Metformin and bone metabolism: unraveling their direct and indirect effects

Abstract

Metformin is the most widely used antihyperglycemic agent for the treatment of a wide range of diseases. Activation of AMP-activated protein kinase (AMPK) is the best-known mechanism by which metformin exerts most of its beneficial effects. In recent years, research and applications of metformin in bone metabolism have made significant progress. The molecular mechanisms of its action are being elucidated with an increasingly complex understanding, raising the question of whether metformin acts directly or indirectly on bone. This review examines the indirect role of metformin in improving the bone marrow microenvironment by regulating autophagy, oxidative stress, inflammation, and skeletal aging. Furthermore, we focus on the direct mechanisms of metformin on osteoblasts, osteocytes, bone marrow adipocytes, and osteoclasts. In summary, metformin has been shown to affect bone in multiple ways and to exert osteoprotective effects. In light of the positive benefits of metformin in preventing osteoporosis, future treatment plans for patients with osteoporosis, particularly those with diabetes who are at high risk for fractures, may consider prioritizing the use of metformin as antidiabetic drug for bone protection. While metformin has been shown to improve bone health, particular attention should be paid to renal function, vitamin B12 status, and individual patient factors.

Plain language summary

Understanding how metformin affects bone metabolism directly and indirectly

Metformin is a common medicine used to treat many health problems, especially diabetes. In the past, it works mainly by activating a protein called AMPK, which helps in many good effects. However, there is a debate about whether the process that protects bones is indirectly affected by changing glucose metabolism or directly by cells in the bone. This review looks at how metformin can improve the environment inside the bone marrow by indirectly controlling processes like self-eating (autophagy), stress from oxygen (oxidative stress), inflammation, and the aging of bones. We also look at how metformin directly affects different types of bone cells, like bone-building cells (osteoblasts), bone cells (osteocytes), fat cells in the bone marrow, and bone-breaking cells (osteoclasts). Since metformin is good for osteoporosis, it might be a good idea to think about adding it to the treatment plan for people with osteoporosis in the future.

Graphical Abstract

Keywords

AMPK-mediated bone remodeling bone marrow niche bone microenvironment metformin osteoporosis skeletal aging

Introduction

Diabetic osteoporosis, a metabolic bone disorder marked by suppressed osteogenesis and enhanced osteoclastogenesis, manifests as progressive skeletal microarchitectural deterioration with preserved bone mineral density (BMD), a paradoxical feature particularly evident in type 2 diabetes mellitus (T2DM).^1,2 While microvascular complications (retinopathy, nephropathy) dominate diabetes research agendas, accumulating epidemiological evidence positions skeletal fragility as an underrecognized yet consequential diabetic complication.³ Pathophysiologically, the diabetic skeletal milieu arises from multiple interplays: (i) advanced glycation end-products (AGEs) accumulation compromising bone material properties; (ii) microangiopathy-driven cortical porosity; (iii) suppressed bone turnover accelerating skeletal aging.⁴ A primary underlying etiology stems from prolonged hyperglycemia disrupting the homeostasis of bone cells, which critically compromises bone regeneration and remodeling processes. Disease progression further engages multidimensional crosstalk between oxidative stress-induced apoptosis in bone cells, senescence-associated secretory phenotypes (SASP), and AGEs-mediated inflammation, synergistically tilting the nuclear factor kappa ligand-receptor activator/osteoprotegerin (RANKL/OPG) axis toward net bone resorption.⁵ These multifactorial mechanisms collectively disrupt the dynamic equilibrium between osteogenesis and bone resorption, ultimately altering bone microarchitecture and diminishing intrinsic bone strength. Interventions targeting one of these mechanisms could theoretically improve the others and have a beneficial effect on the physiologic function of the skeleton. Despite this mechanistic rationale for therapeutic strategies, a critical translational gap persists. While preclinical models suggest that targeting key nodes may yield pleiotropic skeletal benefits, robust clinical validation through randomized controlled trials with bone-specific endpoints remains limited, particularly in evaluating metformin’s bone metabolism effects. This evidence void exacerbates clinical equipoise regarding optimal antidiabetic pharmacotherapy in diabetic osteoporosis, where the skeletal consequences of glucose-lowering agents often conflict with their metabolic efficacy. Consequently, delineating the bone metabolism duality of diabetes interventions, specifically elucidating how glycemic control strategies modulate skeletal homeostasis, emerges as a research imperative to reconcile endocrine management with musculoskeletal preservation.

Metformin, a widely used hypoglycemic agent for over six decades, is the primary oral medication recommended in many clinical guidelines for newly diagnosed T2DM patients.⁶ Beyond its canonical AMP-activated protein kinase (AMPK) activation modulating osteoblast–osteoclast equilibrium, emerging evidence implicates mammalian targets of rapamycin (mTOR) complex 1 (mTORC1) inhibition, sirtuin (SIRT) pathway modulation, inflammation, and mitochondrial redox regulation as pleiotropic mechanisms governing its skeletal effects.^7–10 Notably, its therapeutic duality, wherein direct osteoanabolic action and indirect benefits through glycemic control affect the bone marrow niche, is mechanistically contested. While preclinical models demonstrate metformin-enhanced osteogenic differentiation and suppressed adipogenesis in bone marrow mesenchymal stem cells (BMSCs), translational validation in human trials remains sparse, particularly regarding dose-response relationships and long-term fracture risk reduction. This knowledge gap is exacerbated by the absence of consensus on optimal antidiabetic regimens for osteoporotic T2DM patients, underscoring the exigency for mechanistic reappraisal.

This review synthesizes contemporary insights into metformin’s bone-modulatory axis through three investigative lenses: (i) Direct skeletal effects on bone metabolism and remodeling are mediated through pathways such as AMPK-dependent mechanisms; (ii) Microenvironmental crosstalk, focusing on age-related skeletal fragility, oxidative stress, inflammatory, and autophagy imbalance; (iii) Therapeutic potential across osteoporotic subtypes, with comparative evaluation of diabetic, postmenopausal, and glucocorticoid-induced osteoporosis management. By delineating molecular hierarchies within metformin’s pleiotropic mechanisms, we aim to establish a framework for its repurposing as adjunctive osteoporosis therapy by reviewing the convergence of clinical and basic medicine.

Methods

A comprehensive search was conducted in PubMed, encompassing all articles published up to February 2025, to assess the association of metformin with bone regeneration, fracture, bone metabolism, and BMD. The search terms included “osteoporosis” and “fracture” and “bone metabolism” and “bone mineral density” and “bone” and “bone turnover markers” and “hypoglycemic agents” and “metformin.” A comprehensive search strategy was employed to identify relevant English-language publications, encompassing observational and experimental studies, reviews, clinical trials, and meta-analyses. This yielded a total of 1783 articles. The initial review identified a wide range of publications pertinent to bone metabolism, osteoporosis, and glucose-lowering drugs, especially metformin. The primary focus was on the effect of metformin on bone, and articles that did not align with the subject of this review were excluded. We continued to read the titles and abstracts of articles of interest, and ultimately, 214 articles were included in this review.

