Glucosamine inhibits angiotensin II-induced cytoplasmic Ca2+ elevation in neonatal cardiomyocytes via protein-associated O-linked N-acetylglucosamine

We previously reported that glucosamine and hyperglycemia attenuate the response of cardiomyocytes to inositol 1,4,5-trisphosphate-generating agonists such as ANG II. This appears to be related to an increase in flux through the hexosamine biosynthesis pathway (HBP) and decreased Ca²⁺ entry into the cells; however, a direct link between HBP and intracellular Ca²⁺ homeostasis has not been established. Therefore, using neonatal rat ventricular myocytes, we investigated the relationship between glucosamine treatment; the concentration of UDP-N-acetylglucosamine (UDP-GlcNAc), an end product of the HBP; and the level of protein O-linked N-acetylglucosamine (O-GlcNAc) on ANG II-mediated changes in intracellular free Ca²⁺ concentration ([Ca²⁺]_i). We found that glucosamine blocked ANG II-induced [Ca²⁺]_i increase and that this phenomenon was associated with a significant increase in UDP-GlcNAc and O-GlcNAc levels. O-(2-acetamido-2-deoxy-D-glucopyranosylidene)-amino-N-phenylcarbamate, an inhibitor of O-GlcNAcase that increased O-GlcNAc levels without changing UDP-GlcNAc concentrations, mimicked the effect of glucosamine on the ANG II-induced increase in [Ca²⁺]_i. An inhibitor of O-GlcNAc-transferase, alloxan, prevented the glucosamine-induced increase in O-GlcNAc but not the increase in UDP-GlcNAc; however, alloxan abrogated the inhibition of the ANG II-induced increase in [Ca²⁺]_i. These data support the notion that changes in O-GlcNAc levels mediated via increased HBP flux may be involved in the regulation of [Ca²⁺]_i homeostasis in the heart. hypertrophy; left ventricle; calcium channels; calcium signaling     

Glucosamine protects neonatal cardiomyocytes from ischemia-reperfusion injury via increased protein O-GlcNAc and increased mitochondrial Bcl-2

We have previously reported that glucosamine protected neonatal rat ventricular myocytes against ischemia-reperfusion (I/R) injury, and this was associated with an increase in protein O-linked-N-acetylglucosamine (O-GlcNAc) levels. However, the protective effect of glucosamine could be mediated via pathways other that O-GlcNAc formation; thus the initial goal of the present study was to determine whether increasing O-GlcNAc transferase (OGT) expression, which catalyzes the formation of O-GlcNAc, had a protective effect similar to that of glucosamine. To better understand the potential mechanism underlying O-GlcNAc-mediated cytoprotection, we examined whether increased O-GlcNAc levels altered the expression and translocation of members of the Bcl-2 protein family. Both glucosamine (5 mM) and OGT overexpression increased basal and I/R-induced O-GlcNAc levels, significantly decreased cellular injury, and attenuated loss of cytochrome c. Both interventions also attenuated the loss of mitochondrial membrane potential induced by H₂O₂ and were also associated with an increase in mitochondrial Bcl-2 levels but had no effect on Bad or Bax levels. Compared with glucosamine and OGT overexpression, NButGT (100 µM), an inhibitor of O-GlcNAcase, was less protective against I/R and H₂O₂ and did not affect Bcl-2 expression, despite a 5- to 10-fold greater increase in overall O-GlcNAc levels. Decreased OGT expression resulted in lower basal O-GlcNAc levels, prevented the I/R-induced increase in O-GlcNAc and mitochondrial Bcl-2, and increased cellular injury. These results demonstrate that the protective effects of glucosamine are mediated via increased formation of O-GlcNAc and suggest that this is due, in part, to enhanced mitochondrial Bcl-2 translocation. mitochondria; apoptosis; necrosis, O-linked-N-acetylglucosamine     

Selective decrease of membrane-associated PKC-α and PKC-ε in response to elevated intracellular O-GlcNAc levels in transformed human glial cells

