Increased O-GlcNAc Levels Research Articles

Brain Transcriptome Changes Associated With an Acute Increase of Protein O-GlcNAcylation and Implications for Neurodegenerative Disease.

Enhancing protein O-GlcNAcylation by pharmacological inhibition of the enzyme O-GlcNAcase (OGA) has been considered as a strategy to decrease tau and amyloid-beta phosphorylation, aggregation, and pathology in Alzheimer's disease (AD). There is still more to be learned about the impact of enhancing global protein O-GlcNAcylation, which is important for understanding the potential of using OGA inhibition to treat neurodegenerative diseases. In this study, we investigated the acute effect of pharmacologically increasing O-GlcNAc levels, using the OGA inhibitor Thiamet G (TG), in normal mouse brains. We hypothesized that the transcriptome signature in response to a 3 h TG treatment (50 mg/kg) provides a comprehensive view of the effect of OGA inhibition. We then performed mRNA sequencing of the brain using NovaSeq PE 150 (n = 5 each group). We identified 1234 significant differentially expressed genes with TG versus saline treatment. Functional enrichment analysis of the upregulated genes identified several upregulated pathways, including genes normally down in AD. Among the downregulated pathways were the cell adhesion pathway as well as genes normally up in AD and aging. When comparing acute to chronic TG treatment, protein autophosphorylation and kinase activity pathways were upregulated, whereas cell adhesion and astrocyte markers were downregulated in both datasets. AMPK subunit Prkab2 was one gene in the kinase activity pathway, and the increase after acute and chronic treatment was confirmed using qPCR. Interestingly, mitochondrial genes and genes normally down in AD were up in acute treatment and down in chronic treatment. Data from this analysis will enable the evaluation of the mechanisms underlying the impact of OGA inhibition in the treatment of AD. In particular, OGA inhibitors appear to have downstream effects related to bioenergetics which may limit their therapeutic benefits.

Abstract 4136700: Sodium-Potassium Pump: Newly O-GlcNAc Protein Offering Potential Benefits in Hemorrhagic Shock situation

Hemorrhagic shock(HS) results from significant blood loss, leading to insufficient oxygen delivery, which causes metabolic changes and multi-organ dysfunction.The HS-related acute kidney injury developed by the patient are mainly due to alteration in Na/K ATPase.In previous O-GlcNAcylomic studies, we identified the Na/K ATPase as potentially O-GlcNAcylated.O-GlcNAcylation, a post-translational modification, plays a role in various cellular processes, such as cell survival and stress response.O-GlcNAcylation may have beneficial effect in shock situations. The objective of this study is to ascertain whether Na/K ATPase is O-GlcNAcylated and to elucidate its potential role during HS. Hemorrhagic shock was induced by an exsanguination of rats.They were then randomly treated or not with an O-GlcNAcase inhibitor, the NButGT(10mg/kg), to increase O-GlcNAc level.Blood pressure and heart rate were monitored throughout the experiment.Blood samples were collected for complete blood analysis, including biochemistry and gas measurements. kidneys were immediately frozen for histological and biochemical studies.To study the O-GlcNAcylation of Na/K ATPase an immunoprecipitation and Wheat Germ Agglutinin(WGA) based lectin affinity gel electrophoresis were used. HEK cells were treated with or without NButGT(100µM,6 hours) and/or ouabain(Na/K ATPase inhibitor) and a colorimetric kit was assessed to study O-GlcNAcylation impact on Na/K ATPase activity. O-GlcNAc levels increased by 2 folds in heart and kidney due to the NButGT treatment(p<0.05).Mean arterial pressure(MAP,p<0.01) and bicarbonate(p<0.01) are restored in treated group as kalemia(Sham:4.2 ± 0.1;HS:4.7±0.1;NButGT:4.3 ±0.1;mmol/L;p<0.05) and natremia(Sham:141.5±0.4;HS:139.8±0.5;NButGT:141.9 ±0.4; mmol/L;p<0.01).The study of the O-GlcNAcylation of the Na/K ATPase validated its O-GlcNAcylation.Histological analysis of the kidney showed that HS decreased Na/K ATPase expression, but treatment restored it(Sham:15.1±1.1;HS:10.3±0.6;NButGT:13.78±1.1;%area;p<0.05). An acute increase in O-GlcNAc levels through NButGT enhances Na/K ATPase activity. Increased O-GlcNAcylation during hemorrhagic shock restored MAP and reduced indicators of metabolic acidosis.Treatment with NButGT restored natremia and kalemia.This effect is linked to an increase in the expression and activity of Na/K ATPase.Our study is the first to confirm that Na/K ATPase is regulated by O-GlcNAcylation, particularly in terms of its localization and stability.

Acute increase of protein O-GlcNAcylation in mice leads to transcriptome changes in the brain opposite to what is observed in Alzheimer's Disease.

