O-GlcNAc Signaling in the Cardiovascular System

Gladys A. Ngoh, Heberty T. Facundo, Ayesha Zafir and Steven P. Jones-GlcNAc Signaling in the Cardiovascular SystemO

Print ISSN: 0009-7330. Online ISSN: 1524-4571 Copyright © 2010 American Heart Association, Inc. All rights reserved.is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation Research

doi: 10.1161/CIRCRESAHA.110.2246752010;107:171-185Circ Res.

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Reviews

This Review is the last in a thematic series on Novel Posttranslational Modifications of Proteins and TheirCardiovascular Significance, which includes the following articles:The Emerging Characterization of Lysine Residue Deacetylation on The Modulation of Mitochondrial Function andCardiovascular Biology [2009;105:830–841]Protein Acetylation in the Cardiorenal Axis: The Promise of Histone Deacetylase Inhibitors [2010;106:272–284]Protein S-Nitrosylation and Cardioprotection [2010;106:285–296]Sent to Destroy: The Ubiquitin Proteasome System Regulates Cell Signaling and Protein Quality Control inCardiovascular Development and Disease [2010:106:463–478]S-Nitrosylation in Cardiovascular Signaling [2010;106:633–646]Sumoylation and Regulation of Cardiac Gene Expression [2010;107:19–29]

O-GlcNAc Signaling in the Cardiovascular System

Elizabeth Murphy, Guest Editor

O-GlcNAc Signaling in the Cardiovascular SystemGladys A. Ngoh,* Heberty T. Facundo,* Ayesha Zafir, Steven P. Jones

Abstract: Cardiovascular function is regulated at multiple levels. Some of the most important aspects of suchregulation involve alterations in an ever-growing list of posttranslational modifications. One such modificationorchestrates input from numerous metabolic cues to modify proteins and alter their localization and/or function.Known as the �-O-linkage of N-acetylglucosamine (ie, O-GlcNAc) to cellular proteins, this unique monosaccha-ride is involved in a diverse array of physiological and pathological functions. This review introduces readers to thegeneral concepts related to O-GlcNAc, the regulation of this modification, and its role in primary pathophysiology.Much of the existing literature regarding the role of O-GlcNAcylation in disease addresses the protracted elevationsin O-GlcNAcylation observed during diabetes. In this review, we focus on the emerging evidence of its involvement inthe cardiovascular system. In particular, we highlight evidence of protein O-GlcNAcylation as an autoprotective alarmor stress response. We discuss recent literature supporting the idea that promoting O-GlcNAcylation improves cellsurvival during acute stress (eg, hypoxia, ischemia, oxidative stress), whereas limiting O-GlcNAcylation exacerbatescell damage in similar models. In addition to addressing the potential mechanisms of O-GlcNAc–mediatedcardioprotection, we discuss technical issues related to studying protein O-GlcNAcylation in biological systems. Thereader should gain an understanding of what protein O-GlcNAcylation is and that its roles in the acute and chronicdisease settings appear distinct. (Circ Res. 2010;107:171-185.)

Key Words: myocardial ischemia � glucose � diabetes mellitus � mitochondria

Much has been written about glycolysis, �-oxidation, andthe other major metabolic pathways in cells. Yet, there

are several underinvestigated accessory glycolytic pathwayswhose importance in the cardiovascular system is nowbeginning to be appreciated. Eukaryotic glycosylation repre-sents a highly varied and complex collection of biologicalpathways, which are too broad for serious discussion here.

This review focuses on one unique form of glycosylation, andthe reader should refer to definitive sources1 for informationon other forms of glycosylation. The hexosamine biosyntheticpathway (HBP) exemplifies one such accessory pathway forglucose metabolism. Based on evidence from cell lines,2 theHBP consumes a small fraction of glucose and involves aseries of enzyme-catalyzed reactions ending with the formation

Original received May 20, 2010; resubmission received May 20, 2010; accepted June 10, 2010.From the Institute of Molecular Cardiology, Diabetes and Obesity Center, and Department of Physiology Biophysics, University of Louisville, Ky.*Both authors contributed equally to this work.Correspondence to Steven P. Jones, PhD, Institute of Molecular Cardiology, University of Louisville, 580 South Preston St, 404C, Baxter II-404C,

Louisville, KY, 40202. E-mail [email protected]© 2010 American Heart Association, Inc.

Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.110.224675

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of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc).This pathway (see Figure 1) begins with the rate-limitingconversion of fructose-6-phosphate to glucosamine-6-phosphateby L-glutamine: fructose-6-phosphate amidotransferase (GFAT)using glutamine. The next critical reaction involves the conver-sion of gluocosamine-6-phosphate to N-acetylglucosamine-6-phosphate by the Emeg32 (glucosamine-6-phosphate acetyl-transferase) using acetyl-coenzyme A (CoA). Once formed,UDP-GlcNAc provides the glycoside precursor for glycopro-

teins, glycolipids, proteoglycans, and more germane to thisreview, serves as the nucleotide sugar for the posttranslationalglycosylation of nuclear, cytoplasmic, and mitochondrial pro-teins known as O-GlcNAc. In general, little is known about therelative flux through HBP in the heart or vasculature.

Hexosamine Biosynthetic PathwayThe diverse nature of the precursors of UDP-GlcNAc linksHBP to several metabolic pathways. Once glucose enters thecell, it is phosphorylated to glucose-6-phosphate and metab-olized to fructose-6-phosphate and a fraction is diverted to theHBP.2 Glutamine, which enters the HBP at the rate-limitingstep, is a highly abundant nonessential amino acid in musclecells and potentially links HBP to amino acid metabolism.Acetyl-CoA, a ubiquitous metabolic intermediate, links HBPto lipolysis/lipogenesis, glucose oxidation, and amino acidcatabolism. Finally, the HBP requires ATP at the final step,ie, the conversion of N-acetylglucosamine-1-phosphate toUDP-GlcNAc by UDP-GlcNAc pyrophosphorylase. Becausethe precursors of UDP-GlcNAc are nutrient derived andpotentially from other metabolic pathways, UDP-GlcNAc,and hence the O-GlcNAc posttranslational modification,might act as a nutrient/metabolic sensor.

Little is known about the regulation of HBP flux in theheart. Eukaryotic GFAT is highly conserved and regulatedtranscriptionally3 and posttranslationally by cAMP-dependent protein kinase4,5 and by UDP-GlcNAc feedbackinhibition.6 GFAT exists in 2 isoforms, GFAT1 (highlyexpressed in the pancreas, placenta and testis) and GFAT2(highly expressed in the heart and CNS). Emeg32(glucosamine-6-phosphate acetyltransferase) is critical formaintaining the proper intracellular concentration of UDP-GlcNAc,7 thus, it may indirectly regulate O-GlcNAcylation.Thus, investigations in this area will undoubtedly provideimportant insights into cardiovascular disease.

