O-linked N-acetylglucosamine Transferase Research Articles

Gene Expression and Ultrastructural Evidence for Metabolic Derangement in the Primary Mitral Regurgitation Heart

Abstract Aims Chronic neurohormonal activation and hemodynamic load cause derangement in myocardial substrate utilization. We test the hypothesis that the primary mitral regurgitation heart (PMR) heart shows altered metabolic gene profile and cardiac ultrastructure consistent with decreased fatty acid and glucose metabolism despite LVEF > 60%. Methods and Results Metabolic gene expression in right atrial (RA), left atrial (LA), and left ventricular (LV) biopsies from donor hearts (n = 10) and from patients with moderate to severe PMR (n = 11) at surgery showed decreased mRNA glucose transporter type 4 (GLUT-4), GLUT-1 and insulin receptor substrate 2 and increased mRNA hexokinase 2, O-linked N-acetylglucosamine transferase and O-GlcNAcase, rate-limiting steps in the hexosamine biosynthetic pathway. Pericardial fluid levels of Neuropeptide Y were 4-fold higher than simultaneous plasma indicative of increased sympathetic drive. Quantitative TEM shows glycogen accumulation, glycophagy, increased lipid droplets, and mitochondrial cristae lysis. These findings are associated with increased mRNA for glycogen synthase kinase 3β, decreased carnitine palmitoyl transferase 2, and fatty acid synthase in PMR vs. normals. Cardiac magnetic resonance/positron emission tomography for 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) uptake showed decreased LV [18F]FDG uptake and increased plasma HbA1c, free fatty acids, mtDAMPs in a separate cohort of stable moderate PMR patients with LVEF > 60% (n = 8) vs. normal controls (n = 8). Conclusions The PMR heart has a global ultrastructural and metabolic gene expression pattern of decreased glucose uptake along with increased glycogen and lipid droplets. Further studies must determine whether this presentation is an adaptation or maladaptation in the PMR heart in the clinical evaluation of PMR.

Overexpression of Fatty Acid Synthase Upregulates Glutamine-Fructose-6-Phosphate Transaminase 1 and O-Linked N-Acetylglucosamine Transferase to Increase O-GlcNAc Protein Glycosylation and Promote Colorectal Cancer Growth.

Fatty acid synthesis has been extensively investigated as a therapeutic target in cancers, including colorectal cancer (CRC). Fatty acid synthase (FASN), a key enzyme of de novo lipid synthesis, is significantly upregulated in CRC, and therapeutic approaches of targeting this enzyme are currently being tested in multiple clinical trials. However, the mechanisms behind the pro-oncogenic action of FASN are still not completely understood. Here, for the first time, we show that overexpression of FASN increases the expression of glutamine-fructose-6-phosphate transaminase 1 (GFPT1) and O-linked N-acetylglucosamine transferase (OGT), enzymes involved in hexosamine metabolism, and the level of O-GlcNAcylation in vitro and in vivo. Consistently, expression of FASN significantly correlates with expression of GFPT1 and OGT in human CRC tissues. shRNA-mediated downregulation of GFPT1 and OGT inhibits cellular proliferation and the level of protein O-GlcNAcylation in vitro, and knockdown of GFPT1 leads to a significant decrease in tumor growth and metastasis in vivo. Pharmacological inhibition of GFPT1 and OGT leads to significant inhibition of cellular proliferation and colony formation in CRC cells. In summary, our results show that overexpression of FASN increases the expression of GFPT1 and OGT as well as the level of protein O-GlcNAcylation to promote progression of CRC; targeting the hexosamine biosynthesis pathway could be a therapeutic approach for this disease.

Gene and Protein Expression of Placental Nutrient-Stress Sensor Proteins in Fetal Growth Restriction