Cellular transport and intracellular activation of metformin

Metformin is a positively charged hydrophilic compound that requires a carrier for transport into target cells to exert its pharmacological effects (Figure 1). The SLC22A gene encodes multispecific cell membrane organic cation transporters (OCTs), which facilitate the transport of metformin into cells, including OCT-1, OCT-2, and OCT-3.¹¹ Specifically, OCT-1 is expressed on the basolateral membrane of the small intestine, functioning to transport metformin from enterocytes to the portal vein. In the liver, both OCT-1 and OCT-3 are primary transporters responsible for the uptake of metformin by hepatocytes and the excretion of metformin from the liver to the circulation via multidrug and toxin extrusion transporter (MATE) 1. The majority of metformin is excreted into the urine via the kidneys. Among these, OCT-2 is the major transporter for metformin uptake by renal epithelial cells, whereas OCT-1, MATE1, and MATE2 are involved in the secretion of metformin from renal tubular cells into the urine.¹² Moreover, OCTs also play a crucial role in the skeletal system.¹³ Their expression is a biological prerequisite for promoting intracellular absorption of metformin. Al Jofi et al. demonstrated that inhibiting OCTs expression and function in mesenchymal stem cells (MSCs) significantly reduced intracellular metformin accumulation.¹⁴ Furthermore, OCT-1 is highly expressed in rat mandibular osteoblasts, enabling active transport of metformin into the cells.¹⁵ Consequently, it can be concluded that both MSCs and osteoblasts are capable of transferring metformin into cells through OCTs. In the following sections, we will delve into the mechanism of action of metformin once it enters the cell.

Figure 1.

(a) Expression of OCTs in major organs. The intracellular uptake of metformin is dependent on the action of specific transporter OCTs. In the intestine, metformin is absorbed via OCT-1, while in the liver, it is taken up through both OCT-1 and OCT-3. OCT-2 is the primary transporter responsible for the uptake of metformin into renal cells. Additionally, metformin is secreted into the urine by OCT-1, MATE1, and MATE2. (b) The mechanism of metformin uptake by MSCs and osteoblasts. OCT-1 facilitates the entry of metformin into MSCs and osteoblasts. Upon entering the cell, metformin activates AMPKK, which activates AMPK and inhibits mTOR, thereby regulating bone turnover.

Upon entering the cell, metformin has no effect on the activity of purified AMPK and could only activate AMPK indirectly through the AMPK kinases (AMPKKs). Afterwards, AMPKK activates AMPK through the phosphorylation in the activation loop of the catalytic α-subunit. Three types of AMPKK have been identified: liver kinase B1 (LKB1; also known as STK11), Ca²⁺/calmodulin-dependent protein kinase kinase beta (CaMKKβ), and transforming growth factor-β-activated kinase 1 (TAK1).^16,17 LKB1, one of the most well-characterized upstream AMPKKs, is a target of metformin and induces osteogenesis.^18,19 Additionally, protein kinase D (PKD) and MAPKAPK2 (MK2) represent other targets of metformin.²⁰ The conventional hypothesis of AMPK activation involves its ability to inhibit ATP production by inhibiting mitochondrial complex I of the electron transport chain, leading to the dissociation of oxidative phosphorylation and increasing the AMP/ATP ratio.²¹ AMPK complex is composed of three distinct subunits in a 1:1:1 ratio, including the α-catalytic subunit, β and γ-regulatory subunits (α1, α2, β1, β2, γ1, γ2, and γ3). The isoforms of these three subunits result in 12 possible constituent forms of the AMPK complex, though their different combinations exhibit cell-specific characteristics.²² AMPK plays a regulatory role in the equilibrium between osteogenesis and osteoclastogenesis, and subunit deletion affects bone mass.²³ The α1 subunit stimulates bone nodule formation, while the α2 subunit promotes osteocyte proliferation.²⁴ The α2 subunit has more significant osteogenic potential and a greater capacity to inhibit adipogenesis, as well as a weaker capacity to induce osteoblast-associated osteoclastogenesis.^25–27 This phenomenon can be attributed to the elevated expression levels of α2, which result in diminished secretion of RANKL and macrophage colony-stimulating factor (M-CSF) by the cells. Deleting either β1 or β2 subunits results in a reduction in bone mass but does not affect the number of osteoblasts or osteoclasts.²⁸ In summary, the role of the α2 subunit is found to be more significant in the process of positive bone remodeling in comparison to the α1 subunit.

AMPK activation initiates a signaling cascade that suppresses the mechanistic target of mTOR activity. mTOR assembles into two distinct signaling complexes through binding to a variety of chaperone proteins, collectively known as mTORC1 and mTOR complex 2 (mTORC2).²⁹ Using the Cre/LoxP system, Martin et al. generated primary mouse MSCs lacking Rptor or Rictor, resulting in impaired mTORC1 and mTORC2 signaling.³⁰ Their findings revealed that when mTORC1 was defective, the mice showed reduced bone marrow adipocyte and enhanced mineralization. In contrast, when mTORC2 was impaired, the mice displayed increased lipogenic differentiation.³¹ These findings underscore the mTORC1 and mTORC2 regulatory dichotomy in balancing osteogenic and adipogenic fates. Notably, metformin-driven AMPK phosphorylation at Thr172 exerts bidirectional control over these complexes to steer osteogenic differentiation, including mTORC1 inhibition and mTORC2 potentiation.³² mTORC2 has been shown to exert a more pronounced regulatory effect on bone by inhibiting GSK-3β activity through AKT phosphorylation action to stabilize runt-related transcription factor 2 (RUNX2).³³ Conversely, mTORC1 activation is not conducive to osteogenesis, establishing opposing effects between these complexes in an AMPK-activated manner. Critical to therapeutic interpretation, pharmacological mTORC inhibition using rapamycin preferentially targets mTORC1 through allosteric inhibition. This selectivity explains paradoxical observations where rapamycin treatment impairs bone formation and autophagy, which are now recognized as mTORC1-specific consequences.²⁹ These findings underscore the necessity of compound-specific characterization when interpreting mTOR pathway modulation studies.

In addition to the most extensively studied classical AMPK pathway, metformin has been observed to inhibit mitochondrial glycerophosphate dehydrogenase and to bind to the lysosomal presenilin enhancer 2 (PEN2) protein.³⁴ These discrepancies in the mechanism of action of metformin may be attributable to the varying modes of metformin administration and the divergent doses of metformin utilized in disparate studies. However, metformin inhibition of the AMPK pathway activation due to mitochondrial complex I is only observed at a minimum of 1–5 mM, which does not occur in the clinical setting. In contrast, patients with diabetes mellitus taking daily prescribed doses of 1.5–2 g/day of metformin, with plasma metformin concentrations of 10–40 µM, utilize the PEN2 pathway.³⁵ Recent studies have indicated that rhythmic renal elimination exerts the most significant influence on metformin pharmacokinetics. The rhythms of glomerular filtration rate, renal blood flow, and OCT-2-dependent transit rate have been identified as the primary variables in this relationship.³⁶ Concentrations of metformin in various bodily tissues have been the subject of numerous studies. The findings indicate that concentrations of 5–30 µM are present in muscle, 3–10 µM in fat, and 100–300 µM in kidney when 1 g of metformin is administered three times daily, while bone remains pharmacologically underserved (estimated 1–8 µM) despite requiring over 80 µM for direct AMPK-driven osteogenic effects in placental-derived human mesenchymal stem cell.³⁷ This pharmacokinetic paradox is resolved through indirect niche modulation, as detailed in section “The indirect mechanism of metformin in bone marrow niche.” Metformin ameliorates oxidative stress, attenuates inflammatory cytokine production, and enhances M2 macrophage polarization, collectively optimizing the bone marrow microenvironment to favor osteogenic differentiation while suppressing osteoclastogenesis. To transcend these concentration barriers, future studies may consider the following directions: (i) Investigating whether supraphysiological dosing regimens can achieve therapeutic bone tissue concentrations while systematically evaluating their pharmacokinetic safety profiles through longitudinal clinical trials; (ii) Developing bone-targeted drug delivery systems, such as nanoparticles and bone-targeted peptides, to increase the concentration of the drug in bone tissue; (iii) Combining the metabolism-modifying drug with other bone metabolism-regulating drugs, such as vitamin D and bisphosphonates to enhance the therapeutic effect.

