBCL) and grade 3b follicular lymphoma (FL3b) with significantly better overall survival (OS) than those without metformin [226].

4.1.2.2 Paclitaxel (PTX)

Paclitaxel is a diterpene amide isolated from the Pacific yew. It has been used for multiple cancers by targeting the tubulin, inducing the defects of mitotic spindle assembly, chromosome segregation, and cell division. Metformin in combination with PTX enhances chemosensitivity and inhibits cell proliferation by modulating the AMPK/mTOR pathway in various types of cancers such as endometrial cancer and breast cancer [227,228]. This combination treatment has also induced oxidative stress and activation of mitochondrial-dependent apoptotic pathways in prostate cancer [229]. In addition, metformin and PTX combination was shown to be effective in lung cancer by downregulating the ERCC1 and inhibiting p38-MAPK [230]. Furthermore, this combination has been shown to alter the metabolic target and modulation of mTOR pathway in ovarian cancer [231].

4.1.2.3 Docetaxel (DCX)

DCX is a chemotherapeutic drug with similar chemical structure and mechanisms to PTX. Although it provides benefits in clinical cases, the side effects are significant due to its effect of enhancing aerobic glycolysis [232]. Previous studies showed metformin combined with DCX reduced cancer cell viability in castration-resistant prostate cancer through decreasing AKT phosphorylation, increasing AMPK phosphorylation, and decreasing acetyl-CoA and HMG-CoAR activity [233,234]. However, metformin addition failed to improve the standard DCX regimen in castration-resistant prostate cancer clinically (NCT01796028). Further research targeting tumor cell metabolism should be performed.

4.1.3 Metformin combines with antibiotics and drugs targeting DNA synthesis

Antibiotics, such as dactinomycin D, daunorubicin, or doxorubicin, can exert anticancer activities, but accompanied with adverse effects such as severe cardiotoxicities. These antibiotics affect the structure of DNA through interrupting the structure of DNA and inhibiting the activity of topoisomerase to exert its anti-cancer activities. Combining these antibiotics with metformin may be a new strategy worth investigating. The beneficial effects of metformin may help overcome the side effects of these antibiotics.

4.1.3.1 Doxorubicin (DOX)

DOX exerts anticancer activity accompanied by serious side effects including chemo-resistance and cardiotoxicities. In DOX-resistant breast cancer cells, metformin in combination with DOX shows synergistic activity and overcomes drug resistance by downregulating drug-resistant genes such as P-gp and HIF1α [235,236]. Metformin has been found to prevent DOX-induced cardiotoxicity in vivo with multiple mechanisms including the activation of AMPK pathway, inhibition of oxidative stress, energy starvation, and depletion of intramitochondrial CoA-SH [237–240]. In addition, metformin also enhances the anticancer activity of DOX in breast cancer [241] and reverses drug resistance through inhibiting NF-κB pathway and phosphorylation of STAT3 [242]. Combination treatment of metformin and DOX can prevent cancer relapse and reduce the dosage of chemotherapy [243].

4.1.3.2 Cisplatin

Cisplatin exerts remarkable anticancer activity, but renal toxicity and drug resistance were discovered in clinical settings. Previous studies reported that metformin enhances anticancer effect in thyroid carcinoma [241] and overcomes resistance of cisplatin by downregulating RAD51 expression in triple-negative breast cancer cells [244] and suppressing IL6/STAT3 pathway in lung cancer [245]. Metformin increases chemo-efficacy of cisplatin via AMPK/mTOR pathway in meningioma [200] and has been found to inhibit Jarid1b in NSCLC in the presence of wild type p53 [246]. Metformin combined with cisplatin was found to strongly inhibit the activity of cancers, promote apoptosis in nasopharyngeal carcinoma, and ovarian cancer [247,248]. This combination treatment can induce cell arrest via phosphorylated YAP1 pathway [249] and attenuate cytotoxicity caused by cisplatin via AMPK/FOXO3α pathway [250]. Moreover, metformin prevents cisplatin-induced cognitive impairment and brain damage in mice [200,251].

4.1.3.3 Cyclophosphamide (CYP)

CYP exerts powerful and broad-spectrum anticancer activity, while along with obvious side effects such as immunosuppression, special cystitis, and drug resistance. Metformin combination can ameliorate CYP-induced memory impairment and nephrotoxicity [252,253], suggesting the potential for further application.

4.1.4 Other chemotherapeutic agents

4.1.4.1 Arsenic trioxide (ATO)

ATO is a traditional Chinese medicine that has been used for treating acute promyelocytic leukemia (APL). ATO frequently causes organ toxicity, such as hepatotoxicity, neurotoxicity, and cardiotoxicity. Previous studies showed that metformin ameliorates ATO-induced hepatotoxicity via inhibiting mitochondrial complex I, indicating a possibility for combination drug treatment [254]. Metformin enhances the sensitivity induced by ATO through downregulation of Bcl-2 expression in hepatocellular carcinoma [255]. Metformin also strengthens ATO’s effect of suppressing intrahepatic cholangiocarcinoma through activation of AMPK, upregulation of ERK3, and inhibition of mTORC1 [256].

4.2 Combination of metformin with molecular targeted drugs

Chemotherapy has long been the primary strategy for cancer treatment, but it is not an eradication method and causes inevitable side effects, such as myelosuppression and gastrointestinal reaction. Compared with traditional chemotherapy drugs, molecular targeted drugs with high efficacy and low toxicity specifically target cancer cells without causing obvious damage to normal cells. Interestingly, metformin not only shows synergistic effects with numerous chemotherapy agents, but also improves the efficacy of targeted therapy in preclinical and clinical cases. The following section summarizes the progress of tumor targeting drugs combined with metformin to treat different cancers. This section is divided into two parts: single-target inhibitors and multi-target inhibitors. According to the different targets, the potential mechanism of action is described in detail, and the development prospect of cancer treatment is discussed.

4.2.1 Metformin combines with single targeted inhibitors

4.2.1.1 EGFR inhibitors

Gefitinib, the first epidermal growth factor receptor (EGFR) inhibitor, is a reversible inhibitor with quinazoline structure [179]. Gefitinib is highly effective in NSCLC patients with EGFR mutations and provides complete remission of disease progression, but it was found that patients rapidly developed drug resistance after long-term gefitinib treatment. The mechanisms of developing resistance include amino acid mutation, activation of AKT/mTOR pathway, and upregulation of IGF-1R [257]. Therefore, gefitinib combining with metformin can improve efficacy by inhibiting AKT/mTOR or insulin-related pathways, which may be a promising direction for further investigation. Metformin was found to inhibit the expression of anti-apoptosis protein, thus increasing the sensitivity of gefitinib [258]. Previous studies showed that metformin effectively blocks tumor growth and inhibits tumor recurrence in xenografts of gefitinib-resistant cancer cells [259]. The inhibition of IGF-1R was reported to overcome the drug resistance of EGFR tyrosine kinase receptor inhibitors, thus metformin combined with gefitinib showed their synergistic effects in bladder cancer [260]. Overall, combination of metformin and gefitinib would be a promising strategy for tumor drug therapy.