N-Glycosylation Versus O-GlcNAcylationVersus PhosphorylationO-GlcNAc is a posttranslational modification of nuclear, cyto-plasmic and mitochondrial proteins first described in 1984 by

Non-standard Abbreviations and Acronyms

Ad GFP adenovirus-delivered green fluorescent protein

Ad OGT adenovirus-delivered uridine diphospho-N-acetylglucosamine:polypeptide �-N-acetylglucosaminyltransferase

Ad OGA adenovirus-delivered O-GlcNAcase

DON 6-diazo-5-oxo-L-norleucine

Emeg32 glucosamine-6-phosphate acetyltransferase

eNOS endothelial nitric oxide synthase

GFAT glutamine:fructose amidotransferase

GlcN glucosamine

HBP hexosamine biosynthetic pathway

IL interleukin

NRCM neonatal rat cardiac myocyte

O-GlcNAc �-O-linked N-acetylglucosamine

O-GlcNAcase �-N-acetylglucosaminidase

OGT uridine diphospho-N-acetylglucosamine:polypeptide�-N-acetylglucosaminyltransferase

NF-�B nuclear factor �B

PGC-1� peroxisome proliferator-activated receptor-�coactivator-1

PUGNAc O-(2-acetamido-2-deoxy-d-glycopyranosylidene)amino-N-phenylcarbamate

TNF tumor necrosis factor

UDP-GlcNAc uridine diphospho-�-N-acetylglucosamine

VDAC voltage-dependent anion channel

Figure 1. Hexosamine biosynthetic pathway.Phosphorylated glucose enters either the glycogensynthetic pathway or is further converted tofructose-6-phosphate by glucose-6-phosphateisomerase. The majority of fructose-6-phosphate ischanneled to glycolysis. Less than 5% of glucoseuptake is ultimately channeled to a unique acces-sory pathway for glucose metabolism, the HBP.This pathway begins with the rate-limiting enzyme,GFAT, followed by acetylation of gluocsamine-6-phosphate by Emeg32 (glucosamine-6-phosphate acetyltransferase) to GlcNAc-6-P(N-acetylglucosamine-6-phosphate). Next are 2reversible reactions: the conversion of GlcNAc-6-Pto GlcNAc-1-P by phosphate-acetylglucosaminemutase and then the formation UDP-GlcNAc byUDP-GlcNAc pyrophosphorylase. This high-energymolecule serves as the monosaccharide donor forthe posttranslational modification by O-GlcNActransferase (OGT). O-GlcNAcase (OGA) removesO-GlcNAc modification from proteins.

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Torres and Hart.8 In their study, they attempted to probe forglycans on surface proteins of lymphocytes using �-d-1 to4-galactosylaminyl transferase.8 Most of the proteins labeledwere intracellular proteins and the labels were incorporated onsingle GlcNAc residues rather than complex polysaccharideassociated with cell surface proteins.8 O-GlcNAcylation is inmany ways distinct from “classic” protein glycosylation. First,O-GlcNAc modified proteins are found mostly within thenucleus, cytoplasm, or mitochondria contrary to N-glycosylation,which predominates in cell surfaces, the lumen of membra-nous organelles, the endoplasmic reticulum, and Golgi appa-ratus.9,10 Second, O-GlcNAc is not elongated into complexstructures or further modified with the exception of a nuclearpore protein, unlike the extraordinarily complex array ofglycans found on extracellular glycoproteins.8,11,12 Third,O-GlcNAc rapidly cycles on and off proteins on a time scalesimilar to that of phosphorylation/dephosphorylation butunlike extracellular complex glycans, which are essentiallystatic.13–16 Fourth, there is yet no obvious consensus sequencefor the addition of O-GlcNAc to proteins, whereas N-glyco-sylation has Asn-X-Ser/Thr sequence (where X could be anyamino acid other than proline or aspartic acid). Fifth, GlcNAcis added to proteins through an O-linkage on the hydroxylgroup of Ser/Thr, whereas in N-glycosylation, the monosaccha-rides are added through an N-linkage on the amide group of Asn.

O-GlcNAcylation is one of the most common posttransla-tional modifications17 and is similar to protein phosphoryla-tion in that: Both O-GlcNAcylation and phosphorylationposttranslational modifications are found on serine and thre-onine residues18,19; both modifications are dynamically addedand removed from proteins in response to cellular signals20-22;both alter the functions and/or associations of the modifiedprotein. O-GlcNAc differs from protein phosphorylation inthat only 2 enzymes catalyze the addition and removal ofO-GlcNAc from proteins, whereas more than 600 geneticallydistinct kinases and phosphatases regulate the addition andremoval of phosphorylation in mammalian cells. Even thoughmany phosphorylation sites are also known glycosylationsites,20,21 the view that O-GlcNAc and phosphorylation existin a “yin-yang,”18 or simply reciprocal relationship, likelyrepresents an overly simplistic model.

O-GlcNAc and O-phosphate site-mapping studies suggestthat there are several types of dynamic interplay betweenO-GlcNAc and O-phosphate. There is evidence of competitiveoccupancy at the same site, for example that which occurs in thetranscription factor c-Myc,22–24 estrogen receptor-�,25,26 oncop-rotein SV-40 large T-antigen, and endothelial nitric oxidesynthase27; that is, a site is either O-GlcNAc modified, phos-phorylated, or unmodified. In alternative occupancy occurring atadjacent sites, such as that observed in the tumor suppressorp5328 and synapsin I,29 glycosylation can inhibit phosphorylationat adjacent sites by steric hindrance or modulation of proteinstructure. Other highly complex interactions also likely exist anddo not fall into either of the former categories.21,30 Furthermore,the interplay between O-GlcNAc and O-phosphate is alsounderscored by the recent finding that OGT (uridine diphospho-N-acetylglucosamine:polypeptide �-N-acetylglucosaminyltrans-ferase) transiently forms complexes containing the catalyticsubunit of protein phosphatase 1c (PP1c),31 hence in some

contexts there may exist a single enzyme complex for theaddition of GlcNAc and removal of phosphate.

Enzymatic Regulation of O-GlcNAcylationModulation of protein O-GlcNAcylation is achieved by theconcerted action of 2 highly evolutionarily conserved en-zymes, a uridine diphospho-N-acetylglucosamine: peptide�-N-acetylglucosaminyl transferase (O-GlcNAc transferase;aka OGT) and O-�-N-acetylglucosaminidase (O-GlcNAcase;also known as OGA, GCA, or mgea5). O-GlcNAc transferase(OGT) is a soluble, ubiquitously expressed, and highlyconserved enzyme in all multi-cellular eukaryotic organismsinvolved with the addition of a single �-N-acetylglucosamine(GlcNAc) moiety via an O-linkage to serine/threonine aminoresidues nuclear, cytoplasmic and mitochondrial pro-teins.32–35 OGT is expressed in all tissue types examined andmost abundant in the glucose-sensing cells of the pancreasand in the brain. OGT is primarily located in the nucleus andhas an optimum pH of �6.32,33 OGT is encoded by a singlecopy X-linked gene in mammals, whereas plants have 2 OGThomologs, spy and secret agent.32,33,36,37 Even though OGT iscoded by a single gene in mammals, alternative splicing ofOGT mRNA leads to 3 isoforms: nucleocytoplasmic OGT(ncOGT), mitochondrial OGT (mOGT), and short OGT(sOGT).32,33,38 These isoforms share an identical C-terminalcatalytic domain but have distinct N-terminal domains con-tributing to their differential localization and unique targetingsequences.38–40 Structurally, OGT contains an N-terminaltetratricopeptide repeat (TPR), a linker region and C-terminalcatalytic domains.32,33 TPR domain consists of a 34 aminoacid repeat varying from 3 to 12 involved in intersubunitinteraction, protein-protein interaction, subcellular targeting,substrate recognition, cell cycle regulation, and transcrip-tional control.33,41–46 The linker region is the least conservedsequence of OGT. The catalytic domain of OGT is thought tohave a UDP-GlcNAc binding site and is involved with theglycosylation of proteins.47 Posttranslational modification ofOGT by tyrosine phosphorylation and O-GlcNAc modifica-tion, UDP-GlcNAc concentration, and protein-protein inter-action are thought to regulate OGT activity.32,33,48,49

Recently, insulin signaling was shown to regulate OGT.50,51

In neuro-2a neuroblastoma cells, OGT mRNA and proteinexpression are regulated in an AMP-activated protein kinase-dependent manner, whereas OGT enzymatic activity is reg-ulated in a p38 MAPK-dependent manner.48 Moreover, acti-vated p38 has been shown to interact with OGT and recruit itto specific substrates, such as neurofilament H during glucosedeprivation.48 Tissue specific OGT mutation causes distur-bance in somatic cell function,52 whereas conventional OGTdeletion is embryonic-lethal36; hence O-GlcNAc is importantfor cellular viability.

