Integrated Phenotypic and Transcriptomic Analyses of Osteoporosis in Type 2 Diabetic Mice.

Background: Type 2 diabetes (T2D) is a global metabolic condition associated with complications of multiple organs, including the bone. However, the exact impact of T2D on bone along the disease progression, particularly in the early phase, remains largely unknown. Methods: Four-week and sixteen-week high-fat diet (HFD) feeding-induced T2D mouse models were established, and the glucose metabolic status was examined. Bone mass was evaluated by micro-computed tomography (micro-CT), and immunofluorescent (IF) staining was performed for bone histomorphometry with enzyme-linked immunosorbent assay (ELISA) determining serum markers. RNA sequencing analysis was performed to examine the transcriptome of bone, and single-cell RNA-sequencing (scRNA-seq) analysis was further applied. Bone marrow mesenchymal stem cells (BMMSCs) were isolated and analyzed for functional behaviors. Results: The occurrence of glucose metabolic disorders was confirmed at both four weeks and sixteen weeks of HFD feeding, showing increased blood glucose levels with impaired glucose tolerance and insulin sensitivity. Notably, early T2D osteoporosis symptoms were detected at four weeks, especially in the trabecular bone, demonstrating reduced bone mass and mineral density. Histological analysis confirmed that bone remodeling and immune-related inflammation were also altered in T2D mice, remarkably at the early phase, mainly reflected by suppressed bone formation, stimulated bone resorption, increased macrophages, and elevated tumor necrosis factor-alpha (TNF-α) levels. Transcriptomic sequencing further demonstrated significant yet distinct changes in the gene expression profile of bone during T2D progression, which confirmed the histological findings. Notably, overlapping genes with altered expression at four weeks and sixteen weeks of T2D compared to the respective control were identified, and bone marrow scRNA-seq analysis indicated many of them were expressed in BMMSCs, suggesting BMMSCs critically involved in T2D osteoporosis. Dysregulated molecular profiles and functional abnormalities of BMMSCs in T2D mice were validated by ex vivo assays, showing early and persistent occurrence of impaired colony-forming and proliferative capacities with biased differentiation potential. Conclusions: These findings elucidate the bone lesion phenotype in T2D, particularly at the early phase, uncover changes in gene expression profiles of bone during T2D progression, and clarify the functional alterations in bone stem cells, providing a basis for subsequent research and the development of treatment strategies.