O-GlcNAcylation Levels Research Articles

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.

Sevoflurane postconditioning mitigates neuronal hypoxic-ischemic injury via regulating reactive astrocytic STAT3 protein modification

Astrocyte activation plays a pivotal role in accelerating the cascade of neuroinflammation associated with the development of hypoxic-ischemic brain injury. This study aimed to investigate the mechanism by which sevoflurane postconditioning mitigates neuronal damage through astrocytes by regulating reactive astrocytic Signal Transducer and Activator of Transcription 3 (STAT3) modifications. A modified Rice‒Vannucci model in rats and a conditioned culture system established by subjecting primary astrocytes to oxygen glucose deprivation, followed by using the conditioned medium to culture the neuron cell line SH-SY5Y were used to simulate HI insult in vivo and in vitro, respectively. These models were followed by 30 min of 2.5 % sevoflurane treatment. Stattic was used to inhibit STAT3 phosphorylation, and (Z)-PUGNAc or OSMI-1 was added to regulate O-linked-β-N-acetylglucosamine modification (O-GlcNAcylation) in primary astrocytes in vitro. Neurobehavioral tests, Nissl staining, CCK8 assay, and flow cytometry for apoptosis were used to assess neuronal function. Immunofluorescence staining was used to detect astrocyte reactivity and the intracellular distribution of STAT3. Immunoprecipitation combined with Western blotting was used to evaluate the O-GlcNAcylation of STAT3. Protein expression and phosphorylation levels were detected by Western blotting. ELISA was conducted to detect the detrimental cytokines IL-6 and IL-1β in astrocyte-conditioned medium. Sevoflurane postconditioning enhanced the O-GlcNAcylation of astrocytic STAT3 following HI insult via the manner of OGT. Crosstalk between O-GlcNAcylation and phosphorylation of STAT3 showed that O-GlcNAcylation inhibited STAT3 phosphorylation. The inhibitory effect on astrocytes suppressed STAT3 nuclear translocation, reduced astrocyte reactivity, decreased the release of the inflammatory cytokines IL6 and IL-1β, attenuated neuronal apoptosis following HI insult, and improved neuron viability. Sevoflurane postconditioning increased astrocytic STAT3 O-GlcNAcylation level to competitively inhibit STAT3 phosphorylation. This deactivated downstream inflammation pathways and reduced astrocyte reactivity, thereby mitigating HI insult in neurons both in vivo and in vitro.

O-GlcNAcylation inhibition redirects the response of colon cancer cells to chemotherapy from senescence to apoptosis.

The potential use of pro-senescence therapies, known as TIS (Therapy-Induced Senescence), for the treatment of colorectal cancer (CRC) generated significant interest since they require lower doses compared to those required for inducing apoptosis. However, the senescent cell cycle-arrested cancer cells are long-lived, and studies have revealed escape mechanisms contributing to tumor recurrence. To deepen our understanding of the survival pathways used by senescent cancer cells, we delved into the potential involvement of the hexosamine biosynthetic pathway (HBP). HBP provides UDP-GlcNAc, the substrate for O-GlcNAc transferase (OGT), which catalyzes O-GlcNAcylation, a post-translational modification implicated in regulating numerous cellular functions and aberrantly elevated in CRC. In this study, we demonstrated, in the p53-proficient colon cancer cell lines HCT116 and LS174T, that TIS induced by low-dose SN38 or etoposide treatment was accompanied with a decrease of GFAT (the rate limiting enzyme of the HBP), OGT and O-GlcNAcase (OGA) expression correlated with a slight reduction in O-GlcNAcylation levels. Further decreasing this level of O-GlcNAcylation by knocking-down GFAT or OGT redirected the cellular response to subtoxic chemotherapy doses from senescence to apoptosis, in correlation with an enhancement of DNA damages. Pharmacological inhibition of OGT with OSMI-4 in HCT116 and LS174T cells and in a patient-derived colon tumoroid model supported these findings. Taken together, these results suggest that combing O-GlcNAcylation inhibitors to low doses of conventional chemotherapeutic drugs could potentially reduce treatment side effects while preserving efficacy. Furthermore, this approach may increase treatment specificity, as CRC cells exhibit higher O-GlcNAcylation levels compared to normal tissues.

