Secondary Logo

The Nuclear Receptor Nor-1 Is a Pleiotropic Regulator of Exercise-Induced Adaptations

Pearen, Michael, A.1; Muscat, George, E.O.2

Exercise and Sport Sciences Reviews: April 2018 - Volume 46 - Issue 2 - p 97–104
doi: 10.1249/JES.0000000000000143
Articles
Free

Exercise induces various physical and metabolic changes in skeletal muscle that adaptively reprograms this tissue to current physiological and environmental demands. Underlying these changes are broad modifications to gene expression. We postulate that the nuclear hormone receptor, Nor-1, is activated after exercise, and this transcription factor modifies gene expression to drive the molecular and cellular adaptations associated with contractile reorganization.

Nor-1 is an exercise-induced transcription factor that seems to regulate several of the physiological adaptations that occur after exercise.

1Hepatic Fibrosis Group, QIMR Berghofer Medical Research Institute, Royal Brisbane Hospital, Queensland; and 2Institute for Molecular Bioscience, The University of Queensland, Queensland, Australia

Address for correspondence: Michael Pearen, Ph.D., Hepatic Fibrosis Group, QIMR Berghofer Medical Research Institute, Locked Bag 2000, Royal Brisbane Hospital, Queensland 4029, Australia (E-mail: Michael.pearen@qimrberghofer.edu.au).

Accepted for publication: December 13, 2017.

Associate Editor: Karyn Esser, Ph.D.

Back to Top | Article Outline

Key Points

  • After exercise, physical and metabolic changes can occur in skeletal muscle for future adaptation including changes in muscle size, fiber type, blood supply, and energy source.
  • The expression of the nuclear receptor Nor-1 is increased after aerobic exercise, and this induction seems mediated by calcium ion signaling that occurs during exercise.
  • Transgenic overexpression of Nor-1 in skeletal muscle mimics the adaptations that occur in skeletal muscle after aerobic exercise including a shift to a more oxidative fiber type, hypertrophy, autophagy, increased vascularization, enhanced oxidative metabolism, and increased endurance/fatigue resistance.
  • Transgenic overexpression of Nor-1 in skeletal muscle also mimics the whole body metabolic effects of aerobic exercise such as decreased adiposity, resistance to diet-induced weight gain and improved glucose tolerance.
  • Nor-1 seems to regulate gene expression changes that drive the adaptation of skeletal muscle after aerobic exercise.
Back to Top | Article Outline

INTRODUCTION

Exercise can drive significant physical, functional, molecular, cellular, and metabolic changes within skeletal muscle to adapt to new physical demands and movements. The nature and extent of these changes depends on the type, duration, intensity, and frequency of exercise. The main physical changes that occur after exercise involve the reprogramming of the sarcomere in an attempt to adapt the contractile characteristics to the physical environment. These changes to the contractile apparatus and structures manifest as increased contractile protein mass and changes to skeletal muscle fiber type (associated with differential metabolic and oxidative capacity (1)). Supporting the changes to the contractile apparatus, exercise also initiates modifications to optimize the blood supply network (1) and metabolic modifications associated with changes to lipid and glucose metabolism to attempt to optimize energy homeostasis. Furthermore, beyond skeletal muscle, increased physical activity also has beneficial effects on the whole body such as resistance to obesity, increased insulin sensitivity/glucose tolerance, and preventative effects on cardiovascular and neurodegenerative diseases (reviewed in (3)).

To produce this broad scope of phenotypic changes that occur in response to exercise, skeletal muscle requires significant modifications in the fundamental expression of genes (2). The most direct way to specifically change diverse patterns of gene expression is with the differential expression of transcriptional factors and coregulators (i.e., coactivators and corepressors). Several transcription factors and coregulators are activated/induced in response to exercise [e.g., peroxisome proliferator–activated receptor gamma coactivator 1-alpha (PGC-1α (5)) and peroxisome proliferator-activated receptor alpha (PPARα (6)). However, it is likely that more transcriptional regulators are involved in exercise adaptations given the scope of adaptive responses, heterogeneity of skeletal muscle fiber types, response timing, and observations from rodent gain- and loss-of-function transgenic models.

In this context, the expression of the nuclear hormone receptor, Nor-1, has been shown to be induced by aerobic exercise in humans (2,3), rats (4), and in our work in mice (5). Our recent research on Nor-1 has implicated this transcription factor in the regulation of physical, molecular, cellular, and metabolic processes similar to the adaptations that occur after aerobic exercise/physical activity. This includes a shift toward a more oxidative fiber type, increased endurance (6), myofiber hypertrophy, autophagy (5), increased mitochondrial density (6), increased vascularization (5), enhanced glucose tolerance (6), and resistance to diet-induced weight gain (7).

Back to Top | Article Outline

Novel Hypothesis

We propose that the expression (and activity) of Nor-1 is induced after exercise, and this drives changes in gene expression that mediate the adaptations that occur in skeletal muscle after exercise (this hypothesis is summarized in Fig. 1). This raises the possibility that pharmacological activation of Nor-1 could be used therapeutically to induce (or amplify) the beneficial effects of physical activity.

Figure 1

Figure 1

Back to Top | Article Outline

THE NUCLEAR HORMONE RECEPTOR NOR-1

Nuclear hormone receptors (NRs) function as ligand-dependent transcription factors that regulate the functional expression of key genes involved in growth, metabolism, reproduction, development, morphogenesis, differentiation, and general homeostasis in an organ-specific manner (8). Many NRs are designated as orphans in that they belong to the NR superfamily according to structural homology and characteristics. However, to date, the endogenous native ligands for these receptors have not been identified or do not exist. Many NRs (including orphans) are abundantly expressed in skeletal muscle and regulate various aspects of skeletal muscle physiology, function, and metabolism. NRs seem to play a central role in the adaptation of skeletal muscle to exercise with several NRs such as PPARα (9), PPARδ (2), and the NR coactivator PGC-1α (10), implicated in exercise adaptation, metabolic capacity, and improved exercise capacity and fatigue resistance.

