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Nutrient regulation of signaling and transcription

  • Gerald W. Hart
    Correspondence
    To whom correspondence should be addressed. Tel.:706-583-5550; E-mail:.
    Affiliations
    From the Complex Carbohydrate Research Center and Biochemistry and Molecular Biology Department, University of Georgia, Athens, Georgia 30602
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Open AccessPublished:January 09, 2019DOI:https://doi.org/10.1074/jbc.AW119.003226
      In the early 1980s, while using purified glycosyltransferases to probe glycan structures on surfaces of living cells in the murine immune system, we discovered a novel form of serine/threonine protein glycosylation (O-linked β-GlcNAc; O-GlcNAc) that occurs on thousands of proteins within the nucleus, cytoplasm, and mitochondria. Prior to this discovery, it was dogma that protein glycosylation was restricted to the luminal compartments of the secretory pathway and on extracellular domains of membrane and secretory proteins. Work in the last 3 decades from several laboratories has shown that O-GlcNAc cycling serves as a nutrient sensor to regulate signaling, transcription, mitochondrial activity, and cytoskeletal functions. O-GlcNAc also has extensive cross-talk with phosphorylation, not only at the same or proximal sites on polypeptides, but also by regulating each other’s enzymes that catalyze cycling of the modifications. O-GlcNAc is generally not elongated or modified. It cycles on and off polypeptides in a time scale similar to phosphorylation, and both the enzyme that adds O-GlcNAc, the O-GlcNAc transferase (OGT), and the enzyme that removes O-GlcNAc, O-GlcNAcase (OGA), are highly conserved from C. elegans to humans. Both O-GlcNAc cycling enzymes are essential in mammals and plants. Due to O-GlcNAc’s fundamental roles as a nutrient and stress sensor, it plays an important role in the etiologies of chronic diseases of aging, including diabetes, cancer, and neurodegenerative disease. This review will present an overview of our current understanding of O-GlcNAc’s regulation, functions, and roles in chronic diseases of aging.

      Introduction

      O-GlcNAc was discovered when bovine milk galactosyltransferase and UDP-[3H]galactose were used to probe the surfaces of living cells of the murine immune system for terminal GlcNAc moieties (
      • Torres C.R.
      • Hart G.W.
      Topography and polypeptide distribution of terminal N-acetylglucosamine residues on the surfaces of intact lymphocytes: evidence for O-linked GlcNAc.
      ). Surprisingly, further analyses showed that nearly all of the incorporated [3H]galactose was added to single β-O-linked GlcNAc moieties attached to Ser(Thr) residues on polypeptides. Follow-up experiments showed that O-GlcNAc is highly enriched within the nucleus and cytoplasm (
      • Holt G.D.
      • Hart G.W.
      The subcellular distribution of terminal N-acetylglucosamine moieties: localization of a novel protein-saccharide linkage, O-linked GlcNAc.
      ), particularly on nuclear envelope and chromatin proteins. Thus, the labeling in initial experiments (
      • Torres C.R.
      • Hart G.W.
      Topography and polypeptide distribution of terminal N-acetylglucosamine residues on the surfaces of intact lymphocytes: evidence for O-linked GlcNAc.
      ) detected O-GlcNAcylated proteins on the small percentage of lysed or damaged cells in the cultures, indicating that O-GlcNAc is quite abundant on nucleocytoplasmic proteins. Cytosolic localization of O-GlcNAc was further confirmed by identification of O-GlcNAc on cytoplasmic proteins in human erythrocytes (
      • Holt G.D.
      • Haltiwanger R.S.
      • Torres C.R.
      • Hart G.W.
      Erythrocytes contain cytoplasmic glycoproteins: O-linked GlcNAc on Band 4.1.
      ). Subsequently, O-GlcNAc was found to be particularly enriched on the nuclear and cytosolic faces of the nuclear pore complex (
      • Hanover J.A.
      • Cohen C.K.
      • Willingham M.C.
      • Park M.K.
      O-Linked N-acetylglucosamine is attached to proteins of the nuclear pore: evidence for cytoplasmic and nucleoplasmic glycoproteins.
      ,
      • Holt G.D.
      • Snow C.M.
      • Senior A.
      • Haltiwanger R.S.
      • Gerace L.
      • Hart G.W.
      Nuclear pore complex glycoproteins contain cytoplasmically disposed O-linked N-acetylglucosamine.
      • Park M.K.
      • D'Onofrio M.
      • Willingham M.C.
      • Hanover J.A.
      A monoclonal antibody against a family of nuclear pore proteins (nucleoporins): O-linked N-acetylglucosamine is part of the immunodeterminant.
      ). Similar to its unusual localization in cells, O-GlcNAc was found to be on proteins associated with nucleic acids in viruses (
      • Benko D.M.
      • Haltiwanger R.S.
      • Hart G.W.
      • Gibson W.
      Virion basic phosphoprotein from human cytomegalovirus contains O-linked N-acetylglucosamine.
      ,
      • Greis K.D.
      • Gibson W.
      • Hart G.W.
      Site-specific glycosylation of the human cytomegalovirus tegument basic phosphoprotein (UL32) at serine 921 and serine 952.
      ) and also to be abundant on RNA polymerase II transcription factors (
      • Jackson S.P.
      • Tjian R.
      O-Glycosylation of eukaryotic transcription factors: implications for mechanisms of transcriptional regulation.
      ,
      • Reason A.J.
      • Morris H.R.
      • Panico M.
      • Marais R.
      • Treisman R.H.
      • Haltiwanger R.S.
      • Hart G.W.
      • Kelly W.G.
      • Dell A.
      Localization of O-GlcNAc modification on the serum response transcription factor.
      ), some of which are well-known oncogenic factors (
      • Chou T.Y.
      • Dang C.V.
      • Hart G.W.
      Glycosylation of the c-Myc transactivation domain.
      ,
      • Chou T.Y.
      • Hart G.W.
      • Dang C.V.
      c-Myc is glycosylated at threonine 58, a known phosphorylation site and a mutational hot spot in lymphomas.
      ) or tumor suppressor proteins (
      • Wells L.
      • Slawson C.
      • Hart G.W.
      The E2F-1 associated retinoblastoma-susceptibility gene product is modified by O-GlcNAc.
      ). The IIa (nonphosphorylated) form of RNA polymerase II is heavily O-GlcNAcylated on its C-terminal domain (CTD),
      The abbreviations used are: CTD
      C-terminal domain
      OGT
      O-GlcNAc transferase
      mOGT
      mitochondrial isoform of OGT
      OGA
      O-GlcNAcase
      eNOS
      endothelial nitric-oxide synthase
      CaMKII and CaMKIV
      calcium/calmodulin kinase II and IV, respectively
      TAK1
      transforming growth factor-β–activated kinase
      CDK
      cyclin-dependent kinase
      HBP
      hexosamine biosynthetic pathway
      AD
      Alzheimer's disease
      PFK1
      phosphofructokinase 1
      MMP
      matrix metalloproteinase
      VEGF
      vascular endothelial growth factor
      PDAC
      pancreatic ductal adenocarcinoma.
      in a region that reciprocally becomes heavily phosphorylated during the elongation phase of transcription (
      • Kelly W.G.
      • Dahmus M.E.
      • Hart G.W.
      RNA polymerase II is a glycoprotein: modification of the COOH-terminal domain by O-GlcNAc.
      ). Early studies also suggested that O-GlcNAc is involved in the regulation of protein translation (
      • Datta B.
      • Ray M.K.
      • Chakrabarti D.
      • Wylie D.E.
      • Gupta N.K.
      Glycosylation of eukaryotic peptide chain initiation factor 2 (eIF-2)-associated 67-kDa polypeptide (p67) and its possible role in the inhibition of eIF-2 kinase-catalyzed phosphorylation of the eIF-2 α-subunit.
      ). Analyses of Drosophila polytene chromosomes showed that O-GlcNAc is particularly abundant on chromatin, especially at sites of active gene transcription (
      • Kelly W.G.
      • Hart G.W.
      Glycosylation of chromosomal proteins: localization of O-linked N-acetylglucosamine in Drosophila chromatin.
      ). Pulse–chase studies found that O-GlcNAc cycles rapidly on the HSP27 family of heat shock proteins (
      • Roquemore E.P.
      • Chevrier M.R.
      • Cotter R.J.
      • Hart G.W.
      Dynamic O-GlcNAcylation of the small heat shock protein α B-crystallin.
      ). O-GlcNAc was also shown to be highly dynamic on lymphocyte proteins, cycling rapidly in response to activation (
      • Kearse K.P.
      • Hart G.W.
      Lymphocyte activation induces rapid changes in nuclear and cytoplasmic glycoproteins.
      ).
      Using a synthetic peptide as a substrate, an assay for the O-GlcNAc transferase (OGT) was developed, and properties of the enzyme from rabbit reticulocytes were defined (
      • Haltiwanger R.S.
      • Holt G.D.
      • Hart G.W.
      Enzymatic addition of O-GlcNAc to nuclear and cytoplasmic proteins. Identification of a uridine diphospho-N-acetylglucosamine:peptide β-N-acetylglucosaminyltransferase.
      ). Using the peptide substrate assay, combined with conventional and affinity chromatography, OGT was purified over 30,000-fold from rat liver and found to be a large multimeric enzyme with high affinity for its donor substrate, UDP-GlcNAc (
      • Haltiwanger R.S.
      • Blomberg M.A.
      • Hart G.W.
      Glycosylation of nuclear and cytoplasmic proteins: purification and characterization of a uridine diphospho-N-acetylglucosamine:polypeptide β-N-acetylglucosaminyltransferase.
      ). An assay for O-GlcNAcase (OGA; the enzyme that removes O-GlcNAc) was also developed, and O-GlcNAcase was purified 22,000-fold from rat spleen (
      • Dong D.L.