Inhibition of O-GlcNAc transferase activates type I interferon-dependent antitumor immunity by bridging cGAS-STING pathway.

The O-GlcNAc transferase (OGT) is an essential enzyme that mediates protein O-GlcNAcylation, a unique form of posttranslational modification of many nuclear and cytosolic proteins. Recent studies observed increased OGT and O-GlcNAcylation levels in a broad range of human cancer tissues compared to adjacent normal tissues, indicating a universal effect of OGT in promoting tumorigenesis. Here, we show that OGT is essential for tumor growth in immunocompetent mice by repressing the cyclic GMP-AMP synthase (cGAS)-dependent DNA sensing pathway. We found that deletion of OGT (Ogt-/-) caused a marked reduction in tumor growth in both syngeneic mice tumor models and a genetic mice colorectal cancer (CRC) model induced by mutation of the Apc gene (Apcmin). Pharmacological inhibition or genetic deletion of OGT induced a robust genomic instability (GIN), leading to cGAS-dependent production of the type I interferon (IFN-I) and IFN-stimulated genes (ISGs). As a result, deletion of Cgas or Sting from Ogt-/- cancer cells restored tumor growth, and this correlated with impaired CD8+ T-cell-mediated antitumor immunity. Mechanistically, we found that OGT-dependent cleavage of host cell factor C1 (HCF-1) is required for the avoidance of GIN and IFN-I production in tumors. In summary, our results identify OGT-mediated genomic stability and activate cGAS-STING pathway as an important tumor-cell-intrinsic mechanism to repress antitumor immunity.

High glucose promotes lipopolysaccharide-induced macrophage pyroptosis through GSDME O-GlcNAcylation.

The high glucose (HG) environment in diabetic periodontitis aggravates the damage of periodontal tissue. Pyroptosis has been shown to be positively correlated with the severity of periodontitis, including macrophage pyroptosis. O-GlcNAcylation is a posttranslational modification that is involved in the pathogenesis of periodontitis. However, whether HG regulates macrophage pyroptosis through O-GlcNAcylation remains uncertain. This study aimed to investigate the effect of HG on the O-GlcNAcylation level of a pyroptosis regulator GSDME in macrophages to further probe the mechanisms of diabetic periodontitis. Blood samples were collected from patients with diabetic periodontitis. THP-1 monocytes were induced to differentiate into macrophages by phorbol 12-myristate 13-acetate and then treated with HG to simulate periodontitis invitro. GSDME expression of blood samples and macrophages was measured by quantitative real-time PCR. Pyroptosis was assessed by propidium iodide staining, measurement of cell viability, cytotoxicity, protein levels of inflammation factors, and pyroptosis-related proteins. O-GlcNAcylation of GSDME was analyzed using co-immunoprecipitation (co-IP), IP, and western blot. The results showed that GSDME expression was elevated in patients with periodontitis and HG-treated macrophages. HG inhibited cell viability but increased LDH content, levels of IL-1β, IL-18, TNF-α, NLRP3, GSDMD, and Caspase-1, indicating that HG promoted pyroptosis of macrophages, which was reversed by GSDME knockdown. HG treatment increased O-GlcNAcylation in macrophages. Mechanically, GSDME interacted with OGT, and OGT knockdown suppressed O-GlcNAcylation of GSDME at Ser (S)339 site. Knockdown of OGT inhibited pyroptosis in HG-treated macrophages, while GSDME overexpression partially reversed this inhibition. HG treatment enhanced OGT-mediated GSDME O-GlcNAcylation, thereby augmenting pyroptosis in LPS-induced macrophages. These results may provide a novel sight for the treatment of periodontitis.