The hypothesis of this article focuses on the NR Nor-1 (neuron-derived orphan receptor 1, NR4A3; also known as MINOR, TEC, and CHN), which is an orphan NR found in skeletal muscle and other tissues/organs, including neural tissue/central nervous system (CNS) (11). Nor-1 is part of the NR4A subgroup of NRs along with two closely related orphan NRs: neuron-derived clone 77 (Nur77/NR4A1) and nuclear receptorrelated 1 (Nurr1/NR4A2), which are also expressed in skeletal muscle (2,12). Although the hypothesis of this review focuses on Nor-1, Nur77 also will be discussed at some points because there seems to be functional overlap between these two receptors in skeletal muscle. The function of Nurr1 has not yet been examined in skeletal muscle.

All three members of the NR4A subgroup lack identified endogenous ligands, and these receptors seem to be constitutively active. Regulation of Nor-1 and the NR subgroup seems largely to be associated with increased expression of these receptors in response to physiological and environmental stimuli (11). The expression of Nor-1 and the NR4A subgroup is induced by a wide range of diverse chemical and physical stimuli including fatty acids, physical stress, calcium ion signaling, hormones, growth factors, and neurotransmitters (reviewed in ref. (13)). In most of these examples, stimuli that induces one member of the NR4A subgroup also seem to induce the expression of the other two members in a similar spatiotemporal manner, and this seems to be dependent on the transcription factor cAMP response element binding protein (CREB) driving the common transcriptional induction of all three NR4A members (13).

In our early work on Nor-1, we discovered that siRNA-mediated knockdown of Nor-1 expression in an in vitro skeletal muscle cell culture model attenuated oxidative metabolism (14) and genes involved in fatty acid metabolism and increased lactate production (15). After these observations, we decided to investigate the skeletal muscle–specific function of Nor-1 in vivo.

Back to Top | Article Outline

Nor-1 Expression Is Induced by Exercise and Exercise Signaling

Multiple processes are induced after exercise that initiates downstream signaling to drive adaptations to exercise. Initially, the contraction of skeletal muscle that occurs during exercise is ultimately controlled by neuromuscular motor neuron signaling from the CNS. Furthermore, during exercise, there also does seem to be increased sympathetic nerve activity in skeletal muscle (16). The motor neuron–mediated neuromuscular signaling results in transient surges of calcium ions released by the sarcoplasmic reticulums in each myofiber. These transient surges of calcium ions are central to exercise signaling because not only do they initiate muscle contraction but also activate a range of downstream cell signaling pathways. Downstream mediators of calcium ion signaling that are known to be activated during/after exercise and seem to mediate exercise adaptations include the following: calmodulin, calcineurin, Ca2+/calmodulin-dependent protein kinase (CaMk), and p38 mitogen-activated protein kinase (p38 MAPK; reviewed in ref. (19)). Furthermore, other mechanisms seem to induce exercise adaptation signaling including physical changes caused by movement, stretch, myofiber damage, reactive oxygen production, hypoxia, myokines, and changes in energy metabolite levels such as the NAD/NADH ratio that could activate AMPK (17). Ultimately, exercise-dependent adaptations are mediated by changes in gene expression. Exercise regulates gene expression in multiple ways including modifications to transcription factors (e.g., CREB; 5), transcriptional coregulators (e.g., PGC-1α; 5), histone acetylation (18), and gene promotor epigenetics (e.g., demethylation; 22).

In the context of gene expression, our early studies on the transcription factor Nor-1 reported that Nor-1 mRNA expression in skeletal muscle was highly induced greater than 100-fold in vivo and greater than 30-fold in vitro by β-adrenoreceptor agonists (14,15). This induction of Nor-1 expression after (pan) β-adrenoreceptor stimulation seemed to be mediated by a cAMP-dependent mechanism involving protein kinase A (PKA), p38 MAPK, and phosphorylation/activation of CREB (14). In the context of exercise signaling, the sympathetic nervous system (19), the p38 MAPK pathway, and CREB (10) are known to be activated from/after exercise. In particular, activation of the p38 MAPK pathway and CREB seem to mediate the expression of Pgc-1α mRNA (10). This provided evidence that Nor-1 expression is likely to be induced by exercise on the basis of common upstream pathways that are associated with exercise signaling.

We have recently shown that Nor-1 expression is significantly induced at 30–240 min after a single (40 min) bout of rigorous treadmill aerobic exercise in wild-type mice (5). The induction of Nor-1 mRNA expression has been previously shown in microarray analysis of human skeletal muscle after endurance exercise (2) with a second study revealing a 26-fold increase in Nor-1 expression after exercise in humans (3). This induction also has been observed in rats (4), highlighting the conserved nature of this response in different mammalian species.

Furthermore, microarray-mediated expression profiling analysis of human skeletal muscle also showed increased expression of the other two members of the NR4A subgroup, Nur77 and Nurr1, after endurance exercise (2). Interestingly, in our mouse exercise study, the induction in Nor-1 expression after exercise did not seem to be initiated by β-adrenoreceptor signaling because the induction of Nor-1 expression was not attenuated to any extent by pretreatment with the pan β-adrenoreceptor antagonist/inhibitor, propranolol (5). Because of this, we are uncertain about the physiological role of the induction of Nor-1 by β-adrenoreceptor signaling in the context of aerobic physical activity.

