Levels Of Protein O-GlcNAcylation Research Articles

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

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

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.

NButGT Reinforces the Beneficial Effects of Epinephrine on Cardiac Mitochondrial Respiration, Lactatemia and Cardiac Output in Experimental Anaphylactic Shock.

Anaphylactic shock (AS) is the most severe form of acute systemic hypersensitivity reaction. Although epinephrine can restore patients' hemodynamics, it might also be harmful, supporting the need for adjuvant treatment. We therefore investigated whether NButGT, enhancing O-GlcNAcylation and showing beneficial effects in acute heart failure might improve AS therapy. Ovalbumin-sensitized rats were randomly allocated to six groups: control (CON), shock (AS), shock treated with NButGT alone before (AS+pre-Nbut) or after (AS+post-Nbut) AS onset, shock treated with epinephrine alone (AS+EPI) and shock group treated with combination of epinephrine and NButGT (AS+EPI+preNBut). Induction of shock was performed with an intravenous (IV) ovalbumin. Cardiac protein and cycling enzymes O-GlcNAcylation levels, mean arterial pressure (MAP), heart rate, cardiac output (CO), left ventricle shortening fraction (LVSF), mitochondrial respiration, and lactatemia were evaluated using Western blotting experiments, invasive arterial monitoring, echocardiography, mitochondrial oximetry and arterial blood samples. AS decreased MAP (-77%, p < 0.001), CO (-90%, p < 0.001) and LVSF (-30%, p < 0.05). Epinephrine improved these parameters and, in particular, rats did not die in 15 min. But, cardiac mitochondrial respiration remained impaired (complexes I + II -29%, p < 0.05 and II -40%, p < 0.001) with hyperlactatemia. NButGT pretreatment (AS+pre-Nbut) efficiently increased cardiac O-GlcNAcylation level as compared to the AS+post-Nbut group. Compared to epinephrine alone, the adjunction of NButGT significantly improved CO, LVSF and mitochondrial respiration. MAP was not significantly increased but lactatemia decreased more markedly. Pretreatment with NButGT increases O-GlcNAcylation of cardiac proteins and has an additive effect on epinephrine, improving cardiac output and mitochondrial respiration and decreasing blood lactate levels. This new therapy might be useful when the risk of AS cannot be avoided.

GFPT1 deficiency is a novel cause of a dilated cardiomyopathy and is required for myocardial adaptation to chronic pressure-overload stress-induced heart failure

Abstract Background GFPT1 is the rate-limiting enzyme of a pathway called the hexosamine biosynthesis pathway (HBP) that turns fructose 6-phosphate derived from glucose into another monosaccharide called N-acetylglucosamine (GlcNAc). GlcNAc is an important substrate for modifying diverse cellular proteins on their serine and threonine residues termed O-GlcNAcylation often competing with phosphorylation and altering their activity or stability (Bond &amp; Hanover; 2015). Increased GFPT1 activity is known to stimulate the cellular integrated stress response improving protein quality control and cell survival (Denzel et al 2014). In the heart, GFPT1 expression and O-GlcNAcylation increases in the early stages of chronic cardiac stress but the physiological role of GFPT1 in regulating heart function including its role during chronic stress remains unclear. Identifying molecular pathways that promote adaptive cardiac remodelling is a promising approach to discover novel therapies for heart failure. Methods Cardiomyocyte-specific GFPT1 knockout mice were generated to study the effects of GFPT1 deletion in hearts. Heart function was examined by echocardiography. HBP activity was quantified using Western blotting of O-GlcNAc-modified proteins. Abdominal aortic banding (AAB) was used to induce chronic pressure-overload heart failure. Results Homozygous GFPT1 KO mice spontaneously develop a dilated cardiomyopathy (DCM) with a 30% mortality rate (ejection fraction 32% compared with 62% for floxed controls and end diastolic volume 125 microL versus 60 microL; n=5-6 per group; p &amp;lt;0.05). Hearts showed reduced levels of total protein O-GlcNAcylation suggesting impaired hexosamine biosynthesis pathway activity. Heart function could be rescued by supplementing their diets with oral glucosamine bypassing GFPT1 to provide metabolites required for hexosamine biosynthesis and normalising O-GlcNAcylation (ejection fraction improved to 40% in KO with supplementation versus KO without; n=4-6 per group; p &amp;lt;0.05). Heterozygous KO mice do not develop a DCM but when subject to pressure-overload cardiac stress, develop worse heart failure with a 50% mortality rate over 9 weeks of pressure-overload (ejection fraction 43% in het KO versus 53% in floxed control; n= 3-6 per group; p = 0.0025). Conclusion Deficiency of GFPT1 is a novel cause of a spontaneous dilated cardiomyopathy. GFPT1 is required for physiological growth of the heart that may in part involve the O-GlcNAcylation of proteins. Partial GFPT1 deficiency is associated with adverse cardiac remodelling following pressure-overload stress. Increasing GFPT1 activity may be a key therapeutic target in developing novel heart failure treatments.

