O-GlcNAc Transferase Levels Research Articles

The role of O‐linked β‐N acetylglucosamine transferase in cardiac hypertrophy (LB692)

Cardiomyocytes exhibit diminished re‐activation of the fetal gene program (FGP) in response to hypertrophic stimuli due to elevated post‐translational modification of proteins by O‐linked β‐N‐acetylglucosamine (O‐GlcNAc), catalyzed by a single enzyme, O‐GlcNAc transferase (OGT). Repressor element 1‐silencing transcription factor (REST), which associates with OGT, controls FGP transcription. OGT is also involved in transcription via histone modifications such as monoubiquitination and methylation as well as regulation of transcription factor proteolysis, purportedly by the O‐GlcNAcylation to ubiquitination ratio. The objective of this study was to examine the role of OGT in transcriptional control via the ubiquitin proteasome system (UPS) and histone modifications during cardiac hypertrophy. In preliminary studies, upregulation of O‐GlcNAc in isolated adult cardiomyocytes resulted in increased REST and decreased monoubiquitinated histone H2B and protein ubiquitination. Inducible cardiomyocyte specific OGT knockdown (OGT‐KD) and control mice were tamoxifen treated at 5 weeks and sacrificed at 7, 10 and 14 weeks of age. Diminished OGT and protein O‐GlcNAcylation and elevated mRNA levels of FGP components were observed at 7 weeks. Heart weight:tibia length ratio was elevated in OGT‐KD by 10 weeks, indicating decreased cardiac OGT levels result in hypertrophy. Protein ubiquitination was elevated in OGT‐KD hearts. These data suggest that OGT is involved in transcriptional regulation of cardiac hypertrophy via both histone modifications and the UPS.Grant Funding Source: Supported by NIH/NHLBI HL104549

OGT and OGA expression in postmenopausal skeletal muscle associates with hormone replacement therapy and muscle cross-sectional area

Protein glycosylation via O-linked N-acetylglucosaminylation (O-GlcNAcylation) is an important post-translational regulatory mechanism mediated by O-GlcNAc transferase (OGT) and responsive to nutrients and stress. OGT attaches an O-GlcNAc moiety to proteins, while O-GlcNAcase (OGA) catalyzes O-GlcNAc removal. In skeletal muscle of experimental animals, prolonged increase in O-GlcNAcylation associates with age and muscle atrophy. Here we examined the effects of hormone replacement therapy (HRT) and power training (PT) on muscle OGT and OGA gene expression in postmenopausal women generally prone to age-related muscle weakness. In addition, the associations of OGT and OGA gene expressions with muscle phenotype were analyzed. Twenty-seven 50–57-year-old women participated in a yearlong randomized placebo-controlled trial: HRT (n=10), PT (n=8) and control (n=9). OGT and OGA mRNA levels were measured from muscle samples obtained at baseline and after one year. Knee extensor muscle cross-sectional area (CSA), knee extension force, running speed and vertical jumping height were measured. During the yearlong intervention, HRT suppressed the aging-associated upregulation of OGT mRNA that occurred in the controls. The effects of PT were similar but weaker. HRT also tended to increase the OGA mRNA level compared to the controls. The change in the ratio of OGT to OGA gene expressions correlated negatively with the change in muscle CSA. Our results suggest that OGT and OGA gene expressions are associated with muscle size during the critical postmenopausal period. HRT and PT influence muscle OGT and OGA gene expression, which may be one of the mechanisms by which HRT and PT prevent aging-related loss of muscle mass.