4.2.1.2 mTOR inhibitors

The combination of mTOR inhibitors and metformin shows potential value in terms of molecular mechanism. Everolimus, an oral mTOR inhibitor, was approved in 2011 for advanced pancreatic neuroendocrine tumors [261]. In breast cancer cells, combination treatment of metformin and everolimus enhanced the inhibitory effects on cell proliferation and colony formation [262]. And the combination shows significant inhibitory effects on obesity-induced tumor growth [263]. Clinical studies have also shown that metformin sensitizes patients with pancreatic neuroendocrine tumors to everolimus [264].

4.2.1.3 VEGF inhibitors

Bevacizumab is a recombinant humanized monoclonal antibody (mAb) that targets all isoforms of vascular endothelial growth factor A (VEGF-A). It prevents VEGF-A from binding to VEGFR-1 and VEGFR-2, leading to the degeneration of tumor angiogenesis, thereby inhibiting tumor growth [265,266]. One case report showed that bevacizumab combined with metformin improved the performance of patients with recurring endometrial cancer: computed tomography showed reduced radiation density in the lungs and mediastinal lesions in liver disease, suggesting increased tumor necrosis [267]. In addition, bevacizumab combined with metformin specifically targets cancer stem cells and synergistically inhibits angiogenesis in ovarian cancer [268]. Moreover, the clinical trials of bevacizumab combined with metformin in the domestic and overseas are under way.

4.2.1.4 HER-2 inhibitors

HER2-targeted therapy is effective in breast cancer patients with HER2 overexpression and/or amplification. Trastuzumab, the first anti-HER2 monoclonal antibody developed in 1990, binds to the HER2 receptor in cancer cells and blocks the formation of heterodimers between HER2 and HER1, while also blocks HER3 or HER4, thereby inhibiting signal transduction pathways for cell survival and proliferation (such as PI3K or MAPK) and inducing apoptosis [269]. Trastuzumab has been the standard treatment for stable and early-stage HER2-positive breast cancer for over a decade. However, trastuzumab is also associated with an increased risk of cardiotoxicity and prolonged exposure to trastuzumab can lead to drug resistance [270]. Vazquez-martin et al. found that metformin significantly inhibits the self-renewal and proliferation of trastuzumab resistant breast cancer stem cells [271]. In addition, the proliferation of trastuzumab resistant HER2 overexpressing breast cancer cells was also inhibited by metformin. The mechanism of action involves disrupting the HER2/IGF-1R complex, which is found only in the resistant subpopulation [272].

4.2.2 Combination of metformin and multi-target inhibitors

Classical multi-target tumor inhibitors include sorafenib, imatinib, sunitinib, dasatinib, etc. Sorafenib was approved by the FDA in 2007 for the treatment of advanced hepatocellular carcinoma (HCC), but it is often accompanied with serious side-effects and low efficacy in tumor treatment. It has been shown that metformin sensitizes cancer cells to sorafenib. The two drugs work together to inhibit cancer cell proliferation and reduce sphericity, especially in drug-resistant cancer stem cells [273]. The addition of metformin reduced the dose of sorafenib by 25% without loss of its tumor suppressive effect [274]. Metformin can also enhance the anti-metastasis effect of sorafenib in patients with HCC by downregulating the ERK/JNK-mediated NF-κB-dependent pathway and reducing the expression of urokinase-type plasminogen activator (uPA) and MMP-9 [275]. Some preclinical studies have shown that metformin and sorafenib synergistically promote apoptosis and autophagy in HCC cells, showing synergistic antitumor effects [276]. In conclusion, the combination of metformin and sorafenib for the treatment of HCC shows broad application prospects.

Imatinib (IM) is a member of a class of small molecule tyrosine kinase inhibitors that selectivity targets the Abl kinase and launched a new era of tumor-targeted therapy. However, drug resistance frequently occurs, resulting in a significant reduction of therapeutic efficacy in chronic myelocytic leukemia (CML). Inhibition of mTOR and its effectors plays an important role in the generation of the antileukemic effects of BCR-ABL inhibitors [277]. With this aim, Eliza Vakana et al. found that metformin inhibited the mTOR signaling cascade in BCR-ABL expressing cells and suppressed the growth of different CML-derived cell lines, as well as primitive progenitors from CML patients. Importantly, metformin was also shown to possess growth inhibitory effects that can specifically target cells expressing the T315I-BCR-ABL mutation eliciting drug resistance [278]. These findings are particularly interesting and can lead the development of metformin as an antineoplastic agent in treatment of CML.

4.3 Combination of metformin with immunotherapy

Over the past decade, we have experienced a revolution in cancer treatment, especially in the field of novel cancer immunotherapy represented by immune checkpoint inhibitors (ICIs). ICIs, such as cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) inhibitors, programmed cell death protein (PD-1) inhibitors, and programmed cell death ligand-1 (PD-L1) inhibitors, have been used in the treatment of a variety of malignant tumors [279]. However, despite great success of current clinical oncology treatments, the low response rate of tumor immunotherapy is a major problem [280,281]. Previous study reported that metformin showed better antitumor effect in immunocompetent mouse models than in immunodeficient models, suggesting that metformin’s antitumor effect may be mediated primarily through the immune system [280]. Indeed, metformin could remodel immune cells and immune-related molecules, affecting the tumor microenvironment, and regulating the antitumor immune response [155]. Therefore, we reviewed the clinical benefits of metformin combined with immunotherapy, with emphasis on ICIs therapy, in cancer treatment and the related mechanisms.

4.3.1 Metformin increases the function of CD8⁺ cells

CD8⁺ CTLs, a key component for an effective antitumor response, produce effector cytokines and cytotoxic molecules such as granzyme and perforin, which can directly result in tumor cells death [282]. However, some tumor infiltrating CTLs remain in an “exhausted” functional state, marked by increased expression of various cell surface checkpoint proteins (such as PD-1, CTLA4) and decreased development of effector cytokines, due to long-term exposure to tumor antigens. Checkpoint proteins expressed by “exhausted” T cells bind to PD-L1 on the surface of tumor cells, suppressing the CTLs, and leading to tumor immune escape [283]. Eikawa et al. and Zhang et al. reported that metformin directly increased the number of CD8⁺ tumor-infiltrating lymphocytes (TILs), stimulated the production of TNFα and IFNγ, and decreased the expression of PD-1, thus saving the cells from exhaustion in an AMPK-dependent manner [284,285]. Not surprisingly, several retrospective studies have reported improved clinical outcomes in patients with NSCLC and melanoma who received ICIs in combination with metformin [285,286].

Metformin can also indirectly alter the function of CD8⁺ cells in the tumor immune microenvironment (TIME)(Fig.3). Recently, Cha et al. demonstrated that metformin via AMPK disrupts PD-L1/PD1 axis by reducing PD-L1 stability and membrane localization in tumor cells, hence exhibiting stronger cytotoxic effects [280]. Consistent with these findings, the combination of metformin and CTLA4 inhibitor exhibited stronger anti-tumor effect on breast cancer 4T1, B16F10, and CT26 models [280]. Similarly, blocking FOXO3-mediated c-MYC-PD-L1 and STAT3-PD-L1 dual pathways by metformin also promoted CD8⁺ T cells response and anti-tumor immunity, sensitizing tumors to anti-PD-1 therapy [287]. In addition, metformin could also synergize with tumor vaccine immunotherapy to alter the phenotype and function of CD8⁺ T cells [288]. Although metformin monotherapy had little therapeutic benefit in highly aggressive tumors, combination of metformin with PD-1 blockades resulting in improved intertumoral T cell function and tumor clearance [289].

