1α,25-Dihydroxyvitamin D₃ promotes bone formation by promoting nuclear exclusion of the FoxO1 transcription factor in diabetic mice

1α,25-Dihydroxyvitamin D₃ (1,25(OH)₂D₃) is the active form of vitamin D, which is responsible for reducing the risk for diabetes mellitus (DM), decreasing insulin resistance, and improving insulin secretion. Previous studies have shown that 1,25(OH)₂D₃ inhibited the activity of FoxO1, which has been implicated in the regulation of glucose metabolism. However, its function and mechanism of action in DM-induced energy disorders and also in bone development remains unclear. Here, using in vitro and in vivo approaches including osteoblast-specific, conditional FoxO1-knock-out mice, we demonstrate that 1,25(OH)₂D₃ ameliorates abnormal osteoblast proliferation in DM-induced oxidative stress conditions and rescues the impaired glucose and bone metabolism through FoxO1 nuclear exclusion resulting from the activation of PI3K/Akt signaling. Using alizarin red staining, alkaline phosphatase assay, Western blot, and real-time qPCR techniques, we found that 1,25(OH)₂D₃ promotes osteoblast differentiation and expression of osteogenic phenotypic markers (i.e. alkaline phosphatase (1), collagen 1 (COL-1), osteocalcin (OCN), and osteopontin (OPN)) in a high-glucose environment. Moreover, 1,25(OH)₂D₃ increased both total OCN secretion and levels of uncarboxylated OCN (GluOC) by phosphorylating FoxO1 and promoting its nuclear exclusion, indicated by Western blot and cell immunofluorescence analyses. Taken together, our findings confirm that FoxO1 is a key mediator involved in glucose homeostasis and indicate that 1,25(OH)₂D₃ improves glucose metabolism and bone development via regulation of PI3K/Akt/FoxO1/OCN pathway.