Because β-adrenoreceptor signaling was not responsible for the exercise-induced expression of Nor-1 (5), the induction of Nor-1 expression was examined in the context of calcium ion signaling. The induction of Nor-1 could be blocked by pretreatment of the mice with cyclosporine A before exercise, suggesting that the signaling was mediated by calcineurin and calcium ion signaling (5). Further insights into this process were gained in vitro with the observation that Nor-1 is induced by calcium ion signaling, and this is mediated by p38 and possibility PKC in cultured myotubes. Finally, the phosphorylation (activation of CREB was shown to occur at 30 min after exercise, at the start of increasing Nor-1 expression (5)). This process suggests that the calcium ion signaling during exercise activates a pathway mediated by calcineurin, p38, and CREB, which transactivates Nor-1 expression after exercise (shown in Fig. 2). Similarly, Nur77 expression in myotubes also has been shown to be increased by the activation of calcium ion signaling in vitro (20). However, the induction of Nur77 expression by exercise has not been examined in detail. There also is conflicting evidence that AMPK (which is activated by exercise) is involved in the induction of Nur77 expression (4,20).

Figure 2

Figure 2

Interestingly, skeletal muscle contraction also has been reported to induce demethylation at the Nor-1 promotor to induce transcription of Nor-1 (3), suggesting a secondary mechanism that participates in the induction of Nor-1 expression after exercise. It is possible that this epigenetic process is syngeneic with the transactivation of Nor-1 expression by CREB.

Back to Top | Article Outline

NOR-1 OVEREXPRESSION MIMICS THE BIOLOGICAL ADAPTATIONS TO EXERCISE

After our in vitro studies on Nor-1, which showed that knockdown of Nor-1 expression in skeletal muscle cells caused changes to metabolic gene expression and function (14,15), we decided to explore the skeletal muscle-specific role of Nor-1 in vivo. Partial deletion of Nor-1 had previously been reported as embryonically lethal (21). Therefore, we created a gain-of-function transgenic mouse line with preferential and selective expression of activated Nor-1 in skeletal muscle using the human skeletal muscle α-actin promoter to drive the tissue-specific expression of Nor-1 (6). This promotor produced highly preferential transgene expression in skeletal muscle with transgene mRNA levels more than 25-fold greater in skeletal muscle compared with heart tissue (6). To ensure that Nor-1 was in a physiologically activated state, the small activation domain of the Herpes simplex viral protein 16 (VP16) was fused to the N-terminus of full-length Nor-1 cDNA.

Although the Nor-1 transgenic mouse line did have significant changes in metabolic gene expression and function as expected (6,7), the mouse line also exhibited a phenotype associated with adaptations caused by aerobic exercise (5–7). This section compares the phenotype of the Nor-1 transgenic mouse line to the adaptations that happen after aerobic exercise.

Back to Top | Article Outline

Skeletal Muscle Fiber Type and Exercise Performance

Skeletal muscle consists of a heterogeneous mixture of different skeletal muscle fiber types that have variable functional and metabolic characteristics suited to different types of exercise activity. The different fiber types form a functional continuum in the order of type I, type IIa, type IIx/d, and type IIb. At the type I end of the fiber type continuum, these fibers are suited to low-strength, long-duration usage (endurance exercise) and are fueled predominately by oxidative metabolism. At the opposite end, the type IIb fibers are suited to short-duration, high-force activity (strength exercise) and are predominately fueled by glycolytic anaerobic metabolism. Type IIa and type IIx/d fibers have intermediate kinetic and metabolic properties with a fuel preference for glycolytic oxidative metabolism. Exercise can remodel existing myofibers to adapt the distribution of fiber types to the exercise environment. In this context, aerobic exercise/endurance training seems to shift skeletal muscle toward type I/IIa fiber types (22), and conversely, high-intensity, short-duration resistance exercise drives the formation of type IIa and IIx/d in humans (23) and IIb fibers in rodents (24).

During initial analysis of the Nor-1 mouse model, we observed that the skeletal muscle was unusually red in color (6). This color observation implied a change in skeletal muscle fiber type, and we consequently undertook a comprehensive examination and analysis of fiber type and myosin heavy chain isoform expression. From this, it was clear that elevated Nor-1 expression in skeletal muscle had shifted the predominate fiber type in mice (glycolytic type IIb) toward more oxidative fiber types (IIx/IIa; 10). This was the case in all skeletal muscle types examined except in the soleus, which is an atypical highly aerobic muscle needed for posture and has a predominately type I fiber type in mice. In the soleus, there was a shift from type I to type IIx/IIa fibers, highlighting that Nor-1 is driving the acquisition of type IIa/IIx fibers in both directions (6). The endurance and fatigue-resistant performance of the Nor-1 mouse line was assessed to investigate whether the fiber type transformation and contractile reprogramming influenced exercise performance. In treadmill performance experiments, the Nor-1 transgenic mouse model could run more than twice the distance of wild-type litter mates and displayed a reduction in maximal running speed concordant with a functional oxidative fiber shift favoring endurance over speed (and power; (10)). Given that a shift toward oxidative fiber types is a characteristic of adaptation to aerobic exercise in humans (22) and mice (10), the Nor-1 transgenic mice were examined for the possibility of increased in-cage exercising using measurements of infrared beam obstruction. This showed no difference between Nor-1 transgenic mice and wild-type mice (7), highlighting that the Nor-1 transgenic mouse line had fiber type changes consistent with aerobic exercise without being exercised. Supporting this connection between Nor-1 expression and aerobic/endurance exercise is that Nor-1 protein expression was significantly higher in rats selectively bred for high aerobic running performance compared with rats selected for low aerobic running performance (25).