O-GlcNAcase is a soluble, highly conserved enzyme, andexpressed in all eukaryotic organisms involved with the removalof O-GlcNAc modification from proteins.53 O-GlcNAcase isprimarily located in the cytoplasm with an optimum pH of 5.5to 7 and coded for by a single gene. Two splice variants ofO-GlcNAcase have been reported in rats both lackingO-GlcNAcase activity but retained HAT activity. The splicedvariant detected in Goto–Kakizaki rats (�90 kDa) lacks exon

Ngoh et al O-GlcNAc Signaling in CV Disease 173




8, whereas the spliced variant in Sprague–Dawley (�84 kDa)lacks both exons 8 and 9. Structurally, O-GlcNAcase is a 917amino acid protein with an N-terminal hexosaminidase and aC-terminal histone acetyltransferase domain (HAT).53–57 TheN-terminal domain is similar to hyaluronidase and was origi-nally identified as meningioma expressed antigen 5.53,55–57 Al-though there may be some activity against hyaluron in vitro, thepreferred substrate for O-GlcNAcase is O-GlcNAc.53,58 TheC-terminal HAT domain can acetylate free histones andnucleosomal histone proteins.54 It is of interest to note thatcaspase 3 can cleave O-GlcNAcase into HAT and hex-osaminidase domains with no change in the activity of eachdomain.58 Protein-protein interaction and phosphorylation arealso thought to regulate O-GlcNAcase activity,53,58 thoughthe data in this regard are limited. Interestingly, Hanover andcolleagues59 have described a short form of OGA thatseemingly lacks the HAT domain.

Cell CycleCellular growth, division, and maturation are ordered processesand are tightly controlled by a number of different extracellularevents and genetic programs. To this end, Slawson et al showedconvincing evidence that O-glycosylation was modulated duringthe cell cycle, being lowest at M phase and highest at G1/S andG2/M.60 Most recently, it was demonstrated that OGT andO-GlcNAcase interact transiently with the mitotic kinase AuroraB and the protein phosphatase 1.61 The strongest support for afunctional link between O-GlcNAc and cell cycle is the obser-vation that several proteins are modified by O-GlcNAc in a cellcycle dependent manner, including c-Myc,62 keratins,20 YY1,63

and vimentin.61 Also supporting this notion, OGT knockout cellsbecame growth arrested.52 Interestingly, altering extracellularglucosamine levels has been implicated in growth arrest in somecancer cells,64 further strengthening the observation thatO-GlcNAc cycling is important for regulation of the cell cycle.

Transcriptional RegulationNumerous proteins are responsible for the correct control andmaintenance of transcription in the eukaryotic nucleus. Chro-matin remodeling (stimulated by the activity of histoneacetyltransferase) in response to stimuli, permits transcrip-tional machinery to initiate mRNA synthesis. Posttransla-tional modification of key proteins has distinct roles incontrolling this process. Teleologically, it should not besurprising that O-GlcNAc signaling can influence transcrip-tion because OGT is known to associate with histone deacety-lase complexes.44 O-GlcNAcase also contains a domain withreported histone acetyltransferases (HAT) activity in aminoacids 583 to 917 and shares high homology with HAT inresidues 772 to 899.54 Mutation in aspartic acid and phenyl-alanine residues ablates activity and correlates with evidencesuggesting that O-GlcNAcase is capable of acetylating eithernucleosomal histone proteins or free core histones.54

Experimental evidence supports O-GlcNAcylation as animportant posttranslational modification directly regulatingtranscription. In fact, several transcription factors have beenidentified to be regulated by O-GlcNAc modification.65–68

O-GlcNAcylation can either suppress or enhance transcrip-

tion, depending on the promoter involved and other associ-ated coactivator/repressor proteins. For example, OGT canmediate transcriptional repression after being recruited topromoter regions by association with the transcriptionalcorepressor mSin3A.44 Others have shown that the transcrip-tion factor STAT5A alters gene activation by preferentiallybinding to the coactivator of transcription, CREB-bindingprotein, when O-GlcNAc–modified.69

O-GlcNAc modification of Sp1 has multiple effects on thefunction of Sp1 as a transcription factor.68 Augmented O-GlcNAcmodification of Sp1 drives the transcription of plasminogenactivator and extracellular matrix proteins, which have animportant role in diabetic cardiovascular disease, whereas reduc-tion of Sp1 O-GlcNAcylation increased Sp1 proteasomal sus-ceptibility.70 Several posttranslational modifications are neces-sary to control its activity, among these are 8 O-GlcNAcmodified sites.71 The O-GlcNAcylation of Sp1 seems to havean exquisite logic regarding the modification site of theprotein. O-GlcNAcylation of the DNA-binding domain of theC terminus augments its activity.72,73 Similarly, inhibition ofO-GlcNAcase increased Sp1 activity, whereas overexpres-sion of O-GlcNAcase, RNAi against OGT, or a dominantnegative form of OGT reduced the activity of Sp1.74 Con-versely, if the O-GlcNAc modification occurs in sites locatedin the N terminus, the result is an inhibition of its transacti-vation potential,72 inhibiting protein-protein interaction. The11 TPR repeats of OGT (AA1–485) are the residues essentialfor Sp1 transcriptional repression and protein-protein inter-action between OGT and mSin3A repressor.44 Interestingly,insulin elevates nuclear O-GlcNAcylation of Sp1.75

The ability of OGT to promote O-GlcNAc modification onRNA polymerase II on the same residues as phosphoryla-tion,66 provides a clue regarding how the O-GlcNAc modifi-cation can influence cellular transcriptional status. Morespecifically, O-GlcNAcylation of RNA polymerase II occursin the C terminus of the enzyme, which induces a conforma-tional change, blocks phosphorylation on these residues, andpotentially regulates gene expression.66 The O-GlcNAcylationof RNA polymerase II is facilitated by a recruitment of OGT totranscriptional complexes by the OGT interacting protein OIP-106.76 Conversely, in vitro experiments have revealed that asingle phosphate residue on the C terminus of RNA polymeraseII blocks the activity of OGT on the enzyme.30

Transcriptional repression by OGT does not apply for alltranscriptions factors. For example, the O-GlcNAc modificationon FOXO1 promotes promoter activation of some gluconeo-genic genes.77,78 Similarly, O-GlcNAcylation of the transcrip-tional coactivator CRTC2 induces nuclear translocation andalso contributes for gluconeogenic gene transcription.79 CRTC2is a coactivator for the cAMP response element binding protein(CREB) that has been reported to be O-GlcNAc modified,producing transcriptional repression in vitro.67 Another interest-ing example is the transcription factor YY-1, which is incapableof binding to DNA when in complex with the retinoblastomaprotein (pRb). O-GlcNAcylation of YY-1 relieves the DNAbinding inhibition by releasing the protein from a complex withthe pRb protein and, consequently, activates transcription.63 Asin many complex systems, questions still remain unanswered.For example, how does O-GlcNAc accomplish the task of





activating some transcription factors and inhibiting others? Whathas to be determined is how O-GlcNAc “selects” which genesmust be turned off or on.

