Title: Sustained O-GlcNAcylation reprograms mitochondrial function to regulate energy metabolism
Abstract: Dysfunctional mitochondria and generation of reactive oxygen species (ROS) promote chronic diseases, which have spurred interest in the molecular mechanisms underlying these conditions. Previously, we have demonstrated that disruption of post-translational modification of proteins with β-linked N-acetylglucosamine (O-GlcNAcylation) via overexpression of the O-GlcNAc-regulating enzymes O-GlcNAc transferase (OGT) or O-GlcNAcase (OGA) impairs mitochondrial function. Here, we report that sustained alterations in O-GlcNAcylation either by pharmacological or genetic manipulation also alter metabolic function. Sustained O-GlcNAc elevation in SH-SY5Y neuroblastoma cells increased OGA expression and reduced cellular respiration and ROS generation. Cells with elevated O-GlcNAc levels had elongated mitochondria and increased mitochondrial membrane potential, and RNA-sequencing analysis indicated transcriptome reprogramming and down-regulation of the NRF2-mediated antioxidant response. Sustained O-GlcNAcylation in mouse brain and liver validated the metabolic phenotypes observed in the cells, and OGT knockdown in the liver elevated ROS levels, impaired respiration, and increased the NRF2 antioxidant response. Moreover, elevated O-GlcNAc levels promoted weight loss and lowered respiration in mice and skewed the mice toward carbohydrate-dependent metabolism as determined by indirect calorimetry. In summary, sustained elevation in O-GlcNAcylation coupled with increased OGA expression reprograms energy metabolism, a finding that has potential implications for the etiology, development, and management of metabolic diseases. Dysfunctional mitochondria and generation of reactive oxygen species (ROS) promote chronic diseases, which have spurred interest in the molecular mechanisms underlying these conditions. Previously, we have demonstrated that disruption of post-translational modification of proteins with β-linked N-acetylglucosamine (O-GlcNAcylation) via overexpression of the O-GlcNAc-regulating enzymes O-GlcNAc transferase (OGT) or O-GlcNAcase (OGA) impairs mitochondrial function. Here, we report that sustained alterations in O-GlcNAcylation either by pharmacological or genetic manipulation also alter metabolic function. Sustained O-GlcNAc elevation in SH-SY5Y neuroblastoma cells increased OGA expression and reduced cellular respiration and ROS generation. Cells with elevated O-GlcNAc levels had elongated mitochondria and increased mitochondrial membrane potential, and RNA-sequencing analysis indicated transcriptome reprogramming and down-regulation of the NRF2-mediated antioxidant response. Sustained O-GlcNAcylation in mouse brain and liver validated the metabolic phenotypes observed in the cells, and OGT knockdown in the liver elevated ROS levels, impaired respiration, and increased the NRF2 antioxidant response. Moreover, elevated O-GlcNAc levels promoted weight loss and lowered respiration in mice and skewed the mice toward carbohydrate-dependent metabolism as determined by indirect calorimetry. In summary, sustained elevation in O-GlcNAcylation coupled with increased OGA expression reprograms energy metabolism, a finding that has potential implications for the etiology, development, and management of metabolic diseases. Chronically impaired mitochondrial respiration and uncontrolled reactive oxygen species (ROS) 2The abbreviations used are: ROS, reactive oxygen species, O-GlcNAc, N-acetylglucosamine; OGT, O-GlcNAc transferase; OGA, O-GlcNAcase; HBP, hexosamine biosynthetic