Effect Of O-GlcNAc Modification-mediated Energy Metabolism On Endometrial Cell Function And Pregnancy Outcome

1.BackgroundO-GlcNAc modification is a post-translational modification whereby single Olinked ?-D-N-acetylglucosamine(O-GlcNAc)is added to serine threonine residues in nuclear,cytoplasmic,and mitochondrial proteins.Modification effect of O-GlcNAc on the protein was only dynamically regulated by two enzymes: O-GlcNAc transferase(OGT)was responsible for the addition of O-GlcNAc,and O-GlcNAcase(OGA)was responsible for the removal of O-GlcNAc.The O-GlcNAc modification requires UDPGlcNAc,an end product of the hexosamine biological pathway(HBP),as a donor.Besides,O-GlcNAc can extensively modify more than 4,000 proteins in cells and regulate key cellular processes through the O-GlcNAc modification of these proteins,including regulation of signal transduction,mitochondrial activity,cytoskeleton function,and protein degradation.Increased evidence has shown that an O-GlcNAc modification is involved in a variety of physiological processes,with abnormalities of this modification closely related to the occurrence of a variety of diseases.However,the role of O-GlcNAcylation in pregnancy has rarely been described.Mammalian "embryo implantation" refers to the process in which the blastocyst becomes implanted in the endometrium through a series of behaviors such as recognition,localization,adhesion,and crossing the basement membrane.Successful embryo implantation is a key event in establishing an ongoing pregnancy.The endometrial conditions required for the successful completion of this process mainly consist of the appropriate differentiation of the endometrium at the right time.The endometrium undergoes profound cellular and biochemical changes,from being proliferative to secretory,in the preimplantation of the embryo,which leads to the endometrium entering a receptive state.Studies have shown that multiple pregnancy complications in humans are associated with abnormal glucose metabolism in the uterus.However,the mechanism by which pre-implantation O-GlcNAcylation mediated the effects of glucose metabolism on endometrial cell physiology and the preparation for implantation remained largely unknown.Aquaporin-3(AQP3),a member of the Aquaporin family(AQPs),plays an important role in the transport of water and solutes across cell membranes.According to sequence similarity and substrate selectivity,aquaporins can be divided into three categories(traditional aquaporins,hydroglycerin aquaporins,and super aquaporins),and AQP3 belongs to hydroglycerin aquaporins.There is growing evidence that AQP3 s play a key role in cancer metastasis,signaling transduction.However,research on the role of AQPs in the reproductive system is still in its infancy.The previous results from our laboratory indicate that human secretory-phase endometrium AQP3 is highly expressed and affects cell morphology by altering the cytoskeleton through interaction with Ezrin proteins,leading to EMT,and then involved in the invasion and migration of the endometrium.Given the ability of AQP3 to transport glycerol,it is not clear to us whether it plays a role in the regulation of endometrial metabolism.Specificity protein 1(Sp1)has been identified as a transcription factor that binds to the SV40 promoter.Sp1 regulates several of key factors that are important in many disease states.There is growing evidence that Sp1 can be regulated by post-translational modifications in multiple ways.Our team found that among the predicted possible transcription factors,Sp1 can specifically bind to the promoter region of AQP3 and activate the transcription of AQP3.It has been reported that Sp1 is modified by OGlcNAc and has multiple potential O-GlcNAcylation sites.Therefore,whether OGlcNAcylation of Sp1 is involved in the regulation of AQP3 expression and which specific modification site plays a key role remains to be determined.Successful embryo implantation requires receptive endometrium,which is conducive to the process of embryo recognition,adhesion and invasion within a certain period of time and is inseparable from the dynamic interaction between 17?