Fetal growth restriction (FGR) and low birth weight increase the risk of non-communicable diseases such as type 2 diabetes and heart failure in adulthood. Placental insufficiency is widely considered a major contributor to FGR. Two crucial placental proteins involved in nutrient and stress sensing—O-linked N-acetylglucosamine transferase (OGT) and mechanistic target of rapamycin (mTOR) kinase—play roles in post-translational protein modification and protein translation, influencing cellular growth and metabolism in response to maternal stress, hypoxia, and nutritional status in the placenta. In our study, we examined the gene and protein profiles of OGT and mTOR in FGR and control placentae, comparing those appropriate for gestational age (AGA), while also considering potential confounding effects of fetal sex and delivery mode. Our findings revealed no significant differences in gene expression, protein levels, or activity of OGT, OGA, mTOR, or their associated markers between female AGA and FGR cesarean placentae, nor between female AGA and male AGA cesarean placentae. Additionally, the mode of delivery in female AGA placentae did not affect the levels or activity of these proteins. Overall, our study did not observe significant differences in nutrient sensor protein expression after stratifying by FGR, sex, and delivery mode. Nevertheless, these unbiased results provide a more comprehensive understanding of the complexities of placental gene expression involving OGT and mTOR.

Metabolic reprogramming regulated by TRAF6 contributes to the leukemia progression

TNF receptor associated factor 6 (TRAF6) is an E3 ubiquitin ligase that has been implicated in myeloid malignancies. Although altered TRAF6 expression is observed in human acute myeloid leukemia (AML), its role in the AML pathogenesis remains elusive. In this study, we showed that the loss of TRAF6 in AML cells significantly impairs leukemic function in vitro and in vivo, indicating its functional importance in AML subsets. Loss of TRAF6 induces metabolic alterations, such as changes in glycolysis, TCA cycle, and nucleic acid metabolism as well as impaired mitochondrial membrane potential and respiratory capacity. In leukemic cells, TRAF6 expression shows a positive correlation with the expression of O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT), which catalyzes the addition of O-GlcNAc to target proteins involved in metabolic regulation. The restoration of growth capacity and metabolic activity in leukemic cells with TRAF6 loss, achieved through either forced expression of OGT or pharmacological inhibition of O-GlcNAcase (OGA) that removes O-GlcNAc, indicates the significant role of O-GlcNAc modification in the TRAF6-related cellular and metabolic dynamics. Our findings highlight the oncogenic function of TRAF6 in leukemia and illuminate the novel TRAF6/OGT/O-GlcNAc axis as a potential regulator of metabolic reprogramming in leukemogenesis.

O-GlcNAc transferase regulates collagen deposition and fibrosis resolution in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic pulmonary disease that is characterized by an excessive accumulation of extracellular matrix (ECM) proteins (e.g. collagens) in the parenchyma, which ultimately leads to respiratory failure and death. While current therapies exist to slow the progression, no therapies are available to resolve fibrosis. We characterized the O-linked N-Acetylglucosamine (O-GlcNAc) transferase (OGT)/O-GlcNAc axis in IPF using single-cell RNA-sequencing (scRNA-seq) data and human lung sections and isolated fibroblasts from IPF and non-IPF donors. The underlying mechanism(s) of IPF were further investigated using multiple experimental models to modulate collagen expression and accumulation by genetically and pharmacologically targeting OGT. Furthermore, we hone in on the transforming growth factor-beta (TGF-β) effector molecule, Smad3, by co-expressing it with OGT to determine if it is modified and its subsequent effect on Smad3 activation. We found that OGT and O-GlcNAc levels are upregulated in patients with IPF compared to non-IPF. We report that the OGT regulates collagen deposition and fibrosis resolution, which is an evolutionarily conserved process demonstrated across multiple species. Co-expression of OGT and Smad3 showed that Smad3 is O-GlcNAc modified. Blocking OGT activity resulted in decreased phosphorylation at Ser-423/425 of Smad3 attenuating the effects of TGF-β1 induced collagen expression/deposition. OGT inhibition or knockdown successfully blocked and reversed collagen expression and accumulation, respectively. Smad3 is discovered to be a substrate of OGT and its O-GlcNAc modification(s) directly affects its phosphorylation state. These data identify OGT as a potential target in pulmonary fibrosis resolution, as well as other diseases that might have aberrant ECM/collagen accumulation.