Low-dose deoxynivalenol exposure triggers hepatic excessive ferritinophagy and mitophagy mitigated by hesperidin modulated O-GlcNAcylation

The level and breadth of deoxynivalenol (DON) contamination in foods made with cereals have increased due to global warming. Consumption of DON-contaminated food and feed poses significant risks to human health and animal production. However, the mechanism by which prolonged exposure to low-dose DON leads to liver damage in animals and effective treatments remain unclear. Our investigation focused on the impact of varying DON exposure times on AML12 cells as well as the long-term liver damage caused by low-dose DON exposure in mice. In addition, this article investigated the unique role of hesperidin in mitigating hepatic ferroptosis induced by low-dose DON exposure. Our results imply that DON's suppression of O-GlcNAcylation exacerbated mitophagy by encouraging ferritinophagy and causing labile iron to aggregate within mitochondria. Furthermore, DON could increase NCOA4-mediated ferritinophagy by De-O-GlcNAcylation FTH to trigger ferroptosis-associated liver injury in mice. Notably, hesperidin alleviated the susceptibility to ferroptosis by increasing O-GlcNAcylation levels and effectively attenuated the liver injury induced by low-dose DON exposure. This finding provides a new strategy for dealing with liver injury caused by low-dose DON exposure.

Senegenin suppresses hepatocellular carcinoma by regulating O-GlcNAcylation.

Based on current knowledge, hepatocellular carcinoma (HCC) is a condition with numerous etiologies and risk factors. However, the pathogenesis of HCC remains unclear. To investigate the roles of senegenin and O-GlcNAcylation in the growth and metastasis of HCC. The levels of O-linked N-acetylglucosamine transferase (OGT) and O-GlcNAcylation in HCC cells and tissues were detected using western blot analysis. The effects of senegenin and O-GlcNAcylation on the proliferation of HCC cells were investigated in vitro using cell counting kit-8 and clonogenic assays. The potential effects of senegenin and O-GlcNAcylation on HCC metastasis were examined using the transwell migration assay. O-GlcNAcylation levels were altered via drug treatment and lentiviral infection, and western blot analysis was used to detect proteins involved in various pathways. Western blot analysis revealed that OGT and O-GlcNAcylation levels were significantly elevated in HCC tissues and cells. O-GlcNAcylation levels in HCC cells were significantly altered by drug treatment and lentiviral infection. An increase in the glycosylation level was linked to enhanced proliferation, invasiveness, clonogenicity, and metastatic potential of cancer cells. O-GlcNAcylation induced by senegenin was found to slow the proliferation and migration of HCC cells. The levels of proteins involved in nuclear factor-kappa B (NF-κB) and c-Jun N-terminal kinase (JNK) pathways, which are associated with endoplasmic reticulum stress, were altered. Senegenin lowers O-GlcNAcylation levels, decreases OGT expression, and inhibits cancer cell growth and metastasis by regulating proteins involved in NF-κB and JNK pathways.

The study on the role of O-GlcNAcylation of SIRT3 in regulating mitochondrial oxidative stress during simulate myocardial ischemia-reperfusion.

Myocardial ischemia-reperfusion injury (MIRI) is a significant complication following reperfusion therapy after myocardial infarction. Mitochondrial oxidative stress is a critical factor in MIRI, and Sirtuin 3 (SIRT3), as a major mitochondrial deacetylase, plays a key protective role, with its activity potentially regulated by O-GlcNAcylation. This study used the H9C2 cell line to establish a simulated ischemia/reperfusion (SI/R) model, we utilized co-immunoprecipitated to validate the relationship between O-GlcNAc transferase (OGT) and SIRT3, demonstrated SIRT3 O-GlcNAcylation sites through LC-MS/MS, and performed site mutations using CRISPR/Cas9 technology. The results were validated using immunoblotting. SIRT3 and superoxide dismutase 2 (SOD2) activities were detected using a fluorometric assay, while mitochondrial reactive oxygen species (MROS) levels and cellular apoptosis were assessed using immunofluorescence. We have identified an interaction between SIRT3 and OGT, where SIRT3 undergoes dynamic O-GlcNAcylation at the S190 site, facilitating SIRT3 deacetylase activity. During SI/R, elevated levels of O-GlcNAcylation activate SOD2 by promoting SIRT3 enzyme activity, thereby inhibiting excessive MROS production. This significantly mitigates the occurrence of malignant autophagy in myocardial cells during reperfusion, promoting their survival. Conversely, blocking SIRT3 O-GlcNAcylation at the S190 site exacerbates SI/R injury. We demonstrate that O-GlcNAcylation is a crucial post-translational modification (PTM) of SIRT3 during SI/R, shedding light on a promising mechanism for future therapeutic approaches.