pathway; TMG, thiamet-G; PI, propidium iodide; PARP, poly(ADP-ribose) polymerase; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; MAS, mitochondrial assay solution; CHX, cycloheximide; RER, respiratory exchange ratio; qPCR, quantitative PCR; F, forward; R, reverse; SOD, superoxide dismutase; ETC, electron transport chain; OCR, oxygen consumption rate; RNA-seq, RNA-sequencing; EE, energy expenditure; mOGT, mitochondrial form of OGT; AD, Alzheimer's disease; Avg, average; IPA, ingenuity pathway analysis; GSEA, gene set enrichment analysis; RNS, reactive nitrogen species; CS, citrate synthase; TXN, thioredoxin; ECAR, extracellular acidification rate; TRX, thioredoxin. 2The abbreviations used are: ROS, reactive oxygen species, O-GlcNAc, N-acetylglucosamine; OGT, O-GlcNAc transferase; OGA, O-GlcNAcase; HBP, hexosamine biosynthetic pathway; TMG, thiamet-G; PI, propidium iodide; PARP, poly(ADP-ribose) polymerase; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; MAS, mitochondrial assay solution; CHX, cycloheximide; RER, respiratory exchange ratio; qPCR, quantitative PCR; F, forward; R, reverse; SOD, superoxide dismutase; ETC, electron transport chain; OCR, oxygen consumption rate; RNA-seq, RNA-sequencing; EE, energy expenditure; mOGT, mitochondrial form of OGT; AD, Alzheimer's disease; Avg, average; IPA, ingenuity pathway analysis; GSEA, gene set enrichment analysis; RNS, reactive nitrogen species; CS, citrate synthase; TXN, thioredoxin; ECAR, extracellular acidification rate; TRX, thioredoxin. generation contribute to complex chronic diseases (1.Lezi E. Swerdlow R.H. Mitochondria in neurodegeneration.Adv. Exp. Med. Biol. 2012; 942: 269-286Crossref PubMed Scopus (137) Google Scholar). Interventions that improve mitochondrial function and reduce uncontrolled ROS production will have widespread clinical applications in the treatment of chronic disease. Prolonged treatment with glucosamine (GlcN), a common amino-sugar, improved mitochondrial function and extended the life span in both Caenorhabditis. elegans and mice. GlcN treatment increased mitochondrial respiration and transiently induced mitochondrial ROS generation followed by a significant reduction in ROS (2.Weimer S. Priebs J. Kuhlow D. Groth M. Priebe S. Mansfeld J. Merry T.L. Dubuis S. Laube B. Pfeiffer A.F. Schulz T.J. Guthke R. Platzer M. Zamboni N. Zarse K. Ristow M. d-Glucosamine supplementation extends life span of nematodes and of ageing mice.Nat. Commun. 2014; 5: 3563Crossref PubMed Scopus (138) Google Scholar). The initial transient ROS induction promotes mitochondrial biogenesis and improves anti-oxidant response resulting in life-span extension (2.Weimer S. Priebs J. Kuhlow D. Groth M. Priebe S. Mansfeld J. Merry T.L. Dubuis S. Laube B. Pfeiffer A.F. Schulz T.J. Guthke R. Platzer M. Zamboni N. Zarse K. Ristow M. d-Glucosamine supplementation extends life span of nematodes and of ageing mice.Nat. Commun. 2014; 5: 3563Crossref PubMed Scopus (138) Google Scholar). Of note, GlcN feeds into the hexosamine biosynthetic pathway (HBP) after the pathway rate-limiting enzyme glutamine fructose-6-phosphate aminotransferase 1 (GFAT) leading to increased O-GlcNAcylation; thus, we hypothesize that O-GlcNAc regulates mitochondrial function, the oxidative stress response, and can influence chronic disease progression. O-GlcNAcylation is the addition of a single O-linked β-d-N-acetylglucosamine sugar moiety to serine or threonine residues in nuclear, cytoplasmic, and mitochondrial proteins (3.Hart G.W. Slawson C. Ramirez-Correa G. Lagerlof O. Cross talk between O-GlcNAcylation and phosphorylation: roles in signaling, transcription, and chronic disease.Annu. Rev. Biochem. 