O-GlcNAcylated Protein Research Articles

Glucose and O-GlcNAcylated protein regulation of maternal-placental interactions

Glucose and O-GlcNAcylated protein regulation of maternal-placental interactions

Metformin inhibits cervical cancer cell proliferation via decreased AMPK O-GlcNAcylation

ABSTRACTMetformin is a widely used drug for the treatment of type 2 diabetes. Antidiabetic drugs are also known to influence cancer progression, as high glucose levels affect both cancer and diabetes. Metformin induces cell cycle arrest in cancer cells, but the underlying mechanism remains unclear in cervical cancer system. Here, we examined how metformin affects cell cycle arrest and apoptosis in cervical cancer cells. Western blot analysis showed that levels of O-linked N-acetylglucosamine (O-GlcNAc) and O-GlcNAc transferase (OGT) were increased in cervical cancer cells; these effects were reversed by metformin treatment. Immunoprecipitation analysis was used to examine the interplay between O-GlcNAcylation and phosphorylation in HeLa cells, revealing that metformin decreased O-GlcNAcylated AMP-activated protein kinase (AMPK) and increased levels of phospho-AMPK compared to untreated cells. These results were associated with decreased cell cycle arrest and apoptotic cell death in HeLa cells, as shown by flow cytometry. Moreover, 6-diazo-5-oxo-L-norleucine (a glutamine fructose-6-phosphate aminotransferase inhibitor) or thiamet G (an O-GlcNAcase inhibitor) decreased or increased levels of O-GlcNAcylated AMPK, and increased or decreased levels of phosphorylated AMPK, respectively, suggesting that O-GlcNAc modification affects AMPK activation. Of note, we found that metformin treatment of HeLa cells increased the levels of p21 and p27 (which are AMPK-dependent cell cycle inhibitors), leading to increased cell cycle arrest and apoptosis in HeLa cells compared to untreated cells. These findings suggest that metformin may serve as a useful antiproliferative drug in cervical cancer cells, with potential therapeutic benefit.

Optimization of Chemoenzymatic Mass Tagging by Strain-Promoted Cycloaddition (SPAAC) for the Determination of O-GlcNAc Stoichiometry by Western Blotting.

The dynamic modification of intracellular proteins by O-linked β -N-acetylglucosamine (O-GlcNAcylation) plays critical roles in many cellular processes. Although various methods have been developed for O-GlcNAc detection, there are few techniques for monitoring glycosylation stoichiometry and state (i.e., mono-, di-, etc., O-GlcNAcylated). Measuring the levels of O-GlcNAcylation on a given substrate protein is important for understanding the biology of this critical modification and for prioritizing substrates for functional studies. One powerful solution to this limitation involves the chemoenzymatic installation of polyethylene glycol polymers of defined molecular mass onto O-GlcNAcylated proteins. These "mass tags" produce shifts in protein migration during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)that can be detected by Western blotting. Broad adoption of this method by the scientific community has been limited, however, by a lack of commercially available reagents and well-defined protein standards. Here, we develop a "click chemistry" approach to this method using entirely commercial reagents and confirm the accuracy of the approach using a semisynthetic O-GlcNAcylated protein. Our studies establish a new, expedited experimental workflow and standardized methods that can be readily utilized by non-experts to quantify the O-GlcNAc stoichiometry and state on endogenous proteins in any cell or tissue lysate.

A widely compatible expression system for the production of highly O-GlcNAcylated recombinant protein in Escherichia coli.

O-GlcNAcylation is a ubiquitous and dynamic post-translational modification on serine/threonine residues of nucleocytoplasmic proteins in metazoa, which plays a critical role in numerous physiological and pathological processes. But the O-GlcNAcylation on most proteins is often substoichiometric, which hinders the functional study of the O-GlcNAcylation. This study aimed to improve the production of highly O-GlcNAcylated recombinant proteins in Escherichia coli (E. coli). To achieve this goal, we constructed a bacterial artificial chromosome-based chloramphenicol-resistant expression vector co-expressing O-GlcNAc transferase (OGT) and key enzymes (phosphoglucose mutase, GlmM and N-acetylglucosamine-1-phosphate uridyltransferase, GlmU) of the uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) synthesis pathway in E. coli, which can effectively increase the O-GlcNAcylation of the OGT target protein expressed by another vector. The results revealed that the expression of GlmM and GlmU increases the cellular concentration of UDP-GlcNAc in E. coli, which markedly enhanced the activity of the co-expressed OGT to its target proteins, such as H2B, p53 and TAB1. Altogether, we established a widely compatible E. coli expression system for producing highly O-GlcNAcylated protein, which could be used for modifying OGT target proteins expressed by almost any commercial expression vectors in E. coli. This new expression system provides possibility for investigating the roles of O-GlcNAcylation in the enzymatic activity, protein-protein interaction and structure of OGT target proteins.

