Decarboxylated osteocalcin, a possible drug for type 2 diabetes, triggers glucose uptake in MG63 cells

Figure 1 Western blot analysis demonstrating the protein expression of G protein-coupled receptor family C group 6 subtype A in MG63 cells. HEK-293 cells were selected as the negative control cells, and Jurkat cells were used as the positive control cells. GPRC6A: G protein-coupled receptor family C group 6 subtype A.

Figure 2 Flow cytometric analyses of 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose uptake by MG63 cells after decarboxylated osteocalcin stimulation. A: The distribution of fluorescence intensities which represented 2-Deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG) uptake after decarboxylated osteocalcin (dcOC) stimulation for 1 h; B: The normalized fluorescence intensities corresponding to 2-NBDG uptake after dcOC stimulation for 1 h; C: Distribution of fluorescence intensities which represented 2-NBDG uptake after dcOC stimulation for 72 h; D: The normalized fluorescence intensity corresponding to 2-NBDG uptake after dcOC stimulation for 72 h. The red lines in the histograms in A and C indicate the control group (0 ng/mL), while the other lines indicate the groups treated with dcOC at different concentrations. The data are representative of three independent experiments performed in triplicate. ^aP < 0.05, ^bP < 0.01 vs the control group.

Figure 3 Promotional effect of decarboxylated osteocalcin on glucose uptake after short-term stimulation (1 h) in MG63 cells. A: Protein bands; B: Relative protein levels of GPRC6A (G protein-coupled receptor family C group 6 subtype A), p-Akt (Ser473)/Akt, hypoxia-inducible factor 1 alpha, glucose transporter 1 (GLUT1), and Runx2 after decarboxylated osteocalcin (dcOC) stimulation for 1 h at different concentrations. C: Protein bands; D: Relative protein levels of p-Akt (Ser473)/Akt and GLUT1. E: The distribution of fluorescence intensities which represented 2-Deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG) uptake; F: The normalized fluorescence intensity corresponding to 2-NBDG uptake. The MG63 cells in C-F were treated with dcOC (3 ng/mL) for 1 h and (or) LY294002 (10 µmol/L) for 30 min. The data are representative of three independent experiments performed in triplicate. B: ^aP < 0.05, ^bP < 0.01 vs untreated cells; D and F: ^aP < 0.05, ^bP < 0.01 vs the control group; ^cP < 0.05, ^dP < 0.01 vs the dcOC-only group. dcOC: Decarboxylated osteocalcin; HIF-1α: Hypoxia-inducible factor 1 alpha; GLUT1: Glucose transporter 1; GPRC6A: G protein-coupled receptor family C group 6 subtype A.

Figure 4 Promotional effect of decarboxylated osteocalcin on glucose uptake after long-term stimulation (72 h) in MG63 cells. A: Protein bands; B: Relative protein levels of GPRC6A (G protein-coupled receptor family C group 6 subtype A), p-Akt (Ser473)/Akt, hypoxia-inducible factor 1 alpha (HIF-1α), glucose transporter 1 (GLUT1), and Runx2 after decarboxylated osteocalcin (dcOC) stimulation for 72 h at different concentrations; C: Protein bands; D: Relative protein levels of HIF-1α and GLUT1; E: The distribution of fluorescence intensities which represented 2-Deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG) uptake; F: The normalized fluorescence intensity corresponding to 2-NBDG uptake. The MG63 cells in C-F were treated with dcOC (10 ng/mL) 72 h and (or) were transfected with HIF-1α siRNA. The data are representative of three independent experiments performed in triplicate. B: ^aP < 0.05, ^bP < 0.01 vs untreated cells; D and F: ^aP < 0.05, ^bP < 0.01 vs the control group; ^cP < 0.05, ^dP < 0.01 vs the dcOC-only group. dcOC: Decarboxylated osteocalcin; HIF-1α: Hypoxia-inducible factor 1 alpha; GLUT1: Glucose transporter 1; GPRC6A: G protein-coupled receptor family C group 6 subtype A.

Figure 5 Effects of decarboxylated osteocalcin on the proliferation and alkaline phosphatase activity in MG63 cells. A: Histogram showing the effects of decarboxylated osteocalcin (dcOC) on MG63 cell proliferation. The absorbance value was measured at 24, 48, and 72 h; B: Alkaline phosphatase activity in MG63 cells after 72 h of incubation with different concentrations of dcOC. Significant differences (P < 0.01) were found between the dcOC treatment groups (3 ng/mL and 10 ng/mL) and the control group (0 ng/mL). The data are representative of three independent experiments performed in triplicate. ^aP < 0.05, ^bP < 0.01 vs the control group (0 ng/mL). dcOC: Decarboxylated osteocalcin; ALP: Alkaline phosphatase.