Abstract LB293: O-GlcNAc transferase drives the proliferative state in adaptive drug tolerance

Abstract Drivers of the drug tolerant proliferative persister (DTPP) state have not been well investigated. Histone H3 lysine-4 trimethylation (H3K4me3), an active histone mark, might enable slow cycling drug tolerant persisters (DTP) to regain proliferative capacity. This study aimed to determine H3K4me3 transcriptionally active sites identifying a key regulator of DTPPs in melanoma. Deploying a model of adaptive cancer drug tolerance H3K4me3 ChIP-seq data of DTPPs guided identification of top transcription factor binding motifs. These suggested involvement of O-linked N-acetylglucosamine transferase (OGT), which was confirmed by metabolomics analysis and biochemical assays. OGT impact on DTPPs and adaptive resistance was explored in vitro and in vivo. H3K4me3 remodeling was widespread in CPG island regions and DNA binding motifs associated with O-GlcNAc marked chromatin. Accordingly, we observed an upregulation of OGT, O-GlcNAc and its binding partner TET1 in chronically treated cancer cells. Inhibition of OGT led to loss of H3K4me3 and downregulation of genes contributing to drug resistance. Genetic ablation of OGT prevented acquired drug resistance in vivo. Our findings uncover a fundamental mechanism of adaptive drug resistance that governs cancer cell reprogramming towards acquired drug resistance, a process that can be exploited to improve response duration and patient outcomes. Citation Format: Dinoop Ravindran Menon, Heinz Hammerlindl, Gregory Gimenez, Elmar Zuegner, Christoph Magens, Michael Eccles, Mayumi Fujita, Helmut Schaider. O-GlcNAc transferase drives the proliferative state in adaptive drug tolerance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB293.

407 The OGT/O-GlcNAc Axis Regulates Fibrosis Resolution in Idiopathic Pulmonary Fibrosis

OBJECTIVES/GOALS: Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease characterized by dysregulated collagen accumulation in the lung parenchyma. Our goal is to investigate the role of O-linked N-Acetylglucosamine (O-GlcNAc) transferase (OGT) in pulmonary fibrosis to ultimately discover novel therapies for fibrosis resolution. METHODS/STUDY POPULATION: Lung tissue from IPF and non-IPF donors was subjected to immunohistochemistry (IHC) to assess O-GlcNAc levels. Primary human lung fibroblasts were treated with OGT or O-GlcNAcase (OGA) inhibitors followed by transforming growth factor-beta 1 (TGF-β1) stimulation to assess O-GlcNAc regulation of fibroblast-to-myofibroblast transition (FMT) markers [alpha smooth muscle actin (α-SMA) and type 1 and type 3 collagen (COL1α1, COL3α1)] In Drosophila melanogaster, OGT knockdown (KD)/overexpression (OE) was conditionally induced to assess pericardin, a type IV collagen-like protein, regulation by immunofluorescence. Lastly, a mouse model of bleomycin-induced pulmonary fibrosis was examined following OGT KD and assessed for fibrosis resolution via histology, hydroxyproline assay, and western blotting. RESULTS/ANTICIPATED RESULTS: O-GlcNAc staining was increased in IPF lung tissue compared to non-IPF control lungs. In primary human lung fibroblasts, TGF-α1 administration resulted in increased FMT markers (α-SMA, COL1α1, and COL3α1), which were reduced or increased by OGT or OGA inhibition, respectively. Genetic manipulation in the Drosophila models showed decreased pericardin expression with OGT KD compared to the wild-type, whereas OGT OE increased pericardin compared to control. Additionally, OGT KD in bleomycin treated aged mice resulted in reduced collagen levels at the transcript and protein level and concurrent fibrosis resolution as assessed by Masson’s trichrome staining and total hydroxyproline analysis. Collectively, showing OGT/O-GlcNAc regulating collagen in fibrosis resolution. DISCUSSION/SIGNIFICANCE: These data suggest that the OGT/O-GlcNAc axis regulates collagen deposition in pulmonary fibrosis, and we show that O-GlcNAc is implicated in the pathogenesis of IPF. We identified OGT as a therapeutic target to overcome current drug limitations, opening new horizons for biomedical treatment.

