Introduction

O-linked β-D-N-acetylglucosamine (O-GlcNAc) modification is a post-translational modification in which a single O-GlcNAc is added to serine or threonine residues in nuclear, cytoplasmic, and mitochondrial proteins, and is involved in a variety of physiological processes.

Objectives

In the present study, the role of O-GlcNAcylation in embryo implantation was evaluated. Furthermore, whether O-GlcNAcylation is involved in orchestrating glucose metabolism to influence endometrial cell physiological functions was investigated.

Methods

Different endometrial tissues were detected using immunohistochemistry. Pregnant mouse models were established to verify molecular expression. O-GlcNAc transferase and aquaporin 3 (AQP3) knockdown were used to detect embryo implantation efficiency in vitro and in vivo. Western blotting and immunofluorescence were used to detect protein expression and stability. Dual luciferase reporter assay and chromatin immunoprecipitation (ChIP) were used to verify the binding transcription factor. Glycolysis was detected using bioenergy analyzer, and metabolites were analyzed using isotope 13C-labeled LC-MS. Metabolic-related genes were determined using RNA sequencing.

Results

Activation of endometrial hexosamine biosynthetic pathway (HBP) caused elevated O-GlcNAcylation during the window of implantation, affecting endometrial cell function and embryo implantation. Specifically, elevated O-GlcNAcylation increased glucose uptake via glucose transporter 1 (GLUT1) leading to glucose metabolic flow into the pentose phosphate pathways and HBP, which regulate the metabolic reprogramming of endometrial cells. Furthermore, O-GlcNAcylation mediated the intracellular transport of glycerol to support and compensate for glycolysis through regulation of AQP3. Unexpectedly, elevated AQP3 also increased glucose uptake via GLUT1. These processes maintained higher metabolic requirements for endometrial physiology. Furthermore, the transcription factor SP1 specifically bound to the AQP3 promoter region, and O-GlcNAcylation of SP1 increased its stability and transcriptional regulation of AQP3 which is associated with O-GlcNAcylation of SP1.

Conclusion

Overall, O-GlcNAcylation regulated glucose metabolism in endometrial cells, and AQP3-mediated compensation provides new insights into the communication between glycolysis and O-GlcNAcylation.

中文翻译：

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O-GlcNAc修饰介导水通道蛋白3协调子宫内膜细胞糖酵解并影响胚胎着床

介绍

O-连接的 β-DN-乙酰氨基葡萄糖 ( O - GlcNAc) 修饰是一种翻译后修饰，其中将单个O -GlcNAc 添加到细胞核、细胞质和线粒体蛋白的丝氨酸或苏氨酸残基上，并参与多种生理过程。

目标

在本研究中，评估了O -GlcNAcylation 在胚胎植入中的作用。此外，还研究了O -GlcNAcylation 是否参与协调葡萄糖代谢以影响子宫内膜细胞的生理功能。

方法

使用免疫组织化学检测不同的子宫内膜组织。建立怀孕小鼠模型以验证分子表达。O -GlcNAc 转移酶和水通道蛋白 3 (AQP3) 敲低用于检测体外和体内胚胎植入效率。Western印迹和免疫荧光用于检测蛋白质表达和稳定性。双荧光素酶报告基因测定和染色质免疫沉淀 (ChIP) 用于验证结合转录因子。使用生物能量分析仪检测糖酵解，使用同位素 13C 标记的 LC-MS 分析代谢物。使用 RNA 测序确定代谢相关基因。

结果

子宫内膜己糖胺生物合成途径（HBP）的激活导致着床窗口期间O -GlcNAcylation升高，影响子宫内膜细胞功能和胚胎着床。具体而言，升高的O -GlcNAcylation 增加了通过葡萄糖转运蛋白 1 (GLUT1) 的葡萄糖摄取，导致葡萄糖代谢流入戊糖磷酸途径和 HBP，从而调节子宫内膜细胞的代谢重编程。此外，O -GlcNAcylation 介导甘油的细胞内转运，通过调节 AQP3 来支持和补偿糖酵解。出乎意料的是，升高的 AQP3 也增加了葡萄糖摄取过剩1。这些过程维持了对子宫内膜生理的更高代谢要求。此外，转录因子 SP1 与 AQP3 启动子区域特异性结合， SP1 的O -GlcNAcylation 增加了其稳定性和 AQP3 的转录调控，这与SP1 的O -GlcNAcylation 相关。

结论

总体而言，O -GlcNAcylation 调节子宫内膜细胞中的葡萄糖代谢，AQP3 介导的补偿为糖酵解和O -GlcNAcylation之间的交流提供了新的见解。