      • Hart G.W.
      Purification and characterization of an O-GlcNAc selective N-acetyl-β-d-glucosaminidase from rat spleen cytosol.
      ) and shown to have enzymatic properties similar to those of previously described crude preparations of hexosaminidase C (
      • Braidman I.
      • Carroll M.
      • Dance N.
      • Robinson D.
      • Poenaru L.
      • Weber A.
      • Dreyfus J.C.
      • Overdijk B.
      • Hooghwinkel G.J.
      Characterisation of human N-acetyl-β-hexosaminidase C.
      ,
      • Braidman I.
      • Carroll M.
      • Dance N.
      • Robinson D.
      Separation and properties of human brain hexosaminidase C.
      ). Subsequently, the OGT was cloned and sequenced from both rats and humans (
      • Kreppel L.K.
      • Blomberg M.A.
      • Hart G.W.
      Dynamic glycosylation of nuclear and cytosolic proteins: cloning and characterization of a unique O-GlcNAc transferase with multiple tetratricopeptide repeats.
      ,
      • Lubas W.A.
      • Frank D.W.
      • Krause M.
      • Hanover J.A.
      O-linked GlcNAc transferase is a conserved nucleocytoplasmic protein containing tetratricopeptide repeats.
      ) and was shown to be a unique enzyme with multiple tetratricopeptide repeats (protein-docking domains). OGT was shown to be an X-linked gene located near the centromere, and its sequence is very highly conserved from C. elegans to humans (
      • Shafi R.
      • Iyer S.P.
      • Ellies L.G.
      • O'Donnell N.
      • Marek K.W.
      • Chui D.
      • Hart G.W.
      • Marth J.D.
      The O-GlcNAc transferase gene resides on the X chromosome and is essential for embryonic stem cell viability and mouse ontogeny.
      ). Knockout of OGT showed that it is essential to the viability of murine embryonic stem cells and is required for embryonic development (
      • O'Donnell N.
      • Zachara N.E.
      • Hart G.W.
      • Marth J.D.
      Ogt-dependent X-chromosome-linked protein glycosylation is a requisite modification in somatic cell function and embryo viability.
      ). Bovine brain OGA was purified, partially sequenced, and used to clone and sequence human OGA (
      • Gao Y.
      • Wells L.
      • Comer F.I.
      • Parker G.J.
      • Hart G.W.
      Dynamic O-glycosylation of nuclear and cytosolic proteins: cloning and characterization of a neutral, cytosolic β-N-acetylglucosaminidase from human brain.
      ). OGA was shown to be identical to a previously cloned gene (MGEA5) associated with meningiomas and postulated to be a hyaluronidase (
      • Heckel D.
      • Comtesse N.
      • Brass N.
      • Blin N.
      • Zang K.D.
      • Meese E.
      Novel immunogenic antigen homologous to hyaluronidase in meningioma.
      ). The O-GlcNAcase gene is also unique. It is located on human chromosome 10, is essential in mammals and plants, and is also very highly conserved from C. elegans to humans. Recent structural analyses of OGT have not only led to a much better understanding of its enzymatic mechanism, but also, these studies have helped us to understand how a single catalytic subunit can specifically target thousands of different substrates. These investigations have also led to the development of useful inhibitors (
      • Hurtado-Guerrero R.
      • Dorfmueller H.C.
      • van Aalten D.M.
      Molecular mechanisms of O-GlcNAcylation.
      • Martinez-Fleites C.
      • Macauley M.S.
      • He Y.
      • Shen D.L.
      • Vocadlo D.J.
      • Davies G.J.
      Structure of an O-GlcNAc transferase homolog provides insight into intracellular glycosylation.
      ,
      • Lazarus M.B.
      • Nam Y.
      • Jiang J.
      • Sliz P.
      • Walker S.
      Structure of human O-GlcNAc transferase and its complex with a peptide substrate.
      ,
      • Vocadlo D.J.
      O-GlcNAc processing enzymes: catalytic mechanisms, substrate specificity, and enzyme regulation.
      ,
      • Ma X.
      • Liu P.
      • Yan H.
      • Sun H.
      • Liu X.
      • Zhou F.
      • Li L.
      • Chen Y.
      • Muthana M.M.
      • Chen X.
      • Wang P.G.
      • Zhang L.
      Substrate specificity provides insights into the sugar donor recognition mechanism of O-GlcNAc transferase (OGT).
      ,
      • Pathak S.
      • Alonso J.
      • Schimpl M.
      • Rafie K.
      • Blair D.E.
      • Borodkin V.S.
      • Albarbarawi O.
      • van Aalten D.M.F.
      The active site of O-GlcNAc transferase imposes constraints on substrate sequence.
      ,
      • Trapannone R.
      • Rafie K.
      • van Aalten D.M.
      O-GlcNAc transferase inhibitors: current tools and future challenges.
      ,
      • Aquino-Gil M.
      • Pierce A.
      • Perez-Cervera Y.
      • Zenteno E.
      • Lefebvre T.
      OGT: a short overview of an enzyme standing out from usual glycosyltransferases.
      ,
      • Wang Y.
      • Zhu J.
      • Zhang L.
      Discovery of cell-permeable O-GlcNAc transferase inhibitors via tethering in situ click chemistry.
      • Ghirardello M.
      • Perrone D.
      • Chinaglia N.
      • Sádaba D.
      • Delso I.
      • Tejero T.
      • Marchesi E.
      • Fogagnolo M.
      • Rafie K.
      • van Aalten D.M.F.
      • Merino P.
      UDP-GlcNAc analogs as inhibitors of O-GlcNAc transferase (OGT): spectroscopic, computational and biological studies.
      ) (for a recent review, see Ref.
      • Levine Z.G.
      • Walker S.
      The biochemistry of O-GlcNAc transferase: which functions make it essential in mammalian cells?.
      ). Surprisingly, OGT was also recently shown to use a novel mechanism, involving UDP-GlcNAc at its active site, to proteolytically cleave the important transcription factor, host cell factor 1 (
      • Capotosti F.
      • Guernier S.
      • Lammers F.
      • Waridel P.
      • Cai Y.
      • Jin J.
      • Conaway J.W.
      • Conaway R.C.
      • Herr W.
      O-GlcNAc transferase catalyzes site-specific proteolysis of HCF-1.
      ,
      • Lazarus M.B.
      • Jiang J.
      • Kapuria V.
      • Bhuiyan T.
      • Janetzko J.
      • Zandberg W.F.
      • Vocadlo D.J.
      • Herr W.
      • Walker S.
      HCF-1 is cleaved in the active site of O-GlcNAc transferase.
      ), into its active forms.
      Whereas less is known about how OGA is targeted to it substrates, several recent studies have defined its detailed structure. These studies have also elucidated the molecular mechanisms of the enzyme, and they have led to the development of highly specific and potent OGA inhibitors that work in living cells (
      • Schultz J.
      • Pils B.
      Prediction of structure and functional residues for O-GlcNAcase, a divergent homologue of acetyltransferases.
      • Macauley M.S.
      • Whitworth G.E.
      • Debowski A.W.
      • Chin D.
      • Vocadlo D.J.
      O-GlcNAcase uses substrate-assisted catalysis: kinetic analysis and development of highly selective mechanism-inspired inhibitors.
      ,
      • Dennis R.J.
      • Taylor E.J.
      • Macauley M.S.
      • Stubbs K.A.
      • Turkenburg J.P.
      • Hart S.J.
      • Black G.N.
      • Vocadlo D.J.
      • Davies G.J.
      Structure and mechanism of a bacterial β-glucosaminidase having O-GlcNAcase activity.
      ,
      • Dorfmueller H.C.
      • Borodkin V.S.
      • Schimpl M.
      • Shepherd S.M.
      • Shpiro N.A.
      • van Aalten D.M.
      GlcNAcstatin: a picomolar, selective O-GlcNAcase inhibitor that modulates intracellular O-glcNAcylation levels.
      ,
      • Clarke A.J.
      • Hurtado-Guerrero R.
      • Pathak S.
      • Schüttelkopf A.W.
      • Borodkin V.
      • Shepherd S.M.
      • Ibrahim A.F.
      • van Aalten D.M.
      Structural insights into mechanism and specificity of O-GlcNAc transferase.
      ,
      • 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.
      ,
      • Gloster T.M.
      • Vocadlo D.J.
      Mechanism, structure, and inhibition of O-GlcNAc processing enzymes.
      ,
      • Alonso J.
      • Schimpl M.
      • van Aalten D.M.
      O-GlcNAcase: promiscuous hexosaminidase or key regulator of O-GlcNAc signaling?.
      ,
      • Li B.
      • Li H.
      • Hu C.W.
      • Jiang J.
      Structural insights into the substrate binding adaptability and specificity of human O-GlcNAcase.
      ,
      • Li B.
      • Li H.
      • Lu L.
      • Jiang J.
      Structures of human O-GlcNAcase and its complexes reveal a new substrate recognition mode.
      • Roth C.
      • Chan S.
      • Offen W.A.
      • Hemsworth G.R.
      • Willems L.I.
      • King D.T.
      • Varghese V.
      • Britton R.
      • Vocadlo D.J.
      • Davies G.J.
      Structural and functional insight into human O-GlcNAcase.
      ).
      Perhaps the greatest impediment to understanding the functions of O-GlcNAcylation is the enormous difficulty in detecting and mapping the sites of O-GlcNAc on proteins (
      • Zachara N.E.
      • Vosseller K.
      • Hart G.W.
      Detection and analysis of proteins modified by O-linked N-acetylglucosamine.
      ). Despite its abundance within the nucleus and cytoplasm, O-GlcNAc remained undetected until 1983 for many reasons. 1) Generally, the presence or absence of O-GlcNAc does not alter the electrophoretic migration of a polypeptide, even in two-dimensional electrophoresis; 2) O-GlcNAc is very labile at the source and in the gas phase in MS, making detection of O-GlcNAc peptides and site mapping very difficult (
      • Wang Z.
