Abstract
Our previous studies demonstrated that glimepiride enhanced the proliferation and differentiation of osteoblasts and led to activation of the PI3K/Akt pathway. Recent genetic evidence shows that endothelial nitric oxide synthase (eNOS) plays an important role in bone homeostasis. In this study, we further elucidated the roles of eNOS, PI3K and Akt in bone formation by osteoblasts induced by glimepiride in a high glucose microenvironment. We demonstrated that high glucose (16.5 mM) inhibits the osteogenic differentiation potential and proliferation of rat osteoblasts. Glimepiride activated eNOS expression in rat osteoblasts cultured with two different concentrations of glucose. High glucose-induced osteogenic differentiation was significantly enhanced by glimepiride. Down-regulation of PI3K P85 levels by treatment with LY294002 (a PI3K inhibitor) led to suppression of P-eNOS and P-AKT expression levels, which in turn resulted in inhibition of RUNX2, OCN and ALP mRNA expression in osteoblasts induced by glimepiride at both glucose concentrations. ALP activity was partially inhibited by 10 µM LY294002. Taken together, our results demonstrate that glimepiride-induced osteogenic differentiation of osteoblasts occurs via eNOS activation and is dependent on the PI3K/Akt signaling pathway in a high glucose microenvironment.
Highlights
Type 2 diabetes mellitus (DM) is a metabolic disease with elevated morbidity and mortality
Our findings suggest that glimepiride activates endothelial nitric oxide synthase (eNOS) expression in rat osteoblasts via the PI3K/Akt pathway, and this activation is likely required for glimepiride to stimulate differentiation of rat osteoblasts in a high glucose microenvironment
Dark red mineralized bone matrix was visualized in stained sections (Fig. 1A). We detected their key proteins by immunofluorescence staining, which found the osteoblasts to be positive for alkaline phosphatase (ALP), collagenase 1 (COL-1) and OCN (Fig. 1B,C,D)
Summary
Type 2 diabetes mellitus (DM) is a metabolic disease with elevated morbidity and mortality. Hyperglycemia has various adverse effects on bone metabolism, especially in patients with poorly controlled DM. In addition to its stimulatory effects on pancreatic insulin secretion, glimepiride has been reported to have extrapancreatic functions including activation of PI3K (phosphoinositide 3-kinase) and Akt(v-akt murine thymoma viral oncogene homologue) in rat adipocytes and skeletal muscle [4,5,6]. Osteoblasts are bone forming cells that play an essential role in osteogenesis. Osteoblasts differentiate from mesenchymal stem cells and form bone through endochondral and intramembranous ossification. Many signaling molecules have been identified that positively or negatively regulate osteoblast differentiation. PI3K/Akt signaling is crucial for osteoblast differentiation [9,10], whereas p53 is a negative regulator of osteogenesis [11]
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