Increased flux through the hexosamine biosynthetic pathway (HBP) has been shown to affect the activity and translocation of certain protein kinase C (PKC) isoforms. It has been suggested that this effect is due to increases in the β-O-linked N-acetylglucosamine (O-GlcNAc) modification. Herein, we demonstrate the effect of increasing the O-GlcNAc modification on the translocation of select PKC isozymes in a human astroglial cell line. Treating cells with either 8 mM d-glucosamine (GlcN), 5 mM streptozotocin (STZ), or 80 μM O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) produced a significant increase in the O-GlcNAc modification on both cytosolic and membrane proteins; however, both the level and rate of O-GlcNAc increase varied with the compound. GlcN treatment resulted in a rapid, transient translocation of PKC-βII that was maximal after 3 h (73±8%) and also produced a 48±15% decrease in membrane-associated PKC-ε after 9 h of treatment. Similar to GlcN treatment, STZ and PUGNAc treatment also resulted in decreased levels of PKC-ε in the membrane fraction. Significant decreases were seen as early as 5 h and, by 9 h of treatment, had decreased by 87±6% with STZ and 73±7% with PUGNAc. Unlike GlcN, both STZ and PUGNAc produced a decrease in PKC-α membrane levels by 9 h posttreatment (78±10% with STZ and 66±8% with PUGNAc) while neither compound produced any changes in PKC-βII translocation. In addition, none of the three compounds affected membrane levels of PKC-ι. Altogether, these results demonstrate a novel link between increased levels of the O-GlcNAc modification and the regulation of specific PKC isoforms.     

Impact of Type 2 diabetes and aging on cardiomyocyte function and O-linked N-acetylglucosamine levels in the heart

Increased levels of O-linked attachment of N-acetylglucosamine (O-GlcNAc) on nucleocytoplasmic proteins are implicated in the development of diabetic cardiomyopathy and are regulated by O-GlcNAc transferase (OGT) expression and its substrate UDP-GlcNAc. Therefore, the goal of this study was to determine whether the development of diabetes in the Zucker diabetic fatty (ZDF) rat, a model of Type 2 diabetes, results in defects in cardiomyocyte mechanical function and, if so, whether this is associated with increased levels of O-GlcNAc and increased OGT expression. Six-week-old ZDF rats were hyperinsulinemic but normoglycemic, and there were no differences in cardiomyocyte mechanical function, UDP-GlcNAc, O-GlcNAc, or OGT compared with age-matched lean control rats. Cardiomyocytes isolated from 22-wk-old hyperglycemic ZDF rats exhibited significantly impaired relaxation, compared with both age-matched lean control and 6-wk-old ZDF groups. There was also a significant increase in O-GlcNAc levels in high-molecular-mass proteins in the 22-wk-old ZDF group compared with age-matched lean control and 6-wk-old ZDF groups; this was associated with increased UDP-GlcNAc levels but not increased OGT expression. Surprisingly, there was a significant decrease in overall O-GlcNAc levels between 6 and 22 wk of age in lean, ZDF, and Sprague-Dawley rats that was associated with decreased OGT expression. These results support the notion that an increase in O-GlcNAc on specific proteins may contribute to impaired cardiomyocyte function in diabetes. However, this study also indicates that in the heart the level of O-GlcNAc on proteins appears to be differentially regulated by age and diabetes. hexosamine biosynthesis; protein O-glycosylation; O-linked N-acetylglucosamine transferase     

