Exercise training has been well-documented as an efficient low-cost strategy in prevention and treatment of many chronic diseases and understanding of the mechanisms responsible for health-promoting effects of exercise has advanced markedly. Reactive oxygen species (ROS) produced in the contracting muscle are now known to play multiple beneficial roles in cellular development and function. Moreover, redox signaling intertwines with energy- and stress-sensing pathways modulating cellular metabolism and survival. Hexosamine biosynthesis pathway (HBP) has emerged as an important nutrient and stress sensor, providing a substrate for modification of proteins with O-linked -N-acetylglucosamine (O-GlcNAc). O-GlcNAc is a monosaccharide that binds to intracellular proteins and modifies their structure, function and metabolism. Dynamic cycling of the sugar unit on and off proteins is regulated by two highly conserved enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Over 4000 proteins have been identified so far to be O-GlcNAc-modified and the modification appears to be involved in almost every aspect of cellular function. Aberrant O-GlcNAc modification has been shown to play part in the etiology of several diseases, including type 2 diabetes, cardiovascular diseases, neurodegenerative disorders and cancer. Acute upregulation of the modification, however, exhibited cytoprotective effects and promoted cell survival during exposure to various stressors. O-GlcNAc-modified proteins are abundant in skeletal muscle, but O-GlcNAcylation has not been extensively studied in this tissue, especially in the context of exercise-induced oxidative stress. The aim of this PhD thesis was to investigate the effects of exercise on O-GlcNAc modification of skeletal muscle proteins using different pathophysiological animal models (atherosclerosis and cardiovascular kidney disease) and in combination with treatment to modify antioxidant defenses.
The first study examined the effects of exercise training on O-GlcNAcylation of skeletal muscle proteins in an animal model of atherosclerosis. Apolipoprotein E deficient mice (ApoE-/-) were subjected to 8 weeks of voluntary wheel exercise. The exercise intervention appeared to reduce the characteristics of atherosclerotic vessel morphology and promote macrophage plaque density, however, these effects were not significant. Exercise significantly increased protein O-GlcNAcylation, but no changes were detected in OGT protein expression or at the mRNA level of OGT and OGA enzymes. Moreover, mRNA expression of the two isoforms of HBP's rate-limiting enzyme, glutamine:fructose-6-phosphate amidotransferase (GFAT1 and GFAT2), was not affected by exercise. The findings of this study indicated an interrelationship between O-GlcNAc protein modification and exercise and warranted further investigation into the process of this modification in skeletal muscle.
Study Two investigated the effects of exercise on O-GlcNAc protein modification in a model of cardiovascular kidney disease. Impaired O-GlcNAc modification has been suggested to play a role in the etiology and progression of the disease. This study aimed to determine the effects of renal dysfunction and exercise training on O-GlcNAc modification of skeletal muscle proteins. ApoE-/- mice underwent subtotal nephrectomy or sham surgery and were subjected to 12 weeks of voluntary wheel exercise or no exercise. Nephrectomy was associated with a significant decrease in body weight and increased inflammation. Exercise attenuated atherosclerotic plaque size significantly and indicated a non-significant anti-inflammatory effect in the uremic mice. Nephrectomized mice exhibited significantly reduced O-GlcNAc and OGT protein levels in the soleus muscle. In addition, OGT protein expression in the nephrectomized animals was further suppressed with exercise. No effects of exercise training or nephrectomy on O-GlcNAc and OGT levels were detected in the white gastrocnemius muscle. This study demonstrated that renal dysfunction is associated with downregulated protein O-GlcNAcylation in skeletal muscle exposed to atherosclerotic conditions.
The effects of regular exercise and a single exercise bout on cellular metabolism and function are quite different, with exercise training leading to an improved endogenous defense system as part of the cellular adaptation process. Acute exercise can significantly increase ROS generation, resulting in oxidative stress. Study Three examined the effects of acute exercise and glutathione depletion on O-GlcNAc protein modification in skeletal muscle of healthy rats. Diethyl maleate (DEM) was used to deplete endogenous antioxidant glutathione and animals were subjected to a treadmill run to fatigue and were sacrificed immediately after exercise or after a four hour recovery. Exercise and DEM both reduced intracellular glutathione levels and promoted O-GlcNAcylation. Furthermore, DEM upregulated OGT protein expression. The effects of the two interventions were significant four hours after exercise. The changes in mRNA levels of OGT, OGA, GFAT1 and GFAT2 enzymes were different in the white gastrocnemius and soleus muscles, potentially resulting from different rates of oxidative stress and metabolic demands between the muscle types. These findings show that oxidative environment promotes O-GlcNAcylation in skeletal muscle. Moreover, the changes in O-GlcNAc may play a role in the oxidative stress response and health benefits of exercise.
The final study investigated the role of O-GlcNAc in modulating the response of skeletal muscle cells to oxidative stress. Study Four focused on antioxidant enzymes superoxide dismutase 2 (SOD2), catalase (CAT) and glutathione peroxidase 1 (GPX1) and transcriptional regulators of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1) and forkhead box protein O1 (FOXO1), which play important roles in oxidative stress response and are known to be O-GlcNAc-modified. C2C12 myoblasts were subjected to a 24-hour incubation with different reagents including hydrogen peroxide, DEM, high glucose and glucosamine and the inhibitors of O-GlcNAc cycling enzymes. Surprisingly, O-GlcNAc levels were significantly increased only with glutamine whilst other treatments showed no effect. Significant changes at the mRNA level were observed with concomitant upregulation of the genes for O-GlcNAc enzymes and stress-related proteins with oxidizing agents and downregulation of these genes with agents promoting O-GlcNAcylation. Our findings suggest O-GlcNAc participates in stimulation of the endogenous defense system in skeletal muscle cells. Future studies will continue to investigate the roles of O-GlcNAc signaling in the cellular adaptation to oxidative challenges. In summary, the findings of this thesis demonstrated an interaction between exercise and O-GlcNAc protein modification in skeletal muscle and different responses of this modification under various (patho)physiological conditions. O-GlcNAcylation has been shown to reflect cellular redox status and increase with exercise-induced oxidative stress. Importantly, our data indicate a significant role of O-GlcNAc in modulation of the endogenous antioxidant system. This could represent one of the mechanisms underlying cellular adaptation to oxidative stress and health-promoting effects of exercise, warranting further research into the process of O-GlcNAc cycling in skeletal muscle.