ABSTRACTEnhancing protein O-GlcNAcylation by pharmacological inhibition of the enzyme O-GlcNAcase (OGA) is explored as a strategy to decrease tau and amyloid-beta phosphorylation, aggregation, and pathology in Alzheimer’s disease (AD). There is still more to be learned about the impact of enhancing global protein O-GlcNAcylation, which is important for understanding the mechanistic path of using OGA inhibition to treat AD. In this study, we investigated the acute effect of pharmacologically increasing O-GlcNAc levels, using OGA inhibitor Thiamet G (TG), on normal mouse brains. We hypothesized that the transcritome signature in respones to TG treatment provides a comprehensive view of the effect of OGA inhibition. We sacrificed the mice and dissected their brains after 3 hours of saline or 50 mg/kg TG treatment, and then performed mRNA sequencing using NovaSeq PE 150 (n=5 each group). We identified 1,234 significant differentially expressed genes with TG versus saline treatment. Functional enrichment analysis of the upregulated genes identified several upregulated pathways, including genes normally down in AD. Among the downregulated pathways were the cell adhesion pathway as well as genes normally up in AD and aging. When comparing acute to chronic TG treatment, protein autophosphorylation and kinase activity pathways were upregulated, whereas cell adhesion and astrocyte markers were downregulated in both datasets. Interestingly, mitochondrial genes and genes normally down in AD were up in acute treatment and down in chronic treatment. Data from this analysis will enable the evaluation of the mechanisms underlying the potential benefits of OGA inhibition in the treatment of AD. In particular, although OGA inhibitors are promising to treat AD, their downstream chronic effects related to bioenergetics may be a limiting factor.Abstract Figure

Pharmacological activation of nuclear factor erythroid 2-related factor-2 prevents hyperglycemia-induced renal oxidative damage: Possible involvement of O-GlcNAcylation

Hyperglycemia is a major risk factor for kidney diseases. Oxidative stress, caused by reactive oxygen species, is a key factor in the development of kidney abnormalities related to hyperglycemia. The nuclear factor erythroid 2-related factor-2 (Nrf2) plays a crucial role in defending cells against oxidative stress by activating genes that produce antioxidants. L-sulforaphane (SFN), a drug that activates Nrf2, reduces damage caused by hyperglycemia. Hyperglycemic Wistar rats and HEK 293 cells maintained in hyperglycemic medium exhibited decreased Nrf2 nuclear translocation and reduced expression and activity of antioxidant enzymes. SFN treatment increased Nrf2 activity and reversed decreased renal function, oxidative stress and cell death associated with hyperglycemia. To investigate mechanisms involved in hyperglycemia-induced reduced Nrf2 activity, we addressed whether Nrf2 is modified by O-linked β-N-acetylglucosamine (O-GlcNAc), a post-translational modification that is fueled in hyperglycemic conditions. In vivo, hyperglycemia increased O-GlcNAc-modified Nrf2 expression. Increased O-GlcNAc levels, induced by pharmacological inhibition of OGA, decreased Nrf2 activity in HEK 293 cells. In conclusion, hyperglycemia reduces Nrf2 activity, promoting oxidative stress, cell apoptosis and structural and functional renal damage. Pharmacological treatment with SFN attenuates renal injury. O-GlcNAcylation negatively modulates Nrf2 activity and represents a potential mechanism leading to oxidative stress and renal damage in hyperglycemic conditions.

Abstract P1138: Transcriptomic Insights Into How Previously Increased Cardiac Protein O-GlcNAcylation Enhances Susceptibility To Cardiac Dysfunction In A Response To Pressure-Overload

Increased levels of cardiac protein posttranslational β-linked N-acetylglucosamine (O-GlcNAc) modification are implicated in adverse remodeling seen in hypertrophy and diabetes. Our previous work found elevated O-GlcNAc levels in hearts from diabetic mice, and a chronic increase in O-GlcNAc caused adverse cardiac remodeling. In diabetes, increased cardiac disease risk persists even after patients return to tight glycemic control. Thus, we hypothesize that a transient increase in O-GlcNAc is sufficient to exacerbate adverse cardiac remodeling under subsequent pressure-overload, and we propose that this is due to epigenetic modifications causing persistent changes in gene expression. We used an inducible, cardiomyocyte specific, dominant negative O-GlcNAcase (dnOGA) mouse. After a 2-wk induction (which leads to a 1.8-fold increase in O-GlcNAc levels) and subsequent 2-wk washout, Control (Con) and dnOGA mice undergo transverse-aortic constriction (TAC) or Sham surgery. After 8-wks, cardiac function is measured via echocardiography, gravimetric measures at harvest, and RNA-seq analysis is done on cardiac tissue. In both Con+TAC and dnOGA+TAC vs. Sham groups, systolic function is decreased ( -12%, p < 0.001, -11%, p < 0.01 ), and ventricular weight is increased ( +43%, +69%, both p < 0.0001 ), consistent with cardiac hypertrophy. Interestingly, we see further exacerbation of cardiac hypertrophy ( +29%, p < 0.0001 ) and pulmonary edema ( +36%, p < 0.01 ) between dnOGA+TAC vs. Con+TAC groups. Also, RNA-seq results show distinct transcriptomic profiles between the experimental groups via hierarchical clustering ( |FC| > 1.50, FDR < 0.1 ). A candidate gene approach shows enhanced and exacerbated changes in genes associated with cardiac disease states like heart failure and fibrosis (e.g., Atp2a2, Col1a1, & Nox4 ), consistent with cardiac remodeling. Our results support the hypothesis that transient increase in cardiac O-GlcNAc levels increases susceptibility to subsequent cardiac pathology. Moreover, the exacerbation of pathology is consistent with epigenetic mediated effects of transient O-GlcNAc induction; and pertaining to diabetes, provide a possible mechanism into the continued increased of cardiac disease risk.

Higher levels of O-GlcNAcylation induce aortic stiffness by increasing elastase activity in male, but not female rats.

