Nitric oxide-induced lipophagic defects contribute to testosterone deficiency in rats with spinal cord injury

Introduction Males with acute spinal cord injury (SCI) frequently exhibit testosterone deficiency and reproductive dysfunction. While such incidence rates are high in chronic patients, the underlying mechanisms remain elusive.Methods and results Herein, we generated a rat SCI model, which recapitulated complications in human males, including low testosterone levels and spermatogenic disorders. Proteomics analyses showed that the differentially expressed proteins were mostly enriched in lipid metabolism and steroid metabolism and biosynthesis. In SCI rats, we observed that testicular nitric oxide (NO) levels were elevated and lipid droplet-autophagosome co-localization in testicular interstitial cells was decreased. We hypothesized that NO impaired lipophagy in Leydig cells (LCs) to disrupt testosterone biosynthesis and spermatogenesis. As postulated, exogenous NO donor (S-nitroso-N-acetylpenicillamine (SNAP)) treatment markedly raised NO levels and disturbed lipophagy via the AMPK/mTOR/ULK1 pathway, and ultimately impaired testosterone production in mouse LCs. However, such alterations were not fully observed when cells were treated with an endogenous NO donor (L-arginine), suggesting that mouse LCs were devoid of an endogenous NO-production system. Alternatively, activated (M1) macrophages were predominant NO sources, as inducible NO synthase inhibition attenuated lipophagic defects and testosterone insufficiency in LCs in a macrophage-LC co-culture system. In scavenging NO (2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO)) we effectively restored lipophagy and testosterone levels both in vitro and in vivo, and importantly, spermatogenesis in vivo. Autophagy activation by LYN-1604 also promoted lipid degradation and testosterone synthesis.Discussion In summary, we showed that NO-disrupted-lipophagy caused testosterone deficiency following SCI, and NO clearance or autophagy activation could be effective in preventing reproductive dysfunction in males with SCI.