Effect of vitrification on protein O-GlcNAcylation in mouse metaphase II oocytes.

Oocyte vitrification leads to DNA hypomethylation, which results in defect in early embryo development. This study reveals that oocyte vitrification impairs the DNA methylation pattern by influencing protein O-GlcNAcylation. Oocyte vitrification leads to decreased DNA methylation levels, which impairs the quality and the developmental potential of oocytes. However, the underlying molecular mechanism still need to be further revealed. In this study, mouse metaphase II (M II) oocytes were frozen by vitrification technology, while fresh oocytes were used as the control group. The effect of oocyte vitrification on protein O-GlcNAcylation and its impact on the developmental potential of oocytes were elucidated. We found that the protein O-GlcNAcylation levels were significantly increased in vitrified oocytes. Increase of protein O-GlcNAcylation levels in control oocytes by PUGNAc (an O-GlcNAcase inhibitor) decreases blastocyst rate after parthenogenetic activation (20.82% in PUGNAc-treated group; 53.82% in control group, P < 0.05). We also discovered that DNA methylation was disrupted in two-cell embryos derived from vitrified oocytes, resulting in decreased 5mC and increased 5hmC, showing similar phenotypes to that derived from PUGNAc-treated oocytes. In vitrified and PUGNAc-treated oocytes, O-GlcNAcylated TET3 was significantly increased. Notably, by inhibiting protein O-GlcNAcylation in vitrified oocytes using OSMI1 (an O-GlcNAc transferase inhibitor) we restored the DNA methylation in two-cell embryos and ameliorated the developmental defects in early embryo. Thus, elevated protein O-GlcNAcylation in vitrified oocytes is an essential contributor to their declining embryonic developmental potential. Modulation of protein O-GlcNAcylation improves the developmental potential of vitrified oocytes.

An O-GlcNAc transferase pathogenic variant linked to intellectual disability affects pluripotent stem cell self-renewal.

O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) is an essential enzyme that modifies proteins with O-GlcNAc. Inborn OGT genetic variants were recently shown to mediate a novel type of congenital disorder of glycosylation (OGT-CDG), which is characterised by X-linked intellectual disability (XLID) and developmental delay. Here, we report an OGTC921Y variant that co-segregates with XLID and epileptic seizures, and results in loss of catalytic activity. Colonies formed by mouse embryonic stem cells carrying OGTC921Y showed decreased levels of protein O-GlcNAcylation accompanied by decreased levels of Oct4 (encoded by Pou5f1), Sox2 and extracellular alkaline phosphatase (ALP), implying reduced self-renewal capacity. These data establish a link between OGT-CDG and embryonic stem cell self-renewal, providing a foundation for examining the developmental aetiology of this syndrome.

O-GlcNAcylation is crucial for sympathetic neuron development, maintenance, functionality and contributes to peripheral neuropathy.