O-deGlcNAcylation is required for Entamoeba histolytica-induced HepG2 cell death

Entamoeba histolytica is an enteric tissue-invading protozoan parasite that causes amoebic colitis and occasionally liver abscess in humans. E. histolytica can induce host-cell apoptosis by initiating various intracellular signaling mechanisms closely associated with tissue pathogenesis and parasitic immune evasion. O-GlcNAcylation, similar to phosphorylation, is involved in various cell-signaling processes, including apoptosis and proliferation, with O-GlcNAc addition and removal regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. However, whether O-GlcNAc alterations in host cells affect E. histolytica-induced cell death and which signal molecules participate in E. histolytica-induced deglycosylation remain unknown. In this study, co-incubation of HepG2 cells with E. histolytica increased DNA fragmentation and LDH release as compared with control cells. Additionally, Gal-lectin-mediated amoebic adherence of live trophozoites to HepG2 cells decreased O-GlcNAcylated protein levels within 5 min. We also observed a rapid decrease in cellular OGT protein level, but not OGA, in HepG2 cells in a contact-dependent manner. Furthermore, HepG2 pretreatment with OGA inhibitors or OGA siRNA prevented E. histolytica-induced O-deGlcNAcylation, DNA fragmentation, and LDH release. Our results suggested that E. histolytica-induced O-deGlcNAcylation in HepG2 cells was an important process required for hepatocyte cell death induced by E. histolytica adherence.

WGA-based lectin affinity gel electrophoresis: A novel method for the detection of O-GlcNAc-modified proteins.

Post-translational modification with O-linked β-N-acetylglucosamine (O-GlcNAc) occurs selectively on serine and/or threonine residues of cytoplasmic and nuclear proteins, and dynamically regulates their molecular functions. Since conventional strategies to evaluate the O-GlcNAcylation level of a specific protein require time-consuming steps, the development of a rapid and easy method for the detection and quantification of an O-GlcNAcylated protein has been a challenging issue. Here, we describe a novel method in which O-GlcNAcylated and non-O-GlcNAcylated forms of proteins are separated by lectin affinity gel electrophoresis using wheat germ agglutinin (WGA), which primarily binds to N-acetylglucosamine residues. Electrophoresis of cell lysates through a gel containing copolymerized WGA selectively induced retardation of the mobility of O-GlcNAcylated proteins, thereby allowing the simultaneous visualization of both the O-GlcNAcylated and the unmodified forms of proteins. This method is therefore useful for the quantitative detection of O-GlcNAcylated proteins.

Validation, Identification, and Biological Consequences of the Site-specific O-GlcNAcylation Dynamics of Carbohydrate-responsive Element-binding Protein (ChREBP).

O-GlcNAcylation of carbohydrate-responsive element-binding protein (ChREBP) is believed as an important modulator of ChREBP activities, however little direct evidence of O-GlcNAcylation on ChREBP and no exact O-GlcNAcylation sites have been reported so far. Here, we validate O-GlcNAcylation on ChREBP in cell-free coupled transcription/translation system and in cells by chemoenzymatic and metabolic labeling, respectively. Moreover, for the first time, we identify O-GlcNAcylation on Ser614 in the C-terminus of ChREBP by mass spectrometry and validate two important sites, Thr517 and Ser839 for O-GlcNAcylation and their function via molecular and chemical biological method. Under high glucose conditions, Ser514 phosphorylation enhances ChREBP O-GlcNAcylation, maintaining the transcriptional activity of ChREBP; Ser839 O-GlcNAcylation is essential for Mlx-heterodimerization and DNA-binding activity enhancement, consequently inducing transcriptional activity. Ser839 O-GlcNAcylation is also crucial for ChREBP nuclear export partially by strengthening interactions with CRM1 and 14-3-3. This work is a detailed study of ChREBP O-GlcNAcylation and highlights the biological consequences of the site-specific O-GlcNAcylation dynamics of ChREBP.

