N-acetylglucosamine Modification Research Articles

O-GlcNAcylation of progranulin promotes hepatocellular carcinoma proliferation.

Progranulin (PGRN) is overexpressed and implicated in hepatocellular carcinoma (HCC) development; however, its post-translational modifications and regulatory mechanisms in HCC remain largely unexplored. Here, the expression levels of PGRN, OGT, and O-GlcNAcylation were found to be elevated in both HCC samples and cell lines. LC-MS/MS analysis and immunoprecipitation revealed that PGRN underwent O-linked N-acetylglucosamine (O-GlcNAc) modification at threonine 272 (Thr272). Co-immunoprecipitation and confocal microscopy confirmed the interaction and colocalization of O-GlcNAc transferase (OGT) with PGRN. Reducing O-GlcNAcylation increased the ubiquitination of PGRN, while increasing O-GlcNAcylation inhibited ubiquitination and elevated PGRN stability, as measured by cycloheximide (CHX) chase experiments. This regulation of PGRN stability was directly linked to its expression levels. Moreover, mutation at the primary O-GlcNAc site Thr272 inhibited the activity of the PI3K/AKT/mTOR signaling pathway and suppressed HCC cell proliferation. Together, our findings indicate that O-GlcNAcylation at Thr272 is essential for PGRN-driven HCC cell proliferation.

O-GlcNAcylation promotes astroglial-mesenchymal transition via the connexin43 pathway under high-glucose conditions.

This study aimed to investigate the effects of O-linked N-acetylglucosamine modification (O-GlcNAcylation) on astroglial-mesenchymal transition through connexin43 (Cx43) pathway under high-glucose conditions. The primary rat astrocytes were cultured under normal and high-glucose conditions, and level of GFAP, α-SMA and Cx43 was investigated. To explore the influence of O-GlcNAcylation on astroglial-mesenchymal transition, Thiamet G treatment was employed to enhance O-GlcNAcylation, while Alloxan was used to decrease it. Cx43 knockdown was acquired through lentivirus constructs to explore its role in astrocyte transition. The levels of GFAP and α-SMA expressions were examined, while astrocyte proliferation was evaluated using the CCK-8 assay, and migration was assessed through wound healing assays. The results showed that primary rat astrocytes were identified by GFAP antibody staining. Under high-glucose conditions, the levels of GFAP, α-SMA, and Cx43 increased, as confirmed by Western blot and immunofluorescence. O-GlcNAcylation augmentation induced by Thiamet G treatment significantly increased the expression of GFAP, α-SMA, and Cx43 compared to both normal and high-glucose conditions. Conversely, the inhibition of O-GlcNAcylation reversed the high-glucose-induced increase in GFAP and α-SMA. Cx43 knockout led to the downregulation of GFAP and α-SMA compared to high-glucose and O-GlcNAcylation-augmented conditions. Additionally, levels of O-GlcNAcylation and VEGF were reduced in Cx43 knockout group. Consistently, CCK8 and wound healing assays demonstrated that Cx43 knockout could inhibit astrocyte proliferation and migration compared to the high-glucose and O-GlcNAcylation augmented groups. These findings demonstrate that astroglial-mesenchymal transition occurs under high-glucose conditions, and can be promoted by O-GlcNAcylation augmentation, but suppressed by Cx43 knockout. The study underscores the significant role of Cx43 in this transition and its potential involvement in diabetic complications.

Astragalus polysaccharide treatment relieves cerebral ischemia‒reperfusion injury by promoting M2 polarization of microglia by enhancing O-GlcNAcylation.

Cerebral ischemia‒reperfusion (I/R) injury seriously threatens the lives of patients. Astragalus polysaccharide (APS) is the main active ingredient of Astragalus membranaceus and has a wide range of pharmacological activities. Here, we aimed to explore the impacts of APS on cerebral I/R injury and its specific mechanisms. We established a cerebral I/R injury model using middle cerebral artery occlusion (MCAO)-treated rats and oxygen glucose deprivation/reoxygenation (OGD/R)-treated BV2 cells. The interleukin 1β (IL-1β), interleukin 6 (IL-6) and interleukin (IL-10) levels were determined using corresponding ELISA kits and RT‒qPCR. The levels of M1 microglial markers (INOS and CD16) and M2 microglial markers (Arg-1 and CD206) were measured by RT‒qPCR. The O-linked N-acetylglucosamine modification (O-GlcNAcylation), O-GlcNAc transfer (OGT) and O-GlcNAc glycosidase (OGA) protein levels were measured by Western blot.Our results showed that APS treatment decreased IL-1β (179.72 ± 9.08 vs. 81.33 ± 6.30) and IL-6 (445.56 ± 33.09 vs. 234.75 ± 27.62) levels and increased IL-10 (41.95 ± 4.18 vs. 86.40 ± 7.16) levels in OGD/R-treated BV2 cells (p < 0.001). In addition, APS promoted the M2 polarization of OGD/R-treated BV2 cells, manifested by an increase in Arg-1 (0.43 ± 0.04 vs. 0.76 ± 0.03) and CD206 (0.36 ± 0.03 vs. 0.65 ± 0.06) and a decrease in INOS (2.84 ± 0.39 vs. 1.56 ± 0.19) and CD16 (4.04 ± 0.36 vs. 1.88 ± 0.09) in OGD/R-treated BV2 cells (p < 0.001). Additionally, APS treatment increased the O-GlcNAcylation and OGT levels in OGD/R-treated BV2 cells, while OGT knockdown reversed the effect of APS in OGD/R-treated BV2 cells and MCAO-treated rats (p < 0.05).Our study demonstrated that APS alleviated cerebral I/R injury by promoting the M2 polarization of microglia by enhancing OGT-mediated O-GlcNAcylation.

