Diabetic bone fragility through advanced glycation end product-collagen axis: Mechanisms and therapy of sodium glucose cotransporter 2 inhibitors

Table 1 Fracture risk estimates from the key cohort/meta-analyses of type 1 diabetes mellitus and type 2 diabetes mellitus patients

Ref.	Design and population	Sample size	Fracture endpoint	Effect estimate (95%CI)	Diabetes type
Emanuelsson et al[29]	United Kingdom Biobank + Copenhagen, Mendelian randomization + observational study	507428	Fragility fracture	T1DM: HR = 1.50 (95%CI: 1.19-1.88); T2DM: HR = 1.22 (95%CI: 1.13-1.32)	T1DM; T2DM
Zoulakis et al[1]	Swedish elderly female cohort study	3008	Any fracture	HR = 1.26 (95%CI: 1.04-1.54)	T2DM
Champakanath et al[6]	University of Colorado CACTI cohort	1416	Osteoporotic fracture	HR = 1.08 (95%CI: 1.02-1.04)	T1DM

CACTI: Coronary artery calcification in type 1 diabetes; CI: Confidence interval; HR: Hazard ratio; T1DM: Type 1 diabetes mellitus; T2DM: Type 2 diabetes mellitus.

Table 2 Three metabolic pathways of bone matrix advanced glycation end-product formation and their rate-limiting determinants

Pathway	Key steps	Products/intermediates	Rate-limiting determinants
Classical Maillard reaction	Glucose reacts with epsilon-amino group of lysine, Schiff base, Amadori rearrangement, early Amadori products	Fructosamine, 1-deoxy-1-ketofructose	Blood glucose concentration, temperature, pH
Glyco-oxidative stress	ROS/RNS oxidize sugars or Amadori products, carboxylated side chains	CML, CEL and other “oxidative AGEs”	ROS levels, antioxidant defenses
Carbonyl stress/dicarbonyl pathway	Glucose autoxidation, degradation or lipid peroxidation, MGO, GO, 3-DG, conjugation with lysine/arginine	MG-H1, glucosepane, pentosidine	Glyoxalase-1 activity, glutathione pool

ROS: Reactive oxygen species; RNS: Reactive nitrogen species; MGO: Methylglyoxal; GO: Glyoxal; 3-DG: 3deoxyglucosone; CML: Nepsilon-(carboxymethyl)lysine; CEL: Nepsilon-(carboxyethyl)lysine; AGEs: Advanced glycation end-products; MG-H1: Methylglyoxal-derived hydroimidazolone-1; pH: Potential hydrogen (acidity/alkalinity).

Table 3 Classification and representative structures of advanced glycation end-products

AGE type	Chemical name	Characteristics	Biological activity
CML	CML	One of the most common AGEs; formed via glycoxidation or lipid peroxidation	RAGE agonist; promotes inflammation and fibrosis
CEL	CEL	Structurally similar to CML; derived from pyruvate and other metabolic intermediates	Associated with insulin resistance
Pentosidine	Crosslink product of reducing sugars with lysine or arginine	Signature structure of AGE crosslinks	Strongly associated with bone fragility and arterial stiffness
MG-H1	MG-H1	Key biomarker of diabetes-related AGE	Promotes apoptosis and mitochondrial dysfunction

CML: Nepsilon-(carboxymethyl)lysine; CEL: Nepsilon-(carboxyethyl)lysine; MG-H1: Methylglyoxal-derived hydroimidazolone 1; AGE: Advanced glycation end product; RAGE: Receptor for advanced glycation end products.

Table 4 Comparison of advanced glycation end product detection techniques in bone tissue and their application limitations

Method	Minimum sample size	Spatial resolution	Absolute quantification	Major advantages	Limitations	Ref.
HPLC-FLD/LC-MS	2 mg defatted bone powder		Yes	High sensitivity; can differentiate CML/CEL/MG-H1/Pen	Destructive; requires acid hydrolysis; labor-intensive	[62]
Autofluorescence (Ex 335/Em 385)	5 μm tissue section	Micron level	No (relative)	Rapid, high-throughput; suitable for biopsy screening	Interference from mineral/Lipid autofluorescence; cannot distinguish AGE types	[63]
Raman spectroscopy	Applicable to both in vivo and tissue sections	Approximately 1 μm	No (semiquantitative)	In situ detection; simultaneously captures mineral-matrix information	Sensitive to water; spectrum interpretation requires expertise	[64]
Nano-FTIR/AFM-IR	10 μm tissue section	20-50 nm	No (semiquantitative)	Highest spatial resolution; enables single-fiber localization	Expensive equipment; limited scanning area	[65]

HPLC: High-performance liquid chromatography; FLD: Fluorescence detector; LC-MS: Liquid chromatography-mass spectrometry; Ex: Excitation; Em: Emission; Nano-FTIR: Nanoscale Fourier transform infrared spectroscopy; AFM-IR: Atomic force microscopy-infrared spectroscopy; CML: Nepsilon-(carboxymethyl)lysine; CEL: Nepsilon-(carboxyethyl)lysine; MG-H1: Methylglyoxal-derived hydroimidazolone 1; Pen: Pentosidine; AGE: Advanced glycation end-product.