The Sodium-Glucose Cotransporter 2 Inhibitor Dapagliflozin Prevents Cardiomyopathy in a Diabetic Lipodystrophic Mouse Model.

Type 2 diabetes mellitus (T2DM) is a well-recognized independent risk factor for heart failure. T2DM is associated with altered cardiac energy metabolism, leading to ectopic lipid accumulation and glucose overload, the exact contribution of these two parameters remaining unclear. To provide new insight into the mechanism driving the development of diabetic cardiomyopathy, we studied a unique model of T2DM: lipodystrophic Bscl2-/- (seipin knockout [SKO]) mice. Echocardiography and cardiac magnetic resonance imaging revealed hypertrophic cardiomyopathy with left ventricular dysfunction in SKO mice, and these two abnormalities were strongly correlated with hyperglycemia. Surprisingly, neither intramyocardial lipid accumulation nor lipotoxic hallmarks were detected in SKO mice. [18F]Fludeoxyglucose positron emission tomography showed increased myocardial glucose uptake. Consistently, the O-GlcNAcylated protein levels were markedly increased in an SKO heart, suggesting a glucose overload. To test this hypothesis, we treated SKO mice with the hypoglycemic sodium-glucose cotransporter 2 (SGLT2) inhibitor dapagliflozin and the insulin sensitizer pioglitazone. Both treatments reduced the O-GlcNAcylated protein levels in SKO mice, and dapagliflozin successfully prevented the development of hypertrophic cardiomyopathy. Our data demonstrate that glucotoxicity by itself can trigger cardiac dysfunction and that a glucose-lowering agent can correct it. This result will contribute to better understanding of the potential cardiovascular benefits of SGLT2 inhibitors.

405 - Hyper-O-GlcNAcylation Attenuates Autophagic Flux in an MTOR Dependent Manner and Leads to Accumulation of α-Synuclein in Neurons

O-GlcNAcylation is one of the unique forms of glycosylation which involves the β-O-linkage of N-acetylglucosamine (O-GlcNAc) to cellular proteins. This unique post translational modification is involved in a vast array of physiological and pathological functions. To determine the impact of increased O-GlcNAcylation on neuronal function and survival, we cultured rat primary cortical neurons and exposed them with thiamet G, a highly selective OGA inhibitor. We found that thiamet G significantly increased protein O-GlcNAcylation, increased p-mTOR/mTOR, and decreased autophagic flux. This was accompanied by an accumulation of aggregation-prone, Parkinson’s disease causing α-synuclein protein. To determine whether enhancing autophagy in the presence of hyper-O-GlcNAcylation may attenuate α-synuclein accumulation, we exposed neurons to rapamycin, an mTOR dependent autophagy inducer. Rapamycin alone significantly decreased basal O-GlcNAcylated protein levels, without decreasing thiamet-G induced hyper-O-GlcNAcylation. Interestingly, rapamycin partially rescued hyper-O-GlcNAcylation-induced autophagic inhibition as indicated by a partial decrease of p62 levels in the presence of rapamycin in neurons exposed to thiamet G. Furthermore, thiamet-G induced α-synuclein accumulation was also partially decreased by rapamycin. Taken together the results of this study suggest that mTOR activation may be one of the mechanisms involved in hyper-O-GlcNAcylation induced autophagic inhibition and α-synuclein accumulation.

Semi-synthesis of a tag-free O-GlcNAcylated tau protein by sequential chemoselective ligation.

