Physical activity maintains aortic endothelium-dependent relaxation in the obese type 2 diabetic OLETF rat

Abstract

We tested the hypothesis that physical activity can attenuate the temporal decline of ACh-induced endothelium-dependent relaxation during type 2 diabetes mellitus progression in the Otsuka Long-Evans Tokushima fatty (OLETF) rat. Sedentary OLETF rats exhibited decreased ACh-induced abdominal aortic endothelium-dependent relaxation from 13 to 20 wk of age (20-35%) and from 13 to 40 wk of age (35-50%). ACh-induced endothelium-dependent relaxation was maintained in the physically active OLETF group and control sedentary Long-Evans Tokushima Otsuka (LETO) group from 13 to 40 wk of age. Aortic pretreatment with N(G)-nitro-l-arginine (l-NNA), indomethacin (Indo), and l-NNA + Indo did not alter the temporal decline in ACh-induced endothelium-dependent relaxation. Temporal changes in the protein expression of SOD isoforms in the aortic endothelium or smooth muscle did not contribute to the temporal decline in ACh-induced endothelium-dependent relaxation in sedentary OLETF rats. A significant increase in the 40-wk-old sedentary LETO and physically active OLETF rat aortic phosphorylated endothelial nitric oxide (p-eNOS)-to-eNOS ratio was observed versus 13- and 20-wk-old rats in each group that was not seen in the 40- versus 13- and 20-wk-old sedentary OLETF rats. These results suggest that temporal changes in the antioxidant system, EDHF, and cycloxygenase metabolite production in sedentary OLETF rat aortas do not contribute to the temporal decline in sedentary OLETF rat aortic ACh-induced endothelium-dependent relaxation seen with type 2 diabetes mellitus progression. We also report that physical activity in conjunction with aging in the OLETF rat results in a temporal increase in the aortic endothelial p-eNOS-to-eNOS ratio that was not seen in sedentary OLETF rats. These results suggest that the sustained aortic ACh-induced endothelium-dependent relaxation in aged physically active OLETF rats may be the result of an increase in active aortic eNOS.

Fig. 1.

Concentration-response curves for the ACh-induced relaxation of isolated abdominal aortic rings obtained from sedentary Long-Evans Tokushima Otsuka sedentary (LETO) rats (LSED), physically active Otsuka Long-Evans Tokushima fatty (OLETF) rats (OPA), and sedentary OLETF rats (OSED) at 13, 20, and 40 wk of age. Data were obtained in the absence (A, C, and E) or presence (B, D, and F) of N^G-nitro-l-arginine [L-NNA (LN); 3 e⁻⁴ M]. See methods for more details. Data are means ± SE; n = 6–8 rats per group or treatment combination. *P < 0.05, 20 vs. 13 wk; #P < 0.05, 40 vs. 13 wk; †P < 0.05, 40 vs. 20 wk.

Fig. 2.

Concentration-response curves for the ACh-induced relaxation of isolated abdominal aortic rings obtained from LSED, OPA, and OSED rats at 13, 20, and 40 wk of age. Data were obtained in the presence of indomethacin (Indo; 5 e⁻⁶ M; A, C, and E) or combined l-NNA + Indo (B, D, and F). See methods for more details. Data are means ± SE; n = 6–8 rats per group or treatment combination. *P < 0.05, 20 vs. 13 wk; #P < 0.05, 20 vs. 40 wk.

Fig. 3.

Concentration-response curves for the sodium nitroprusside (SNP)-induced relaxation of isolated abdominal aortic rings obtained from LSED, OPA, and OSED rats at 13, 20, and 40 wk of age. Data were obtained in the absence (A, C, and E) or presence (B, D, and F) of l-NNA (3 e⁻⁴ M). See methods for more details. Data are means ± SE; n = 6–8 rats per group or treatment combination. *P < 0.05, 40 vs. 13 wk; #P < 0.05, 40 vs. 20 wk.

Fig. 4.

Concentration-response curves for the SNP-induced relaxation of isolated abdominal aortic rings obtained from LSED, OPA, and OSED rats at 13, 20, and 40 wk of age. Data were obtained in the presence of Indo (5 e⁻⁶ M; A, C, and E) or combined l-NNA + Indo (B, D, and F). See methods for more details. Data are means ± SE; n = 6–8 rats per group or treatment combination. *P < 0.05, 40 vs. 13 wk; #P < 0.05, 40 vs. 20 wk.

Fig. 5.

Analysis of SOD2 (A) and SOD1 (B) protein expression in thoracic aorta segments scraped free of endothelial cells obtained from LSED, OSED, and OPA rats at 13, 20, and 40 wk of age. Top: representative Western blots for SOD2 and SOD1 in each rat group at each age. Bottom: bands were quantified as described in methods. Ratios were calculated for the optical density of each SOD isoform at 13, 20, or 40 wk of age over that of 13-wk-old rats in each respective group. Values are means ± SE of 6–8 determinations. *P < 0.05 vs. 13 wk in each respective group; #P < 0.05 vs. 20 wk in each respective group.

Fig. 6.

Analysis of SOD2 (A) and SOD1 (B) protein expression in endothelial cells scraped from segments of the thoracic aorta obtained from LSED, OSED, and OPA rats at 13, 20, and 40 wk of age. Top: representative Western blots for SOD2 and SOD1 in each rat group at each age. Bottom: bands were quantified as described in methods. Ratios were calculated for the optical density of each SOD isoform at 13, 20, or 40 wk of age over that of 13-wk-old rats in each respective group. Values are means ± SE of 6–8 determinations. *P < 0.05 vs. 13 wk in each respective group; #P < 0.05 vs. 40 wk in each respective group.

Fig. 7.

Analysis of endothelial nitric oxide synthase (eNOS; A) and phosphorylated eNOS (p-eNOS; Ser¹¹⁷⁷; B) protein expression in endothelial cells scraped from segments of the thoracic aorta obtained from LSED, OSED, and OPA rats at 13, 20, and 40 wk of age. Top: representative Western blots for eNOS and p-eNOS in each rat group at each age. Bottom: bands were quantified as described in methods section. Ratios were calculated for the optical density of eNOS or p-eNOS at 13, 20, or 40 wk over that of 13-wk-old rats in each respective group. Values are means ± SE of 6–8 determinations. *P < 0.05 vs. 13 wk in each respective group; #P < 0.05 vs. 20 wk in each respective group.

Fig. 8.

A: representative Western immunoblots of p-eNOS (Ser¹¹⁷⁷; top) and eNOS (bottom) protein expression in endothelial cells scraped from segments of the thoracic aorta obtained from LSED, OSED, and OPA rats at 13, 20, and 40 wk of age. B: analysis of eNOS and p-eNOS (Ser¹¹⁷⁷) protein expression in endothelial cells scraped from segments of the thoracic aorta. Ratios were calculated for the optical density of eNOS or p-eNOS at 13, 20, or 40 wk over that of 13-wk-old rats in each respective group and are expressed as the ratio of p-eNOS to eNOS. Values are means ± SE of 6–8 determinations. *P < 0.05 vs. 13 wk in each respective group; #P < 0.05 vs. 20 wk in each respective group.