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REVIEW Role of O-GlcNAcylation in nutritional sensing, insulin resistance and in mediating the benefits of exercise Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by San Diego (UCSD) on 09/13/14 For personal use only.

Jason P. Myslicki, Darrell D. Belke, and Jane Shearer

Abstract: The purpose of this review is to highlight the role of O-linked ␤-N-acetylglucosamine (O-GlcNAc) protein modification in metabolic disease states and to summarize current knowledge of how exercise affects this important post-translational signalling pathway. O-GlcNAc modification is an intracellular tool capable of integrating energy supply with demand. The accumulation of excess energy associated with obesity and insulin resistance is mediated, in part, by the hexosamine biosynthetic pathway (HBP), which results in the O-GlcNAcylation of a myriad of proteins, thereby affecting their respective function, stability, and localization. Insulin resistance is related to the excessive O-GlcNAcylation of key metabolic proteins causing a chronic blunting of insulin signalling pathways and precipitating the accompanying pathologies, such as heart and kidney disease. Lifestyle modifications such as diet and exercise also modify the pathway. Exercise is a front-line and cost-effective therapeutic approach for insulin resistance, and recent work shows that the intervention can alter O-GlcNAc gene expression, signalling, and protein modification. However, there is currently no consensus on the effect of frequency, intensity, type, and duration of exercise on O-GlcNAc modification, the HBP, and its related enzymes. On one end of the spectrum, mild, prolonged swim training reduces O-GlcNAcylation, while on the other end, higher intensity treadmill running increases cardiac protein O-GlcNAc modification. Clearly, a balance between acute and chronic stress of exercise is needed to reap the benefits of the intervention on O-GlcNAc signalling. Key words: insulin resistance, signalling, exercise training, post-translational modification. Résumé : Cette analyse documentaire se propose de mettre en évidence le rôle de la modification de la protéine O-GlcNAc (␤-N-acétylglucosamine liée en O) en présence d’une maladie métabolique et de présenter brièvement l’état des connaissances au sujet de l’effet de l’exercice sur cette importante voie de signalisation post-traductionnelle. La modification d’O-GlcNAc constitue un moyen intracellulaire d’intégrer l’approvisionnement énergétique a` la demande. L’accumulation du surplus d’énergie associée a` l’obésité et a` l’insulinorésistance est médiée en partie par la voie de la biosynthèse de l’hexosamine (HBP) pour aboutir a` l’O-GlcNAcylation d’une kyrielle de protéines et en modifier leur fonction, stabilité et localisation respective. L’insulinorésistance est associée a` l’O-GlcNAcylation excessive de protéines métaboliques clés, ce qui cause l’émoussement chronique des voies de signalisation de l’insuline et les pathologies correspondantes du cœur et des reins, par exemple. Les modifications du mode de vie telles que le régime alimentaire et la pratique de l’activité physique ont aussi un effet sur la voie. L’exercice physique est une approche de première ligne et rentable contre l’insulinorésistance; d’après des travaux récents, l’exercice physique peut modifier l’expression du gène O-GlcNAc, la signalisation et la modification protéinique. Toutefois, il n’y a pas de consensus sur l’effet de la fréquence, de la durée, de l’intensité et de la nature de l’exercice physique sur la modification de l’O-GlcNAc, l’HBP et des enzymes connexes. À une extrémité du continuum, un léger entraînement prolongé a` la nage diminue l’O-GlcNAcylation; a` l’autre extrémité, la course d’intensité élevée sur un tapis roulant accroit la modification de la protéine cardiaque O-GlcNAc. De toute évidence, un juste équilibre entre une brève séance et un stress prolongé a` l’effort est requis pour recueillir les fruits de l’intervention sur la signalisation de l’O-GlcNAc. [Traduit par la Rédaction] Mots-clés : insulinorésistance, signalisation, entraînement physique, modification post-traductionnelle.

Introduction Nutritional sensing is vital to the survival of all organisms. Individuals must not only be able to sense the need for nutrients, but also the rate of energy utilization and feedback indicating the status of energy stores. This ability is particularly important during exercise and in situations where there exists an energetic imbalance, such as in metabolic disease states, including obesity, type 2 diabetes, and cardiovascular disease. While the body has many redundant mechanisms for nutrient sensing, an oftenoverlooked mechanism involves O-linked ␤-N-acetylglucosamine (O-GlcNAc) protein modification.

O-GlcNAcylation describes a biochemical process analogous to protein phosphorylation in which a single N-acetylglucosamine moiety is attached to cytosolic, mitochondrial, and nuclear proteins. The attachment can affect protein function, stability, and localization. This review highlights the general aspects of O-GlcNAcylation, provides an overview of current literature as it relates to this post-translational modification, and examines how it is altered in metabolic disease states. Following this overview, we discuss how increasing fuel usage through exercise may restore energy balance and aberrant O-GlcNAc signalling. Where appropriate, the impact of reducing fuel intake by dietary means is also addressed.