In this paper, the first semi-synthesis of the Alzheimer-relevant tau protein carrying an O-GlcNAcylation is demonstrated by using sequential chemoselective ligation. The 52-amino acid C-terminus of tau was obtained by native chemical ligation between two synthetic peptide fragments, one carrying the O-GlcNAc moiety on Ser400, which has recently been demonstrated to inhibit tau phosphorylation and to hinder tau oligomerization, and the other equipped with a photocleavable biotin handle. After desulfurization to deliver a native alanine at the ligation junction, the N-terminal cysteine was unmasked, and the peptide was further used for expressed protein ligation to generate the full-length tau protein, which was purified by a photocleavable biotin tag. We thus provide a synthetic route to obtain a homogenous tag-free O-GlcNAcylated tau protein that can further help to elucidate the significance of posttranslational modification on the tau protein and pave the way for evaluating possible drug targets in Alzheimer's disease. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

A highly effective and adjustable dual plasmid system for O-GlcNAcylated recombinant protein production in E. coli.

O-GlcNAcylation is a ubiquitous, dynamic and reversible post-translational protein modification in metazoans, and it is catalysed and removed by O-GlcNAc transferase (OGT) and O-GlcNAcase, respectively. Prokaryotes lack endogenous OGT activity. It has been reported that coexpression of mammalian OGT with its target substrates in Escherichia coli produce O-GlcNAcylated recombinant proteins, but the plasmids used were not compatible, and the expression of both OGT and its target protein were induced by the same inducer. Here, we describe a compatible dual plasmid system for coexpression of OGT and its target substrate for O-GlcNAcylated protein production in E. coli. The approach was validated using the CKII and p53 protein as control. This compatible dual plasmid system contains an arabinose-inducible OGT expression vector with a pUC origin and an isopropyl β-d-thiogalactopyranoside-inducible OGT target substrate expression vector bearing a p15A origin. The dual plasmid system produces recombinant proteins with varying O-GlcNAcylation levels by altering the inducer concentration. More importantly, the O-GlcNAcylation efficiency was much higher than the previously reported system. Altogether, we established an adjustable compatible dual plasmid system that can effectively yield O-GlcNAcylated proteins in E. coli.

In vivo putative O-GlcNAcylation of human SCP1 and evidence for possible role of its N-terminal disordered structure.

RNA polymerase II carboxyl-terminal domain (RNAPII CTD) phosphatases are responsible for the dephosphorylation of the C-terminal domain of the small subunit of RNAPII in eukaryotes. Recently, we demonstrated the identification of several interacting partners with human small CTD phosphatase1 (hSCP1) and the substrate specificity to delineate an appearance of the dephosphorylation catalyzed by SCP1. In this study, using the established cells for inducibly expressing hSCP1 proteins, we monitored the modification of β-O-linked N-acetylglucosamine (O-GlcNAc). O-GlcNAcylation is one of the most common post-translational modifications (PTMs). To gain insight into the PTM of hSCP1, we used the Western blot, immunoprecipitation, succinylayed wheat germ agglutininprecipitation, liquid chromatography-mass spectrometry analyses, and site-directed mutagenesis and identified the Ser41 residue of hSCP1 as the O-GlcNAc modification site. These results suggest that hSCP1 may be an O-GlcNAcylated protein in vivo, and its N-terminus may function a possible role in the PTM, providing a scaffold for binding the protein(s). [BMB Reports 2014; 47(10): 593-598]

Extracellular O-linked β-N-acetylglucosamine: Its biology and relationship to human disease.

The O-linked β-N-acetylglucosamine (O-GlcNAc)ylation of cytoplasmic and nuclear proteins regulates basic cellular functions and is involved in the etiology of neurodegeneration and diabetes. Intracellular O-GlcNAcylation is catalyzed by a single O-GlcNAc transferase, O-GlcNAc transferase (OGT). Recently, an atypical O-GlcNAc transferase, extracellular O-linked β-N-acetylglucosamine (EOGT), which is responsible for the modification of extracellular O-GlcNAc, was identified. Although both OGT and EOGT are regulated through the common hexosamine biosynthesis pathway, EOGT localizes to the lumen of the endoplasmic reticulum and transfers GlcNAc to epidermal growth factor-like domains in an OGT-independent manner. In Drosophila, loss of Eogt gives phenotypes similar to those caused by defects in the apical extracellular matrix. Dumpy, a membrane-anchored apical extracellular matrix protein, was identified as a major O-GlcNAcylated protein, and EOGT mediates Dumpy-dependent cell adhesion. In mammals, extracellular O-GlcNAc was detected on extracellular proteins including heparan sulfate proteoglycan 2, Nell1, laminin subunit alpha-5, Pamr1, and transmembrane proteins, including Notch receptors. Although the physiological function of O-GlcNAc in mammals has not yet been elucidated, exome sequencing identified homozygous EOGT mutations in patients with Adams-Oliver syndrome, a rare congenital disorder characterized by aplasia cutis congenita and terminal transverse limb defects. This review summarizes the current knowledge of extracellular O-GlcNAc and its implications in the pathological processes in Adams-Oliver syndrome.

