Metformin's many effects from diabetes to degenerative diseases

Alzheimer's disease (AD) is the most prevalent form of neurodegeneration, and its incidence is projected to rise with population aging. While numerous therapeutic strategies have been proposed and tested, effective and curative interventions for AD remain elusive. Recent epidemiology studies have revealed a significant correlation between the incidence of AD and T2DM (Table 1) [62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77], providing a rationale for investigating metformin as a potential treatment for dementia [78]. Being commonly prescribed as a hypoglycemic drug, metformin is safe and well-tolerated, with no significant side effects reported in individuals with Mild Cognitive Impairment (MCI) or AD [72,73]. Thanks to its ability to efficiently cross the blood–brain barrier (BBB), metformin rapidly accumulates in the brain, where it exerts anti-inflammatory and antioxidant effects [79,80]. In this regard, it is crucial to note that, in AD patients, the typical elevated cholesterol levels, coupled with abnormal sphingolipid and phospholipid production, damage key cells in the brain, including pericytes, vascular endothelial cells, microglia, and astrocytes [81,82]. Thus, the neuroprotective effects of metformin might be significantly beneficial in mitigating these damages. This is particularly relevant in diabetic patients, where hyperglycemia triggers OS, with consequent accumulation of amyloid beta (Aβ) oligomers, neuronal toxicity, and brain inflammation [79,80]. Molecularly (Figure 2), metformin acts as a modulator of the AKT/ERK/GSK-3β signaling pathway and leverages GSK-3β suppression to reduce tau phosphorylation [83]. Though Nrf2 stabilization, metformin also mitigates oxidative stress and reduces cellular apoptosis [84]. Furthermore, by regulating the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) signaling cascades, metformin stimulates autophagy and counteracts mitochondrial dysfunction in the brain, thereby reducing AD risk [85,86]. The parallel modulation of IGF-1 and mTOR pathways further explains the positive effect of metformin intake in reducing AD occurrence, positioning this caloric restriction mimetic a strong candidate for clinical investigation [87,88]. Deeper insights gained from mouse studies have significantly advanced the mechanistic understanding of metformin in brain diseases. Notably, single-cell RNA sequencing in drug-treated amyloid precursor protein/presenilin 1 (APP/PS1) transgenic mice showed that negative regulation of apoptosis, enhanced astrocyte differentiation from stem cells, together with increased expression of neural stem cell and oligodendrocyte precursor cell self-renewal pathways resulted in improved spatial memory, reduced anxiety scores, and enhanced cognition [89]. Consistently, functional analysis demonstrated decreased hippocampal CA1 neuronal loss and reduced levels of the pro-apoptotic proteins caspase-3 and poly ADP ribose polymerase in animal models of AD treated with metformin [90,91,92,93]. In parallel, metformin improved cholinergic function by counteracting the reduced hippocampal acetylcholinesterase (AChE) activity typical of AD, thereby maintaining the neurotransmitter levels required to sustain memory and learning [90,93,94]. This was accompanied by reduced levels of Aβ oligomers and phosphorylated (p)-tau in the corpus callosum and hippocampus of treated animals compared to their untreated counterparts, which instead exhibited higher dementia scores [91,95,96]. Mechanistically, enhanced chaperone-mediated autophagy and phagocytosis in microglia are considered putative mechanisms contributing to Aβ plaque and tau protein clearance upon metformin administration [97,98]. Additionally, diminished neuroinflammation, through lowering levels of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6), in the hippocampus of AD mice and rats under metformin therapy significantly reduced microgliosis and astrogliosis, with minimal glial fibrillary acidic protein (GFAP) and ionized calcium-binding adapter molecule 1 (IBA1) immunoreactivity [90,91,92,93,99,100]. Given the established correlation between inflammation and OS, it is noteworthy that while AD animals exhibited elevated OS parameters, as evidenced by increased levels of hippocampal inducible nitric oxide synthase (iNOS), neuronal nitric oxide synthase (nNOS), reactive oxygen species (ROS), malondialdehyde (MDA), and diminished superoxide dismutase (SOD), metformin reversed these effects [90,91,93,95,101]. Finally, and more intuitively, metformin mitigated insulin resistance by stimulating AMPK signaling, p-protein kinase B (p-AKT), protein kinase C (PKC), and p-glycogen synthase kinase (p-GSK)—all pathways and proteins whose activation promotes insulin sensitivity [91,95,96]. However, the role of metformin's glucose-lowering effect in its therapeutic efficacy for AD remains debated, and further investigations are needed to understand whether blood sugar control is necessary or dispensable for its benefits in this context [96,99]. Nevertheless, animal studies across a variety of mouse and rat AD models consistently present compelling evidence supporting the use of metformin in clinical trials.