Age-associated structural and functional changes in the aorta, including stiffness, loss of elasticity, and reduced vasodilatation, are known to hamper stable peripheral circulation and lead to organ dysfunction. The extracellular matrix (ECM) is a network of macromolecules produced by resident cells. Remodeling of ECM components, include fragmentation of elastin and excessive deposition and crosslinking of collagens, and is a hallmark of aging which leads to stiffening of the aorta. Recently, O-linked beta-N-acetyl glucosamine (O-GlcNAc) has emerged as an essential regulator of cell stress and survival. Interestingly, higher levels of O-GlcNAc are a biomarker for cardiovascular risks (e.g., hypertension and diabetes). However, it is unknown whether O-GlcNAcylation contributes to age-associated aortic stiffness. The goal of these studies was divided into two parts: 1. To determine how the higher levels of O-GlcNAc impact aortic stiffness, and 2. To investigate any potential sex differences. To address this aims, the thoracic aorta was isolated from male and female Wistar and Fisher 344 rats at 3 (3M) and 16 months of age (16M) and mounted on a tissue puller (DMT) to evaluate vessel strain/stress. Blood pressure was measured directly in the carotid artery under anesthesia, and western blot, immunofluorescence, and histology were performed to measure O-GlcNAc levels, elastase expression, and elastin fragmentation, respectively. To induce O-GlcNAc, aorta from 3M Wistar rats were treated with Thiamet G, a pharmacological inhibitor of OGA (1 uM, 24h). The aorta from 3M males presented with a higher strain than age-matched females, indicating sex difference in stiffness (ß-coefficient: 2.5±0.9 vs. 1.6±0.6, p<0.05). However, this difference was abolished in old male and female rats (5.1±0.9 vs. 4.6±0.6 p<0.05 n= duplicates of 4-5). The systolic blood pressure in 16 MO males was increased by ~ 30 mmHg compared to 3M (87±3 vs. 117±4 p=3-4), while only increasing ~ 16 mmHg when compared to 3M and 16 MO females (83±3 vs. 99±4 p=3-4). We observed elevation in O-GlcNAc protein levels from aortas of 16 MO males and females, modestly higher in females. Counter-intuitively, the Thiamet G increased O-GlcNAc levels and induced aortic stiffness in males (2.1± 0.9 vs. 3.6± 0.9 p<0.05 n= duplicates 10), but not in females (2.6± 0.9 vs. 2.7±0.8 n= duplicates 5). The aortic stiffness in males was associated with increased elastin fragmentation and elastase expression (Control 100±10 vs. Thiamet G 400±70 UA p<0.05 n=duplicate 3), which was not observed in females. Collectively, these findings indicate that higher levels of O-GlcNAc contributed to sex differences in aortic stiffness and blood pressure during aging possibly by inducing elastin fragmentation. NIH (R00HL151889, RO1DK132948), NIA (K01AG061263), and American Heart Association (AHA-916031) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Benefice of the stimulation of O-GlcNAcylation on sodium potassium pump during hemorrhagic shock

Hemorrhagic shock (HS) is the consequence of an important loss in blood volume leading to an insufficient delivery in oxygen. HS causes metabolic changes, multi-organ dysfunction and inflammation. The acute kidney injury related to HS is associated with hyperkalemia mainly through an alteration of Na/K ATPase pump. In a previous O-GlcNAcylomic study, we have identified Na/K ATPase as potentially O-GlcNAcylated protein. O-GlcNAcylation is a post-translational modification with a putative beneficial effect in shock situation. This study aims at evaluating if Na/K ATPase is O-GlcNAcylated and its potential impact during the early phase of hemorrhagic shock. Wistar rats were subjected to a HS protocol induced via exsanguination and then randomly treated or not with NButGT (10 mg/kg), a O-GlcNAcase inhibitor, to increase O-GlcNAc levels. Blood pressure and heart rate were monitored throughout the experiment. Blood was collected from the animals to perform a complete blood count and a blood gas analysis. Kidney was immediately frozen for histological and biochemical studies. Immunoprecipitation and Wheat Germ Agglutinin (WGA) based lectin affinity gel electrophoresis were used to study the O-GlcNAcylation of Na/K ATPase. NButGT increased by 2 O-GlcNAc levels in heart and kidney. This increase in O-GlcNAc restored mean arterial pressure (MAP) (P < 0.01) and bicarbonates (P < 0.01). In treated group, kalemia (Sham: 4.2 ± 0.1; HS: 4.7 ± 0.1; NButGT: 4.3 ± 0.1 mmol/L; P < 0.05) and natremia (Sham: 141.5 ± 0.4; HS: 139.8 ± 0.5; NButGT: 141.9 ± 0.4 mmol/L; P < 0.01) were restored. The study of the O-GlcNAcylation of the Na/K ATPase by immunoprecipitation and WGA gel validated its O-GlcNAcylation. Histological analysis of kidney revealed that HS reduced NA/K ATPase expression during HS and treatment by NButGT restored it (Sham: 15.1 ± 1.1; HS: 10.3 ± 0.6; NButGT: 13.78 ± 1.1; % area; P < 0.05). Our results demonstrate that increase O-GlcNAcylation during hemorrhagic shock restored MAP and reduced markers of metabolic acidosis. In our study, animal treatment with NButGT to increase O-GlcNAc levels, restored natremia and kalemia. This effect is associated with an increase in Na/K ATPase expression. To our knowledge, our study validates the O-GlcNAcylation of the Na/K ATPase. With regard to our results, now it will be relevant to study the effect of O-GlcNAcylation on the activity and localisation.