Involvement in Diabetes and Insulin SignalingDiabetes remains a primary risk factor for the development ofheart disease. Numerous reports have implicated alterationsin O-GlcNAc signaling in diabetic pathophysiology. Fromsimple approaches like exposing cells to glucosamine orhyperglycemia, or, in a more advanced system such as usingdiet-induced or genetic animal models, data support thenotion that O-GlcNAc may contribute to diabetes.80,81 Mostrecently, studies have described potential mechanisms relat-ing high glucose78,79 and the enzyme OGT51 to insulinresistance (a hallmark of type II diabetes). Montminy’s groupfound that the transducer of regulated cAMP response el-ement–binding protein 2 (TORC2 or CRTC2) is a substratefor OGT and is O-GlcNAcylated at Ser70 and Ser171, whichare known phosphorylation sites.79 Phosphorylation ofCRTC2 prevents its nuclear translocation via interaction withthe chaperone protein 14:3:3.82 The O-glycosylation ofCRTC2 impairs its phosphorylation and releases it from thecomplex with 14:3:3 protein79 (Figure 2). In addition to thiseffect, the O-GlcNAc modification of CRTC2 promotesactivation of a conserved cyclic adenosine 3�-5� monophos-phate (cAMP) response element (CRE) on the glucose-6-phosphatase (G6Pase) promoter,79 which is required forG6Pase transcription83 in response to glucose. So, it appearsthat a balance between O-GlcNAc and phosphorylation mustexist to avoid disturbances in insulin signaling.

CRTC2 is not the only O-GlcNAc modified protein thataugments expression of gluconeogenic genes after exposingcells to glucose. Housley and colleagues demonstrated thatthe transcription factor FOXO1 is also O-GlcNAc modifiedin diabetes, resulting in increased expression of gluconeo-genic genes PEPCK and G6Pase. These authors also foundthat O-GlcNAc elevated expression of several antioxidantgenes in association with elevated glucose production byhepatocytes.77,78

Recently, an innovative hypothesis regarding insulin sig-naling has emerged (see Figure 2). Yang et al, showed thatinsulin induces nuclear to cytoplasmic translocation of OGT,where OGT binds the lipid phosphatidylinositol-3,4,5-trisphosphate at the plasma membrane.51 This interaction is notconstitutively active, but may underlie an important mecha-nism in insulin signaling. Such dynamic trafficking of OGTresults in altered phosphorylation of key insulin signalingmolecules and in attenuation of insulin signaling. Based onthe fact that OGT shares some limited, regional homologywith protein phosphatase 5, which exhibits affinity for lipids,they hypothesized the interaction of OGT with lipids. More-over, the authors identified the regulatory domain, necessaryto bind lipids, in the C terminus of OGT as being rich inlysine residues (K981, K982, K986, K989). The N terminusof OGT may contribute by making OGT specific tophosphatidylinositol-3,4,5-trisphosphate compared to otherslipids of the same class.51 Sustained activity of OGT bynutrient excess, for example, impairs insulin signaling.51

Moreover, augmenting O-GlcNAc levels via OGT overex-pression, or inhibiting O-GlcNAcase (via PUGNAc) alsorecapitulates the phenomenon of insulin resistance.51,79,84–86

Others have specifically investigated the potential contri-bution of O-GlcNAcylation because of the pathogenesis ofdiabetes/hyperglycemia. In the intact animal, Hu et al,87 werefirst able to link cardiac dysfunction in streptozotocin-induced diabetic mice to excessive O-GlcNAcylation. Workfrom McClain et al,81 showed that overexpressing OGTinduces insulin resistance in myocytes. OGT upregulation hasalso been reported in other diabetic mouse models.81 Unfor-tunately, limited clinical insights exist regarding O-GlcNAcsignaling. However, insights from some small studies relatecases of human diabetes to impairments in O-GlcNAc sig-naling. Namely, one study examining type II diabetes inMexican-Americans found reduced O-GlcNAcase expressionwith the progression of the disease.88 It is then plausible thatthe O-GlcNAc network rearrangements (with increased OGTlevels or decreased O-GlcNAcase, or both) are the majorcontributors to the development and progression of thedisease. More recently, Hart’s laboratory has demonstrated

Figure 2. O-GlcNAc Affects Insulin Signaling.Insulin binds to tyrosine kinase receptor and rap-idly activates intracellular signaling. PI3K gives riseto PIP3, which serves to anchor PDK1 and AKT onthe membrane. According to Yang et al,51 PIP3also recruits OGT to the plasma membranewhere OGT attenuates insulin signaling byO-glycosylation of Thr308, consequently inhibitingphosphorylation of Akt at the same residue.Excessive activation of OGT could disturb insulinsignaling. Hyperglycemia may also stimulate theO-GlcNAcylation of CRTC2 and its migration intothe nucleus, where this coactivator binds toCREB:CBP and stimulates transcription of glu-coneogenic genes (PEPCK and G6Pase).79 Thetranscription factor FOXO1 is also O-GlcNAcylatedunder similar conditions and stimulates gluconeo-genic gene transcription.77,78





the concept of using blood samples for diagnoses in humanpatients with diabetes.89 So, if O-GlcNAc contributes to thepathogenesis of diabetes, is there a potential mechanism?Work from Dillmann’s group suggests that hyperglycemiareduces the levels of the sarcoplasmic reticulum Ca-ATPase2A (SERCA2A), which was correlated with prolonged Ca2�

transients and reversed by O-GlcNAcase.90 Indeed, morework is needed to determine the potential mechanisms.

Vascular dysfunction represents an important aspect ofinsulin resistance and diabetes. Limited data exists in pa-tients, but Federici et al found that O-GlcNAcylation waselevated in carotid plaques from patients with diabetescompared to patients without.91 Interestingly, some groupshave identified endothelial nitric oxide synthase (eNOS) to bean important target of O-GlcNAcylation in this process.27,91,92

O-GlcNAcylation modification of eNOS apparently inhibitsits ability to generate NO, promote vasodilation, and limitplatelet aggregation, and, contributes to mitochondrial oxida-tive stress.92 Although eNOS may directly be modified byO-GlcNAc, its impaired activation may also be attributable toimpaired Akt phosphorylation at serine 1177, which isapparently sensitive to GFAT inhibition.27 Many questionsremain regarding the interplay among diabetes, O-GlcNAc,and cardiovascular function but existing evidence supportsthe notion that chronic elevations in O-GlcNAc participate inthe etiology and the pathogenesis of diabetes. However, thenext section of this review also demonstrates the context ofthe O-GlcNAc signal, particularly its duration, determines itsrole in pathophysiology.