In humans, despite multiple meta-analyses and systematic reviews, including recent observational studies, reporting a lower risk of dementia associated with metformin intake, conflicting studies also exist [102,103,104]. For instance, while some studies, such as one involving 1873 T2DM patients with AD, showed a negative correlation between metformin use and the rate of cognitive decline [measured by mini-mental state examination (MMSE) score] [67], other research indicates that metformin intake may be even detrimental in aged AD patients, where alternative anti-diabetic drugs appear more effective in dementia prevention [69,70,74]. Critically, dose, duration, timings, and treatment formulations are crucial points to be considered when assessing the benefits of metformin across studies. This is further complicated by the fact that AD clinical trials primarily included T2DM patients, thus leaving the assessment of metformin intake in non-diabetic and healthy aged subjects unclear [105]. Furthermore, studies in mice subgroups have yielded conflicting results; indeed, while metformin improved attention, learning, and memory in healthy young mice, these effects were reversed upon chronic treatment in aged animals. Adding to these discrepancies, negative effects were observed in 3xTg-AD mice, where metformin correlated with higher levels of p-tau, Aβ plaques, and GSK-3β [106]. Similarly, enhanced p-tau, lipogenesis, and gliosis were associated with metformin administration in apolipoprotein E (APOE)-deficient mice [107]. Within this APOE genetic background, sex differences also appear to play a role, as metformin was reported to improve cognitive scores and behaviour in old female mice but proved detrimental in males [108]. In humans, studies indicate improved cognition in normal individuals taking metformin, but not in AD patients, with APOE genotype possibly acting as a confounder [68]. Beyond its potential as a curative treatment, the use of metformin in dementia prevention also remains debated [102,103,104]. For instance, no reduction in dementia occurrence was found in older diabetic patients taking metformin as a preventive strategy [77]. Nevertheless, this is likely due to the use of non-standardized definitions for risk/protection scores when evaluating therapeutic outcomes, which vary across therapeutic centres [63]. Moreover, the study's limitation to Canadian participants introduces demographic, genetic background, environmental, and lifestyle biases, all recognized confounding factors. In this respect, a meta-analysis conducted on 100 reviews and 27 clinical studies reported a significant bias favouring metformin in Western countries, particularly the US, while no effect was observed in the Eastern populations [109]. Similar findings were reported in an independent meta-analysis, where no association between dementia risk and metformin intake was found among Europeans [110]. Conversely, metformin significantly decreased the risk of AD occurrence among US veterans under insulin therapy, as shown in an independent retrospective longitudinal study of 5528 participants [71]. In stark contrast, however, data from an observational study conducted on the Korean National Health Insurance Service DM cohort documented an increased risk of AD in T2DM patients undergoing metformin therapy, which was even worse among depressed participants [111]. Not only antidepressants but also non-steroidal anti-inflammatory drugs and neuropsychiatric conditions appear to alter the ability of metformin to reduce dementia risk, as reported by a longitudinal study involving 50 T2DM patients [76]. Lastly, it is important to consider that, as metabolism is shaped by various genetic, lifestyle, and environmental factors, geographical location might impact key molecular and functional targets of metformin, which can serve as a potential biomarker for sample stratification [112,113,114]. For instance, while cerebrospinal fluid (CSF) p-tau, total (t)-tau and Aβ-42 are potential biomarkers for metformin's effect in diabetic patients [62], plasma and CSF levels of 7 proteins regulating inflammation and cell death (namely, REG1A, AZU1, PRTN3, CCL11, CCL20, CASP-2 and IL32, see Table 1) can monitor AD progression in non-diabetic MCI individuals [64]. Conversely, NDUFA2, a gene encoding for the nicotinamide adenine dinucleotide (NADH): ubiquinone oxidoreductase of mitochondrial complex I and a metformin target, was negatively correlated with AD risk in a Mendelian randomized cohort of over 500,000 participants [65].

Time-related assumptions made in observational studies, such as immortal time bias, time-window, or lag times, can further distort the result of clinical studies, leading to either an overestimation or underestimation of treatment effects [115]. In addition to these methodological challenges, the optimal doses and delivery methods are unclear and require standardization. Indeed, although oral administration is prevalent, intranasal delivery offers a potential advantage by increasing metformin concentration in the hippocampus compared to systemic circulation, which might be beneficial in AD patients without T2DM [116]. Even better, innovative formulations with improved BBB penetration, selective uptake by damaged neurons, and enhanced bioavailability, are now available and hold promise for future therapy optimization. These include, but are not limited to, supramolecular nanodrugs capable to release on-demand donepezil and metformin upon Aβ overexpression [117], mixed solid–liquid formulations [83], nanocarriers [118], and nanoflower multifunctional selenium carriers [119]. On this line, synergistic therapies combining metformin with reported natural compounds hold promise for further boosting therapeutic efficacy. For example, co-administration of the plant-derived cyanidin 3-O-galactoside with metformin showed synergistic activity in senescence-accelerated mouse-prone 8 (SAMP8) mice, reducing ketones, indoles, and Aβ-aggregation while increasing SCFA and fatty acid biosynthesis, thereby restoring memory and spatial learning capacity [120]. Similarly, reduced endoplasmic reticulum (ER) stress, achieved via protein kinase RNA-like ER kinase modulation, along with improved mitochondrial and metabolic functions was observed in SAMP8 mice through co-delivery of the alkaloid Dendrobium nobile Lindl. and metformin, respectively. In this study, reduced ER dilation coefficients and decreased calpain1, GSK-3β, and cyclin-dependent kinase 5 activity were identified as key markers of therapeutic efficacy [121]. Again, low doses of lupeol and metformin proved more effective than high-dose monotherapies, resulting in higher levels of the antioxidants nuclear factor erythroid 2-related factor 2 (Nrf2) and glutathione (GSH), along with lower myeloperoxidase, neutrophil neuroinflammation, OS, and AChE activity, ultimately improving Morris water maze scores in streptozotocin-induced mouse models of dementia [122].

A cross-sectional study in 50 individuals found that dipeptidyl peptidase 4 inhibition increased serum brain-derived neurotrophic factor (BDNF) and improved cognitive functions (as measured by standardized MMSE scores) in T2DM patients treated with metformin, calling for larger studies [123]. The ongoing MET-FINGER randomized double-blind phase IIb clinical trial, exploring the combination of multimodal lifestyle interventions (FINGER 2.0) with metformin in 600 participants aged between 60 and 79, promises to yield valuable insights. MET-FINGER will assess changes in memory, spatial learning, cognitive processing speed, metabolic and vascular status, and lifestyle wellbeing over 2 years, with a planned phase 3 clinical trial based on the results [124]. This trial is expected to confirm or disprove preliminary findings in diabetic AD rats, where aerobic exercise coupled with metformin was shown to improve cognitive function and hippocampal neurogenesis by reducing microgliosis and astrogliosis [125]. Therefore, although human studies are scarce, synergistic multimodal targeting appears as a promising approach for further enhancing metformin's clinical benefits in dementia. In addition, innovative machine learning software applying artificial intelligence is emerging as a valuable tool to improve unbiased AD evaluation. As proof, a study conducted in 15,428 Swedish AD patients using unsupervised clustering to monitor cognitive performance demonstrated a significant correlation between metformin intake and cognitive function [75]. Given their unbiased evaluations, these tools are expected to play a crucial role in patient stratification and clinical monitoring, with the aim of improving standardization and supporting clinical decisions.