Received 7 April 2014. Accepted 21 July 2014. J.P. Myslicki and D.D. Belke. Faculty of Kinesiology, University of Calgary, Calgary, AB T2N 1N4, Canada. J. Shearer.* Faculty of Kinesiology, University of Calgary, Calgary, AB T2N 1N4, Canada; Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, Calgary, AB T2N 1N4, Canada. Corresponding author: Jane Shearer (e-mail: [email protected]). *All editorial decisions for this paper were made by John Thyfault and Terry Graham. Appl. Physiol. Nutr. Metab. 39: 1–9 (2014) dx.doi.org/10.1139/apnm-2014-0122

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What is intracellular protein O-GlcNAcylation?

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Fig. 1. Interplay between protein O-GlcNAcylation and phosphorylation. In some proteins, specific sites either can be O-GlcNAcated or phosphorylated (e.g Thr-58). Alternatively, a protein can be both O-GlcNAcated and phosphorylated on adjacent sites (e.g Thr-58 and Ser-68 in the above). These modifications can occur on the same protein at the same time and results in competitive inhibition or co-stimulation. In addition, the order of glycosylation and phosphorylation can be reversed. G, O-GlyNAcylation; OGT, O-linked GlcNAc transferase; OGA, N-acetylglucosaminidase; P, phosphorylation.

First discovered in 1984, protein O-GlcNAc modification is a specific type of protein glycosylation that is both abundant and dynamic, consisting of a single N-acetylglucosamine moiety attached by an O-␤-glycosidic linkage on serine and threonine residues of nuclear and cytosolic proteins (Fig. 1) (Torres and Hart 1984). Unlike classical protein glycosylation, which is characterized by stable and complex elongated oligosaccharide structures, O-GlcNAc is highly reversible (Hu et al. 2010), therefore lending itself to altering cellular processes such as signal transduction, protein turnover, protein–protein interactions, differential gene expression, and metabolism among others (Zachara and Hart 2004; Slawson et al. 2006; Zachara and Hart 2006). A schematic of the attachment, pathway, and the general effects of O-GlcNAc is shown in Fig. 2. O-GlcNAc acts on serine and threonine residues, which are also key sites of phosphorylation on many proteins. Given this, O-GlcNAc signalling is ubiquitous, appearing in all cellular compartments on a wide array of proteins. An initial O-GlcNAc database created by Wang et al. (2011) identified 404 O-GlcNAc sites on 172 different proteins and subsequently inferred, with protein modelling, that O-GlcNAcylation takes place at over 1139 different residues on over 1163 different proteins. However, new enrichment techniques and more advanced mass spectrometers are enabling researchers to identify less abundant proteins and as a result over 4000 O-GlcNAcylated proteins have been identified (Ma and Hart 2014). Advances in peptide enrichment techniques as opposed to the more traditional reliance on whole-protein enrichment have brought focus to the development of the lectin weak affinity chromatography technique (Vosseller et al. 2006; Chalkley et al. 2009) and enabled Trinidad et al. (2012) to distinguish 1750 unique O-GlcNAc sites, far out-distancing the initial mass spectrometry and protein modelling predictions of Wang et al. (2011). Unlike phosphorylation, which is controlled by hundreds of kinases and a handful of phosphatases, protein O-GlcNAcylation is regulated by only 2 reciprocal enzymes, O-linked ␤-N-acetylglucosamine (OGT), which catalyzes the addition of the sugar residue onto nucleocytoplasmic proteins, and O-GlcNAcase (OGA), which hydrolyzes the removal of the moiety (Figs. 1, 2). Both enzymes are encoded by single genes and are highly conserved among species (Hart et al. 2007). Initially called hexosaminidase C, OGA is inhibited by the common diabetes-inducing agent streptozotocin, which increases levels of cellular O-GlcNAc, with continued exposure resulting in pancreatic ␤-cell death (Konrad et al. 2001; Toleman et al. 2006). Similarly, OGT is also necessary for cell viability (Shafi et al. 2000). The OGT knockout mouse is embryonic lethal, while loss of OGT as a consequence of conditional knockout in cardiomyocytes in adult mice leads to the rapid development of heart failure (Watson et al. 2014). These studies demonstrate the need for a certain amount of O-GlcNAc for normal cellular function. In contrast, deletion of OGT in Caenorhabditis elegans results in profound insulin resistance, glycogen, and triglyceride accumulation, highlighting the metabolic role of this enzyme (Hanover et al. 2005). OGT itself is modified by O-GlcNAc and by tyrosine phosphorylation, suggesting it may be cooperatively regulated by kinase-mediated signal transduction (Kreppel et al. 1997).

