Figure 8. Increased endothelial nitric oxide synthase (NOS3)/nitic oxide (NO) pathway endothelial cells treated with ADK inhibitors.
A. Representative Western blot results of phospho-NOS3 (Ser1177) (p-NOS3S1177), total NOS3 (NOS3), phospho-AKT (Ser473) (p-AKTS473), total AKT (AKT), ADK and β-actin in HUVECs (human umbilical vein endothelial cells) treated with vehicle (Ctrl) and ITU (10 μM) for 4 h (left), and relative ratio of p-NOS3S1177/β-actin (right) were quantitated by densitometric analysis of the corresponding Western blots (n = 3). B. Quantification of relative intracellular adenosine concentration in HUVECs treated with vehicle (Ctrl) and ABT702 (ABT) (10 μM) for 6 h (n = 6). C. Representative Western blot results of phospho-NOS3 (Ser1177) (p-NOS3S1177) and β-actin in HUVECs treated with ABT at 10 μM for 24 h (top) and relative ratio of p-NOS3S1177/β-actin were quantitated by densitometric analysis of the corresponding Western blots (bottom) (n = 5). D. Quantification of relative NO concentration in the culture medium of HUVECs treated with increasing concentrations of ABT (2–20 μM) for 24 h (n = 6). E. Representative Western blot results of phospho-VASP (Ser239) (p-VASPS239) and total VASP (VASP) in HUVECs treated with ABT at 10 μM for 24 h (top) and relative ratio of p-VASPS239/VASP were quantitated by densitometric analysis of the corresponding Western blots (bottom) (n = 4). Data (A-E) are represented as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.001 for indicated comparisons; unpaired two-tailed Student’s t test for (A-C and E), one-way ANOVA with Bonferroni’s post-hoc test for (D). F. Schematic of the proposed mechanism for endothelial ADK deficiency-mediated improved insulin sensitivity. Increased NOS3 activity in ADK deficient ECs is achieved through adenosine receptor A2b -dependent NOS3 protein upregulation and p-AKT/p-NOS3 dependent NOS3 activation. Elevated NO production protects mice from diet-induced insulin resistance through vasodilation, anti-inflammation and angiogenesis. AMP, adenosine monophosphate; ADK, adenosine kinase; ENT, equilibrative nucleoside transporter; ADORA2B, adenosine receptor A2b; p-NOS3, phospho-NOS3 (Ser1177); p-AKT, phospho-AKT (Ser473); NO, nitric oxide.