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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.
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 (
). 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 (
) 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 (
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 (
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 (
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 (
Glycosylation of nuclear and cytoplasmic proteins: purification and characterization of a uridine diphospho-N-acetylglucosamine:polypeptide β-N-acetylglucosaminyltransferase.
Dynamic glycosylation of nuclear and cytosolic proteins: cloning and characterization of a unique O-GlcNAc transferase with multiple 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 (
Dynamic O-glycosylation of nuclear and cytosolic proteins: cloning and characterization of a neutral, cytosolic β-N-acetylglucosaminidase from human brain.
). 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 (
). 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 (
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 (
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 (
). 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 (
Enrichment and site mapping of O-linked N-acetylglucosamine by a combination of chemical/enzymatic tagging, photochemical cleavage, and electron transfer dissociation 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 (
Enrichment and site mapping of O-linked N-acetylglucosamine by a combination of chemical/enzymatic tagging, photochemical cleavage, and electron transfer dissociation mass spectrometry.
). 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 (
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) (
Dynamic glycosylation of nuclear and cytosolic proteins: cloning and characterization of a unique O-GlcNAc transferase with multiple 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 (
) 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 (
). Hyperglycemia also inhibits vascular endothelial nitric-oxide synthase (eNOS) by O-GlcNAcylation, blocking its key regulatory AKT phosphorylation site (
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 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.
O-GlcNAcylation of a large subset of proteins increases rapidly in response to almost any type of cellular stress, and this increased O-GlcNAcylation protects cells from cellular damage (
Glucosamine cardioprotection in perfused rat hearts associated with increased O-linked N-acetylglucosamine protein modification and altered p38 activation.
Am. J. Physiol. Heart Circ. Physiol.2007; 292 (17208994): H2227-H2236
Hexosamine biosynthetic pathway flux and cardiomyopathy in type 2 diabetes mellitus: focus on “Impact of type 2 diabetes and aging on cardiomyocyte function and O-linked N-acetylglucosamine levels in the heart”.
Am. J. Physiol. Cell Physiol.2007; 292 (17151141): C1243-C1244
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.
). Nearly all RNA polymerase II transcription factors are O-GlcNAcylated, often at multiple sites, and the functions of the modification depend not only on the specific transcription factor but also on the specific sites to which the sugar is attached (
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.
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.
Protein O-N-acetylglucosaminylation modulates promoter activities of cyclic AMP response element and activator protein 1 and enhances E-selectin expression on HuH-7 human hepatoma cells.
O-GlcNAc modification of the runt-related transcription factor 2 (Runx2) links osteogenesis and nutrient metabolism in bone marrow mesenchymal stem cells.
Modification of histones by sugar β-N-acetylglucosamine (GlcNAc) occurs on multiple residues, including histone H3 serine 10, and is cell cycle-regulated.
). 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 (
). 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 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.
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 (
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 (
). 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 (
). 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 (
). 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 (
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 (
). 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 (
). 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 (
). 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 (
). 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 (
). 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 (
). 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 (
Alternative O-glycosylation/O-phosphorylation of serine-16 in murine estrogen receptor β: post-translational regulation of turnover and transactivation activity.
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.
Glycosylation sites flank phosphorylation sites on synapsin I: O-linked N-acetylglucosamine residues are localized within domains mediating synapsin I interactions.
Phosphorylation and glycosylation interplay: protein modifications at hydroxy amino acids and prediction of signaling functions of the human β3 integrin family.
Alternative O-GlcNAcylation/O-phosphorylation of Ser16 induce different conformational disturbances to the N terminus of murine estrogen receptor beta.
). 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 (
). Glycomic/proteomic analyses have shown that in terms of cross-talk between phosphorylation and O-GlcNAcylation at the site level, all possibilities exist (
). 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 (
). 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 (
). 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 (
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 (
). 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 (
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 (
). 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 (
). In Arabidopsis, O-GlcNAcylation of DELLA transcription factors, which are master growth repressors in plants, regulates and coordinates multiple signaling pathways during development (
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 (
). 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 (
). 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 (
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 (
). 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) (
). 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 (
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.
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 (
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 (
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 (
). 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 (
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 (
). 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 (
Increased flux through the hexosamine biosynthetic pathway also appears to be involved in glucose toxicity and insulin resistance in humans with diabetes (
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 (
). 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 (
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 (
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.
). 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 (
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 (
), 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 (
Insulin-dependent activation of endothelial nitric oxide synthase is impaired by O-linked glycosylation modification of signaling proteins in human coronary endothelial cells.
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 (
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