Preclinical and clinical studies

Numerous cellular and animal studies have demonstrated that metformin has a bone protection effect in T2DM, ovariectomized, and glucocorticoid-induced animal models.^38,39 As illustrated in Table 1, the most common rodent models of metformin interventions in recent years include gavage, subcutaneous injection, and intraperitoneal injection. Metformin alleviates disorders of glucose and bone metabolism⁴⁰ and decreases the risk of fractures.^41,42 In rodents, metformin increases systemic BMD, as shown by the decreased trabecular separation (Tb.Sp), higher bone volume/tissue volume (BV/TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th).^43–45 Stunes et al. indicated that metformin treatment led to a 10% rise in femoral BMD, improved trabecular microstructure, and increased bone resilience in ovariectomized rats.⁴⁶ Additionally, metformin could reduce β-CTX and tartrate-resistant acid phosphatase (TRAP) levels, elevate osteocalcin (OCN) and alkaline phosphatase (ALP) concentrations,^47–50 and decrease inflammation and oxidative stress. It is important to note that many of these animal models do not have diabetes, yet the observation that metformin was able to enhance bone microstructure suggests a potential direct effect of the drug on bone. However, research on the effectiveness of metformin in enhancing bone density in animal models remains scarce. The necessity for the utilization of additional animal models is a consideration for future research endeavors.

Table 1.

Rodent studies of metformin on bone metabolism.

Model	Species	Metformin dose	Route of administration	Duration/frequency	Bone microarchitecture	Bone metabolism	Mechanism	Study
Type 2 diabetes mellitus	Sprague Dawley rats	100 mg/kg/day	Gavage	12 weeks	BMD ↑, Tb.Th ↑	OPG ↑, OCN ↑, ALP ↑	–	47
	Goto-Kakizaki spontaneous rats	200 mg/kg/day	Oral	20 weeks	vBMD ↑, Conn-D ↑, BV/TV ↑, Tb/Ar ↑, Tb/N ↑, Maximum load ↑, Fracture load ↑, stiffness↑, MAR ↑, Tb.Sp ↓	ALP ↓, TRAP ↓	MDA ↑, CHO ↑, GSP ↑	51
	Ob/Ob mice	200 mg/kg/day	Oral	16 weeks	Adipocyte area ↑	RUNX2 ↑, OPN ↑, OCN ↑	PPAR-γ ↑, C/EBPα ↑, Scd1 ↑	40
	db/db mice	125 mg/kg/day	NR	4 weeks	BV/TV ↑, Tb.Th ↑, Tb/N ↑, Tb.Sp ↓, SMI ↓	RUNX2 ↑, Osterix ↑, COL1A1 ↑	Nrf2 ↑, HO-1 ↑	52
	C57 BL/KS db/db mice	113.75 mg/kg/day	Gavage	12 weeks	Spine Tb.Sp ↓, Tb/N ↑	OCN ↑, GLP-1 ↑, ALP ↑	GPR43 ↑, GPR41 ↑, PC1/3 ↑	44
Postmenopausal osteoporosis	Mice	100 mg/kg/day	Oral	8 weeks	BV/TV ↑, Tb.Th ↑, BS/BV ↓, Tb.Sp ↓	Collagen1 ↑, OCN ↑, RUNX2 ↑	SOD ↑,T-AOC ↑	53
	Sprague Dawley rats	100 mg/kg/day	Gavage	10 weeks	BV/TV ↑	Sclerostin ↓, RANKL/OPG ↓, TRAP ↓	–	54
	C57BL/6 mice	100 mg/kg	I.P. injection	3×/week for 4 weeks	BMD ↑, BV/TV ↑, Tb.N ↑, BS/TV ↑, Tb/Ar ↑	TRAP ↓, β-CTX ↓	LC3 ↓	55
	C57BL/6J mice	100 mg/kg/day	Gavage	8 weeks	Tb.Th ↑, BV/TV ↑, Tb.Sp ↓, BS/BV ↓	–	SOD 1 ↑, CAT ↑	56
Glucocorticoid-induced osteoporosis	Sprague Dawley rats	200 mg/kg/day	Subcutaneous	9 weeks	BMD ↑, BV/TV ↑, %L.Pm ↑, MAR ↑, BFR/BV ↑	OCN ↑, TRAP-5b ↓	–	49
	Sprague Dawley rats	200 mg/kg/day	Oral	6 weeks	BV/TV ↑, Tb.Th ↑, Tb.N ↑, Tb.Sp ↓	–	P16 ↓, p21 ↓, p53 ↓, SIRT1 ↑	45

In vivo rodent studies discussed in this review are included here with the species, metformin dose, route of administration, duration of treatment listed for each study. The upward and downward arrows indicate substantial disparities (p < 0.05) in bone microarchitecture and bone metabolism compared to the modeled animals (type 2 diabetes mellitus, postmenopausal osteoporosis, and glucocorticoid-induced osteoporosis). Bone microarchitecture, bone metabolism, and mechanism are also listed if they are reported in the original research.

ALP, alkaline phosphatase; β-CTX, β-Cross-linked C-telopeptide of type I collagen; BFR/BV, bone formation rate per bone volume; BMD, bone mineral density; BS/TV, bone surface/total volume; BV/TV, bone volume/tissue volume; CAT, catalase; C/EBPα, CCAAT/enhancer binding protein-alpha; CHO, cholesterol; Conn-D, connectivity density; GLP-1, glucagon-like peptide-1; GPR41, G-protein coupled receptor 41; GPR43, G-protein coupled receptor 43; GSP, glutathione S-transferase; HO-1, heme oxygenase-1; LC3, microtubule-associated protein light chain 3; MAR, marrow adiposity ratio; MDA, malondialdehyde; Nrf2, nuclear factor erythroid 2-related factor 2; OCN, osteocalcin; OPG, osteoprotegerin; OPN, osteopontin; PC1/3, proconvertase 1/3; RANKL, nuclear factor kappa ligand-receptor activator; RUNX2, runt-related transcription factor 2; Scd1, stearoyl-Coenzyme A desaturase 1; SMI, structure model index; SIRT1, Sirtuin1; SOD, superoxide dismutase; T-AOC, total antioxidant capacity; Tb/Ar, trabecular bone area; Tb/N, trabecular number; Tb.Sp, trabecular separation; Tb.Th, trabecular thickness; TRAP, tartrate-resistant acid phosphatase; vBMD, vertebral bone mineral density; %L.Pm, percent labeled perimeter.

With the increasing research on T2DM osteoporosis, exploring the clinical significance of metformin as a preventive and treatment becomes crucial. Recent epidemiological research has shown that metformin positively affects osteoporosis patients (Tables 2 –4), lowers bone fracture risks, and influences bone turnover markers (BTMs). Overall, it increases the osteogenic marker P1NP and decreases the osteoclastic marker β-CTX and TRACP-5b. Despite the positive benefits of patients with T2DM and patients treated with glucocorticoids, the therapeutic value of metformin in middle-aged and older women is not significant. This may be attributed to the pronounced decline in estrogen levels observed in perimenopausal and menopausal women.

Table 2.

Clinical study of metformin on bone mineral density, fracture, and bone metabolism in T2DM patients.

