Biochemistry Primer for Exercise Science

Compared with endurance exercise, resistance exercise consists of much higher intensity contractions (e.g., usually 70% to 80% of one repetition maximum) repeated at high frequencies (referring again to motoneuron firing frequency) that can only be sustained for short durations due to the fatiguing nature of the exercise. Therefore, the pattern of cytoplasmic [Ca2+] oscillations and changes in [AMP]/[ATP] and ROS production with skeletal muscle contractions is different between endurance and resistance exercise. For example, endurance exercise likely results in extended periods of moderately elevated [Ca2+], while resistance exercise would generate short cycles of significantly higher intracellular [Ca2+] in skeletal muscle (Chin 2010). Therefore, differences in the magnitude and pattern of these primary signals generated by endurance and resistance exercise will result in the activation of different signaling pathways and different gene-expression and protein-synthesis responses in skeletal muscle. A number of studies have shown that resistance exercise results in increased rates of muscle-protein synthesis, about two- to fivefold after exercise for periods up to 48 h before declining to baseline values. Although acute resistance and endurance-type exercise result in a similar global anabolic response in untrained skeletal muscle, increases in muscle mass (i.e., hypertrophy) and strength are significantly greater following chronic resistance training compared with chronic endurance training. This means that chronic resistance training, but not endurance training, increases the rate of muscle-protein synthesis to levels above the rate of protein degradation.

Activation and differentiation of muscle satellite cells into new muscle cells that fuse with existing muscle fibers can also contribute significantly to the hypertrophic response to resistance exercise. Muscle fibers are large multinucleated cells. They are also postmitotic cells in that they no longer have the ability to divide and reproduce themselves. Additionally, in order to significantly increase in size or hypertrophy, muscles have to add more nuclei, since a nucleus is only able to supply mRNA for new protein synthesis to a limited amount of cytoplasm in its vicinity. Hence, a specific ratio of nuclei to cytoplasm must be maintained. In order to do this, skeletal muscles have small specialized satellite cells,or myogenic precursor cells, located at the periphery of their outer membranes. When stimulated by resistance training or by exercise-induced muscle damage, these specialized cells will be activated and induced to create daughter cells or proliferate. These new satellite cells will then fuse with the existing muscle fibers and add their nuclei to the cells to support greater protein synthesis and increase muscle hypertrophy or repair.

It is well known that the primary factor determining the hypertrophic response to contractile activity is the load across the muscle or the mechanical stretch/strain imposed on the muscle fibers, which is higher in resistance exercise than in endurance exercise. Ultimately, these mechanical/force signals are transduced by signaling pathways that regulate transcriptional and translational processes and satellite-cell activation.

For several years, researchers have focused on the role of insulin-like growth factor (IGF)-1 as a primary signaling molecule that mediates skeletal-muscle growth in response to resistance exercise, given that resistance exercise stimulates the secretion of IGF-1. IGF-1 is known to induce muscle hypertrophy by binding to its receptor on the muscle-cell surface and activating the classical growth factor pathway (see figure 3.27). IGF-1 binding to the receptor activates phosphatidylinositol 3-kinase (PI3K), which leads to the activation of Akt, a serine/threonine protein kinase. Akt phosphorylates and inactivates tuberosclerosis complex (TSC2), resulting in the activation of the ras homologous protein enriched in brain (Rheb) and mammalian target of rapamycin (mTOR). mTOR phosphorylates and suppresses the eukaryotic initiation factor 4E binding protein (4E-BP1) to blunt 4E-BP1 inhibition of translation-initiation cap-binding protein eIF4E (see figure 3.21). mTOR also phosphorylates the 70KDa ribosomal S6 protein kinase (p70S6K1), resulting in an increase in protein synthesis.

It is well accepted that mTOR signaling plays a dominant role in the adaptive response to resistance training; however, researchers have questioned the importance of IGF-1 signaling in mediating this response, based on evidence from several studies employing pharmacological and knockout-mouse approaches to systematically manipulate the IGF-1-PI3K-Akt pathway (Philp, Hamilton, and Baar 2011). It appears that the IGF-1 signaling pathway is not required for mTOR activation or increased protein synthesis that is induced by resistance-type exercise. It is possible that mechanical signals working through stretch-activated membrane channels, for example, might be able to activate mTOR and its downstream targets. However, this hypothesis needs to be examined experimentally (Philp, Hamilton, and Baar 2011).

