Discovery and identification of semaphorin 4D as a bioindicator of high fracture incidence in type 2 diabetic mice with glucose control

Bone fracture is increasing in patients with type 2 diabetes mellitus (T2DM) due to skeletal fragility. Most antidiabetics are expected to reduce the incidence of fracture in patients with T2DM, however the results are disappointing. Metformin and GLP-1 receptor agonists have a neutral or minor positive effect in reducing fractures. We aim to reveal the mechanism of fracture in patients with T2DM treated with metformin or exendin-4, explore the key regulators responsible for bone fragility in T2DM. Trabecular and cortical masses in mice with T2DM were analyzed using micro-computed tomography. Biomechanical strength of bone was determined according to three-point bending, and the expression of bone-associated factors was examined with enzyme-linked immunosorbent assays. Important proteins and miRNAs were identified using proteomics analysis and deep screening analysis. Lastly, immunoprecipitation-mass spectrometry and dual-luciferase reporter analysis were used to identify key molecular signals. We found that sermaphorin 4D (Sema4D) is the key regulator of bone fragility in T2DM. Exendin-4 increased the biomechanical properties of bone by decreasing serum Sema4D levels, and metformin has little effect on Sema4D. Anti-sema4D treatment could improve bone strength in T2DM mice compared with metformin or exendin-4. The biomechanical properties of bone were comparable between anti-Sema 4D and the combination of metformin and exendin-4. Exendin-4 promoted osteogenesis of BMSCs by activating CRMP2 to reverse the effect of sema4D. Metformin increased miR-140-3p levels, which decreased plexin B1 expression in bone mesenchymal stem cells. Metformin increased the effect of exendin-4 with more GLP-1 receptor expression to increase the biomechanical strength of bone via miR-140-3p-STAT3-miR-3657 signaling. Blood glucose level is not the major factor contributing to impairment in bone remodeling. Sema4D is responsible for the increase in the incidence of bone fractures in T2DM. Accordingly, we proposed an effective therapeutic strategy to eliminate the effect of sema4D.