Role of O-GlcNAc As an Alarm/Stress SignalPhysiological or chemical stress normally induces signaltransduction events that involve activation and/or productionof molecules and proteins that diminish the effects of dele-terious signaling pathways.93,94 Protein phosphorylation istypically the mechanism associated with these signal trans-duction pathways but in 2004, Zachara et al showed thatO-GlcNAc might be a stress-induced signal. This was apivotal study for those interested in basic mechanisms ofcellular survival. In their study, when multiple cell lines weresubjected to diverse stressors (heat shock, ethanol, UV,hypoxia, reductive, oxidative, and osmotic stress), there wasa rapid and global increase in O-GlcNAc levels.95 Moreover,diminished O-GlcNAc levels via OGT knockout (in MEFcells) or OGT knockdown (in Cos-7 and Neuro-2A cells)sensitized cells to stress, whereas increased O-GlcNAc levelsvia O-GlcNAcase inhibition (in Neuro-2A cells) or OGToverexpression (in Cos-7 cells) augmented stress tolerance.Since then, several studies have reinforced the idea thatO-GlcNAc may mediate stress-induced signal transductionpathways in various systems.

O-GlcNAc is an Acute Stress Signal in the HeartDoes protein O-GlcNAcylation change in the acutely stressedheart? Several groups have shown that O-GlcNAc signalingis altered in vitro when cardiac myocytes are subjected tooxidative, hypoxic, and ER stress, and in vivo following acutemyocardial ischemic and trauma-hemorrhagic shock.10,96–113

In a bid to characterize changes in the O-GlcNAc profilefollowing short-term induction of oxidative stress, Jones et alshowed that O-GlcNAc levels increased early on induction ofoxidative stress and decreased by 45 minutes.109 Such decre-ment in O-GlcNAc levels corresponded to exacerbated mito-chondrial dysfunction and cellular injury. How O-GlcNAclevels change during hypoxia–reoxygenation still remainsunclear. However, data from Champattanachai et al showedthat O-GlcNAc levels rise following 4 hours hypoxia andearly reoxygenation,113 which was confirmed in a follow-upstudy by the same group.112 Whether such findings areconsistent for all durations of hypoxia and reoxygenationremains to be seen. Finally, Ngoh et al recently showed thatpharmacological induction of ER stress (with tunicamycin orbrefeldin A) in NRCMs augmented O-GlcNAc signaling.103

Work in isolated, perfused rat hearts in Chatham’s laboratoryrevealed that simulated ischemia alone augments O-GlcNAclevels during the reflow phase.105 A follow-up study from thesame group also showed that low-flow global ischemiaaugmented both UDP-HexNAc and O-GlcNAc levels. Theincrease in O-GlcNAc levels occurred early in the low-flowphase and then declined during reflow.110 Whether this is truefor the intact myocardium remains to be tested. The differ-ential response of O-GlcNAc levels to different models ofisolated heart ischemia and in vivo myocardial ischemia mayprovide insights into the regulation of O-GlcNAc signaling inthe hypoxic myocardium.

Severe injury such as trauma-hemorrhagic shock inducesstress hormones, leading to a hypermetabolic state. An earlyresponse to severe injury is systemic hyperglycemia andenhanced peripheral glucose uptake.114 Even though theeffects of hyperglycemia in trauma are still debatable, Mizocket al showed that stress-induced hyperglycemia, or the pro-vision of additional glucose, could be beneficial by providingan adequate supply of glucose necessary for energy produc-tion in critical organs.114 Considering the protective effect ofaugmented O-GlcNAc signaling shown by Zachara et al,95 itis plausible that the increase in glucose uptake occurringduring trauma-hemorrhage may boost flux through the HBPand consequently augment O-GlcNAc levels. Indeed, recentreports by Chatham’s group showed that trauma-hemorrhagemodels reduced O-GlcNAc signaling in rats.96–98

Inducing brief nonlethal episodes of ischemia and reperfu-sion to the heart before an episode of sustained lethalmyocardial ischemia has the capacity to dramatically reducemyocardial injury. This phenomenon, termed ischemic pre-conditioning,115 is a transient, self-defense mechanism pres-ent in the heart and many other organs, including the kidney,liver, and brain. The ability of ischemic preconditioning toreduce myocardial infarct size is significant and reproducible,and, serves as the gold standard for studies of cardioprotec-tion.116 Intense investigation of the mechanisms responsiblefor the protective effects of preconditioning has revealednumerous potential mediators and downstream effectors ofpreconditioning, but cause and effect relationships have notbeen fully delineated. However, several groups have success-fully demonstrated that ischemic preconditioning enhancesglucose uptake.117–119 Because the monosaccharide donor forO-GlcNAcylation of proteins, ie, UDP-GlcNAc, is derived





from an accessory pathway for glucose metabolism (HBP), itis possible that the increase in glucose uptake occurringduring preconditioning may boost flux through the HBP andconsequently augment O-GlcNAc levels. Indeed, a recentreport showed that either early or delayed ischemic precon-ditioning can augment cardiac O-GlcNAc levels in vivo.109

Although it was not determined whether the changes inO-GlcNAcylation of proteins observed in preconditioningcontributes to its protective effects, pharmacological augmen-tation of O-GlcNAc levels is sufficient to reduce infarct sizein vivo.109 It would be interesting to determine whetherischemic preconditioning relies on O-GlcNAc signaling forcardioprotection, and also determine whether preconditioningof other organs like the brain and liver elevates O-GlcNAcsignaling. Clearly, O-GlcNAc represents an acute stresssignal in the heart. The next question is: Does nutrient/pharmacological/genetic alteration of O-GlcNAc signalinginfluence cell survival?

Hexosamine Biosynthetic Pathway Flux inCardiomyocyte SurvivalThis section of the review focuses on approaches to alterO-GlcNAc signaling via manipulating HBP flux/activity. Therate-limiting step of the HBP requires glutamine, a nonessentialamino acid abundant in muscle tissues, to form glucosamine-6-phosphate. Hence, glutamine contributes to the formation ofUDP-GlcNAc, thereby driving the O-GlcNAc modification ofproteins. In isolated perfused hearts, augmentation of fluxthrough the HBP with glutamine or before hypoxia–reoxygen-ation increased O-GlcNAc levels reduced cardiac damage andpreserved posthypoxic contractile function during the reflowphase.107 Conversely, perfusion of the hypoxic isolated heartwith azaserine, blocked the HBP-mediated increase inO-GlcNAc levels, diminished functional recovery, and exacer-bated posthypoxic tissue injury.105–107 Though the cardioprotec-tive effect of glutamine had been reported by others before thisstudy,120 the mechanism was not attributed to alterations in fluxthrough HBP or O-GlcNAc signaling. However, results from anin vivo porcine model of myocardial ischemia-reperfusion injurydid not demonstrate protection.121

Glucosamine contributes to the formation of UDP-GlcNAc,thereby driving the O-GlcNAc modification of proteins. Usingan approach similar to the aforementioned glutamine studies,Chatham’s group perfused isolated rat hearts with glu-cosamine,105,107,110 which likely enters the HBP downstreamof GFAT and depends on phosphorylation by hexokinase.Glucosamine treatment reduced injury resulting from calciumparadox and hypoxia–reoxygenation.105,107,110,111,122 In neona-tal cardiac myocytes, Champattanachai et al showed thataugmentation of O-GlcNAc levels with high glucose orglucosamine improved posthypoxic cellular viability andattenuated necrosis and apoptosis.113 Conversely, euglycemiccardiomyocytes or hyperglycemic cardiomyocytes treatedwith azaserine (ie, a GFAT inhibitor) were more sensitive tohypoxic stress.113 Interestingly, in vivo augmentation ofO-GlcNAc levels using glucosamine after severe injury suchas hemorrhagic shock has been shown to improve cardiacfunction and peripheral organ perfusion in rats. Taken together,these results provide evidence that the protective effect of

glucosamine is associated with augmented O-GlcNAc signalingand can be translated to the in vivo environment. Clearly,approaches to boost hexosamine flux promote cardiomyocytesurvival.