Nisin resistance is increased through GtcA mutation induced loss of cell wall teichoic acid N-acetylglucosamine modifications in Listeria monocytogenes

Nisin resistance development is one of food safety challenges posed by Listeria monocytogenes, an important foodborne pathogen that causes human listeriosis. The GtcA flippase enzyme is functionally crucial in two separate pathways that glycosylate cell envelope wall teichoic acids (WTA) with N-acetylglucosamine (NAG) and lipoteichoic acids (LTA) with galactose, respectively. This study investigated phenotypic roles and molecular mechanisms underlying GtcA involvement in L. monocytogenes nisin resistance. A GtcAA65V mutation was linked with increased nisin resistance in a food processing environment associated L. monocytogenes strain. Examination of nisin stress survival and growth phenotypes among L. monocytogenes gtcA mutants in different genetic backgrounds showed that GtcA function promoted sensitivity and loss of its function through genetic deletion (ΔgtcA) and a natural GtcAA65V mutation increased nisin resistance. Individual contributions of GtcA WTA NAG and LTA galactose glycosylation functions to nisin resistance modulation were examined through nisin sensitivity analysis of genetic deletion mutants and L. monocytogenes strains complemented using functionally altered GtcA mutants. This revealed WTA NAG glycosylation to be the main functional mechanism that determines GtcA dependent nisin phenotypic sensitization. An examination for mechanisms underlying GtcA involvement in nisin sensitivity revealed that the loss of GtcA function induces changes in the cell envelope carbohydrate composition profiles reducing cell surface hydrophobicity. Overall, our results showed that cell envelope WTA NAG glycosylation promotes nisin susceptibility through facilitation of hydrophobic interactions between nisin and the Listeria cell envelope. There may be practical implications from our observations since nisin resistance could be gained in food associated L. monocytogenes strains that develop phage resistance through acquisition of mutations in genes that cause loss of cell envelope WTA NAG modifications.

Glut3 promotes cellular O-GlcNAcylation as a distinctive tumor-supportive feature in Treg cells.

Regulatory T cells (Tregs) establish dominant immune tolerance but obstruct tumor immune surveillance, warranting context-specific mechanistic insights into the functions of tumor-infiltrating Tregs (TIL-Tregs). We show that enhanced posttranslational O-linked N-acetylglucosamine modification(O-GlcNAcylation) of cellular factors is a molecular feature that promotes a tumor-specific gene expression signature and distinguishes TIL-Tregs from their systemic counterparts. We found that altered glucose utilization through the glucose transporter Glut3 is a major facilitator of this process. Treg-specific deletion of Glut3 abrogates tumor immune tolerance, while steady-state immune homeostasis remains largely unaffected in mice. Furthermore, by employing mouse tumor models and human clinical data, we identified the NF-κB subunit c-Rel as one such factor that, through Glut3-dependent O-GlcNAcylation, functionally orchestrates gene expression in Tregs at tumor sites. Together, these results not only identify immunometabolic alterations and molecular events contributing to fundamental aspects of Treg biology, specifically at tumor sites but also reveal tumor-specific cellular properties that can aid in the development of Treg-targeted cancer immunotherapies.

Phosphorylation activates master growth regulator DELLA by promoting histone H2A binding at chromatin in Arabidopsis

DELLA proteins are conserved master growth regulators that play a central role in controlling plant development in response to internal and environmental cues. DELLAs function as transcription regulators, which are recruited to target promoters by binding to transcription factors (TFs) and histone H2A via their GRAS domain. Recent studies showed that DELLA stability is regulated post-translationally via two mechanisms, phytohormone gibberellin-induced polyubiquitination for its rapid degradation, and Small Ubiquitin-like Modifier (SUMO)-conjugation to increase its accumulation. Moreover, DELLA activity is dynamically modulated by two distinct glycosylations: DELLA-TF interactions are enhanced by O-fucosylation, but inhibited by O-linked N-acetylglucosamine (O-GlcNAc) modification. However, the role of DELLA phosphorylation remains unclear as previous studies showing conflicting results ranging from findings that suggest phosphorylation promotes or reduces DELLA degradation to others indicating it has no effect on its stability. Here, we identify phosphorylation sites in REPRESSOR OF ga1-3 (RGA, an AtDELLA) purified from Arabidopsis by mass spectrometry analysis, and show that phosphorylation of two RGA peptides in the PolyS and PolyS/T regions enhances RGA activity by promoting H2A binding and RGA association with target promoters. Notably, phosphorylation does not affect RGA-TF interactions or RGA stability. Our study has uncovered a molecular mechanism of phosphorylation-induced DELLA activity.

