067 Rapamycin Suppresses NADPH Oxidase-mediated Reactive Oxygen Species Production and Reverses Erectile Dysfunction in Western Diet-fed Mice

Abstract

The mammalian 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-sugar 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 activator of Nox in the penis in response to the Western diet (WD) and to determine if this pathway is relevant in WD-induced ED. Young male C57Bl/6J 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 or the mTOR inhibitor rapamycin (Group 1: 1 mg/kg; Group 2: 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. ROS convert the reagents to a fluorescent byproduct resorufin, which was measured in the outflowing dialysate. Three replicate samples were collected, after which 300 μM apocynin, a Nox inhibitor, was added to the perfusate, and additional samples were collected. Nox-mediated ROS were determined by calculation of the ROS that was inhibited by apocynin. Significant differences between groups were determined by two-way ANOVA with Tukey’s multiple comparisons post-hoc analysis, with an alpha level of 0.05.