competitively inhibits the phosphorylation of Tyr608, a key regulatory site (Whelan et al. 2010). This inhibition prevents phosphorylation and the activation of IRS-1, downregulating protein kinase B and glucose transporter-4 signalling. As a result, glucose uptake is blunted to protect the cell from further metabolic insult. In addition, some proteins can be concomitantly phosphorylated and O-GlcNAcylated, which is feasible because of a protein’s capacity to be simultaneously modified at each serine/threonine site within the same protein. Examples include glycogen synthase (Parker et al. 2003), IRS-1 (Ball et al. 2006), and cardiac myosin light chain (MLC) (Ramirez-Correa et al. 2008) (Fig. 1). O-GlcNAcylation and O-phosphorylation of myofibril proteins both impact cardiac function (Ramirez-Correa et al. 2008). However, this relationship is not well understood. In cardiomyocytes, MLC phosphorylation by MLC kinase increases the rate of cross-bridge formation and force production (Stelzer et al. 2006). In contrast, increased O-GlcNAc expression is associated with decreased cardiac contractile force, similar to the effects of ischemia or diabetes. So it seems that while phosphorylation and O-GlcNAcylation of MLC should have contradictory effects, and that their interaction is difficult to predict. This highlights the potential conflict between these 2 post-translational modifications at serine and (or) threonine residues. Finally, protein O-GlcNAcylation and phosphorylation share their ability to react to stimuli, such as the case with acute ischemic stress, which can increase both uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) and phosphorylation simultaneously (Fulop et al. 2007b).

O-GlcNAc reciprocity with protein phosphorylation

O-GlcNAcylation and nutritional sensing

Similar to phosphorylation, O-GlcNAc modification has emerged as a key regulatory mechanism in numerous molecular signalling pathways. Furthermore, a complex interplay exists between protein O-GlcNAc modification and phosphorylation (Fig. 1). The O-GlcNAc modification interacts with, and can effectively compete against, phosphorylated sites on proteins (Whelan and Hart 2003), with approximately two-thirds of potential sites being in conflict (Hu et al. 2010). For example, an elevated glucose level results in O-GlcNAc modification of insulin receptor substrate-1 (IRS-1) that

O-GlcNAc modification depends on circulating concentrations of substrates, principally glucose. Upon entering the cell, glucose is quickly converted to glucose-6-phosphate by hexokinase. From there, the vast majority of glucose is either stored as glycogen or undergoes isomerization to fructose 6-phosphate (F-6-P) by phosphoglucose isomerase, which enters into glycolysis. However, a small fraction of F-6-P (2%–5%) is diverted into the hexosamine biosynthetic pathway (HBP) (Fig. 2) (McClain and Crook 1996). The pathway gained fame in the early 1990s, when its activity was Published by NRC Research Press

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Fig. 2. The hexosamine biosysnthesis pathway. Flux through the pathway is dependent on incoming glucose (G-6-P), glycogen concentration, and glycolytic demands. Glucosamine is converted to glucosamine-6-phosphate (glucosamine-6-P) by the rate-limiting enzyme glutamine fructose-6phosphate amidotransferase (GFAT), the rate-limiting enzyme for the pathway. Uridine diphosphate N-acteylglucosamine (UDP-GlcNAc) is produced and subsequently acts as a substrate, which is added to serine or threonine residues of target proteins by O-linked GlcNAc transferase (OGT) and removed by N-acetylglucosaminidase (OGA). The resulting O-GlcNAc modification can affect numerous sites as indicated. GS, glycogen synthase; GP, glycogen phosphorylase; Ism, glucose-6-phosphate isomerase.

strongly linked to insulin resistance (Marshall et al. 1991). Despite this, the specific contributions of the HBP towards the initiation, development, and progression of insulin resistance remained unknown until a decade ago when it was realized that the pathway was key to transmitting signals linked to nutrient overabundance in numerous tissues (McClain 2002). In the HBP, F-6-P is converted to glucosamine-6-phosphate by the rate-limiting enzyme D-fructose-6-phosphate amidotransferase (GFAT), which controls flux through the HBP (Marshall et al. 1991). In the process, glutamine is converted to glutamate, highlighting the input from amino acid metabolism. Glucosamine-6-phosphate then combines with a sugar donor (uridine diphosphate, UDP) to produce UDP-GlcNAc, a high-energy substrate that is catalyzed by OGT and subsequently modifies proteins at serine and threonine residues (Fig. 2). One remarkable aspect linking the HBP with protein

O-GlcNAcylation is that the activity of OGT is increased with UDPGlcNAc concentration, and the enzyme shows no “saturation kinetics” (Kreppel and Hart 1999). That is to say, protein O-GlcNAcylation will increase without limit as the HBP becomes more active (no upper-limit to OGT activation). This makes protein O-GlcNAcylation a highly sensitive mechanism for sensing the nutritional state. Excessive O-GlcNAcylation has been linked to insulin resistance, and translocation of OGT to the plasma membrane is known to impede insulin signalling (Yang et al. 2008). Once translocated to the plasma membrane from the nucleus, OGT catalyzes the O-GlcNAcylation of the insulin signalling pathway, thereby competitively inhibiting the phosphorylation of key signalling molecules and subsequently reducing the insulin sensitivity of the cell (Yang et al. 2008). As previously mentioned, O-GlcNAc is nutrient-responsive and is influenced by nutritional intake (Hawkins et al. 1997; Gazdag et al. Published by NRC Research Press