Structure and Inhibition of Insect UDP-N-acetylglucosamine Pyrophosphorylase: A Key Enzyme in the Hexosamine Biosynthesis Pathway.

UDP-N-acetylglucosamine pyrophosphorylase (UAP) catalyzes the last step in the hexosamine biosynthesis pathway to directly produce UDP-N-acetylglucosamine (UDP-GlcNAc). Because UAPs play important physiological and pathological roles in organisms, they are considered potential targets for drug and pesticide development. However, the lack of efficient and selective inhibitors is a bottleneck that must be overcome. This study reports the first crystal structure of the insect UAP from Spodoptera frugiperda (SfUAP) in complex with UDP-GlcNAc. SfUAP has two insect-specific structural characteristics in the active pocket, namely, a free Cys (Cys334) and a Mg2+ binding site, which differentiate it from human UAP (HsAGX1) and fungal UAP (AfUAP) in terms of substrate and inhibitor binding. N-(4-Nitrophenyl)maleimide (pNPMI) and myricetin are discovered as potent covalent and noncovalent inhibitors of SfUAP, respectively. Moreover, myricetin can significantly reduce the level of cellular O-GlcNAcylation by inhibiting both UAP and O-GlcNAc transferase. These findings provide novel insights into the development of UAP-based drugs and pesticides.

FOXM1 Upregulates O-GlcNAcylation Level Via The Hexosamine Biosynthesis Pathway to Promote Angiogenesis in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) presents significant challenges in treatment and prognosis because of its aggressive nature and high metastatic potential. This study aims to investigate the role of the hexosamine biosynthesis pathway (HBP) and its association with HCC progression and prognosis. We identified SPP1 and FOXM1 as hub genes within the HBP pathway, showing their correlation with poor prognosis and late-stage progression. In addition, the analysis uncovered the complex participation of the HBP pathway in nutrients and oxygen reactions, PI3K-AKT signaling, AMPK activation, and angiogenesis regulation. The disruption of these pathways is pivotal in influencing the growth and progression of HCC. Targeting the HBP presents a promising therapeutic approach to modulate the tumor microenvironment, thereby enhancing the efficacy of immunotherapy. In addition, FOXM1 was identified as the HBP pathway regulator, influencing cellular O-GlcNAcylation level and VEGF secretion, thereby promoting angiogenesis in HCC. Inhibition of O-GlcNAcylation significantly hindered angiogenesis, which is suggested as a potential avenue for therapeutic intervention. Our research demonstrates the practicality of using the HBP-related gene as a prognostic marker in liver cancer patients and suggests targeting FOXM1 as a novel avenue for personalized therapy.

SMAD4 promotes EMT in COPD airway remodeling induced by cigarette smoke through interaction with O-GlcNAc transferase

Cigarette smoke (CS) is a prevalent chemical indoor air contaminant known to be the primary cause of EMT during airway remodeling in COPD. While some evidence indicates the involvement of SMAD4 in EMT across certain diseases, its specific role in CS-induced EMT in airway remodeling associated with COPD is not established. In our research, we observed a substantial upregulation in SMAD4 expression, O-GlcNAcylation and EMT in patients with COPD, as well as in vitro and in vivo COPD models induced by CS, than those of the controls. Downregulation of SMAD4 resulted in a reduction in CS-induced EMT in vitro and in vivo. As a post-translational modification of proteins, O-GlcNAcylation is dynamically controlled by the duo of enzymes: O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) and O-GlcNAcase (OGA). We further discovered the enhancement of O-GlcNAcylation levels induced by CS was due to an elevated OGT expression, as the expression of OGA remained unchanged. Using an OGT inhibitor (OSMI-1) counteracted the effects of SMAD4 on EMT. Whereas, overexpressing OGT increased SMAD4 expression and promoted EMT. OGT-mediated SMAD4 O-GlcNAcylation shielded SMAD4 from proteasomal degradation by reducing its ubiquitination, thereby aiding in SMAD4 stabilization in response to EMT induced by CS. Overall, this research uncovers a fresh pathway for CS-induced EMT in the airway remodeling of COPD and offers valuable insights.

