Interaction between advanced glycation end products formation and vascular responses in femoral and coronary arteries from exercised diabetic rats

Abstract

Background: The majority of studies have investigated the effect of exercise training (TR) on vascular responses in diabetic animals (DB), but none evaluated nitric oxide (NO) and advanced glycation end products (AGEs) formation associated with oxidant and antioxidant activities in femoral and coronary arteries from trained diabetic rats. Our hypothesis was that 8-week TR would alter AGEs levels in type 1 diabetic rats ameliorating vascular responsiveness.

Methodology/principal findings: Male Wistar rats were divided into control sedentary (C/SD), sedentary diabetic (SD/DB), and trained diabetic (TR/DB). DB was induced by streptozotocin (i.p.: 60 mg/kg). TR was performed for 60 min per day, 5 days/week, during 8 weeks. Concentration-response curves to acetylcholine (ACh), sodium nitroprusside (SNP), phenylephrine (PHE) and tromboxane analog (U46619) were obtained. The protein expressions of eNOS, receptor for AGEs (RAGE), Cu/Zn-SOD and Mn-SOD were analyzed. Tissues NO production and reactive oxygen species (ROS) generation were evaluated. Plasma nitrate/nitrite (NO(x)⁻), superoxide dismutase (SOD), catalase (CAT), thiobarbituric acid reactive substances (TBARS) and N(ε)-(carboxymethyl) lysine (CML, AGE biomarker). A rightward shift in the concentration-response curves to ACh was observed in femoral and coronary arteries from SD/DB that was accompanied by an increase in TBARS and CML levels. Decreased in the eNOS expression, tissues NO production and NO(x)⁻ levels were associated with increased ROS generation. A positive interaction between the beneficial effect of TR on the relaxing responses to ACh and the reduction in TBARS and CML levels were observed without changing in antioxidant activities. The eNOS protein expression, tissues NO production and ROS generation were fully re-established in TR/DB, but plasma NO(x)⁻ levels were partially restored.

Conclusion: Shear stress induced by TR fully restores the eNOS/NO pathway in both preparations from non-treated diabetic rats, however, a massive production of AGEs still affecting relaxing responses possibly involving other endothelium-dependent vasodilator agents, mainly in coronary artery.

Figure 1. Blood glucose, insulin tolerance test, water and food intake.

Blood glucose (panel A), Insulin tolerance test (Kitt, panel B) water (panel C) and food (panel D) intake. Data are mean ± SEM. The number of animals per group is indicated in the figure. *p<0.05 compared to C/SD; ⁺p<0.05 compared to SD/DB.

Figure 2. Concentration-response curves to vasodilator agents.

Femoral (panel A) and coronary (panel B) artery diameters. Concentration-response curves to acetylcholine (ACh, panels C and D) and sodium nitroprusside (SNP, panels E and F), in rats femoral and coronary arteries, respectively, with intact endothelium from control sedentary (C/SD), sedentary diabetic (SD/DB) and trained diabetic (TR/DB). Maximal response values are inserted in the figure. Data are mean ± SEM. The number of animals per group is indicated in the figure. *p<0.05 compared to C/SD; ⁺p<0.05 compared to SD/DB.

Figure 3. Concentration-response curves to contractile agents.

Concentration-response curves to phenylephrine (PHE, panels A and B) and tromboxane mimetic 9,11-dideoxy-11α,9α-epoxy methanoprostaglandin F_2α (U46619, panels C and D) in rats femoral and coronary arteries, respectively, with intact endothelium from control sedentary (C/SD), sedentary diabetic (SD/DB) and trained diabetic (TR/DB). Maximal response values are inserted in the figure. Data are mean ± SEM. The number of animals per group is indicated in the figure.

Figure 4. Protein expression of endothelial nitric oxide synthase (eNOS) and receptor for advanced glycation end products (RAGE).

Protein expression of endothelial nitric oxide synthase (eNOS, panels A and B) and receptor for advanced glycation end products (RAGE, panels C and D) from isolated rats femoral and coronary arteries, respectively. Bottom panel representative Western Blot and top panel quantitative analysis. Data are mean ± SEM. The number of animals per group is indicated in the bars. *p<0.05 compared to C/SD and ⁺p<0.05 compared to SD/DB. Negative control (NC).

Figure 5. Protein expression of cytosolic Cu/Zn-superoxide dismutase (Cu/Zn-SOD) and mitochondrial Mn-superoxide dismutase (Mn-SOD).

Protein expression of cytosolic Cu/Zn-superoxide dismutase (Cu/Zn-SOD, panels A and B) and mitochondrial Mn-superoxide dismutase (Mn-SOD, panels C and D) from isolated rats femoral and coronary arteries, respectively. Bottom panel representative Western Blot and top panel quantitative analysis. Data are mean ± SEM. The number of animals per group is indicated in the bars. Negative control (NC).

Figure 6. Nitric oxide (NO) production in femoral and coronary arteries.

Upper panel: Representative fluorographs of DAF-2-treated sections without (BASAL) or with acetylcholine (ACh, 10 µM)-stimulation of femoral (panel A) and coronary (panel B) arteries from control sedentary (C/SD), sedentary diabetic (SD/DB) and trained diabetic (TR/DB) rats. Lower panel: Quantitative analysis of the NO production measured by DAF-2 (delta of ACh integrative density minus basal integrative density) in transverse sections of femoral (panel A) and coronary (panel B) arteries. Data are mean ± SEM. The number of animals per group is indicated in the figure. *p<0.05 compared to C/SD and ⁺p<0.05 compared to SD/DB.

Figure 7. Reactive oxygen species in femoral and coronary arteries.

Representative fluorographs (upper panel) and quantitative analysis (lower panel) of the ethidium-bromide-positive nuclei in transverse sections of femoral (panel A) and coronary arteries (panel B, the images with (left) and without (right) rhodamine filter) from control sedentary (C/SD), sedentary diabetic (SD/DB) and trained diabetic (TR/DB) rats. Data are mean ± SEM. The number of animals per group is indicated in the figure. *p<0.05 compared to C/SD and ⁺p<0.05 compared to SD/DB.