Research objects	Research contents	Research results	References
11,458 T2DM patients	A retrospective single center study: the association between metformin and BMD	Metformin was positively associated with higher BMD in men, lumbar, femoral neck, and total hip scores	57
1259 Women aged 40 or more who were not on anti-osteoporotic drugs and were on metformin	A cross-sectional study: the association between metformin and BMD	Regardless of the presence of type 2 diabetes or obesity, metformin was associated with a lower risk of osteoporosis	58
1367 T2DM patients	A prospective randomized controlled trial: the association between metformin and BMD for 12 years	Femoral neck BMD was higher in the metformin compared to placebo group	59
415 T2DM patients	A double-blind randomized controlled trial: metformin versus placebo combined with different insulins effect on BMC and BMD for 18 months	Compared with placebo, the administration of metformin combined with insulin had more positive effects on BMC and BMD in peripheral bones	60
464 T2DM patients	A prospective randomized controlled trial: the effects of metformin compared with placebo on BMD and TBS for 18 months	Compared with a placebo, metformin had no significant effect on BMD in the spine and hip or TBS in patients with T2DM	61
64,878 T2DM patients	A retrospective cohort study: the association between metformin and the risk of hip fracture	No significant association	62
14,611 T2DM patients	A retrospective single center study: the effect of metformin on the risk of osteoporosis and/or vertebral fracture (VF)	Metformin significantly reduced the risk of both osteoporosis and VF by 30%–40%	63
7827 Patients with carcinoma in situ (CIS) and diabetes were treated with metformin and 23,481 were not treated	A retrospective single center study: the efficacy of metformin in the treatment of osteoporosis in diabetic patients with CIS	Metformin attenuates the rate of osteoporosis in diabetic patients with CIS	64
371 T2DM patients	A prospective randomized controlled trial: the effect of metformin on bone metabolism	Metformin treatment decrease P1NP	65

BMC, bone mineral content; BMD, bone mineral density; T2DM, type 2 diabetes mellitus; TBS, trabecular bone score.

Table 3.

Clinical study of metformin on bone mineral density, fracture, and bone metabolism in postmenopausal women with T2DM.

Research objects	Research contents	Research results	References
16 Postmenopausal women with T2DM	A retrospective study: the association between metformin and BMD and bone metabolism	Compared with the control group, metformin increased BMD and decreased serum levels of β-CTX and TRACP-5b	55
124 Peri-menopausal women with T2DM	A prospective cohort study: the association between metformin and BMD	Taking metformin was not associated with changes in BMD	66
40 Postmenopausal women with T2DM	A randomized controlled trial: the association between metformin and bone metabolism	Only serum OC decreased slightly	67

BMD, bone mineral density; OC, osteoclast; T2DM, type 2 diabetes mellitus.

Table 4.

Clinical study of metformin on bone mineral density, fracture, and bone metabolism in patients treated with glucocorticoid.

Research objects	Research contents	Research results	References
849 Inflammatory disease patients continuously were treated with prednisolone	A randomized, double-blind, multi-center controlled trial: the association between metformin and BMD and bone metabolism	Metformin reduced β-CTX and increased hip BMD in glucocorticoid-treated patients, but P1NP and osteocalcin did not change significantly	68
26 Patients with endogenous Cushing’s syndrome	A prospective cohort study: the association between metformin and BMD and bone metabolism	Patients in the metformin increased BMD and decreased β-CTX levels	69

BMD, bone mineral density.

Metformin not only improves bone health in T2DM patients but also helps in reducing the occurrence of cancer and complications. A further retrospective cohort study comprising 673,532 T2DM individuals demonstrated that those who adhered to metformin therapy exhibited a markedly reduced risk of developing primary bone cancer.⁷⁰ Zi et al. explored metformin suppressing the growth of myeloma cells via the IGF-1R/PI3K/AKT/mTOR signaling in both laboratory and live xenograft models.⁷¹ Furthermore, individuals suffering from colorectal adenocarcinoma and simultaneous T2DM, treated with metformin, showed decreased distant metastases and enhanced survival rates.⁷² Additionally, T2DM patients receiving metformin showed a markedly reduced likelihood of cardiovascular disease compared to those on non-metformin or insulin.⁷³ Patients on metformin showed a decreased probability of developing sarcopenia.⁷⁴ Furthermore, metformin proved effective in improving the orthodontic movement of T2DM rats undergoing orthodontic treatment.⁷⁵ Latest research has shown metformin for periodontal disease in individuals without diabetes, is capable of markedly averting bone degradation and altering microbial metabolism and inflammation.⁷⁶

The indirect mechanism of metformin in bone marrow niche

The bone marrow niche represents a dynamic ecosystem where cellular senescence, oxidative stress, and chronic inflammation converge to disrupt skeletal integrity. Metformin emerges as a pleiotropic modulator of this microenvironment through coordinated actions on four interlinked axes: slowing down the aging process, redox equilibrium, immune recalibration, and autophagic flux restoration. These processes engage with various cellular signaling, with AMPK as a central mechanism (Figure 2).

Figure 2.

Summarize the indirect mechanism of metformin in bone marrow nich (created with BioRender.com). Metformin primarily modulates BMSCs’ senescence, alleviates oxidative stress and inflammation, and promotes autophagy, thereby improving the balance of the bone marrow microenvironment to regulate bone metabolism.

Senescence reprogramming

Cellular senescence in the bone marrow niche drives age-related skeletal deterioration through SASP-mediated bystander effects, where aged BMSCs exhibit reduction in osteogenic differentiation capacity, and increased adipogenic propensity. Metformin counteracts this senescent cascade,⁷⁷ encompassing conditions of the musculoskeletal system like osteoporosis, osteoarthritis, sarcopenia, and frailty.⁷⁸

BMSCs can differentiate into various cell types including osteoblasts, adipocytes, and chondrocytes. Nonetheless, the ability of BMSCs to self-renew and differentiate functionally diminishes as they age. The processes of skeletal aging and the increased effects of oxidative stress exert a mutual influence upon on another, potentially resulting in osteoporosis or osteopenia.⁷⁹ Research has shown that metformin suppresses aging in BMSCs by enhancing their proliferation and regulating various factors linked to senescence, such as Sirtuin1 (SIRT1) and prelamin A.⁸⁰ Metformin is capable of preserving anti-aging molecule SIRT1 expression and reducing the expression of senescence-associated genes to mitigate the aging of BMSCs caused by glucocorticoids.⁴⁵ Furthermore, metformin reduces the levels of prelamin A and its shortened variant by suppressing the splicing factor SRSF1.⁸¹ This has been shown to curtail the upstream DNA damage response signaling, thereby hindering the aging process of BMSCs.

Metformin is closely associated with lysosomes in the regulation of aging and lifespan. As cells age, the lysosome ruptures, triggering the release of hydrolytic enzymes that aid in the digestion of the cell as a whole. Furthermore, the lysosome acts as the key controller in the process of cellular signal transmission and metabolism. Metformin reinstates AMPK phosphorylation (Ser485/491), rekindles autophagosome-lysosome fusion reliant on FOXO1, and amplifies its anti-aging properties.^82–84 Mechanistically speaking, PEN2, attaching to minimal amounts of metformin, creates a complex with ATP6AP1, a component of v-ATPase on the lysosome. The activity of v-ATPase is suppressed by this complex, which in turn activates AMPK.⁸⁵ Consequently, it is suggested that metformin could prolong life via the lysosomal AMPK. These coordinated actions reduce SA-β-gal⁺ cells, senescence mediators p16Ink4a, p21WAF1, and the activation of p53 in aged BMSCs cultures, which resulted in a reduction in the expression of several genes associated with SASP.⁸⁶

Skeletal redox rebalancing

Mounting evidence implicates oxidative stress as a pivotal mediator in osteoporotic pathogenesis, primarily through mitochondrial dysfunction-induced redox imbalance. In the bone marrow milieu, compromised antioxidant defenses coupled with diminished mitochondrial mass and membrane potential collapse create a pernicious cycle of reactive oxygen species (ROS) accumulation.⁸⁷ Metformin’s cationic properties drive its selective mitochondrial enrichment (1000:1 concentration gradient), positioning it as a targeted redox modulator within bone cells.