Neuro-protective effects of increased O-GlcNAcylation by glucosamine in an optic tectum traumatic brain injury model of adult zebrafish.

This study investigated the behavioral and molecular changes in the telencephalon following needle stab-induced injury in the optic tectum of adult zebrafish. At 3 days post-injury (dpi), there was noticeable structural damage to brain tissue and reduced neuronal proliferation in the telencephalon that persisted until 30 dpi. Neurobehavioral deficits observed at 3 dpi included decreased exploratory and social activities and impaired learning and memory (L/M) functions; all of these resolved by 7 dpi. The injury led to a reduction in telencephalic phosphorylated cAMP response element-binding protein and O-GlcNAcylation, both of which were restored by 30 dpi. There was an increase in GFAP expression and nuclear translocation of NF-κB p65 at 3 dpi, which were not restored by 30 dpi. The injury caused decreased O-GlcNAc transferase and increased O-GlcNAcase levels at 3 dpi, normalizing by 30 dpi. Glucosamine (GlcN) treatment at 3 dpi significantly restored O-GlcNAcylation levels and L/M function, also reducing GFAP activation. Glucose treatment recovered L/M function by 7 dpi, but inhibition of the hexosamine biosynthetic pathway by 6-diazo-5-oxo-L-norleucine blocked this recovery. These findings suggest that the O-GlcNAc pathway is a potential therapeutic target for addressing L/M impairment following traumatic brain injury in zebrafish.

O-GlcNAcylation of SERCA protects skeletal muscle in hibernating Spermophilus dauricus from disuse atrophy

Long-term inactivity of skeletal muscle results in muscular disuse atrophy; however, hibernating animals do not experience muscular disuse atrophy during the hibernation period. The molecular mechanism underlining the anti-atrophy effect in these animals is unclear. O-linked N acetyl-β-D-glucosaminylation (O-GlcNAcylation) and its effect on cell signaling pathways are important mechanisms underlying muscular disuse atrophy; thus, in this study, we investigated O-GlcNAcylation changes during hibernation in Spermophilus dauricus to explore the role of O-GlcNAcylation in the muscle disuse atrophy resistance of hibernating animals. The results showed that during hibernation, the muscle fiber cross-sectional area and ratio of muscle fiber did not change, and the morphological structure of the muscle remained intact, with normal contractile function. The level of O-GlcNAcylation decreased during hibernation, but quickly returned to normal in the periodic arousal stage. The O-GlcNAcylation level of sarcoplasmic/endoplasmic reticulum calcium ATPase 1 (SERCA1) decreased, whereas its activity increased. The decrease in O-GlcNAcylation of SERCA could result in the decreased binding of phospholamban to SERCA1, thus decreasing its inhibition to SERCA1 activity. This in turn can inhibit muscle cell calcium overload, maintain muscle cell calcium homeostasis, and stabilize the calpain proteolytic pathway, ultimately inhibiting skeletal muscle atrophy. Our results demonstrate that periodic arousal along with returning O-GlcNAcylation level to normal are important mechanisms in preventing disuse atrophy of skeletal muscle during hibernation.

Discovery of 4-(Arylethynyl)piperidine Derivatives as Potent Nonsaccharide O-GlcNAcase Inhibitors for the Treatment of Alzheimer's Disease.

Inhibiting O-GlcNAcase and thereby up-regulation of the O-GlcNAc levels of tau was a potential approach for discovering AD treatments. Herein, a series of novel highly potent OGA inhibitors embracing a 4-(arylethynyl)piperidine moiety was achieved by capitalizing on the substrate recognition domain. Extensive structure-activity relationships resulted in compound 81 with significant enzymatic inhibition (IC50 = 4.93 ± 2.05 nM) and cellular potency (EC50 = 7.47 ± 3.96 nM in PC12 cells). It markedly increased the protein O-GlcNAcylation levels and reduced the phosphorylation on Ser199, Thr205, and Ser396 of tau in the OA-injured SH-SY5Y cell model, suggesting its potential role for AD treatment. In fact, an in vivo efficacy of ameliorating cognitive impairment was observed following treatment of APP/PS1 mice with compound 81 (100 mg/kg). Additionally, the appropriate plasma PK and beneficial BBB penetration properties were also observed. Compound 81 deserves to be further explored as an anti-AD agent.