High Glucose-induced O-GlcNAcylated Carbohydrate Response Element-binding Protein (ChREBP) Mediates Mesangial Cell Lipogenesis and Fibrosis: THE POSSIBLE ROLE IN THE DEVELOPMENT OF DIABETIC NEPHROPATHY

Carbohydrate response element-binding protein (ChREBP) is a transcription factor responsible for carbohydrate metabolism in the liver. However, the role of ChREBP in diabetic nephropathy has not been elucidated. Thus, we investigated the role of ChREBP in mesangial cells in diabetic nephropathy. Treatment with 25 mM glucose (high glucose; HG) increased cellular O-GlcNAc and O-GlcNAcylated ChREBP in mesangial cells compared with normal 5.5 mM glucose. O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino N-phenylcarbamate (PUGNAc), a drug that increases O-GlcNAc, augmented the expression of ChREBP targets, whereas DON, a drug that decreases O-GlcNAc and O-GlcNAcase overexpression, mitigated the increase with HG. O-GlcNAc augmented the protein stability, transcriptional activity, and nuclear translocation of ChREBP. HG treatment also stimulated lipid accumulation and the contents of triglyceride and cholesterol in mesangial cells. In addition, HG triggered expression of hypoxia-inducible factor 1-α, vascular endothelial growth factor, and extracellular matrix components related to nephrosclerosis. The ChREBP mutant, W130A, did not exhibit HG-induced lipid accumulation and fibrotic proteins, suggesting that the Trp-130 residue in the MCR3 domain is important in the development of glomerulosclerosis. O-GlcNAcylated ChREBP was elevated in mesangium cells of streptozotocin-induced diabetic rats. In conclusion, HG increased the O-GlcNAcylated ChREBP level, which resulted in lipid accumulation and up-regulation of fibrotic proteins in mesangial cells. These effects may lead mesangial cells to an ultimately pathological state.

Hyperglycaemic conditions perturb mouse oocyte in vitro developmental competence via beta-O-linked glycosylation of Heat shock protein 90

What is the effect of beta-O-linked glycosylation (O-GlcNAcylation) on specific proteins in the cumulus-oocyte complex (COC) under hyperglycaemic conditions? Heat shock protein 90 (HSP90) was identified and confirmed as being O-GlcNAcylated in mouse COCs under hyperglycaemic conditions (modelled using glucosamine), causing detrimental outcomes for embryo development. O-GlcNAcylation of proteins occurs as a result of increased activity of the hexosamine biosynthesis pathway, which provides substrates for cumulus matrix production during COC maturation, and also for O-GlcNAcylation. COCs matured under hyperglycaemic conditions have decreased developmental competence, mediated at least in part through the mechanism of increased O-GlcNAcylation. This study was designed to examine the effect of hyperglycaemic conditions (using the hyperglycaemic mimetic, glucosamine) on O-GlcNAc levels in the mouse COC, and furthermore to identify potential candidate proteins which are targets of this modification, and their roles in oocyte maturation. COCs from 21-day-old superovulated CBA × C57BL6 F1 hybrid female mice were matured in vitro (IVM). Levels of O-GlcNAcylated proteins, HSP90 and O-GlcNAc transferase (OGT, the enzyme responsible for O-GlcNAcylation) in COCs were measured using western blot, and localization observed using immunocytochemistry. For glycosylated HSP90 levels, and to test OGT-HSP90 interaction, immunoprecipitation was performed prior to western blotting. Embryo development was assessed using in vitro fertilization and embryo culture post-maturation. Addition of the hyperglycaemic mimetic glucosamine to IVM medium for mouse COCs increased detectable O-GlcNAcylated protein levels (by western blot and immunocytochemistry), and this effect was reversed using an OGT inhibitor (P < 0.05). HSP90 was identified as a target of O-GlcNAcylation in the COC, and inhibition of HSP90 during IVM reversed glucosamine-induced decreases in oocyte developmental competence (P < 0.05). We also demonstrated the novel finding of an association between HSP90 and OGT in COCs, suggesting a possible client-chaperone relationship. In vitro maturation of COCs was used so that treatment time could be limited to the 17 h of maturation prior to ovulation. Additionally, glucosamine, a hyperglycaemic mimetic, was used because it specifically activates the hexosamine pathway which provides the O-GlcNAc moieties. The results in this study should be confirmed using in vivo models of hyperglycaemia and different HSP90 inhibitors. This study leads to a new understanding of how diabetes influences oocyte competence and provides insight into possible therapeutic interventions based on inhibiting HSP90 to improve oocyte quality. This work was supported by a programme grant from the National Health and Medical Research Council, Australia, ID 453556. J.G.T. is a recipient of funding from and a consultant to Cook Medical Pty Ltd. The other authors have no conflicts of interest to declare.