An in-silico analysis of OGT gene association with diabetes mellitus

O-GlcNAcylation is a nutrient-sensing post-translational modification process. This cycling process involves two primary proteins: the O-linked N-acetylglucosamine transferase (OGT) catalysing the addition, and the glycoside hydrolase OGA (O-GlcNAcase) catalysing the removal of the O-GlCNAc moiety on nucleocytoplasmic proteins. This process is necessary for various critical cellular functions. The O-linked N-acetylglucosamine transferase (OGT) gene produces the OGT protein. Several studies have shown the overexpression of this protein to have biological implications in metabolic diseases like cancer and diabetes mellitus (DM). This study retrieved 159 SNPs with clinical significance from the SNPs database. We probed the functional effects, stability profile, and evolutionary conservation of these to determine their fit for this research. We then identified 7 SNPs (G103R, N196K, Y228H, R250C, G341V, L367F, and C845S) with predicted deleterious effects across the four tools used (PhD-SNPs, SNPs&Go, PROVEAN, and PolyPhen2). Proceeding with this, we used ROBETTA, a homology modelling tool, to model the proteins with these point mutations and carried out a structural bioinformatics method– molecular docking– using the Glide model of the Schrodinger Maestro suite. We used a previously reported inhibitor of OGT, OSMI-1, as the ligand for these mutated protein models. As a result, very good binding affinities and interactions were observed between this ligand and the active site residues within 4Å of OGT. We conclude that these mutation points may be used for further downstream analysis as drug targets for treating diabetes mellitus.

Compound SJ-12 attenuates streptozocin-induced diabetic cardiomyopathy by stabilizing SERCA2a

Heart failure (HF) is one of the major causes of death among diabetic patients. Although studies have shown that curcumin analog C66 can remarkably relieve diabetes-associated cardiovascular and kidney complications, the role of SJ-12, SJ-12, a novel curcumin analog, in diabetic cardiomyopathy and its molecular targets are unknown. 7-week-old male C57BL/6 mice were intraperitoneally injected with single streptozotocin (STZ) (160 mg/kg) to develop diabetic cardiomyopathy (DCM). The diabetic mice were then treated with SJ-12 via gavage for two months. Body weight, fast blood glucose, cardiac utrasonography, myocardial injury markers, pathological morphology of the heart, hypertrophic and fibrotic markers were assessed. The potential target of SJ-12 was evaluated via RNA-sequencing analysis. The O-GlcNAcylation levels of SP1 were detected via immunoprecipitation. SJ-12 effectively suppressed myocardial hypertrophy and fibrosis, thereby preventing heart dysfunction in mice with STZ-induced heart failure. RNA-sequencing analysis revealed that SJ-12 exerted its therapeutic effects through the modulation of the calcium signaling pathway. Furthermore, SJ-12 reduced the O-GlcNAcylation levels of SP1 by inhibiting O-linked N-acetylglucosamine transferase (OGT). Also, SJ-12 stabilized Sarcoplasmic/Endoplasmic Reticulum Calcium ATPase 2a (SERCA2a), a crucial regulator of calcium homeostasis, thus reducing hypertrophy and fibrosis in mouse hearts and cultured cardiomyocytes. However, the anti-fibrotic effects of SJ-12 were not detected in SERCA2a or OGT-silenced cardiomyocytes, indicating that SJ-12 can prevent DCM by targeting OGT-dependent O-GlcNAcylation of SP1.These findings indicate that SJ-12 can exert cardioprotective effects in STZ-induced mice by reducing the O-GlcNAcylation levels of SP1, thus stabilizing SERCA2a and reducing myocardial fibrosis and hypertrophy. Therefore, SJ-12 can be used for the treatment of diabetic cardiomyopathy.

O-GlcNAcylation regulates long-chain fatty acid metabolism by inhibiting ACOX1 ubiquitination-dependent degradation

BackgroundCold as a common environmental stress, causes increased heat production, accelerated metabolism and even affects its production performance. How to improve the adaptability of the animal organism to cold has been an urgent problem. As a key hub of lipid metabolism, the liver can regulate lipid metabolism to maintain energy balance, and O-GlcNAcylation is a kind of important PTMs, which participates in a variety of signaling and mechanism regulation, and at the same time, is very sensitive to changes in stress and nutritional levels, and is the body's “stress receptors” and “nutrient receptors”. Therefore, the aim of this experiment was to investigate the effect of cold-induced O-GlcNAcylation on hepatic lipid metabolism, and to explore the potential connection between O-GlcNAcylation and hepatic lipid metabolism. MethodsTo investigate the loss of O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) and the precise impacts of additional cold-induced circumstances on liver mass, shape, and metabolic profile, C57 mice were used as an animal model. Using the protein interactions approach, the mechanism of O-GlcNAcylation, as well as the degradation pathway of acyl-Coenzyme A oxidase 1 (ACOX1), were clarified. Additional in vitro analyses of oleic acid (OA) and OGT inhibitor tetraoxan (Alloxan) (Sigma, 2244-11-3) on lipid breakdown in AML-12 cells. ResultsIn C57BL/6 mice, deletion of O-GlcNAcylation disrupted lipid metabolism, caused hepatic edema and fibrosis, and altered mitochondrial apoptosis. This group of modifications was made worse by cold induction. The accumulation of medium- and long-chain fatty acids is a hallmark of lipolysis, which is accelerated by the deletion of O-GlcNAcylation, whereas lipid synthesis is slowed down. The association between ACOX1 and OGT at the K48 gene precludes ubiquitinated degradation.