Despite emergent positive results, many cross-section studies and meta-analysis did not find any advantage in using metformin against AD. Indeed, while safe and tolerated, metformin was not associated with any improvement of AD or protection against dementia risk when adjusted for biases [72,73,103,115,126,127]. Moreover, the preferential inclusion for obese and diabetic participants further adds confounding factors, which makes the outcome difficult to interpret.

Overall, these data propose metformin as a safe, well-tolerated, commercially available, and effective therapy for reducing cognitive decline, sustaining memory and learning, and boosting attention. However, while mouse studies are generally positive, human clinical trials remain inconclusive, are often limited to diabetic populations, and exhibit significant discordance. This variability points to genetic background, geographical location, lifestyle, environmental factors, comorbidities, co-treatments/treatment history, age, and sex as potential confounding factors biasing clinical trial results. Additionally, the lack of standardized disease monitoring criteria and treatment regimens further complicate study outcomes, pointing to the urgent need of standardized clinical interventions for future large-scale investigations. To date, a dose ranging from 500 mg/day to 2000 mg/day has been adopted by most clinical trials [72,73,128], but synergism and/or interaction with other age-related drugs has been poorly addressed.

It is possible that the use of biomarkers for patient stratification and an unbiased machine learning algorithm will facilitate these efforts.

Parkinson's disease (PD), one of the most common age-related neurodegenerative disorders, has a significant increase in incidence with aging [129]. Its pathological hallmarks, including the progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta, as well as the presence of Lewy bodies composed of misfolded α-synuclein (SNCA) protein, lead to both motor (tremor, muscle rigidity, bradykinesia, and postural instability) and non-motor (including apathy, anhedonia, depression, and cognitive dysfunction) symptoms [130]. While the exact pathogenic mechanisms remain elusive, a growing body of evidence highlights the key role of OS, mitochondrial dysfunction, neuroinflammation, dysregulated autophagy, and aging in PD pathogenesis [131,132]. To date, there are no disease-modifying therapies for PD; the current pharmacological interventions focus on symptom alleviation rather than slowing down or preventing neuronal death progression [133]. Consequently, the global rise in the elderly population emphasizes the urgent need to explore new therapeutic approaches.

To date, there is evidence that mice with T2DM may have dopaminergic neurodegeneration [134] and patients with diabetes mellitus have an increased PD risk [135]. In this context, metformin may offer potential neuroprotective effects against PD development and progression. This hypothesis is corroborated by several in vitro and in vivo studies that document the encouraging impact of metformin in PD pathogenesis (Table 2) [71,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154].

In vitro, metformin has been found to modulate various cellular pathways involved in PD pathogenesis (Figure 3). By regulating AMPK activity, metformin influences several subsequent pathways involved in autophagy, mitochondrial quality control (by regulating mitochondrial complex I activity, succinate dehydrogenase complex subunit A, pyruvate dehydrogenase, and voltage-dependent anion channel), and antioxidant processes (by increasing SOD1, SOD2, glutathione peroxidase-1, cationic amino acid transporter-1, nuclear respiratory factor-1, mitochondrial transcription factor A, and uncoupling protein-2), all dysregulated in PD [155]. As mentioned above, one of PD's hallmarks is the presence of SNCA protein aggregates, whose formation is due to several post-translational modifications, including phosphorylation at Ser129, Ser87, and Tyr125 [156]. Of note, the protein phosphatase 2A (PP2A), the primary SNCA phosphatase, is dysregulated in the post mortem brain of PD patients [157]. Interestingly, metformin has been observed to activate PP2A and thus reduce SNCA phosphorylation by suppressing mTOR signalling, through AMPK-dependent and -independent mechanisms (in SH-SY5Y and HeLa cellular models, respectively) [145]. The presence of SNCA aggregation has been associated with impaired autophagy, a biological process crucial for degrading aggregated proteins [158]. In this regard, the pro-autophagic activity of metformin has been well documented [159]. Indeed, Lu and coworkers demonstrated the protective role of metformin on dopaminergic neurons via activation of the AMPK-autophagy pathway [140].

The potential role of metformin in counteracting mitochondrial dysfunction and OS, both key features of PD, has also been extensively investigated [136,138,140,142,160,161]. Interestingly, metformin has been reported to inhibit mitochondrial complex I activity, which is one of the major sources of superoxide and other ROS [162]. Specifically, Lu and coworkers demonstrated the ability of metformin to reduce the number of dysfunctional mitochondria and ROS production in 1-methyl-4-phenylpyridinium ion (MPP⁺)-treated SH-SY5Y in an AMPK-dependent manner, thus suggesting its neuroprotective effects [140]. Furthermore, another study underlined metformin's capability of upregulating the expression of several mitochondrial proteins, including succinate dehydrogenase complex subunit A, pyruvate dehydrogenase, and voltage-dependent anion channel, in SH-SY5Y cells in a dose-dependent manner [142]. This mechanism appears to be AMPK-independent and involves the activation of the transcription factor 2 (ATF2)/CREB pathway, which induces the upregulation of the PGC-1α. Of note, PGC-1α is a transcriptional cofactor downregulated in PD brains and crucial for mitochondrial biogenesis and antioxidant defence [142,163]. In turn, the activation of this pathway results in the expression of PGC1α target genes, such as SOD1, SOD2, glutathione peroxidase-1, cationic amino acid transporter-1, nuclear respiratory factor-1, mitochondrial transcription factor A, and uncoupling protein-2 [142], thus suggesting metformin's potential beneficial effects. In line with this study, Katila and colleagues revealed the neuroprotective effects of metformin in SH-SY5Y rotenone-treated cells. In detail, metformin protects these cells from rotenone-mediated death via reduction of ROS levels and restoration of mitochondrial function, by upregulating the expression of PGC-1α, as well as some antioxidant molecules and pathways (i.e., GSH, SOD, Nrf2) [150]. Nevertheless, it is worth noting the adverse effects of metformin in the MPP⁺-N27 dopaminergic cell line derived from rat ventral mesencephalon [138]. Indeed, the authors observed that the increase in ROS production and the reduction in ATP levels were aggravated when this PD cellular model was treated with 100 μM of MPP⁺ combined with metformin [138].