O-GlcNAc in cancer biology

O-linked β-N-actylglucosamine (O-GlcNAc) is a carbohydrate post-translational modification on hydroxyl groups of serine and/or threonine residues of cytosolic and nuclear proteins. Analogous to phosphorylation, O-GlcNAcylation plays crucial regulatory roles in a variety of cellular processes. O-GlcNAc was termed a nutritional sensor, as global levels of the modification are elevated in response to increased glucose and glutamine flux into the hexosamine biosynthetic pathway. A unique feature of cancer cell energy metabolism is a shift from oxidative phosphorylation to the less efficient glycolytic pathway (Warburg effect), necessitating greatly increased glucose uptake. Additionally, to help meet increased biosynthetic demands, cancer cells also up-regulate glutamine uptake. This led us to hypothesize that the universal feature of increased glucose and glutamine uptake by cancer cells might be linked to increased O-GlcNAc levels. Indeed, recent work in many different cancer types now indicates that hyper-O-GlcNAcylation is a general feature of cancer and contributes to transformed phenotypes. In this review, we describe known/potential links between hyper-O-GlcNAcylation and specific hallmarks of cancer, including cancer cell proliferation, survival, cell stresses, invasion and metastasis, aneuploidy, and energy metabolism. We also discuss inhibition of hyper-O-GlcNAcylation as a potential novel therapeutic target for cancer treatment.     

Glucose Deprivation-induced Increase in Protein O-GlcNAcylation in Cardiomyocytes Is Calcium-dependent

The posttranslational modification of nuclear and cytosolic proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) has been shown to play an important role in cellular response to stress. Although increases in O-GlcNAc levels have typically been thought to be substrate-driven, studies in several transformed cell lines reported that glucose deprivation increased O-GlcNAc levels by a number of different mechanisms. A major goal of this study therefore was to determine whether in primary cells, such as neonatal cardiomyocytes, glucose deprivation increases O-GlcNAc levels and if so by what mechanism. Glucose deprivation significantly increased cardiomyocyte O-GlcNAc levels in a time-dependent manner and was associated with decreased O-GlcNAcase (OGA) but not O-GlcNAc transferase (OGT) protein. This response was unaffected by either the addition of pyruvate as an alternative energy source or by the p38 MAPK inhibitor SB203580. However, the response to glucose deprivation was blocked completely by glucosamine, but not by inhibition of OGA with 2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate. Interestingly, the CaMKII inhibitor KN93 also significantly reduced the response to glucose deprivation. Lowering extracellular Ca²⁺ with EGTA or blocking store operated Ca²⁺ entry with {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365 also attenuated the glucose deprivation-induced increase in O-GlcNAc. In C2C12 and HEK293 cells both glucose deprivation and heat shock increased O-GlcNAc levels, and CaMKII inhibitor KN93 attenuated the response to both stresses. These results suggest that increased intracellular calcium and subsequent activation of CaMKII play a key role in regulating the stress-induced increase in cellular O-GlcNAc levels.     

Up-regulation of O-GlcNAc Transferase with Glucose Deprivation in HepG2 Cells Is Mediated by Decreased Hexosamine Pathway Flux