Aberrant O-GlcNAc-modified proteins expressed in primary colorectal cancer

O-GlcNAcylation is a post-translational modification of serine and threonine residues which is dynamically regulated by 2 enzymes; O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) that catalyze the addition and removal of a single N-acetylglucosamine (GlcNAc) molecule, respectively. This modification is thought to be a nutrient sensor in highly proliferating cells via the hexosamine biosynthesis pathway, a minor branch of glycolysis. Although emerging evidence suggests that O-GlcNAc modification is associated with many types of cancer, identification of O-GlcNAc-modified proteins and their role in cancer remain unexplored. In the present study, we demonstrated that O-GlcNAcylation is increased in primary colorectal cancer tissues, and that this augmentation is associated with an increased expression of OGT levels. Using 2-dimensional O-GlcNAc immunoblotting and LC-MS/MS analysis, 16 proteins were successfully identified and 8 proteins showed an increase in O-GlcNAcylation, including cytokeratin 18, heterogeneous nuclear ribonucleoproteins A2/B1 (hnRNP A2/B1), hnRNP H, annexin A2, annexin A7, laminin-binding protein, α-tubulin and protein DJ-1. Among these identified proteins, annexin A2 was further confirmed to show overexpression of O-GlcNAc in all cancer samples. The results, therefore, indicate that aberrant O-GlcNAcylation of proteins is associated with colorectal cancer and that identification of O-GlcNAc-modified proteins may provide novel biomarkers of cancer.

Epigenetic regulation of physiological cardiac hypertrophy by O‐GlcNAc and histone deacetylases in diabetes

Exercise causes physiological cardiac hypertrophy, and type 2 diabetes blunts hypertrophic signaling. Diabetic hearts have elevated levels of O‐linked β‐N‐acetylglucosamine (O‐GlcNAc), which post‐translationally modifies a complex of mSin3A and histone deacetylases (HDACs) 1 and 2. mSin3A/HDAC1/2 regulates hypertrophy and is targeted to genes by REST and O‐GlcNAc transferase (OGT). We examined the role of O‐GlcNAc in these mechanisms in 8‐week‐old diabetic (db/db) and non‐diabetic C57BL/6J mice randomized to exercise or sedentary groups for 4 weeks. Db/db mice had higher heart weight to tibia length (5.4±0.1 vs 4.7±0.1 mg/mm), fasting glucose (10.1±3.2 vs 5.5±1.5 mmol/L), and body weight (39.1±2.5 vs 20.1±1.2 g). Db/db hearts had higher levels of O‐GlcNAc and OGT, but lower mSin3A, HDAC1, and HDAC2. Conversely, HDAC2 expression and HDAC1/2 activity were higher in db/db hearts, and α‐actin and BNP expression were lower. In db/db hearts, immunoprecipitation showed that mSin3A was more O‐GlcNAcylated and more associated with REST, and OGT was less associated with HDAC2. Exercise corrected these changes, showing that diabetes and exercise have interacting effects on the epigenetic control of hypertrophy.

O-GlcNAc transferase (OGT) as a placental biomarker of maternal stress and reprogramming of CNS gene transcription in development

Maternal stress is a key risk factor for neurodevelopmental disorders, including schizophrenia and autism, which often exhibit a sex bias in rates of presentation, age of onset, and symptom severity. The placenta is an endocrine tissue that functions as an important mediator in responding to perturbations in the intrauterine environment and is accessible for diagnostic purposes, potentially providing biomarkers predictive of disease. Therefore, we have used a genome-wide array approach to screen placental expression across pregnancy for gene candidates that are sex-biased and stress-responsive in mice and translate to human tissue. We identifed O-linked-N-acetylglucosamine (O-GlcNAc) transferase (OGT), an X-linked gene important in regulating proteins involved in chromatin remodeling, as fitting these criteria. Levels of both OGT and its biochemical mark, O-GlcNAcylation, were significantly lower in males and further reduced by prenatal stress. Examination of human placental tissue found similar patterns related to X chromosome dosage. As a demonstration of the importance of placental OGT in neurodevelopment, we found that hypothalamic gene expression and the broad epigenetic microRNA environment in the neonatal brain of placental-specific hemizygous OGT mice was substantially altered. These studies identified OGT as a promising placental biomarker of maternal stress exposure that may relate to sex-biased outcomes in neurodevelopment.

O-GlcNAcylation: A New Cancer Hallmark?

