Endocrine Abstracts

Introduction: Diabetes leads to skeletal fragility and an increased fracture risk. As a master regulator of bone-remodeling, osteocyte dysfunction may play a role in the pathogenesis of diabetes-related skeletal fragility. To study the effects of hyperglycemia on the osteocyte in-isolation, we used the osteocyte-like cell line IDG-SW3 taking an unbiased multi-omics-based approach.

Methods: IDG-SW3 cells were plated in a differentiation osteogenic medium under 3-conditions: normal glucose (5mM; NG), high-glucose (25mM; HG) and mannitol 20mM with glucose 5mM to control for the hyperosmolarity induced by HG. Media was changed twice weekly for 28 days, with cells harvested at 1,3,7,14, 21 and 28-days post differentiation induction. 3-biological-replicates were used for each condition with the entire experiment repeated 3-times. RNA was extracted from cell lysates 14 days after induction to osteogenesis with bulk RNA-SEQ performed. Protein was extracted at day 16 after induction to osteogenesis with proteomics analysis by LCMS.

Results: HG conditions led to lower PH in the supernatant and a delay in markers of osteocyte differentiation. RNA-SEQ analysis revealed 1340 differently expressed genes (767 up 573 down; DEGs). Interestingly, mannitol led to a much smaller transcriptional change with only 103 DEGS vs NG (cut-off of FC>2 and p.adj <0.05) with just 38 DEGs in common between the comparisons. Ingenuity Pathway Anaylsis (IPA) of DEGs (HG vs NG) revealed significant deactivation of the autophagy related pathways CLEAR Signaling (Z-score -3.015, -log[B-H P-value] 8.31) and Phagosome Formation (Z-score -3.43, -log[B-H P-value] 1.458); a pattern not observed in mannitol vs NG. Proteomics analysis revealed HG led to activation of the senescence pathway (Z-score 1.213 -log[B-H P-value] 3.66) and deactivation of the CLEAR signaling pathway Z-score -0.755 -log[B-H P-value] 3.03).

Conclusion: These results reveal the differential effects of hyperosmolarity and hyperglycemia on transcriptional changes and cell differentiation in the osteocyte, which may play a role in the pathophysiology of diabetes-related skeletal fragility. Specifically, the deactivation of autophagy and activation of senescence pathways induced by hyperglycemia in osteocytes warrant future research, as they may serve potential therapeutic targets for prevention and treatment of diabetes-related skeletal fragility.