Abstract 5280: O-GlcNAcylation of progesterone receptor promotes mammary tumorigenesis

Abstract The role of hormone receptors in breast cancer has been well established as a key contributor to breast cancer tumorigenesis. Estrogen receptor and progesterone receptors are present in nearly 70% of breast cancers. The role of estrogen receptor and estrogens in breast cancer has been extensively studied and targeted effectively through endocrine therapy, however the impact of progesterone and the progesterone receptor (PR) independent of estrogen is not well understood. We have previously demonstrated that PR promotes tumor growth and drives an immune suppressive environment, therefore understanding the biology of PR in breast cancer is critical. We have also demonstrated that PR attenuates type 1 interferon signaling via inhibition of STAT1 phosphorylation and increased degradation of STAT2. Post-translational modifications such as phosphorylation and SUMOylation can influence PR target gene promoter selectivity thereby altering its function. Our recent work indicates PR is modified by O-GlcNAc, a single N-acetyl-glucosamine sugar that cycles on and off and serine or threonine amino acids in nuclear, cytoplasmic and mitochondrial proteins. Levels of total O-GlcNAc staining are higher in breast cancer tissue compared to adjacent normal tissue. Active O-GlcNAcylation, as evidenced by immunohistochemistry staining of O-GlcNAc-transferase (OGT), is elevated in patients with PR+ tumors compared to PR-. Using T47D breast cancer cells (an ER/PR-positive tumor line), mass spectrometry analysis revealed an O-GlcNAc site at S499 on PR. We then used a naturally occurring PR-negative variant of T47D cells to introduce stable expression of a mutant PR with serine to alanine substitution at the O-GlcNAc site (S499A), thereby blocking O-GlcNAc flux, in addition to wt PR as a control. RNA-Seq analysis of these cells following treatment with progesterone revealed several significant differential gene expression patterns, including rescue of PR dependent interferon signaling attenuation. T47D-mutant PR cell lines implanted into immune deficient mice had significantly decreased growth as compared to WT-PR control tumors. In syngeneic mice harboring PR-positive and PR-negative tumors, loss of O-GlcNAc flux and increased O-GlcNac levels increased tumor growth only when tumors were expressing PR, corroborating the influence of O-GlcNAc in immunodeficient mice. Together these findings indicate O-GlcNAcylation of PR is potentially a mechanism of PR driven breast cancer tumorigenesis and immune evasion. Citation Format: Harmony Ivanna Saunders, Sean M. Holloran, Gloria Trinca, Chad Slawson, Christy Hagan. O-GlcNAcylation of progesterone receptor promotes mammary tumorigenesis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5280.

First comprehensive identification of cardiac proteins with putative increased O-GlcNAc levels during pressure overload hypertrophy.

Protein posttranslational modifications (PTMs) by O-GlcNAc globally rise during pressure-overload hypertrophy (POH). However, a major knowledge gap exists on the specific proteins undergoing changes in O-GlcNAc levels during POH primarily because this PTM is low abundance and easily lost during standard mass spectrometry (MS) conditions used for protein identification. Methodologies have emerged to enrich samples for O-GlcNAcylated proteins prior to MS analysis. Accordingly, our goal was to identify the specific proteins undergoing changes in O-GlcNAc levels during POH. We used C57/Bl6 mice subjected to Sham or transverse aortic constriction (TAC) to create POH. From the hearts, we labelled the O-GlcNAc moiety with tetramethylrhodamine azide (TAMRA) before sample enrichment by TAMRA immunoprecipitation (IP). We used LC-MS/MS to identify and quantify the captured putative O-GlcNAcylated proteins. We identified a total of 700 putative O-GlcNAcylated proteins in Sham and POH. Two hundred thirty-three of these proteins had significantly increased enrichment in POH over Sham suggesting higher O-GlcNAc levels whereas no proteins were significantly decreased by POH. We examined two MS identified metabolic enzymes, CPT1B and the PDH complex, to validate by immunoprecipitation. We corroborated increased O-GlcNAc levels during POH for CPT1B and the PDH complex. Enzyme activity assays suggests higher O-GlcNAcylation increases CPT1 activity and decreases PDH activity during POH. In summary, we generated the first comprehensive list of proteins with putative changes in O-GlcNAc levels during POH. Our results demonstrate the large number of potential proteins and cellular processes affected by O-GlcNAc and serve as a guide for testing specific O-GlcNAc-regulated mechanisms during POH.

Disruption of O-GlcNAcylation Homeostasis Induced Ovarian Granulosa Cell Injury in Bovine

O-linked β-N-acetylglucosamine (O-GlcNAc) modification is a ubiquitous, reversible, and highly dynamic post-translational modification, which takes charge of almost all biological processes examined. However, little information is available regarding the molecular regulation of O-GlcNAcylation in granulosa cell function and glucose metabolism. This study focused on the impact of disrupted O-GlcNAc cycling on the proliferation and apoptosis of bovine granulosa cells, and further aimed to determine how this influenced glucose metabolism. Pharmacological inhibition of OGT with benzyl-2-acetamido-2-deoxy-α-D-galactopyranoside (BADGP) led to decreased cellular O-GlcNAc levels, as well as OGT and OGA protein expressions, whereas increasing O-GlcNAc levels with the OGA inhibitor, O-(2-acetamido-2-deoxy--gluco-pyranosylidene) (PUGNAc), resulted in elevated OGA protein expression and decreased OGT protein expression in granulosa cells. Dysregulated O-GlcNAc cycling reduced cell viability, downregulated the proliferation-related genes of CDC42 and PCNA transcripts, upregulated the pro-apoptotic genes of BAX and CASPASE-3 mRNA and the ratio of BAX/BCL-2, and increased the apoptotic rate. Glycolytic enzyme activities of hexokinase and pyruvate kinase, metabolite contents of pyruvate and lactate, mitochondrial membrane potential, ATP levels, and intermediate metabolic enzyme activities of succinate dehydrogenase and malate dehydrogenase involved in the tricarboxylic acid cycle, were significantly impaired in response to altered O-GlcNAc levels. Moreover, inhibition of OGT significantly increased the expression level of thioredoxin-interacting protein (TXNIP), but repression of OGA had no effect. Collectively, our results suggest that perturbation of O-GlcNAc cycling has a profound effect on granulosa cell function and glucose metabolism.