O-linked N-acetylglucosaminylation (O-GlcNAcylation) is a reversible post-translational modification consisting in the addition of a sugar moiety to serine/threonine residues of cytosolic or nuclear proteins. Catalyzed by O-GlcNAc-transferase (OGT) and removed by O-GlcNAcase, this dynamic modification is dependent on environmental glucose concentration. O-GlcNAcylation regulates the activities of a wide panel of proteins involved in almost all aspects of cell biology. As a nutrient sensor, O-GlcNAcylation can relay the effects of excessive nutritional intake, an important cancer risk factor, on protein activities and cellular functions. Indeed, O-GlcNAcylation has been shown to play a significant role in cancer development through different mechanisms. O-GlcNAcylation and OGT levels are increased in different cancers (breast, prostate, colon…) and vary during cell cycle progression. Modulating their expression or activity can alter cancer cell proliferation and/or invasion. Interestingly, major oncogenic factors have been shown to be directly O-GlcNAcylated (p53, MYC, NFκB, β-catenin…). Furthermore, chromatin dynamics is modulated by O-GlcNAc. DNA methylation enzymes of the Tet family, involved epigenetic alterations associated with cancer, were recently found to interact with and target OGT to multi-molecular chromatin-remodeling complexes. Consistently, histones are subjected to O-GlcNAc modifications which regulate their function. Increasing number of evidences point out the central involvement of O-GlcNAcylation in tumorigenesis, justifying the attention received as a potential new approach for cancer treatment. However, comprehension of the underlying mechanism remains at its beginnings. Future challenge will be to address directly the role of O-GlcNAc-modified residues in oncogenic-related proteins to eventually propose novel strategies to alter cancer development and/or progression.

Critical Role of O-Linked β-N-Acetylglucosamine Transferase in Prostate Cancer Invasion, Angiogenesis, and Metastasis

Cancer cells universally increase glucose and glutamine consumption, leading to the altered metabolic state known as the Warburg effect; one metabolic pathway, highly dependent on glucose and glutamine, is the hexosamine biosynthetic pathway. Increased flux through the hexosamine biosynthetic pathway leads to increases in the post-translational addition of O-linked β-N-acetylglucosamine (O-GlcNAc) to various nuclear and cytosolic proteins. A number of these target proteins are implicated in cancer, and recently, O-GlcNAcylation was shown to play a role in breast cancer; however, O-GlcNAcylation in other cancers remains poorly defined. Here, we show that O-GlcNAc transferase (OGT) is overexpressed in prostate cancer compared with normal prostate epithelium and that OGT protein and O-GlcNAc levels are elevated in prostate carcinoma cell lines. Reducing O-GlcNAcylation in PC3-ML cells was associated with reduced expression of matrix metalloproteinase (MMP)-2, MMP-9, and VEGF, resulting in inhibition of invasion and angiogenesis. OGT-mediated regulation of invasion and angiogenesis was dependent upon regulation of the oncogenic transcription factor FoxM1, a key regulator of invasion and angiogenesis, as reducing OGT expression led to increased FoxM1 protein degradation. Conversely, overexpression of a degradation-resistant FoxM1 mutant abrogated OGT RNAi-mediated effects on invasion, MMP levels, angiogenesis, and VEGF expression. Using a mouse model of metastasis, we found that reduction of OGT expression blocked bone metastasis. Altogether, these data suggest that as prostate cancer cells alter glucose and glutamine levels, O-GlcNAc modifications and OGT levels become elevated and are required for regulation of malignant properties, implicating OGT as a novel therapeutic target in the treatment of cancer.

Alterations in O‐linked‐β‐N‐acetylglucosamine levels during acute exercise

Acute bouts of exercise reduce damage to cardiomyocytes during ischemic events, including a reduction in infarct size and improvement in heart function. The mechanism underlying exercise-induced cardioprotection is still unknown. Previous reports have demonstrated increases in O-linked-β-N-aceytlglucosamine (O-GlcNAc) provide protection against ischemia-reperfusion injury, yet the role of O-GlcNAc in exercise has not been investigated. Our lab proposes O-GlcNAc may be a major factor in exercise-induced cardioprotection. We predicted an increase in O-GlcNAc levels in both cellular and nuclear compartments of male CD-1 mouse hearts following acute exercise (EX) compared to a sedentary control (SED). O-GlcNAc levels were not altered in either cytosolic or nuclear fractions of the 30 min EX group and SED group. O-GlcNAc transferase (OGT), the enzyme that attaches O-GlcNAc to proteins, was not different between 30 min groups. The 15 min groups did not demonstrate a difference in cytosolic levels of OGT, however, there was a significant decrease in OGT levels in the nuclear compartment of the EX group. Overall, the data suggest that cardiac O- GlcNAc and OGT levels either do not change or decrease during acute exercise.