2011; 80: 825-858Crossref PubMed Scopus (915) Google Scholar). O-GlcNAcylation is facilitated by two functionally opposing enzymes, O-GlcNAc transferase (OGT) catalyzes the addition of O-GlcNAc and O-GlcNAcase (OGA) hydrolyzes the modification (3.Hart G.W. Slawson C. Ramirez-Correa G. Lagerlof O. Cross talk between O-GlcNAcylation and phosphorylation: roles in signaling, transcription, and chronic disease.Annu. Rev. Biochem. 2011; 80: 825-858Crossref PubMed Scopus (915) Google Scholar). During catalysis, OGT uses the nucleotide sugar UDP-GlcNAc generated from the HBP. GlcN feeds into the HBP downstream of GFAT1 and increases cellular O-GlcNAcylation by elevating the synthesis of UDP-GlcNAc (4.Marshall S. Nadeau O. Yamasaki K. Dynamic actions of glucose and glucosamine on hexosamine biosynthesis in isolated adipocytes: differential effects on glucosamine 6-phosphate, UDP-N-acetylglucosamine, and ATP levels.J. Biol. Chem. 2004; 279: 35313-35319Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Therefore, GlcN influence on mitochondrial function and life-span extension could be mediated by O-GlcNAc. Because UDP-GlcNAc synthesis relies on the integration of multiple metabolic pathways, O-GlcNAcylation can sense changes in nutrients (5.Hart G.W. Three decades of research on O-GlcNAcylation–a major nutrient sensor that regulates signaling, transcription and cellular metabolism.Front. Endocrinol. (Lausanne). 2014; 5: 183Crossref PubMed Scopus (81) Google Scholar). Importantly, chronic disruptions in O-GlcNAcylation due to nutrient imbalance influence metabolic diseases (6.Bond M.R. Hanover J.A. O-GlcNAc cycling: a link between metabolism and chronic disease.Annu. Rev. Nutr. 2013; 33: 205-229Crossref PubMed Scopus (207) Google Scholar, 7.Dias W.B. Hart G.W. O-GlcNAc modification in diabetes and Alzheimer's disease.Mol. Biosyst. 2007; 3: 766-772Crossref PubMed Scopus (204) Google Scholar). O-GlcNAc is a key regulator of mitochondrial function (8.Love D.C. Kochan J. Cathey R.L. Shin S.H. Hanover J.A. Kochran J. Mitochondrial and nucleocytoplasmic targeting of O-linked GlcNAc transferase.J. Cell Sci. 2003; 116: 647-654Crossref PubMed Scopus (154) Google Scholar, 9.Tan E.P. Villar M.T. E L. Lu J. Selfridge J.E. Artigues A. Swerdlow R.H. Slawson C. Altering O-linked β-N-acetylglucosamine cycling disrupts mitochondrial function.J. Biol. Chem. 2014; 289: 14719-14730Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Both OGT (8.Love D.C. Kochan J. Cathey R.L. Shin S.H. Hanover J.A. Kochran J. Mitochondrial and nucleocytoplasmic targeting of O-linked GlcNAc transferase.J. Cell Sci. 2003; 116: 647-654Crossref PubMed Scopus (154) Google Scholar) and OGA localize to the mitochondria (10.Banerjee P.S. Ma J. Hart G.W. Diabetes-associated dysregulation of O-GlcNAcylation in rat cardiac mitochondria.Proc. Natl. Acad. Sci. U.S.A. 2015; 112: 6050-6055Crossref PubMed Scopus (130) Google Scholar), and the pyrimidine nucleotide carrier 1 (PNC1) transports UDP-GlcNAc into the mitochondria providing substrate for OGT (10.Banerjee P.S. Ma J. Hart G.W. Diabetes-associated dysregulation of O-GlcNAcylation in rat cardiac mitochondria.Proc. Natl. Acad. Sci. U.S.A. 2015; 112: 6050-6055Crossref PubMed Scopus (130) Google Scholar). Several electron transport chain (ETC) subunits are modified by O-GlcNAc (11.Ma J. Liu T. Wei A.C. Banerjee P. O'Rourke B. Hart G.W. O-GlcNAcomic profiling identifies widespread O-linked β-N-acetylglucosamine modification (O-GlcNAcylation) in oxidative phosphorylation system regulating cardiac mitochondrial function.J. Biol. Chem. 