O-GlcNAc stimulation is beneficial in septic shock in the young rat without affecting gene expression

O-GlcNAcylation, a post-translational modification, has been associated with an improvement of the cell survival and the stress response through a modulation of gene expression. We have previously demonstrated that O-GlcNAc stimulation in different models of sepsis is associated with a reduction in mortality and an improvement of different biological markers. Evaluate how O-GlcNAcylation stimulation improves sepsis outcomes in young rats. Endotoxemic shock was induced in 28 days old rats by lipopolysaccharides injection (O111:B4, 20 mg.kg −1 ) and compared to control rats (NaCl 0.9%). One hour after lipopolysaccharides injection, rats were randomly assigned to no therapy (LPS), fluidotherapy (NaCl 0.9%, 10 mg.kg −1 -LPS + R) ± NButGT (10 mg.kg −1 ) to increase O-GlcNAc levels. Physiological functions were evaluated 2 hours later, as well as mortality over 36 hours. Cardiac O-GlcNAcylated proteins were mapped by untargeted mass spectrometry and compared to genes transcription via 3′ SRP analysis. The impact of O-GlcNAc has been evaluated on cardiac tissue and HL1 cells (Thiamet G (O-GlcNAc stimulator): 10-8 to 10-5 M). LPS induced a shock with a decrease in mean arterial pressure ( P < 0.05). LPS + R had no beneficial effect while NButGT improves mean arterial pressure and median time of survival ( P < 0.05). The mRNA expression was not impacted 2 hours after treatment in LPS + R and NButGT group. However, 33 proteins are differentially O-GlcNAcylated in all groups. Among them, 60% are involved in metabolism and metabolic pathways. ATP-citrate lyase (ACLY) a key enzyme in fatty acid biosynthesis, is the only protein less O-GlcNAcylated in the NButGT group. O-GlcNAc stimulation on HL1 cells increases expression of tetrameric form of ACLY ( P < 0.05). Acute O-GlcNAc stimulation improves outcome in young septic rat. Stimulate the O-GlcNAcylation increased cardiac O-GlcNAcylated proteins notably those involved in metabolic pathways. Surprisingly, the transcription is not impacted by the O-GlcNAc stimulation. Study the impact of the O-GlcNAcylation on the activity or the interactome of ACLY is one issue to decipher the involvment of the O-GlcNAcylation during sepsis.

116-LB: Interaction between Iron Stores and Metformin Action

The mechanism of action of MET (MET) remains controversial. MET induces an iron starvation response in yeast, so we hypothesized that tissue iron might influence MET responsiveness in higher organisms. We reviewed electronic health records to correlate MET responsiveness in diabetic humans with serum ferritin, a measure of iron stores. Both higher and subnormal serum ferritin were associated with decreased HbA1c response to MET (N=80, p&amp;lt;0.001) , and MET responsiveness was lost entirely at higher than normal ferritin. Mice were placed on diabetogenic diets with different iron contents, and treated or not with MET. MET responses of fasting glucose values were superior in mice on low-normal and normal iron diets compared to those on high iron: Fasting glucose decreased 60.4 mg/dL on low-normal iron (N=9, p&amp;lt;0.001) , 40.5 mg/dL on normal iron (N=23, p&amp;lt;0.01) and 2.6 mg/dL on high-normal iron (N=27, p=NS) , with significant interaction between iron and MET (p=0.0015, ANOVA) . Other effects of iron (e.g. leptin expression) are known to be mediated by protein modification by O-linked N-acetylglucosamine (O-GlcNAc) . MET increased O-GlcNAc levels 200% in mice on normal iron (p=0.04) but not high-normal iron diets (-8%, p=NS) . In mice with elevated O-GlcNAc resulting from heterozygous deletion of the O-GlcNAcase gene, MET’s ability to decrease fasting glucose levels in mice on high iron was restored, with differences between MET and placebo being 30.3, 61.3, and 56.5 mg/dL on low-normal, normal, and high-normal iron diets (p&amp;lt;0.0001 by ANOVA) ; in contrast to wild type mice, the significant effects of iron and its interaction with MET were lost (p=0.13 and 0.47) . We conclude that tissue iron affects glycemic responses to MET, supporting the hypothesis that MET induces an iron starvation response but that high iron overwhelms that signal. The results on protein O-GlcNAc modification suggest that it mediates the interaction of iron and MET. MET responsiveness in patients with diabetes could be optimized by modulating iron stores. Disclosure D. A. Mcclain: None. C. O. Usoh: None. F. Lorenzo: None. A. V. Harrison: None. Funding VA Research Service 2I01 BX001140; NIH 1P30DK12472

The Identification of a Novel Calcium-Dependent Link Between NAD+ and Glucose Deprivation-Induced Increases in Protein O-GlcNAcylation and ER Stress.