4.3.2 Metformin enhances the killing effect of NK cells

While T cells remain a key component of an effective antitumor response, NK cells, as the “first responders,” exhibit broad tumor cytotoxicity without the requirement for major histocompatibility complex (MHC) restrictions [290]. The MHC class I polypeptide-related sequence A (MICA), subgroup of MHC class I molecules, serves as a natural ligand of NK cells. Cancer cells with abnormal expression of MICA can escape from anticancer effect of NK cells. Metformin treatment upregulates MICA on the surface of human cervical cancer cells through PI3K/Akt pathway, and downregulates PD-L1 on the surface of breast cancer cells through Foxo3-PD-L1 axis, which indirectly enhances the cytotoxicity of NK cells [287,291]. In addition, low-dose metformin can also activate the AKT/mTOR pathway in a P38 MAPK-dependent manner, triggering the polarization and secretion of cytolytic granules in NK cells [292]. Therefore, the combination of metformin and anti-PD-1 antibodies can augment the cytotoxic function of NK cells and improve the therapeutic efficacy [287,292].

4.3.3 Metformin inhibits suppressive immune cells

4.3.3.1 Myeloid-derived suppressor cells (MDSCs)

Remodeling or removing suppressive immune cells in TIME is an important way to improve immunotherapy. Myeloid-derived suppressor cells (MDSCs) produce immunosuppressive cytokines that allow tumor immune escape [293]. Several studies have investigated metformin treatment which could prevent PMN-MDSC or M-MDSC or both accumulation in TIME, thus providing potential preclinical benefits for antitumor immunotherapy [294–296].

4.3.3.2 Tumor-associated macrophages (TAMs)

In addition to MDSCs, tumor-associated macrophages (TAMs) also contribute to an immunosuppressive tumor microenvironment. In most solid tumors, M1 phenotype macrophages are tumor-resistant whereas M2 leads to tumor promotion. Metformin appears to inhibit M2 polarization of macrophages induced by inflammatory cytokines in vitro in an AMPK-dependent manner [297–299]. Furthermore, preclinical, and clinical evidences indicated that M1 macrophages increased and M2 macrophages decreased in the TIME after metformin administration in vivo [297,300,301]. In short, metformin may play a beneficial role in combination with tumor immunotherapy by blocking the accumulation of M2-like TAMs in the tumor and affecting TAMs polarization.

4.3.3.3 Regulatory T cells (Tregs)

Metformin can also target Tregs, which negatively regulates CTL functions needed for tumor elimination. Physiologic concentrations of metformin could inhibit the differentiation of CD4⁺ naive T cells to inducible Tregs induced by TGF-β in vitro and reduce CD4⁺CD25⁺ Tregs in tumors, both appearing to be correlated with reducing Forkhead Box P3 (FOXP3) protein caused by mTORC1 activation and metabolic reprogramming of glycolysis [302]. Unsurprisingly, a preclinical trial showed that metformin combined treatment with anti-PD-1/anti-CTLA4 increased the tumor-infiltrating CD8⁺/Treg ratio and significantly suppressed tumor growth compared with ICIs monotherapy [303].

Cancer immunotherapy has improved the outcomes of some patients, but its low response rate remains a major problem. In fact, due to the complexity of TIME, it is often necessary to use a cocktail of inhibitors or cytokines to achieve a powerful therapeutic effect, bringing unknown side effects and increase the economic burden of patients. Metformin can target different types of cells in TIME and profoundly reshape the inhibitory microenvironment at multiple levels through various mechanisms, thus it is promising to combine metformin with immunotherapy.

4.4 Summary and future perspectives

In the development of antitumor drugs, it has experienced three different stages of change: chemotherapy, targeted therapy, and immunotherapy. Metformin, as one of the most widely recognized metabolic modulators, shows prominent anticancer effects. Interestingly, although it is effective as a standalone drug, metformin is more effective in combination with other antineoplastic drugs and can be incorporated into traditional and emerging strategies. There are two main benefits of using metformin as an effective strategy for cancer treatment, the first is that the combination synergistically and additively enhances efficacy thereby reducing side effects due to lower doses. The second benefit is that when metformin is used in combination with other therapies it can potentially overcome or reverse drug resistance, thereby enhancing therapeutic anticancer benefits. Herein, we summarized and discussed the combination strategy of metformin in cancer, as an impressive and interesting approach for cancer therapy. However, attention should be paid to the potential side effects due to potential drug–drug interactions such as drug amalgamation. In addition, the effectiveness and feasibility of metformin alone or in combination with other treatments may vary between diabetic or non-diabetic patients. The anticancer effects of metformin generally increase with higher doses, which is much higher than the conventional concentration in diabetic patients, thus resulting in intolerable side effects and potential drug toxicity in actual clinical use. Meanwhile, the efficacy of metformin is highly dependent on the concentration of glucose in the tumor microenvironment. Other factors such as the pharmacokinetics of metformin, microenvironment, drug resistance after long-term administration and metabolic environment also should be considered when using metformin alone or in combination with other drugs. In conclusion, although the combination of metformin with clinically available antitumor agents shows strong synergistic effects, its future clinical application requires more sophisticated design and experimental investigation in clinical trials.

5 Metformin and aging

Aging is a natural process in which multiple function of living organisms gradually decline as people getting older [304]. Aging is considered as a risk factor for a number of age-related chronic diseases, including osteoporosis, cardiovascular disease (CVD), cancer, and neurodegenerative diseases [305]. It is predicted that by 2050, there will be 2.1 billion people aged 60 years or older [306], leading to the growing financial burden of age-related diseases worldwide. Therefore, there is an urgent need for effective drugs to prevent and delay aging. In this section, we will focus on the effects and mechanisms of metformin alleviating aging and the potential mechanisms inside.

5.1 Benefits of metformin in attenuating physiologic aging and age-related diseases

Metformin is also proposed as an “anti-aging” drug. A retrospective analysis of diabetics medicated with metformin showed longer survival than those who were treated with other anti-sugar drugs [32,307]. More importantly, diabetics on long-term metformin therapy survived longer, even when compared with healthy people [307]. Metformin can also effectively extend the healthy life span of different model organisms, including C. elegans, Drosophila melanogaster, and rodents, despite that some studies used high concentrations of metformin [111,308,309]. Based on these, clinical trials, including MILES (Metformin In Longevity Study), and TAME (Targeting Aging with Metformin), have been designed to determine if metformin can offset aging and extend lifespan (Tab.5). Data from the MILES trial suggest that metformin modifies a variety of pathways associated with aging, including metabolic pathways, collagen trimerization and extracellular matrix (ECM) remodeling, adipose tissue and fatty acid metabolism, mitochondrial and DNA mismatch repair which declines with age [310]. This study provides the first evidence that, in older adults, metformin has metabolic and nonmetabolic effects linked to aging. Metformin is now the focus of the first proposed clinical trial of an anti-aging drug—TAME program [311,312]. The goals of TAME are to measure the clinical outcomes by the emergence of new age-related chronic diseases and functional outcomes, such as athletic ability and cognitive impairment. In addition, the biomarkers of aging, such as inflammation and aging, are also considered. TAME trial results will provide more insight into whether metformin can reduce the risk of age-dependent diseases (excluding diabetes) in non-diabetic individuals, and may provide tools to target aging itself rather than related diseases.