Another key process with a pivotal role in PD pathogenesis is neuroinflammation mediated by microglia [164]. In this context, metformin has been shown to partially reduce the mRNA of the pro-inflammatory cytokine IL-1β in BV2 cells treated with lipopolysaccharide (LPS) [136]. Despite these improvements, the authors did not find any alteration in mRNA levels of iNOS and TNF-α.

Based on these in vitro studies, which revealed the ability of metformin to modulate several key features of PD, the potential neuroprotective effects of this drug have been asserted. However, the controversial findings underline the need for further studies to better understand the molecular mechanisms of metformin and its possible use to counteract/prevent neurodegeneration.

In in vivo studies, metformin has been reported to prevent the degeneration of dopaminergic neurons (by upregulating BDNF and activating its downstream signalling pathways, including AKT, extracellular regulated protein kinase 1/2, GSK-3β, CREB); reduce neuroinflammation (lower values of TNF-α, IL-1β, IL-6, iNOS) and OS (by increasing Nrf2, SOD, catalase, GSH, thioredoxin, and HO-1, as well as sod3); and regulate autophagy (by suppressing the mTOR signalling pathway), SNCA phosphorylation (by suppressing the mTOR signalling pathway), and mitochondrial function (by increasing SDHA, PDHA, VDAC, and COXIV proteins). These mechanisms are summarized in Figure 2 (Figure 3; Table 2).

Regarding the behavioural deficits, metformin has been shown to ameliorate locomotor and muscular impairment in various PD animal models, including 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice, 6-hydroxydopamine (6-OHDA)-lesioned mice, and rotenone mouse models [137,140,142,143,146,147]. However, other studies revealed conflicting results [139,149]. For example, Adedeji and coworkers found no effect of metformin on motor coordination, observing only a reduced duration in catalepsy score in a haloperidol-induced PD mouse model [139]. This discrepancy might be partially explained by the huge variability of animal models and metformin doses used across different studies.

As mentioned before, another hallmark of PD is the progressive loss of dopaminergic neurons and the consequent decrease in dopamine content within the brain [165]. While some studies have underlined the ability of metformin to prevent dopaminergic cell loss [137,140,141,142,143,144,147,148,149], others have reported contrasting results [136,138,146]. For instance, Patil and coauthors reported significant protection of tyrosine hydroxylase-positive cells, as well as increased levels of BDNF (a neurotrophic factor with neuroprotective and neurorestorative effects on dopaminergic neurons), in MPTP mice treated with metformin compared to the MPTP-only group [137,166]. Similarly, other works revealed the upregulation of BDNF and the activation of its downstream signalling pathways (AKT, extracellular regulated protein kinase 1/2, GSK-3β, CREB) in MPTP and 6-OHDA mice treated with metformin [143,146]. Conversely, other experiments performed on the LPS-induced rat neuroinflammation model and on MPTP-treated mice indicated that not only metformin failed to protect dopaminergic neurons from death, but also exacerbated neuronal damage [136,138]. Notably, MPTP and metformin may have an additive inhibitory impact on mitochondrial complex I, resulting in reduced ATP levels. This may explain the detrimental effect of the drug in neurons, which are particularly sensitive to complex I inhibition and get easily damaged [138]. Moreover, the neuroprotective efficacy of metformin could be influenced by the dosage and the treatment scheme. Nevertheless, other experiments have documented increased dopamine levels [140,143] and upregulation of the tyrosine hydroxylase cat2 gene (necessary for dopamine synthesis) following metformin administration [148].

Some studies have also reported metformin's ability to prevent SNCA phosphorylation and aggregation [140,143,145,148,149]. In this context, metformin has been observed to lower phospho-Ser129 SNCA levels in the brain of mice [145] and attenuate SNCA accumulation in MPTP-plus-probenecid PD mice, through a mechanism involving AMPK phosphorylation and autophagy [143].

Neuroinflammation and OS are other two key hallmarks of PD [167]. In this context, a substantial body of evidence highlights the promising anti-inflammatory and antioxidant effects of metformin, possibly contributing to neuroprotection [136,137,138,139,140,147,148,149]. Of note, this drug has been demonstrated to reduce microglial activation and the consequent release of pro-inflammatory markers (mRNA and protein), including TNF-α, IL-1β, IL-6, iNOS. Simultaneous increase of anti-inflammatory cytokines, such as IL-10, was also observed [136,138,140]. Metformin can then attenuate lipid peroxidation, MDA and nitric oxide production, and increase the expression of the OS markers Nrf2, SOD, catalase (CAT), GSH, thioredoxin, and HO-1 in different PD animal models [137,139,147,148,149].

Despite the limited number of in vivo studies and some reported inconsistencies, substantial evidence supports the potential neuroprotective effects of metformin in PD neuropathology. However, further research is crucial to confirm these promising results and better clarify the possible role of metformin in managing this complex disease.