      • Udeshi N.D.
      • O'Malley M.
      • Shabanowitz J.
      • Hunt D.F.
      • Hart G.W.
      Enrichment and site mapping of O-linked N-acetylglucosamine by a combination of chemical/enzymatic tagging, photochemical cleavage, and electron transfer dissociation mass spectrometry.
      ,
      • Ma J.
      • Hart G.W.
      Analysis of protein O-GlcNAcylation by mass spectrometry.
      ); 3) in mixtures, ion suppression of O-GlcNAc peptides by unmodified peptides in MS masks the presence of the O-GlcNAcylated species. Fortunately, pan-specific O-GlcNAc monoclonal antibodies have greatly improved methods for detection of O-GlcNAc (
      • Holt G.D.
      • Snow C.M.
      • Senior A.
      • Haltiwanger R.S.
      • Gerace L.
      • Hart G.W.
      Nuclear pore complex glycoproteins contain cytoplasmically disposed O-linked N-acetylglucosamine.
      ,
      • Snow C.M.
      • Senior A.
      • Gerace L.
      Monoclonal antibodies identify a group of nuclear pore complex glycoproteins.
      ,
      • Comer F.I.
      • Vosseller K.
      • Wells L.
      • Accavitti M.A.
      • Hart G.W.
      Characterization of a mouse monoclonal antibody specific for O-linked N-acetylglucosamine.
      ), and specific enrichment methods have been developed to circumvent the ion suppression problem (
      • Wang Z.
      • Udeshi N.D.
      • O'Malley M.
      • Shabanowitz J.
      • Hunt D.F.
      • Hart G.W.
      Enrichment and site mapping of O-linked N-acetylglucosamine by a combination of chemical/enzymatic tagging, photochemical cleavage, and electron transfer dissociation mass spectrometry.
      ,
      • Alfaro J.F.
      • Gong C.X.
      • Monroe M.E.
      • Aldrich J.T.
      • Clauss T.R.
      • Purvine S.O.
      • Wang Z.
      • Camp 2nd, D.G.
      • Shabanowitz J.
      • Stanley P.
      • Hart G.W.
      • Hunt D.F.
      • Yang F.
      • Smith R.D.
      Tandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets.
      ). Perhaps the most important breakthrough in site mapping for O-GlcNAc on polypeptides has been the development of electron transfer dissociation fragmentation MS, which does not result in the cleavage of the very labile O-GlcNAc glycosidic linkage to serine or threonine (
      • Mikesh L.M.
      • Ueberheide B.
      • Chi A.
      • Coon J.J.
      • Syka J.E.
      • Shabanowitz J.
      • Hunt D.F.
      The utility of ETD mass spectrometry in proteomic analysis.
      ,
      • Myers S.A.
      • Daou S.
      • Affar E.B.
      • Burlingame A.
      Electron transfer dissociation (ETD): the mass spectrometric breakthrough essential for O-GlcNAc protein site assignments—a study of the O-GlcNAcylated protein host cell factor C1.
      ). Like other post-translational modifications, the functions of O-GlcNAc must be understood at the individual site level, making site mapping a key first step to elucidate its biological functions. Whereas inhibitors of OGT and OGA and genetic knockout experiments of OGA or OGT have allowed us to make great strides in understanding the functions of O-GlcNAc, the lack of methods to alter O-GlcNAcylation levels on a single protein or at a single site has greatly limited progress in this area. Site-directed mutagenesis of O-GlcNAc sites to alanine is useful, but effects are difficult to interpret if the same or proximal site is also subjected to phosphorylation or other modifications.

      O-GlcNAc as a sensor of nutrients and stress

      The concentration of UDP-GlcNAc (the donor substrate for OGT) in cells is highly responsive to nutrients and flux through the major metabolic pathways via their connectivity to the hexosamine biosynthetic pathway, including glucose metabolism, nitrogen metabolism, nucleotide metabolism, and fatty acid metabolism (Fig. 1) (
      • Zachara N.E.
      • Hart G.W.
      O-GlcNAc a sensor of cellular state: the role of nucleocytoplasmic glycosylation in modulating cellular function in response to nutrition and stress.
      ,
      • Hart G.W.
      • Slawson C.
      • Ramirez-Correa G.
      • Lagerlof O.
      Cross talk between O-GlcNAcylation and phosphorylation: roles in signaling, transcription, and chronic disease.
      ). Both the activity of OGT and its substrate selectivity are highly responsive to UDP-GlcNAc concentrations across a large range (
      • Kreppel L.K.
      • Blomberg M.A.
      • Hart G.W.
      Dynamic glycosylation of nuclear and cytosolic proteins: cloning and characterization of a unique O-GlcNAc transferase with multiple tetratricopeptide repeats.
      ,
      • Kreppel L.K.
      • Hart G.W.
      Regulation of a cytosolic and nuclear O-GlcNAc transferase: role of the tetratricopeptide repeats.
      ), indicating that O-GlcNAcylation at specific sites on polypeptides is highly responsive to the metabolic state of the cell. O-GlcNAcylation of nuclear pore proteins (
      • Han I.
      • Oh E.S.
      • Kudlow J.E.
      Responsiveness of the state of O-linked N-acetylglucosamine modification of nuclear pore protein p62 to the extracellular glucose concentration.
      ) and O-GlcNAcylation of many polypeptides within β-cells of the pancreas (
      • Liu K.
      • Paterson A.J.
      • Chin E.
      • Kudlow J.E.
      Glucose stimulates protein modification by O-linked GlcNAc in pancreatic beta cells: linkage of O-linked GlcNAc to beta cell death.
      ) are very responsive to extracellular glucose concentrations. Hyperglycemia qualitatively and quantitatively alters the O-GlcNAcylation or expression of many O-GlcNAc–modified proteins within the nucleus of rat aorta or smooth muscle cells (
      • Akimoto Y.
      • Kreppel L.K.
      • Hirano H.
      • Hart G.W.
      Hyperglycemia and the O-GlcNAc transferase in rat aortic smooth muscle cells: elevated expression and altered patterns of O-GlcNAcylation.
      ). Hyperglycemia also inhibits vascular endothelial nitric-oxide synthase (eNOS) by O-GlcNAcylation, blocking its key regulatory AKT phosphorylation site (
      • Du X.L.
      • Edelstein D.
      • Dimmeler S.
      • Ju Q.
      • Sui C.
      • Brownlee M.
      Hyperglycemia inhibits endothelial nitric oxide synthase activity by posttranslational modification at the Akt site.
      ). Elevated glucose and insulin both stimulate increased O-GlcNAcylation in L6 myotubes (a model of skeletal muscle) (
      • Walgren J.L.
      • Vincent T.S.
      • Schey K.L.
      • Buse M.G.
      High glucose and insulin promote O-GlcNAc modification of proteins, including α-tubulin.
      ). Hyperglycemia-induced O-GlcNAcylation mediates plasminogen activator inhibitor-1 gene expression and Sp1 transcriptional activity in glomerular mesangial cells (
      • Goldberg H.J.
      • Whiteside C.I.
      • Hart G.W.
      • Fantus I.G.
      Posttranslational, reversible O-glycosylation is stimulated by high glucose and mediates plasminogen activator inhibitor-1 gene expression and Sp1 transcriptional activity in glomerular mesangial cells.
      ).
      Figure thumbnail gr1
      Figure 1The HBP links flux through major metabolic pathways, allowing O-GlcNAcylation to serve as a “rheostat” that modulates most cellular processes in response to nutrients. The biosynthesis of UDP-GlcNAc, the donor for the OGT, is directly coupled to flux through glucose, amino acid, fatty acid, and nucleotide metabolic pathways. OGT is highly sensitive to UDP-GlcNAc concentrations, both in terms of activity and selectivity. O-GlcNAcylation has extensive cross-talk with phosphorylation. Shown is the universal symbol for a rheostat, indicating that unlike phosphorylation, which is more analogous to a switch, O-GlcNAc serves in a more analog fashion as a rheostat to modulate processes in response to nutrients and stress. GFAT, glutamine:fructose-6-phosphate amidotransferase. Modified from Refs.
      • Hart G.W.
      • Slawson C.
      • Ramirez-Correa G.
      • Lagerlof O.
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      Nutrient regulation of gene expression

      Cells must closely regulate gene expression in response to their metabolic state and the availability of building blocks and fuels. It is now clear that O-GlcNAcylation plays key roles in nutrient regulation of transcription, yet we know very little about the molecular mechanisms involved. Recent studies indicate that O-GlcNAcylation affects nearly every step of transcription (Fig. 2) (for reviews, see Refs.
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      β-Catenin is O-GlcNAc glycosylated at serine 23: implications for β-catenin’s subcellular localization and transactivator function.
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      O-GlcNAc modification of the runt-related transcription factor 2 (Runx2) links osteogenesis and nutrient metabolism in bone marrow mesenchymal stem cells.
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      O-GlcNAc modification of Sp1 mediates hyperglycaemia-induced ICAM-1 up-regulation in endothelial cells.
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      ). Assembly of the preinitiation complex in the transcription cycle requires O-GlcNAcylation of the C-terminal domain of RNA polymerase II (
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      Human RNA polymerase II promoter recruitment in vitro is regulated by O-linked N-acetylglucosaminyltransferase (OGT).
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      ). Phosphorylation of the CTD is required and precedes elongation (
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      Transcription: another mark in the tail.
      ). O-GlcNAc is part of the histone code (
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      Modification of histones by sugar β-N-acetylglucosamine (GlcNAc) occurs on multiple residues, including histone H3 serine 10, and is cell cycle-regulated.