Increased O-GlcNAcylation by Upregulation of Mitochondrial O-GlcNAc Transferase (mOGT) Inhibits the Activity of Respiratory Chain Complexes and Controls Cellular Bioenergetics.

O-linked β-N-acetylglucosamine (O-GlcNAc) is a reversible post-translational modification involved in the regulation of cytosolic, nuclear, and mitochondrial proteins. The interplay between O-GlcNAcylation and phosphorylation is critical to control signaling pathways and maintain cellular homeostasis. The addition of O-GlcNAc moieties to target proteins is catalyzed by O-linked N-acetylglucosamine transferase (OGT). Of the three splice variants of OGT described, one is destined for the mitochondria (mOGT). Although the effects of O-GlcNAcylation on the biology of normal and cancer cells are well documented, the role of mOGT remains poorly understood. In this manuscript, the effects of mOGT on mitochondrial protein phosphorylation, electron transport chain (ETC) complex activity, and the expression of VDAC porins were investigated. We performed studies using normal and breast cancer cells with upregulated mOGT or its catalytically inactive mutant. Proteomic approaches included the isolation of O-GlcNAc-modified proteins of the electron transport chain, followed by their analysis using mass spectrometry. We found that mitochondrial OGT regulates the activity of complexes I-V of the respiratory chain and identified a group of 19 ETC components as mOGT substrates in mammary cells. Furthermore, we observed that the upregulation of mOGT inhibited the interaction of VDAC1 with hexokinase II. Our results suggest that the deregulation of mOGT reprograms cellular energy metabolism via interaction with and O-GlcNAcylation of proteins involved in ATP production in mitochondria and its exchange between mitochondria and the cytosol.

Transcriptome analysis of Chinese mitten crabs (Eriocheir sinensis) gills in response to ammonia stress.

The Chinese mitten crab (Eriocheir sinensis) is an important commercial species in China. E. sinensis is typically farmed in rice-crab symbiosis, as an important ecological farming model. However, E. sinensis is often exposed to a high ammonia environment due to the application of nitrogen fertilizers essential for rice growth. We investigated the molecular mechanisms in the gills of E. sinensis exposed to high ammonia at transcriptional and histological levels. We randomly assigned E. sinensis to two groups (control group, CG; ammonia stress group, AG), and gill samples were excised from the CG and AG groups for histopathological and transcriptome analyses. The histopathological evaluation revealed that ammonia stress damaged the gills of E. sinensis. The transcriptome analysis showed that some essential genes, including Xanthine dehydrogenase (XDH), Ubiquitin C-terminal hydrolase-L3 (UCHL3), O-linked N-acetylglucosamine transferase (OGT), Cathepsin B (CTSB), and Ubiquitin-conjugating enzyme E2 W (UBE2W) changed significantly during ammonia exposure. These genes are related to ammonia detoxification, the immune response, and apoptosis. This study demonstrated the molecular response mechanism of E. sinensis gills to ammonia stress at the transcriptional and histological levels. This study provides insight for further study on the molecular mechanism of ammonia stress in crustaceans and supplies technical support for rice crab symbiosis.

Androgen-repressed lncRNA LINC01126 drives castration-resistant prostate cancer by regulating the switch between O-GlcNAcylation and phosphorylation of androgen receptor.