2015; 290: 29141-29153Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) suggesting that O-GlcNAc modulates ETC activity. For example, short-term high-glucose treatment elevates O-GlcNAcylation but suppresses ETC activity and ATP production in rat cardiomyocytes (12.Hu Y. Suarez J. Fricovsky E. Wang H. Scott B.T. Trauger S.A. Han W. Hu Y. Oyeleye M.O. Dillmann W.H. Increased enzymatic O-GlcNAcylation of mitochondrial proteins impairs mitochondrial function in cardiac myocytes exposed to high glucose.J. Biol. Chem. 2009; 284: 547-555Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar); however, short-term OGA inhibition in rats increased oxygen consumption in isolated mitochondria (11.Ma J. Liu T. Wei A.C. Banerjee P. O'Rourke B. Hart G.W. O-GlcNAcomic profiling identifies widespread O-linked β-N-acetylglucosamine modification (O-GlcNAcylation) in oxidative phosphorylation system regulating cardiac mitochondrial function.J. Biol. Chem. 2015; 290: 29141-29153Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). OGT or OGA overexpression in SH-SY5Y neuroblastoma cells alters the mitochondrial proteome, including several TCA cycles and ETC proteins (9.Tan E.P. Villar M.T. E L. Lu J. Selfridge J.E. Artigues A. Swerdlow R.H. Slawson C. Altering O-linked β-N-acetylglucosamine cycling disrupts mitochondrial function.J. Biol. Chem. 2014; 289: 14719-14730Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). The loss of ETC protein expression adversely affected mitochondrial shape and impaired respiration. These data indicate O-GlcNAc is essential for the proper regulation of mitochondrial function and energy metabolism. Here, we report that increasing O-GlcNAcylation in cell lines and mice by inhibiting OGA or with GlcN supplementation reprograms mitochondrial function and changes energy metabolism. Sustained elevation in O-GlcNAc lowered respiration and reduced ROS generation. These changes were followed by transcriptional reprogramming of the proteome and a decrease in the transcription factor nuclear factor (erythroid-derived)-like 2 (NRF2)-mediated oxidative stress response. In contrast, OGT liver knockdown severely impaired mitochondrial function, increased ROS levels, and activated the NRF2 antioxidant response. Together, these data demonstrate distinct roles for O-GlcNAc in modulating mitochondrial function and provide new insights into how pharmacological targeting of O-GlcNAc protects against aging and metabolic diseases. Prolonged treatment with GlcN improves mitochondrial function and extends the life span in both C. elegans and mice (2.Weimer S. Priebs J. Kuhlow D. Groth M. Priebe S. Mansfeld J. Merry T.L. Dubuis S. Laube B. Pfeiffer A.F. Schulz T.J. Guthke R. Platzer M. Zamboni N. Zarse K. Ristow M. d-Glucosamine supplementation extends life span of nematodes and of ageing mice.Nat. Commun. 2014; 5: 3563Crossref PubMed Scopus (138) Google Scholar). We hypothesized that sustained elevation of O-GlcNAcylation would alter energy metabolism. To determine how O-GlcNAcylation affects energy metabolism, we treated SH-SY5Y neuroblastoma cells with either TMG (10 μm) or GlcN (0.35 mm) for at least 3 weeks prior to harvesting these cells. TMG is a highly selective OGA inhibitor (13.Yuzwa S.A. Macauley M.S. Heinonen J.E. Shan X. Dennis R.J. He Y. Whitworth G.E. Stubbs K.A. McEachern E.J. Davies G.J. Vocadlo D.J. A potent mechanism-inspired O-GlcNAcase inhibitor that blocks phosphorylation of tau in vivo.Nat. Chem. Biol. 2008; 4: 483-490Crossref PubMed Scopus (490) Google Scholar), whereas GlcN feeds into the hexosamine biosynthetic pathway after GFAT1 increasing cellular