-estradiol(E2)and progesterone(P4).Proper glucose metabolism is critical for the profound physiological changes in the endometrium entering the receptive state.And glucose transporters(GLUTs)are responsible for intracellular uptake of glucose and are the first step in glucose metabolism.Prior literature has reported the presence of GLUTs in the endometrium.However,we still do not understand the specific mechanisms of this process.The aim of this study was to clarify the effect of O-GlcNAc modification on the endometrium during embryo implantation,to investigate whether O-GlcNAc modification regulates changes in endometrial energy metabolism and whether AQP3 is involved in this process,and to clarify which of the potential Sp1 modification sites plays a key role in regulating AQP3.Finally,whether glucose metabolism is regulated by hormones are discussed.This study reveals the molecular mechanisms underlying the effects of O-GlcNAc modification-mediated changes in energy metabolism on endometrial cell function and pregnancy outcome.2.Methods1.Elevated O-GlcNAc modifications induced by GLUT1 through HBP during implantation affect endometrial changes and pregnancy outcome1)Expression of O-GlcNAc,GLUT1,GFAT and Glycogen in human endometrial tissues was analyzed by immunohistochemistry.2)CCK-8 and Transwell were used to detect cell proliferation,migration and invasion.3)Adhesion efficiency was detected by JAR cells(simulated embryos)in vitro.4)Effectivity of embryo implantation was detected by siOGT injection of the uterine horn.5)QPCR was used to detect OGT mRNA in endometrial tissues of spontaneous abortion and induced abortion.6)The expression levels of GLUT1,GFAT and Glycogen in the D1-D5 uterus of the pregnant mouse model were detected by immunohistochemistry.2.O-GlcNAc modification regulates metabolic reprogramming by AQP3 compensating glycolysis1)Extracellular acidification rate(ECAR,approximate glycolysis)and oxygen consumption rate(OCR,approximate mitochondrial respiration)were measured using a biological energy analyzer(Seahorse).2)The metabolites of glycolysis,PPP,HBP,and TCA cycle were analyzed by LC-MS of labeled with C13-glucose.3)The contents of glycerol,pyruvate,lactate and ATP in endometrial tissues and cells were detected by the assay kits.4)Transcriptome sequencing was used to detect metabolism-related genes and pathways regulated by OGT.5)The glucose intake of cells was detected by 2-NBDG.6)The expression of GYK in human and mouse endometrial tissues during implantation was analyzed by immunohistochemistry.7)Effectivity of embryo implantation was detected by siAQP3 injection of the uterine horn.3.O-GlcNAc modification at S491 of Sp1 activates the expression of AQP31)Immunoprecipitation was used to detect the O-GlcNAcylation level of Sp1.2)Expression and localization of Sp1 were detected by immunofluorescence assay.3)Mutate all or each of the potential O-GlcNAc modification sites of Sp1(S491,S612,T640,S641,S698,S702)to alanine to construct mutant plasmids.4)Cells were transfected with the mutant plasmid,Sp1 and AQP3 were detected by Western blotting and qPCR,and the effects of O-GlcNAc modification on self-stability and transcription activity of AQP3 were detected.4.Progesterone promotes endometrial receptivity by regulating glucose metabolism through GLUT11)The effect of progesterone on GLUT1 was determined by immunohistochemistry of ovariectomized mice.2)WB was used to detect the expression of GLUT1 and G6 PD in cells treated with different concentrations of progesterone.3)2-BNDG was used to detect glucose intake in cells treated with different concentrations of progesterone.4)The contents of pyruvate,lactate and ATP in progesterone-treated cells were detected by assay kits.5)CCK-8 and Transwell were used to detect cell proliferation and invasion.6)The extracellular acidification rate(ECAR,similar to glycolysis)was measured by a biological energy analyzer(Seahorse).7)Adhesion efficiency was detected by JAR cells(simulated embryos)in vitro.8)Effectivity of embryo implantation was detected by siGLUT1 injection of the uterine horn.3.Results(1).Elevated O-GlcNAc modifications induced by GLUT1 through HBP during implantation affect endometrial changes and pregnancy outcome1.O-GlcNAc modification is important for cell function and pregnancy outcomes1)The level of O-GlcNAc modification in the human endometrium was higher in the secretory phase than in the proliferative phase.2)In HEC-1A,RL95-2 cells,modulating the level of O-GlcNAc modification affects cell proliferation,migration,and invasion ability.3)Adhesion efficiency of JAR cells to HEC-1A and RL95-2 can be affected by regulating the level of O-GlcNAc modification in vitro.4)The injection of OGT siRNA into the uterine horn of mice significantly reduced the rate of embryo implantation.5)The level of OGT mRNA in endometrial tissue of human spontaneous abortion was significantly lower than that of induced abortion.2.Elevated GLUT1 increases O-GlcNAcylation through HBP during the “window of implantation”1)Human endometrial samples and pregnant mouse models showed that GLUT1 was elevated during the window of implantation.2)Human endometrial samples and pregnant mouse models showed glycogen accumulation during the window period of implantation.3)High Glucose(15 mM,25 mM)increased the level of O-GlcNAc modification in HEC-1A,RL95-2 cells.4)Activation and inhibition of the HBP affect the level of O-GlcNAc modification.5)Human endometrial samples and pregnant mouse models showed that the key enzyme GFAT of HBP was elevated during the window of implantation.