O-GlcNAcylation is a post-translational modification (PTM) that regulates a wide range of cellular functions and has been associated with multiple metabolic diseases in various organs. The sympathetic nervous system (SNS) is the efferent portion of the autonomic nervous system that regulates metabolism of almost all organs in the body. How much the development and functionality of the SNS are influenced by O-GlcNAcylation, as well as how such regulation could contribute to sympathetic neuron (symN)-related neuropathy in diseased states, remains unknown. Here, we assessed the level of protein O-GlcNAcylation at various stages of symN development, using a human pluripotent stem cell (hPSC)-based symN differentiation paradigm. We found that pharmacological disruption of O-GlcNAcylation impaired both the growth and survival of hPSC-derived symNs. In the high glucose condition that mimics hyperglycemia, hPSC-derived symNs were hyperactive, and their regenerative capacity was impaired, which resembled typical neuronal defects in patients and animal models of diabetes mellitus. Using this model of sympathetic neuropathy, we discovered that O-GlcNAcylation increased in symNs under high glucose, which lead to hyperactivity. Pharmacological inhibition of O-GlcNAcylation rescued high glucose-induced symN hyperactivity and cell stress. This framework provides the first insight into the roles of O-GlcNAcylation in both healthy and diseased human symNs and may be used as a platform for therapeutic studies.

ATP-CITRATE LYASE expression and activity are modulated by O-GlcNAc levels

The enzyme ATP-citrate lyase (ACLY) is a nucleocytoplasmic protein that cleaves citrate into oxaloacetate and acetyl-CoA. It is described as a central enzyme of cellular metabolism and has an important role in the stress response particularly in sepsis. Its activity depends on its phosphorylation (particularly that of phospho Ser447-Thr451-Ser455) and multimerization. We have identified ACLY among the cardiac O-GlcNAcylated proteins in young septic rats. Interestingly, ACLY was less O-GlcNAcylated when global O-GlcNAcylation levels were stimulated with NButGT treatment. The O-GlcNAcylation (O-GlcNac) is a post-translational modification (PTM) notably involved in cell survival, metabolism and stress response. This PTM is regulated by a unique enzyme couple : O-GlcNac Transferase (OGT) and O-GlcNacAse (OGA) and could compete with phosphorylation on proteins. O-GlcNAc stimulation improves outcomes in young septic rats. Yet, no studies have attempted to understand the potential link between O-GlcNAc and ACLY. Decipher the impact of the O-GlcNAcylation on the expression and activity of ACLY. Chinese hamster ovary cells (CHO) were treated for 24 h with 10 μM Thiamet G (OGA inhibitor) or 50 μM OSMI (OGT inhibitor). O-GlcNAc levels, ACLY, P-ACLY Ser 455 et P-ACLY T447-S451 protein expression were evaluated by Western blot. ACLY activity was measured using the malate dehydrogenase coupled method. All results were compared to untreated CHO. Treatment with Thiamet G increases protein O-GlcNAcylation levels by 36% while treatment with OSMI decreases protein O-GlcNAcylation by 2 folds. Thiamet G does not affect ACLY's expression, phosphorylation or activity, while OSMI increases the expression of ACLY by 44% and in particular its multimers by 3 folds and all site of phosphorylation (P447-451-455). Accordingly, ACLY activity is also increased (+19%). In CHO cells, increasing the levels of O-GlcNAcylation of proteins with an OGA inhibitor has no impact on ACLY expression and activity, whereas decreasing the levels of O-GlcNac results in a significant increase in the expression of ACLY, its phosphorylation, multimerization and activity. We report for the first time the regulation of ACLY via the O-GlcNAcylation.

OGT Binding Peptide-Tagged Strategy Increases Protein O-GlcNAcylation Level in E. coli.

O-GlcNAcylation is a single glycosylation of GlcNAc mediated by OGT, which regulates the function of substrate proteins and is closely related to many diseases. However, a large number of O-GlcNAc-modified target proteins are costly, inefficient, and complicated to prepare. In this study, an OGT binding peptide (OBP)-tagged strategy for improving the proportion of O-GlcNAc modification was established successfully in E. coli. OBP (P1, P2, or P3) was fused with target protein Tau as tagged Tau. Tau or tagged Tau was co-constructed with OGT into a vector expressed in E. coli. Compared with Tau, the O-GlcNAc level of P1Tau and TauP1 increased 4~6-fold. Moreover, the P1Tau and TauP1 increased the O-GlcNAc-modified homogeneity. The high O-GlcNAcylation on P1Tau resulted in a significantly slower aggregation rate than Tau in vitro. This strategy was also used successfully to increase the O-GlcNAc level of c-Myc and H2B. These results indicated that the OBP-tagged strategy was a successful approach to improve the O-GlcNAcylation of a target protein for further functional research.