pools of UDP-GlcNAc (4.Marshall S. Nadeau O. Yamasaki K. Dynamic actions of glucose and glucosamine on hexosamine biosynthesis in isolated adipocytes: differential effects on glucosamine 6-phosphate, UDP-N-acetylglucosamine, and ATP levels.J. Biol. Chem. 2004; 279: 35313-35319Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). In contrast to GlcN, which can influence a variety of metabolic pathways, TMG precisely manipulates O-GlcNAcylation by targeting only OGA. Both TMG- and GlcN-treated SH-SY5Y cells increased total and mitochondrial O-GlcNAc levels (Fig. 1, a and b). Although not significant, OGT protein and transcript levels were trending lower; while OGA protein and transcript levels were significantly increased in response to elevated O-GlcNAcylation (Fig. 1, a and d) (14.Zhang Z. Tan E.P. VandenHull N.J. Peterson K.R. Slawson C. O-GlcNAcase expression is sensitive to changes in O-GlcNAc Homeostasis.Front. Endocrinol. (Lausanne). 2014; 5: 206Crossref PubMed Scopus (79) Google Scholar). GFAT1 protein level was decreased in TMG-treated cells (Fig. 1a). OGA-specific activity was increased in GlcN-treated SH-SY5Y cells (Fig. 1e). Similar results were found in NT2 human teratocarcinoma cells treated with TMG or GlcN (Fig. 1c). Prolonged elevation of O-GlcNAc altered cellular respiration in SH-SY5Y cells (Fig. 2a). TMG lowered baseline oxygen consumption rates (OCR), whereas GlcN increased baseline OCR (Fig. 2b). Under basal conditions, in vivo ATP production was lower in the presence of TMG- but higher in GlcN-treated cells (Fig. 2f). Neither treatment affected the proton leak rate (Fig. 2c). The maximum respiratory rate was increased in GlcN cells, whereas TMG was similar to control cells (Fig. 2d). SH-SY5Y cells grown in oxidative media regardless of TMG or GlcN treatment have no reserve capacity (Fig. 2e), showing that these cells were respiring at their maximum capacity. To eliminate any cell type-specific effects, we performed the respiration assays in NT2 cells. TMG-treated NT2 cells replicated the data from SH-SY5Y cells (Fig. 2, i–n). In NT2 cells, GlcN-treated mirrored the TMG-treated results from SH-SY5Y/NT2 cells showing lower respiration rates (Fig. 2, i–n). NT2 cell mitochondria had much more reserve capacity than the SH-SY5Y cells suggesting that these cells were not respiring at a maximal rate. The total cellular ATP levels in both TMG- and GlcN-treated SH-SY5Y cells and in TMG-treated NT2 cells were lower (Fig. 2, g and o). TMG treatment caused both SH-SY5Y and NT2 cells to produce significantly less ATP. Likely, GlcN-treated SH-SY5Y cells produced and consumed more energy due to increased phosphorylation of GlcN to GlcN-6-P by hexose kinase (4.Marshall S. Nadeau O. Yamasaki K. Dynamic actions of glucose and glucosamine on hexosamine biosynthesis in isolated adipocytes: differential effects on glucosamine 6-phosphate, UDP-N-acetylglucosamine, and ATP levels.J. Biol. Chem. 2004; 279: 35313-35319Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Because oxidative phosphorylation and glycolysis are interdependent energy-producing pathways (15.Fernie A.R. Carrari F. Sweetlove L.J. Respiratory metabolism: glycolysis, the TCA cycle and mitochondrial electron transport.Curr. Opin. Plant Biol. 2004; 7: 254-261Crossref PubMed Scopus (782) Google Scholar), we examined glycolytic energy production by measuring cellular glycolytic flux. Both TMG- and GlcN-treated SH-SY5Y cells had lower basal glycolytic rates (Fig. 2h), and TMG-treated NT2 cells had a lower basal glycolytic rate. GlcN-treated NT2 cells showed no changes (Fig. 2p). Because prolonged TMG