Increased Expression of β-N-Acetylglucosaminidase in Erythrocytes From Individuals With Pre-diabetes and Diabetes

OBJECTIVEO-linked β-N-acetylglucosamine (O-GlcNAc) plays an important role in the development of insulin resistance and glucose toxicity. O-GlcNAcylation is regulated by O-GlcNAc transferase (OGT), which attaches O-GlcNAc to serine and/or threonine residues of proteins and by O-GlcNAcase, which removes O-GlcNAc. We investigated the expression of these two enzymes in erythrocytes of human subjects with diabetes or pre-diabetes.RESEARCH DESIGN AND METHODSVolunteers with normal condition, pre-diabetes, and diabetes were recruited through a National Institutes of Health (National Institute of Diabetes and Digestive and Kidney Diseases) study and at the Johns Hopkins Comprehensive Diabetes Center. Erythrocyte proteins were extracted and hemoglobins were depleted. Global O-GlcNAcylation of erythrocyte proteins was confirmed by Western blotting using an O-GlcNAc–specific antibody. Relative OGT and O-GlcNAcase protein amounts were determined by Western blot analysis. Relative expression of O-GlcNAcase was compared with the level of A1C.RESULTSErythrocyte proteins are highly O-GlcNAcylated. O-GlcNAcase expression is significantly increased in erythrocytes from both individuals with pre-diabetes and diabetes compared with normal control subjects. Unlike O-GlcNAcase, protein levels of OGT did not show significant changes.CONCLUSIONSO-GlcNAcase expression is increased in erythrocytes from both individuals with pre-diabetes and individuals with less well-controlled diabetes. These findings, together with the previous study that demonstrated the increased site-specific O-GlcNAcylation of certain erythrocyte proteins, suggest that the upregulation of O-GlcNAcase might be an adaptive response to hyperglycemia-induced increases in O-GlcNAcylation, which are likely deleterious to erythrocyte functions. In any case, the early and substantial upregulation of O-GlcNAcase in individuals with pre-diabetes may eventually have diagnostic utility.

Abstract 1465: Cardiomyocyte Hypertrophy Induces O-linked-β-N-acetylglucosamine Modification of PGC-1α and Augments its Transcriptional Activity

Background and Hypothesis: PGC-1α (peroxisome proliferator activated receptor-gamma coactivator-1α) coordinately regulates fatty acid metabolism. The O-linked β-N-acetylglucosamine post-translational modification (O-GlcNAc) of proteins is a glucose-derived metabolic signal. We hypothesized that metabolic changes during cardiomyocyte hypertrophy might involve interaction between glycolysis and fatty acid metabolism, specifically via O-GlcNAc modification of PGC-1α. Methods and Results: Mechanical stretch (24 h at 4%; Flexercell FX-4000) in neonatal rat cardiomyocytes (n > 4/group) induced a significant (p<0.05) increase (113 ± 35% over No Stretch) in ANP mRNA, confirming induction of hypertrophy. Mechanical stretch significantly augmented (p<0.001; n = 5) global O-GlcNAcylation of several proteins, which was completely reversed by adenoviral overexpression of the deglycosylating enzyme (O-GlcNAcase). Mechanical stretch also augmented mRNA levels of O-GlcNAc transferase (OGT: adds O-GlcNAc to proteins) and glutamine:fructose aminotransferase (GFAT: rate-limiting step for the O-GlcNAc sugar donor), further indicating recruitment of O-GlcNAc signaling. Immunoprecipitation identified PGC-1α as an O-GlcNAc target in this cardiomyocyte hypertrophy model. Real-time (q)-PCR revealed that O-GlcNAc modification of PGC-1α correlated with elevated mRNA levels (n=4/group) of MCAD and COXIV-5b, implying transcriptional activation of PGC-1α. Conclusions: Cardiomyocyte hypertrophy induces O-GlcNAcylation of PGC-1α and represents a surprising and novel potential regulatory interaction between glycolytic and fatty acid metabolism. This research has received full or partial funding support from the American Heart Association, AHA National Center.

