Rapamycin Suppresses Penile NADPH Oxidase Activity to Preserve Erectile Function in Mice Fed a Western Style High‐Fat, High‐Sucrose Diet

Abstract

IntroductionThe mechanistic target of rapamycin (mTOR) is a nutrient‐sensitive cellular signaling kinase that has been shown to be activated by high‐fat and/or high‐sugar feeding in highly metabolic tissues such as adipose tissue, liver, and skeletal muscle. mTOR activation has been implicated in the excess production of reactive oxygen species (ROS) resulting from overnutrition, which has been associated with activation of NADPH oxidase (Nox) as a source of excess ROS production. A Western style high‐fat, high‐sucrose diet has recently been used to induce erectile dysfunction (ED) in rodents, in which elevated Nox has been implicated in ED pathogenesis. The objective of this study was to determine if mTOR is an upstream mediator of Nox in the penis in response to the Western diet (WD) and to determine if this pathway is relevant in WD‐induced ED.MethodsYoung male C57Bl/6 mice (n = 90) were fed a control diet (CD) or WD ad libitum for 12 weeks. For the final four weeks of the dietary intervention, mice were intraperitoneally injected with either vehicle (Veh) or the mTOR inhibitor rapamycin (Rap; 2 mg/kg) three days/week. Following the intervention, erectile function was assessed by measuring intracavernosal pressure (ICP) and mean arterial pressure (MAP) during cavernous nerve stimulation. In separate mice following the same intervention, in vivo ROS production was measured in the penis utilizing a microdialysis approach. Microdialysis probes were inserted into the penis of anesthetized mice and perfused with saline containing 100 μM Amplex Ultrared, 1 U/ml horseradish peroxidase, and 10 U/ml superoxide dismutase, and fluorescence was measured from three dialysate replicates. 300 μM apocynin, a Nox inhibitor, was then added to the perfusate, and additional dialysate samples were analyzed. Nox‐mediated ROS were determined by calculation of the ROS that was inhibited by apocynin. Western blots were performed on corpus cavernosum (CC) tissue for Nox subunits, phosphorylation of the mTOR active site, and phosphorylation of p70S6K, a downstream mediator of mTOR Complex 1 (mTORC1) signaling. Significant differences between groups were determined by two‐way ANOVA with Tukey’s multiple comparisons post‐hoc analysis.ResultsErectile function was significantly impaired in mice fed the WD in the vehicle condition, while Rap restored erectile function in the WD condition. P‐mTOR/mTOR and P‐p70S6K/p70S6K were increased in CC from WD‐Veh relative to CD‐Veh mice, while both of these measures were markedly suppressed by Rap treatment regardless of diet. Penile production of the ROS hydrogen peroxide and superoxide were elevated in vehicle treated WD‐fed mice, which was normalized in WD‐fed mice with Rap treatment. Similarly, Nox‐mediated penile ROS was elevated in vehicle treated WD‐fed mice, which was normalized in WD‐fed mice with Rap treatment. The Nox subunits Nox2, p47, and p22 were significantly elevated in CC from WD‐Veh mice, while Rap significantly blunted these expressions. The p67 and Nox4 subunits were not different amongst any groups.ConclusionsThe mTORC1/p70S6K axis appears to be an upstream mediator of Nox2 in the corpus cavernosum in response to chronic consumption of a high‐fat, high‐sucrose, Western pattern diet, which has a deleterious effect on erectile function.