Increased O-GlcNAcylation promotes IGF-1 receptor/PhosphatidyI Inositol-3 kinase/Akt pathway in cervical cancer cells

O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) is a reversible post-translational modification on serine and threonine residues of cytosolic, nuclear and mitochondrial proteins. O-GlcNAcylation level is regulated by OGT (O-GlcNAc transferase), which adds GlcNAc on proteins, and OGA (O-GlcNAcase), which removes it. Abnormal level of protein O-GlcNAcylation has been observed in numerous cancer cell types, including cervical cancer cells. In the present study, we have evaluated the effect of increasing protein O-GlcNAcylation on cervical cancer-derived CaSki cells. We observed that pharmacological enhancement of protein O-GlcNAcylation by Thiamet G (an inhibitor of OGA) and glucosamine (which provides UDP-GlcNAc substrate to OGT) increases CaSki cells proliferation, migration and survival. Moreover, we showed that increased O-GlcNAcylation promotes IGF-1 receptor (IGF1R) autophosphorylation, possibly through inhibition of protein tyrosine-phosphatase 1B activity. This was associated with increased IGF-1-induced phosphatidyl-Inositol 3-phosphate production at the plasma membrane and increased Akt activation in CaSki cells. Finally, we showed that protein O-GlcNAcylation and Akt phosphorylation levels were higher in human cervical cancer samples compared to healthy cervix tissues, and a highly positive correlation was observed between O-GlcNAcylation level and Akt phosphorylation in theses tissues. Together, our results indicate that increased O-GlcNAcylation, by activating IGF1R/ Phosphatidyl inositol 3-Kinase (PI-3K)/Akt signaling, may participate in cervical cancer cell growth and proliferation.

PRMT5 Prevents Dilated Cardiomyopathy via Suppression of Protein O-GlcNAcylation.

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A computational strategy for metabolic network construction based on the overlapping ratio: Study of patients’ metabolic responses to different dialysis patterns

BackgroundUremia is a worldwide epidemic disease and poses a serious threat to human health. Both maintenance hemodialysis (HD) and maintenance high flux hemodialysis (HFD) are common treatments for uremia and are generally used in clinical applications. In-depth exploration of patients’ metabolic responses to different dialysis patterns can facilitate the understanding of pathological alterations associated with uremia and the effects of different dialysis methods on uremia, which may be used for future personalized therapy. However, due to variations of multiple factors (i.e., genetic, epigenetic and environment) in the process of disease treatments, identification of the similarities and differences in plasma metabolite changes in uremic patients in response to HD and HFD remains challenging. MethodsIn this study, a computational strategy for metabolic network construction based on the overlapping ratio (MNC-OR) was proposed for disease treatment effect research. In MNC-OR, the overlapping ratio was introduced to measure metabolic reactions and to construct metabolic networks for analysis of different treatment options. Then, MNC-OR was employed to analyze HD-pattern-dependent changes in plasma metabolites to explore the pathological alterations associated with uremia and the effectiveness of different dialysis patterns (i.e., HD and HFD) on uremia. Based on the networks constructed by MNC-OR, two network analysis techniques, namely, similarity analysis and difference analysis of network topology, were used to find the similarity and differences in metabolic signals in patients under treatment with either HD or HFD, which can facilitate the understanding of pathological alterations associated with uremia and provide the guidance for personalized dialysis therapy. ResultsSimilarity analysis of network topology suggested that abnormal energy metabolism, gut metabolism and pyrimidine metabolism might occur in uremic patients, and maintenance of both HFD and HD therapies have beneficial effects on uremia. Then, difference analysis of network topology was employed to extract the crucial information related to HD-pattern-dependent changes in plasma metabolites. Experimental results indicated that the amino acid metabolism was closer to the normal status in HFD-treated patients; however, in HD-treated patients, the ability of antioxidation showed greater reduction, and the protein O-GlcNAcylation level was higher. Our findings demonstrate the potential of MNC-OR for explaining the metabolic similarities and differences of patients in response to different dialysis methods, thereby contributing to the guidance of personalized dialysis therapy.

O-GlcNAcylation inHyperglycemic Pregnancies: Impact on Placental Function.