While preclinical studies have largely shown the beneficial effects of metformin in PD, clinical studies remain scarce. Even when conducted, those have primarily focused on the impact of metformin intake on PD risk in patients with T2DM, yielding conflicting results [71,151,152,153,154]. For instance, Wahlqvist and coauthors performed a cohort study investigating the potential effects of oral antihyperglycemic agents (OAAs), namely, sulfonylureas and metformin, on PD development. In detail, they found an increased PD risk in T2DM patients treated solely with sulfonylureas compared to those without OAAs therapy. Conversely, metformin alone, or in combination with sulfonylureas, did not affect PD risk [151]. Similarly, another study reported a reduced PD risk in elderly T2DM patients associated with prolonged (>4 years) metformin treatment [71]. In contrast, Kuan and colleagues found a higher PD development risk in the metformin-treated group in a retrospective cohort study involving 4651 T2DM patients treated with metformin and 4651 T2DM patients without metformin therapy [152]. These contradictory findings may be partially explained by the possible synergistic neuroprotective effects of metformin when combined with other anti-diabetic drugs with a different mechanism of action. Supporting this, Brakedal and coworkers reported a reduced risk of PD development with glitazone therapy compared to metformin-only treatment [153]. Lastly, a cross-sectional study found a dose–response relationship between metformin use and PD risk in T2DM patients. In detail, low dosage and intensity of metformin use were associated with a reduced probability of developing PD, while higher dosage and intensity showed no neuroprotective effect [154].

One key side effect to consider in the context of metformin use in PD therapy is the risk of developing vitamin B12 deficiency in diabetic patients treated with high metformin doses [168]. Given the association between low vitamin B12 levels and PD [169], it is plausible that high doses of metformin could increase PD risk by decreasing serum vitamin B12 levels [152]. Besides the dose, it is crucial to highlight that prolonged use of metformin also leads to vitamin B12 and folate deficiency together with higher homocysteine levels. These irregularities are linked with the risk of cognitive impairment and peripheral neuropathy [170,171]. Of note, homocysteine induces OS, neuronal apoptosis, and DNA damage [172]. Epidemiological studies revealed that hyperhomocysteinemia was associated with developing neurodegenerative disorders, including PD [173,174]. In this regard, some studies reported that higher plasma homocysteine levels are associated with poorer clinical outcomes and greater motor severity in PD patients [172,175]. In addition, a prospective study of 680 PD patients showed that low vitamin B12 levels at baseline were linked to greater worsening of motor symptoms in the early stages of the disease [176]. Notably, low vitamin B12 levels in patients with PD are associated with cholinergic transmission dysfunction and motor severity [175]. Therefore, metformin-induced vitamin B12 and folate deficiency with increased homocysteine levels could be potential links in the development and progression of PD. Thus, supplementation with folate and B12 in T2DM patients on metformin therapy might be a preventive measure to prevent cognitive impairment and PD development.

Based on this evidence, it is noticeable that metformin could have beneficial and harmful effects in PD depending on the duration and dose. Further clinical studies are mandatory to better clarify the effects of metformin on PD risk. Moreover, as existing studies have focused on T2DM patients without PD, it is also crucial to expand the investigations including patients already diagnosed with this neurodegenerative disease in order to assess whether metformin may protect against PD progression.

Cardiovascular disease (CVD) is a broad term encompassing a range of disorders affecting the heart and blood vessels, including coronary artery disease (CAD), cerebrovascular diseases, peripheral artery disease, and aortic atherosclerosis. CVD remains a leading cause of death worldwide, despite being largely preventable. Also known as coronary heart disease, CAD, the most prevalent type of CVD, results from decreased myocardial perfusion that causes angina (chest pain due to reduced blood flow to the heart), myocardial infarction (occurring when a blood vessel supplying the heart is abruptly blocked, potentially damaging the heart muscle), and heart failure (HF; the heart's inability to pump blood effectively, leading to fluid buildup in the lungs and breathing difficulties). Stroke is one of the most common cerebrovascular diseases and occurs when a blood vessel supplying the brain is either blocked (ischaemic stroke) or ruptures and bleeds (hemorrhagic stroke). Peripheral artery disease affects the limbs, while aortic atherosclerosis involves thoracic and abdominal aneurysms [178]. Abdominal aortic aneurysms (AAAs) are the most common type of aneurysm, and a "watch and wait" approach with regular surveillance is the standard of care for small aneurysms, as there are currently no effective drug treatments available to limit their growth [179]. Several risk factors lead to CVD, including smoking, hypertension, high cholesterol, high lipoproteins, obesity, and diabetes mellitus [180]. The treatments for CVD are diverse and tailored to the specific type of heart disease, encompassing lifestyle changes, medications (including anticoagulants, antiplatelet therapies, angiotensin-converting enzyme inhibitors, beta-blockers, calcium channel blockers, cholesterol-lowering medications, and diuretics), and/or medical procedures [181].

The beneficial effects of metformin have been demonstrated in various animal models of cardiovascular disorders, especially atherosclerosis, myocardial injury, and HF [6,182]. Recent studies have identified additional novel molecular targets contributing to metformin's cardiovascular protection (see below). These findings provide crucial insights into the molecular mechanisms underlying metformin's cardioprotective action, thereby expanding the foundation for further clinical trials to conclusively establish its efficacy in the management of specific cardiovascular disorders. In particular, some cardioprotective effects of metformin are mediated by AMPK (see Figure 4). AMPK kinase plays an important role in regulating myocardial energy metabolism and reducing I/R injury [183,184]. Of note, metformin can protect against apoptosis induced by cardiac I/R injury through AMPK activation. Metformin was shown to reduce the size of the infarct and inhibit cardiac fibrosis through the reduction of proinflammatory cytokines (TNF-α, IL-6, IL-1β) and the suppression of the activation of the NLR family pyrin domain containing 3 (NLRP3) inflammasome in isolated rat hearts subjected to I/R [185]. Concerning OS, metformin upregulates the phosphorylation of AMPK and decreases the NOX4 gene expression, leading to the decrease in myocardial oxidative damage. This is accompanied with higher levels of antioxidant enzymes such as MnSOD which alleviate reperfusion injury [184]. Metformin has also been reported to protect cardiomyocytes against H₂O₂-induced apoptosis through the AMPK/CCAAT-enhancer-binding protein (C/EBP) beta/miR-1a-3p/grpq4 (β/miR-1a-3p/GRPQ4) pathway. In detail, miR-1a-3p follows protein GRP94, which results in the accumulation of unfolded or misfolded proteins, generating ER stress. C/EBP β directly induces the upregulation of miR-1a-3p by binding to its promoter. According to the research, metformin has a positive impact on AMPK and also decreases the levels of C/EBP β and miR-1a-3p, especially when compared to the control group in the study [186]. These results confirm the positive effect of metformin for the treatment of cardiac I/R injury by attenuating cell apoptosis. Figure 4 summarizes the different pathways through which metformin exerts cardioprotective effects in cardiomyocytes.