In addition to biological aging, epidemiological studies have shown that the use of metformin may reduce the incidence of age-related diseases, such as cancers and neurodegenerative diseases [313]. Metformin was found to induce favorable metabolic and nonmetabolic pathways in skeletal muscle and subcutaneous adipose tissues of older adults with impaired glucose tolerance, the most common form of glucose dysregulation in this age group [310]. Clinical trial results have shown that metformin can slow the development of age-related cardiovascular disease and reduce the incidence of cardiovascular disease in diabetics [314,315]. This result is believed to be a result of metformin’s pleiotropic effects, which extend beyond glycemic management [32]. Indeed, a recent meta-analysis showed significant reductions in cardiovascular mortality, all-cause mortality, and cardiovascular events in patients with coronary artery disease treated with metformin [316]. Age is the single biggest risk factor for many cancers [317]. Metformin has been widely studied as a promising anticancer drug, which can be used alone or in combination with other anticancer drugs for the treatment of malignant tumors [318], as mentioned in the above part. Aging people become highly prone to neurodegenerative diseases [319], including Alzheimer’s disease. The experimental and clinical data showed that metformin can help prevent AD, including lowering risk of AD and enhancing cognitive function [320–322]. These clinically relevant data suggest that metformin can provide a strong protective effect in age-related diseases.

5.2 The primary mechanisms of metformin action in attenuating aging

Despite clinical use for metformin as an anti-diabetic even anti-aging drug, the exact cellular mechanisms by which metformin exerts its actions remain unclear. In the following part, we will focus on the main mechanisms of anti-aging action of metformin (Fig.4).

5.2.1 Metformin improves the deregulated nutrient-sensing

With age increasing, the organism is characterized by deregulated nutrient-sensing and metabolic disorder, further leading to the aging phenotype [323]. These highly conserved nutrient sensing and signal regulation pathways mainly include: (1) the insulin/IGF-1 signaling (IIS) pathway; (2) other nutrient-sensing systems: mTOR, AMPK, and sirtuins. These trophic pathways were mentioned in the 2013 review and supported by omics data [324]. Metformin exerts beneficial effects on energy metabolism and aging by directly regulating key energy sensors. Metformin activates AMPK through a LKB1-dependent mechanism, and long-term administration of low concentration metformin (0.1% w/w in diet) can improve health span in male mice [309]. Long-term treatment of female isolated SHR mice with metformin (100 mg/kg drinking water) also increased their average life expectancy by 37.8% [325]. In summary, the researchers suggest that metformin therapy mimics similar benefits of calorie restriction, such as improved physical performance, increased insulin sensitivity, and reduced LDL and cholesterol levels without reducing caloric intake [326].

5.2.2 Metformin alleviates genomic instability

Accumulation of genetic damage is a commonly accepted cause of aging, which may disrupt cellular homeostasis and lead to genomic instability [327]. As we age, defects in DNA repair mechanisms affect the expression and transcription of essential genes, leading to cell dysfunction. A large number of preclinical studies have shown that impaired DNA repair ability would lead to premature aging syndromes, such as Werner syndrome and Bloom syndrome [328]. In addition, mitochondrial DNA mutations and nuclear lamina defects during aging can also lead to genomic instability [329]. There are several mechanisms by which metformin alleviates the response to genomic instability in the context of aging and various types of cancer. Studies have shown that metformin exerts the genomic protective effect by reducing oxidative stress, DNA damage, regulating ataxia telangiectasia mutated (ATM) protein, and epigenetic effects [330]. Our team used to report that AMPK-mediated phosphorylation on 53BP1 promotes classic non-homologous end joining (c-NHEJ), suggesting that metformin as a putative activator of AMPK may also regulate this process [331]. A recent clinical trial [332] in Kazakhstan was the first to investigate the changes in lymphocyte DNA damage before and after the use of metformin in obese people. It was found that the use of metformin 850 mg/day for 3 months in the treatment of obesity (BMI greater than 30 kg/m²) reduced the DNA breakage of lymphocytes. Previous clinical studies have also shown that short-term administration of metformin in the elderly can induce BRCA-mediated DNA damage response and DNA repair in skeletal muscle [310]. The antioxidant properties of metformin are currently controversial. 25 mg/kg metformin can significantly improve the concentrations of antioxidant enzymes (SOD, CAT) and GSH in the treatment of alloxacil-induced diabetic rats, indicating that metformin can help to protect oxidative stress-induced damage in diabetic complications [333]. In an elderly rat model of another study by Allard et al. [334], long-term administration of metformin decreased the transcription and activation of the antioxidant regulator Nrf2. The authors explain that the dose and timing of metformin are important factors to consider in rodent studies, and they combined long-term use of metformin with a relatively high daily dose. Anisimov and colleagues [335] reported that the life-prolonging ability of metformin decreased with increasing age of metformin treatment. Although there is no consensus on the mechanism of how metformin regulates oxidative damage or attenuates genomic instability, more evidences indicate that metformin prevents or reverses age-induced disorders via regulating nuclear and mitochondrial genome stability and chromosome structure.

5.2.3 Metformin downregulates SASP and reduces the burden of senescent cells

Cellular senescence can be defined as a stable arrest of the cell cycle and the accumulation of senescent cells aggravates aging. Senescent cells exhibit dramatic changes in their secretory group, some of which are enriched with pro-inflammatory cytokines and matrix metalloproteinases, known as the “senescence-associated secretory phenotype” (SASP). Although metformin could not work as senolytics, it can effectively inhibit cell senescence and SASP in many age-related diseases. Chronic low-dose metformin delays cellular senescence through NRF2-mediated upregulation of glutathione peroxidase 7 (Gpx7), characterized by the reduced percentage of senescence-associated β-galactosidase (SA-β-Gal)-positive cells [336]. In addition, metformin inhibits the SASP by preventing NF-κB translocation into the nucleus, thereby interfering with IKK/NF-κB activation [337]. In addition to this, metformin can reduce the RNA levels of SASP markers including IL-6 and IL-8 and the protein expression levels of p16 and p21 in human fibroblasts through a DICER1-dependent mechanism [338]. Thus, metformin can function as senomorphics to regulate SASP via varied mechanisms. In the future, drug combinations of metformin can be considered to alleviate aging and extend lifespan.

5.2.4 Metformin downregulates pro-inflammation responses

Aging is associated with a chronic, low-grade inflammatory state called “inflamm-aging,” which is associated with age-related diseases [339]. The definition of “inflamm-aging” is based on cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) [340]. Despite the fact that there is no clear understanding about the causes of “inflamm-aging,” it is a highly significant risk factor for morbidity and mortality in the elderly [341,342].