O-Linked N-acetylglucosamine (O-GlcNAc) is a post-translational modification of proteins that functions as a nutrient sensing mechanism. We have previously shown a significant induction of O-GlcNAc modification under conditions of glucose deprivation. Increased O-GlcNAc modification was mediated by increased mRNA for nucleocytoplasmic O-linked N-acetylglucosaminyltransferase (ncOGT). We have investigated the mechanism mediating ncOGT induction with glucose deprivation. The signal does not appear to be general energy depletion because no differences in AMP-dependent kinase protein levels or phosphorylation were observed between glucose-deprived and normal glucose-treated cells. However, treatment of glucose-deprived cells with a small dose (1 mm) of glucosamine blocked the induction of ncOGT mRNA and subsequent increase in O-GlcNAc protein modification, suggesting that decreased hexosamine flux is the signal for ncOGT up-regulation. Consistent with this, treatment of glucose-deprived cells with an inhibitor of O-GlcNAcase (O-(2-acetamido-2-deoxy-d-glucopyranosylidene) amino N-phenyl carbamat) completely prevented the subsequent up-regulation of ncOGT. Glucosamine treatment also resulted in a 40% rescue of the down-regulation of glycogen synthase activity normally seen after glucose deprivation. We conclude that deglycosylation of proteins within the first few hours of glucose deprivation promotes ncOGT induction. These findings suggest a novel negative feedback regulatory loop for OGT and O-GlcNAc regulation.Dynamic O-linked N-acetylglucosamine (O-GlcNAc)² modification is a critical modulator of the fate and function of diverse nuclear and cytoplasmic proteins. O-GlcNAcylation of target proteins is dependent upon substrate synthesis in the hexosamine biosynthetic pathway (HBP) coupled with O-linked N-acetylglucosaminyltransferase (OGT)-mediated protein modification. The HBP converts a portion of imported glucose to uridine 5′-diphospho (UDP)-GlcNAc. OGT catalyzes GlcNAc transfer to serine and threonine residues of target proteins, whereas O-GlcNAcase catalyzes O-GlcNAc removal (1). HBP flux is known to parallel substrate (glucose) availability, making the HBP a nutrient sensor (2–5).O-GlcNAcylation is regulated principally by substrate availability. Previous work has indicated that protein O-GlcNAcylation is proportional to substrate (glucose) availability (8). However, we have shown that human hepatocellular carcinoma (HepG2) cells demonstrate a robust O-GlcNAc increase when deprived of glucose, and this O-GlcNAc induction is mediated not by substrate-driven HBP flux increase but instead by increased OGT expression and O-GlcNAcase down-regulation (6). It has subsequently been shown that glucose deprivation of Neuro-2a neuroblastoma cells also results in OGT and O-GlcNAc induction (7). We have therefore investigated the mechanism for regulation of OGT in HepG2 cells and determined that the signal responsible for the induction of OGT mRNA in glucose deprivation is an early decrease in HBP flux and O-GlcNAc modification of proteins. Thus, the levels of O-GlcNAc in these cells are maintained through a feedback mechanism responsive to the degree of protein O-GlcNAc modification.     

Excessive O-GlcNAcylation of proteins suppresses spontaneous cardiogenesis in ES cells

Increased modification of proteins with O-linked N-acetylglucosamine (O-GlcNAc) has been implicated in the development of diabetic cardiomyopathy. We used the well-characterized ES cells (Nkx2.5GFP knock-in ES cells), to investigate the role of O-GlcNAcylation in cardiomyocyte development. O-GlcNAcylation decreased in differentiating ES cells, as did the expression of O-GlcNAc transferase. Increasing O-GlcNAcylation with glucosamine or by inhibiting N-acetylglucosaminidase (streptozotocin or PUGNAc) decreased the number of cardiomyocyte precursors and cardiac-specific gene expression. On the other hand, decreasing O-GlcNAcylation with an inhibitor of glutamine fructose-6-phosphate amidotransferase (6-diazo-5-oxo-norleucine) increased cardiomyocyte precursors. These results suggest that excessive O-GlcNAcylation impairs cardiac cell differentiation in ES cells.     

Inhibition of a bacterial <Emphasis Type="Italic">O</Emphasis>-GlcNAcase homologue by lactone and lactam derivatives: structural,kinetic and thermodynamic analyses

The dynamic, intracellular, O-GlcNAc modification is of continuing interest and one whose study through targeted “chemical genetics” approaches is set to increase. Of particular importance is the inhibition of the O-GlcNAc hydrolase, O-GlcNAcase (OGA), since this provides a route to elevate cellular O-GlcNAc levels, and subsequent phenotypic evaluation. Such a small molecule approach complements other methods and potentially avoids changes in protein–protein interactions that manifest themselves in molecular biological approaches to O-GlcNAc transferase knockout or over-expression. Here we describe the kinetic, thermodynamic and three-dimensional structural analysis of a bacterial OGA analogue from Bacteroides thetaiotaomicron, BtGH84, in complex with a lactone oxime (LOGNAc) and a lactam form of N-acetylglucosamine and compare their binding signatures with that of the more potent inhibitor O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino N-phenyl carbamate (PUGNAc). We show that both LOGNAc and the N-acetyl gluconolactam are significantly poorer inhibitors than PUGNAc, which may reflect poorer mimicry of transition state geometry and steric clashes with the enzyme upon binding; drawbacks that the phenyl carbamate adornment of PUGNAc helps mitigate. Implications for the design of future generations of inhibitors are discussed.     