O-GlcNAc Modification Is a Promising Therapeutic Target for Diabetic Retinopathy.

Diabetic retinopathy (DR) is a very serious diabetes complication. Changes in the O-linked N-acetylglucosamine (O-GlcNAc) modification are associated with many diseases. However, its role in DR is not fully understood. In this research, we explored the effect of O-GlcNAc modification regulation by activating AMP-activated protein kinase (AMPK) in DR, providing some evidence for clinical DR treatment in the future. Bioinformatics was used to make predictions from the database, which were validated using the serum samples of diabetic patients. As an in vivo model, diabetic mice were induced using streptozotocin (STZ) injection with/without an AMPK agonist (metformin) or an AMPK inhibitor (compound C) treatment. Electroretinogram (ERG) and H&E staining were used to evaluate the retinal functional and morphological changes. In vitro, 661 w cells were exposed to high-glucose conditions, with or without metformin treatment. Apoptosis was evaluated using TUNEL staining. The protein expression was detected using Western blot and immunofluorescence staining. The angiogenesis ability was detected using a tube formation assay. The levels of O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) in the serum changed in the DR patients in the clinic. In the diabetic mice, the ERG wave amplitude and retinal thickness decreased. In vitro, the apoptotic cell percentage and Bax expression were increased, and Bcl2 expression was decreased in the 661 w cells under high-glucose conditions. The O-GlcNAc modification was increased in DR. In addition, the expression of GFAT/TXNIP O-GlcNAc was also increased in the 661 w cells after the high-glucose treatment. Additionally, the Co-immunoprecipitation(CO-IP) results show that TXNIP interacted with the O-GlcNAc modification. However, AMPK activation ameliorated this effect. We also found that silencing the AMPKα1 subunit reversed this process. In addition, the conditioned medium of the 661 w cells may have affected the tube formation in vitro. Taken together, O-GlcNAc modification was increased in DR with photoreceptor cell degeneration and neovascularization; however, it was reversed after activating AMPK. The underlying mechanism is linked to the GFAT/TXNIP-O-GlcNAc modification signaling axis. Therefore, the AMPKα1 subunit plays a vital role in the process.

RAD18 O-GlcNAcylation promotes translesion DNA synthesis and homologous recombination repair

RAD18, an important ubiquitin E3 ligase, plays a dual role in translesion DNA synthesis (TLS) and homologous recombination (HR) repair. However, whether and how the regulatory mechanism of O-linked N-acetylglucosamine (O-GlcNAc) modification governing RAD18 and its function during these processes remains unknown. Here, we report that human RAD18, can undergo O-GlcNAcylation at Ser130/Ser164/Thr468, which is important for optimal RAD18 accumulation at DNA damage sites. Mechanistically, abrogation of RAD18 O-GlcNAcylation limits CDC7-dependent RAD18 Ser434 phosphorylation, which in turn significantly reduces damage-induced PCNA monoubiquitination, impairs Polη focus formation and enhances UV sensitivity. Moreover, the ubiquitin and RAD51C binding ability of RAD18 at DNA double-strand breaks (DSBs) is O-GlcNAcylation-dependent. O-GlcNAcylated RAD18 promotes the binding of RAD51 to damaged DNA during HR and decreases CPT hypersensitivity. Our findings demonstrate a novel role of RAD18 O-GlcNAcylation in TLS and HR regulation, establishing a new rationale to improve chemotherapeutic treatment.

Resveratrol alleviates inflammatory bowel disease by inhibiting JAK2/STAT3 pathway activity via the reduction of O-GlcNAcylation of STAT3 in intestinal epithelial cells