When the related NR4A nuclear receptor Nur77 was overexpressed in mouse skeletal muscle, a similar oxidative fiber type shift is observed, with the fiber type distribution moving from type IIb to type IIx (26). Supporting this, the inverse of this shift (IIx to IIb) happens in the skeletal muscle–specific Nur77 knockout (27). Interestingly, Nur77 (unlike Nor-1) seems to have no effect on type IIa fibers (26). This suggests that the NR4A subgroup differentially regulates the mechanisms that selectively control the diversity of skeletal muscle fiber type.

Back to Top | Article Outline

Nor-1, Sarcomeric Binding Proteins and Exercise Performance

One key change observed in the Nor-1 transgenic mouse line was that differential expression was observed in several critical genes encoding sarcomeric and calcineurin-binding proteins that regulate fatigue resistance. We identified that the expression of α-actinin-3 (Actn3) was decreased in the skeletal muscle of this mouse line. In skeletal muscle, α-actinins are important constituents of the Z-disc that help anchor the myofibrillar actin filaments. In humans, approximately 18% of whites are deficient in α-actinin-3 because of a homozygous null R577X polymorphism (28). Interestingly, the frequency of this homozygous null polymorphism is significantly enriched in endurance athletes, suggesting a functional association between α-actinin-3 deficiency and endurance driving the phenotypic enrichment (28). Supporting this association, α-actinin-3 knockout mice display increased endurance and enhanced activity of oxidative mitochondrial enzymes (29). This phenotype resembles and is consistent with the reduction of α-actinin-3 in the Nor-1 transgenic mouse line (5).

Transgenic Nor-1 expression also induced the expression of Striated muscle activator of Rho signaling (STARS). STARS is a Z-disc actin-binding protein with upregulated expression after exercise that mediates downstream transcription factors such as serum response factor (30). Overexpression of STARS in skeletal muscle cells in vitro shifts these cells to a more oxidative phenotype (31), and this is consistent with STARS being a transcriptional target gene of Nor-1.

One key function of the Z-disc of the sarcomere is calcineurin signaling and the regulation of contractile characteristics. Associated with calcineurin are the calsarcin proteins. In the Nor-1 transgenic, increased expression was observed in the mRNAs encoding Calsarcin-1 (Myoz2) and Calsarcin-3 (Myoz3), which may play a role in calcineurin signaling and therefore myofiber remodeling (32).

Back to Top | Article Outline

Skeletal Muscle Hypertrophy

Not only can exercise modify the composition and therefore function of the muscle sarcomere, but exercise also can lead to fiber hypertrophy that drives increased contractile force output. In particular, hypertrophy occurs from high-intensity, short-duration resistance exercise where there is demand for increased contractile force (33). However, limited hypertrophy has been observed from aerobic exercise in humans (34).

In our skeletal muscle–specific Nor-1 overexpression mouse line, there was a shift from type IIb myofibers toward more oxidative fiber types (6). Given that these type IIx/IIa skeletal myofibers should have a significantly smaller cross-sectional area compared with IIb (35), it would be expected that from the Nor-1 skeletal muscle fiber phenotype, there would be decreases in muscle size given the proportionally increased abundance of IIx/IIa skeletal myofibers. However, when examined, the Nor-1 transgenic mice actually displayed a small but significant increase in skeletal muscle mass (5). This suggested that skeletal muscle fiber hypertrophy was occurring despite the fiber type shift toward what should be smaller myofibers. The myofiber hypertrophy was confirmed by examining skeletal muscle myofiber cross-sectional area, which revealed a significant increase in the abundance of fibers with larger cross-sectional areas (5). Although hypertrophy is a classical adaptation to resistance/anaerobic exercise (33), aerobic endurance exercise seems to produce some skeletal muscle hypertrophy in humans (reviewed in (37)), and therefore, this phenotype is likely consistent with the phenotypic adaptation to either resistance or aerobic exercise.

In terms of the molecular mechanisms underlying skeletal muscle hypertrophy, the expression of myostatin and Mothers against decapentaplegic homolog 3 (Smad3) were both reduced in the Nor-1 overexpression mouse line (5). Both myostatin and SMAD3 are negative regulators of skeletal muscle mass, and therefore, this is consistent with the observed hypertrophy (36). Supporting this, a significant decrease in the expression of Muscle RING-finger protein-1 (MuRF1) was observed, suggesting that a reduction in contractile protein degradation via the ubiquitin-proteasome system may occur (5). Interestingly, loss of MuRF1 alone does not seem to produce hypertrophy in global knockout mice; however, MuRF1-deficient mice were protected against skeletal muscle atrophy (37). This possibly suggests that increased protein synthesis is required in coordination with decrease protein degradation for skeletal muscle hypertrophy. However, there did not seem to be an activation of mTOR-mediated protein synthesis in the Nor-1 overexpression mouse line, with a slight decrease in the ratio of phosphorylated mTOR (Ser2448) relative to total mTOR expression, which is indicative of reduced mTOR activity (5).

Supporting the relation between Nor-1 and hypertrophy is the observation that overexpression of Nor-1 upregulates small muscle protein X-linked (SMPX) in human muscle cells (38). SMPX is upregulated by stretch in skeletal muscle and has been suggested as a mechanical regulator of hypertrophy based on downstream gene expression, although the functional role is currently unclear.

Skeletal muscle hypertrophy also has been shown to develop from Nur77 overexpression in skeletal muscle, whereas conversely, reduced muscle mass occurs in Nur77-deficient mice (26). Again, this shows the overlapping function of the NR4A subgroup in relation to skeletal muscle hypertrophy.