Mitochondrial respiratory chain complex I (NADH: ubiquinone oxidoreductase) aids in transforming NADH into ATP synthesis energy via oxidative phosphorylation and concurrently creates a proton gradient along with ROS. Nonetheless, excess ROS may lead to considerable disruption in the mitochondrial respiratory chain. Under normal physiological circumstances, cellular metabolism naturally generates low levels of ROS as a byproduct. These ROS are instrumental in sustaining bone metabolism, osteoblast differentiation, and bone matrix formation. However, elevated levels of ROS result in the formation of oxidative stress, which in turn disturbs the bone marrow microenvironmental homeostasis.⁸⁸ Metformin could orchestrate mitochondrial ROS homeostasis and be broadly categorized into the following mechanisms.

First, metformin regulates the expression of oxidative-related enzymes, increasing the levels of antioxidant enzymes such as superoxide dismutase (SOD), catalase, and glutathione peroxidase (GSH-Px) and decreasing the levels of ROS-producing enzymes.⁸⁹ Oral metformin positively impacts postmenopausal women by alleviating the hyperoxidative state and boosting antioxidant enzyme levels in bone tissue.⁵⁶ Within osteocytes, the activation of AMPK by metformin reduces the levels of ROS-producing enzymes NADPH oxidase 1 (Nox1) and Nox2, subsequently leading to the reduction of ROS.⁹⁰ Consequently, the presence of metformin enhances the activity of mitochondrial oxidative-related enzymes.

Secondly, nitric oxide (NO), an antioxidant, is produced by nitric oxide synthase (NOS). Both NOS isoforms, inducible NOS (iNOS) and endothelial NOS (eNOS), are expressed in osteoblasts. Prior research has demonstrated that mice lacking eNOS exhibit diminished bone formation and mineral accumulation. It has been shown that metformin influences NO synthesis through the activation of e/iNOS expression, indirectly promoting osteogenic proliferation, differentiation, and mineralization.^91–94

Thirdly, administering metformin led to an increase in mitochondrial mass. Furthermore, there was a rise in the levels of mitochondrial transcription factor A (Tfam) and Cpt1a.⁹⁵ The increased expression of Tfam serves to maintain the stability of the mitochondrial genome, while the increased expression of Cpt1a facilitates the transport of long-chain fatty acids from the cytoplasm to the mitochondria for β-oxidation.

Fourthly, metformin has been demonstrated to enhance mitochondrial transmembrane potential, which is often reduced due to oxidative stress. A standard mitochondrial membrane potential transforms ADP and phosphate into ATP, thereby supplying the necessary energy for the cell. Metformin has been demonstrated to maintain mitochondrial dynamics and inhibit mitochondrial fission in retinal pigment epithelial cells following UVA induction.⁹⁶ Furthermore, standard mitochondrial membrane potential functions to block calcium ion entry, preserving cellular homeostasis. It can therefore be postulated that the mechanism of metformin’s anti-oxidative stress action occurs at the mitochondrial level.⁹⁷

It is well established that elevated glucose levels are a significant contributor to oxidative stress in bone tissue, thereby irreversibly generating AGEs. The serum AGE levels of osteoporotic patients were higher than those of healthy individuals, and a negative correlation was identified between AGE levels and BMD. In addition, the cellular receptor for advanced glycation end-products (RAGEs) expression varies, showing suppression in pre-diabetic conditions to heightened expression in T2DM. Consequently, diabetic patients at various phases show diverse levels of osteogenic symptoms.⁹⁸ Furthermore, RAGE overexpression was observed in BMSCs with impaired osteogenic differentiation capacity, but not in those with enhanced osteogenic differentiation potential. This suggests a link between postponed osteogenic differentiation in T2DM patients and the heightened expression of RAGE.⁹⁹ Metformin further counters hyperglycemia-induced oxidative damage through AGE-RAGE axis modulation.¹⁰⁰ It obstructs the RAGE-JAK2-STAT1 signaling pathway to enhance the bone-forming differentiation of BMSCs in a high-glucose microenvironment.⁵¹ Furthermore, metformin impeded AGE-induced cellular ferroptosis and mitigated the disruption of osteoblast function.^101,102 In addition, metformin also mitigates oxidative stress through other mechanisms in bone.¹⁰³ It facilitates osteogenesis by controlling the endoplasmic reticulum stress (ERS) pathway, AKT-mTOR signaling, and FoxO3A signaling pathway through activating AMPK.^42,104–106

Metformin reprograms bone inflammatory landscapes

Chronic inflammation disrupts skeletal equilibrium by skewing osteoblast–osteoclast crosstalk toward excessive resorption. Metformin counteracts this imbalance through multi-tiered immunomodulation by diminishing inflammatory cytokines.^107–112 Metformin exerts broad-spectrum anti-inflammatory effects by regulating the expression of master regulatory nodes such as nuclear factor kappa-B (NF-κB) p65, and Hmgb1 genes in inflammatory signaling.^113,114 Administering metformin to annular fibroblast stem cells results in the retention of HMGB1 in the nucleus, thus hindering its release into the extracellular space. Consequently, this leads to a decrease in the generation of inflammatory cytokines such as prostaglandin E2 (PGE2), IL-1β, IL-6, and tumor necrosis factor-alpha (TNF-α).¹¹⁵ AMPK activates peroxisome proliferator-activated receptor gamma (PPAR-γ) coactivator-1 alpha (PGC-1α), subsequently suppressing the inflammatory pathway of NF-κB.¹¹⁶ Metformin alters the NF-κB inflammatory pathway, coupled with a decrease in soluble dipeptidyl peptidase-4 (sDPP4), which in turn reduces inflammation.¹⁰⁹ The drug also attenuates monocyte-to-macrophage differentiation by reducing STAT3 phosphorylation.¹¹⁷ This factor exerts control over the signaling of pro-inflammatory events and the establishment of an inflammatory microenvironment. Upon activation by elevated glucose levels, TLR4 attracts NF-κB, triggering the inflammation-related genes, such as TNF-α and chemokine CXCL1/KC, which are attenuated by metformin.¹¹⁸ As an illustration, metformin proved effective in mitigating the suppression of osteoblast apoptosis and differentiation caused by hyperglycemia through the inhibition of TLR4/MyD88/NF-κB.¹¹⁹

The effect of metformin on the AMPK and mTOR pathways has been demonstrated to inhibit Th17 cells and enhance Treg cells to alleviate inflammation.^92,120 Significantly, Th17 cells interact with osteoclast precursors and produce the cytokine IL-17, which activates osteoclastogenesis. On the other hand, it has been shown that metformin lowers the plasma concentration of IL-17 and impedes bone degradation.¹²¹

Varying states of macrophage polarization exert distinct impacts on bone metabolism. The polarization of macrophages determines its function, which can be classified as M1 pro-inflammatory and M2 anti-inflammatory. Consequently, M1 macrophages reduce the capacity for osteogenesis, while M2 macrophages are known to aid in the osteogenic transformation of BMSCs.^122,123 Metformin reshapes bone immune microenvironments through reprogramming macrophage polarization. It distinctly suppresses the release of pro-inflammatory cytokines by M1 macrophages, while promoting anti-inflammatory cytokines toward the M2 phenotype.¹²⁴ As a result, metformin can induce osteogenic effects by altering M2 macrophages, thus converting an inflammatory environment and immune microenvironment into one favorable for osteogenesis in diabetic individuals.^125,126 The polarization shift is achieved through the activation of AMPK and the PI3K/AKT/mTOR pathway, coupled with the concurrent inhibition of the SIRT1/NF-κB and RAGE/NF-κB pathways.^127–129 Additionally, it has been observed that metformin promotes M2 macrophage polarization by upregulating the TLR4/NF-κB signaling pathway.¹³⁰

Metformin modulates bone-related inflammation through inflammation-associated non-coding RNA networks. Specifically, the pro-inflammatory cytokines IL-1β and TNF-α, along with various TLR ligands, trigger the activation of miR-155 and miR-146a, thereby intensifying inflammatory signaling. Conversely, metformin was observed to reverse the elevated expression of miR-155 and miR-146a, and it also reduced the levels of the apoptosis regulator miR-34a in the bone tissues of arthritic rats.¹³¹ Additionally, metformin contributes to the attenuation of the inflammatory response by mitigating the progression of miR-601 activity.⁴⁵