The modification of proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) is associated with the regulation of numerous cellular processes. Despite the importance of O-GlcNAc in mediating cellular function our understanding of the mechanisms that regulate O-GlcNAc levels is limited. One factor known to regulate protein O-GlcNAc levels is nutrient availability; however, the fact that nutrient deficient states such as ischemia increase O-GlcNAc levels suggests that other factors also contribute to regulating O-GlcNAc levels. We have previously reported that in unstressed cardiomyocytes exogenous NAD+ resulted in a time and dose dependent decrease in O-GlcNAc levels. Therefore, we postulated that NAD+ and cellular O-GlcNAc levels may be coordinately regulated. Using glucose deprivation as a model system in an immortalized human ventricular cell line, we examined the influence of extracellular NAD+ on cellular O-GlcNAc levels and ER stress in the presence and absence of glucose. We found that NAD+ completely blocked the increase in O-GlcNAc induced by glucose deprivation and suppressed the activation of ER stress. The NAD+ metabolite cyclic ADP-ribose (cADPR) had similar effects on O-GlcNAc and ER stress suggesting a common underlying mechanism. cADPR is a ryanodine receptor (RyR) agonist and like caffeine, which also activates the RyR, both mimicked the effects of NAD+. SERCA inhibition, which also reduces ER/SR Ca2+ levels had similar effects to both NAD+ and cADPR on O-GlcNAc and ER stress responses to glucose deprivation. The observation that NAD+, cADPR, and caffeine all attenuated the increase in O-GlcNAc and ER stress in response to glucose deprivation, suggests a potential common mechanism, linked to ER/SR Ca2+ levels, underlying their activation. Moreover, we showed that TRPM2, a plasma membrane cation channel was necessary for the cellular responses to glucose deprivation. Collectively, these findings support a novel Ca2+-dependent mechanism underlying glucose deprivation induced increase in O-GlcNAc and ER stress.

Thiamet G effects on mitochondria and AD pathological hallmarks in human iPSC derived models.

Thiamet-G (TMG) has recently been examined in human Alzheimer's Disease (AD) clinical trials. TMG increases total O-GlcNAc levels through inhibition of O-GlcNAcase (OGA). Modulation of OGA through genetic (overexpression or knockdown) or chemical means (TMG) has shown positive effects in transgenic AD animal models. Here, we sought to examine the effects of TMG and increased O-GlcNAc levels on induced pluripotent stem cell (iPSC) models of AD using both neurons and cerebral organoids. Human iPSCs were derived from the KU ADC fibroblast repository or obtained from WiCell. Twelve age and sex matched iPSC lines were used (6 non-demented/ND, 2 familial AD/FAD with APP V717I mutation, and 4 sporadic AD/SAD). Neurons or cerebral organoids were derived and matured from iPSCs using StemCell Technologies StemDiff protocols and reagents. iPSC derived neurons (iNeurons) and cerebral organoids were treated with 20 µM or 40 µM TMG for 14 days. Cell culture supernatants were collected and Aβ/sAPP measured. Protein lysates were used to measure O-GlcNAc, mitochondrial proteins, tau, and cell signaling proteins. Seahorse Bioanalyzer was used to measure oxygen consumption rates and fluorescent imaging for mitochondrial membrane potential, mitochondrial superoxide, and mitochondrial number/turnover. ND and SAD iNeurons treated with TMG had lower Aβ40 . SAD iNeurons had reduced Aβ42 with TMG treatment. PHF1 tau and total tau were increased in TMG treated SAD iNeurons. TMG treatment in ND and SAD iNeurons increased mitochondrial membrane potential and mitochondrial superoxide. TMG treatment increased mitochondrial mass for both new and old mitochondria in SAD iNeurons. In cerebral organoids, TMG treatment increased Aβ40 in ND and SAD samples, increased Aβ42 in SAD samples, and increased the ratio of Aβ42 / Aβ40 in ND and SAD samples. Overall, no changes were observed in FAD TMG treated iNeurons or cerebral organoids. Modulation of O-GlcNAc robustly effects AD pathological hallmarks and mitochondrial function in SAD but not FAD derived iNeurons and cerebral organoids. Further study is warranted to understand the mechanisms and lack of effect in FAD derived cell and organoid models.

Temporal regulation of protein O-GlcNAc levels during pressure-overload cardiac hypertrophy.

Protein posttranslational modifications (PTMs) by O‐linked β‐N‐acetylglucosamine (O‐GlcNAc) rise during pressure‐overload hypertrophy (POH) to affect hypertrophic growth. The hexosamine biosynthesis pathway (HBP) branches from glycolysis to make the moiety for O‐GlcNAcylation. It is speculated that greater glucose utilization during POH augments HBP flux to increase O‐GlcNAc levels; however, recent results suggest glucose availability does not primarily regulate cardiac O‐GlcNAc levels. We hypothesize that induction of key enzymes augment protein O‐GlcNAc levels primarily during active myocardial hypertrophic growth and remodeling with early pressure overload. We further speculate that downregulation of protein O‐GlcNAcylation inhibits ongoing hypertrophic growth during prolonged pressure overload with established hypertrophy. We used transverse aortic constriction (TAC) to create POH in C57/Bl6 mice. Experimental groups were sham, 1‐week TAC (1wTAC) for early hypertrophy, or 6‐week TAC (6wTAC) for established hypertrophy. We used western blots to determine O‐GlcNAc regulation. To assess the effect of increased protein O‐GlcNAcylation with established hypertrophy, mice received thiamet‐g (TG) starting 4 weeks after TAC. Protein O‐GlcNAc levels were significantly elevated in 1wTAC versus Sham with a fall in 6wTAC. OGA, which removes O‐GlcNAc from proteins, fell in 1wTAC versus sham. GFAT is the rate‐limiting HBP enzyme and the isoform GFAT1 substantially rose in 1wTAC. With established hypertrophy, TG increased protein O‐GlcNAc levels but did not affect cardiac mass. In summary, protein O‐GlcNAc levels vary during POH with elevations occurring during active hypertrophic growth early after TAC. O‐GlcNAc levels appear to be regulated by changes in key enzyme levels. Increasing O‐GlcNAc levels during established hypertrophy did not restart hypertrophic growth.