Back to Top | Article Outline

Skeletal Muscle Autophagy

In response to exercise, skeletal muscle needs to remove damaged components. Furthermore, the process of myofiber remodeling (in terms of fiber type and hypertrophy) that occurs after exercise also requires the removal of existing contractile proteins with subsequent protein synthesis to modify the type and abundance of contractile proteins. Exercise is one key factor that controls the balance between contractile protein degradation/synthesis. In this context, autophagy is an intracellular degradation (and recycling) mechanism that is activated by exercise (39). Furthermore, activation of autophagy after exercise may mediate the improvements in glucose tolerance that occurs from exercise because defective autophagy seems to disrupt this exercise-induced adaptation (39).

The Nor-1 transgenic mouse line displayed several protein markers of active autophagy including increased abundance of structural proteins involved in autophagy [microtubule-associated protein 1 light chain 3A (LC3A), autophagy protein 5 and 12 (ATG5/12)] and decreased p62 expression indicative of increased autophagolysosome assembly (5). This was supported by in vitro studies in myotubes showing that autophagolysosome formation was significantly increased by ectopic Nor-1 expression (5). Other mechanisms involved in contractile protein homeostasis including the ubiquitin-proteasome degradation system and the rate of protein synthesis have not been examined in the Nor-1 transgenic mouse line.

Back to Top | Article Outline

Enhanced Skeletal Muscle Vascularization

During exercise, skeletal muscle requires significant blood supply for gas exchange to supply metabolic substrates and to remove waste. In particular, oxygen availability forms a significant bottleneck during exercise. Exercise can induce several adaptive mechanisms to increase the availability of oxygen to skeletal myofibers including 1) enhancing the capillary network (1), 2) increasing the expression of the oxygen storage molecule myoglobin (40), and 3) increasing cardiac output.

In the context of the Nor-1 overexpression model, we observed significant increases in the capillary network surrounding the myofibers (5), which is consistent with the adaptations that occur after aerobic exercise (1) and the observed oxidative shift in fiber type. In the skeletal muscle, we also observed statistically significant increases in the expression of several genes implicated in neovascularization including Epas1, Pecam1, Col4a3, Flt1, and Itgav (5)

In relation to oxygen storage, myoglobin expression was increased in skeletal muscle from the Nor-1 overexpression model, and this also was evident from the deep red color of the skeletal muscle (6).

Back to Top | Article Outline

Mitochondrial Biogenesis

Aerobic muscle movements require a large turnover to ATP to supply contractions. To meet this requirement, skeletal muscle adapted for aerobic activity contains a large number of mitochondria. As would be expected, increased mitochondrial biogenesis is a key feature of the skeletal muscle adaptation to aerobic exercise (40).

In the skeletal muscle of the Nor-1 transgenic mouse line, we observed increases in mitochondrial: number, genome DNA, proteins, and gene expression (6). This included increases in genes encoding the subunits of the electron transport chain complexes and increased levels of mitochondrial enzyme activity assessed via skeletal muscle section staining (e.g., NADH/SDH). Also observed was increased expression of the mitochondrial fusion protein mitofusin-2 (Mfn2).

A similar increase in mitochondria number and function of isolated mitochondria has been observed from the skeletal muscle–specific overexpression of Nur77 (41), highlighting a key functional overlap between the two NR4A receptors and the association of these two receptors with exercise-induced mitochondrial biogenesis.

Back to Top | Article Outline

Changes in Skeletal Muscle Metabolism and Gene Expression

Exercise also modifies skeletal muscle metabolism at the myofiber level to balance the exercise environment with energy substrates and storage. Aerobic exercise tends to push skeletal muscle toward oxidative metabolism (22), whereas high-intensity, short-duration anaerobic exercise tends to push skeletal muscle toward glycolic metabolism (23). These changes also correlate with changes in fiber type and fiber type distribution. In aerobic metabolism, energy is predominately supplied by the oxidation of fatty acids and carbohydrates, whereas anaerobic metabolism is fueled by the anaerobic glycolysis of carbohydrates into lactate. The reason for this is that during high-intensity anaerobic exercise, only glycolysis can create ATP fast enough to supply the contractile proteins compared with the slower but highly efficient oxidative phosphorylation. In this context, aerobic exercise induces genes and pathways involved in oxidative energy metabolism (2).

Similar to the adaptation to aerobic exercise, expression analysis of the Nor-1 transgenic line revealed that Nor-1 overexpression in skeletal muscle causes increased expression of multiple enzymes involved in glycolysis, the tricarboxylic acid cycle, oxidative phosphorylation, fatty acid oxidation, and glycogen synthesis (7). This included an induction of the genes involved in the malate-aspartate shuttle and a decrease in the glycerol 3-phosphate shuttle. The transition in gene expression is associated with maximal oxidative phosphorylation and aerobic ATP production. Of interest, endurance training has been shown to increase the levels of malate-aspartate shuttle enzymes in human skeletal muscle (42).

These comprehensive changes to gene expression suggest metabolic optimization for the aerobic oxidation of both fatty acids and carbohydrates, which is consistent with the metabolic nature of the observed type IIa/IIx fiber type shift. Similar (although often different at the gene level) changes have been observed in Nur77 mouse models including Nur77 transgenic expression muscle specifically favoring fatty acid oxidation over glucose oxidation (41).