O‐GlcNAcylation modifies the metastatic properties of prostate cancer cells (789.5)

Prostate cancer is one of the most common forms of cancer in men and the second leading cause of cancer deaths in men in the United States. O‐linked β‐N‐acetylglucosamine (O‐GlcNAc) is a ubiquitous and dynamic post‐translational modification that exists on serine and threonine residues of nuclear and cytoplasmic proteins. This modification is regulated by O‐GlcNAc transferase (OGT), which attaches O‐GlcNAc to proteins, and O‐GlcNAcase (OGA), which removes O‐GlcNAc. O‐GlcNAc serves as a nutrient sensor to regulate virtually all cellular processes as well as playing roles in the various diseases, including Alzheimer's disease, diabetes, and cancer. The roles of O‐GlcNAc in the regulation of prostate cancer remains unclear. Herein, we investigate how the regulation of O‐GlcNAc may affect prostate cancer, progression using normal prostate epithelial cell (PrEc) and prostate cancer cell lines (LNCaP and PC‐3) as models. Using western blotting techniques, we show that O‐GlcNAc, OGT, and OGA are highly elevated in LNCaP and PC‐3 when compared to PrEc. LNCaP exhibited the highest levels of O‐GlcNAc, OGT, and OGA. OGT and OGA are differentially localized in lines with different metastatic properties. Additionally, 2D‐gel electrophoretic analyses show significant differences in O‐GlcNAcylated protein profiles among the three cell lines. In order to answer how O‐GlcNAc affects the biological properties of these cancer cells, including shapes, proliferation, invasion and metastasis, several assays were used. The result of soft agar assays indicate that alterations in O‐GlcNAc influence the metastasis ability of prostate cancer.

Entamoeba histolytica induces cell death in HepG2 cells via the change of protein O-GlcNAcylation (P3355)

Abstract Entamoeba histolytica is an enteric tissue-invasion protozoan parasite that causes amoebic colitis and liver abscess in humans. E. histolytica can induce host cell apoptosis to evade host immune responses. Protein O-GlcNAcyation has been thought to contribute to the various cellular signal processes including apoptosis and proliferation. O-GlcNAc addition and removal are regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. However, it is unknown whether the alteration of O-GlcNAcylated protein level in host cells can affect E. histolytican-induced cell death. In this study, co-incubation of HepG2 cells with virulent amoeba HM-1 strain remarkably increased DNA fragmentation and LDH release compared to cells incubated with medium alone. In addition, Entamoeba HM-1 induced activation of caspases-3 and calpain in HepG2 cells. Amoebic adherence to the HepG2 cells showed the decrease of O-GlcNAcylated protein levels in HepG2 cells within 2 min, while non-virulent Rahman strain did not. Indeed, pretreatment of host cells with O-GlcNAcase inhibitors (PUGNAc or Thi-Met G) or OGA siRNA prevented the E. histolytica-induced protein deGlcNAcylation and cell death in HepG2 cells. These results suggest that decrease of protein O-GlcNAcylation is an important process required for hepatocyte cell death induced by adherence of E. histolytica.