treatment lowered cellular respiration, we postulated that the ETC complex activity was lower; however, mitochondria isolated from both TMG- and GlcN-treated SH-SY5Y cells had increased complex I activity (Fig. 3a). TMG also elevated complex IV activity (Fig. 3, b–d). Because isolated ETC complex activity did not correspond to the in vitro respiration measurements, we considered whether the observed decrease in OCR was due to reduced concentration of NADH available for respiration. The NAD+ concentration was elevated in TMG but reduced in GlcN-treated SH-SY5Y cells (Fig. 3e), whereas the NADH concentration was decreased in both TMG- and GlcN-treated SH-SY5Y cells (Fig. 3f). The NAD+ and NADH ratio skewed toward NAD+ for both TMG- and GlcN-treated cells (Fig. 3g). To ascertain whether altered cellular respiration was due to changes in mitochondrial membrane potential, we probed for mitochondrial membrane polarization differences in TMG- or GlcN-treated SH-SY5Y cells. TMG significantly enhanced mitochondrial hyperpolarization (Fig. 3h). To assess whether mitochondrial morphology was changed in TMG- or GlcN-treated SH-SY5Y cells, we used transmission electron microscopy to visualize the mitochondria. The approximate average length of mitochondria from both treated cells was longer compared with control cells (Fig. 3, i and j). A typical length of a mitochondrion ranges from ∼0.5 to 2 μm (16.Baffou G. Rigneault H. Marguet D. Jullien L. A critique of methods for temperature imaging in single cells.Nat. Methods. 2014; 11: 899-901Crossref PubMed Scopus (169) Google Scholar). We found that both treatments increased the percentage of mitochondria that were longer than 2 μm (Fig. 3k). Confocal imaging data agreed with the electron microscopy data showing longer mitochondria in TMG- or GlcN-treated cells (Fig. 3l). Next, we measured the proteins involved in regulating mitochondrial fission and fusion. Mitofusin 1 (MFN1), mitofusin 2 (MFN2), and dynamin-like proteins (DLP1), which facilitate mitochondrial fusion and fission, were lower in TMG-treated cells; however, GlcN treatment increased the DLP1 protein levels, whereas MFN1 and MFN2 levels were unaffected (Fig. 3m). We considered whether the observed mitochondrial changes were mediated by alterations in the transcriptome. Our Next Generation RNA-sequencing (RNA-seq) analysis found 8793 genes in TMG- or GlcN-treated SH-SY5Y cells with high sequencing counts (cpm of >10 in at least two samples) (Fig. 4, a–c, and supplemental Table S1; RNA-seq data are available on the Gene Expression Omnibus). A total of 240 genes for TMG treatment and 48 genes for GlcN treatment were elevated, whereas 152 genes for TMG treatment and 257 genes for GlcN treatment were reduced (p value < 0.01) (Fig. 4d). Among these genes, 230 genes from TMG and 48 genes from GlcN treatment were increased 1.5-fold or more, whereas 149 genes from TMG and 257 genes from GlcN decreased 1.5-fold or more (Fig. 4d). The tissue factor pathway inhibitor 2 (TFPI2), guanine nucleotide-binding protein, γ subunit 1 (GNG1), activating transcription factor 4 (ATF4), and insulin-like growth factor 2 (IGF2) were selected for orthogonal validation and agreed with the RNA sequencing results (Fig. 4, e and f). As a positive control (Fig. 1d), the RNA-seq data showed that OGA (MGEA5) gene expression was increased in both TMG and GlcN treatment (supplemental Tables S2 and S3) (14.Zhang Z. Tan E.P. VandenHull N.J. Peterson K.R. Slawson C. O-GlcNAcase expression is sensitive to changes in O-GlcNAc Homeostasis.Front. Endocrinol. (Lausanne). 