Inhibition of O-GlcNAc transferase activates type I interferon-dependent antitumor immunity by bridging cGAS-STING pathway.

The O-GlcNAc transferase (OGT) is an essential enzyme that mediates protein O-GlcNAcylation, a unique form of posttranslational modification of many nuclear and cytosolic proteins. Recent studies observed increased OGT and O-GlcNAcylation levels in a broad range of human cancer tissues compared to adjacent normal tissues, indicating a universal effect of OGT in promoting tumorigenesis. Here, we show that OGT is essential for tumor growth in immunocompetent hosts by repressing the cyclic GMP-AMP synthase (cGAS)-dependent DNA sensing pathway. We found that deletion of OGT (Ogt -/- ) caused a marked reduction in tumor growth in both syngeneic tumor models and a genetic colorectal cancer (CRC) model induced by mutation of the Apc gene (Apc min ). Pharmacological inhibition or genetic deletion of OGT induced a robust genomic instability (GIN), leading to cGAS-dependent production of the type I interferon (IFN-I) and IFN-stimulated genes (ISGs). As a result, deletion of Cgas or Sting from Ogt -/- cancer cells restored tumor growth, and this correlated with impaired CD8+ T cell-mediated antitumor immunity. Mechanistically, we found that OGT-dependent cleavage of host cell factor C1 (HCF-1) is required for the avoidance of GIN and IFN-I production in tumors. In summary, our results identify OGT-mediated genomic stability and activate cGAS-STING pathway as an important tumor cell-intrinsic mechanism to repress antitumor immunity.

Dysfunction of astrocytic glycophagy exacerbates reperfusion injury in ischemic stroke

Glycophagy has evolved from an alternative glycogen degradation pathway into a multifaceted pivot to regulate cellular metabolic hemostasis in peripheral tissues. However, the pattern of glycophagy in the brain and its potential therapeutic impact on ischemic stroke remain unknown. Here, we observed that the dysfunction of astrocytic glycophagy was caused by the downregulation of the GABA type A receptor-associated protein like 1 (GABARAPL1) during reperfusion in ischemic stroke patients and mice. PI3K-Akt pathway activation is involved in driving GABARAPL1 downregulation during cerebral reperfusion. Moreover, glycophagy dysfunction-induced glucosamine deficiency suppresses the nuclear translocation of specificity protein 1 and TATA binding protein, the transcription factors for GABARAPL1, by decreasing their O-GlcNAcylation levels, and accordingly feedback inhibits GABARAPL1 in astrocytes during reperfusion. Restoring astrocytic glycophagy by overexpressing GABARAPL1 decreases DNA damage and oxidative injury in astrocytes and improves the survival of surrounding neurons during reperfusion. In addition, a hypocaloric diet in the acute phase after cerebral reperfusion can enhance astrocytic glycophagic flux and accelerate neurological recovery. In summary, glycophagy in the brain links autophagy, metabolism, and epigenetics together, and glycophagy dysfunction exacerbates reperfusion injury after ischemic stroke.

O-GlcNAcylation levels remain stable regardless of the anesthesia procedure in healthy rats

O-GlcNAcylation levels remain stable regardless of the anesthesia procedure in healthy rats

Mild therapeutic hypothermia upregulates the O-GlcNAcylation level of COX10 to alleviate mitochondrial damage induced by myocardial ischemia-reperfusion injury.