The role of O-linked N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation) in the pathogenesis of inflammatory bowel disease (IBD) has been increasingly highlighted in recent studies. It's been reported that signal transducer and activator of transcription 3 (STAT3) O-GlcNAcylation can affect the activity of the Janus kinase2 (JAK2)/STAT3 pathway.Our recent study showed that resveratrol repairsIBDin mice.On this basis,the present study aimed to explore whether the mechanism of IBD repair by resveratrol is associated with STAT3 O-GlcNAcylation. Pretreatment of colitis mice and intestinal epithelial cells with an O-GlcNAcylation promoter (Thiamet G, or Glucosamine) and an O-GlcNAcylation inhibitor (OSMI-1) showed that increased O-GlcNAcylation promoted colitis in mice.The pro-inflammatory cytokines interleukin (IL) -6, IL-1β, and tumor necrosis factor-α (TNF-α) were increased, while the anti-inflammatory cytokine IL-10 was decreased. Moreover, the downstream target proteins of JAK2/STAT3, cyclooxygenase-2 and nitric oxide synthase 2 were up-regulated, Resveratrol treatment mitigated the inflammation by decreasing JAK2/STAT3 activity, as well as STAT3 O-GlcNAcylation. Finally, the correlation between STAT3 glycosylation and phosphorylation in intestinal epithelial cells under the effect of resveratrol was investigated by Immunofluorescence co-localization and immunoprecipitation.The results showed that resveratrol inhibited STAT3 O-GlcNAcylation, thereby inhibiting its phosphorylation, reducing JAK2/STAT3 pathway activity, and alleviating IBD.

HSF1 Increases EOGT-Mediated Glycosylation of Notch1 to Promote IL-1β-Induced Inflammatory Injury of Chondrocytes.

Osteoarthritis (OA) is the most common arthritic disease in humans. Nevertheless, the pathogenic mechanism of OA remains unclear. This study aimed to explore that heat-shock transcription factor 1 (HSF1) facilitated interleukin-1 beta (IL-1β) chondrocyte injury by increasing Notch1 O-linked N-acetylglucosamine (O-GlcNAc) modification level. Human chondrocytes were incubated with 5 ng/ml interleukin-1 beta (IL-1β) for 24 h to establish OA cell model. The messenger RNA (mRNA) or protein expressions were assessed using reverse transcription-quantitative polymerase chain reaction, western blot, or immunofluorescence. Chondrocyte viability was examined by Cell Counting Kit-8 assay. Enzyme-linked immunosorbent assay was employed to detect the secretion levels of interleukin-6 (IL-6) and interleukin-8 (IL-8). Immunoprecipitation was adopted to detect Notch1 O-GlcNAc modification level. The interaction between HSF1 and epidermal growth factor-like (EGF) domain-specific O-GlcNAc transferase (EOGT) promoter was analyzed by dual-luciferase reporter gene and chromatin immunoprecipitation assays. Herein, our results demonstrated that HSF1, EOGT, Notch1, and Notch1 intracellular domain (NICD1) expressions in chondrocytes were markedly increased by IL-1β stimulation. EOGT elevated Notch1 expression in IL-1β-treated chondrocytes by increasing Notch1 O-GlcNAc modification level. EOGT silencing reduced IL-1β-induced chondrocyte inflammatory injury. In addition, HSF1 knockdown relieved IL-1β-induced chondrocyte inflammatory injury. Molecular interaction experiment proved that HSF1 transcriptionally activated EOGT expression in IL-1β-treated chondrocytes. HSF1 promoted IL-1β-induced inflammatory injury in chondrocytes by increasing EOGT-mediated glycosylation of Notch1.

O-GlcNAcylation Inhibition Upregulates Connexin43 Expression in the Endothelium to Protect the Tight Junction Barrier in Diabetic Retinopathy.

This study aimed to investigate the effects of O-linked N-acetylglucosamine modification (O-GlcNAcylation) on connexin43 (Cx43) expression and its subsequent effects on tight junction properties in diabetic retinopathy (DR). O-GlcNAcylation levels in primary human retinal vascular endothelial cells (HRVECs) and retinas from rats with diabetes were regulated by treatment with Thiamet G or alloxan. Immunoprecipitation was used to examine the relationship between O-GlcNAcylation and Cx43 expression. Stable overexpression and knockdown of Cx43 in HRVECs were achieved using lentivirus constructs; further, their effects on occludin and zonula occluden-1 (ZO-1) expression and tight junction barrier function were determined. O-GlcNAcylation level increased significantly, whereas Cx43 expression decreased in retinas from rats with diabetes and HRVECs cultured under high-glucose conditions. Immunoprecipitation revealed that Cx43 was modified by O-GlcNAcylation and phosphorylation simultaneously. O-GlcNAcylation inhibition negatively regulated both total Cx43 and phosphorylated Cx43 expression, subsequently disrupting tight junction properties. Conversely, Cx43 overexpression reversed the disruption of tight junction properties and downregulated vascular endothelial growth factor expression. Consistently, Cx43 overexpression increased transendothelial electrical resistance values in HRVEC layers. O-GlcNAcylation negatively regulated Cx43 expression, contributing to the disruption of the blood retinal barrier. However, O-GlcNAcylation inhibition and Cx43 overexpression could reverse the tight junction disruption. Therefore, O-GlcNAcylation inhibition is a potential target for avoiding tight junction disruption through the Cx43 pathway in DR.