Preclinical and clinical findings on metformin's cardiovascular protection are summarized in Table 3 [187,188,189,190,191,192,193,194,195,196,197,198,199,200,201,202,203,204,205,206,207,208,209].

Emerging preclinical studies have identified new metformin's modes of action, including the activation of the AMPK/autophagy axis, the suppression of mitochondrial ROS via AMPK-independent pathway, the activation of sirtuin signaling, as well as autophagy-dependent pathway [187,188,189,190,191,192,193,194,195,196,197]. Concerning the first two mechanisms related to its atheroprotective role, some works performed on different animal models have shown that metformin attenuates atheromatous plaque formation, suppresses atherogenesis, improves autophagy-mediated cholesterol efflux in vascular smooth muscle cells-derived foam cells (a significant component of atherosclerotic lesions), and reduces glucose production [187,188,189,190]. Conversely, through the activation of the latter two mechanisms, metformin was reported to ameliorate myocardial damage, protect against cardiotoxicity and cardiac hypertrophy, and prevent cardiomyopathy [192,194,195,196]. However, despite these promising results, the studies are limited. Therefore, further preclinical investigations are necessary to confirm and better clarify the molecular mechanisms underlying metformin's effects. Nevertheless, recent clinical studies have corroborated the atheroprotective effects of metformin. In this regard, several randomized controlled trials (RCTs) have demonstrated the ability of metformin to improve the metabolic status and reduce the progression of atherosclerosis in non-diabetic and diabetic patients with T1DM and T2DM [198,199,200,201,202,203]. This consistent positive evidence from both preclinical and clinical studies prompted the development of large-scale RCTs to investigate the atheroprotective effects of metformin in specific patient populations. The multicenter VA-IMPACT trial, involving over 7400 participants and currently in the recruiting phase, aims to evaluate the effects of metformin treatment on reducing mortality and cardiovascular morbidity in patients with pre-diabetes and established atherosclerotic cardiovascular disease, compared with placebo (NCT02915198). Furthermore, a few small-scale RCTs have also demonstrated positive effects of this drug on myocardial metabolic and functional parameters, even in patients with HF [204,205,206,207,208,209]. In this latter context, while the observational studies have been limited to patients with diabetes, the results suggest improved cardiovascular outcomes with metformin treatment [205,206,207]. Consequently, randomized trials are mandatory to further explore these findings in patients both with and without diabetes. These studies highlight the cardiovascular and metabolic benefits of metformin; however, large-scale RCTs, such as the VA-IMPACT trial, are needed to assess the cardiovascular outcomes of metformin use, particularly in patients at high risk of adverse cardiovascular events and HF.

Regarding aneurysms, the current body of evidence concerning metformin is characterized by a significant divergence between observational and interventional data. A systematic review and meta-analysis of 11 articles, comprising over 110,000 patients, found that metformin use was associated with a statistically significant reduction in the annual progression of AAA diameter, with a weighted mean difference of −0.84 mm. The pooled data also suggested an association with a decrease in AAA-related events, such as rupture and mortality. Although promising, these findings are based on observational studies, which can show association but are prone to confounding factors. For instance, healthier patients may be more likely to be prescribed metformin, leading to a healthier cohort overall [210]. This is where the importance of the ongoing and future randomized controlled trials becomes clear. The completed, underpowered trial demonstrated no significant effect on aneurysm growth, but it could not definitively rule out a small, clinically relevant effect due to its small sample size. The ongoing, large-scale LIMIT (NCT04500756) and MAT trials are designed precisely to address this evidence gap. By enrolling a large cohort and following participants over a period of years in a placebo-controlled, blinded setting, these trials will provide the robust, high-level evidence necessary to confirm or refute the observational findings. A positive outcome would be a transformative event in vascular medicine, providing the first pharmacological treatment for a disease that currently has no drug therapy.

Observational data and specific trial findings have led to a well-established reputation of the drug for providing cardioprotection. However, a meticulous review of the literature reveals a fundamental paradox: compelling evidence from multiple large-scale studies and meta-analyses demonstrates a lack of a statistically significant effect on broad, composite CVD endpoints [211]. This ambiguity is not a sign of the drug's ineffectiveness but rather a reflection of the intricate relationship between its mechanism of action, the heterogeneity of patient populations, and the statistical limitations of composite endpoints in clinical trials. This controversy is supported by data from a large meta-analysis performed by Shabil and coworkers [211], a critical re-evaluation of the foundational UK Prospective Diabetes Study (UKPDS) [212], and findings from trials in non-diabetic populations [213]. Furthermore, a recent meta-analysis of 22 studies, encompassing over 612,000 participants [211] found that metformin treatment may not have a clear, significant effect on composite CVD outcomes as a whole. Given these data, the authors concluded that metformin may not have a significant effect on a composite of events such as stroke, myocardial infarction (MI), heart failure (HF), and major adverse cardiovascular events (MACE) [211].

The hypothesis that metformin has direct cardioprotective properties, independent of its glucose-lowering effects, led to its investigation in non-diabetic populations. However, the results from these trials have been mixed and largely disappointing [213]. The GIPS-III trial, which studied metformin in patients with ST-segment elevation myocardial infarction who did not have diabetes, showed as "little or no effect". This failure suggests that the direct cardioprotective effects alone may be insufficient to produce a statistically significant clinical outcome. It is the synergistic effect of these direct mechanisms combined with the systemic improvements of glycemic control (e.g., reduced inflammation, improved endothelial function, better lipid profiles) that likely drives the positive outcomes observed in studies of diabetic patients.