Autophagic flux orchestration

Autophagy serves as a critical quality-control mechanism in bone marrow homeostasis, selectively eliminating senescent, damaged, and oxidatively modified cells and organelles to mitigate age-related bone loss.¹³² Central to this process is AMPK, the energy-sensing regulator that initiates autophagic flux.^133,134 Genetic evidence reveals the AMPKα2 isoform’s non-redundant role, as metformin activates autophagy to regulate osteogenesis and osteoclastogenesis.^135–137 However, the AMPK mechanism activated by metformin to enhance autophagy appears to be inconsistent with its effects on osteoclast differentiation. Xie et al. revealed that metformin suppresses osteoclast-autophagic flux via E2F1-dependent transcriptional repression of BECN1/BCL2, a mechanism divergent from the classical AMPK/mTOR axis.⁵⁵ Mechanistically, metformin orchestrates autophagosome-lysosome fusion and reduces autophagic vacuoles in diabetic peripheral blood mononuclear cells.¹³⁸ Meanwhile, metformin enhances the production of autophagy-related proteins, encompassing microtubule-associated protein light chain 3-II (LC3-II), autophagy protein 5 (Atg 5), and BECN1.^139–142 Metformin activates autophagy via the AMPK/mTOR/p70S6K signaling. As described in section “Cellular transport and intracellular activation of metformin,” metformin orchestrates mTOR biphasic regulation through an AMPK-mediated mechanism, inhibition of mTORC1, and activation of mTORC2 to maintain osteogenic differentiation. This biphasic regulation enables metformin to counteract glucocorticoid-induced osteoblast apoptosis while rescuing high glucose-impaired mineralization.¹⁴³ Additionally, metformin aids in counteracting osteogenesis suppression in environments rich in glucose through the suppression of mTOR.¹³⁹ Clinical relevance is underscored by improved dental implant osseointegration in osteoporotic patients receiving metformin.¹³³ Notably, metformin’s autophagic effects extend beyond waste clearance. Metformin promotes the production and release of extracellular vesicles containing osteogenic miRNAs through the initiation of autophagy.¹⁴¹ Metformin reverses IL-1β-induced AMPK suppression, restoring autophagic flux in inflammatory milieus.¹⁴⁴ Collectively, these mechanisms establish metformin as a multimodal regulator of skeletal autophagy, balancing bone formation and resorption through precision control of the AMPK/mTOR/ULK1 axis,¹³⁷ followed by the delayed activation of the Akt/mTOR signaling pathway.¹⁴⁵

The direct mechanism of metformin in bone marrow niche

The bone marrow niche harbors a hierarchical cellular ecosystem comprising stem cells and progenitor cells that differentiate into osteoblasts, osteocytes, osteoclasts, and adipocytes. Osteoblasts and adipocytes are derived from BMSCs, with osteoblasts undergoing partial encapsulation of the bone matrix to become osteocytes upon maturation. Osteoclasts, differentiated from hematopoietic stem cell (HSC) precursors, serve as principal bone-resorbing cells that secrete critical regulators to maintain bone marrow homeostasis through coupled bone remodeling cycles. Metformin accumulates in bone marrow compartments, where it exerts pleiotropic osteoanabolic effects by modulating niche cell fate determination. Following cellular internalization, metformin activates AMPK-dependent signaling cascades that directly regulate osteoblastogenesis, inhibit adipogenesis, and suppress osteoclast differentiation as detailed in Figure 3.

Figure 3.

The direct role of metformin in the bone marrow niche (created with BioRender.com). Metformin exerts its effects on a variety of bone-associated cells, including BMSCs, osteoblasts, osteocytes, bone marrow adipocytes, and osteoclasts and related signaling pathways.

Metformin and osteogenesis

Metformin orchestrates a dose-dependent osteogenic program in BMSCs through coordinated modulation of key signaling cascades.^146,147 During the early stage, metformin enhances BMSC proliferation at pharmacologically relevant concentrations (0–500 μM). Metformin initiates extracellular matrix remodeling by upregulating Col1a1 expression to synthesize collagen¹²⁷ and reduces matrix-degrading enzyme production to preserve matrix stabilization.¹⁴⁸ During differentiation, metformin exhibits temporal boosting of osteogenic markers ALP, RUNX2, and bone morphogenic proteins (BMPs) and enhancing mineralization markers osteopontin (OPN), OCN, and bone sialoprotein (BSP).^51,133,149 Research has shown that metformin facilitates the mineralization of osteoblasts. Upon the activation of AMPK, the markers of the initial stages of calcification, ALP, and the late stages of calcification, osterix, are increased significantly.¹⁵⁰ Metformin triggers various transcription factors, encompassing RUNX2, osterix, and BMPs.^41,51,52 This multilayered regulation spanning from progenitor expansion to terminal mineralization positions metformin as a pleiotropic modulator of bone formation. Crucially, its therapeutic window is defined by concentration-dependent effects, with high concentrations (>1 mM) paradoxically suppressing proliferation.¹⁴⁶ Consequently, it is plausible to infer that metformin could be instrumental in facilitating the entire process of osteoblast proliferation, differentiation, and mineralization.

Metformin and osteocyte differentiation

Osteocytes, the master regulators of bone mechanotransduction, emerge as pivotal cellular targets in diabetic osteoporosis pathophysiology. Metformin could modulate osteocyte homeostasis through AMPKα2-dependent mechanisms, as evidenced by functional studies in mature osteocyte models.¹⁵¹ In MLO-Y4 cells, AMPKα2 silencing reduced proliferation and attenuated expression of osteogenic marker OPG, OPN, OCN, and BMP6.²⁴ Pharmacologically, metformin restores bone remodeling equilibrium through dual regulation of the OPG/RANKL axis, simultaneously enhancing OPG expression while suppressing RANKL production.⁵⁴

Metformin and adipogenesis

Emerging evidence highlights metformin’s dual skeletal benefits promoting osteogenesis while potently suppressing adipogenesis, such as marrow adipose tissue (MAT), brown adipose tissue, and white adipose tissue.¹⁵² This part intends to conduct an in-depth analysis of how metformin affects the reduction of MAT.

Acting through AMPK activation, metformin redirects the lineage commitment of BMSCs, inhibiting adipocyte differentiation in both clinical and preclinical models of T2DM and diet-induced obesity.^153,154 Mechanistically, metformin downregulates adipogenic transcription factors PPAR-γ, LPL, and CCAAT/enhancer binding protein-alpha (C/EBPα) in BMSCs, disrupting lipid droplet formation.¹⁴⁶ Crucially, PPAR-γ suppression attenuates RANKL production, thereby inhibiting osteoclastogenesis driven by MAT-derived signals. Furthermore, another study attributed the observed increase in MAT to the filling of adipose tissue following metformin-induced apoptosis of BMSCs.⁴⁰ Collectively, these actions create an osteogenic-permissive microenvironment, favoring BMSCs differentiation into osteoblasts over adipocytes while mitigating MAT-associated bone loss.