Although gene and protein expression suggested a fiber type shift toward a more oxidative fiber type distribution in the Nor-1 overexpression mouse line, these mice also displayed significantly higher levels of skeletal muscle glycogen relative to wild-type litter mates (7). This was unexpected given that glycogen storage is associated with glycolytic fiber types. However, because endurance training also seems to induce glycogen storage capacity (43), this result is consistent with the effects of aerobic exercise on glycolytic energy storage. Enhanced glycogen storage also is apparent with Nur77 overexpression in myotubes in vitro (20) and in vivo with transgenic (44) and electrotransfer-mediated overexpression (20). Both Nor-1 and Nur77 seem to be mediating enhanced glycogen storage capacity by directly upregulating glycogen synthase (Gys1) and downregulating the glycogen synthesis inhibitor protein phosphatase 1 regulatory subunit 1A (Ppp1r1a) (7,41,44).

Back to Top | Article Outline

Beneficial Effects on Body Metabolic Function

Skeletal muscle, on average, accounts for approximately 40% of total body mass and approximately 30% of the resting metabolic rate (45). Because of the proportionally large size, skeletal muscle has a large influence over the health of the whole body. In this context, exercise is associated with health effects across the entire body including beneficial effects on cardiovascular, metabolic, bone, mental, and neurological health (reviewed in (3)). Exercise is associated with positive effects on a broad range of metabolic parameters including glucose tolerance/insulin sensitivity, cholesterol/lipoprotein levels, metabolic rate, and resistance to obesity (reviewed in (3)). The majority of these beneficial whole-body alterations that are associated with exercise are thought to originate from metabolic changes occurring within the skeletal muscle and from myokines, which are endocrine factors secreted from skeletal muscle.

In our Nor-1 transgenic skeletal muscle overexpression mouse model, we observed a range of positive metabolic effects outside skeletal muscle. The most obvious change was the significantly decreased body adiposity relative to wild-type littermates, with significant reductions in all adipose depots examined (7). The transgenic animals also were more resistant to weight gain when placed on a high-fat diet. Because food input and exercise levels remained similar between transgenic and wild-type groups, this suggested that Nor-1 transgenic mice have a higher metabolic rate. Supporting this, we observed increased oxygen consumption (6) and carbon dioxide production, which is consistent with a higher metabolic rate (7). Looking at glucose tolerance, the Nor-1 transgenic mouse line also displayed enhanced glucose tolerance and increased protein levels of the insulin-stimulated glucose transporter GLUT4 (6). Therefore, the whole-body metabolic phenotype of our Nor-1 transgenic mouse line clearly mimics some of the metabolic adaptations that occur after aerobic exercise.

Interestingly, transgenic overexpression of the related receptor Nur77 in skeletal muscle did not seem to reduce adiposity or modify glucose tolerance (41). However, conversely, Nur77 knockout mice displayed increased sensitivity to diet-induced obesity and insulin resistance (46).

Back to Top | Article Outline

SUMMARY

We hypothesized that the NR transcription factor Nor-1 is activated after exercise, and that this activation modifies gene expression to drive the adaptation of skeletal muscle to exercise (summarized in Fig. 2). Using a reductionist approach, the hypothesis is well supported. Nor-1 expression is increased after exercise, and this seems to be initiated by calcium ion signaling, which is the fundamental driver of skeletal muscle contraction. Supporting this, calcium ions signaling increases Nor-1 expression in vitro, and this process seems to be mediated by the calcium ion sensor calcineurin followed by p38 MAPK/CREB to transactivate Nor-1 expression. Activated CREB is a well-characterized exercised-induced transcription factor that drives expression of Nor-1 along with Pgc-1α, and Nur77 (5,10,13). This suggests that CREB is coordinating the expression of several exercise-induced transcriptional regulators that seem to drive exercise-induced gene expression (see Fig. 2). The increased expression of Nor-1 would lead to increased transcription of Nor-1 target genes, and the downstream effect of this has been demonstrated in the Nor-1 overexpression mouse model. In this mouse model, downstream gene expression changes from Nor-1 produce a broad range of physiological and metabolic changes that are similar to the adaptations that occur in skeletal muscle after exercise. This includes: a switch toward a more oxidative myofiber distribution, increased running endurance, fiber hypertrophy, increased autophagy, enhanced glycogen storage, increased vascularization, mitochondrial biogenesis, and increased glycolytic/oxidative gene expression. Furthermore, the Nor-1 overexpression mouse model exhibits whole-body characteristics of exercise including reduced adiposity and increased glucose tolerance.

More research is needed to confirm our overall hypothesis that Nor-1 is driving the adaptation of skeletal muscle in response to exercise. Examining the adaptive response of a skeletal muscle tissue-specific Nor-1 knockout mouse line to exercise could answer that question. However, one thing to consider is that loss of Nor-1 expression/function can be compensated for by some functional redundancy with Nur77, which could possibly mask any phenotype. Such compensatory increases have been previously noted within the NR4A subgroup (47), and therefore, the overlapping and unique functions of Nor-1 and Nur77 will need to be elucidated. Furthermore, given that one key molecule in exercise signaling, Pgc-1α, is an NR coactivator (10), it is possible that Pgc-1α is a coactivator of Nor-1 or Nur77, although this has not been examined.

If the hypothesis is correct, and Nor-1 is partly responsible for broadly regulating exercise adaptations, then this raises the exciting possibility that activating Nor-1 function could be used to mimic the health benefits of exercise for the treatment of metabolic diseases such as obesity/type II diabetes or skeletal muscle atrophy. Currently, there are no identified endogenous ligands for Nor-1 (or other members of the NR4A subgroup). However, several synthetic/supraphysiological agonists for Nor-1 and Nur77 have been identified (reviewed in (13)). This demonstrates that the NR4A subgroup has excellent potential for the discovery of pharmacological activators.

Back to Top | Article Outline

Acknowledgment

This study was supported by an NHMRC project grant.

George Muscat is a Principal Research Fellow of the National Health and Medical Research Council of Australia (NHMRC).

