GALNT3-mediated AKT1 glycosylation activates the AKT1/CREB signaling pathway to inhibit high glucose-induced spermatogenic cell apoptosis and mitochondrial dysfunction

Diabetes-induced testicular damage (DITD) is a common complication of diabetes mellitus (DM), impairing spermatogenesis and contributing to male infertility, a condition associated with polypeptide N-acetylgalactoacyltransferase 3 (GALNT3), a critical enzyme for protein glycosylation. However, the role of GALNT3 in DITD progression remains elusive. This study elucidated the action of GALNT3 on DITD pathogenesis and investigated the potential underlying mechanism. Mouse spermatogenic GC-1 spg cells were stimulated by high glucose (HG) to establish the in vitro model of DITD. Cell viability and Apoptosis were evaluated utilizing the CCK-8 method and flow cytometry, respectively. Reactive Oxygen Species (ROS) production and mitochondrial membrane potential (MMP) levels were measured utilizing flow cytometry. Coimmunoprecipitation was performed to measure the binding of GALNT3 to Akt1. The GalNAc-type O-glycosylation of Akt1 was defined utilizing the lectin pull-down assay. The ChIP-PCR and dual-luciferase reporter assays were performed to determine the binding of cAMP response element-binding (CREB) to the GLANT3 promoter. GLANT3 expression was reduced in HG-induced GC-1 spg cells. Overexpressed Glant3 restrained HG-stimulated Apoptosis and ROS production and elevated the HG-induced decrease of the MMP levels of GC-1 spg cells. Besides, Akt1 signaling pathway inactivation mediated the induction of silenced Glant3 on Apoptosis and mitochondrial dysfunction. Further investigation found that GLANT3 induced Akt1 glycosylation to suppress HG-induced Apoptosis and mitochondrial dysfunction. Moreover, transcription factor CREB could bind to the Glant3 promoter and regulate promoter activity. GALNT3-mediated Akt1 glycosylation activated the Akt1/CREB signaling pathway to inhibit HG-induced spermatogenic cell Apoptosis and mitochondrial dysfunction. Therefore, GLANT3 might be a valuable target for DITD management.

AKT1; Apoptosis; CREB; Diabetes-induced testicular damage; GALNT3; Glycosylation; Mitochondrial dysfunction.