O-GlcNAc cycling mediates energy balance by regulating caloric memory

Caloric need has long been thought a major driver of appetite. However, it is unclear whether caloric need regulates appetite in environments offered by many societies today where there is no shortage of food. Here we observed that wildtype mice with free access to food did not match calorie intake to calorie expenditure. While the size of a meal affected subsequent intake, there was no compensation for earlier under- or over-consumption. To test how spontaneous eating is subject to caloric control, we manipulated O-linked β-N-acetylglucosamine (O-GlcNAc), an energy signal inside cells dependent on nutrient access and metabolic hormones. Genetic and pharmacological manipulation in mice increasing or decreasing O-GlcNAcylation regulated daily intake by controlling meal size. Meal size was affected at least in part due to faster eating speed. Without affecting meal frequency, O-GlcNAc disrupted the effect of caloric consumption on future intake. Across days, energy balance was improved upon increased O-GlcNAc levels and impaired upon removal of O-GlcNAcylation. Rather than affecting a perceived need for calories, O-GlcNAc regulates how a meal affects future intake, suggesting that O-GlcNAc mediates a caloric memory and subsequently energy balance.

O-GlcNAcylation blood levels are increased in response to stress induced by cardiopulmonary bypass

Cardiopulmonary bypass (CPB) is a common procedure to maintain body perfusion during cardiac surgery. It is associated with a systemic inflammatory response, hemostasis and circulatory dysfunction that can lead to organ dysfunction and seldomly to death. O-GlcNAcylation is a post-translational modification modulated in response to stress in different acute and chronic situations. Decipher if blood O-GlcNAc levels are modified during CPB induced stress. Vingt adult patients with CPB time > 60 minutes were included. Blood samples were collected on EDTA at (i) anesthesia induction (Ind), (ii) aortic declamping (AD) and (iii) 5 hours post-declamping (AD + 5). Whole blood and plasma were recovered from samples. O-GlcNAc levels in blood were evaluated by western blot while plasmatic biological markers were measured on automatic laboratory analysis system to correlate O-GlcNAc level with systemic response to CPB. Demographics and One-Way ANOVA analysis were performed with RStudio. CPB induced an early increase of plasmatic inflammatory marker IL-6 (Ind: 5.7 ± 3.2, AD: 120.0 ± 26.5 pg/mL, P < 0.01) that was sustained after 5 hrs. Systemic stress was confirmed via an increase in Creatine Kinase (Ind: 95.2 ± 12.2, AD: 271.9 ± 41.1 U/L, P < 0.001) and Lactate Dehydrogenase (AD: 316.3 ± 32.2, AD + 5: 541.2 ± 64.3 U/L, P < 0.001) release. Heart and renal markers demonstrated a dysfunction with Troponin T (AD: 656 ± 148, AD + 5: 1899 ± 568 pg/mL, P < 0.05) and Creatinine (AD: 94.8 ± 6.6, AD + 5:106.0 ± 7.14 μM, P < 0.05) respectively. CPB induced a significant 20% increase in O-GlcNAc levels ( P < 0.01). In this preliminary study, we highlighted that the inflammation and tissue stress consecutive to CPB are associated with an increase in blood O-GlcNAc levels 5 hours after CPB. Inclusion of more patients and correlations between O-GlcNAc level variation and demographics and/or patients’ outcome might reveal a new biomarker of risk after CPB.

Protective Roles of O‐GlcNAc in Neurodegenerative Diseases

O-linked N-acetylglucosamine (O-GlcNAc) is an abundant form of protein O-glycosylation found in the nucleus, cytoplasm, and mitochondria of all multicellular eukaryotes. This modification is found on several hundred proteins but is surprisingly regulated by only two enzymes; the glycosyltransferase O-GlcNAc transferase (OGT) and the glycoside hydrolase O-GlcNAcase (OGA). Because the substrate of OGT is derived from glucose, the levels of O-GlcNAc within cells reflect environmental glucose availability. O-GlcNAc has also emerged as part of a coordinated response to cellular stresses and levels of this modification increase markedly on exposure to diverse stresses ranging from heat shock to oxidative stress. These observations have stimulated interest in the potential protective roles of increased O-GlcNAc in a range of tissues including in brain, where impaired nutrient utilization has been linked to various neurodegenerative diseases. Remarkably, brain permeable small molecule inhibitors of OGA have been shown to be surprisingly well tolerated in a range of animal models. Moreover, pharmacological agents that increase O-GlcNAc levels in the brains of various transgenic models of Alzheimer Disease (AD) strikingly reduce the progression of disease pathology and neurodegeneration. While these effects have been shown to be remarkably reproducible, and OGA inhibitors have now been found to be generally well tolerated in early clinical trials, the mechanisms by which O-GlcNAc protect against these pathologies and neurodegeneration remain less clear. Here we will discuss new advances in the biochemistry of OGA inhibitors, efforts to create improved inhibitors, and the protective effects of OGA in transgenic models of neurodegenerative diseases. Finally, we will discuss recent progress on various possible protective mechanisms by which increased O-GlcNAc levels confer protection against various neurodegenerative diseases ranging from blockade of aggregation of toxic proteins to the induction of autophagy.