Back to Top | Article Outline

References

1. Brodal P, Ingjer F, Hermansen L. Capillary supply of skeletal muscle fibers in untrained and endurance-trained men. Am. J. Physiol. 1977; 232(6):H705–12.
2. Mahoney DJ, Parise G, Melov S, Safdar A, Tarnopolsky MA. Analysis of global mRNA expression in human skeletal muscle during recovery from endurance exercise. FASEB J. 2005; 19(11):1498–500.
3. Pattamaprapanont P, Garde C, Fabre O, Barres R. Muscle contraction induces acute hydroxymethylation of the exercise-responsive gene Nr4a3. Front. Endocrinol. (Lausanne). 2016; 7:165.
4. Kawasaki E, Hokari F, Sasaki M, Sakai A, Koshinaka K, Kawanaka K. Role of local muscle contractile activity in the exercise-induced increase in NR4A receptor mRNA expression. J. Appl. Physiol. 2009; 106(6):1826–31.
5. Goode JM, Pearen MA, Tuong ZK, et al. The nuclear receptor, Nor-1, induces the physiological responses associated with exercise. Mol. Endocrinol. 2016; 30(6):660–76.
6. Pearen MA, Eriksson NA, Fitzsimmons RL, et al. The nuclear receptor, Nor-1, markedly increases type II oxidative muscle fibers and resistance to fatigue. Mol. Endocrinol. 2012; 26(3):372–84.
7. Pearen MA, Goode JM, Fitzsimmons RL, et al. Transgenic muscle-specific Nor-1 expression regulates multiple pathways that effect adiposity, metabolism, and endurance. Mol. Endocrinol. 2013; 27(11):1897–917.
8. Chawla A, Repa JJ, Evans RM, Mangelsdorf DJ. Nuclear receptors and lipid physiology: opening the X-files. Science. 2001; 294(5548):1866–70.
9. Horowitz JF, Leone TC, Feng W, Kelly DP, Klein S. Effect of endurance training on lipid metabolism in women: a potential role for PPARalpha in the metabolic response to training. Am. J. Physiol. Endocrinol. Metab. 2000; 279(2):E348–55.
10. Akimoto T, Pohnert SC, Li P, et al. Exercise stimulates Pgc-1alpha transcription in skeletal muscle through activation of the p38 MAPK pathway. J. Biol. Chem. 2005; 280(20):19587–93.
11. Pearen MA, Muscat GE. Minireview: nuclear hormone receptor 4A signaling: implications for metabolic disease. Mol. Endocrinol. 2010; 24(10):1891–903.
12. Pearen MA, Ryall JG, Lynch GS, Muscat GE. Expression profiling of skeletal muscle following acute and chronic beta2-adrenergic stimulation: implications for hypertrophy, metabolism and circadian rhythm. BMC Genomics. 2009; 10:448.
13. Darragh J, Soloaga A, Beardmore VA, et al. MSKs are required for the transcription of the nuclear orphan receptors Nur77, Nurr1 and Nor1 downstream of MAPK signaling. Biochem. J. 2005; 390(Pt 3):749–59.
14. Pearen MA, Myers SA, Raichur S, Ryall JG, Lynch GS, Muscat GE. The orphan nuclear receptor, NOR-1, a target of beta-adrenergic signaling, regulates gene expression that controls oxidative metabolism in skeletal muscle. Endocrinology. 2008; 149(6):2853–65.
15. Pearen MA, Ryall JG, Maxwell MA, Ohkura N, Lynch GS, Muscat GE. The orphan nuclear receptor, NOR-1, is a target of beta-adrenergic signaling in skeletal muscle. Endocrinology. 2006; 147(11):5217–27.
16. Seals DR, Victor RG. Regulation of muscle sympathetic nerve activity during exercise in humans. Exerc. Sport Sci. Rev. 1991; 19:313–49.
17. Bassel-Duby R, Olson EN. Signaling pathways in skeletal muscle remodeling. Annu. Rev. Biochem. 2006; 75:19–37.
18. McGee SL, Fairlie E, Garnham AP, Hargreaves M. Exercise-induced histone modifications in human skeletal muscle. J. Physiol. 2009; 587(Pt 24):5951–8.
19. Christensen NJ, Galbo H. Sympathetic nervous activity during exercise. Annu. Rev. Physiol. 1983; 45:139–53.
20. Kanzleiter T, Wilks D, Preston E, Ye J, Frangioudakis G, Cooney GJ. Regulation of the nuclear hormone receptor nur77 in muscle: influence of exercise-activated pathways in vitro and obesity in vivo. Biochim. Biophys. Acta. 2009; 1792(8):777–82.
21. DeYoung RA, Baker JC, Cado D, Winoto A. The orphan steroid receptor Nur77 family member Nor-1 is essential for early mouse embryogenesis. J. Biol. Chem. 2003; 278(47):47104–9.
22. Ricoy JR, Encinas AR, Cabello A, Madero S, Arenas J. Histochemical study of the vastus lateralis muscle fibre types of athletes. J. Physiol. Biochem. 1998; 54(1):41–7.
23. Jansson E, Esbjornsson M, Holm I, Jacobs I. Increase in the proportion of fast-twitch muscle fibres by sprint training in males. Acta. Physiol. Scand. 1990; 140(3):359–63.
24. Yarasheski KE, Lemon PW, Gilloteaux J. Effect of heavy-resistance exercise training on muscle fiber composition in young rats. J. Appl. Physiol. 1990; 69(2):434–7.
25. Stephenson EJ, Stepto NK, Koch LG, Britton SL, Hawley JA. Divergent skeletal muscle respiratory capacities in rats artificially selected for high and low running ability: a role for Nor1? J. Appl. Physiol. 2012; 113(9):1403–12.