Multiple studies in patients with T2DM provide evidence of the anti-inflammatory effects of metformin. In T2DM patients, metformin monotherapy was associated with significantly lower levels of inflammatory molecules, including TNF-α, soluble TNF receptor 1 (sTNFRI), and sTNFRII compared with other anti-diabetic monotherapy [343]. Chen et al. found that metformin reduced blood IL-6 and urine MCP-1 levels in patients with T2DM in a time- and dose-dependent manner [344]. In another T2D study, metformin treatment significantly reduced levels of NF-κB, IL-1β, and niacin receptor GPR109A in peripheral blood leukocytes [345]. Notably, in patients with atherosclerotics, most of whom were free from diabetes, plasma levels of inflammatory factors (e.g., IL-6 and TNF-α) also reduced after metformin treatment [346]. Together, these clinically relevant data suggest a promising role of metformin in targeting age-related chronic inflammation, but whether the effect varies between diabetics and non-diabetics remain further study.

The mechanism by which metformin alleviates inflammation has been extensively studied in animal models. The anti-inflammatory effects of metformin initiated with AMPK activation followed by downstream inhibition of the mTOR and NF-κB signaling cascades [337,347,348]. In addition to AMPK-mediated effects, several effects have been described that appear to be unrelated to AMPK. Metformin upregulated miR-146a and miR-155 to improve endothelial cell inflammation [349]. Metformin increased the expression of miR-34a-5p and miR-125b-5p, which in turn inhibits the production of pro-inflammatory cytokines from macrophages [350]. In addition, metformin treatment did not change the phosphorylation of AMPK, but it did enhance the expression of PFKFB3/iPFK2 axis in adipocytes, which is associated with the anti-inflammatory effects of metformin [91].

5.2.5 Metformin enhances autophagy

Autophagy-mediated protein degradation is deteriorated with aging, leading to an imbalance in protein abundance and loss of proteostasis [351]. Dysregulation of the autophagy pathway hinders the removal of dysfunctional, damaged, or extra organelles, has a major impact on cellular health, and can lead to the development of metabolic and aging-related diseases [352,353].

Evidence from different model species supports the role of autophagy in the regulation of aging and age-related diseases. For example, the autophagy-related gene (ATG) was needed to extend longevity in long-lived C. elegans with daf-2 mutation [354]. By preventing Beclin-1 from interacting with the negative regulator Bcl-2, Becn1F121A/F121A mutant mice exhibited higher levels of basal autophagic flux, and thus lived longer and experienced fewer age-related symptoms in vivo [355]. These findings suggest that individuals with impaired autophagy are susceptible to age-related diseases and shortened lifespan.

Clinically, limited paper found that metformin upregulated mitochondrial autophagy of mononuclear cells in T2DM patients, and subsequently improved mitochondrial morphology and function [356]. Metformin enhanced autophagy in CD4⁺ T cells from older subjects and improved mitochondrial bioenergetics and T cell inflammation [357]. One study also showed that metformin improves polycystic ovary syndrome (PCOS) by activating autophagy and reducing testosterone levels [358]. Experimental data from animal models fully compensate for the lack of clinical data. Depending on AMPK-autophagy axis, metformin improved age-related pathological phenotypes, such as hepatic steatosis [359], atherosclerosis [360] and neurodegeneration [361]. In summary, these studies suggest that autophagy plays a role in metformin prolonging lifespan and improving age-related diseases.

5.3 Additional potential mechanisms of metformin against aging

Progressive telomere attrition and shortening is associated with biological aging and aging-related diseases [362]. Metformin was reported to reduce telomere attrition in leukocytes of patients with mild aging-related diabetes, showing a significant anti-aging effect [363]. Similar studies were also processed and found that metformin prevented shortening of leukocyte telomere length in diabetic individuals [364,365]. However, the mechanisms by which metformin reduces telomere attrition remain elusive. Further studies are needed to illustrate how metformin prevents telomere attrition and maintains telomere integrity.

Mitochondrial dysfunction caused by various pathological factors affects cell metabolism and impairs body function and health [366]. It is reported that metformin improved the physiologic function of the elderly by eliminating exercise mediated increase in mitochondrial respiration of skeletal muscle [367]. ROS accumulation and metabolic disorder resulted from mitochondrial dysfunction during aging are both the potential targets of metformin [368]. Metformin treatment of in vitro-aged adipose-derived stromal cells (ASCs) from older women decreased oxidative stress and mitochondrial dysfunction, via activation of AMPK, which resulted in decreased senescence, implying that metformin might be a useful drug to target age-related adipose tissue dysfunction [369]. Metformin was reported to regulate mitochondrial biogenesis and senescence through AMPK mediated H3K79 methylation, thus alleviating age-associated vascular dysfunction [370]. Metformin also inhibits mitochondrial ETC complex I, thereby directly affecting mitochondrial induced oxidative stress by reducing ROS or other indirect mechanisms to scavenge ROS [371]. However, there is no consensus conclusion on whether improving mitochondrial dysfunction by metformin is tissue specific and the appropriate treatment, metformin monotherapy or in combination with other interventions.

With aging, there is a systemic decline the regenerative capacity of tissues, represented by the disruption of homeostasis of stem and progenitor cells [372]. Metformin was found to restore CNS remyelination capacity by rejuvenating aged stem cells via AMPK [373]. Furthermore, in Drosophila melanogaster midgut stem cells, metformin was demonstrated to attenuate aging-related phenotypes by regulating autophagy-related genes and AKT/mTOR signaling pathway [374]. Ma et al. recently discovered a novel target of low-dose metformin [81]. These findings revealed that metformin binds PEN2 and initiates a signaling route that intersects, through ATP6AP1, the lysosomal glucose-sensing pathway for AMPK activation. The signaling pathway ensures the beneficial effect of metformin, including lifespan extension in nematodes. This ensures that metformin exerts its therapeutic benefits in patients without substantial adverse effects.

Metformin was found to induce hormesis and improve resilience under stress conditions in various organs, cells, and endpoints, possibly via AMPK-Nrf2 axis, thus preventing numerous aging-related health conditions. But the detailed mechanisms remain further illustrated. These distinct biomedical effects of metformin have important implications for future studies, showing the potential to influence development of study design, patient dose optimization, and dose interval [375].

The microbiome is widely recognized as a core regulator of host health [376]. A high-throughput method was applied to investigate host-microbe-drug-nutrient interactions, where metformin host effects are identified to be regulated by a bacterial nutrient signaling pathway. Metabolic modeling of human gut microbiomes links metformin to microbial agmatine and metformin-bacterial interactions engage host lipid metabolism to extend lifespan [111].

5.4 Summary and future perspectives

Most clinical data on the health advantages of metformin came from studies of people with diabetes, insulin resistance, and obesity who were also frequently taking numerous drugs to treat co-occurring conditions. Metformin therapy appears to be relatively safe over the long-term, enables effective glycemic control, and provides additional health benefits not directly related to glycemia management. Metformin affects neuroendocrine control, mediates weight loss, enhances insulin sensitivity, and favorably alters lipid profiles, which are all beneficial for ameliorating aging.