N-linked glycosylation of flavivirus E protein contributes to viral particle formation.

In the case of the Japanese encephalitis virus (JEV), the envelope protein (E), a major component of viral particles, contains a highly conserved N-linked glycosylation site (E: N154). Glycosylation of the E protein is thought to play an important role in the ability of the virus to attach to target cells during transmission; however, its role in viral particle formation and release remains poorly understood. In this study, we investigated the role of N-glycosylation of flaviviral structural proteins in viral particle formation and secretion by introducing mutations in viral structural proteins or cellular factors involved in glycoprotein transport and processing. The number of secreted subviral particles (SVPs) was significantly reduced in N154A, a glycosylation-null mutant, but increased in D67N, a mutant containing additional glycosylation sites, indicating that the amount of E glycosylation regulates the release of SVPs. SVP secretion was reduced in cells deficient in galactose, sialic acid, and N-acetylglucosamine modifications in the Golgi apparatus; however, these reductions were not significant, suggesting that glycosylation mainly plays a role in pre-Golgi transport. Fluorescent labeling of SVPs using a split green fluorescent protein (GFP) system and time-lapse imaging by retention using selective hooks (RUSH) system revealed that the glycosylation-deficient mutant was arrested before endoplasmic reticulum (ER)- Golgi transport. However, the absence of ERGIC-53 and ERGIC-L, ER-Golgi transport cargo receptors that recognize sugar chains on cargo proteins, does not impair SVP secretion. In contrast, the solubility of the N154A mutant of E or the N15A/T17A mutant of prM in cells was markedly lower than that of the wild type, and proteasome-mediated rapid degradation of these mutants was observed, indicating the significance of glycosylation of both prM and E in proper protein folding and assembly of viral particles in the ER.

Abstract P1138: Transcriptomic Insights Into How Previously Increased Cardiac Protein O-GlcNAcylation Enhances Susceptibility To Cardiac Dysfunction In A Response To Pressure-Overload

Increased levels of cardiac protein posttranslational β-linked N-acetylglucosamine (O-GlcNAc) modification are implicated in adverse remodeling seen in hypertrophy and diabetes. Our previous work found elevated O-GlcNAc levels in hearts from diabetic mice, and a chronic increase in O-GlcNAc caused adverse cardiac remodeling. In diabetes, increased cardiac disease risk persists even after patients return to tight glycemic control. Thus, we hypothesize that a transient increase in O-GlcNAc is sufficient to exacerbate adverse cardiac remodeling under subsequent pressure-overload, and we propose that this is due to epigenetic modifications causing persistent changes in gene expression. We used an inducible, cardiomyocyte specific, dominant negative O-GlcNAcase (dnOGA) mouse. After a 2-wk induction (which leads to a 1.8-fold increase in O-GlcNAc levels) and subsequent 2-wk washout, Control (Con) and dnOGA mice undergo transverse-aortic constriction (TAC) or Sham surgery. After 8-wks, cardiac function is measured via echocardiography, gravimetric measures at harvest, and RNA-seq analysis is done on cardiac tissue. In both Con+TAC and dnOGA+TAC vs. Sham groups, systolic function is decreased ( -12%, p &lt; 0.001, -11%, p &lt; 0.01 ), and ventricular weight is increased ( +43%, +69%, both p &lt; 0.0001 ), consistent with cardiac hypertrophy. Interestingly, we see further exacerbation of cardiac hypertrophy ( +29%, p &lt; 0.0001 ) and pulmonary edema ( +36%, p &lt; 0.01 ) between dnOGA+TAC vs. Con+TAC groups. Also, RNA-seq results show distinct transcriptomic profiles between the experimental groups via hierarchical clustering ( |FC| &gt; 1.50, FDR &lt; 0.1 ). A candidate gene approach shows enhanced and exacerbated changes in genes associated with cardiac disease states like heart failure and fibrosis (e.g., Atp2a2, Col1a1, &amp; Nox4 ), consistent with cardiac remodeling. Our results support the hypothesis that transient increase in cardiac O-GlcNAc levels increases susceptibility to subsequent cardiac pathology. Moreover, the exacerbation of pathology is consistent with epigenetic mediated effects of transient O-GlcNAc induction; and pertaining to diabetes, provide a possible mechanism into the continued increased of cardiac disease risk.

O-linked N-acetylglucosamine modification is essential for physiological adipose expansion induced by high-fat feeding.