26. Tontonoz P, Cortez-Toledo O, Wroblewski K, et al. The orphan nuclear receptor Nur77 is a determinant of myofiber size and muscle mass in mice. Mol. Cell. Biol. 2015; 35(7):1125–38.
27. Cortez-Toledo O, Schnair C, Sangngern P, Metzger D, Chao LC. Nur77 deletion impairs muscle growth during developmental myogenesis and muscle regeneration in mice. PLoS One. 2017; 12(2):e0171268.
28. Yang N, MacArthur DG, Gulbin JP, et al. ACTN3 genotype is associated with human elite athletic performance. Am. J. Hum. Genet. 2003; 73(3):627–31.
29. MacArthur DG, Seto JT, Chan S, et al. An Actn3 knockout mouse provides mechanistic insights into the association between alpha-actinin-3 deficiency and human athletic performance. Hum. Mol. Genet. 2008; 17(8):1076–86.
30. Wallace MA, Hock MB, Hazen BC, Kralli A, Snow RJ, Russell AP. Striated muscle activator of Rho signaling (STARS) is a PGC-1α/oestrogen-related receptor-α target gene and is upregulated in human skeletal muscle after endurance exercise. J. Physiol. 2011; 589(Pt 8):2027–39.
31. Wallace MA, Russell AP. Striated muscle activator of Rho signaling is required for myotube survival but does not influence basal protein synthesis or degradation. Am. J. Physiol. Cell. Physiol. 2013; 305(4):C414–26.
32. Seto JT, Quinlan KG, Lek M, et al. ACTN3 genotype influences muscle performance through the regulation of calcineurin signaling. J. Clin. Invest. 2013; 123(10):4255–63.
33. McCall GE, Byrnes WC, Dickinson A, Pattany PM, Fleck SJ. Muscle fiber hypertrophy, hyperplasia, and capillary density in college men after resistance training. J. Appl. Physiol. 1996; 81(5):2004–12.
34. Konopka AR, Harber MP. Skeletal muscle hypertrophy after aerobic exercise training. Exerc. Sport. Sci. Rev. 2014; 42(2):53–61.
35. Delp MD, Duan C. Composition and size of type I, IIA, IID/X, and IIB fibers and citrate synthase activity of rat muscle. J. Appl. Physiol. 1996; 80(1):261–70.
36. Sartori R, Milan G, Patron M, et al. Smad2 and 3 transcription factors control muscle mass in adulthood. Am. J. Physiol. Cell. Physiol. 2009; 296(6):C1248–57.
37. Bodine SC, Latres E, Baumhueter S, et al. Identification of ubiquitin ligases required for skeletal muscle atrophy. Science. 2001; 294(5547):1704–8.
38. Ferran B, Marti-Pamies I, Alonso J, et al. The nuclear receptor NOR-1 regulates the small muscle protein, X-linked (SMPX) and myotube differentiation. Sci. Rep. 2016; 6:25944.
39. He C, Bassik MC, Moresi V, et al. Exercise-induced BCL2-regulated autophagy is required for muscle glucose homeostasis. Nature. 2012; 481(7382):511–5.
40. Holloszy JO. Biochemical adaptations to exercise: aerobic metabolism. Exerc. Sport Sci. Rev. 1973; 1:45–71.
41. Chao LC, Wroblewski K, Ilkayeva OR, et al. Skeletal muscle Nur77 expression enhances oxidative metabolism and substrate utilization. J. Lipid Res. 2012; 53(12):2610–9.
42. Schantz PG, Sjoberg B, Svedenhag J. Malate-aspartate and alpha-glycerophosphate shuttle enzyme levels in human skeletal muscle: methodological considerations and effect of endurance training. Acta. Physiol. Scand. 1986; 128(3):397–407.
43. Greiwe JS, Hickner RC, Hansen PA, Racette SB, Chen MM, Holloszy JO. Effects of endurance exercise training on muscle glycogen accumulation in humans. J. Appl. Physiol. 1999; 87(1):222–6.
44. Chao LC, Zhang Z, Pei L, Saito T, Tontonoz P, Pilch PF. Nur77 coordinately regulates expression of genes linked to glucose metabolism in skeletal muscle. Mol. Endocrinol. 2007; 21(9):2152–63.
45. Zurlo F, Larson K, Bogardus C, Ravussin E. Skeletal muscle metabolism is a major determinant of resting energy expenditure. J. Clin. Invest. 1990; 86(5):1423–7.
46. Chao LC, Wroblewski K, Zhang Z, et al. Insulin resistance and altered systemic glucose metabolism in mice lacking Nur77. Diabetes. 2009; 58(12):2788–96.
47. Kanzleiter T, Schneider T, Walter I, et al. Evidence for Nr4a1 as a cold-induced effector of brown fat thermogenesis. Physiol. Genomics. 2005; 24(1):37–44.
48. Ruegsegger GN, Booth FW. Health benefits of exercise. Cold Spring Harb. Perspect. Med. 2017. doi: 10.1101/cshperspect.a029694.
    49. Hawley JA, Hargreaves M, Zierath JR. Signaling mechanisms in skeletal muscle: role in substrate selection and muscle adaptation. Essays Biochem. 2006; 42:1–12.
    50. Barres R, Yan J, Egan B, et al. Acute exercise remodels promoter methylation in human skeletal muscle. Cell Metab. 2012; 15(3):405–11.
    Keywords:

    exercise; Nor-1; NR4A3; NR4A; skeletal muscle; hypertrophy; fiber type

    © 2018 American College of Sports Medicine