However, there also emerged some negative or contradictory results. Complementary findings from UK Biobank and meta-analyses revealed that the neurocognitive impact of T2DM suggests marked acceleration of normal brain aging, but metformin treatment did not improve neurocognitive outcomes [377]. A series of sometimes controversial data triggered a debate, that is, whether the beneficial effect of metformin extends people’s lifespan or enhances health span via its anti-hyperglycemic effect remains elusive. Despite the data in support of anti-aging benefits, whether metformin has a protective effect in those subjects without disease also remains controversial. It is also worth noting that anti-aging effects of metformin on various species appear to be age-dependent [335,378]. Metformin even was reported to shorten lifespan when provided in late life, contrary to its positive effects in young organisms in C. elegans and human primary cells, since metformin exacerbated aging-associated mitochondrial dysfunction, causing respiratory failure [379]. Another clinical trial, MASTERS study, also did not support the use of metformin to enhance the benefits of physical activity in healthy elderly people [380]. This data implied that metformin induces deleterious changes of conserved metabolic pathways in late life, which could bring into question its benefits for older individuals without diabetes. Several other clinical trials (NCT04264897, NCT02570672, NCT03107884) on the anti-aging effect of metformin are currently underway, which may address some of the concerns and the results of these studies will better assess the benefits of metformin on healthy longevity. Metformin’s safety and effectiveness data in young, healthy people are still lacking. It is necessary to conduct well-designed randomized controlled trials to determine whether metformin medication at a younger age is risk-free and indeed promotes longevity and optimal health maintenance. Again, we must also consider that aging is a heterogeneous phenomenon and that different individuals in the same population may respond differently to metformin. The use of metformin in clinical settings has certain limitations due to its short half-life and relatively low bioavailability. We also can not ignore the side effects of long-term using metformin, including vitamin B12 deficiency, gastrointestinal complaints, lactic acidosis and even nervous system disorders mentioned below. Despite the issues with side effects addressed in many ways for translation, more research is needed to calibrate the balance between safety and anti-aging efficacy of metformin [381].

6 Metformin and neurological disorder

Metformin has many applications and is one of the most prescribed drugs in the world [154]. Recent studies of this drug have found that metformin can affect neurons in the brain through influencing the activation of AMPK and activate different pathways to enhance mitochondrial functions including activation of transcriptional coactivator peroxisome proliferator-activated receptor-λ coactivator 1-α (PGC-1α) [382–384]. Metformin induces phosphorylation and activation of AMPK in neurons can also inhibit downstream effectors such as acetyl-CoA carboxylases (ACCs) 1 and 2 which suppress fatty acid synthesis and promote fatty acid oxidation [385]. Administration of metformin was found to inactivate downstream effector ACC in human stem cells and dorsal root ganglia (DRG) [386,387]. Although metformin has been a well-known activator of AMPK, there was a controversial study that showed a lowering of AMPK activation after metformin administration in the hypothalamus of rats [125]. This implies that metformin can have various effects on neurons in different brain regions. Although there are different mechanisms in play regarding AMPK, administration of metformin has been found to reduce neuronal damage and promote neuroblast proliferation [388].

Metformin is a drug that has been approved for use in clinical settings. It has potential to modulate and protect neurons under certain conditions and has been used in reprogramming neurons [389,390]. It has been suggested that dysregulation of insulin function contributes greatly to aging and the development of neurological diseases such as Alzheimer’s disease (AD), Parkinson’s disease, and many others [391,392]. There have been many studies linking diabetes and insulin resistance to development of neurological disorders, however the mechanism is currently unknown [393–395]. There have been many studies regarding metformin and its effect on neurodegenerative disease in both humans and animals (Fig.5), however, the studies have yielded conflicting results, therefore metformin has not yet been recognized for therapeutic use [391].

6.1 Metformin and neurodegenerative diseases

6.1.1 Alzheimer’s disease

AD is one of the most common types of dementia in the world, effecting 45 million people worldwide [391]. Dementia is a common neurological disorder characterized by the loss of two or more cognitive abilities and has a drastic negative impact on those afflicted [396]. The patients suffering from AD possess signs of progressive memory loss and decline of cognitive function with neurofibrillary tangles (NFT) and amyloid-β plaques (Aβ) expressing throughout the brain [397]. The tau protein is associated with microtubules and is responsible for their stabilization [398]. Abnormal tau hyperphosphorylation is associated with NFTs in AD [399]. The Aβ plaques are thought to be due to β-secretase β-site amyloid precursor protein cleaving enzyme 1 (BACE1) and γ-secretase complex cleaving the membrane of the amyloid precursor protein (APP) [400]. Dysregulation and buildup of these proteins in the brain are believed to be one of the underlying causes of AD. In recent research, metformin has been used to counteract AD and improve spatial memory in diabetic mice through preventing p-tau and amyloid plaque load, preventing neuronal death [401]. However, another research has found that metformin may cause an increased risk of AD through increasing production of both intracellular and extracellular Aβ production [402]. The increase of Aβ production may be due to AMPK effecting the APP metabolism in the lipid rafts through altering cholesterol and sphingomyelin homeostasis [403].

In clinical settings, metformin was mostly found to reduce diabetes associated cognitive impairment and AD in type 2 diabetes mellitus (T2DM) patients [38,404,405]. Incidence involving dementia was lowered in T2DM patients receiving metformin or a combination of metformin and sulfonylurea than patients receiving sulfonylurea or thiazolidinediones alone in two studies [406–408]. Although most studies showed the benefit of metformin on lowering AD, one long-term study of using metformin alone for treatment of T2DM found that it had higher risk of developing AD [409]. This signified that T2DM patients that receive metformin may have varying effects. Later research supported this theory through use of latent class analysis which was able to group different T2DM patients who have received metformin into various risk-profiles of developing AD [410]. This varying effect may be due to metformin’s effect on T2DM alone which has been known to cause memory impairments [411]. Therefore, after recovery, patients with T2DM should abstain from further treatment of metformin. Furthermore, AD patients with T2DM should not take metformin in the long term, as it may exacerbate AD progression through increasing Aβ production, although this still need further research. Due to the varying effects of metformin, there is still much research that needs to be conducted to ensure its efficacy in treatment of AD and T2DM associated AD.