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2000). However, this input extends beyond glucose, encompassing numerous aspects of cellular metabolism as the HBP sits at the nexus, receiving input from glucose, amino acid, fatty acid, and nucleotide metabolic pathways (Wells et al. 2003; Ngoh et al. 2010). For instance, high-fat diets increase liver and cardiac O-GlcNAcylation (Li et al. 2005). Acetyl-groups from fatty acids, glutamate, and highenergy phosphate-labelled nucleic acids (uridine triphosphate O requiring nucleic acid synthesis and energetic capacity) also influence the HBP (Fig. 2). Relevant to insulin resistance, Hawkins et al. (1997) assessed HBP flux under various euglycemic–hyperinsulinemic conditions in rats. When free fatty acids levels were maintained with intralipid infusion during the clamp, they noted that UDP-GlcNAc levels were doubled in skeletal muscle. Of interest, an infusion of either glucosamine or uridine alone also elevated UDP-GlcNAc and the subsequent impairment in insulin-stimulated glucose uptake demonstrated the sensitivity of the HBP pathway to multiple substrates (Hawkins et al. 1997).

Regulation by glucose homeostasis By virtue of the HBP pathway, O-GlcNAc is linked to circulating glucose levels (Fig. 2). Protein O-GlcNAcylation is thought to be protective in states of acute glucose flux O regulating uptake and metabolism thereby protecting against glucotoxicity. However, chronically elevated glucose levels result in persistent global protein O-GlcNAcylation that is thought to perpetuate metabolic abnormalities (Hart et al. 2011; Zachara et al. 2011; Ma and Hart 2013). Such is the case in type 2 diabetes where a profound, whole-body insulin resistance causes a recurrent elevation in circulating glucose levels. Perhaps the most studied tissue in the examination of the relationship between O-GlcNAc, metabolism and function is the heart. Prolonged consumption of a diet high in saturated fat and sugar, also known as a “western or cafeteria style diet”, increases protein O-GlcNAcylation in the heart (Medford et al. 2012). Similarly, elevated O-GlcNAc levels contribute to cardiac dysfunction (or diabetic cardiomyopathy) as seen with type 2 diabetes (Clark et al. 2003; Chess and Stanley 2008). It is estimated that 60% of individuals with type 2 diabetes have some form of cardiac dysfunction or diabetic cardiomyopathy (Poirier et al. 2001; Redfield et al. 2003). As a result, it is the number 1 complication of the disease with individuals being 4 to 6 times more likely to have heart disease or a stroke when compared with healthy controls (Taegtmeyer et al. 2002; Young et al. 2002a, 2002b; Diamant et al. 2003). Cardiac function and metabolism are tightly coupled with the beating heart continually matching energy production (ATP) to demand. As such, small metabolic improvements can result in functional enhancements. As a demonstration of this, Hu et al. (2005) administered an adenovirus expressing OGA into the hearts of diabetic mice. Upon increased OGA expression in the heart, there was a reduction in overall cellular O-GlcNAcylation, improved contractile function, and enhanced calcium handling (Hu et al. 2005). Taken together, these results show indisputable evidence of the O-GlcNAc signalling pathway in the aetiology of diabetic cardiomyopathy. The pathway is also involved in regulating cardiac hypertrophy, a compensatory response to mechanical stress placed on the heart. Increased protein O-GlcNAcylation in type 2 diabetes decreases the hypertrophic signalling response of cardiomyocytes in diabetic mice (Marsh et al. 2011). This blunted hypertrophic response would be expected to further stress an already dysfunctional system, leading to the development of heart failure. Pathologic conditions, such as obesity, diabetes, and insulin resistance are associated with excess energy levels that lead to increased HBP flux and ultimately manifest as aberrant O-GlcNAcylation, including the modification of transcription factors involved in gene expression. There is evidence that O-GlcNAc can alter tissue-specific gene expression by modifying transcription factors in response to nutritional status (Ozcan et al. 2010). Most transcription factors that are O-GlcNAc–modified also undergo other post-translational