Adipose tissues accumulate excess energy as fat and heavily influence metabolic homeostasis. O-linked N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation), which involves the addition of N-acetylglucosamine to proteins by O-GlcNAc transferase (Ogt), modulates multiple cellular processes. However, little is known about the role of O-GlcNAcylation in adipose tissues during body weight gain due to overnutrition. Here, we report on O-GlcNAcylation in mice with high-fat diet (HFD)-induced obesity. Mice with knockout of Ogt in adipose tissue achieved using adiponectin promoter-driven Cre recombinase (Ogt-FKO) gained less body weight than control mice under HFD. Surprisingly, Ogt-FKO mice exhibited glucose intolerance and insulin resistance, despite their reduced body weight gain, as well as decreased expression of de novo lipogenesis genes and increased expression of inflammatory genes, resulting in fibrosis at 24 weeks of age. Primary cultured adipocytes derived from Ogt-FKO mice showed decreased lipid accumulation. Both primary cultured adipocytes and 3T3-L1 adipocytes treated with OGT inhibitor showed increased secretion of free fatty acids. Medium derived from these adipocytes stimulated inflammatory genes in RAW 264.7 macrophages, suggesting that cell-to-cell communication via free fatty acids might be a cause of adipose inflammation in Ogt-FKO mice. In conclusion, O-GlcNAcylation is important for healthy adipose expansion in mice. Glucose flux into adipose tissues may be a signal to store excess energy as fat.NEW & NOTEWORTHY We evaluated the role of O-GlcNAcylation in adipose tissue in diet-induced obesity using adipose tissue-specific Ogt knockout mice. We found that O-GlcNAcylation in adipose tissue is essential for healthy fat expansion and that Ogt-FKO mice exhibit severe fibrosis upon long-term overnutrition. O-GlcNAcylation in adipose tissue may regulate de novo lipogenesis and free fatty acid efflux to the degree of overnutrition. We believe that these results provide new insights into adipose tissue physiology and obesity research.

D-Glucosamine induces circadian phase delay by promoting BMAL1 degradation through AMPK/mTOR pathway

Circadian rhythms are closely linked to the metabolic network through circadian feedback regulation. The hexosamine biosynthetic pathway (HBP) is a branch of glucose metabolism that affects circadian rhythms through the O-linked N-acetylglucosamine modification (O-GlcNAcylation) of clock proteins. Here, we found out that, among the downstream metabolites regulated by d-glucosamine (GlcN) in HBP salvage pathway, only GlcN is able to induce circadian phase delay both in vitro and in vivo. Mechanistic studies indicated that the phase-shift induced by GlcN is independent of O-GlcNAcylation. Instead, GlcN selectively up-regulates p-AMPK activity, leading to the inhibition of mTOR signaling pathway, and thus down-regulation of p-BMAL1 both in human cell line and mouse tissues. Moreover, GlcN promoted BMAL1 degradation via proteasome pathway. These findings reveal a novel molecular mechanism of GlcN in regulating clock phase and suggest the therapeutic potential of GlcN as new use for an old drug in the future treatment of shift work and circadian misalignment.

On the Role of Glycolysis in Early Tumorigenesis—Permissive and Executioner Effects

Reprogramming energy production from mitochondrial respiration to glycolysis is now considered a hallmark of cancer. When tumors grow beyond a certain size they give rise to changes in their microenvironment (e.g., hypoxia, mechanical stress) that are conducive to the upregulation of glycolysis. Over the years, however, it has become clear that glycolysis can also associate with the earliest steps of tumorigenesis. Thus, many of the oncoproteins most commonly involved in tumor initiation and progression upregulate glycolysis. Moreover, in recent years, considerable evidence has been reported suggesting that upregulated glycolysis itself, through its enzymes and/or metabolites, may play a causative role in tumorigenesis, either by acting itself as an oncogenic stimulus or by facilitating the appearance of oncogenic mutations. In fact, several changes induced by upregulated glycolysis have been shown to be involved in tumor initiation and early tumorigenesis: glycolysis-induced chromatin remodeling, inhibition of premature senescence and induction of proliferation, effects on DNA repair, O-linked N-acetylglucosamine modification of target proteins, antiapoptotic effects, induction of epithelial-mesenchymal transition or autophagy, and induction of angiogenesis. In this article we summarize the evidence that upregulated glycolysis is involved in tumor initiation and, in the following, we propose a mechanistic model aimed at explaining how upregulated glycolysis may play such a role.

O-linked N-acetylglucosamine modification induced by lipopolysaccharide is involved in inflammatory signaling pathway in endothelial cells