6.1.2 Metformin and Parkinson’s disease

Parkinson’s disease (PD) is the second most common neurodegenerative disease that exists in the elderly population [412]. It effects about 1% of the elderly population over the age of 60 years and 4% in those population over the age of 80 years [413]. Patients suffering from PD suffer from progressive loss of dopaminergic neurons in the sub-nigra (SN) region of the brain [412,414]. Insoluble proteins known as Lewy bodies were found in various brain regions, consisting of aggregated α synuclein (aSyn) [415,416]. These Lewy bodies can spread throughout several brain region along with disease progression [417,418]. Metformin has been shown to reduce mean platelet volume (MPV) in patients with PD that have platelet dysfunctions demonstrating antiatherogenic effect [419,420]. Recent research using metformin in 1-methyl-4-fenyl-1,2,3,6-tetrahydropyridin (MPTP) induced acute PD mice models showed many varied degrees of success despite similar experimental design [421–425]. In most studies, metformin was found to reduce the effect of MPTP on dopaminergic neurons in the SN [421–424]. There were two studies that contradicted earlier studies, reporting that metformin had no protective effects against MPTP induced neuronal cell loss and the loss of dopaminergic neurons in the SN, however, in one study, protection of dopaminergic neurons in the striatum was found [423,425]. These contradicting studies show that metformin may have very different functions in different brain regions of MPTP induced PD model or the fact that there may be different AMPK effects in dopaminergic neurons in certain regions of the brain. Furthermore, the metformin may have varying effects that are dependent on the different stages of the disease, such that it may prevent or inhibit neuronal loss in early stages of PD, however, the effects are negligible in the later stages of the disease.

In clinical setting, metformin is often used in combination with other drugs instead of its standalone to treat PD [391]. A clinical study to evaluate the effects of metformin on type 2 diabetes and its relation with PD, was used to determine the efficacy of sulfonylurea, metformin, and in combination [426]. The test found that the patients who received sulfonylurea had increased risk of PD compared to those who received anti-hyperglycemic agents, metformin, or a combination of metformin and sulfonylurea [391]. This suggests that metformin may have been able to counteract the adverse effects of sulfonylurea [426]. Metformin has also shown to aggravate PD when taken alone, signifying the potential usefulness as a supplementary drug. Incorporation of metformin in treatments for PD may have potential due to its neuron protection properties which may help eliminate potential adverse effects elicited by other medications. Although there is potential in combination drug use of metformin with PD medications, extensive studies are still required to determine the overall effects it will have on patients.

6.1.3 Metformin and Huntington’s disease

Huntington’s disease (HD) is a neurodegenerative disease that causes involuntary jerky, irregular, and spasmodic movement. This disease causes progressive cognitive decline and is often diagnosed around 30–40 years of age. HD is a dominantly inherited genetic disorder caused by CAG trinucleotide repeat expansions in the HTT gene which encodes the Huntingtin protein [427]. The defect in the HTT gene often causes production of abnormally long versions of the Huntingtin protein which are broken down by the cell into toxic fragments that aggregate and accumulate in neurons causing neurodegeneration [391]. Reports of metabolic changes in patients with HD have been contradictory [391]. Clinical studies of patients with HD have been reported to have altered glucose metabolism and increased T2DM [428,429]. However, other studies found no changes in metabolism through comparing the glucose tolerance test and examining the changes in pancreatic tissues between patients with HD and control [430,431]. Metformin has been used in clinical settings to treat HD patients and showed that HD patients with T2DM showed improved cognitive results compared to HD patients that do not have T2DM [391]. In contrast, non-HD T2DM patients that took metformin performed worse on cognitive tests compared with non-diabetic controls [432]. The improvement of cognitive functionality may only be due to recovery of T2DM and not solely HD itself. Furthermore, the mutation in the HTT gene in those with HD has been found to cause abnormal activation of AMPKα1 under oxidative stress causing neurotoxicity to surrounding neurons [433]. This would explain why taking metformin worsened cognitive functionality of HD patients, contributing to increased neurotoxicity and neurodegeneration. Taken together, metformin has shown poor influence in restoring cognitive functionality in patients with HD and may even worsen this condition. Thus, metformin should be used with caution and best be avoided when dealing with HD. This signifies that metformin has limited influence on restoring cognitive functionality in patients with HD.

6.1.4 Metformin and multiple sclerosis

Multiple sclerosis (MS) is a chronic demyelination of the neurons in the central nervous system (CNS) caused by the immune system [434]. Progression of MS has been shown to affect the normal functionality of B and T cells through the dysfunction of regulatory T cells (TR) [435–439]. Th17 cells secrete pro-inflammatory cytokines such as IL-17A, IL-17F, IL-21 and tumor necrosis factor α (TNF-α) and through infiltration of the CNS can disrupt the blood brain barrier (BBB) [440,441]. Metformin has shown therapeutic effect on animal model of systemic lupus erythematosus (SLE), an autoimmune disease of the skin, through inhibition of oxidative phosphorylation (OXPHOS) and lowering the CD4⁺ T cell activation [442]. Metformin has shown to have a positive effect on fibrinolysis and thus regulate neuroinflammation [443–445]. Research has also found that metformin reduces platelet aggregation, lowers platelet adhesion, and decreases adenosine diphosphate (ADP) induced adhesion to fibrinogen [434,446]. A study has shown that elevating AMPK expression can affect the expression of IFN-γ and IL-17 [447]. Furthermore, TR has shown increased expression in metformin supplemented patients [447]. Increased TR can suppress inflammation in those with MS which can reduce swelling and help reduce damage to adjacent undamaged neurons thereby reducing size of the brain lesions. This was reflected in clinical research where patients given 6–24 months of treatment using metformin showed significant reduction in the number of new or expanding T2 brain lesions [434]. Metformin activating AMPK can also affect neurogenesis and may be able to repair damaged neurons caused by MS [448]. In recent research metformin was found to restore regenerative ability of oligodendrocyte progenitor cells which play important roles in remyelination processes which become impaired in MS [373,449]. Taken together, metformin has shown to have many benefits in treating patients with MS with little adverse effects. Treatment of MS using metformin may also be considered for combination drug therapy as it has shown to have potential in this aspect.

6.2 Metformin and other neurological disorders

6.2.1 Metformin and epilepsy

Epilepsy is a neurological disorder that causes uncontrolled involuntary spasms. It is characterized by hyper-excitatory seizures caused by imbalance of excitatory and inhibitory neurons [450]. Progression of epilepsy has been shown to cause neuronal loss in the brain due to excitotoxicity [451,452]. The overall mechanism of epilepsy is still poorly understood; however, it is accepted that the spontaneous discharge of epilepsy is caused by either the increased excitability of excitatory neurons or decreased excitability of inhibitory neurons [453]. In animal models, metformin was found anti-seizure properties. In one experiment metformin was able to attenuate pentylenetetrazol (PTZ) induced seizures in mice and increase the latency to seizure onset while decreasing seizure duration, suggesting that metformin may modulate epileptogenesis through ameliorating the expression of C/EBP homologous protein and suppress the endoplasmic reticulum (ER) stress through AMPK-PI3K-c-Jun kinase pathway and thereby protect against BBB disruption and reduce neuronal apoptosis [454]. Furthermore, administration of metformin was able to lower the seizure stage, suppress kindle seizure progression, and protect against learning memory impairment in PTZ kindled mice model [455]. Metformin has also demonstrated anti-seizure effect on kainic acid (KA) induced chronic seizures, shortening epilepsy duration, suggesting that AMPK activation may play an important role in seizure regulation [456]. A study showed that AMPK activation can upregulate Bcl-2-modifying factor (Bmf), which can alleviate seizure-induced cell death in the hippocampus, suggesting that metformin may also impact seizure progression through this pathway [457]. Evaluation of metformin’s effect on epilepsy has also found that seizure progression was reduced in response to metformin administration 45 days after induction of status epilepticus (SE) in rats [458]. Furthermore, another study found that treatment with metformin decreased levels of phosphorylated mTOR protein and elevated levels of phosphorylated AMPK protein in pilocarpine induced epileptic mice model [458]. Metformin treatment has also been found to suppress the expression of BDNF and its receptor, trkB, which has been reported to be elevated in brains of epileptic mice [458,459]. In clinical studies, 500 mg of metformin was given orally to patients with tuberous sclerosis, and the seizure frequency was found to decrease by 44% as opposed to the placebo group [460]. In patients with Lafora disease, the patients who were given metformin showed a reduction in seizure frequency, however, there was lack of clinical response in late stages of the disease [461]. This is probably due to the increased threshold required to mitigate the late-stage seizures. The use of metformin in treating seizures may be applied in early stages of the disease to prevent seizure progression. In late-stage seizures, metformin may be combined with other seizure medication to help alleviate different adverse effect while maintaining anti-seizure effects. Taken together, metformin has shown great potential in treating epilepsy and diseases that may result in epilepsy.