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      ). Whereas several O-GlcNAc sites on histones are in the tail regions, along with other epigenetic marks, some O-GlcNAc moieties are located at the histone:DNA interface. O-GlcNAc regulates both ubiquitination and methylation of histones (
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      GlcNAcylation of histone H2B facilitates its monoubiquitination.
      ). OGT and O-GlcNAc also regulate DNA methylation via their interactions and via regulation and modification of the TET proteins. Very recent studies have shown that the TATA-binding protein’s (TBP’s) cycling on and off of DNA is regulated by its O-GlcNAcylation.
      S. Hardivillé, P. S. Banerjee, E. S. Selen Alpergin, G. Han, J. Ma, C. C. Talbot, Jr., P. Hu, M. J. Wolfgang, and G. W. Hart, submitted for publication.
      When bound to DNA, TBP is O-GlcNAcylated, and this modification reduces its interaction with the TFIID complex, specifically preventing binding to the BTAF1 subunit. Reducing the BTAF1:TBP interaction increases TBP's residence time at promoters, increasing overall promoter occupancy on several promoters and resulting in major changes in the expression of many metabolic enzymes.
      Figure thumbnail gr2
      Figure 2O-GlcNAcylation serves as a nutrient sensor to modulate nearly every step in transcription. Nearly every transcription factor is O-GlcNAcylated, often at multiple sites. OGT is a polycomb gene. Assembly of the preinitiation complex requires O-GlcNAcylation of RNA polymerase II. Elongation of mRNA requires removal of O-GlcNAc from RNA polymerase II. O-GlcNAc is part of the histone code. O-GlcNAc regulates ubiquitinylation and methylation of histones. O-GlcNAc regulates DNA methylation by the TET proteins. TATA-binding protein’s residence time at promoters is regulated by O-GlcNAcylation. HDAC, histone deacetylase; Pol, polymerase; TF, transcription factor; H3K4me3, histone H3 Lys-4 trimethylation. Modified from Ref.
      • Hardivillé S.
      • Hart G.W.
      Nutrient regulation of signaling, transcription, and cell physiology by O-GlcNAcylation.
      . This research was originally published in Cell Metabolism. Hardivillé, S., and Hart, G. W. Nutrient regulation of signaling, transcription, and cell physiology by O-GlcNAcylation. Cell Metab. 2014; 20:208–213. © Cell Press.
      Despite many descriptive studies of the roles of O-GlcNAc in transcription, there remain many questions about the functions of OGT, OGA, and O-GlcNAc in transcription. For example, 1) there are limited data suggesting that O-GlcNAcylation of transcription factors may affect their interactions with other components of the transcription machinery and alter their promoter specificity, but detailed studies of this possibility are lacking. This topic is of particular importance to molecular mechanisms underlying glucose toxicity, where abnormal gene expression occurs in many tissues exposed to prolonged hyperglycemia. 2) How O-GlcNAcylation regulates the basal machinery and the transcription cycle is not understood. 3) Are so-called “housekeeping transcription factors” O-GlcNAcylated differently in different cell types or in different metabolic states of the same cell type? These are but a few of the questions that need study. Clearly, how nutrients regulate transcription will be a fertile area for future research that will impact our understanding of disease etiologies.
      We know even less about how dynamic O-GlcNAcylation regulates protein translation. However, data are emerging from several studies suggesting that nutrients also regulate protein synthesis via O-GlcNAcylation. Early studies suggested that O-GlcNAcylation of p67 protein plays a required role in p67’s regulation of phosphorylation of the eIF-2 α subunit (
      • Datta B.
      • Ray M.K.
      • Chakrabarti D.
      • Wylie D.E.
      • Gupta N.K.
      Glycosylation of eukaryotic peptide chain initiation factor 2 (eIF-2)-associated 67-kDa polypeptide (p67) and its possible role in the inhibition of eIF-2 kinase-catalyzed phosphorylation of the eIF-2 α-subunit.
      ). Reticulocyte extracts, which are often used for the study of in vitro protein translation, are very efficient at O-GlcNAcylation of nascent polypeptides (
      • Starr C.M.
      • Hanover J.A.
      Glycosylation of nuclear pore protein p62. Reticulocyte lysate catalyzes O-linked N-acetylglucosamine addition in vitro.
      ). It was proposed that OGT and O-GlcNAcylation protect proteins from aggregation during heat stress (
      • Sohn K.C.
      • Lee K.Y.
      • Park J.E.
      • Do S.I.
      OGT functions as a catalytic chaperone under heat stress response: a unique defense role of OGT in hyperthermia.
      ,
      • Lim K.H.
      • Chang H.I.
      O-linked N-acetylglucosamine suppresses thermal aggregation of Sp1.
      ). An unbiased RNA-mediated interference-based screen showed extensive O-GlcNAcylation of stress granules, which are ribonucleoprotein granules that regulate translation and mRNA decay, during cellular stress. O-GlcNAcylation of the translational machinery is required for aggregation of untranslated messenger ribonucleoproteins in the formation of stress granules (
      • Ohn T.
      • Kedersha N.
      • Hickman T.
      • Tisdale S.
      • Anderson P.
      A functional RNAi screen links O-GlcNAc modification of ribosomal proteins to stress granule and processing body assembly.
      ). Gycomic analyses identified many O-GlcNAcylated translation factors and ribosome proteins (
      • Zeidan Q.
      • Wang Z.
      • De Maio A.
      • Hart G.W.
      O-GlcNAc cycling enzymes associate with the translational machinery and modify core ribosomal proteins.
      ). Over 20 core ribosome proteins are O-GlcNAcylated, and both OGT and OGA are tightly bound to purified ribosomal preparations. Even though most OGT is nuclear in dividing cells, the transferase is completely excluded from the nucleolus, the site of ribosome biogenesis (
      • Zeidan Q.
      • Wang Z.
      • De Maio A.
      • Hart G.W.
      O-GlcNAc cycling enzymes associate with the translational machinery and modify core ribosomal proteins.
      ). A mild overexpression of OGT causes some of the enzyme to “leak” into the nucleolus, resulting in profound disruption of nucleolar structure and an accumulation of 60S subunits and 80S monosomes. Upon inhibition of the proteasome, both OGT and OGA become very tightly bound to ribosomes. O-GlcNAcylation of many ribosome-associated proteins dramatically increases, and protein synthesis stops for a period of time. The significance of these observations needs further investigation. Recently, O-GlcNAcylation was found to be more extensive on nascent polypeptide chains, presumably protecting them from premature degradation by blocking co-translational ubiquitination, suggesting that O-GlcNAc might play a role in the cytosolic compartment that is similar to the role of N-glycans in the calnexin/calreticulin system for proteins in the secretory pathway (
      • Zhu Y.
      • Liu T.W.
      • Cecioni S.
      • Eskandari R.
      • Zandberg W.F.
      • Vocadlo D.J.
      O-GlcNAc occurs cotranslationally to stabilize nascent polypeptide chains.
      ).

      Nutrient regulation of signaling

      It is now clear that O-GlcNAcylation’s interplay with phosphorylation plays a key role in modulating signaling pathways in response to nutrients and stress (
      • Zachara N.E.
      • Hart G.W.
      Cell signaling, the essential role of O-GlcNAc!.
      ). One of the earliest studies suggesting interplay between protein phosphorylation and O-GlcNAcylation showed that the IIa form of RNA polymerase II is abundantly O-GlcNAcylated in its CTD, yet the heavily phosphorylated IIo form of RNA polymerase II CTD is completely lacking in O-GlcNAc moieties (
      • Kelly W.G.
      • Dahmus M.E.
      • Hart G.W.
      RNA polymerase II is a glycoprotein: modification of the COOH-terminal domain by O-GlcNAc.
      ). In vitro assays using synthetic CTD repeats showed that O-GlcNAcylation and phosphorylation are mutually exclusive, with the presence of a single O-GlcNAc completely blocking the activity of CTD kinases and the presence of a single phosphate moiety completely blocking OGT’s activity on CTD (
      • Comer F.I.
      • Hart G.W.
      Reciprocity between O-GlcNAc and O-phosphate on the carboxyl terminal domain of RNA polymerase II.
      ). O-GlcNAcylation at Ser-1177 of eNOS blocks its phosphorylation at this site, thus preventing its activation by AKT kinase (
      • Du X.L.
      • Edelstein D.
      • Dimmeler S.
      • Ju Q.
      • Sui C.
      • Brownlee M.
      Hyperglycemia inhibits endothelial nitric oxide synthase activity by posttranslational modification at the Akt site.
      ). Short-term treatment of cells with the broad-spectrum phosphatase inhibitor, okadaic acid, or with phorbol esters, which activate protein kinase C, or treatment with adenosine monophosphate, which activates protein kinase A, all lead to global decreased O-GlcNAcylation (
      • Haltiwanger R.S.
      • Busby S.
      • Grove K.
      • Li S.
      • Mason D.
      • Medina L.
      • Moloney D.
      • Philipsberg G.
      • Scartozzi R.
      O-Glycosylation of nuclear and cytoplasmic proteins: regulation analogous to phosphorylation?.
      ). In contrast, treatment of cells with the nonspecific kinase inhibitor, staurosporine, increases global O-GlcNAcylation, supporting a “yin-yang” relationship between the two modifications on many proteins. One complexity in these types of studies is that if such treatments are performed at a high dose or for too long, they induce a stress response, which by itself elevates global O-GlcNAcylation.
      Activation of protein kinase A or C in cerebellar neurons of post-natal mice results in reduced levels of O-GlcNAc, specifically in cytoskeletal and cytoskeleton-associated proteins, whereas inhibition of the same kinases results in increased levels of O-GlcNAc (
      • Griffith L.S.
      • Schmitz B.
      O-Linked N-acetylglucosamine levels in cerebellar neurons respond reciprocally to pertubations of phosphorylation.