O-GlcNAc modification of FoxO1 increases its transcriptional activity: A role in the glucotoxicity phenomenon?

O-GlcNAc glycosylations on serines or threonines are reversible post-translational modifications that control the localisation, the activity or the stability of cytosolic and nuclear proteins. These dynamic modifications are tightly dependent on the availability of glucose and on its flux through the hexosamine biosynthetic pathway. We recently showed that treatments that increase protein O-GlcNAc glycosylation (high-glucose concentrations, glucosamine) or inhibit their deglycosylation (PUGNAc), induced O-GlcNAc modification of FoxO1 in HEK293 cells. O-GlcNAc glycosylation of FoxO1 resulted in an increased of its activity towards a glucose 6-phosphatase promoter-luciferase reporter gene (G6Pase-luc). This effect appeared to be independent of FoxO1 sub-cellular re-localisation, since it was also observed with the constitutively nuclear FoxO1-AAA mutant. In liver-derived HepG2 cells, glucosamine and PUGNAc increased the expression of G6Pase mRNA, and synergistic effects were observed when both agents were present together. In addition, the expression of PGC1 alpha gene, which is known to be under the control of FoxO1, was also increased by glucosamine and PUGNAc. In HepG2 cells stably expressing the G6Pase-luc reporter gene, glucosamine and PUGNAc also increased the activity of the G6Pase promoter. The stimulation of the G6Pase reporter gene by these agents was abolished by two different FoxO1 siRNAs, thereby demonstrating the involvement of endogenous FoxO1 in the observed effects. Since G6Pase plays a key role in glucose production by the liver, increased in its expression through FoxO1 O-GlcNAc modification may be of considerable importance in the context of glucotoxicity associated with chronic hyperglycaemia. Moreover, since FoxO1 also plays important roles in several aspects of cell biology, including cell proliferation, survival and apoptosis, the regulation of FoxO1 activity by O-GlcNAc modification may have implications for other crucial biological processes.     

Increased O-GlcNAc levels during reperfusion lead to improved functional recovery and reduced calpain proteolysis

We have previously shown that preischemic treatment with glucosamine improved cardiac functional recovery following ischemia-reperfusion, and this was mediated, at least in part, via enhanced flux through the hexosamine biosynthesis pathway and subsequently elevated O-linked N-acetylglucosamine (O-GlcNAc) protein levels. However, preischemic treatment is typically impractical in a clinical setting; therefore, the goal of this study was to investigate whether increasing protein O-GlcNAc levels only during reperfusion also improved recovery. Isolated perfused rat hearts were subjected to 20 min of global, no-flow ischemia followed by 60 min of reperfusion. Administration of glucosamine (10 mM) or an inhibitor of O-GlcNAcase, O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc; 200 microM), during the first 20 min of reperfusion significantly improved cardiac functional recovery and reduced troponin release during reperfusion compared with untreated control. Both interventions also significantly increased the levels of protein O-GlcNAc and ATP levels. We also found that both glucosamine and PUGNAc attenuated calpain-mediated proteolysis of alpha-fodrin as well as Ca(2+)/calmodulin-dependent protein kinase II during reperfusion. Thus two independent strategies for increasing protein O-GlcNAc levels in the heart during reperfusion significantly improved recovery, and this was correlated with attenuation of calcium-mediated proteolysis. These data provide further support for the concept that increasing cardiac O-GlcNAc levels may be a clinically relevant cardioprotective strategy and suggest that this protection could be due, at least in part, to inhibition of calcium-mediated stress responses.     