Prostate cancer (PCa) initially shows satisfactory response to therapies targeting the androgen receptor (AR). However, progression to a castration-resistant stage indicates poor prognosis in PCa patients. AR signalling still plays a central role in most castration-resistant prostate cancers (CRPC). Therefore, unveiling the mechanisms of AR reactivation under androgen-deprived conditions is imperative to discover novel therapeutic targets for CRPC. Using an integrative analysis of the transcriptomics of three independent PCa cohorts and a published landscape of AR-regulated long non-coding RNA (lncRNA), lncRNA LINC01126 was selected as a candidate gene that could drive CRPC progression for further study. Quantitative reverse transcription polymerase chain reaction, in situ hybridisation (ISH) and fluorescent ISH were performed to detect LINC01126 in PCa tissues and cells. The functional role and mechanism of LINC01126 were further investigated using in vitro and in vivo gain and loss of function assays. LINC01126, identified as an AR-repressed lncRNA, was significantly upregulated after AR-targeted therapies. In addition, we found that LINC01126 was upregulated in CRPC and was associated with poor prognosis. We also proved that LINC01126 stabilised AR protein and enhanced AR nuclear translocation and transactivation by promoting the transition from O-GlcNAcylation at threonine 80 to phosphorylation at serine 81 (S81) within the AR protein. Mechanism analysis revealed that LINC01126 facilitates the interaction of CDK9 with AR and impedes the binding of O-linked N-acetylglucosamine (O-GlcNAc) transferase to AR. Consequently, LINC01126 expression was sufficient to activate AR signalling without androgen. LINC01126 overexpression increased, whereas LINC01126 knockdown decreased castration resistance traits in PCa cells in vitro and in vivo. Furthermore, our data showed that LINC01126-targeting antisense oligonucleotides (ASO) substantially inhibited CRPC cells in vitro. Our research expands the functions of AR-regulated lncRNA in sustaining androgen-independent AR activity and promoting CRPC progression and reveals that LINC01126 may be a new therapeutic target for PCa.

H3K4me3 remodeling induced acquired resistance through O-GlcNAc transferase.

Drivers of the drug tolerant proliferative persister (DTPP) state have not been well investigated. Histone H3 lysine-4 trimethylation (H3K4me3), an active histone mark, might enable slow cycling drug tolerant persisters (DTP) to regain proliferative capacity. This study aimed to determine H3K4me3 transcriptionally active sites identifying a key regulator of DTPPs. Deploying a model of adaptive cancer drug tolerance, H3K4me3 ChIP-Seq data of DTPPs guided identification of top transcription factor binding motifs. These suggested involvement of O-linked N-acetylglucosamine transferase (OGT), which was confirmed by metabolomics analysis and biochemical assays. OGT impact on DTPPs and adaptive resistance was explored in vitro and in vivo. H3K4me3 remodeling was widespread in CPG island regions and DNA binding motifs associated with O-GlcNAc marked chromatin. Accordingly, we observed an upregulation of OGT, O-GlcNAc and its binding partner TET1 in chronically treated cancer cells. Inhibition of OGT led to loss of H3K4me3 and downregulation of genes contributing to drug resistance. Genetic ablation of OGT prevented acquired drug resistance in in vivo models. Upstream of OGT, we identified AMPK as an actionable target. AMPK activation by acetyl salicylic acid downregulated OGT with similar effects on delaying acquired resistance. Our findings uncover a fundamental mechanism of adaptive drug resistance that governs cancer cell reprogramming towards acquired drug resistance, a process that can be exploited to improve response duration and patient outcomes.

EOGT enables residual Notch signaling in mouse intestinal cells lacking POFUT1

Notch signaling determines cell fates in mouse intestine. Notch receptors contain multiple epidermal growth factor-like (EGF) repeats modified by O-glycans that regulate Notch signaling. Conditional deletion of protein O-fucosyltransferase 1 (Pofut1) substantially reduces Notch signaling and markedly perturbs lineage development in mouse intestine. However, mice with inactivated Pofut1 are viable, whereas complete elimination of Notch signaling in intestine is lethal. Here we investigate whether residual Notch signaling enabled by EGF-domain-specific O-linked N-acetylglucosamine transferase (Eogt) permits mice conditionally lacking Pofut1 in intestine to survive. Mice globally lacking Eogt alone were grossly unaffected in intestinal development. In contrast, mice lacking both Eogt and Pofut1 died at ~ 28 days after birth with greater loss of body weight, a greater increase in the number of goblet and Paneth cells, and greater downregulation of the Notch target gene Hes1, compared to Pofut1 deletion alone. These data reveal that both O-fucose and O-GlcNAc glycans are fundamental to Notch signaling in the intestine and provide new insights into roles for O-glycans in regulating Notch ligand binding. Finally, EOGT and O-GlcNAc glycans provide residual Notch signaling and support viability in mice lacking Pofut1 in the intestine.