OGT in Cardiomyocyte SurvivalAdditional support for cytoprotection associated with globalaugmentation of O-GlcNAc levels can be found in studiesusing genetic and molecular approaches to evaluate theenzymes controlling the presence of O-GlcNAc on proteins.Studies involving pharmacological inhibition of O-GlcNActransferase (OGT) have been limited, likely because the fewinhibitors described have not been well characterized andmay exhibit some level of toxicity. The most popular OGTinhibitor, alloxan, is a uracil and UDP-GlcNAc analog andmay be an irreversible inhibitor of OGT.123 Nevertheless, useof such compounds has yielded consistent results. In onestudy, alloxan not only blocked the glucosamine-mediatedincrease in O-GlcNAc levels but also inhibited angiotensinII-induced increase in intracellular Ca2� in NRCMs.104 Al-though results from these studies are cautiously interpretedbecause of the high concentration of alloxan used, its toxicity,its lack of specificity,124 and because of the recent revelationthat alloxan may also inhibit O-GlcNAcase,125 they provideimportant insight into the necessity of OGT signaling in thecontext of elevated hexosamine biosynthesis. “Compound 4”and “compound 5,” were recently described to be potent OGTinhibitors.126,127 Ngoh et al showed that both compounds 4and 5 (referred to as TT04 and TT40 in their article) reducedO-GlcNAc levels and exacerbated posthypoxic cardiomyocyteinjury.102 Moreover, inhibition of OGT also exaggerated theposthypoxic collapse of mitochondrial membrane potential.102

Genetic manipulation of OGT has been shown to altercardiomyocyte survival after hypoxia. Ngoh et al showedthat adenoviral-mediated OGT overexpression (AdOGT)significantly elevated OGT protein expression, augmentedO-GlcNAc levels, and reduced posthypoxic cardiac myocytedeath, whereas OGT knockdown (via short interfering RNA)or knockout (via cre-lox recombination) reduced O-GlcNAclevels and exacerbated posthypoxic cell death.102 In addition,posthypoxic mitochondrial membrane potential was alsobetter preserved in the AdOGT group compared with theposthypoxic control virus (AdGFP). This prosurvival role ofOGT was supported by findings from Champattanachai et alshowing that OGT overexpression attenuated H2O2-inducedloss of mitochondrial membrane potential and hypoxia-induced apoptosis, whereas OGT knockdown (via siRNA)sensitized NRCMs to H2O2-induced loss of mitochondrialmembrane potential and hypoxia-induced apoptosis.112 Thus,OGT seems essential in the constitutive, as well as inducible,abilities of the cell to withstand lethal stressors. In otherwords, OGT promotes cell survival during acute cardiomyo-cyte stress.

O-GlcNAcase in Cardiomyocyte SurvivalUnlike OGT, there are several pharmacological inhibitorsof O-GlcNAcase, including streptozotocin,128 PUGNAc,129

1,2-dideoxy-2-methyl-d-glucopyranoso[2,1-d]-2-thiazoline-





(NButGT),130 GlcNAcstatin,131 and Thiamet-G.132 PUGNAc, aGlcNAc analog, is the most widely studied inhibitor ofO-GlcNAcase and prevents the binding of O-GlcNAcase toGlcNAc. Thus, PUGNAc prevents the removal of O-GlcNAcleading to a rapid increase in O-GlcNAc levels. Even thoughPUGNAc lacks the cytotoxic effects of STZ,133 it potentiallyinhibits other lysosomal hydrolases and shows limited specific-ity for O-GlcNAcase more than �-hexosaminidase.130

In NRCMs, augmenting O-GlcNAc levels using PUGNAcattenuated posthypoxic113,134 and oxidative stress-induced109

injury and inhibited both posthypoxic134 and H2O2-induced109

mitochondrial depolarization. Similar findings were observedin isolated perfused hearts where PUGNAc administrationearly in reperfusion improved cardiac functional recovery,reduced troponin release, and attenuated calpain-mediatedproteolysis of �-fodrin and Ca2�/calmodulin-dependent proteinkinase II compared to untreated control.106,107 The cytoprotectiveeffects of PUGNAc seen in NRCMs and isolated perfusedhearts, can be replicated in an in vivo setting. Jones et al showedin an in vivo murine model of myocardial ischemia-reperfusioninjury that PUGNAc reduced infarct size following acute myo-cardial ischemia reperfusion.109

NAG-thiazoline130 inhibits O-GlcNAcase and has 1500-foldgreater specificity for O-GlcNAcase over �-hexosaminidasethan PUGNAc, but has not been as widely studied as PUGNAcand is not widely available. Recently, Champattanachai et alshowed that NButGT can attenuate cardiac myocyte deathfollowing hypoxia and oxidative stress.112 Few studies haveaddressed the role of O-GlcNAcase inhibition in cellular injurybeyond the use of PUGNAc or NButGT. As with inhibitors usedin the OGT work above, concern remains regarding the off-target effects of the putative inhibitors of O-GlcNAcase. Thus,the use of other approaches, such as RNA interference oradenoviruses, could assuage concerns of some of the off-targeteffects of the aforementioned pharmacological approaches. In-deed, Ngoh et al recently showed that siRNA knockdown ofO-GlcNAcase augmented O-GlcNAc levels, preserved posthy-poxic cardiomyocyte membrane potential, and mitigated cellularinjury, whereas overexpression of O-GlcNAcase reducedO-GlcNAc levels, sensitized NRCMs to loss of mitochondrialmembrane potential, and exacerbated cellular injury.134 In short,O-GlcNAcase activity antagonizes cell survival during acutecardiomyocyte stress.

Mechanisms of O-GlcNAc Signalingin CytoprotectionAlthough the specific mechanisms underlying the cytoprotec-tion associated with O-GlcNAc signaling remain to be deter-mined, several putative mechanisms have been advanced toexplain augmented stress tolerance. O-GlcNAc signalinglikely involves numerous intracellular targets, which maycontribute to varying degrees during myocardial preservation.Progress in the area of target protein identification has beenrestricted because of the limitations of the tools necessary forthe identification of O-GlcNAc targets. Zachara et al showedthat increased O-GlcNAc signaling activated transcription ofheat shock proteins HSP40 and HSP70.95 Because the car-dioprotective effects of glucosamine observed in isolatedperfused hearts occurred early,105,110 there may be mecha-

nisms other than de novo protein synthesis contributing toO-GlcNAc–mediated cytoprotection. Recent studies from theJones laboratory revealed that modulating O-GlcNAc levelsmay alter O-GlcNAc modification on (at least) the mitochon-drial voltage-dependent anion channel (VDAC), which mayrepresent a unique mechanism of cytoprotection.102,109,134,135

Activation of mPTP formation is a critical step in mitochon-drial mediated death pathway, and, although the molecularidentity of the mitochondrial permeability transition pore(mPTP) remains debatable, VDAC has been widely recog-nized as a putative component, or at least a modulator ofmPTP. In these studies, treatment of mice with PUGNAc (anO-GlcNAcase inhibitor), increased O-GlcNAc modificationof VDAC and produced resistance of isolated, adult cardiacmitochondria to calcium-induced mitochondrial swelling.109