The dynamic cycling of N-acetylglucosamine, termed as O-GlcNAcylation, is a post-translational modification of proteins and is involved in the regulation of fundamental cellular processes. It is controlled by two essential enzymes, O-GlcNAc transferase and O-GlcNAcase. O-GlcNAcylation serves as a modulator in placental tissue; furthermore, increased levels of protein O-GlcNAcylation have been observed in women with hyperglycemia during pregnancy, which may affect the short-and long-term development of offspring. In this review, we focus on the impact of O-GlcNAcylation on placental functions in hyperglycemia-associated pregnancies. We discuss the following topics: effect of O-GlcNAcylation on placental development and its association with hyperglycemia; maternal-fetal nutrition transport, particularly glucose transport, via the mammalian target of rapamycin and AMP-activated protein kinase pathways; and the two-sided regulatory effect of O-GlcNAcylation on inflammation. As O-GlcNAcylation in the placental tissues of pregnant women with hyperglycemia influences near- and long-term development of offspring, research in this field has significant therapeutic relevance.

Protein O-GlcNAcylation alleviates small intestinal injury induced by ischemia-reperfusion and oxygen-glucose deprivation

Protein O-GlcNAcylation is a dynamic post-translational protein modification that regulates fundamental cellular functions in both normal physiology and diseases. The levels of protein O-GlcNAcylation are determined by flux of the hexosamine biosynthetic pathway (HBP), which is a branch of glycolysis, and are directly controlled by a pair of enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). An increase in protein O-GlcNAcylation has been shown to have protective effects on ischemia-related insults in the heart and brain. To determine whether O-GlcNAcylation plays a beneficial role in ischemia-reperfusion (IR)-induced intestinal injury, we used pharmacological manipulation of O-GlcNAc to induce loss- and gain-of-function conditions and evaluated the viability and apoptosis of intestinal epithelioid cells in an in vitro oxygen-glucose deprivation (OGD) model and tissue injury grade in a small intestinal ischemia-reperfusion (SIIR) mouse model. We found that 1) Upregulation of O-GlcNAcylation induced by glucosamine (GlcN, increase in HBP flux) or thiamet G (an OGA inhibitor) enhanced intestinal cell survival in the OGD model. In contrast, downregulation of O-GlcNAcylation induced by DON (due to a reduction in HBP flux) or OMSI-1 (an OGT inhibitor) made the cells more susceptible to hypoxia injury. 2) Reducing the increase in O-GlcNAcylation levels with a combination of either GlcN with DON or thiamet G with OMSI-1 partly canceled its protective effect on OGD-induced cell injury. 3) In the in vivo SIIR mouse model, GlcN augmented intestinal protein O-GlcNAcylation and significantly alleviated intestinal injury by inhibiting cell apoptosis. These results indicate that acute increases in protein O-GlcNAcylation confer protection against intestinal ischemia insults, suggesting that O-GlcNAcylation, as an endogenous stress sensor, could be a universal protective mechanism and could be a potential therapeutic target for intestinal ischemic disease.

Cardiomyocyte Oga haploinsufficiency increases O-GlcNAcylation but hastens ventricular dysfunction following myocardial infarction.

RationaleThe beta-O-linkage of N-acetylglucosamine (i.e., O-GlcNAc) to proteins is a pro-adaptive response to cellular insults. To this end, increased protein O-GlcNAcylation improves short-term survival of cardiomyocytes subjected to acute injury. This observation has been repeated by multiple groups and in multiple models; however, whether increased protein O-GlcNAcylation plays a beneficial role in more chronic settings remains an open question.ObjectiveHere, we queried whether increasing levels of cardiac protein O-GlcNAcylation would be beneficial during infarct-induced heart failure.Methods and resultsTo achieve increased protein O-GlcNAcylation, we targeted Oga, the gene responsible for removing O-GlcNAc from proteins. Here, we generated mice with cardiomyocyte-restricted, tamoxifen-inducible haploinsufficient Oga gene. In the absence of infarction, we observed a slight reduction in ejection fraction in Oga deficient mice. Overall, Oga reduction had no major impact on ventricular function. In additional cohorts, mice of both sexes and both genotypes were subjected to infarct-induced heart failure and followed for up to four weeks, during which time cardiac function was assessed via echocardiography. Contrary to our prediction, the Oga deficient mice exhibited exacerbated—not improved—cardiac function at one week following infarction. When the observation was extended to 4 wk post-MI, this acute exacerbation was lost.ConclusionsThe present findings, coupled with our previous work, suggest that altering the ability of cardiomyocytes to either add or remove O-GlcNAc modifications to proteins exacerbates early infarct-induced heart failure. We speculate that more nuanced approaches to regulating O-GlcNAcylation are needed to understand its role—and, in particular, the possibility of cycling, in the pathophysiology of the failing heart.