O-GlcNAc stimulation is beneficial in sepsis in the young rat, involvement of the ATP-citrate lyase

The young population, particularly at risk of septic shock, is rarely studied. The O-GlcNAcylation is a post-translational modification involved in cell survival, metabolism and stress response. We have shown that O-GlcNAc stimulation at the early phase of septic shock is beneficial in adult rats. Considering that O-GlcNAc levels are higher in the young, the impact of O-GlcNAc stimulation on sepsis in the young should be tested. Evaluate if O-GlcNAc stimulation could improve sepsis outcomes in young. Endotoxemic shock was induced in 28 days old rats by injection of LPS (O111 :B4, 20 mg.kg −1 –LPS) and compared to control rats (injection of saline–CTRL). One hour after LPS injection rats were randomly assigned to no therapy (LPS), fluidotherapy (saline, 10 mL.kg −1 –LPS + R) ± NButGT (10 mg.kg −1 –NButGT) to increase O-GlcNAc levels ( n = 11 per group). Physiological functions and plasmatic markers were evaluated 2 hours later. Impact of treatment was evaluated with an adapted Pediatric RISk of Mortality score (PRISM score) and mortality was evaluated over 36 hours ( n = 16 per group). Untargeted mass spectrometry was performed to identify cardiac O-GlcNAcylated proteins. LPS induced a shock (mean arterial pressure (MAP): CTRL: 67.2 ± 1.9; LPS: 50.7 ± 2.1 mmHg; P < 0.05), altered biological parameter (lactates: CTRL: 3.92 ± 0.26; LPS: 6.42 ± 0.45 mmol.L −1 ; P < 0.05) and PRISM score ( P < 0.05). LPS + R has no beneficial effect while NButGT improves MAP ( P < 0.05), PRISM score ( P < 0.05) and the median survival (NButGT: 36.0; LPS + R: 13.65; hours; P < 0.001). Most of proteins identified by mass spectrometry (60 %) are involved in the metabolism. Strinckingly, the ATP-citrate lyase (ACLY) is the only protein less O-GlcNAcylated in the NButGT group. O-GlcNAc stimulation acutely improves outcome in young septic shock rat. The impact of O-GlcNAcylation on ACLY is a potential issue to decipher the role of this protein during sepsis.

Abstract 14066: O -GlcNAc Levels Stimulation is a New Potential Therapeutic Strategy at the Early Phase of Septic Shock in the Young

Background: We have shown that increase in O -GlcNAc levels, a post-translational modification involved in the stress response, at the early phase of septic shock in adult (84 days old rats) is a potential new therapeutic strategy. Most studies focus in adults while the population most affected by septic shock, young children, is rarely studied. Considering that O- GlcNAc levels are higher in the young, the impact of O- GlcNAc on septic shock in the young should be tested. Purpose: Evaluate if O- GlcNAc stimulation could improve sepsis outcomes in young. Methods: Endotoxemic shock was induced in 28 days old rats with an i.v. injection of saline (CTRL, n=10) or LPS (O111:B4, 20mg.kg -1 - LPS, n=9). 1 hour after LPS rats were randomly assigned to no therapy (LPS), fluidotherapy (saline, 10ml.kg -1 - LPS+R, n=10) ± NButGT (10 mg.kg -1 - NButGT, n=11) to increase O- GlcNAc levels. 2 hours later, physiological functions and markers of severity were measured and used in adapted Pediatric RISk of Mortality score (PRISM score). The impact of treatment on survival was evaluated on n=16 per group. Mass spectrometry (MS) study was performed to identify O -GlcNAcylated proteins. Results: LPS induce a shock (mean arterial pressure (MAP): CTRL: 67.2 ± 1.9; LPS: 50.7 ± 2.1; mmHg; p&lt;0.05), alter biological parameters (lactates: CTRL: 3.92 ± 0.26; LPS: 6.42 ± 0.45; mmol.l -1 ; pH: CTRL: 7.27 ± 0.02; LPS: 7.15 ± 0.02; p&lt;0.05), PRISM score (p&lt;0.05) and is associated with multi organs dysfunction (troponin T: CTRL: 19.7 ± 4.0; LPS: 45.4 ± 11.4; ng.l -1 ; creatinine: CTRL: 15.9 ± 1.5; LPS: 25.3 ± 2.6; μmol.l -1 ; p&lt;0.05). LPS+R has no beneficial effect while NButGT improves MAP (p&lt;0.05), PRISM score (p&lt;0.05) and the median survival (NButGT: 36.0; LPS+R: 13.65; hours; p&lt;0.001) compared to LPS+R treatment. MS highlight important variations of O- GlcNAcylation particularly that of mitochondrial proteins. Conclusions: Despite higher O- GlcNAc levels, we demonstrate that O- GlcNAc stimulation is also a potential new therapeutic strategy for septic shock in young. Our results show that it is the difference between the basal levels and the post-stimulation levels which induces a protection against sepsis. Proteins identify by MS will need to be specifically studied to decipher their impact in septic shock.