Conversely, treating mice with compound 4 (a putative OGTinhibitor) reduced O-GlcNAc modification of VDAC andsensitized isolated, adult cardiac mitochondria to mPTPformation.102 Augmented O-GlcNAc levels via OGT overex-pression or O-GlcNAcase inhibition (with PUGNAc orsiRNA) preserved posthypoxic mitochondrial membrane po-tential.102,134 Moreover, boosting HBP flux (with glu-cosamine), OGT overexpression, and O-GlcNAcase inhibi-tion (with NButGT) attenuate H2O2-induced loss ofmitochondrial membrane potential and cytochrome c releasein NRCMs.109,112,135 O-GlcNAc levels affect posthypoxicmitochondrial Ca2� overload in NRCMs. Overexpression ofO-GlcNAcase exacerbated hypoxia-induced Ca2� overload,whereas inhibition of O-GlcNAcase mitigated hypoxia-induced Ca2� overload. Several studies have implicated Ca2�

overload as a key contributor to mitochondrial permeabilitytransition leading to ischemia-reperfusion injury and inhibit-ing the rise in mitochondrial [Ca2�] has been shown to confercardioprotection following acute myocardial ischemia.136,137

Therefore, it is possible that blocking mitochondrial Ca2�

overload may be an upstream action of O-GlcNAc signalingto prevent mPTP formation in addition to direct effects onmPTP components (Figure 3). Finally, augmented O-GlcNAclevels have been shown to increase mitochondrial Bcl-2 inNRCMs subjected to hypoxia–reoxygenation. Because Bcl-2is thought to inhibit mPTP formation by interacting withVDAC, it is possible that augmented O-GlcNAc levels wouldactivate Bcl-2 translocation to the mitochondria, increasingthe interaction of Bcl-2 with VDAC and subsequently block-ing mPTP formation and the release of death factor from themitochondria. Thus, there is a potential mechanistic linkamong a likely target of O-GlcNAc signaling, mitochondrialpreservation, and cell viability.

Using glucosamine to boost O-GlcNAc levels, others haveshown activation of p38 MAPK and reduce calpain pro-teolytic activity, reduced ischemic contracture, and atten-uated reperfusion induced arrhythmias in isolated perfusedhearts.107,110 Such effects could be related to alterations incalcium handling87,90,104,105,134 and/or heat shock protein ac-tivation.95,109 Considering the evidence for O-GlcNAc–medi-ated regulation of the ubiquitin-proteasome system,70,138–140

modulation of UPS activity could represent yet another targetin the portfolio of O-GlcNAc–mediated cytoprotection. In-deed, just as the targets of O-GlcNAc modification are





numerous, so too are the potential mechanisms responsiblefor cytoprotection. Regardless of the mechanisms (and thereare likely multiple targets), the salient feature is that acute,global changes in cellular O-GlcNAcylation reflect a proad-aptive stress response.

O-GlcNAc Signaling andVascular Injury/InflammationArterial hypertension is a multifactorial condition considereda major risk factor for cardiovascular disease. Hypertension ischaracterized by abnormal vascular reactivity, impaired en-dothelium-dependent relaxation, and enhanced sensitivity tovasoconstrictors. Several proteins involved in vascular func-tion have been identified to be O-GlcNAc modified,27,91 suchas eNOS and protein kinase B (PKB/Akt). Even though it iswell established that O-GlcNAc is critical for cellular func-tion, very few studies have addressed the vascular effects ofO-GlcNAcylation. Recently, Lima et al showed that the aortaand mesentery of deoxycorticosterone acetate-salt hyperten-sive rats have augmented O-GlcNAc levels compared tocontrol.141 Such elevated O-GlcNAc signaling was associatedwith increased reactivity to constrictor stimuli, phenyleph-rine, and impaired endothelium-dependent vasodilatation toacetylcholine. Whether the change in O-GlcNAc signalingobserved with DOCA hypertension is true for all forms ofhypertension remains to be tested, though this same group142

has continued to extend their findings.Inflammation contributes to the pathogenesis of numerous

cardiovascular diseases. Even though our understanding ofO-GlcNAc signaling in most of cardiovascular pathophysiol-ogy remains limited, several recent studies indicate its poten-tial impact extends beyond the cardiomyocyte. Oparil’s grouptested the hypothesis that in vivo arterial injury may beaffected by alterations in O-GlcNAc signaling.99 Using ovari-ectomized rats, balloon injury of the carotid artery producedthe expected inflammation and vascular pathology in thismodel. However, treatment with either glucosamine (en-hances HBP flux) or PUGNAc (which inhibits O-GlcNAcaseand increases O-GlcNAc levels) reduced leukocyte infiltra-tion, inhibited tumor necrosis factor (TNF)-�–stimulated

chemokine and adhesion molecule (ICAM-1 and VCAM-1)expression, I�B-� phosphorylation and nuclear factor(NF)-�B activation. These data indicate antiinflammatoryeffects of augmented O-GlcNAc signaling in their model.Conversely, Tostes’ group has found a potential role forO-GlcNAcylation in the pathophysiology of hypertension,141–143

implying a complex interplay in the vasculature.Severe injury such as trauma-hemorrhagic shock has been

shown to alter O-GlcNAc signaling and that enhancedO-GlcNAc signaling improved functional recovery in theheart following hemorrhagic shock.96–98 In series of in vivostudies from Chatham’s group, glucosamine administrationduring resuscitation significantly attenuated hemorrhagicshock induced increase in circulating TNF-� and interleukin(IL)-6 levels, ICAM expression, I�B-� phosphorylation,NF-�B expression, and NF-�B DNA-binding activity in ratheart.97,98 The same group also confirmed this finding with anO-GlcNAcase inhibitor (PUGNAc). In addition, intravenousadministration of PUGNAc 30 minutes after the onset ofresuscitation following trauma-hemorrhage in rats showedattenuated circulating TNF-� and IL-6 levels supporting theprotective effect of O-GlcNAc signaling on stress-mediatedinflammation.96

Technical Limitations/SuggestionsSince its discovery more than 2 decades ago, several analyticprocedures have been evaluated to identify O-GlcNAc modifiedproteins.144 Because of its substoichiometric concentration, la-bile nature, lack of charge, and small mass, identification ofO-GlcNAc modification has been difficult. The initial tool usedenzymatic labeling of terminal O-GlcNAc residues with radio-active uridine diphospho-galactose (UDP[3H]Gal) using galac-tosyltransferase.8,145,146 Because O-linkage of GlcNAc to aprotein is resistant to peptide/N-glycosidase F (PNGase F),nonspecifically UDP[3H]Gal tagged N-linked oligosaccharidesare cleaved by PNGase F treatment. The main limitation of thistechnique is that O-GlcNAc is not very accessible to galactosyl-transferase, thereby limiting its utility in many instances.

Others have also used succinylated wheat germ agglutinin(sWGA)48,49,147–149 to identify O-GlcNAc–modified proteins.

Figure 3. Mitochondria and O-GlcNAc sig-naling. Myocardial ischemia induces mito-chondrial Ca2� overload and ROS generation,with subsequent mitochondrial permeabilitytransition pore formation (mPTP). Formationof mPTP causes loss of mitochondrialtrans–inner membrane potential, mitochon-drial swelling, rupture of mitochondrialmembrane, and cytochrome c release.Augmentation of O-GlcNAc levels beforemyocardial ischemia attenuates ischemia-induced Ca2� overload, ROS generation,and subsequent mPTP formation. Aug-mented O-GlcNAc signaling also mitigatesmPTP formation by possibly augmentingO-GlcNAcylation of VDAC and/or BCl-2interaction with VDAC. This is an exampleof one potential mechanism; there are likelyseveral. Moreover, augmented O-GlcNAclevels diminish mPTP-mediated mitochon-drial swelling, loss of mitochondrial mem-brane potential, and cytochrome c release.





sWGA binds to any terminal GlcNAc residue; hence thistechnique is not particularly selective. The specificity of thistechnique may be improved using PNGase F to removenonspecific N-linked modifications bound to sWGA. Anotherdisadvantage of this method is that sWGA is less sensitiveand only proteins with multiple O-GlcNAc residues arereadily detected. Sensitivity may be improved by usingsWGA-conjugated Sepharose column to isolate and enrichO-GlcNAc–modified proteins from cell extracts.