Metformin and osteoclast differentiation

Osteoclasts, derived from HSCs, are central to pathological bone resorption in metabolic disorders. Metformin exerts potent anti-osteoclastic effects by targeting the AMPK/mTOR axis, with mechanistic studies revealing its dual inhibition of upstream CaMKK and downstream mTOR signaling.^110,155

The RANKL/RANK/OPG governs osteoclast differentiation, a process dynamically regulated by metformin. Metformin upregulates OPG expression, competitively binding RANKL to prevent receptor activation, and suppresses nuclear factor kappa-B (receptor activator for nuclear factor kappa-B (RANK)) surface density on osteoclasts. The binding of RANK to RANKL triggers various transcription factors, encompassing NF-κB and mitogen-activated protein kinase (MAPK)-related cytokines, like extracellular regulated protein kinases (ERK), c-Jun N-terminal kinase (JNK), and p38. Metformin suppresses the NF-κB/MAPK pathway for osteoclast differentiation.^156–158 Consequently, there is a reduction in the expression of the nuclear factor of activated T-cell cytoplasmic 1 (NFATc1) by metformin, and its movement from the cytoplasm to the nucleus.¹⁵⁹ Eventually, this mechanism triggers specific genes linked to osteoclastogenesis, such as TRAP, cathepsin K (CTSK), β3 integrin, and matrix metalloproteinase 9 (MMP-9).¹⁶⁰ Metformin down-regulates the expression of the osteoclast precursor fusion gene dendritic dell-specific transmembrane protein (DC-STAMP), hindering the fusion of the osteoclast precursor.¹⁶¹ In addition, metformin inhibited osteoclast activation by decreasing the expression of several factors, including TNF-α and M-CSF. The equilibrium of various cytokines is of vital importance for the normal development of osteoclasts, while metformin is capable of inhibiting osteoclast differentiation by multilayered regulation.

Related signaling pathways in bone cells

Sirtuins

SIRTs, a family of NAD⁺-dependent histone deacetylases, orchestrate skeletal homeostasis by integrating metabolic and epigenetic signaling pathways (Figure 4). Emerging evidence positions metformin as a master modulator of SIRT signaling, particularly through SIRT1, SIRT3, and SIRT6—key regulators of osteogenic differentiation.¹⁶² SIRT1 has significant anti-inflammatory and anti-aging effects, which are imperative for preserving bone mass.¹⁶³ SIRT1 significantly enhances the Wnt/β-catenin and BMP-2 signaling, crucial for the differentiation of MSCs and osteoblasts. Employing a SIRT1 agonist alongside mineral-coated acellular matrix microcarriers boosts osteogenic mineralization and concurrently diminishes excessive osteoclastogenesis, thereby increasing OPN and RUNX2 levels.¹⁶⁴ Furthermore, elevated SIRT1 levels resulting from metformin treatment promote autophagy, thereby preventing the premature aging and death of stem cells.¹⁶⁵ Metformin alleviates trauma-triggered heterotopic ossification by blocking the NF-κB signaling pathway dependent on SIRT1.¹²⁸ In addition, metformin leverages SIRT3 to combat age-related osteoporosis.^166,167 SIRT3 systemically regulates AGEs-induced BMSCs senescence and senile osteoporosis by triggering mitochondrial autophagy.^168,169 Metformin induces PINK1/Parkin-mediated mitophagy to eliminate oxidative stress and loss of cell viability.¹⁷⁰ Another study has shown that metformin reduces osteoblast apoptosis through phosphatidylinositol 3-kinase/threonine kinase (PI3K/AKT)-mediated antioxidant enzyme production.⁵⁶ SIRT6 also belongs to the SIRTs family, and metformin enhances SIRT6 to govern epigenetic osteoblast programming.¹⁷¹ In osteoblasts precursor, metformin promotes the expression of SIRT6 both before and during differentiation in the presence of high levels of glucose, thereby inhibiting NF-κB and octamer-binding transcription factor 4 (OCT4).³² Maintains physiological expression balance SIRT6 removes acetyl groups from lysine 9 (H3K9) in histone H3 at the promoters of RUNX2, Osx, Dkk1, and OPG, leading to a reduction in their overexpression. This occurrence leads to hindering normal osteoblastogenesis and osteoclastogenesis.¹⁷² Collectively, by modulating the SIRTs network that spans epigenetic regulation (SIRT1/6) and mitochondrial homeostasis (SIRT3), metformin emerges as a multimodal epigenetic modifier of skeletal metabolism.

Figure 4.

SIRTs that are stimulated by metformin and play a role in bone (created with BioRender.com). Metformin has been demonstrated to promote osteogenic differentiation and inhibit osteoblastic differentiation. This occurs via its effect on the sirtuins SIRT1, SIRT3, and SIRT6, which in turn affect bone-related signaling pathways.

Osteogenesis-related signaling pathways

Metformin coordinates osteogenic differentiation through synergistic modulation of key transcriptional and signaling cascades, establishing a pro-osteogenic niche within BMSCs. Various signaling plays a pivotal role in the differentiation of osteoblasts.¹⁷³ RUNX2, for example, whose expression increases proportionally with metformin concentration.¹⁷⁴ AMPK directly targets RUNX2, leading to the phosphorylation (Ser118) in its DNA-binding domain. Such phosphorylation is associated with osteogenic commitment, conversely, a lack of this phosphorylation results in adipogenesis.¹⁷⁵ Observations show that T2DM patients and diabetic rats secreting BMP-4 when stimulated by metformin increase the phosphorylation of Smad1/5/8, which in turn enhances RUNX2 expression, promoting osteogenic differentiation in BMSCs.^176,177 Additionally, metformin promotes osteoblast differentiation by controlling RUNX2 via AMPK/USF-1/SHP and Shh/Gli1 signaling pathway.^178,179 Metformin activates the canonical Wnt/β-catenin pathway, thereby promoting osteogenic proliferation and differentiation through the EGFR/GSK-3β/calcium pathway.^{54,97,180,181} Metformin downregulates sclerostin and DKK1 expression, synergistically enhancing Wnt signaling routes (detailed in section “Osteocyte differentiation-associated pathways”). In addition, activation of the Notch pathway by metformin, including Notch1 and its downstream factors Hes1 and Hey1, was observed to promote both osteogenesis and angiogenesis in human umbilical vein endothelial cells.¹⁸² Consequently, metformin multimodal regulates crucial signaling pathways related to osteogenic differentiation.

Osteocyte differentiation-associated pathways

Osteocytes secrete a variety of factors to control the activities of osteoblasts and osteoclasts, such as sclerostin, and DKK1, and secrete the fibroblast growth factor 23 (FGF23). Both sclerostin and DKK1 are osteogenic inhibitors that impede the Wnt/β-catenin signaling pathway by binding to LRP5/6, thereby preventing the formation of Frizzled/LRP receptor complexes.⁵⁴ Metformin counteracts this inhibitory axis by reducing sclerostin and DKK1 secretion in mature osteocytes, a process mechanistically linked to the mevalonate pathway.^183–185 Concurrently, metformin enhances FGF23-Klotho endocrine signaling, a critical bone-kidney axis regulating phosphate homeostasis and vitamin D metabolism.¹⁸⁶ With advancing age, renal function declines and Klotho levels diminish.¹⁸⁷ Research has shown that metformin boosts Klotho and aids in the elimination of phosphorus from the kidneys.¹⁸⁸ Interestingly, other antidiabetic drugs, such as glucagon-like peptide-1 (GLP-1)-based, gamma-aminobutyric acid (GABA), and PPAR-γ agonists, also demonstrate efficacy in this regard. AMPK regulated the Nox2 signaling pathway and afforded protection to osteocyte viability from dexamethasone-induced apoptosis.¹⁸⁹ However, fundamental mechanisms underlying these processes remain elusive due to limited mechanistic studies.