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modifications, such as phosphorylation, acetylation, and sumoylation, thereby adding to the complexity of understanding the paradigm of altered gene expression (Ozcan et al. 2010). Ozcan et al. (2010) also suggest that transcription factors that feature multiple O-GlcNAcylation sites may require multiple modifications to modulate transcription factor function, as is the case with the zinc finger transcription factor, specificity protein 1 (Sp1), which further complicates determining the impact of O-GlcNAcylation on gene expression. For instance, the complexity of Sp1 O-GlcNAc modification is evident by its association with both stimulation (Goldberg et al. 2006; Chung et al. 2008) and inhibition (Kudlow 2006) in regulating cell growth (Li et al. 2004; Wierstra 2008). With respect to diabetes, it is known that increased O-GlcNAc modification of transcription factor Sp1 is detrimental to diabetic blood vessels (Du et al. 2000), and McClain et al. (1992) concluded that elevated O-GlcNAc signalling stimulates the transcription of growth factors in vascular smooth muscle cells, likely contributing to the vascular complications associated with diabetes. The O-GlcNAc modification of other transcription factors, such as pancreatic and duodenal homeobox 1, has been linked to decreased insulin production (Gao et al. 2003). It has been suggested that alterations in genetic programming may serve as a mechanism to enable the development of insulin resistance (Rossetti et al. 1995; Vosseller et al. 2002). Cellular glucose starvation or hypoglycemia, also leads to elevated global O-GlcNAc modification, generally through an increase in OGT and a decrease in OGA protein expression rather than the increase in UDP-GlcNAc levels as discussed previously (Taylor et al. 2008). Zou et al. (2012) suggest that activation of the Ca2+/calmodulin-dependent protein kinase II may be a mechanism for regulating aberrant cellular O-GlcNAc levels. This study demonstrated that increasing substrate energy (pyruvate) did not mediate O-GlcNAc levels in glucose-deprived cardiomyocytes, supporting the notion that an alternative mechanism is responsible for this stress-induced post-translational modification. An alternative mechanism to explain how both hyper- and hypoglycemia lead to protein O-GlcNAcylation is the unfolded protein response (UPR) proposed by Wang et al. (2014). Engagement of the UPR increases the activity of X-Box–binding protein 1, which is a direct promoter of HBP activity via increased genetic coding for GFAT. Wang et al. (2014) demonstrated that a wide array of stresses lead to UPR, including glucose deprivation. In addition, activation of the UPR is highly correlated with cellular O-GlcNAc modification and, in fact, endoplasmic reticulum (ER) stress is a trigger of this protein O-GlcNAcylation. In the case of glucose deprivation, the UPR and subsequent O-GlcNAc modification prove to be cardioprotective as cellular apoptosis is attenuated in the face of the detrimental effects of ER stress (Palorini et al. 2013).

O-GlcNAcylation during rest and exercise Exercise remains the simplest and most cost-effective intervention to moderate the impact of diabetes (Pan et al. 1997; Bassuk and Manson 2005; Snowling and Hopkins 2006; Firestone and Mold 2009). Although some of the effects of exercise on molecular signalling pathways are well established, we are just beginning to understand others, including the impact of post-translational protein O-GlcNAcylation. Exercise studies related to O-GlcNAc modification are few and far between with a lack of consensus on the impact of exercise on the post-translational modification of various tissues (Belke 2011; Cox and Marsh 2013; Johnsen et al. 2013; Medford et al. 2013). A summary of studies examining the relationship between O-GlcNAc modification and exercise is shown in Table 1. Extensive gaps in the field are evident given the inconsistencies of protocols with regards to the frequency, intensity, type, and time of exercise that result in changes in O-GlcNAc expression. Although exercise might be expected to increase flux through the Published by NRC Research Press

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Table 1. Summary of studies examining the impact of exercise on protein O-GlcNAcylation (O-GlcNAc). Study

Animal model

Exercise

Main findings

Nelson et al. 1997

Wistar rat

Swimming, acute 3–6 h

Belke 2011

Mouse CD-1

Swimming, 3 h per day for 6 wk

Medford et al. 2013

Mouse CD-1

Treadmill/acute 15 or 30 min at 17 m·min–1

Bennett et al. 2013

Mouse CD-1, STZ – type 1 diabetic

Swimming/3 h per day for 6 wk

Johnson et al. 2013

Rat, outbred SD

21 generations selected for high and low running capacity

Cox and Marsh 2013

db/db mouse, model of type 2 diabetes

Treadmill/10 m·min–1 for 40 min·d–1 for 1 or 4 wk

Skeletal muscle: No effect of exercise on GFAT activity, but UDP–hexosamine concentration is increased up to 16 h after exercise. Heart: GFAT2, OGT and OGA expression and O-GlcNAc levels are all decreased in swim-trained mice. Swim training leads to improved cardiac function and physiological hypertrophy. Heart: Decreased cytosolic O-GlcNAc levels after 15 min but not 30 min of exercise. No change in OGT expression; however, decreased OGT–REST interaction promotes physiological hypertrophy. Heart: Swimming leads to decreased protein O-GlcNAc levels despite persistent hyperglycemia. OGT expression not affected by exercise but OGA expression and activity is increased. Swim training leads to improved cardiac function. Heart: Mitochondrial Complex I and IV and VDAC and SERCA all show higher levels of protein O-GlcNAcylation in the low-capacity running group. There were no differences in OGT an OGA expression between groups. Heart: O-GlcNAc levels increased in db/db exercised group at 1 and 4 wk of exercise. There was no change in OGT or OGA expression after 1 wk but both are increased after 4 wk. Diabetes and exercise have opposing effects on cardiac hypertrophy.