To explore whether the lipopolysaccharide (LPS)-induced modification of O-linked N-acetylglucosamine (O-GlcNAc) is involved in the inflammatory signaling pathway of endothelial cells. Human umbilical vein endothelial cells (HUVEC) were cultured in vitro, and cells in logarithmic growth phase were used for experiments. Cells were divided into blank control group, LPS group (2 000 mg/L LPS), O-GlcNAc transferase (OGT) overexpression (OGT-OE)+LPS group (plasmid transfection OGT+2 000 mg/L LPS), protein kinase C (PKC) inhibitor+LPS group (10 μmol/L Go 6983+2 000 mg/L LPS), RhoA inhibitor+LPS group (40 μmol/L Rhoin hydrochloride+2 000 mg/L LPS), phosphatidylinositol-3-kinase (PI3K) inhibitor+LPS group (1 μmol/L SL-2052+2 000 mg/L LPS), serine/threonine kinase (Akt) inhibitor+LPS group (10 μmol/L PP2+2 000 mg/L LPS) and small interfering RNA (siRNA) treated Akt (si-AKT)+LPS group (si-Akt+2 000 mg/L LPS). After 24 hours of LPS treatment, real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was used to detect the transcription levels of inflammatory cytokines [interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1)]. The protein expression or phosphorylation of OGT, O-GlcNAc, Akt, extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38MAPK), nuclear factor-κB p65 (NF-κB p65), and signal transducer and activator of transcription 3 (STAT3) were determined by Western blotting. Compared with the blank control group, the expression of OGT and the modification of O-GlcNAc in the LPS group were decreased, while the expressions of phosphorylated ERK, p38MAPK, and STAT3 were increased, and the transcript levels of inflammatory cytokines were also significantly increased [IL-6 mRNA (2-ΔΔCt): 4.71±0.60 vs. 1.03±0.29, TNF-α mRNA (2-ΔΔCt): 1.89±0.11 vs. 1.04±0.35, ICAM-1 mRNA (2-ΔΔCt): 2.06±0.18 vs. 1.02±0.21, VCAM-1 mRNA (2-ΔΔCt): 2.94±0.57 vs. 1.01±0.17, all P < 0.05], indicating that LPS could decrease O-GlcNAc modification, activate inflammatory signaling pathways and increase inflammatory cytokines expression. Compared with the LPS group, the expressions of phosphorylated ERK, p38MAPK, NF-κB p65, and STAT3 in the endothelial cells of the OGT-OE+LPS group were decreased, and the expression of inflammatory factors were significantly decreased [IL-6 mRNA (2-ΔΔCt): 0.12±0.01 vs. 0.90±0.17, TNF-α mRNA (2-ΔΔCt): 0.31±0.01 vs. 0.91±0.14, ICAM-1 mRNA (2-ΔΔCt): 0.64±0.02 vs. 1.13±0.16, VCAM-1 mRNA (2-ΔΔCt): 0.11±0.01 vs. 0.93±0.11, all P < 0.05], indicating that the increase of OGT level could inhibit the partial activation of the endothelial inflammatory signal pathway under the LPS stimulation. Compared with the blank control group, the phosphorylation level of Akt in the LPS group was increased. Compared with the LPS group, both OGT expression and O-GlcNAc modification were down-regulated after pretreatment of PKC inhibitor, RhoA inhibitor, PI3K inhibitor, or Akt inhibitor. Compared with the LPS group, the transcript levels of IL-6, TNF-α and ICAM-1 in the PP2+LPS group were significantly decreased [IL-6 mRNA (2-ΔΔCt): 1.46±0.16 vs. 3.55±0.87, TNF-α mRNA (2-ΔΔCt): 0.98±0.14 vs. 1.76±0.10, ICAM-1 mRNA (2-ΔΔCt): 1.39±0.24 vs. 2.04±0.13, all P < 0.05], but there was no significant change in VCAM-1. Compared with the LPS group, the expression of OGT and O-GlcNAc modification in the si-Akt+LPS group were decreased, while the transcript levels of inflammatory cytokines were also significantly decreased [IL-6 mRNA (2-ΔΔCt): 0.75±0.03 vs. 0.99±0.09, TNF-α mRNA (2-ΔΔCt): 0.69±0.01 vs. 1.10±0.08, ICAM-1 mRNA (2-ΔΔCt): 0.76±0.01 vs. 0.99±0.02, VCAM-1 mRNA (2-ΔΔCt): 0.93±0.08 vs. 1.20±0.21, all P < 0.05], indicating that Akt participated in the action process of LPS on OGT and affected the inflammatory factor expression. The decreased level of O-GlcNAc modification in endothelial cells stimulated with LPS promotes partial activation of inflammatory signaling pathways, mainly involving ERK, p38MAPK, and STAT3, and affects the expression of inflammatory factors. AKT may be involved in the effect of LPS on the inhibition of O-GlcNAc modification.