2014; 5: 206Crossref PubMed Scopus (79) Google Scholar). Using gene set enrichment analysis (GSEA) (17.Subramanian A. Tamayo P. Mootha V.K. Mukherjee S. Ebert B.L. Gillette M.A. Paulovich A. Pomeroy S.L. Golub T.R. Lander E.S. Mesirov J.P. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles.Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 15545-15550Crossref PubMed Scopus (26549) Google Scholar) to compare TMG-treated with control gene expression data sets, we identified genes from the insulin-signaling pathway, aging, and cancer metastasis enriched in prolonged TMG-treated cells (Fig. 4, g–i, and supplemental Table S4). These data suggested that prolonged O-GlcNAc elevation modulates the progression of chronic metabolic disease. To define biological functions of the altered genes, we performed Ingenuity Pathway Analysis (IPA). A stringent threshold (cpm of >100) was applied to the gene set to prevent false positives. 22 canonical pathways for TMG-treated (Fig. 5a) and 26 canonical pathways for GlcN-treated cells (Fig. 5b) were identified (supplemental Table S5). 13 pathways overlapped in both TMG and GlcN-treated cells and changed in the same predicted manner (see Fig. 5, a and b). Interestingly, the NRF2-mediated oxidative stress response was identified as down-regulated in both TMG- and GlcN-treated SH-SY5Y cells. These data suggest that both TMG and GlcN treatments could modulate ROS levels. TMG or GlcN could affect cellular ROS levels, including reactive nitrogen species (RNS), hydroxyl radical (OH•), hydrogen peroxide (H2O2), and superoxide (O2̇̄) (Fig. 6a). Total ROS (RNS•, OH•, and H2O2) was reduced in both TMG- and GlcN-treated SH-SY5Y cells (Fig. 6b). Superoxide levels were reduced in TMG-treated cells (Fig. 6c). Total ROS and superoxide levels were lower in TMG-treated NT2 cells, whereas GlcN-treated NT2 cells trended lower (Fig. 6, d and e). Because NADPH oxidase is a major source of cytoplasmic ROS (18.Mueller C.F. Laude K. McNally J.S. Harrison D.G. ATVB in focus: redox mechanisms in blood vessels.Arterioscler. Thromb. Vasc. Biol. 2005; 25: 274-278Crossref PubMed Scopus (248) Google Scholar), we measured and found that both TMG and GlcN treatments decreased NADPH oxidase protein levels in SH-SY5Y cells (Fig. 6f). Together, these data indicated that prolonged elevations of O-GlcNAcylation suppress ROS production. We hypothesized that O-GlcNAc could be directly modulating NRF2 function. TMG treatment lowered NRF2 protein and NRF2 transcript levels (Fig. 7, a and b). Additionally, TMG treatment lowered the NRF2 target gene thioredoxin reductase 1 (TXNRD1) transcript level (Fig. 7c) and reduced superoxide dismutase 1 (SOD1) and thioredoxin (TXN) protein levels (Fig. 7d). However, TXNRD1 and peroxidase 1 (PRX1) protein expression was not altered (Fig. 7e). Interestingly, both TMG and GlcN treatments increased NRF2 O-GlcNAcylation (Fig. 7f). Because O-GlcNAcylation of transcription factors influences their cytolocalization (19.Housley M.P. Rodgers J.T. Udeshi N.D. Kelly T.J. Shabanowitz J. Hunt D.F. 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Cell Biol. 2006; 8: 1074-1083Crossref PubMed Scopus (339) Google Scholar), we pretreated SH-SY5Y cells with cycloheximide (CHX) to inhibit protein translation (22.Obrig T.G. Culp W.J. McKeehan W.L. Hardesty B. The mechanism by which cycloheximide and related glutarimide antibiotics inhibit peptide synthesis on reticulocyte ribosomes.J. Biol. Chem. 1971; 246: 174-181Abstract Full Text PDF PubMed Google Scholar) and harvested the cells at different time points after CHX treatment. We observed no change in NRF2 protein levels in O-GlcNAc-elevated cells between 0 h