Note: The study authors, year, animal model, type and duration of exercise are included along with the tissue studied and the main outcome. CD-1, mouse strain; db/db, the leptin receptor deficient mouse, model of type 2 diabetes; GFAT, rate-limiting enzyme glutamine fructose-6-phosphate amidotransferase; OGA, O-linked N-acetylglucosaminase; OGT, O-linked N-acetylglucosamine transferase; REST, repressor element 1 – silencing transcription factor; SERCA, sarco/endoplasmic reticulum Ca2+-ATPase; SD, Sprague–Dawley rat; VDAC, voltage-dependent anion channel; UDP, uridine diphosphate.

HBP, the specific effects of acute exercise and chronic exercise training on O-GlcNAc are not well characterized. Aerobic capacity and O-GlcNAcylation Emerging evidence shows that differences in intrinsic aerobic capacity play a critical role in the development of perturbed metabolism, chronic disease, morbidity and all-cause mortality. To date, 2 studies have examined the relationship of O-GlcNAc with aerobic capacity. Johnsen et al. (2013) investigated cardiac protein O-GlcNAc modification in rats that were artificially selected for low running capacity and high running capacity. High-capacity runners (HCR) exhibit higher aerobic capacity, resistance to environmental stress, improved sensitivity to insulin, and protection against oxidative damage (Barbato et al. 1998; Tweedie et al. 2011). In contrast, low-capacity runners (LCR) are hyperlipidemic, glucose intolerant, hypertensive, and exhibit elevated oxidative stress (Rivas et al. 2011). These differences have also been revealed by transcriptomic and proteomic analysis and continuously diverge with age (Groves-Chapman et al. 2011; Keller et al. 2011). Examination of cardiac muscle from these animals showed no difference between HCR and LCR groups with respect to global O-GlcNAc expression, OGT, or OGA. However, when individual proteins were examined, there was a significant difference between HCR and LCR groups in O-GlcNAc expression of selected mitochondrial proteins, including mitochondrial complex I, complex IV, voltage-dependent anion channel (VDAC), and sarcoendoplasmic reticulum (SR) calcium transport ATPase (SERCA) (Johnsen et al. 2013). Mitochondrial complex I and IV, as well as VDAC, are mitochondrial proteins that are actively involved in energy production and transport. Indirectly, the increased O-GlcNAc modification of several mitochondrial proteins supports the theory that the O-GlcNAc modification is linked to metabolism and perhaps the development of insulin resistance. SERCA is a key mechanism

to move calcium ions from the cytoplasm into the SR during muscle relaxation and therefore plays a very important role in muscle contraction. Both hyperglycemia and increased O-GlcNAc levels are associated with impaired calcium excitation–contraction (EC) rates (Davidoff and Ren 1997; Ren et al. 1997; Clark et al. 2003). Since Dutta et al. (2002) demonstrated that glucose-induced cardiomyopathy and impaired EC are not associated with changes in SERCA levels, Fulop et al. (2007a) speculated that O-GlcNAcylation of calcium-handling proteins may contribute to adult cardiomyocytes dysfunction. On the other hand, hyperglycemia-induced O-GlcNAcylation of neonatal cardiomyocytes reduced SERCA mRNA and protein expression (Clark et al. 2003). However, it is noteworthy that the impaired calcium cycling associated with O-GlcNAcylation can be mediated with adeno-viral overexpression of OGA (Clark et al. 2003; Hu et al. 2005). Myslicki et al.1 recently examined the relationship between aerobic capacity and O-GlcNAcylation in the whole blood of young males. Existing reports show that erythrocytes and leukocytes of healthy, prediabetic, and overtly diabetic individuals are increasingly O-GlcNAcylated with increasing severity of the disease (Wang et al. 2009; Springhorn et al. 2012). Studies have demonstrated that analysis of protein O-GlcNAc modification is more sensitive than hemoglobin A1c at threshold levels of prediabetes and diabetes onset (Park et al. 2010; Springhorn et al. 2012), thereby supporting its potential use as a screening tool for the earlier detection of metabolic disturbances. Examination of protein O-GlcNAcylation in relation to a panel of anthropometric and metabolic measures showed a strong relation to the homeostatic model of assessment for insulin resistance ([fasting glucose (mmol·L−1) × fasting insulin (pmol)]/22.5), but only a weak relation to aerobic capacity as measured by oxygen uptake. This suggests that circulating glucose and insulin directly regulate O-GlcNAc

J.P. Myslicki, J. Shearer, D.S. Hittel, C.C. Hughey, and D. Belke. O-GlcNAc modification in whole blood of healthy young adult males in relation to insulin sensitivity. Diabetology and Metabolic Syndrome, 1058330539126572. In press.