ROS scavenging before 27 degrees C ischemia protects hearts and reduces mitochondrial ROS, Ca2+ overload, and changes in redox state

We have shown that cold perfusion of hearts generates reactive oxygen and nitrogen species (ROS/RNS). In this study, we determined 1) whether ROS scavenging only during cold perfusion before global ischemia improves mitochondrial and myocardial function, and 2) which ROS leads to compromised cardiac function during ischemia and reperfusion (I/R) injury. Using fluorescence spectrophotometry, we monitored redox balance (NADH and FAD), O₂^– levels and mitochondrial Ca²⁺ (m[Ca²⁺]) at the left ventricular wall in 120 guinea pig isolated hearts divided into control (Con), MnTBAP (a superoxide dismutase 2 mimetic), MnTBAP (M) + catalase (C) + glutathione (G) (MCG), C+G (CG), and N^G-nitro-L-arginine methyl ester (L-NAME; a nitric oxide synthase inhibitor) groups. After an initial period of warm perfusion, hearts were treated with drugs before and after at 27°C. Drugs were washed out before 2 h at 27°C ischemia and 2 h at 37°C reperfusion. We found that on reperfusion the MnTBAP group had the worst functional recovery and largest infarction with the highest m[Ca²⁺], most oxidized redox state and increased ROS levels. The MCG group had the best recovery, the smallest infarction, the lowest ROS level, the lowest m[Ca²⁺], and the most reduced redox state. CG and L-NAME groups gave results intermediate to those of the MnTBAP and MCG groups. Our results indicate that the scavenging of cold-induced O₂^– species to less toxic downstream products additionally protects during and after cold I/R by preserving mitochondrial function. Because MnTBAP treatment showed the worst functional return along with poor preservation of mitochondrial bioenergetics, accumulation of H₂O₂ and/or hydroxyl radicals during cold perfusion may be involved in compromised function during subsequent cold I/R injury. hypothermic ischemia; mitochondrial Ca²⁺; reactive oxygen species     

O-GlcNAc Protein Modification in Cancer Cells Increases in Response to Glucose Deprivation through Glycogen Degradation

When cellular glucose concentrations fall below normal levels, in general the extent of protein O-GlcNAc modification (O-GlcNAcylation) decreases. However, recent reports demonstrated increased O-GlcNAcylation by glucose deprivation in HepG2 and Neuro-2a cells. Here, we report increased O-GlcNAcylation in non-small cell lung carcinoma A549 cells and various other cells in response to glucose deprivation. Although the level of O-GlcNAc transferase was unchanged, the enzyme contained less O-GlcNAc, and its activity was increased. Moreover, O-GlcNAcase activity was reduced. The studied cells contain glycogen, and we show that its degradation in response to glucose deprivation provides a source for UDP-GlcNAc required for increased O-GlcNAcylation under this condition. This required active glycogen phosphorylase and resulted in increased glutamine:fructose-6-phosphate amidotransferase, the first and rate-limiting enzyme in the hexosamine biosynthetic pathway. Interestingly, glucose deprivation reduced the amount of phosphofructokinase 1, a regulatory glycolytic enzyme, and blocked ATP synthesis. These findings suggest that glycogen is the source for increased O-GlcNAcylation but not for generating ATP in response to glucose deprivation and that this may be useful for cancer cells to survive.     

O-GlcNAc modification of proteins affects volume regulation in Jurkat cells

An increasing amount of recent research has demonstrated that the hexosamine biosynthesis pathway (HBP) plays a significant role in the modulation of intracellular signaling transduction pathways, and affects cellular processes via modification of protein by O-linked β-N-acetylglucosamine (O-GlcNAc). Besides the many known and postulated effects of protein O-GlcNAc modifications, there is little available data on the role of O-GlcNAc in cellular volume regulation. Our objective was to test the effect of increased O-GlcNAc levels on hypotonia-induced volume changes in Jurkat cells. We pretreated Jurkat cells for 1 h with glucosamine (GlcN), PUGNAc (O-(2-acetamido-2-deoxy-d-glucopyranosylidene)-amino-N-phenylcarbamate) an inhibitor of O-GlcNAcase, or a high level of glucose to induce elevated levels of O-GlcNAc. We found that the response of Jurkat cells to hypotonic stress was significantly altered. The hypotonia induced cell-swelling was augmented in both GlcN and PUGNAc-treated cells and, to a lesser extent, in high glucose concentration-treated cells. Evaluated by NMR measurements, GlcN and PUGNAc treatment also significantly reduced intracellular water diffusion. Taken together, increased cell swelling and reduced water diffusion caused by elevated O-GlcNAc show notable analogy to the regulatory volume changes seen by magnetic resonance methods in nervous and other tissues in different pathological states. In conclusion, we demonstrate for the first time that protein O-GlcNAc could modulate cell volume regulation.     