6.2.2 Metformin and depression

Major depressive disorder (MDD) is a complex neurological disorder that is characterized by lack of energy, dysfunction of cognition and memory [462]. The pathology of MDD is still unclear, it is often associated with chronic stress resulting from either environmental or physiologic factors [463]. Imbalance of excitatory and inhibitory (EI) inputs were found in the brain of depressed mice model [464]. Furthermore, MDD were found to have altered synaptic signaling due to structural alterations in both hippocampus and the prefrontal cortex (PFC) and decrease of both glutamate and GABA neurotransmitters [465]. Disruption of glutamate excitatory projection neurons which are responsible for circuit level transmissions in the brain region may cause structural changes in the brain [465]. GABAergic functionality is also disrupted in MDD resulting in alteration in synaptic transmission [466–469]. Alterations to serotonergic neuron transmissions are present in those afflicted with MDD [470]. Metformin is able to increase the frequency of excitatory postsynaptic current (EPSC) which may alleviate disruption of depression caused by MDD [471]. Furthermore, metformin can also stimulate serotoninergic neurons excitability and modulate serotonergic transmission [472,473]. Regulation of serotonergic neurons signifies that metformin may be able to regulate mood in those afflicted by MDD. In evaluating metformin’s effect on T2DM and its associated mood disorders, administration of metformin in T2DM rats showed decreased depressive-like behaviors [474]. Furthermore, it was found that metformin was able to promote neurogenesis in mice and humans [475]. In clinical settings, metformin was found to have antidepressant effects in diabetic patients through improving their cognitive functions [476]. Metformin’s activation of AMPK can increase neuronal activity, which shows anti-depression effect similar with physical exercise ameliorating depressive-like behaviors via AMPK, although this mechanism remains elusive [477]. In a case study, metformin was found to lower the diabetic patient’s risk of developing MDD [478]. This showed that metformin can counteract diabetic related depression through increasing metabolism. Furthermore, metformin’s increase of neuronal activity and generation of ATP may be a contributing factor. Metformin has demonstrated potential in treating depression in T2DM, however, whether metformin is able to treat patients that do not have T2DM remains to be seen.

6.2.3 Metformin and stroke

Stroke is a common neurological disease that is caused by cardiac dysfunction that can lead to cerebral ischemia [479]. Ischemia results in mitochondrial deficits, oxidative stress, disruption of the BBB, increased neuron apoptosis, and neuroinflammation [479]. Metformin may be able to activate M2 microglia to promote clearance of cellular debris and heighten the expression of growth factors [479–481]. It is also able to further activate anti-inflammatory molecules such as interleukin-4, 10, and TGF-β [479–481]. These activated M2 microglia are neuroprotective in response to ischemic injury, however, they are temporary, and will revert back to M1 phenotype [481]. In an earlier study, metformin upregulated AMK and released elevated levels of interleukin-10 and anti-inflammatory cytokine [482]. Administration of metformin for 30 days showed improvement in neurogenesis, angiogenesis, and functional recovery in mice [482]. This was further supported in another study, where rats receiving metformin prior to stroke induction showed that quantity of viable neurons was significantly higher in metformin pre-treatment group compared with the control group [483]. Metformin treated group also showed increased autophagic response through AMPK activation which can help reduce cellular protein accumulation and thereby improve recovery [483,484]. Although there are animal studies of using metformin in treating, it is still in its infancy. Although pretreatment with metformin may reduce the risk of stroke, its clinical data are still limited. A follow-up clinical study in diabetic patients prescribed with metformin show that metformin can lower patient’s risk of stroke by almost 50% [485]. In light of this, there is still a lot of clinical research that needs to be done for evaluating the efficacy of metformin in stroke. Overall, metformin’s effect of neurogenesis, neuronal protection, and anti-inflammatory effects are undeniable. Metformin’s activation of AMPK may also provide energy to neurons to help negate the negative effect induced by the lack of energy due to oxygen deprivation. Therefore, metformin may be a promising candidate in treatment of ischemic stroke.

6.3 Future prospects

Metformin is an oral anti-diabetic drug that has shown potential in treating many neurological disorders. It has shown to have potential to have an effect on strokes, seizures, and MS. However, many more clinical tests are needed to ensure their efficacy. The current findings of metformin, mainly show its benefits in alleviating symptoms caused by diabetes, however, its neuroprotection and its effect on neuron recovery are undoable. There are many controversial findings of metformin that suggest that not all of metformin and AMPK functionalities have been identified. Although there are many findings that metformin may cause the worsening neuro-degenerative diseases through alternative pathways, it has definitely demonstrated its effect in neuron protection and protecting against adverse effects in other medication when used in conjunction making metformin a promising candidate for clinical consideration. As such, more pre-clinical experiments of metformin in treating other neurological disorders such as AD, PD, MDD, and HD should be conducted to ensure its efficacy and its long-term safety to debunk the controversies before clinical trials. Furthermore, these tests should also involve those who possesses these neurological disorders, but not as a result of T2DM. Overall, metformin has shown to have a wide range of beneficial effects on neurological disorders.

7 Discussion

In this review, we systematically summarize and discuss the development history of metformin, its protective role and molecular mechanism in various diseases, including metabolism and metabolic related diseases, cancer, and the combination strategy, aging and aging related diseases, neurodegenerative diseases, and other neurological disorders. But in view of this, we can not help asking this question: is metformin a drug for all diseases? It is unclear whether all these putative beneficial effects are secondary to its role as an antihyperglycemic and insulin-sensitizing agent, or stem from other cellular effects. Whether metformin can improve these diseases in normal patients instead of diabetics, remains to be further studied. Moreover, approximate 30% of individuals may experience metformin adverse effects, necessitating careful monitoring along with dose and formulation (extended-release versions) adjustments [486]. It is important to pay attention to the concentration and dose of metformin used in preclinical studies, with many of that have employed concentrations in excess of 10 to 1000-fold of maximal plasma levels observed in humans [487]. Therefore, more studies including clinical trials, are required to determine the impact of metformin on multiple diseases and illustrate the indicated mechanisms.

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