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levels, but that this does not necessarily relate to aerobic performance. O-GlcNAcylation in response to acute exercise A few groups have explored the impact of an acute exercise bout on O-GlcNAcylation. Medford et al. (2013) examined the effects of a single bout of exercise on protein O-GlcNAcylation of cardiac tissue. Nondiabetic CD1 mice either remained sedentary or were exposed to moderate or intense treadmill running for either 15 or 30 min at a rate of 17 m·min–1, which is a relatively high speed for an adult mouse. Cardiac muscle was examined for O-GlcNAc and its related enzymes. Pathologic cardiac hypertrophy is regulated by a complex that comprises repressor element 1 – silencing transcription factor (REST) and a corepressor mSin3A, which in conjunction recruit histone deacetylases (HDACs), change chromatin construction, and alter epigenetic expression, ultimately causing gene silencing of hypertrophic mediators (Kuwahara et al. 2001). Results demonstrate that exercise may benefit pathological hypertrophy of the heart as indicated by a reduction in O-GlcNAc, a change in intracellular protein O-GlcNAc distribution, and in the interaction with regulators of cardiac hypertrophy. The acute exercise intervention resulted in a decreased interaction between OGT and REST, but the interaction between OGT and mSin3A remained unchanged, suggesting that as little as 15 min of exercise leads to reduced intracellular cardiac O-GlcNAcylation and reduced interaction between OGT and REST (Medford et al. 2013). The O-GlcNAc modification of OGT at rest promotes pathological hypertrophy, but the removal of the O-GlcNAc modification with exercise leads to the initiation of physiological hypertrophic signalling (Medford et al. 2013). In contrast, 30 min of exercise resulted in no difference in total O-GlcNAc levels of cellular proteins. However, the authors did observe a shift in the O-GlcNAc levels of cytosolic and nuclear proteins with a decrease in cytosol O-GlcNAc modification, and an increase in low molecular weight nuclear protein O-GlcNAc modification as a result of short-term exercise. This effect was not due to changes in the expression of OGT or OGA, but rather attributed to alterations in UDP-GlcNAc substrate availability, an observation that conforms to other studies (Zachara et al. 2004; Taylor et al. 2008). In this study, the authors acknowledged the limitation of forced running at a relatively high speed, which surely stimulates psychological and physiological stresses in mice that can confound and potentially affect results (Brown et al. 2007). It is possible that O-GlcNAc modifications seen in this study were a result of distress because of lack of exercise acclimatization and not associated with the beneficial effects of exercise as often seen in chronic exercise regimes. Finally, Nelson et al. (1997) examined acute swimming and feeding studies in relation to HBP activity in the hind limb skeletal muscle of swim-trained Wistar rats. Although protein O-GlcNAcylation was not specifically examined in this study, GFAT activity in hind limb skeletal muscle was examined after feeding and up to 16 h after acute swim exercise. The specific activity of GFAT remained unchanged postexercise; however, the UDP–hexosamine concentration was increased as a result of feeding, leading the authors to conclude that the HBP in skeletal muscle is responsive to nutritional state but not acute exercise. In agreement, McClain and Crook (1996) hypothesized that products of the HBP may serve as “cellular satiety signals”, which may limit excessive glucose entry and storage postexercise. O-GlcNAcylation and chronic exercise Although acute exercise can lead to many biochemical and intracellular signalling pathway reactions, the beneficial effects of exercise are more evident with chronic exercise regimes. To date, few studies have examined the impact of chronic exercise training on O-GlcNAc modification. Exercise training comprising 6 weeks of swim training twice daily for 1.5 h has been shown to