(2).O-GlcNAc modification regulates metabolic reprogramming through AQP3 compensation for glycolysis1.O-GlcNAcylation regulates metabolic reprogramming1)Modulation of O-GlcNAcylation of endometrial cells affects glycolysis levels.2)Down-regulation of O-GlcNAcylation increases cellular OCR(approximate aerobic respiration).3)13C-labeled glucose metabolites revealed that elevated O-GlcNAcylation generally increased glycolysis,PPP,and HBP metabolites and decreased TCA cycling metabolites.4)The ATP,lactate content of the secretory phase of human endometrium was significantly higher than the proliferative phase.2.O-GlcNAcylation regulates GLUT1 and AQP3 by transcriptome analysis.1)A total of 26,429 transcripts were identified in RL95-2 cells with downregulated OGT,of which 1587 were up-regulated and 2266 were downregulated.2)GO analysis showed that most of the regulated genes belong to ribosome metabolism,nucleotide biosynthesis,energy metabolite biosynthesis,and protein stabilization.3)Enrichment analysis of the KEGG pathway showed that most of the regulated genes were involved in the central carbon metabolism pathway.4)Among the transcripts of the SLC2 family(GLUTs)detected by sequencing in RL95-2 cells with downregulated OGT,SLC2A1(encoding GLUT1)was most significantly downregulated.5)O-GlcNAcylation regulates GLUT1 through c-Myc.6)Inhibition of OGT and OGA can affect glucose intake of cells,and GLUT1 deficiency reduces glycolysis levels.7)The injection of GLUT1 siRNA into the uterine horn of mice significantly reduced the rate of embryo implantation.8)Among the transcripts of the AQPs family detected by sequencing in RL95-2cells with down-regulated OGT,only AQP3 expression was reduced.3.AQP3 provides compensation for glycolytic metabolism.1)Activation and inhibition of HBP affect the expression of AQP3.2)The glycerol content of the secretory phase of the human endometrium was significantly higher than that of the proliferative phase.3)Regulation of O-GlcNAcylation affects the cellular uptake of glycerol.4)Glycerol increases the level of glycolysis in cells5)Human endometrial samples and a model of pregnant mice showed that the glycerol kinase was elevated during the window of implantation.6)In vivo intrauterine horn injection of AQP3 siRNA reduces the rate of embryo implantation but is less detrimental to pregnancy outcome than OGT.7)In RL95-2 cells with down-regulated AQP3,glycerol also increased the level of glycolysis8)Regulation of AQP3 affects glucose uptake and also regulates the expression of c-Myc and GLUT1.(3).O-GlcNAc modification at S491 of transcription factor SP1 activates the expression of AQP31.O-GlcNAcylation of Sp1 promotes the expression of AQP31)Transcription factor Sp1 specifically regulates the expression of AQP3.2)OGT,OGA inhibitors Alloxan and PUGNAc affected the O-GlcNAcylation level of Sp1.3)O-GlcNAcylation affects the nuclear translocation of Sp1.4)Expression of AQP3 was affected by the regulation of O-GlcNAcylation of Sp1 by the mutant plasmid.2.O-GlcNAcylation of Sp1 at the S491 site affects its stability1)Activation and inhibition of HBP affect the expression of AQP3.2)O-GlcNAcylation of Sp1 increases its stability.3)O-GlcNAcylation of Sp1 at S491 affected its stability and transcriptional activity for AQP3.(4).Progesterone promotes endometrial receptivity by regulating glucose metabolism through GLUT11.Progesterone induces GLUT1 expression and affects glucose metabolism and cell function1)GLUT1 expression is elevated in the endometrium during implantation.2)Progesterone induced GLUT1 expression in ovariectomized mice.3)Progesterone promotes cellular glycolysis and pentose phosphate pathways.4)Progesterone promotes cell proliferation and invasion.2.Downregulation of GLUT1 inhibits glycolysis and embryo implantation1)Downregulation of GLUT1 inhibits cell glucose intake and glycolysis.2)Downregulation of GLUT1 inhibited the adhesion of JAR cells to intimal cells.3)The injection of GLUT1 siRNA into the uterine horn of mice significantly reduced the rate of embryo implantation.4.Conclusions1.Elevated endometrial O-GlcNAcylation increases metabolites of glycolysis,PPP,and HBP,decreases metabolites of the TCA cycle,and affects cellular metabolic reprogramming.2.O-GlcNAcylation affects endometrial glycolytic metabolism by regulating GLUT1 and AQP3.3.AQP3 provides compensation for glycolytic metabolism through glycerol transport and increased expression of GLUT1.4.O-GlcNAcylation of Sp1 affects self-stabilization and transcription to AQP3.5.Progesterone regulates glucose metabolism and affects cell function through GLUT1,and GLUT1 deficiency inhibits glycolysis and leads to implantation failure.