Metformin promotes cell proliferation and osteogenesis under high glucose condition by regulating the ROS‑AKT‑mTOR axis

Abstract

Metformin, a cost‑effective and safe orally administered antidiabetic drug used by millions of patients, has exhibited great interest for its potential osteogenic‑promoting properties in different types of cells, including mesenchymal stem cells (MSCs). Diabetic osteopathy is a common comorbidity of diabetes mellitus; however, the underlying molecular mechanisms of metformin on the physiological processes of MSCs, under high glucose condition, remain unknown. To determine the effects of metformin on the regulatory roles of proliferation and differentiation in MSCs, under high glucose conditions, osteogenesis after metformin treatment was detected with Alizarin Red S and ALP staining. The results demonstrated that high glucose levels significantly inhibited cell proliferation and osteogenic differentiation under high glucose conditions. Notably, addition of metformin reversed the inhibitory effects induced by high glucose levels on cell proliferation and osteogenesis. Furthermore, high glucose levels significantly decreased mitochondrial membrane potential (MMP), whereas treatment with metformin helped maintain MMP. Further analysis of mitochondrial function revealed that metformin significantly promoted ATP synthesis, mitochondrial DNA mass and mitochondrial transcriptional activity, which were inhibited by high glucose culture. Furthermore, metformin significantly scavenged reactive oxygen species (ROS) induced by high glucose levels, and regulated the ROS‑AKT‑mTOR axis inhibited by high glucose levels, suggesting the protective effects of metformin against high glucose levels via regulation of the ROS‑AKT‑mTOR axis. Taken together, the results of the present study demonstrated the protective role of metformin on the physiological processes of MSCs, under high glucose condition and highlighted the potential molecular mechanism underlying the effect of metformin in promoting cell proliferation and osteogenesis under high glucose condition.

Figure 1.

Effects of high, medium and regular glucose conditions on osteogenic differentiation of BMSCs. (A) ALP and (B) Alizarin Red S staining assays were performed following incubation for 3 and 5 days (magnification, ×40; scale bar=50 µm). (C) mRNA expression levels of ALP, OCN, OPG and RUNX2, of BMSCs cultured in differentiating medium were detected via reverse transcription-quantitative PCR analysis. *P<0.05 vs. regular group at 3 days; ^#P<0.05 vs. regular group at 5 days. BMSCs, bone marrow stromal cells; ALP, alkaline phosphatase; OCN, osteocalcin; OPG, osteoprotegerin; RUNX2, runt-related transcription factor 2; IOD, integrated optical density.

Figure 2.

Metformin reverses the inhibitory effect induced by high glucose condition on cell proliferation. (A) Cell viability was assessed via the Cell Counting Kit-8 assay following incubation under regular or high glucose conditions for 1–5 days, respectively. (B) Cell cycle phases were measured via propidium iodide staining, followed by flow cytometric analysis. *P<0.05 vs. regular glucose group; ^#P<0.05 vs. high glucose group. OD, optical density; PE-A, phycoerythrin label.

Figure 3.

Metformin promotes osteogenic differentiation of BMSCs under high glucose/differentiating condition. (A) ALP and (B) Alizarin Red S staining assays were performed following incubation in differentiating medium under high glucose condition for 14 days, with or without metformin (magnification, ×40; scale bar=50 µm). (C) mRNA expression levels of ALP, OCN, OPG and RUNX2, of BMSCs cultured in differentiating medium under regular and high glucose conditions were detected via reverse transcription-quantitative PCR analysis. *P<0.05 vs. regular glucose group; ^#P<0.05 vs. high glucose group. BMSCs, bone marrow stromal cells; ALP, alkaline phosphatase; OCN, osteocalcin; OPG, osteoprotegerin; RUNX2, runt-related transcription factor 2; IOD, integrated optical density.

Figure 4.

Metformin reverses mitochondrial dysfunction induced by high glucose condition in differentiating medium. (A) JC-1 staining was performed to detect mitochondrial membrane potential (magnification, ×40; scale bar=50 µm). (B) ATP synthesis was analyzed. (C) Mitochondrial DNA mass was measured via reverse transcription- quantitative PCR. (D) Mitochondrial transcriptional activity was measured by detecting mitochondrial genome-coded genes. *P<0.05 vs. high glucose group.

Figure 5.

Metformin scavenges ROS accumulation induced by high glucose condition. (A) ROS accumulation was quantitatively measured following incubation for 48 h at 37°C. (B) Western blot analysis was performed to detect the protein levels of mTOR, p-mTOR (Ser2448), AKT and p-AKT (Thr308). Β-actin was used as the internal control. The ratios of (C) p-AKT/AKT and (D) p-mTOR/mTOR were measured using ImageJ software. *P<0.05 vs. regular glucose group; ^#P<0.05 vs. high glucose group. ROS, reactive oxygen species; NAC, n-Acetyl Cysteine; mTOR, mammalian target of rapamycin; AKT, serine/threonine kinase 1.