Vascular endothelial growth factor B inhibits insulin secretion in MIN6 cells and reduces Ca2+ and cyclic adenosine monophosphate levels through PI3K/AKT pathway

Figure 1 Decreased insulin secretion in MIN6 cells is related to vascular endothelial growth factor B function. A: Treatment with a gradient concentration of vascular endothelial growth factor B (VEGF-B, VB) protein promotes MIN6 cell proliferation. ^aP < 0.05 vs VB-0, ^bP < 0.01 vs VB-0; B: MIN6 cells with VEGF-B knockdown show an increasing trend in proliferation. ^aP < 0.05 vs Control; C: Stimulation with a gradient concentration of VEGF-B protein inhibits insulin secretion by MIN6 cells. ^aP < 0.05 vs VB-0 in the presence of 5.5 mmol/L glucose; ^bP < 0.01, ^cP < 0.001 vs VB-0 in the presence of 25 mmol/L glucose; D: MIN6 cells with VEGF-B knockdown exhibit increased insulin secretion. ^aP < 0.05 vs Control in the presence of 5.5 mmol/L glucose; ^bP < 0.05 Control in the presence of 25 mmol/L glucose; E: The ratio of insulin secretion by MIN6 cells to their corresponding proliferation upon stimulation with a gradient concentration of VEGF-B protein. ^aP < 0.05, ^cP < 0.001 vs VB-0 in the presence of 5.5 mmol/L glucose; ^bP < 0.01, ^dP < 0.001 vs Control in the presence of 25 mmol/L glucose; F: The ratio of insulin secretion in MIN6 cells to its corresponding cell proliferation after knocking down VEGF-B; G and H: Western blot analysis revealing relative VEGF-B protein expression in cells transfected with VEGF-B siRNA and corresponding statistical data. ^bP < 0.01 vs Control; I: Reverse transcription-polymerase chain reaction revealing relative VEGF-B mRNA expression in cells transfected with VEGF-B siRNA and corresponding statistical data. ^cP < 0.001 vs Control; J: Flow cytometry results indicating that VEGF-B does not affect the apoptosis of MIN6 cells. Student's t-test was performed. VEGF-B: Vascular endothelial growth factor B; VB: VEGF-B; Con: Control; Si: Small interfering RNA; MIN6: MIN6 cell; OD: Optical density.

Figure 2 Exogenous vascular endothelial growth factor B stimulation increases the expression of vascular endothelial growth factor B receptor 1, while vascular endothelial growth factor B small interfering RNA transfection decreases the expression of vascular endothelial growth factor B receptor 1 and neuropilin 1. A-C: Western blot analysis revealing the relative protein expression of vascular endothelial growth factor B receptor 1 (VEGFR1) and neuropilin 1 in cells stimulated with exogenous VEGF-B and corresponding statistical data; D-F: Western blot analysis indicating the relative protein expression of VEGFR1 and neuropilin 1 in cells transfected with VEGF-B small interfering RNA and corresponding statistical data. Student's t-test was performed. ^aP < 0.05 vs Control; ^cP < 0.001 vs Control. NRP1: Neuropilin 1; VEGF-B: Vascular endothelial growth factor B; VB: VEGF-B; Con: Control; Si: Small interfering RNA; MIN6: MIN6 cell.

Figure 3 Exogenous vascular endothelial growth factor B inhibits the expression of PI3K-AKT pathway proteins in MIN6 cells, and knockdown of vascular endothelial growth factor B promotes the expression of these pathway proteins. A-C: Western blot analysis revealing the relative protein expression of phosphatidylinositol 3-kinase (PI3K)-P85 and PI3K-P110γ in cells treated with exogenous vascular endothelial growth factor B (VEGF-B, VB) and corresponding statistical data. ^aP < 0.05 vs VB-0, ^bP < 0.01 vs VB-0; D and E: Western blot analysis indicating the relative protein expression of phosphorylated (p)-serine/threonine kinase (AKT) and AKT in cells treated with exogenous VEGF-B and corresponding statistical data. ^aP < 0.05 vs VB-0 about p-AKT; F: The effect of exogenous VEGF-B protein stimulation on AKT phosphorylation. ^aP < 0.05 vs VB-0; G-I: Western blot analysis revealing the relative protein expression of PI3K-P85 and PI3K-P110γ in cells transfected with VEGF-B small interfering RNA (siRNA) and corresponding statistical data. ^aP < 0.05 vs Control, ^bP < 0.01 vs Control; J and K: Western blot analysis indicating the relative protein expression of p-AKT and AKT in cells transfected with VEGF-B siRNA and corresponding statistical data. ^aP < 0.05 vs Control, ^bP < 0.01 vs Control for p-AKT levels; ^cP < 0.01 vs Control, ^dP < 0.001 vs Control for AKT levels; L: Effect of VEGF-B knockdown on AKT phosphorylation. ^bP < 0.01 vs Con vs Control. Student's t-test was performed. PI3K: Phosphatidylinositol 3-kinase; AKT: Serine/threonine kinase; p-AKT: Phosphorylated-serine/threonine kinase; VB: Vascular endothelial growth factor B; Con: Control; Si: Small interfering RNA; MIN6: MIN6 cell.

Figure 4 Vascular endothelial growth factor B inhibits Ca²⁺ and cyclic adenosine monophosphate levels and phospholipase C gamma 1 protein expression in MIN6 cells. A: Exogenous vascular endothelial growth factor B (VEGF-B, VB) protein reduces cyclic adenosine monophosphate (cAMP) levels in MIN6 cells. ^bP < 0.01 vs VB-0 in the presence of 5.5 mmol/L glucose; ^dP < 0.01 vs VB-0 in the presence of 25 mmol/L glucose; B: cAMP levels in MIN6 cells increase after VEGF-B is knocked down. ^bP < 0.01, ^cP < 0.001 vs Control in the presence of 5.5 mmol/L glucose; ^dP < 0.05 vs Control in the presence of 25 mmol/L glucose; C: Exogenous VEGF-B protein reduces Ca²⁺ levels in MIN6 cells. ^bP < 0.01 vs VB-0 in the presence of 5.5 mmol/L glucose; D: Ca²⁺ levels in MIN6 cells increase after VEGF-B is knocked down. ^cP < 0.001 vs Con in the presence of 5.5 mmol/L glucose; ^dP < 0.01, ^eP < 0.001 vs Con in the presence of 25 mmol/L glucose; E and F: Western blot analysis revealing the relative protein expression of phospholipase C gamma 1 (PLCγ1) and phosphorylated (p)-PLCγ1 in cells stimulated with exogenous VEGF-B and corresponding statistical data. ^aP < 0.05 vs VB-0 for P-PLCγ1; ^bP < 0.05 vs VB-0 for PLCγ1; G: The effect of exogenous VEGF-B protein stimulation on PLCγ1 phosphorylation; H and I: Western blot analysis revealing the relative protein expression of PLCγ1 and p-PLCγ1 in cells transfected with VEGF-B siRNA and corresponding statistical data. ^aP < 0.05 vs Control for p-PLCγ1, ^bP < 0.05 vs Control for PLCγ1; J: Effect of VEGF-B knockdown on PLCγ1 phosphorylation. Student's t-test was performed. VEGF-B: Vascular endothelial growth factor B; VB: VEGF-B; cAMP: Cyclic adenosine monophosphate; Con: Control; Si: Small interfering RNA; MIN6: MIN6 cell; PLCγ1: Phospholipase C gamma 1; p-PLCγ1: Phosphorylated phospholipase C gamma 1.

Figure 5  Possible mechanism for VEGF-B to regulate insulin secretion.