Early effects of high-fat diet on neurovascular function and focal ischemic brain injury

Obesity is a risk factor for stroke, but the early effects of high-fat diet (HFD) on neurovascular function and ischemic stroke outcomes remain unclear. The goal of this study was to test the hypotheses that HFD beginning early in life 1) impairs neurovascular coupling, 2) causes cerebrovascular dysfunction, and 3) worsens short-term outcomes after cerebral ischemia. Functional hyperemia and parenchymal arteriole (PA) reactivity were measured in rats after 8 wk of HFD. The effect of HFD on basilar artery function after middle cerebral artery occlusion (MCAO) and associated O-GlcNAcylation were assessed. Neuronal cell death, infarct size, hemorrhagic transformation (HT) frequency/severity, and neurological deficit were evaluated after global ischemia and transient MCAO. HFD caused a 10% increase in body weight and doubled adiposity without a change in lipid profile, blood glucose, and blood pressure. Functional hyperemia and PA relaxation were decreased with HFD. Basilar arteries from stroked HFD rats were more sensitive to contractile factors, and acetylcholine-mediated relaxation was impaired. Vascular O-GlcNAcylated protein content was increased with HFD. This group also showed greater mortality rate, infarct volume, HT occurrence rate, and HT severity and poor functional outcome compared with the control diet group. These results indicate that HFD negatively affects neurovascular coupling and cerebrovascular function even in the absence of dyslipidemia. These early cerebrovascular changes may be the cause of greater cerebral injury and poor outcomes of stroke in these animals.

A dynamic view to the modulation of phosphorylation and O-GlcNAcylation by inhibition of O-GlcNAcase

Protein phosphorylation and O-GlcNAcylation are reciprocally regulated. As hyperphosphorylation is implicated in tau pathology, approaches have been exploited to reduce the magnitude of tau phosphorylation by increasing the level of tau O-GlcNAcylation. With mathematic models constructed to describe different kinetic scenarios, we analyzed the temporal change of an O-GlcNAcylated protein in contrast to that of the phosphorylated form upon inhibition of O-GlcNAcase (OGA). The analyses indicate that when degradation of the modified protein is negligible relative to the naked one, the magnitude of O-GlcNAcylated protein increase is proportional to the level of inhibition, while the extent of phosphorylated protein decline varies due to other factors. Furthermore, the increase of O-GlcNAcylated protein parallels with the decrease of phosphorylated form upon acute or short-term inhibition of OGA, as observed in many in vitro and short term in vivo studies. However, phosphorylated protein is predicted to return to its initial level while O-GlcNAcylated protein to achieve a higher steady level under sustained inhibition. This simulated result is in line with a recent report on long-term inhibition of OGA in transgenic mice. Noticeably, inhibition withdrawal is anticipated to cause a transient rise of phosphorylated protein. If degradation of modified proteins proceeds in addition to the naked one, the characteristic temporal profiles of each form in response to OGA inhibition would depend on the relative importance of individual degradation pathways. The models described herein may serve as a useful investigational tool that will provide insight into pharmacological intervention for tauopathies in particular and for reciprocally modulated reactions in general.

A correlation between altered O-GlcNAcylation, migration and with changes in E-cadherin levels in ovarian cancer cells

O-GlcNAcylation is a dynamic and reversible posttranslational modification of nuclear and cytoplasmic proteins. In recent years, the roles of O-GlcNAcylation in several human malignant tumors have been investigated, and O-GlcNAcylation was found to be linked to cellular features relevant to metastasis. In this study, we modeled four diverse ovarian cancer cells and investigated the effects of O-GlcNAcylation on ovarian cancer cell migration. We found that total O-GlcNAcylation level was elevated in HO-8910PM cells compared to OVCAR3 cells. Additionally, through altering the total O-GlcNAcylation level by OGT silencing or OGA inhibition, we found that the migration of OVCAR3 cells was dramatically enhanced by PUGNAc and Thiamet G treatment, and the migration ability of HO-8910PM cells was significantly inhibited by OGT silencing. Furthermore, we also found that the expression of E-cadherin, an O-GlcNAcylated protein in ovarian cancer cells, was reduced by OGA inhibition in OVCAR3 cells and elevated by OGT silencing in HO-8910PM cells. These results indicate that O-GlcNAcylation could enhance ovarian cancer cell migration and decrease the expression of E-cadherin. Our studies also suggest that O-GlcNAcylation might become another potential target for the therapy of ovarian cancer.