Glutamine promotes O-GlcNAcylation of G6PD and inhibits AGR2 S-glutathionylation to maintain the intestinal mucus barrier in burned septic mice

Mucus forms the first line of defence of the intestinal mucosa barrier, and mucin is its core component. Glutamine is a vital energy substance for goblet cells; it can promote mucus synthesis and alleviate damage to the intestinal mucus barrier after burn injury, but its mechanism is not fully understood. This study focused on the molecular mechanisms underlying the effects of glutamine on the synthesis and modification of mucin 2 (MUC2) by using animal and cellular models of burn sepsis. We found that anterior gradient-2 (AGR2) plays a key role in the posttranslational modification of MUC2. Oxidative stress induced by burn sepsis enhanced the S-glutathionylation of AGR2, interfered with the processing and modification of MUC2 precursors by AGR2 and blocked the synthesis of mature MUC2. Further studies revealed that NADPH, catalysed by glucose-6-phosphate dehydrogenase (G6PD), is a key molecule in inhibiting oxidative stress and regulating AGR2 activity. Glutamine promotes O-linked N-acetylglucosamine (O-GlcNAc) modification of G6PD via the hexosamine pathway, which facilitates G6PD homodimer formation and increases NADPH synthesis, thereby inhibiting AGR2 S-glutathionylation and promoting MUC2 maturation, ultimately reducing damage to the intestinal mucus barrier after burn sepsis. Overall, we have demonstrated that the central mechanisms of glutamine in promoting MUC2 maturation and maintaining the intestinal mucus barrier are the enhancement of G6PD glycosylation and inhibition of AGR2 S-glutathionylation.

Hypoxia-triggered O-GlcNAcylation in the brain drives the glutamate–glutamine cycle and reduces sensitivity to sevoflurane in mice

BackgroundHypersensitivity to general anaesthetics predicts adverse postoperative outcomes in patients. Hypoxia exerts extensive pathophysiological effects on the brain; however, whether hypoxia influences sevoflurane sensitivity and its underlying mechanisms remain poorly understood. MethodsMice were acclimated to hypoxia (oxygen 10% for 8 h day−1) for 28 days and anaesthetised with sevoflurane; the effective concentrations for 50% of the animals (EC50) showing loss of righting reflex (LORR) and loss of tail-pinch withdrawal response (LTWR) were determined. Positron emission tomography–computed tomography, O-glycoproteomics, seahorse analysis, carbon-13 tracing, site-specific mutagenesis, and electrophysiological techniques were performed to explore the underlying mechanisms. ResultsCompared with the control group, the hypoxia-acclimated mice required higher concentrations of sevoflurane to present LORR and LTWR (EC50LORR: 1.61 [0.03]% vs 1.46 [0.04]%, P<0.01; EC50LTWR: 2.46 [0.14]% vs 2.22 [0.06]%, P<0.01). Hypoxia-induced reduction in sevoflurane sensitivity was correlated with elevation of protein O-linked N-acetylglucosamine (O-GlcNAc) modification in brain, especially in the thalamus, and could be abolished by 6-diazo-5-oxo-l-norleucine, a glutamine fructose-6-phosphate amidotransferase inhibitor, and mimicked by thiamet-G, a selective O-GlcNAcase inhibitor. Mechanistically, O-GlcNAcylation drives de novo synthesis of glutamine from glucose in astrocytes and promotes the glutamate–glutamine cycle, partially via glycolytic flux and activation of glutamine synthetase. ConclusionsIntermittent hypoxia exposure decreased mouse sensitivity to sevoflurane anaesthesia through enhanced O-GlcNAc-dependent modulation of the glutamate–glutamine cycle in the brain.

The beneficial effect of global O-GlcNAcylation on odontogenic differentiation of human dental pulp cells via mTORC1 pathway

ObjectiveTo investigate whether and how global O-linked N-Acetylglucosamine modification (O-GlcNAcylation), a prevalent nutrient-sensitive post-translation modification, regulates odontogenic differentiation and mineralization in human dental pulp cells (hDPCs). DesignFirst, immunostaining assays on sections of dental pulp tissue were performed to detect the distributions of O-GlcNAcylation and its exclusive enzyme set O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Then global O-GlcNAcylation was determined by anti O-linked N-Acetylglucosamine (RL2) Western blot during odontogenesis of hDPCs. Further, inhibition or knockdown of OGT and OGA were achieved by specific inhibitors or siRNA in vitro, respectively. The odonto-induction effect of O-GlcNAcylation ex vivo was investigated by a subcutaneous transplantation experiment. Moreover, the O-GlcNAc modification of RAPTOR was confirmed by immunoprecipitation. Odontogenic differentiation assays also investigated the indispensable role of RAPTOR during enhanced global O-GlcNAcylation. ResultsThe signals of O-GlcNAc became more enriched in the odontoblasts compared to pulp fibroblasts. During odontogenesis of hDPCs, global O-GlcNAcylation was significantly increased. An increase or decrease of O-GlcNAcylation significantly boosted or blunted odontogenic differentiation, respectively. The fluctuation of O-GlcNAcylation continuously impacted the downstream targets of mTORC1. Consistently, RAPTOR was modified by O-GlcNAcylation, which was necessary for inducing odontogenesis. ConclusionsGlobal O-GlcNAcylation participated in and affected the odontogenic differentiation of hDPCs, which was mediated by the mTORC1 pathway. Thus, targeting O-GlcNAcylation might be a potential therapeutic intervention for pulp repair and regeneration.