      ). Likewise, treatment of neuronal cells with the broad-spectrum phosphatase inhibitor, okadaic acid, which induces protein hyperphosphorylation, decreases the levels of O-GlcNAc in both nuclear and cytoplasmic proteins, but with a greater effect in the nuclear fraction (
      • Lefebvre T.
      • Alonso C.
      • Mahboub S.
      • Dupire M.J.
      • Zanetta J.P.
      • Caillet-Boudin M.L.
      • Michalski J.C.
      Effect of okadaic acid on O-linked N-acetylglucosamine levels in a neuroblastoma cell line.
      ). Other studies suggest that O-GlcNAc limits nucleoporin hyperphosphorylation during M-phase and hastens the resumption of regulated nuclear transport at the completion of cell division (
      • Miller M.W.
      • Caracciolo M.R.
      • Berlin W.K.
      • Hanover J.A.
      Phosphorylation and glycosylation of nucleoporins.
      ). Whereas there are numerous examples of phosphate and O-GlcNAc competing for the same hydroxyl moiety on a polypeptide (
      • Chou T.Y.
      • Dang C.V.
      • Hart G.W.
      Glycosylation of the c-Myc transactivation domain.
      ,
      • Chou T.Y.
      • Hart G.W.
      • Dang C.V.
      c-Myc is glycosylated at threonine 58, a known phosphorylation site and a mutational hot spot in lymphomas.
      ,
      • Cheng X.
      • Cole R.N.
      • Zaia J.
      • Hart G.W.
      Alternative O-glycosylation/O-phosphorylation of the murine estrogen receptor β.
      ,
      • Cheng X.
      • Hart G.W.
      Alternative O-glycosylation/O-phosphorylation of serine-16 in murine estrogen receptor β: post-translational regulation of turnover and transactivation activity.
      • Arnold C.S.
      • Johnson G.V.
      • Cole R.N.
      • Dong D.L.
      • Lee M.
      • Hart G.W.
      The microtubule-associated protein Tau is extensively modified with O-linked N-acetylglucosamine.
      ), competition also occurs when they are located proximal to each other (
      • Juang Y.T.
      • Solomou E.E.
      • Rellahan B.
      • Tsokos G.C.
      Phosphorylation and O-linked glycosylation of Elf-1 leads to its translocation to the nucleus and binding to the promoter of the TCR ζ-chain.
      ,
      • Tsokos G.C.
      • Nambiar M.P.
      • Juang Y.T.
      Activation of the Ets transcription factor Elf-1 requires phosphorylation and glycosylation: defective expression of activated Elf-1 is involved in the decreased TCR ζ chain gene expression in patients with systemic lupus erythematosus.
      ,
      • Dong D.L.
      • Xu Z.S.
      • Hart G.W.
      • Cleveland D.W.
      Cytoplasmic O-GlcNAc modification of the head domain and the KSP repeat motif of the neurofilament protein neurofilament-H.
      • Cole R.N.
      • Hart G.W.
      Glycosylation sites flank phosphorylation sites on synapsin I: O-linked N-acetylglucosamine residues are localized within domains mediating synapsin I interactions.
      ,
      • Lefebvre T.
      • Ferreira S.
      • Dupont-Wallois L.
      • Bussière T.
      • Dupire M.J.
      • Delacourte A.
      • Michalski J.C.
      • Caillet-Boudin M.L.
      Evidence of a balance between phosphorylation and O-GlcNAc glycosylation of Tau proteins–a role in nuclear localization.
      ,
      • Liu F.
      • Iqbal K.
      • Grundke-Iqbal I.
      • Hart G.W.
      • Gong C.X.
      O-GlcNAcylation regulates phosphorylation of Tau: a mechanism involved in Alzheimer's disease.
      ,
      • Ahmad I.
      • Hoessli D.C.
      • Walker-Nasir E.
      • Choudhary M.I.
      • Rafik S.M.
      • Shakoori A.R.
      • Nasir-ud-Din
      Phosphorylation and glycosylation interplay: protein modifications at hydroxy amino acids and prediction of signaling functions of the human β3 integrin family.
      ,
      • Chen Y.X.
      • Du J.T.
      • Zhou L.X.
      • Liu X.H.
      • Zhao Y.F.
      • Nakanishi H.
      • Li Y.M.
      Alternative O-GlcNAcylation/O-phosphorylation of Ser16 induce different conformational disturbances to the N terminus of murine estrogen receptor beta.
      ,
      • Deng Y.
      • Li B.
      • Liu F.
      • Iqbal K.
      • Grundke-Iqbal I.
      • Brandt R.
      • Gong C.X.
      Regulation between O-GlcNAcylation and phosphorylation of neurofilament-M and their dysregulation in Alzheimer disease.
      ,
      • Kang M.J.
      • Kim C.
      • Jeong H.
      • Cho B.K.
      • Ryou A.L.
      • Hwang D.
      • Mook-Jung I.
      • Yi E.C.
      Synapsin-1 and Tau reciprocal O-GlcNAcylation and phosphorylation sites in mouse brain synaptosomes.
      • Kaasik K.
      • Kivimäe S.
      • Allen J.J.
      • Chalkley R.J.
      • Huang Y.
      • Baer K.
      • Kissel H.
      • Burlingame A.L.
      • Shokat K.M.
      • Ptáček L.J.
      • Fu Y.H.
      Glucose sensor O-GlcNAcylation coordinates with phosphorylation to regulate circadian clock.
      ). The Stokes radius of an O-GlcNAc moiety is about 5 times that of a phosphate residue.
      OGT occurs in a functional complex with protein phosphatases, suggesting that in some instances, the same protein complex both dephosphorylates and concomitantly O-GlcNAcylates polypeptides (
      • Wells L.
      • Kreppel L.K.
      • Comer F.I.
      • Wadzinski B.E.
      • Hart G.W.
      O-GlcNAc transferase is in a functional complex with protein phosphatase 1 catalytic subunits.
      ). Glycomic/proteomic analyses have shown that in terms of cross-talk between phosphorylation and O-GlcNAcylation at the site level, all possibilities exist (
      • Woo C.M.
      • Lund P.J.
      • Huang A.C.
      • Davis M.M.
      • Bertozzi C.R.
      • Pitteri S.J.
      Mapping and quantification of over 2000 O-linked glycopeptides in activated human T cells with isotope-targeted glycoproteomics (Isotag).
      ,
      • Trinidad J.C.
      • Barkan D.T.
      • Gulledge B.F.
      • Thalhammer A.
      • Sali A.
      • Schoepfer R.
      • Burlingame A.L.
      Global identification and characterization of both O-GlcNAcylation and phosphorylation at the murine synapse.
      ). In proteomic analyses of murine synaptosomes, 7% of mapped O-GlcNAc sites were modified reciprocally by phosphate at the same hydroxyl moiety (
      • Trinidad J.C.
      • Barkan D.T.
      • Gulledge B.F.
      • Thalhammer A.
      • Sali A.
      • Schoepfer R.
      • Burlingame A.L.
      Global identification and characterization of both O-GlcNAcylation and phosphorylation at the murine synapse.
      ). Just these two common post-translational modifications, which often occur at multiple sites on a polypeptide, greatly increase the molecular diversity of proteins. Even though both phosphorylation and O-GlcNAcylation are sub-stoichiometric at any single site on a polypeptide, it is likely that their competition does affect each other's cycling rates, which seems to be the most biologically relevant parameter.
      Another aspect of cross-talk between protein phosphorylation and O-GlcNAcylation is the regulation of each other’s cycling enzymes by the other modification. OGT and OGA are both regulated by phosphorylation. Calcium/calmodulin kinase IV (CaMKIV) activates OGT, and phosphorylation of OGT has an essential role in CaMKIV-dependent AP-1 activation upon depolarization of neuronal cells (
      • Song M.
      • Kim H.S.
      • Park J.M.
      • Kim S.H.
      • Kim I.H.
      • Ryu S.H.
      • Suh P.G.
      O-GlcNAc transferase is activated by CaMKIV-dependent phosphorylation under potassium chloride-induced depolarization in NG-108-15 cells.
      ). As part of a feedback loop, OGT in turn O-GlcNAcylates CAMKIV in its ATP-binding pocket, inactivating the kinase (
      • Dias W.B.
      • Cheung W.D.
      • Wang Z.
      • Hart G.W.
      Regulation of calcium/calmodulin-dependent kinase IV by O-GlcNAc modification.
      ). Insulin stimulates tyrosine phosphorylation of OGT by the insulin receptor and activates OGT’s activity on many specific substrates (
      • Whelan S.A.
      • Lane M.D.
      • Hart G.W.
      Regulation of the O-linked β-N-acetylglucosamine transferase by insulin signaling.
      ). Other kinases also modify OGT and activate its catalytic activity, including Src, GSK3β, and CaMKII. OGA is phosphorylated on at least 10 different sites (Phosphosite Plus), but the functions of these modifications are unexplored.
      Early studies identified at least 42 kinases that are substrates for OGT (
      • Dias W.B.
      • Cheung W.D.
      • Hart G.W.
      O-GlcNAcylation of kinases.
      ). Recent screens have shown that about 80% of all human kinases are substrates for OGT. To date, more than 100 kinases have been confirmed to be O-GlcNAcylated in living cells. Proteomic analyses found over 46 kinases modified by O-GlcNAc in murine synaptosomes alone (
      • Trinidad J.C.
      • Barkan D.T.
      • Gulledge B.F.
      • Thalhammer A.
      • Sali A.
      • Schoepfer R.
      • Burlingame A.L.
      Global identification and characterization of both O-GlcNAcylation and phosphorylation at the murine synapse.
      ).
      Most importantly, every O-GlcNAcylated kinase studied to date, is regulated in some manner by the cycling sugar. O-GlcNAc at the ATP-binding pocket of CaMKIV completely inhibits the enzyme (
      • Dias W.B.