Age-related macular degeneration (AMD) represents the primary cause of vision loss among adults older than 65 years and can result in permanent blindness. AMD causes pathological alterations in the deeper retinal layers of the macula and the adjacent vasculature, resulting in impaired central vision [214,215,216].

In 2020, approximately 200 million individuals worldwide were affected by AMD, and this number is projected to increase to around 288 million by 2040. The prevalence of AMD is expected to continue rising in parallel with global population aging, as aging is the main risk factor for AMD [217].

AMD is clinically categorized into two primary types: dry (atrophic) and wet (neovascular). Dry AMD is defined by the lack of serum or blood leakage and is often identified by the formation of drusen, retinal deposits that act as an early sign of the disease's dry stage. Dry AMD is the most prevalent form and can progress to wet AMD, wherein the formation of choroidal neovascular membranes leads to bleeding and exudation within the retina, causing substantial vision loss [217,218]. Therapeutic options for AMD remain limited, particularly for the dry form. While some vitamin formulations have demonstrated efficacy in slowing the progression of dry AMD, there are currently no available treatments capable of reversing or curing this condition. Evidence suggests that lifestyle modifications, such as smoking cessation, regular physical activity, and maintaining a nutritious diet, may reduce the risk of developing AMD or slow its progression. For wet AMD, anti-vascular endothelial growth factor (anti-VEGF) agents represent the primary therapeutic approach by inhibiting VEGF, a protein responsible for promoting abnormal blood vessel growth in the retina, thereby preventing further macular damage and vision loss. However, these treatments are invasive, costly, and cannot reverse existing retinal damage. Given the limitations of current therapies for AMD, prevention and novel therapeutic strategies are critical for reducing disease prevalence and improving patient outcomes [219,220]. One promising approach is drug repurposing, which involves identifying new clinical applications for established medications. Metformin, a well-tolerated agent with an extensive safety record in the treatment of T2DM as previously underscored, has attracted growing interest as a potential candidate for AMD management due to its diverse pharmacological properties, including antioxidants, anti-inflammatory, antiangiogenic, and antifibrotic effects [221,222,223,224]. These mechanisms are relevant to pathways implicated in AMD pathogenesis. Furthermore, metformin has been postulated to delay aging and mitigate age-related diseases, making it particularly appealing for conditions such as AMD [225]. Preliminary research indicates that metformin may modulate cellular processes involved in AMD progression, highlighting its potential as a therapeutic option to slow or prevent the onset of this vision-threatening disorder, specifically in the elderly [225,226].

To the best of our knowledge, only one randomized clinical trial has evaluated the effect of metformin on AMD in patients without DM. This study enrolled 66 participants aged 55 years or older without DM, all diagnosed with geographic atrophy (GA), an advanced form of dry AMD, in one or both eyes. Among these, 34 participants (57 eyes) were allocated to the observation group, whereas 32 people (53 eyes) were given oral metformin. The main outcome was the annualized rate of GA advancement, which showed no significant difference between the metformin and observation groups. It should be highlighted that the restricted sample size may have diminished the statistical power to identify a possible treatment effect [227]. Despite GA development being a late-stage AMD [228], metformin may still offer benefits by affecting early disease processes, including the onset and progression of hypertransmission abnormalities, drusen accumulation, or neovascularization. In this regard, although no additional randomized clinical trials have been reported, several cohort studies, case–control studies, and case reports have investigated the association between metformin use and the development or progression of AMD. However, only a limited number of these studies have specifically examined the effect of metformin on AMD development in elderly individuals without T2DM. For instance, Aggarwal et al. [214], in a case–control study, reported that metformin use was associated with a 17% reduction in the odds of developing AMD among participants with a mean age of 74 years. Importantly, this association did not appear to be dose-dependent. Specifically, for any form of AMD, a risk reduction was observed across all groups. However, this protective effect was limited to participants in the two lowest quartiles of cumulative metformin use for dry AMD. Furthermore, in a case–control study involving patients aged 60 years and older with newly diagnosed GA, metformin use was evaluated in a subgroup without DM. In this subgroup, metformin use was associated with a 47% reduction in the odds of developing GA (95% CI, 0.33–0.83) [229].

Nevertheless, it should be again underscored that, since metformin is primarily prescribed for diabetes, the majority of cohort, case–control, and cross-sectional studies have included patients with DM. This makes it challenging to distinguish the effects of metformin on AMD from the inherent risks associated with DM itself. Diabetes is a recognized risk factor for both forms of AMD due to its impact on the retinal-endothelial system. Thus, evaluating the effect of metformin on AMD in individuals with DM may introduce confounding, as any observed protective effect could be influenced by the underlying diabetic condition [230,231]. Table 4 [232,233,234,235,236,237,238,239] summarizes some of these recent studies. While current observational evidence suggests a potential protective role of metformin (especially low dose) in AMD, randomized clinical trials are necessary to confirm these findings and to establish the clinical relevance and optimal dosing of metformin as a preventive therapy for AMD.

As mentioned, metformin affects different cellular pathways and has shown beneficial effects in many age-related disorders. Its potential impact on AMD is believed to be mediated through several distinct molecular mechanisms. In this regard, metformin has been proposed to exert both anti-inflammatory and antioxidative properties [13,240] that may contribute to its therapeutic benefit in AMD. In an in vitro study utilizing hydrogen peroxide (H₂O₂)-cultured ARPE-19 cells (an immortalized cell line of human retinal pigment epithelial cells) as a model of AMD, metformin was shown to activate the Nrf2 pathway—a central regulator of cellular antioxidant defence—by promoting both nuclear translocation and DNA-binding activity of Nrf2, resulting in increased expression of downstream antioxidant enzymes [241].