Mild therapeutic hypothermia (MTH) is an important method for perioperative prevention and treatment of myocardial ischemia-reperfusion injury (MIRI). Modifying mitochondrial proteins after protein translation to regulate mitochondrial function is one of the mechanisms for improving myocardial ischemia-reperfusion injury. This study investigated the relationship between shallow hypothermia treatment improving myocardial ischemia-reperfusion injury and the O-GlcNAcylation level of COX10. We used in vivo Langendorff model and in vitro hypoxia/reoxygenation (H/R) cell model to investigate the effects of MTH on myocardial ischemia-reperfusion injury. Histological changes, myocardial enzymes, oxidative stress, and mitochondrial structure/function were assessed.Mechanistic studies involved various molecular biology methods such as ELISA, immunoprecipitation (IP), WB, and immunofluorescence. Our research results indicate that MTH upregulates the O-GlcNACylation level of COX10, improves mitochondrial function, and inhibits the expression of ROS to improve myocardial ischemia-reperfusion injury. In vivo, MTH effectively alleviates ischemia-reperfusion induced cardiac dysfunction, myocardial injury, mitochondrial damage, and redox imbalance. In vitro, the OGT inhibitor ALX inhibits the OGT mediated O-GlcNA acylation signaling pathway, downregulates the O-Glc acylation level of COX10, promotes ROS release, and counteracts the protective effect of MTH. On the contrary, the OGA inhibitor ThG showed opposite effects to ALX, further confirming that MTH activated the OGT mediated O-GlcNAcylation signaling pathway to exert cardioprotective effects. In summary, MTH activates OGT mediated O-glycosylation modified COX10 to regulate mitochondrial function and improve myocardial ischemia-reperfusion injury, which provides important theoretical basis for the clinical application of MTH.

O-GlcNAcylation levels remain stable regardless of the anaesthesia in healthy rats

Anaesthetics are used daily in human and veterinary medicine as well as in scientific research. Anaesthetics have an impact on cell homeostasis especially through modulation of protein post-translational modifications. O-GlcNAcylation, a ubiquitous post-translational modification, plays a role in many biological processes. The aims of this study were to evaluate whether (1) anaesthesia influences O-GlcNAcylation and (2) its stimulation affects physiological parameters. Male Wistar rats (n = 38) were anaesthetized with ketamine-xylazine or isoflurane. They randomly received either an intravenous injection of Ringer's lactate or NButGT (10mg/kg) in order to increase O-GlcNAcylation levels. One hour after induction of anaesthesia, haemodynamic parameters and plasmatic markers were evaluated. Heart, brain and lungs were harvested and O-GlcNAcylation levels and O-GlcNAc-related enzymes were evaluated by western blot. Cardiac and pulmonary O-GlcNAcylation levels and cardiac, cerebral and pulmonary O-GlcNAc associated enzyme expression were not impacted with anaesthesia. Compared with ketamine-xylazine, isoflurane had a lower impact on blood pressure, heart rate and glycaemia. Pharmacological stimulation of O-GlcNAcylation by NButGT did not affect the physiological parameters. This study offers unprecedented insights into the regulation of O-GlcNAcylation and O-GlcNAc related enzymes during anaesthesia. Pharmacological stimulation of O-GlcNAcylation over a 1-h period did not disrupt the physiological balance in healthy anaesthetized rats.

O-GlcNAcylation in ischemic diseases.

Protein glycosylation is an extensively studied field, with the most studied forms being oxygen or nitrogen-linked N-acetylglucosamine (O-GlcNAc or N-GlcNAc) glycosylation. Particular residues on proteins are targeted by O-GlcNAcylation, which is among the most intricate post-translational modifications. Significantly contributing to an organism's proteome, it influences numerous factors affecting protein stability, function, and subcellular localization. It also modifies the cellular function of target proteins that have crucial responsibilities in controlling pathways related to the central nervous system, cardiovascular homeostasis, and other organ functions. Under conditions of acute stress, changes in the levels of O-GlcNAcylation of these proteins may have a defensive function. Nevertheless, deviant O-GlcNAcylation nullifies this safeguard and stimulates the advancement of several ailments, the prognosis of which relies on the cellular milieu. Hence, this review provides a concise overview of the function and comprehension of O-GlcNAcylation in ischemia diseases, aiming to facilitate the discovery of new therapeutic targets for efficient treatment, particularly in patients with diabetes.