Table 5 Research progress on anti-glycation compounds

Intervention	Model and type	Dosage and duration	Evaluation indicators	Summary of main findings	Ref.
Aminoguanidine (AGE formation inhibitor)	db/db genetic T2DM mice (in vivo)	100 mg/kg/day, intraperitoneal injection, 8 weeks (estimated)	Femoral BMD, microarchitecture, biomechanical strength	Reduced AGE accumulation in bone matrix; increased BMD and trabecular number; significantly improved 3-point bending load	[8]
Pyridoxamine (AGE formation inhibitor)	STZ-induced T1DM bone defect model (in vivo); MC3T3-E1 osteoblasts (in vitro)	1 g/L in drinking water for 4 weeks; 50-500 μM for cells	Bone defect CT imaging, histology; ALP activity	Accelerated bone defect healing; increased bone density in defect site within 7-14 days; rescued MGO-induced ALP suppression in vitro	[91]
Metformin (AGE inhibition/antihyperglycemic)	db/db T2DM mice (in vivo)	200 mg/kg/day, oral gavage, 12 weeks (estimated)	Bone volume fraction (BV/TV), biomechanical strength	Increased trabecular bone volume and cortical thickness; improved bending strength; inhibited AGE accumulation in bone	[8]
ALT-711 (AGE crosslink breaker)	Cy/+ chronic kidney disease rats (diabetic osteoporosis-like, in vivo)	3 mg/kg/day, intraperitoneal injection, 10 weeks	Bone AGE content, porosity, mechanical strength	Decreased total bone AGE levels and cortical porosity; no significant improvement in biomechanical strength	[104]
FPS-ZM1 (RAGE small-molecule antagonist)	High-glucose-treated bone marrow mesenchymal stem cells (in vitro)	5 μM for 24 hours	Inflammatory markers (e.g., IL-6), osteogenic markers	Inhibited RAGE and TXNIP/NLRP3 inflammasome; reduced IL-1β and IL-6; upregulated ALP and osteogenic gene expression	[107]
Silybin (natural flavonolignan)	STZ-induced diabetic rats (in vivo); MC3T3-E1 cells (in vitro)	50 mg/kg/day intraperitoneal injection, 6 weeks; 100 μM in cells	BMD, bone strength; osteoblast apoptosis rate	Attenuated diabetic bone loss, increased BMD; inhibited AGE-induced apoptosis by downregulating RAGE and mitochondrial pathway	[111]
Resveratrol (natural polyphenol)	STZ-induced diabetic bone defect model (in vivo)	10 mg/kg/day oral gavage, 8 weeks	Bone regeneration (μCT), serum AGE levels	Promoted mineralized bone formation in defect site; reduced AGE deposition in bone; improved bone matrix quality	[112]

AGE: Advanced glycation end-product; T2DM: Type 2 diabetes mellitus; STZ: Streptozotocin; T1DM: Type 1 diabetes mellitus; MC3T3-E1: Mouse calvaria preosteoblast cell line; Cy/+: Heterozygous cyclophilin mutant (chronic kidney disease model); BMD: Bone mineral density; CT: Computed tomography; ALP: Alkaline phosphatase; BV/TV: Bone volume/total volume; IL-6: Interleukin-6; μCT: Micro-computed tomography; MGO: Methylglyoxal; RAGE: Receptor for advanced glycation end-products; TXNIP: Thioredoxin-interacting protein; NLRP3: NACHT, LRR and PYD domains-containing protein 3; IL-1β: Interleukin-1β.

Table 6 Relative effects of common antidiabetic medications on fracture risk

Drug class	Effect estimates and 95%CI	Study type	Ref.
SGLT-2 inhibitors vs placebo	HR = 0.98 (95%CI: 0.70-1.37)	Randomized controlled trial	Perkovic et al[153]
SGLT-2 inhibitors vs DPP-4 inhibitors	HR = 0.90 (95%CI: 0.73-1.11)	Cohort study	Zhuo et al[17]
SGLT-2 inhibitors vs GLP-1 RAs	HR = 1.00 (95%CI: 0.80-1.25)	Cohort study	Zhuo et al[17]
GLP-1 receptor agonists vs placebo	OR = 1.27 (95%CI: 0.88-1.83)	Systematic review/network meta-analysis	Chai et al[157]
DPP-4 inhibitors vs placebo	RR = 1.44 (95%CI: 1.04-1.98)	Network meta-analysis	Tsai et al[159]
TZDs (e.g., pioglitazone) vs placebo	RR = 1.21 (95%CI: 1.01-1.45)	Systematic review/meta-analysis	Azhari and Dawson[162]

SGLT-2: Sodium-glucose cotransporter 2; DPP-4: Dipeptidyl peptidase-4; GLP-1 RA: Glucagon-like peptide-1 receptor agonist; TZD: Thiazolidinedione; CI: Confidence interval; HR: Hazard ratio; OR: Odds ratio; RR: Risk ratio.