Lipopolysaccharide Induces GFAT2 Expression to Promote O-Linked β-N-Acetylglucosaminylation and Attenuate Inflammation in Macrophages

Glycosylation with O-linked β-N-acetylglucosamine (O-GlcNAcylation) is a reversible posttranslational modification that regulates the activity of intracellular proteins according to glucose availability and its metabolism through the hexosamine biosynthesis pathway. This modification has been involved in the regulation of various immune cell types, including macrophages. However, little is known concerning the mechanisms that regulate the protein O-GlcNAcylation level in these cells. In the present work, we demonstrate that LPS treatment induces a marked increase in protein O-GlcNAcylation in RAW264.7 cells, bone marrow-derived and peritoneal mouse macrophages, as well as human monocyte-derived macrophages. Targeted deletion of OGT in macrophages resulted in an increased effect of LPS on NOS2 expression and cytokine production, suggesting that O-GlcNAcylation may restrain inflammatory processes induced by LPS. The effect of LPS on protein O-GlcNAcylation in macrophages was associated with an increased expression and activity of glutamine fructose 6-phosphate amidotransferase (GFAT), the enzyme that catalyzes the rate-limiting step of the hexosamine biosynthesis pathway. More specifically, we observed that LPS potently stimulated GFAT2 isoform mRNA and protein expression. Genetic or pharmacological inhibition of FoxO1 impaired the LPS effect on GFAT2 expression, suggesting a FoxO1-dependent mechanism. We conclude that GFAT2 should be considered a new LPS-inducible gene involved in regulation of protein O-GlcNAcylation, which permits limited exacerbation of inflammation upon macrophage activation.

Effect of Peripheral Insulin Administration on Phosphorylation of Tau in the Brain.

Abnormally hyperphosphorylated tau is the major protein of neurofibrillary tangles in Alzheimer's disease. Insulin activates PI3K-AKT signaling and regulates tau phosphorylation. Impaired brain insulin signaling is involved in Alzheimer's disease pathogenesis. However, the effect of peripheral insulin on tau phosphorylation is controversial. In the present study, we determined the effect of peripheral insulin administration on tau phosphorylation in brain. We intraperitoneally injected a super physiological dose of insulin to mice and analyzed PI3K-AKT signaling and tau phosphorylation in brains by western blots. We found that peripherally administered insulin activated the PI3K-AKT signaling pathway immediately in the liver, but not in the brain. Tau phosphorylation in the mouse brain was found to be first decreased (15 min) and then increased (30 min and 60 min) after peripheral insulin administration and these changes correlated inversely with body temperature and the level of brain protein O-GlcNAcylation. Maintaining body temperature of mice post peripheral insulin administration prevented the insulin/hypoglycemia-induced tau hyperphosphorylation after peripheral insulin administration. These findings suggest that peripheral insulin can induce tau hyperphosphorylation through both hypothermia and downregulation of brain protein O-GlcNAcylation during hypoglycemia.

OR14-07 Association Between Placental Glucose Uptake and Protein O-Glcnacylation and Birth Outcomes in Obese Non-Diabetic Mothers