O-GlcNAcylation-Inducing Treatments Inhibit Estrogen Receptor α Expression and Confer Resistance to 4-OH-Tamoxifen in Human Breast Cancer-Derived MCF-7 Cells

O-GlcNAcylation (addition of N-acetyl-glucosamine on serine or threonine residues) is a post-translational modification that regulates stability, activity or localization of cytosolic and nuclear proteins. O-linked N-acetylgluocosmaine transferase (OGT) uses UDP-GlcNAc, produced in the hexosamine biosynthetic pathway to O-GlcNacylate proteins. Removal of O-GlcNAc from proteins is catalyzed by the β-N-Acetylglucosaminidase (OGA). Recent evidences suggest that O-GlcNAcylation may affect the growth of cancer cells. However, the consequences of O-GlcNAcylation on anti-cancer therapy have not been evaluated. In this work, we studied the effects of O-GlcNAcylation on tamoxifen-induced cell death in the breast cancer-derived MCF-7 cells. Treatments that increase O-GlcNAcylation (PUGNAc and/or glucosoamine) protected MCF-7 cells from death induced by tamoxifen. In contrast, inhibition of OGT expression by siRNA potentiated the effect of tamoxifen on cell death. Since the PI-3 kinase/Akt pathway is a major regulator of cell survival, we used BRET to evaluate the effect of PUGNAc+glucosamine on PIP₃ production. We observed that these treatments stimulated PIP₃ production in MCF-7 cells. This effect was associated with an increase in Akt phosphorylation. However, the PI-3 kinase inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, which abolished the effect of PUGNAc+glucosamine on Akt phosphorylation, did not impair the protective effects of PUGNAc+glucosamine against tamoxifen-induced cell death. These results suggest that the protective effects of O-GlcNAcylation are independent of the PI-3 kinase/Akt pathway. As tamoxifen sensitivity depends on the estrogen receptor (ERα) expression level, we evaluated the effect of PUGNAc+glucosamine on the expression of this receptor. We observed that O-GlcNAcylation-inducing treatment significantly reduced the expression of ERα mRNA and protein, suggesting a potential mechanism for the decreased tamoxifen sensitivity induced by these treatments. Therefore, our results suggest that inhibition of O-GlcNAcylation may constitute an interesting approach to improve the sensitivity of breast cancer to anti-estrogen therapy.     

Ac4GlcNAcF3, an OGT-tolerated but OGA-resistant regulator for O-GlcNAcylation

O-Linked N-acetylglucosamine (O-GlcNAc) is an abundant posttranslational monosaccaride-modification found on Ser or Thr residues of intracellular proteins in most eukaryotes. The dynamic nature of O-GlcNAc has enabled researchers to modulate the stoichiometry of O-GlcNAc on proteins in order to investigate its function. Cell permeable small moleculars have proven invaluable tools to increase O-GlcNAc levels. Herein, using in vitro substrate screening, we identified GlcNAcF₃ as an OGT-accepted but OGA-resistant sugar mimic. Cellular experiments with cell-permeable peracetylated-GlcNAcF₃ (Ac₄GlcNAcF₃) displayed that Ac₄GlcNAcF₃ was a potent tool to increase O-GlcNAc levels in several cell lines. Further, NIH3T3 cells interfered with OGT (siOGT) showed significant decreasing of O-GlcNAc levels with Ac₄GlcNAcF₃ treatment, indicating O-GlcNAcF₃ was an OGT-dependent modification. In addition, cellular toxic assay confirmed O-GlcNAcF₃ production has no significant effect on cell proliferation or viability. Thus, Ac₄GlcNAcF₃ represents a safe and dual regulator for both OGT and OGA, which will benefit the study of O-GlcNAc.