O-Linked N-Acetylglucosamine Transferase Ensures Survival of Mouse Fetal Liver Hematopoietic Progenitors Partly by Regulating Bcl-xL and Oxidative Phosphorylation.

O-linked N-acetylglucosamine transferase (OGT) critically regulates wide variety of biological processes such as gene expression, metabolism, stress response, signaling and proteostasis. In adult hematopoiesis, OGT is crucial for differentiation of B and T cells and the maintenance of hematopoietic stem cells (HSCs). However, a role for OGT in fetal liver (FL) hematopoiesis remains unknown. To investigate a role for OGT in FL hematopoiesis, we conditionally disrupted OGT in hematopoietic cells in developing FLs. Hematopoietic specific disruption of OGT resulted in embryonic lethality in late stage of gestation due to severe anemia and growth retardation. OGT loss led to profound reduction of differentiating erythroid cells and erythroid progenitors in FLs due to massive apoptosis. In addition, clonogenic capacity of FL cells was severely impaired by OGT loss. Interestingly, expression of BCL-XL, a well-known inhibitor of apoptosis in FL cells, dramatically decreased, and the levels of reactive oxygen species (ROS) were increased in OGT-deficient FL cells. Overexpression of Bcl-xL and reduction of ROS significantly restored the colony formation of OGT-deficient FL cells. This study revealed a novel role for OGT during embryogenesis, which ensures survival of FL hematopoietic cells partly by regulating Bcl-xL and oxidative phosphorylation.

High dietary fructose promotes hepatocellular carcinoma progression by enhancing O-GlcNAcylation via microbiota-derived acetate

Emerging studies have addressed the tumor-promoting role of fructose in different cancers. The effects and pathological mechanisms of high dietary fructose on hepatocellular carcinoma (HCC) remain unclear. Here, we examined the effects of fructose supplementation on HCC progression in wild-type C57BL/6 mice using a spontaneous and chemically induced HCC mouse model. We show that elevated uridine diphospho-N-acetylglucosamine (UDP-GlcNAc) and O-GlcNAcylation levels induced by high dietary fructose contribute to HCC progression. Non-targeted metabolomics and stable isotope tracing revealed that under fructose treatment, microbiota-derived acetate upregulates glutamine and UDP-GlcNAc levels and enhances protein O-GlcNAcylation in HCC. Global profiling of O-GlcNAcylation revealed that hyper-O-GlcNAcylation of eukaryotic elongation factor 1A1 promotes cell proliferation and tumor growth. Targeting glutamate-ammonia ligase or O-linked N-acetylglucosamine transferase (OGT) remarkably impeded HCC progression in mice with high fructose intake. We propose that high dietary fructose promotes HCC progression through microbial acetate-induced hyper-O-GlcNAcylation.

Abstract P3020: O-GlcNAc Transferase Regulates Vascular Smooth Muscle Cell Cycle Progression In Diabetes

Diabetic patients show increased proclivity for vascular smooth muscle cell (VSMC) migration and proliferation. Hyperglycemia, hallmark of diabetes, increases intracellular glucose signaling via O-linked N-acetylglucosamine transferase (OGT), a key regulator of protein O-GlcNAcylation. We previously showed that smooth muscle OGT (smOGT) deletion prevents hyperglycemia-induced atherosclerosis. Goal of this work was to interrogate the role of smOGT in VSMC cell cycle progression in diabetes. For this, we used conditional SMC-specific OGT knockout (smOGT KO ) and wild-type (smOGT WT ) mice generated via tamoxifen (tmx)-inducible VSMC-restricted Cre driver mice ( Itga8-CreER T2 ). Briefly, following tmx injection, 8-wks-old smOGT KO and smOGT WT mice were placed on Western diet feeding regimen for study duration. At 14-wks-age, mice were injected with 50mg/Kg/day streptozotocin i.p. once daily for 5 consecutive days. Mice were harvested at 16-18-wks-age and plasma and aortic tissues were collected for biochemical and molecular studies. Immunoblotting of aortic lysates revealed a significant decrease (59%) in OGT and O-GlcNAc expression, validating the smOGT KO mice. Loss of smOGT reduced (40%) CDK6 expression (regulator of G1-S phase cell cycle progression) concomitant to enhanced (62%) p21 Cip1 expression (cyclin-dependent kinase inhibitor) in aortic vessels from diabetic smOGT KO vs. smOGT WT mice. This was further accompanied with 69% attenuation in SRF expression (transcriptional regulator of SM proliferation) in diabetic smOGT KO vs. smOGT WT aortic vessels. Similar results were obtained with OGT loss-of-function studies in human coronary artery SMC (HCASMC) primary cultures under diabetic conditions in vitro . Briefly, control (siCtrl) and OGT siRNA (siOGT)-transfected HCASMC were treated with or without 25mM glucose+100μM PUGNAc for 48 hours. Immunoblotting revealed that reduced OGT-mediated O-GlcNAcylation in siOGT cells in a diabetic milieu significantly attenuated SRF expression coupled with augmented p21 Cip1 expression compared to diabetic siCtrl cells. Together, our results suggest a direct regulatory role of smooth muscle OGT in VSMC cell cycle progression in diabetes.