Increased transport of nutrients such as glucose, across the placenta, has been linked to abnormal fetal growth and pregnancy complications in obese non-diabetic mothers (1); however, the underlying mechanisms are poorly understood. We hypothesized that in placenta, the metabolic and nutrient sensor O-GlcNAc transferase (OGT), highly sensitive to glucose flux through the hexosamine biosynthetic pathway (HBP), responds to maternal obesogenic environment by increasing O-GlcNAc post-translational modification of nucleocytoplasmic proteins in the placenta altering fetal growth trajectories. Tissue biopsies were isolated from placentas collected at term from 26 non-diabetic mothers alongside routine biochemistry and anthropometric data (maternal fasting glucose, glycated hemoglobin (HbA1c), early pregnancy body weight (BMI) and birth weight). OGT and glucose transporter 1 (GLUT1) protein expression as well as tissue levels of O-GlcNAcylation were determined by immunoblotting using specific antibodies. The BeWo choriocarcinoma cell line was also used as an in vitro model of trophoblast to test the effect of high glucose and GLUT1 silencing on the OGT activity by immunoblotting. Maternal BMI was positively correlated to birth weight centile (BWC) (p=0.0130, R2=0.231), maternal fasting glucose (p=0.0177, R2=0.221) and HbA1c levels (p= 0.0156, R2=0.229) as well as placental OGT protein expression (p=0.0294, R2=0.183). The latter was positively associated to the levels of protein O-GlcNAcylation (p=0.0023, R2=0.326). Interestingly, GLUT1 protein levels were positively correlated to BWC (p=0.0056, R2=0.279) and strongly correlated to protein O-GlcNAcylation (p<0.0001, R2=0.507), suggesting an increase in the placental flux of glucose. In agreement with findings in placenta biopsies, in BeWo cells total protein O-GlcNAcylation levels were altered by cell exposure to different glucose levels (5 mM vs 15 mM, p<0.01). This was prevented by downregulating OGT or GLUT1 expression (p<0.001) using gene silencing. In addition, OGT protein levels were negatively associated to AMP-activated protein kinase (AMPK) activation (p=0.0005, R2=0.402) in placenta biopsies identifying a novel cross-talk between two placental nutrient sensors, OGT and AMPK, previously shown in other tissues (2). Accordingly, the silencing of OGT promoted the activation of AMPK (p<0.01) and its downstream target acetyl-CoA carboxylase (ACC) (p<0.01) in BeWo cells, as demonstrated by increased phosphorylation of residues Thr172 and Ser79 for AMPK and ACC respectively. Such obesity-associated cross talk between metabolic and nutrient sensors might disrupt multiple cellular pathways involved in fetal development and growth.

Glucose regulates tissue-specific chondro-osteogenic differentiation of human cartilage endplate stem cells via O-GlcNAcylation of Sox9 and Runx2

BackgroundThe degenerative disc disease (DDD) is a major cause of low back pain. The physiological low-glucose microenvironment of the cartilage endplate (CEP) is disrupted in DDD. Glucose influences protein O-GlcNAcylation via the hexosamine biosynthetic pathway (HBP), which is the key to stem cell fate. Thiamet-G is an inhibitor of O-GlcNAcase for accumulating O-GlcNAcylated proteins while 6-diazo-5-oxo-l-norleucine (DON) inhibits HBP. Mechanisms of DDD are incompletely understood but include CEP degeneration and calcification. We aimed to identify the molecular mechanisms of glucose in CEP calcification in DDD.MethodsWe assessed normal and degenerated CEP tissues from patients, and the effects of chondrogenesis and osteogenesis of the CEP were determined by western blot and immunohistochemical staining. Cartilage endplate stem cells (CESCs) were induced with low-, normal-, and high-glucose medium for 21 days, and chondrogenic and osteogenic differentiations were measured by Q-PCR, western blot, and immunohistochemical staining. CESCs were induced with low-glucose and high-glucose medium with or without Thiamet-G or DON for 21 days, and chondrogenic and osteogenic differentiations were measured by Q-PCR, western blot, and immunohistochemical staining. Sox9 and Runx2 O-GlcNAcylation were measured by immunofluorescence. The effects of O-GlcNAcylation on the downstream genes of Sox9 and Runx2 were determined by Q-PCR and western blot.ResultsDegenerated CEPs from DDD patients lost chondrogenesis, acquired osteogenesis, and had higher protein O-GlcNAcylation level compared to normal CEPs from LVF patients. CESC chondrogenic differentiation gradually decreased while osteogenic differentiation gradually increased from low- to high-glucose differentiation medium. Furthermore, Thiamet-G promoted CESC osteogenic differentiation and inhibited chondrogenic differentiation in low-glucose differentiation medium; however, DON acted opposite role in high-glucose differentiation medium. Interestingly, we found that Sox9 and Runx2 were O-GlcNAcylated in differentiated CESCs. Finally, O-GlcNAcylation of Sox9 and Runx2 decreased chondrogenesis and increased osteogenesis in CESCs.ConclusionsOur findings demonstrate the effect of glucose concentration on regulating the chondrogenic and osteogenic differentiation potential of CESCs and provide insight into the mechanism of how glucose concentration regulates Sox9 and Runx2 O-GlcNAcylation to affect the differentiation of CESCs, which may represent a target for CEP degeneration therapy.

