Autophagy is vital for maintaining cellular homeostasis through removing impaired organelles. It has recently been found to play pivotal roles in diabetes mellitus (DM), which is associated with increased bone fracture risk and loss of bone density. However, the mechanism whereby autophagy modulates DM-induced bone loss is not fully elucidated. Previous work has shown that 1α,25-Dihydroxyvitamin D3 (1,25D) exerts positive effects on autophagy, thus affecting bone metabolism. Here, we investigated whether autophagy was involved in the regulation of diabetic bone metabolism. Using Micro-CT, Elisa, histology and histomorphometry analysis, we demonstrated that 1,25D rescues glucose metabolism dysfunction and ameliorates bone loss in diabetic mice. In vitro, 1,25D alleviated primary osteoblast dysfunction and intracellular oxidative stress through reducing prolonged high glucose mediated excessive autophagy in primary osteoblasts, reflected by decreased protein level of Beclin1 and LC3. Of note, the autophagy activator rapamycin (RAP) ablated the positive effects of 1,25D in diabetic environment, leading to a marked increase in autolysosomes and autophagosomes, examined by mRFP-GFP-LC3 fluorescence double labeling. The excessive autophagy induced by high glucose was deleterious to proliferation and differentiation of primary osteoblasts. Additionally, biochemical studies identified that PI3K/Akt signaling could be activated by 1,25D, resulting in the inhibition of FoxO1. We confirmed that FoxO1 deficiency alleviated high glucose-induced autophagy and improved biological functions of primary osteoblasts. Together, our results suggest that the PI3K/Akt/FoxO1 signaling pathway is involved in the osteoprotective effect of 1,25D by attenuating autophagy in diabetes, providing a novel insight for the prevention and treatment of diabetes-caused bone loss.

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.

Autophagy is essential for the maintenance of intracellular homeostasis, implicated in various biological processes. Forkhead box protein O1 (FOXO1) is regarded as a key mediator regulating skeletal development. Recent studies indicate that FOXO1 has a multifaceted role in autophagy regulation and dysregulation. Here, we aimed to elucidate the role of FOXO1-autophagy axis in osteogenesis. Osteoblast conditional Foxo1-knockout mice (Foxo1_OB^−/−, KO) and FOXO1 lentivirus overexpression (Len-FoxO1) model were constructed in vivo. Primary osteoblasts were isolated from KO and their wild-type (WT) littermates. And we also applied overexpression lentivirus to investigate the effects of FOXO1 in vitro. Using Micro-CT, fluorescence labeling detection, real-time qPCR and western blot analyses, we found that bone formation was promoted in Len-FOXO1 mice, which was impaired in KO group. Similarly, FOXO1 overexpression enhanced proliferation, migration and differentiation of osteoblasts, while FOXO1 ablation resulted in poor biological functions of osteoblasts. Through the investigation of autophagic process using mRFP-GFP-LC3 fluorescence labeling and co-immunoprecipitation, we observed that overexpression of FOXO1 initiated autophagy induction, with enhanced FOXO1 interaction with autophagy-related protein 7 (ATG7). On the contrary, FOXO1 knockout in osteoblasts impeded FOXO1-ATG7 conjugation, leading to impaired autophagic activity. Furthermore, inhibition of autophagy by chloroquine (CQ) could reverse favorable influences in bone formation induced by FOXO1 overexpression. Our findings confirmed that FOXO1 was an important regulator of bone formation and autophagy might be part of the underlying mechanisms, offering a significant avenue for the potential strategy in the treatment of bone-related disorders.