      • Cheung W.D.
      • Wang Z.
      • Hart G.W.
      Regulation of calcium/calmodulin-dependent kinase IV by O-GlcNAc modification.
      ). The phosphorylated and O-GlcNAcylated forms of casein kinase II have different substrate selectivity (
      • Tarrant M.K.
      • Rho H.S.
      • Xie Z.
      • Jiang Y.L.
      • Gross C.
      • Culhane J.C.
      • Yan G.
      • Qian J.
      • Ichikawa Y.
      • Matsuoka T.
      • Zachara N.
      • Etzkorn F.A.
      • Hart G.W.
      • Jeong J.S.
      • Blackshaw S.
      • et al.
      Regulation of CK2 by phosphorylation and O-GlcNAcylation revealed by semisynthesis.
      ). O-GlcNAc regulates the activity of the energy-sensing kinase, AMP-activated protein kinase, in skeletal muscle (
      • Bullen J.W.
      • Balsbaugh J.L.
      • Chanda D.
      • Shabanowitz J.
      • Hunt D.F.
      • Neumann D.
      • Hart G.W.
      Cross-talk between two essential nutrient-sensitive enzymes: O-GlcNAc transferase (OGT) and AMP-activated protein kinase (AMPK).
      ). Several members of the protein kinase C family are negatively regulated by O-GlcNAcylation (
      • Robles-Flores M.
      • Meléndez L.
      • García W.
      • Mendoza-Hernández G.
      • Lam T.T.
      • Castañeda-Patlán C.
      • González-Aguilar H.
      Posttranslational modifications on protein kinase C isozymes: effects of epinephrine and phorbol esters.
      ). O-GlcNAcylation of phosphofructokinase II inhibits this key enzyme and increases flux into the pentose phosphate pathway, contributing to the “Warburg effect” in cancer cells (
      • Yi W.
      • Clark P.M.
      • Mason D.E.
      • Keenan M.C.
      • Hill C.
      • Goddard 3rd, W.A.
      • Peters E.C.
      • Driggers E.M.
      • Hsieh-Wilson L.C.
      Phosphofructokinase 1 glycosylation regulates cell growth and metabolism.
      ). AKT (protein kinase B) is regulated by O-GlcNAcylation in hepatocytes (
      • Soesanto Y.A.
      • Luo B.
      • Jones D.
      • Taylor R.
      • Gabrielsen J.S.
      • Parker G.
      • McClain D.A.
      Regulation of Akt signaling by O-GlcNAc in euglycemia.
      ). Activation of neurons increases O-GlcNAcylation of cyclin-dependent kinase 5, blocking its binding to p53 to suppress apoptosis (
      • Chen R.
      • Gong P.
      • Tao T.
      • Gao Y.
      • Shen J.
      • Yan Y.
      • Duan C.
      • Wang J.
      • Liu X.
      O-GlcNAc glycosylation of nNOS promotes neuronal apoptosis following glutamate excitotoxicity.
      ). O-GlcNAcylation of p27 blocks cyclin/CDK–p27 binding, contributing to regulation of the cell cycle (
      • Qiu H.
      • Liu F.
      • Tao T.
      • Zhang D.
      • Liu X.
      • Zhu G.
      • Xu Z.
      • Ni R.
      • Shen A.
      Modification of p27 with O-linked N-acetylglucosamine regulates cell proliferation in hepatocellular carcinoma.
      ). O-GlcNAcylation of transforming growth factor-β–activated kinase (TAK1) regulates pro-inflammatory activation and M1 polarization of macrophages (
      • Pathak S.
      • Borodkin V.S.
      • Albarbarawi O.
      • Campbell D.G.
      • Ibrahim A.
      • van Aalten D.M.
      O-GlcNAcylation of TAB1 modulates TAK1-mediated cytokine release.
      ). O-GlcNAcylation of PKAcα and PKAcβ activates the enzymes in the brain and regulates their subcellular localizations (
      • Xie S.
      • Jin N.
      • Gu J.
      • Shi J.
      • Sun J.
      • Chu D.
      • Zhang L.
      • Dai C.L.
      • Gu J.H.
      • Gong C.X.
      • Iqbal K.
      • Liu F.
      O-GlcNAcylation of protein kinase A catalytic subunits enhances its activity: a mechanism linked to learning and memory deficits in Alzheimer's disease.
      ). Loss of O-GlcNAc on these PKAs leads to impaired learning and memory associated with Alzheimer’s disease. Elevated O-GlcNAcylation, as occurs in diabetes, causes CaMKII in the heart to become constitutively active and directly contributes to diabetes-associated cardiomyopathy and arrhythmias (
      • Erickson J.R.
      • Pereira L.
      • Wang L.
      • Han G.
      • Ferguson A.
      • Dao K.
      • Copeland R.J.
      • Despa F.
      • Hart G.W.
      • Ripplinger C.M.
      • Bers D.M.
      Diabetic hyperglycaemia activates CaMKII and arrhythmias by O-linked glycosylation.
      ). Whereas only a handful of the over 400 O-GlcNAcylated kinases have been studied for the effects of O-GlcNAcylation on their functions or on their enzymatic activities, it is already evident that nutrients regulate kinase signaling in large part by modulating their O-GlcNAcylation, which affects kinase functions in many different ways.
      Genetic studies also showed that O-GlcNAcylation regulates signaling in plants, especially the Gibbererellin growth hormone signaling pathway (
      • Thornton T.M.
      • Swain S.M.
      • Olszewski N.E.
      Gibberellin signal transduction presents … the SPY who O-GlcNAc’d me.
      ,
      • Olszewski N.E.
      • West C.M.
      • Sassi S.O.
      • Hartweck L.M.
      O-GlcNAc protein modification in plants: evolution and function.
      ). In Arabidopsis, O-GlcNAcylation of DELLA transcription factors, which are master growth repressors in plants, regulates and coordinates multiple signaling pathways during development (
      • Zentella R.
      • Hu J.
      • Hsieh W.P.
      • Matsumoto P.A.
      • Dawdy A.
      • Barnhill B.
      • Oldenhof H.
      • Hartweck L.M.
      • Maitra S.
      • Thomas S.G.
      • Cockrell S.
      • Boyce M.
      • Shabanowitz J.
      • Hunt D.F.
      • Olszewski N.E.
      • Sun T.P.
      O-GlcNAcylation of master growth repressor DELLA by SECRET AGENT modulates multiple signaling pathways in Arabidopsis.
      ). Plant OGT (Secret Agent) regulates flowering by activating histone methylation in Arabidopsis (
      • Xing L.
      • Liu Y.
      • Xu S.
      • Xiao J.
      • Wang B.
      • Deng H.
      • Lu Z.
      • Xu Y.
      • Chong K.
      Arabidopsis O-GlcNAc transferase SEC activates histone methyltransferase ATX1 to regulate flowering.
      ).

      Nutrient regulation of cytokinesis and the cytoskeleton

      Early studies showed that human Band 4.1, a protein that serves as a bridge joining the cytoskeleton to the inner surface of the plasma membrane in erythrocytes, is modified by O-GlcNAc (
      • Holt G.D.
      • Haltiwanger R.S.
      • Torres C.R.
      • Hart G.W.
      Erythrocytes contain cytoplasmic glycoproteins: O-linked GlcNAc on Band 4.1.
      ). Cytokeratins 8 and 18 are O-GlcNAcylated at multiple sites, and pulse–chase analyses showed that the sugar is dynamically cycling on these intermediate filaments (
      • Chou C.F.
      • Smith A.J.
      • Omary M.B.
      Characterization and dynamics of O-linked glycosylation of human cytokeratin 8 and 18.
      ). Synapsin I, which anchors synaptic vesicles to the cytoskeleton at nerve terminals via a phosphorylation-regulated process, has at least seven O-GlcNAcylation sites clustered around its five phosphorylation sites. However, further analyses suggest that O-GlcNAc’s roles in synapsin I’s functions are more direct than simply controlling phosphorylation (
      • Cole R.N.
      • Hart G.W.
      Glycosylation sites flank phosphorylation sites on synapsin I: O-linked N-acetylglucosamine residues are localized within domains mediating synapsin I interactions.
      ).
      Several studies have shown that O-GlcNAcylation is involved in regulation of the cell cycle and cytokinesis (Fig. 3). Increased O-GlcNAcylation (induced pharmacologically or genetically) results in delayed G2/M progression, altered mitotic phosphorylation, and altered cyclin expression. Decreasing O-GlcNAcylation by overexpression of OGA induces a mitotic exit phenotype accompanied by a delay in mitotic phosphorylation, altered cyclin expression, and pronounced disruption in nuclear organization (
      • Slawson C.
      • Zachara N.E.
      • Vosseller K.
      • Cheung W.D.
      • Lane M.D.
      • Hart G.W.
      Perturbations in O-linked β-N-acetylglucosamine protein modification cause severe defects in mitotic progression and cytokinesis.
      ). Overexpression of OGT results in a polyploid phenotype with faulty cytokinesis, as is often seen in cancer cells (Fig. 3). Strikingly, at M-phase, OGT is highly concentrated at the mitotic spindle and mid-body, and a significant portion of OGT is transiently in a large molecular complex with cell cycle-regulated kinases, phosphatases, and OGA. Glycomic analyses identified 141 previously unknown O-GlcNAc sites on proteins that function in spindle assembly and cytokinesis. Many of these O-GlcNAcylation sites are either identical to known phosphorylation sites or are in close proximity to them. Increased O-GlcNAcylation also altered the phosphorylation of key proteins associated with the mitotic spindle and midbody. Overexpression of OGT increased the inhibitory phosphorylation of cyclin-dependent kinase 1 (CDK1) and reduced the phosphorylation of CDK1 target proteins. Increased phosphorylation of CDK1 resulted from increased activation of its upstream kinase, MYT1, and from a concomitant reduction in transcription of CDK1 phosphatase, CDC25C. OGT overexpression also caused a reduction in both mRNA expression and protein abundance of Polo-like kinase 1, which is upstream of both MYT1 and CDC25C. Pathway analyses of these data uncovered a cascade series of events that illustrate how nutrients regulate cell division via the complex interplay between O-GlcNAcylation and phosphorylation (Fig. 3) (
      • Wang Z.