P1606Glutamine-fructose-6-phosphate amidotransferase 2 mediates isoproterenol-induced cardiac hypertrophy by increasing Akt O-GlcNAcylation through hexosamine biosynthesis pathway

Abstract Background Cardiac hypertrophy is an independent risk factor for heart failure and cardiac death. Hexosamine biosynthesis pathway (HBP), an accessory pathways of glycolysis, is known to be involved in the attachment of O-linked N-acetylglucosamine motif (O-GlcNAcylation) to proteins, a post-translational modification. However, the role of HBP has not been determined in pathological cardiac hypertrophy. Purpose The purpose of this study to examine whether glutamine-fructose-6-phosphate amidotransferase 2 (GFAT2), a critical enzyme of HBP, mediates cardiac hypertrophy by protein O-GlcNAcylation and activating hypertrophic signaling in cardiomyocytes. Methods and results C57BL/6J mice were treated with isoproterenol (ISO: 15 mg/kg/day, 1 week) with or without 6-Diazo-5-oxo-L-norleucine (DON, an inhibitor of GFAT: 500 μg/kg/day, 1week). ISO-treated mice (ISO+vehicle) showed cardiac hypertrophy, which were attenuated in ISO and DON-treated mice (ISO+DON) (heart weight to tibial length ratio: 7.70±0.09 vs. 7.11±0.15 mg/mm, n=12, p&lt;0.05, left ventricular wall thickness: 1.05±0.02 vs. 0.86±0.03 mm, n=6, p&lt;0.05). Cardiomyocyte cross-sectional area was also decreased in ISO+DON compared with ISO+vehicle (309±25 vs. 252±13 mm2, n=,3 p&lt;0.05). Whereas expression levels of GFAT2 and protein O-GlcNAcylation in the heart were increased in ISO+vehicle compared with control+vehicle by 3.3 and 1.5 folds, respectively (n=9 and n=9, p&lt;0.05), expression levels of O-GlcNAc transferase (OGT) and the β-N-acetylglucosaminidase (OGA), other enzymes regulating O-GlcNAcylation, were not altered in both groups, indicating that ISO activated HBP by GFAT2. Protein O-GlcNAcylation in ISO+DON was lower than that in ISO+vehicle by 83% (n=9, p&lt;0.05). In addition, phosphorylation of Akt, a critical mediator of cardiac hypertrophy, but not other mediators of cardiac hypertrophy such as ERK, JNK, or p38MAPK, was significantly decreased in ISO+DON by 76% (n=9, p&lt;0.05). In cultured neonatal rat ventricular myocytes, treatment with ISO (1μM, 12h) increased the expression levels of GFAT2 and protein O-GlcNAcylation by 1.3 and 1.5 folds, respectively (n=6 and n=6, p&lt;0.05), but not GFAT1. Furthermore, ISO stimulation increased a direct O-GlcNAcylation of Akt by 1.4 folds (n=3, p&lt;0.05). Downregulation of GFAT2 by RNA silencing decreased cell size by 82% (n=6, p&lt;0.05) and protein O-GlcNAcylation and phosphorylation of Akt by 76% and 54%, respectively (n=9 and n=9, p&lt;0.05) in cardiomyocyte treated with ISO. Conversely, administration of glucosamine, a substrate of HBP, increased protein of O-GlcNAcylation and phosphorylation of Akt by 1.3 and 1.8 folds, respectively (n=6 and n=6, p&lt;0.05). Conclusions GFAT2, a limiting enzyme of HBP, mediates pathological cardiac hypertrophy by Akt activation probably due to its O-GlcNAcylation. GFAT2-O-GlcNAcylation-Akt pathway might be a potential novel therapeutic target for cardiac hypertrophy.