In another study, ARPE-19 cells pre-treated with metformin showed significantly reduced mitochondrial damage following exposure to antimycin A (a highly effective inhibitor of cellular respiration), with metformin pre-treatment resulting in an 84.5% reduction in damage. These findings indicate that metformin possesses both anti-inflammatory and antioxidant properties and promotes mitophagy in human ARPE subjected to mitochondrial injury [242]. To more accurately simulate AMD in vitro, primary cultured RPE cells can be utilized. Accordingly, in vitro experiments using D407 cells (a human RPE cell line) showed that metformin treatment upregulated autophagy, as indicated by increased LC3B expression and decreased p62 levels. These changes are characteristic of enhanced autophagic activity. Indeed, inhibition of autophagy abrogated metformin-induced alterations in LC3B and p62, as well as its protective effects against H₂O₂-induced loss of cell viability and apoptosis. Similarly, knockdown of the essential autophagy genes Beclin1 and LC3B also prevented metformin's protective effects, suggesting that metformin confers resistance to H₂O₂-induced oxidative damage in D407 cells primarily through the stimulation of autophagy [243].

Metformin has also been shown to exert anti-angiogenic effects. Several studies have demonstrated that metformin inhibits angiogenesis in human retinal vascular endothelial cells both in vitro and in vivo in a dose-dependent manner [244,245]. In a separate study utilizing a neovascular AMD mouse model, intravitreal administration of metformin significantly reduced new vessel growth in choroidal explants, again exhibiting a dose-dependent effect. Intravitreal metformin treatment also suppressed choroidal neovascularization (CNV) and decreased peripheral infiltration of IBA1+ macrophages/microglia. Furthermore, metformin downregulated the expression of genes associated with angiogenesis and inflammation, including DLL4 (delta-like canonical Notch ligand 4) and IBA1 in the choroid and retinal pigment epithelium, two key processes implicated in the progression of neovascular AMD [223]. However, it is crucial to underscore that while current observational studies indicate a potential protective effect of metformin against AMD, these findings predominantly originate from diabetic populations, which limits their direct applicability to non-diabetic individuals. As mentioned earlier, only one case–control study has examined metformin use in non-diabetic individuals, finding it to be associated with reduced odds of developing AMD. However, this association was not dose-dependent, with protective effects observed primarily at lower cumulative metformin intake, raising questions about consistency and biological plausibility [214]. Similar findings in diabetic populations suggest protective effects of metformin at lower doses, while higher doses yield mixed results [246].

Randomized clinical trials are essential to confirm these effects, establish optimal dosing, and evaluate metformin's potential as a preventive or therapeutic agent for AMD, particularly in non-diabetic individuals. If proven effective, metformin could serve as an affordable and accessible adjunct for managing AMD, addressing the limited treatment options currently available.

Overall, metformin use in the elderly, including individuals without DM, may offer a promising approach for both early and late stages of AMD. Its proven efficacy in geriatrics, ease of administration, and favourable safety profile position it as an alternative option in AMD. Nonetheless, contraindications, especially at higher doses, require careful consideration in this population.

Osteoporosis (OP) is one of the most prevalent disorders affecting the elderly. It is characterized by a reduction in bone mineral density and a progressive deterioration of bone microarchitecture, which collectively result in increased bone fragility and a significantly elevated risk of fractures [247,248].

The prevalence of OP increases with age, making it a significant health concern among older adults [249]. It is predicted that one in three women and one in five men over 50 will experience osteoporotic fractures [250]. Notably, 20% of patients suffering from a pelvic fracture die within a year [251]. Further, bone fractures, especially hip fractures, significantly impair daily functioning, with only one-third of patients eventually requiring long-term care in nursing facilities [252].

OP is associated with numerous complications that can profoundly impact patients' quality of life, increase the risk of hospitalization, long-term disability, and raise mortality rates. Moreover, OP imposes a significant financial burden on the healthcare system, with estimated annual costs ranging between $13–22 billion in the U.S. alone [250,253].

Globally, the burden of OP is estimated to range between 19.7% and 21.7% among individuals aged 50 years and older. Women exhibit higher prevalence rates primarily due to a significant decrease in estrogen levels after menopause, as estrogen is essential for preserving bone density and inhibiting bone loss [248,254,255]. Sex differences in metformin's effects on bone health are most pronounced in postmenopausal women, where estrogen deficiency accelerates bone loss and alters bone remodeling dynamics. In this population, metformin may be associated with a lower risk of osteoporosis and higher bone mineral density (BMD), independent of diabetes status or obesity, with the effect being particularly significant in women compared to men [256,257]. Mendelian randomized clinical studies further support a causal relationship between metformin use and reduced osteoporosis risk, as well as increased BMD at the lumbar spine and femoral neck, with the protective effect most evident in women [257,258,259]. Estrogen-related mechanisms modulate the magnitude of metformin's impact on bone health and fracture risk, with the greatest benefit observed in estrogen-deficient states such as postmenopause, but metformin's actions are not dependent on estrogen itself. Estrogen deficiency amplifies oxidative stress and osteoclast activity, both of which are targeted by metformin's actions. The drug's efficacy appears greater in the context of low estrogen, as seen in postmenopausal women and animal models, suggesting that metformin's bone-protective effects are at least partially estrogen-independent but may be enhanced by the altered bone microenvironment following menopause [257,260,261].

A higher prevalence of OP has also been observed in certain regions, with the condition being more common in developing countries compared to developed ones. This is partially due to factors such as poor dietary habits, limited access to healthcare, and other related risk factors [248,254,255].

Beyond its antidiabetic properties, emerging evidence suggests that metformin may also benefit bone health and OP, particularly in the elderly population [257,258]. These protective effects are mediated through several key mechanisms, among which AMPK is a central regulator of cellular energy homeostasis and plays a critical role in bone metabolism by influencing both osteoblast and osteoclast activity [262]. As an AMPK activator, metformin may exert significant effects on bone remodeling through multiple pathways, supported by findings from both in vitro and in vivo studies [263].