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The mechanistic target of rapamycin (mTOR) is a nutrient-sensitive cellular signaling kinase that has been implicated in the excess production of reactive oxygen species (ROS). NADPH oxidase-derived ROS have been implicated in erectile dysfunction pathogenesis. The objective of this study was to determine if mTOR is an activator of NADPH oxidase in the penis and to determine the functional relevance of this pathway in a translationally relevant model of diet-induced erectile dysfunction. Male mice were fed a control diet or a high-fat, high-sucrose Western style diet (WD) for 12 weeks and treated with vehicle or rapamycin for the final 4 weeks of the dietary intervention. Following the intervention, erectile function was assessed by cavernous nerve-stimulated intracavernous pressure measurement, in vivo ROS production was measured in the penis using a microdialysis approach, and relative protein contents from the corpus cavernosum were determined by Western blot. Erectile function was impaired in vehicle treated WD-mice and was preserved in rapamycin treated WD-mice. Penile NADPH oxidase-mediated ROS were elevated in WD-mice and suppressed by rapamycin treatment. Western blot analysis suggests mTOR activation with WD by increased active site phosphorylation of mTOR and p70S6K, and increased expression of NADPH oxidase subunits, all of which were suppressed by rapamycin. These data suggest that mTOR is an upstream mediator of NADPH oxidase in the corpus cavernosum in response to a chronic Western diet, which has an adverse effect on erectile function.

Erectile Dysfunction (ED) is the most common sexual dysfunction in men and is characterized by the inability to achieve or maintain an erection satisfactory for sexual intercourse. Normal erectile function is highly dependent on testosterone so any dysfunction in testosterone production may accelerate ED pathogenesis. Hypogonadism is a direct cause of ED and is characterized by the diminishing functionality of the testes in producing testosterone. Other pathological conditions such as obesity and diabetes, share similar complications of ED due to decreased testosterone levels. Furthermore, these conditions exhibit increased oxidative stress. Decreased testosterone can weaken antioxidant defenses and increase oxidative stress that then leads to endothelial dysfunction by reducing nitric oxide bioavailability and increasing fibrosis which ultimately leads to ED. Current therapies for ED do not directly target antioxidant defenses to reduce oxidative stress damage. Two prospective drugs, Resveratrol and Mitoquinone (MitoQ), have antioxidant properties that limit oxidative stress and may improve erectile function. They have shown to be successful in decreasing oxidative stress damage and improving endothelial function in cardiovascular disease models, so the objective of this study was to explore the effect resveratrol and MitoQ has on antioxidant defense gene expression and erectile function in a model of hypogonadism. We hypothesized that resveratrol and MitoQ treatment will improve antioxidant defenses and improve erectile function. In this study, we used a surgically castrated mouse model to induce ED. We treated mice with resveratrol or MitoQ for eight weeks. Following treatment, we sacrificed and harvested mice corpus cavernosum (CC). We measured the expression of genes related to antioxidant defenses by performing qRT‐PCR. Additionally, ex vivo vasoreactivity of the internal pudendal artery (IPA) and CC were assessed in response to concentration ranges of multiple agonists with DMT myograph system. One‐way ANOVA and Two‐way ANOVA, respectively, was used to compare differences between groups. Upon castration, vasorelaxation of the IPA and CC significantly declined. Antioxidant defenses were reduced with castration as seen by reduced expression in: Gclc, Gpx1, Prdx3, Prdx5, Nqo1, SOD2, SOD3, and Hmox1. After treatment, vasorelaxation did not improve however there were improvements in antioxidant gene expression. Resveratrol significantly increased expression of Gstm, CAT, SOD1. Additionally, MitoQ and resveratrol trended to increase several antioxidant genes. There was a partially restoration of Gpx1, Prdx3, Nqo1 and SOD2 levels and Hmox1 was restored back to sham levels. To conclude, neither long‐term administration of MitoQ nor resveratrol improved relaxation responses of the IPA or CC, however, they were able to stimulate and improve antioxidant gene expression. Even though these drugs were unable to improve erectile function on their own, they may be paired with other treatments such as PDE‐5 inhibitors, to ...

Introduction The 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. Methods Young 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. Results Erectile 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 diffe...