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reduce global cardiac protein O-GlcNAc expression and to lead to physiological hypertrophy and improved cardiac contractile performance in CD-1 mice (Belke 2011; Bennett et al. 2013). Belke (2011) found that GFAT2 and OGT expression, as well as global protein O-GlcNAcylation, were all significantly decreased after a 6-week swim training intervention. Of note, the expression and activity of the enzyme responsible for the removal of O-GlcNAc, OGA, was also reduced, suggesting that exercise led to a coordinated downregulation of the entire system for regulating protein O-GlcNAcylation (GFAT, OGT, and OGA). Beyond general changes in protein O-GlcNAcylation, this study demonstrated a specific reduction in the O-GlcNAc levels of specificity protein 1 transcription factor (SP1) within cardiac muscle, showing that exercise can impact specific proteins as well. Modification of SP1 is important as this transcription factor directly influences the transcription of several genes that impact cardiac function and metabolism (Flesch 2001). Perhaps most significantly, these results are in contrast with pathological cardiac hypertrophy (heart failure), where a substantial increase in O-GlcNAc levels, GFAT, OGT, and OGA expression occurs, illustrating yet another opposing dichotomy between physiological and pathological hypertrophy. Bennett et al. (2013) exhibited similar findings, including improved cardiac contractile function, with the same 6-week swim exercise regime, but in a streptozotocin type 1 diabetic CD-1 mouse model. However, despite exercise training in that study, hyperglycemia persisted and OGT expression remained unchanged while OGA expression and activity was increased. This observation is similar to a previous study where cardiac performance in diabetic mice was improved, not by exercise, but through viral gene therapy involving the overexpression of OGA in the heart (Hu et al. 2005). Together, these studies demonstrate the potential for exercise to lower protein O-GlcNAc levels in heart tissue by modifying the capacity for the HBP-OGT-OGA signalling axis to affect protein O-GlcNAcylation. The studies presented above represent an intensive exercise protocol in healthy and type 1 diabetic mouse models, but how exercise might impact protein O-GlcNAcylation in a type 2 diabetic has only recently been examined by Cox and Marsh (2013). In this study they used an electric treadmill to exercise db/db mice for 1 and 4 weeks at a moderate (10 m·min–1) intensity. Despite exercise intervention, the db/db mouse exhibited an obese phenotype, in addition to cardiac hypertrophy and chronically elevated glucose levels as they inevitably developed overt diabetes, which is consistent with previous studies (Lee et al. 2011; Shearer et al. 2011). Interestingly, the mice demonstrated an increase in circulating blood glucose levels after 4 weeks of exercise. One week of exercise training increased protein O-GlcNAc levels in db/db mice; however, the regulator enzymes OGA and OGT were not different between groups. Upon completion of the 4-week exercise regime, the db/db exercise group global O-GlcNAc levels were greater than the sedentary group. Further stratification of the O-GlcNAc signal according to molecular weight revealed that the db/+ (control mice) and the db/db mice showed an increase in global and high molecular weight proteins O-GlcNAcylation over time, whereas the db/db model had an increase in low- and mid-level protein O-GlcNAcylation with the exercise intervention. The authors examined the interaction of OGT with nuclear transcription factors mSin3A, REST, and HDACs to understand the impact of exercise on factors known to influence gene expression related to cardiac hypertrophy. Although exercise did not impact mSin3a, HDAC1, or HDAC2 levels in the diabetic heart, the OGT:HDAC2 association was restored with exercise in the diabetic heart. Moderate exercise induced adaptations to the mSin3a/HDAC1/2, suggesting that this complex, a key regulator of cardiac hypertrophy, may be instrumental in mediating the beneficial effects of exercise for diabetic patients (Cox and Marsh 2013). Importantly, the author hypothesized that changes in O-GlcNAc in response to exercise Published by NRC Research Press

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Myslicki et al.

training may contribute to other physiological changes associated with exercise such as increased insulin sensitivity. Although research to date has only tackled the impact of exercise on cardiac O-GlcNAc modifications, it is expected that reductions in blood glucose levels associated with exercise (Boule et al. 2001) would also decrease HBP flux and subsequent skeletal muscle protein O-GlcNAcylation. Proteins such as SERCA and MLC O amongst many other proteins that are common in skeletal and cardiac muscle and have been shown to be O-GlcNAc-modified in the heart O warrant further investigation in skeletal muscle with respect to exercise intervention as improved calcium handling and rate of cross-bridge formation would optimize glucose utilization globally, thereby reducing insulin resistance. More research into the effect of both acute and chronic exercise is needed to elicit the exact impact of exercise intervention on insulin resistance related to diabetes and obesity, which are in themselves chronic stressors on the body. Extensive gaps remain in understanding the relationship between protein O-GlcNAc modification and other traditional nutrient sensing mechanisms, such as AMPAMPK (kinase) “fuel-gauge”, which links immediate energy needs with long-term glucose and fatty acid metabolism. Both nutrient sensing mechanisms are affected by metabolic disease and exercise, and both modify the energy/metabolism utilization of the cell through protein modification.

Conclusions Thus far, research into the relationship between exercise training and the adaptations of protein O-GlcNAc modification, including its related enzymes OGT, OGA, and GFAT, remains inconclusive. Chronic swim training reduced protein O-GlcNAcylation in healthy and type 1 diabetic mice (Ramirez-Correa et al. 2008; Belke 2011; Bennett et al. 2013), whereas chronic running treadmill exercise had the opposite effect in type 2 diabetic mice O it instead increased cardiac protein O-GlcNAc modification (Cox and Marsh 2013). Various combinations of changes in the enzymes that contribute to changes in O-GlcNAc expression, OGT, OGA, and GFAT have resulted from the different exercise prescriptions examined to date. Clearly, if OGT and O-GlcNAc levels are decreased with exercise and levels are increased under pathological conditions, then there exists a gradient from health to disease. When OGT and O-GlcNAc levels are increased in the disease state does this represent a “spill-over” of O-GlcNAc signalling to other targets, which would not normally be modified in healthy individuals? While the high level of protein O-GlcNAcylation often observed in various forms of heart disease may be considered as protective against further heart damage, does it also provide a barrier to the recovery of normal heart function? Questions such as these remain to be answered before the final verdict on protein O-GlcNAcylation in relation to health and disease can be reached, and remain a future challenge for researchers the field of nutrition and exercise. Conflict of interest statement The authors have no competing interests to declare.

Acknowledgements Supported by the National Science and Engineering Council of Canada. J.S. holds salary support awards from the Alberta Innovates Health Solutions.

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Role of O-GlcNAcylation in nutritional sensing, insulin resistance and in mediating the benefits of exercise.

The purpose of this review is to highlight the role of O-linked β-N-acetylglucosamine (O-GlcNAc) protein modification in metabolic disease states and ...
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