and up to 6 h after CHX treatment in relation to control cells (Fig. 7h). The NRF2-binding partner KEAP1–E3–ligase complex (Kelch-like ECH associated protein 1) promotes degradation of the transcription factor. However, KEAP1 protein levels did not change after TMG and GlcN treatment (Fig. 7i), suggesting that the TMG-mediated decrease in NRF2 protein levels was not due to altered stability of the protein but due to the decrease in the transcript. MnSOD, a non-NRF2 controlled antioxidant enzyme, is a major antioxidant enzyme present in mitochondria and is essential for the removal of superoxide generated by the ETC complexes (23.Sullivan L.B. Chandel N.S. Mitochondrial reactive oxygen species and cancer.Cancer Metab. 2014; 2: 17Crossref PubMed Google Scholar). MnSOD protein level was decreased in both TMG- and GlcN-treated SH-SY5Y cells (Fig. 7j). Because several antioxidant response pathways were down-regulated with TMG and GlcN treatment, we treated SH-SY5Y cells with 50 or 100 μm H2O2 for upwards of 7 h to induce oxidative stress (24.Tangmansakulchai K. Abubakar Z. Kitiyanant N. Suwanjang W. Leepiyasakulchai C. Govitrapong P. Chetsawang B. Calpastatin overexpression reduces oxidative stress-induced mitochondrial impairment and cell death in human neuroblastoma SH-SY5Y cells by decreasing calpain and calcineurin activation, induction of mitochondrial fission and destruction of mitochondrial fusion.Mitochondrion. 2016; 30: 151-161PubMed Google Scholar), and we measured cell viability. TMG increased cell viability in 100 μm H2O2 (Fig. 7k). The total O-GlcNAcylation level was robustly increased after oxidative stress induction in control cells (Fig. 7l), and the apoptosis marker, cleaved poly(ADP-ribose) polymerase (PARP) protein, was increased after oxidative stress (Fig. 7l). Catalase, TXNRD1, PRDX1, and TXN protein levels were not significantly altered (Fig. 7l). Likely, the increased cell viability in TMG-treated cells was due to increased O-GlcNAcylation mediating other stress-response pathways (25.Groves J.A. Lee A. Yildirir G. Zachara N.E. Dynamic O-GlcNAcylation and its roles in the cellular stress response and homeostasis.Cell Stress Chaperones. 2013; 18: 535-558Crossref PubMed Scopus (95) Google Scholar). Because long-term TMG treatment reduced mitochondrial respiration in cell lines, we asked the questions whether TMG-treated mice replicated the cell line data. We intraperitoneally injected TMG into C57B6J mice at a concentration of 50 mg/kg (13.Yuzwa S.A. Macauley M.S. Heinonen J.E. Shan X. Dennis R.J. He Y. Whitworth G.E. Stubbs K.A. McEachern E.J. Davies G.J. Vocadlo D.J. A potent mechanism-inspired O-GlcNAcase inhibitor that blocks phosphorylation of tau in vivo.Nat. Chem. Biol. 2008; 4: 483-490Crossref PubMed Scopus (490) Google Scholar) every other day for up to 15 days prior to sacrificing the animals. Liver and brain tissues were then used for subsequent experiments (Fig. 8a). TMG robustly elevated brain O-GlcNAcylation and OGA expression and reduced OGT protein levels (Fig. 8b). Similar results were obtained in the livers of TMG-treated mice (Fig. 8c). Next, we purified and pooled mitochondria from four TMG and four control mouse brains and performed ex vivo respiration assays (26.Rogers G.W. Brand M.D. Petrosyan S. Ashok D. Elorza A.A. Ferrick D.A. Murphy A.N. High throughput microplate respiratory measurements using minimal quantities of isolated mitochondria.PLoS ONE. 2011; 6: e21746Crossref PubMed Scopus (329) Google Scholar). TMG lowered basal respiration (basal, in the presence of substrate but no ADP) as well as phospho-relating respiration (State 3