Low-fat diet and exercise preserve eNOS regulation and endothelial function in the penis of early atherosclerotic pigs: A molecular analysis

Introduction. Diet and exercise affect endothelial function in the penis, but the molecular mechanisms underlying their effects are not understood. Aims. We evaluated endothelial nitric oxide synthase (eNOS)interaction with its negative regulator caveolin-1 and eNOS uncoupling as molecular targets in the penis associated with the beneficial effects of low-fat diet and chronic exercise. Methods. The penes were obtained from adult male Yucatan pigs fed a normal-fat or high-fat diet on exercised or sedentary regimen for 24 weeks. Markers of endothelial function (guanosine 3′, 5′-monophosphate [cGMP]production), endothelial dysfunction (eNOS uncoupling and eNOS interaction with caveolin-1), and oxidative stress (thiobarbituric acid reactive substances [TBARS]) were measured in the penes. The concentrations of cGMP and TBARS were determined using commercial kits. eNOS uncoupling was determined by low-temperature sodium dodecyl sulfate polyacrylamide gel electrophoresis. eNOS binding to caveolin-1, eNOS phosphorylation (Ser-1177), and protein expression of eNOS and caveolin-1 were measured by Western blot analysis in penes purified for NOS and in homogenates, respectively. Main Outcome Measures. Molecular parameters of endothelial function including eNOS regulatory function. Results. Relative to normal-fat diet, high-fat diet significantly (P < 0.05) reduced cGMP levels and significantly (P < 0.05) increased eNOS uncoupling, eNOS binding to caveolin-1, and TBARS production in the penis of sedentary pigs. Exercise of pigs on high-fat diet reversed (P < 0.05) the abnormalities in cGMP levels, eNOS uncoupling, and eNOS binding to caveolin-1, but not TBARS levels. Exercise of pigs on normal-fat diet did not affect any of these parameters. Protein expressions of caveolin-1, phosphorylated (Ser-1177), and total eNOS were unaffected by diet or exercise. Conclusion. Low-fat diet and chronic exercise preserve endothelial function in the pig penis by sustaining active eNOS in its dimeric form and by limiting eNOS interaction with its negative regulator caveolin-1.