Survey of O-GlcNAc level variations in Xenopus laevis from oogenesis to early development

Little is known about the impact of O-linked-N-acetylglucosaminylation (O-GlcNAc) in gametes production and developmental processes. Here we investigated changes in O-GlcNAc, UDP-GlcNAc and O-GlcNAc transferase (OGT) levels in Xenopus laevis from oogenesis to embryo hatching. We showed that in comparison to stage VI, stages I-V oocytes expressed higher levels of O-GlcNAc correlating changes in OGT expression, but not in UDP-GlcNAc pools. Upon progesterone stimulation, an O-GlcNAc level burst occurred during meiotic resumption long before MPF and Mos-Erk2 pathways activations. Finally, we observed high levels of O-GlcNAc, UDP-GlcNAc and OGT during segmentation that decreased concomitantly at the onset of gastrulation. Nevertheless, no correlation between the glycosylation, the nucleotide-sugar and the glycosyltransferase was observed after neurulation. Our results show that O-GlcNAc is regulated throughout oogenesis and development within a complex pattern and suggest that dysfunctions in the dynamics of this glycosylation could lead to developmental abnormalities.

Aging leads to increased levels of protein O-linked N-acetylglucosamine in heart, aorta, brain and skeletal muscle in Brown-Norway rats

Changes in the levels of O-linked N-acetyl-glucosamine (O-GlcNAc) on nucleocytoplasmic protein have been associated with a number of age-related diseases such as Alzheimer's and diabetes; however, there is relatively little information regarding the impact of age on tissue O-GlcNAc levels. Therefore, the goal of this study was to determine whether senescence was associated with alterations in O-GlcNAc in heart, aorta, brain and skeletal muscle and if so whether there were also changes in the expression of enzymes critical in regulating O-GlcNAc levels, namely, O-GlcNAc transferase (OGT), O-GlcNAcase and glutamine:fructose-6-phosphate amidotransferase (GFAT). Tissues were harvested from 5- and 24-month old Brown-Norway rats; UDP-GlcNAc, a precursor of O-GlcNAc was assessed by HPLC, O-GlcNAc and OGT levels were assessed by immunoblot analysis and GFAT1/2, OGT, O-GlcNAcase mRNA levels were determined by RT-PCR. In the 24-month old animals serum insulin and triglyceride levels were significantly increased compared to the 5-month old group; however, glucose levels were unchanged. Protein O-GlcNAc levels were significantly increased with age (30-107%) in all tissues examined; however, paradoxically the expression of OGT, which catalyzes O-GlcNAc formation, was decreased by approximately 30% in the heart, aorta and brain. In the heart increased O-GlcNAc was associated with increased UDP-GlcNAc levels and elevated GFAT mRNA while in other tissues we found no difference in UDP-GlcNAc or GFAT mRNA levels. These results demonstrate that senescence is associated with increased O-GlcNAc levels in multiple tissues and support the notion that dysregulation of pathways leading to O-GlcNAc formation may play an important role in the development of age-related diseases.

AGING-RELATED ACCUMULATION OF O-GLCNACYLATED PROTEINS IN THE CARDIOVASCULAR SYSTEM.

Background Excessive post-translational modification of nucleocytoplasmic proteins by the addition of O-linked β-N-acetylglucosamine (O-GlcNAc) to Ser and Thr residues by the enzyme O-GlcNAc transferase (OGT) is a proposed mechanism for diabetogenic injury to the cardiovascular system. The current study tested the hypothesis that O-GlcNAcylation exhibits aging-related alterations in the cardiovascular system in normal aging rats. Methods Young (10 weeks old) and aged (22 months old) female Sprague-Dawley rats were sacrificed, serum was collected, and tissues (carotid artery, heart, liver, and brain) were harvested for assessment of serum glucose, insulin, estradiol, and lipid profiles and for quantification of tissue OGT and O-GlcNAc levels by Western blot analysis. Results Serum glucose and tissue OGT expression were not significantly different in young and aged animals. Aged animals demonstrated significantly greater O-GlcNAcylated protein expression compared with young rats in carotid arteries and heart but not in liver and brain. Conclusions There are tissue-specific, aging-related accumulations of O-GlcNAcylated proteins in cardiovascular tissues that are not accounted for by changes in glucose or OGT expression. The chronic accumulation of O-GlcNAcylated intracellular proteins may contribute to the adverse effects of age in the cardiovascular system.