      • Udeshi N.D.
      • Slawson C.
      • Compton P.D.
      • Sakabe K.
      • Cheung W.D.
      • Shabanowitz J.
      • Hunt D.F.
      • Hart G.W.
      Extensive crosstalk between O-GlcNAcylation and phosphorylation regulates cytokinesis.
      ). Collectively, these studies indicate that O-GlcNAc cycling is a pivotal regulatory component of nutrient regulation of the cell cycle, controlling cell cycle progression by regulating mitotic phosphorylation, cyclin expression, and cell division (
      • Slawson C.
      • Zachara N.E.
      • Vosseller K.
      • Cheung W.D.
      • Lane M.D.
      • Hart G.W.
      Perturbations in O-linked β-N-acetylglucosamine protein modification cause severe defects in mitotic progression and cytokinesis.
      ).
      Figure thumbnail gr3
      Figure 3Nutrients regulate cytokinesis and the cell cycle by O-GlcNAcylation. A, OGT is highly concentrated at the mid-body during the late stages of cytokinesis. B, O-GlcNAcylated proteins are enriched at the midbody and at the nascent nuclear envelope during the late stages of cytokinesis. C, overexpression of OGT causes defective cytokinesis, resulting in polyploidy. D, during the late stages of cytokinesis, OGT, OGA, protein phosphatase I (PP1c), Polo-like kinase (PLK1), and Aurora kinase B (among other proteins) are in a transient molecular complex that modifies proteins involved in cell division.

      Abnormal O-GlcNAcylation underlies the etiologies of chronic diseases associated with aging (Fig. 4)

      Fundamental roles in diabetes

      Data from several laboratories indicate that abnormal O-GlcNAcylation, such as occurs in hyperglycemia associated with diabetes, underlies molecular mechanisms of glucose toxicity, insulin resistance, mitochondrial dysfunction, and abnormal insulin synthesis and secretion by β-cells (for reviews, see Refs.
      • Hart G.W.
      • Slawson C.
      • Ramirez-Correa G.
      • Lagerlof O.
      Cross talk between O-GlcNAcylation and phosphorylation: roles in signaling, transcription, and chronic disease.
      ,
      • Hardivillé S.
      • Hart G.W.
      Nutrient regulation of signaling, transcription, and cell physiology by O-GlcNAcylation.
      , and
      • McClain D.A.
      Hexosamines as mediators of nutrient sensing and regulation in diabetes.
      • Wells L.
      • Vosseller K.
      • Hart G.W.
      A role for N-acetylglucosamine as a nutrient sensor and mediator of insulin resistance.
      ,
      • Akimoto Y.
      • Hart G.W.
      • Hirano H.
      • Kawakami H.
      O-GlcNAc modification of nucleocytoplasmic proteins and diabetes.
      ,
      • Issad T.
      • Masson E.
      • Pagesy P.
      O-GlcNAc modification, insulin signaling and diabetic complications.
      ,
      • Bond M.R.
      • Hanover J.A.
      O-GlcNAc cycling: a link between metabolism and chronic disease.
      ,
      • Ma J.
      • Hart G.W.
      Protein O-GlcNAcylation in diabetes and diabetic complications.
      ,
      • Banerjee P.S.
      • Lagerlöf O.
      • Hart G.W.
      Roles of O-GlcNAc in chronic diseases of aging.
      • Peterson S.B.
      • Hart G.W.
      New insights: a role for O-GlcNAcylation in diabetic complications.
      ). Since the 1950s, there have been over 1400 papers linking the hexosamine biosynthetic pathway to the etiology of diabetes. Marshall et al. (
      • Marshall S.
      • Bacote V.
      • Traxinger R.R.
      Discovery of a metabolic pathway mediating glucose-induced desensitization of the glucose transport system: role of hexosamine biosynthesis in the induction of insulin resistance.
      ) showed that conversion of glucose to glucosamine by the hexosamine biosynthetic pathway (Fig. 1) is required for the desensitization of the insulin-responsive glucose transport system in adipocytes. Pre-exposure of isolated rat skeletal muscle to glucosamine induces insulin resistance of both glucose transport and glycogen synthesis (
      • Robinson K.A.
      • Sens D.A.
      • Buse M.G.
      Pre-exposure to glucosamine induces insulin resistance of glucose transport and glycogen synthesis in isolated rat skeletal muscles: study of mechanisms in muscle and in rat-1 fibroblasts overexpressing the human insulin receptor.
      ). Increasing flux through the hexosamine biosynthetic pathway (HBP) in otherwise normal rats mimics the hallmarks of glucose toxicity, such as the inhibition of glucose-induced insulin secretion and reduced insulin stimulation of both glycolysis and glycogen synthesis (
      • Giaccari A.
      • Morviducci L.
      • Zorretta D.
      • Sbraccia P.
      • Leonetti F.
      • Caiola S.
      • Buongiorno A.
      • Bonadonna R.C.
      • Tamburrano G.
      In vivo effects of glucosamine on insulin secretion and insulin sensitivity in the rat: possible relevance to the maladaptive responses to chronic hyperglycaemia.
      ). In a streptozotocin rat model of type I diabetes, prolonged hyperglycemia increased the flux through the hexosamine biosynthetic pathway, as determined by the UDP-hex/UDP-HexNAc ratio, by over 40% in skeletal muscle (
      • Robinson K.A.
      • Weinstein M.L.
      • Lindenmayer G.E.
      • Buse M.G.
      Effects of diabetes and hyperglycemia on the hexosamine synthesis pathway in rat muscle and liver.
      ). Overexpression of glutamine:fructose-6-phosphate amidotransferase, the first and rate-limiting enzyme of the HBP, in skeletal muscle and adipose tissue of mice mimics the adverse regulatory and metabolic effects of hyperglycemia, specifically with respect to insulin resistance of glucose disposal (
      • Hebert Jr, L.F.
      • Daniels M.C.
      • Zhou J.
      • Crook E.D.
      • Turner R.L.
      • Simmons S.T.
      • Neidigh J.L.
      • Zhu J.S.
      • Baron A.D.
      • McClain D.A.
      Overexpression of glutamine:fructose-6-phosphate amidotransferase in transgenic mice leads to insulin resistance.
      ). Even modest transgenic overexpression of OGT in muscle and fat of mice leads to insulin resistance and hyperleptinemia (
      • McClain D.A.
      • Lubas W.A.
      • Cooksey R.C.
      • Hazel M.
      • Parker G.J.
      • Love D.C.
      • Hanover J.A.
      Altered glycan-dependent signaling induces insulin resistance and hyperleptinemia.
      ).
      Figure thumbnail gr4
      Figure 4O-GlcNAcylation is directly involved in etiologies of chronic diseases associated with aging. Prolonged elevation of O-GlcNAcylation contributes directly to glucose toxicity, insulin resistance, and β-cell dysfunctions in diabetes. Every cancer type studied to date has elevated O-GlcNAc cycling, and blocking O-GlcNAcylation prevents cancer progression. Decreased O-GlcNAcylation in the brain is associated with both Alzheimer’s disease and Parkinson’s disease.
      OGT has a phosphoinositide-binding domain. Upon insulin stimulation, phosphatidylinositol 3,4,5-trisphosphate recruits OGT from the nucleus to the plasma membrane, where OGT catalyzes increased O-GlcNAcylation of the insulin signaling pathway. This increased O-GlcNAcylation results in the altered phosphorylation on these signaling molecules and results in attenuated insulin signaling (
      • Yang X.
      • Ongusaha P.P.
      • Miles P.D.
      • Havstad J.C.
      • Zhang F.
      • So W.V.
      • Kudlow J.E.
      • Michell R.H.
      • Olefsky J.M.
      • Field S.J.
      • Evans R.M.
      Phosphoinositide signalling links O-GlcNAc transferase to insulin resistance.
      ). In addition, hepatic overexpression of OGT reduces the expression of insulin-responsive genes and causes insulin resistance and dyslipidemia (
      • Yang X.
      • Ongusaha P.P.
      • Miles P.D.
      • Havstad J.C.
      • Zhang F.
      • So W.V.
      • Kudlow J.E.
      • Michell R.H.
      • Olefsky J.M.
      • Field S.J.
      • Evans R.M.
      Phosphoinositide signalling links O-GlcNAc transferase to insulin resistance.
      ). Increased O-GlcNAcylation of glycogen synthase results in the retention of the enzyme in a glucose 6-phosphate–dependent state and contributes to reduced activation of the enzyme associated with insulin resistance (
      • Parker G.J.
      • Lund K.C.
      • Taylor R.P.
      • McClain D.A.
      Insulin resistance of glycogen synthase mediated by O-linked N-acetylglucosamine.
      ).
      Increased flux through the hexosamine biosynthetic pathway also appears to be involved in glucose toxicity and insulin resistance in humans with diabetes (
      • Yki-Järvinen H.
      • Daniels M.C.
      • Virkamäki A.
      • Mäkimattila S.
      • DeFronzo R.A.
      • McClain D.
      Increased glutamine:fructose-6-phosphate amidotransferase activity in skeletal muscle of patients with NIDDM.
      ). Nucleotide polymorphisms in OGA are associated with diabetes in Mexican Americans (
      • Lehman D.M.
      • Fu D.J.
      • Freeman A.B.
      • Hunt K.J.
      • Leach R.J.