The development of monoclonal antibodies that react withO-GlcNAc in the context of protein structure has significantlyincreased the efficiency of identifying O-GlcNAc–modifiedproteins.150–154 CTD 110.6 was raised against an O-GlcNAc–modified peptide from the large subunit RNA polymerase IICTD,154 whereas RL2 antibody was raised against O-Glc-NAc–modified peptide from nuclear pore proteins.150–153

Despite their broad immunoreactivity to the O-GlcNAc mod-ification, they are somewhat restrictive in their target speci-ficity and may require more than one O-GlcNAc site, espe-cially for low molecular weight proteins. Recently, newantibodies have been described, which may improve targetidentification.155 To further confirm the specificity of anti-body based approaches (Figure 4), O-GlcNAc immunoblotsshould be incubated with exogenous GlcNAc and O-GlcNAcantibody. Moreover, it would also be useful to demonstrate aloss of signal in parallel aliquots of protein sample todemonstrate that the “O-GlcNAc immunoreactivity” is sen-sitive to O-GlcNAcase (think of using a phosphatase toconfirm a specific phosphorylation signal).

Several chemical approaches have also been developed toanalyze O-GlcNAc residues.58,146,156–162 Initial attempts at

O-GlcNAc site mapping were time consuming and compli-cated by numerous HPLC peptide purifications and manualEdman degradation reactions.145,156,157 Moreover, the lowstoichiometry of the O-GlcNAc modification required ahigher starting concentration of purified protein. Because ofthis, a combination of alkaline �-elimination, collision-induced dissociation (CID), and electrospray ionization massspectrometry have been used158 by some experts. Eventhough alkaline �-elimination reduced the CID energyneeded to ionize and fragment the peptide for sequencing byreducing the size of the glycopeptide and CID fragment whilepreserving O-GlcNAc modification, it causes significantpeptide degradation. Another more recent approach, mild�-elimination followed by Michael addition with dithiothre-itol (BEMAD),159 tags the �,�-unsaturated carbonyl (theproduct of �-elimination) with a nucleophilic tag stabilizingthe O-linkage during collision-induced dissociation. Taggingallowed for site identification by LC-MS/MS, makingBEMAD useful for mass spectrometry.159 Not only does theBEMAD method allow for simultaneous study of O-GlcNAcand O-phosphate quantitatively, it also allows the enrichmentof either posttranslational modification in the study of normalversus diseased states.

Recently, Bertozzi’s group160 reported that OGT andO-GlcNAcase could tolerate analogs of their natural sub-strates. OGT incorporated can incorporate an azide modifiedGlcNAc (GlcNAz) into protein targets. Once labeled, theseGlcNAz-modified proteins can be covalently derivatized withvarious biochemical probes at the site of protein glycosyla-tion using Staudinger ligation. This strategy could identifyO-GlcNAc–modified proteins, as well as map protein targetsites that bear O-GlcNAc modification. Because UDP-GlcNAc is incorporated into several classes of glycoconju-gates, specificity must be demonstrated with properly con-trolled experiments when cells are labeled metabolically.Similar, more specific and improved techniques, tagging-via-substrate161 and Click-chemistry162 have been described.These techniques are highly sensitive and especially usefulfor lower molecular weight proteins. Despite the developmentof such techniques, the lack of a recognizable consensus motifsomewhat complicates the analyses of O-GlcNAc function andlimits predictive capabilities. Currently, it appears that aminoacids modified by O-GlcNAc often are surrounded by serine/threonine residues, with a proline often 3 amino acids to theN-terminal side of the modification.17,163,164 However, there arenumerous exceptions to such guidelines. Should a clear consen-sus sequence be established, this field would experience evenmore growth than its present rate.

ConclusionsMost aspects of cardiovascular biology remain untapped forthe potential involvement of O-GlcNAc signaling. The cur-rent status of O-GlcNAc in cardiac biology represents anexciting time for discovery. Indeed, multiple phenomena willlikely be identified as regulated by O-GlcNAc signaling inthe immediate future. Moreover, our limited understanding ofdiabetes and its impact on metabolism in the cardiovascularsystem will remain important areas of investigation. Based on

Figure 4. Common control measures for O-GlcNAc immuno-blots. O-GlcNAc immunoblots show multiple bands because itis a posttranslational modification of numerous proteins. Theblot on the left is the result of a CTD110.6 antibody coincubatedwith N-acetylglucosamine, which competes for binding with theantibody. The blot on the right is the same membrane and pri-mary antibody (CTD110.6), without GlcNAc. In addition, thelysate loaded in lane 6 is the result of a parallel aliquot of lane 5incubated with O-GlcNAcase (in vitro), showing the loss of im-munopositivity and validating the signal is O-GlcNAc. Such sim-ple measures can confirm the fidelity of the O-GlcNAc signal viaWestern blots.





existing evidence, it appears that O-GlcNAc signaling partic-ipates in the pathophysiology of diabetes. Although signifi-cant advances in O-GlcNAc proteomics have occurred in thelast 5 years, a long road of technological innovation anddissemination remains.

For the uninitiated, many questions likely remain, whichmay relate to consensus sequence (not clearly identified), thepromoters for OGT/O-GlcNAcase (not reported), and the bio-physical impact of O-GlcNAcylation on proteins (no uniformanswer). The existing literature suggests that O-GlcNAcylationis a metabolic sensor, but this review emphasizes an emergingrole for O-GlcNAcylation as a robust stress response. Of course,such a possibility is not mutually exclusive with the current“metabolic sensor” function of O-GlcNAc. Although readers(and reviewers) want to know exactly what the targets ofO-GlcNAcylation are in each specific context, it is our opinionthat the absence of such information should not be the solebarrier to publication or funding. After all, we have collec-tively accepted targeted mutagenesis studies of Ser/Thr resi-dues to be definitive evidence of the importance of specifickinase targets. Were we inadvertently precluding the possi-bility of O-GlcNAcylation? Should we collectively reevalu-ate some of our conclusions regarding phosphorylation ofSer/Thr in this new context?

What is clear is that in addition to its purported functionsas a metabolic sensor, O-GlcNAcylation of proteins appar-ently can also reflect cellular stress in the cardiovascularsystem.

AcknowledgmentsWe thank the members of the Jones Laboratory for technical support.

Sources of FundingThis laboratory has been supported by grants from the NIH NationalHeart, Lung, and Blood Institute (R01 HL083320 and R01HL094419), American Heart Association National Center(0535270N), and NIH National Center for Research Resources (P20RR024489) and a Kentucky Science and Engineering Foundationgrant (KSEF-1677-RDE-011) (to S.P.J.). G.A.N. was an AmericanHeart Association Predoctoral Fellow (0715493B). H.T.F. is anAmerican Heart Association Postdoctoral Fellow (0825643D). A.Z.is supported by an American Recovery and Reinvestment ActSupplement from the NIH.

DisclosuresNone.

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O-GlcNAc Signaling in the Cardiovascular System