Osteoclast differentiation-associated pathways

Relative to osteoblasts, there is a paucity of literature on the effect of metformin on osteoclasts. Notably, metformin treatment inhibits the formation of TRAP-positive multinucleated cells in Raw264.7 cells. In contrast to the conventional AMPK pathway, metformin reduces osteoclast autophagy using E2F1-dependent down-regulation of BECN1 and BCL2, ultimately leading to a reduction in osteoclastic differentiation.⁵⁵

Metformin and other drugs or substances

The effect of various antidiabetic medications on bone risk differs. The use of insulin, sulfonylureas, and thiazolidinediones (TZDs) has been linked to an elevated risk of fracture,^39,190 whereas the utilization of metformin, dipeptidyl peptidase-4 (DPP-4) inhibitors, and GLP-1 receptor agonists improves BMD in those with T2DM.^191,192 The evidence for the efficacy of sodium-glucose linked transporter 2 (SGLT2) inhibitors in the treatment of fractures and BMD is inconclusive, with the majority of studies indicating neutral or negative results.^193,194 The metformin and insulin group exhibited diminished levels of β-CTX and P1NP in comparison to the TZDs group,¹⁹⁵ whereas TZDs exhibited a significant rise. The administration of alpha-glucosidase inhibitors and GLP-1 receptor agonists has been demonstrated to attenuate postprandial inhibition of bone resorption.¹⁹⁶ Nevertheless, the therapy of insulin and SGLT2 inhibitors does not influence BTMs.^65,197

Metformin can be combined with various drugs to enhance bone health. The combination of metformin with GLP-1 receptor agonists or DPP-4 inhibitors decreased the risk of major osteoporotic fracture, and improved BMD, BMP-2, and osteosin.^47,183,198 Furthermore, metformin has been demonstrated to mitigate the deleterious effects of TZDs on bone tissue when administered orally in conjunction with rosiglitazone. When metformin and hydroxyapatite are combined, they synergistically enhance osteogenesis.^199,200 The addition of 1% metformin to rabbit bone defects filled with bovine-derived hydroxyapatite particles and sodium hyaluronate resulted in a higher rate of new bone development than that observed in the control group.²⁰¹ Adding 0.1% metformin to calcium phosphate bone cement scaffolds significantly improved bone healing and blood vessel formation in human periodontal ligament stem cells.²⁰² The inhibition of YAP1/TAZ by metformin promotes type H vessel formation, thereby accelerating fracture healing.²⁰³ Merging omega-3 fatty acids with metformin results in a more positive impact on overall ALP, P1NP, estradiol, and the calcium-phosphorus balance.²⁰⁴ Metformin enhances the healing of extraction sockets and diminishes the area of bone necrosis for patients receiving bisphosphonate treatment alongside tooth removal.^205,206 Collectively, these findings position metformin as a versatile adjuvant for bone capable of multi-target synergy.

A notable finding is the association between metformin use and vitamin B12 (VB12) deficiency, which has been demonstrated in a series of multinational clinical investigations. VB12 deficiency is associated with both extended metformin treatment duration and accumulated therapeutic dosage.^207,208 While the precise mechanisms through which metformin leads to VB12 depletion and its effects on bone remain to be elucidated, several potential mechanisms have been postulated. Upon integration of the hydrophobic tail of metformin within the hydrocarbon core of the membrane, the positively charged end of the drug undergoes a modification of the cell membrane’s surface charge. This alteration, in turn, results in the disruption of the function of divalent cations, such as calcium, thereby rendering metformin a potential calcium channel blocker.²⁰⁹ An alternative mechanism posits that metformin exerts its effects directly on intestinal bacteria, thereby augmenting their uptake and consumption of VB12 and influencing bone metabolism via the intestinal flora.²¹⁰ It is widely accepted that metformin may displace calcium in the surface membrane of the ileum, thereby interrupting intestinal calcium-dependent intrinsic factor uptake of VB12. A deficiency of VB12 has been demonstrated to increase levels of homocysteine and methylmalonic acid (MMA). In vitro studies have shown that these elevated levels of homocysteine and MMA can stimulate osteoclastogenesis, suggesting a potential indirect mechanism through which VB12 deficiency may contribute to increased osteoclastogenesis by affecting MMA and homocysteine levels.²¹¹ In addition, metformin protects against homocysteine elevation-induced osteocyte apoptosis,⁹⁰ while VB12 add-back influences the proliferation of osteoblasts by an augmentation in ALP activity. These findings advocate for VB12 co-supplementation as a dual therapeutic strategy that corrects deficiency while amplifying metformin’s bone-protective efficacy through metabolic crosstalk.²¹²

Safety considerations

Prolonged utilization of metformin has been associated with an elevated risk of VB12 deficiency, a condition that can result in irreversible clinical manifestations, including neurological impairment and anemia. It is therefore recommended that patients undergoing long-term metformin therapy undergo regular monitoring of their VB12 levels and adhere to supplementation as needed. In patients with diminished renal function, it is recommended that consideration be given to adjusting the metformin dosage or initiating replacement therapy. Dosage adjustment is imperative when the eGFR is 30–60 mL/min/1.73 m², and sick-days education is crucial. Discontinuation of metformin is mandatory when the eGFR is <30 mL/min/1.73 m².²¹³ A severe overdose of metformin has the potential to result in lactic acidosis.²¹⁴ Consequently, metformin is not recommended for individuals with heart failure, chronic kidney disease, hepatic impairment, and other risk factors for lactic acidosis. Moreover, gastrointestinal side effects, such as nausea and diarrhea, may affect treatment compliance. Although the risk of hypoglycemia is low with metformin alone, caution is necessary when using it in combination with other hypoglycemic agents. During pregnancy, metformin has been shown to reduce the incidence of pregnancy complications, particularly in obese women. Nevertheless, concerns persist regarding its placental transfer and its association with a reduced mean birth weight compared with insulin. Maternal and infant risks, as well as individual differences, should be taken into consideration, and the use of metformin in pregnant women is not recommended. In conclusion, while metformin is expected to improve bone health, special attention should be paid to renal function, VB12 status, and individual patient factors. Clinicians often recommend a gradual increase in metformin dosage, or a transition to an extended-release formulation, in which the adverse effects are typically mitigated. In long-term metformin users, regular monitoring of VB12 levels and renal function is essential to ensure safety and efficacy. For patients with T2DM who are at elevated risk for osteoporotic fractures, glucose-lowering agents with neutral or beneficial effects on bone metabolism should be prioritized in therapeutic regimens, especially metformin. Additionally, T2DM patients should adhere to non-pharmacological osteoporosis prevention strategies, including lifestyle modifications such as regular physical activity, smoking cessation, moderate alcohol consumption, and adequate dietary intake of calcium and vitamin D. Rapid weight loss should be avoided due to its adverse impact on fracture risk. Comprehensive glycemic management in T2DM requires an integrated approach emphasizing holistic care, with individualized glycemic control strategies guided by patient-specific clinical profiles.

Conclusion

Metformin exerts protective effects on bone tissue via a selective distribution mechanism mediated by OCT/MATE transporters. It primarily targets three key bone cell types: BMSCs, osteoblasts, and osteoclasts. The drug promotes osteogenesis and suppresses bone resorption through several signaling pathways, including AMPK-mTOR-autophagy, Wnt/β-catenin, BMP/Smad, and RANKL/OPG. Although oral metformin mainly accumulates in the gut and liver, the disparity between high-dose in vitro outcomes and actual in vivo exposure highlights the need for more efficient bone-targeted delivery approaches. Metformin’s multifaceted properties, including antioxidant, anti-inflammatory, and anti-aging effects, render it a suitable treatment option for patients with osteoporosis who also have concomitant glucose and lipid metabolism disorders. While rodent studies have indicated the potential benefits of metformin on BMD, the findings from clinical trials have been inconclusive, particularly in the context of postmenopausal osteoporosis. This underscores the necessity to investigate its interaction with estrogen receptors. Before the clinical repurposing of metformin for diabetes-associated skeletal disease, future work should map transporter expression across marrow niches, refine localized bone delivery strategies, and monitor potential adverse effects—including VB12 deficiency.

Footnotes

Acknowledgements

We would like to thank the BioRender website () for its templates.

Declarations

ORCID iDs

Yanping Liu

Xiuwen Wang

Jialu Wu

Aijia Wu

Xijie Yu

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