      • Johnson-Pais T.
      • Hamlington J.
      • Dyer T.D.
      • Arya R.
      • Abboud H.
      • Göring H.H.
      • Duggirala R.
      • Blangero J.
      • Konrad R.J.
      • Stern M.P.
      A single nucleotide polymorphism in MGEA5 encoding O-GlcNAc-selective N-acetyl-β-d-glucosaminidase is associated with type 2 diabetes in Mexican Americans.
      ). As might be expected from the role of the HBP as a central node of metabolism (Fig. 1), fat-induced insulin resistance is also associated with increased end products of the HBP, suggesting that elevated free fatty acids induce skeletal muscle insulin resistance by increasing the flux of fructose 6-phosphate into the hexosamine pathway (
      • Hawkins M.
      • Barzilai N.
      • Liu R.
      • Hu M.
      • Chen W.
      • Rossetti L.
      Role of the glucosamine pathway in fat-induced insulin resistance.
      ). Palmitate also activates the HBP in human myotubes (
      • Weigert C.
      • Klopfer K.
      • Kausch C.
      • Brodbeck K.
      • Stumvoll M.
      • Häring H.U.
      • Schleicher E.D.
      Palmitate-induced activation of the hexosamine pathway in human myotubes: increased expression of glutamine:fructose-6-phosphate aminotransferase.
      ). Expression of the ob gene to make leptin, a potent adipokine released by adipocytes in response to increased energy storage, is controlled by end products of the HBP (
      • Wang J.
      • Liu R.
      • Hawkins M.
      • Barzilai N.
      • Rossetti L.
      A nutrient-sensing pathway regulates leptin gene expression in muscle and fat.
      ,
      • Considine R.V.
      • Cooksey R.C.
      • Williams L.B.
      • Fawcett R.L.
      • Zhang P.
      • Ambrosius W.T.
      • Whitfield R.M.
      • Jones R.
      • Inman M.
      • Huse J.
      • McClain D.A.
      Hexosamines regulate leptin production in human subcutaneous adipocytes.
      ,
      • McClain D.A.
      • Alexander T.
      • Cooksey R.C.
      • Considine R.V.
      Hexosamines stimulate leptin production in transgenic mice.
      ).
      β-Cells of the pancreas have the highest relative amounts of OGT and O-GlcNAc of any tissue (
      • Hanover J.A.
      • Lai Z.
      • Lee G.
      • Lubas W.A.
      • Sato S.M.
      Elevated O-linked N-acetylglucosamine metabolism in pancreatic beta-cells.
      ,
      • Akimoto Y.
      • Kreppel L.K.
      • Hirano H.
      • Hart G.W.
      Increased O-GlcNAc transferase in pancreas of rats with streptozotocin-induced diabetes.
      ). Prolonged elevation of O-GlcNAcylation contributes to β-cell death by apoptosis in diabetes (
      • Liu K.
      • Paterson A.J.
      • Chin E.
      • Kudlow J.E.
      Glucose stimulates protein modification by O-linked GlcNAc in pancreatic beta cells: linkage of O-linked GlcNAc to beta cell death.
      ). Elevated O-GlcNAc leads to deterioration of glucose-stimulated insulin secretion by the pancreas of diabetic Goto–Kakizaki rats (
      • Akimoto Y.
      • Hart G.W.
      • Wells L.
      • Vosseller K.
      • Yamamoto K.
      • Munetomo E.
      • Ohara-Imaizumi M.
      • Nishiwaki C.
      • Nagamatsu S.
      • Hirano H.
      • Kawakami H.
      Elevation of the post-translational modification of proteins by O-linked N-acetylglucosamine leads to deterioration of the glucose-stimulated insulin secretion in the pancreas of diabetic Goto-Kakizaki rats.
      ). Key transcription factors that control expression of proinsulin are dynamically regulated by O-GlcNAcylation. Glucose regulates the nuclear transport of NeuroD1 via its O-GlcNAcylation. O-GlcNAc regulates the DNA binding by PDX-1 (
      • Gao Y.
      • Miyazaki J.
      • Hart G.W.
      The transcription factor PDX-1 is post-translationally modified by O-linked N-acetylglucosamine and this modification is correlated with its DNA binding activity and insulin secretion in min6 beta-cells.
      ), and glucose controls the expression of the MAF-1 transcription factor via O-GlcNAcylation of unknown nuclear proteins (
      • Vanderford N.L.
      • Andrali S.S.
      • Ozcan S.
      Glucose induces MafA expression in pancreatic beta cell lines via the hexosamine biosynthetic pathway.
      ). Collectively, these studies indicate a direct role for O-GlcNAcylation in regulating the production and secretion of insulin. It has been proposed that a chronic increase in O-GlcNAcylation may be a major factor leading to the deterioration of β-cell function (
      • Zraika S.
      • Dunlop M.
      • Proietto J.
      • Andrikopoulos S.
      The hexosamine biosynthesis pathway regulates insulin secretion via protein glycosylation in mouse islets.
      ).
      Hyperglycemia qualitatively and quantitatively alters the O-GlcNAcylation or expression of many proteins within the nucleus. Abnormal O-GlcNAcylation of transcription factors, especially Sp1, appears to play a significant role in the abnormal expression of proteins observed in diabetes (
      • Issad T.
      • Kuo M.
      O-GlcNAc modification of transcription factors, glucose sensing and glucotoxicity.
      ). Hyperglycemia increases O-GlcNAc on Sp1 and induces expression of plasminogen activator inhibitor-1 (
      • Du X.L.
      • Edelstein D.
      • Rossetti L.
      • Fantus I.G.
      • Goldberg H.
      • Ziyadeh F.
      • Wu J.
      • Brownlee M.
      Hyperglycemia-induced mitochondrial superoxide overproduction activates the hexosamine pathway and induces plasminogen activator inhibitor-1 expression by increasing Sp1 glycosylation.
      ), which leads to the expression of genes that contribute to the pathogenesis of diabetic complications. In contrast, activation of PPARγ, a ligand-activated nuclear receptor that increases insulin sensitivity, reduces O-GlcNAcylation of Sp1 (
      • Chung S.S.
      • Kim J.H.
      • Park H.S.
      • Choi H.H.
      • Lee K.W.
      • Cho Y.M.
      • Lee H.K.
      • Park K.S.
      Activation of PPARγ negatively regulates O-GlcNAcylation of Sp1.
      ). Enhanced O-GlcNAcylation is associated with insulin resistance in GLUT1-overexpressing muscles (
      • Buse M.G.
      • Robinson K.A.
      • Marshall B.A.
      • Hresko R.C.
      • Mueckler M.M.
      Enhanced O-GlcNAc protein modification is associated with insulin resistance in GLUT1-overexpressing muscles.
      ). Hyperglycemia, via O-GlcNAcylation, impairs activation of the IR/IRS/PI3K/AKT pathway, resulting in deregulation of eNOS activity (
      • Federici M.
      • Menghini R.
      • Mauriello A.
      • Hribal M.L.
      • Ferrelli F.
      • Lauro D.
      • Sbraccia P.
      • Spagnoli L.G.
      • Sesti G.
      • Lauro R.
      Insulin-dependent activation of endothelial nitric oxide synthase is impaired by O-linked glycosylation modification of signaling proteins in human coronary endothelial cells.
      ). Elevated O-GlcNAc also results in insulin resistance associated with defects in AKT activation in 3T3-L1 adipocytes (
      • Vosseller K.
      • Wells L.
      • Lane M.D.
      • Hart G.W.
      Elevated nucleocytoplasmic glycosylation by O-GlcNAc results in insulin resistance associated with defects in Akt activation in 3T3-L1 adipocytes.
      ).
      Increased O-GlcNAcylation has also been implicated in diabetic complications of the eye. The elevated expression of O-GlcNAc–modified proteins and O-GlcNAc transferase plays a causative role in the corneal epithelial disorders of diabetic GK rats (
      • Akimoto Y.
      • Kawakami H.
      • Yamamoto K.
      • Munetomo E.
      • Hida T.
      • Hirano H.
      Elevated expression of O-GlcNAc-modified proteins and O-GlcNAc transferase in corneas of diabetic Goto-Kakizaki rats.
      ). There is growing evidence that increased O-GlcNAcylation contributes to the pathogenesis of diabetic retinopathy (
      • Semba R.D.
      • Huang H.
      • Lutty G.A.
      • Van Eyk J.E.
      • Hart G.W.
      The role of O-GlcNAc signaling in the pathogenesis of diabetic retinopathy.
      ), including the early loss of retinal pericytes (
      • Gurel Z.
      • Sheibani N.
      O-Linked β-N-acetylglucosamine (O-GlcNAc) modification: a new pathway to decode pathogenesis of diabetic retinopathy.
      ).
      Abnormal O-GlcNAc modification of nucleocytoplasmic proteins appears to also be involved in glucose toxicity in vascular tissues (
      • Akimoto Y.
      • Kreppel L.K.
      • Hirano H.
      • Hart G.W.
      Hyperglycemia and the O-GlcNAc transferase in rat aortic smooth muscle cells: elevated expression and altered patterns of O-GlcNAcylation.
      ,
      • Nakamura M.
      • Barber A.J.
      • Antonetti D.A.
      • LaNoue K.F.
      • Robinson K.A.
      • Buse M.G.
      • Gardner T.W.
      Excessive hexosamines block the neuroprotective effect of insulin and induce apoptosis in retinal neurons.
      ). O-GlcNAcylation of AKT kinase promotes vascular calcification (
      • Heath J.M.
      • Sun Y.
      • Yuan K.
      • Bradley W.E.
      • Litovsky S.
      • Dell'Italia L.J.
      • Chatham J.C.
      • Wu H.
      • Chen Y.
      Activation of AKT by O-linked N-acetylglucosamine induces vascular calcification in diabetes mellitus.