Hepatocyte-specific O-GlcNAc transferase downregulation ameliorates nonalcoholic steatohepatitis by improving mitochondrial function

ObjectiveO-GlcNAcylation is a post-translational modification that directly couples the processes of nutrient sensing, metabolism, and signal transduction, affecting protein function and localization, since the O-linked N-acetylglucosamine moiety comes directly from the metabolism of glucose, lipids, and amino acids. The addition and removal of O-GlcNAc of target proteins are mediated by two highly conserved enzymes: O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) and O-GlcNAcase (OGA), respectively. Deregulation of O-GlcNAcylation has been reported to be associated with various human diseases such as cancer, diabetes, and cardiovascular diseases. The contribution of deregulated O-GlcNAcylation to the progression and pathogenesis of NAFLD remains intriguing, and a better understanding of its roles in this pathophysiological context is required to uncover novel avenues for therapeutic intervention. By using a translational approach, our aim is to describe the role of OGT and O-GlcNAcylation in the pathogenesis of NAFLD. MethodsWe used primary mouse hepatocytes, human hepatic cell lines and in vivo mouse models of steatohepatitis to manipulate O-GlcNAc transferase (OGT). We also studied OGT and O-GlcNAcylation in liver samples from different cohorts of people with NAFLD. ResultsO-GlcNAcylation was upregulated in the liver of people and animal models with steatohepatitis. Downregulation of OGT in NAFLD-hepatocytes improved diet-induced liver injury in both in vivo and in vitro models. Proteomics studies revealed that mitochondrial proteins were hyper-O-GlcNAcylated in the liver of mice with steatohepatitis. Inhibition of OGT is able to restore mitochondrial oxidation and decrease hepatic lipid content in in vitro and in vivo models of NAFLD. ConclusionsThese results demonstrate that deregulated hyper-O-GlcNAcylation favors NAFLD progression by reducing mitochondrial oxidation and promoting hepatic lipid accumulation.

Enzymatic assay for UDP-GlcNAc and its application in the parallel assessment of substrate availability and protein O-GlcNAcylation

O-linked N-acetylglucosaminylation (O-GlcNAcylation) is a ubiquitous and dynamic non-canonical glycosylation of intracellular proteins. Several branches of metabolism converge at the hexosamine biosynthetic pathway (HBP) to produce the substrate for protein O-GlcNAcylation, the uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). Availability of UDP-GlcNAc is considered a key regulator of O-GlcNAcylation. Yet UDP-GlcNAc concentrations are rarely reported in studies exploring the HBP and O-GlcNAcylation, most likely because the methods to measure it are restricted to specialized chromatographic procedures. Here, we introduce an enzymatic method to quantify cellular and tissue UDP-GlcNAc. The method is based on O-GlcNAcylation of a substrate peptide by O-linked N-acetylglucosamine transferase (OGT) and subsequent immunodetection of the modification. The assay can be performed in dot-blot or microplate format. We apply it to quantify UDP-GlcNAc concentrations in several mouse tissues and cell lines. Furthermore, we show how changes in UDP-GlcNAc levels correlate with O-GlcNAcylation and the expression of OGT and O-GlcNAcase (OGA).