Cellular Damage From RONS

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are sometimes collectively referred to as reactive oxygen and nitrogen species (RONS). RONS, and particularly the highly reactive hydroxyl radical, can react with many molecules in cells, including DNA, proteins, and lipids. The slow buildup of threats to mitochondrial and nuclear DNA over years of oxidative damage is thought to be a factor in aging. The relatively rapid turnover (breakdown and resynthesis) of proteins is also partly due to their susceptibility to oxidative damage and their need to ensure structural integrity to maintain optimal function. The free radical theory of aging was first proposed in the 1950s by the American scientist Denham Harman. It has continued to receive scientific support over the years, and it is now well established as an integral component of the aging process. Polyunsaturated fatty acids, which are important components of the phospholipids found in cell membranes, are particularly susceptible to damage by RONS. It has been known for more than 200 years that exposure of fats to air or oxygen will turn them rancid. This rancidity is caused by peroxidation of the fatty acids by RONS. During peroxidation, RONS such as the hydroxyl radical can react with the phospholipids by abstracting a hydrogen from one of the double bonded hydrogen molecules in the unsaturated fat, and in so doing, forming water. In the following equation, LH represents the lipid and OH* represents the hydroxyl radical, with the * representing the electron that is donated:

LH + OH* → L* + H2O

The lipid radical (L*) can then spontaneously propagate a chain reaction, which can result in peroxidation and breakdown of a number of phospholipids in a cell membrane and damage and disruption of cellular function. Similar damage also results from RONS-induced oxidation of proteins and DNA. As will be discussed in the following section, endogenous antioxidants (e.g., glutathione) and antioxidant vitamins (e.g., vitamin E) can help stop these reactions and prevent peroxidative damage to membranes and other cellular molecules, including proteins and DNA. Limiting oxidative stress may be important in limiting various conditions associated with aging (Pourova et al. 2010)

Protection From Reactive Oxygen and Nitrogen Species

Our bodies produce a number of endogenous oxidants that we have categorized as RONS. Added to this is a host of exogenous substances, such as pollution, ozone, automobile exhaust, solvents, pesticides, and cigarette smoke. Foreign molecules that may pose a hazard to us are generally categorized as xenobiotics. Some xenobiotics are free radicals or can produce RONS. Three approaches to maintaining control over RONS are principal. First, reducing the formation of or exposure to RONS and xenobiotics is an essential step. Second, our endogenous scavenging systems, including antioxidant enzymes and other antioxidant molecules, can scavenge those RONS already present. Finally, mechanisms in cells can upregulate antioxidant defenses in the face of persistent problems (Halliwell and Gutteridge 1999). A failure to maintain a balance between the formation of oxidants (e.g., RONS) and their removal by our various antioxidant systems produces a state known as oxidative stress. Powerful oxidants such as those just discussed can damage proteins, DNA, and the unsaturated fatty acid molecules in membranes. It is no wonder, then, that aging and a variety of diseases, such as cancers, heart disease, neurodegenerative diseases, and type 2 diabetes, have a relationship to oxidative
stress.

Figure 5.20 summarizes a number of important reactions that produce and remove ROS, such as SOD, catalase, and GPX. In addition to specific antioxidant enzymes, the body contains a number of nonprotein antioxidant molecules. For example, the essential nutrients vitamin C, vitamin E, and carotenoids such as b-carotene can by themselves scavenge free radicals. Minerals such as selenium, zinc, manganese, and copper are constituents of antioxidant enzymes. Finally, a host of non-nutrient antioxidants from a wide variety of foods of plant origin are important. The general name phytochemical is applied to those molecules that can function in our bodies as antioxidants. Fruits, vegetables, and grains, as well as common products derived from these such as tea and wine, are the major sources. Flavonoids, polyphenols, and lycopene are common examples of phytochemicals.

Exercise and Oxidative Stress

It is extremely difficult to directly measure free radical formation in a human being. Rather, we use indirect measures to point to increased or decreased ROS formation; from this, we make inferences. From a theoretical perspective, during exercise, we should expect a greater formation of superoxide, and therefore hydrogen peroxide and hydroxyl radicals, simply because of an increased flux through the electron transport chain with the increased need for ATP. Since we can greatly increase oxygen consumption and oxidative phosphorylation during exercise, it has been postulated that exercise can increase the generation of oxygen radicals and the possibility of oxidative damage or stress in muscles and other tissues.

Even though the proportion of oxygen that undergoes one- instead of four-electron reduction should decrease with increased activity of the electron transport chain, the fact that the overall flux may increase 10- to 20-fold above resting levels when we exercise should lead us to suspect an increase in superoxide formation. Bailey and colleagues (2003) were the first to actually measure increased formation of free radicals in venous blood of humans during a single-leg exercise task, using an expensive technique known as electron paramagnetic resonance spectroscopy. In this study, the researchers noted that the outflow of free radical species in the venous blood increased as exercise intensity increased.

The active muscle is not the only source of increased RONS formation during exercise. It has been observed that RONS production by white blood cells increases with exercise. However, the effect of RONS production by white blood cells is increasingly blunted the more trained the person is for the particular exercise activity (Mooren, Lechtermann, and Völker 2004). This is likely a consequence of increased antioxidant enzyme activity in white blood cells with training (Elosua et al. 2003). Some types of exercise, such as intense isometric or eccentric contractions, can create inflammation in muscle. Part of the inflammatory response is caused by neutrophils (white blood cell subset) accumulating in the muscle. As mentioned earlier, these cells produce superoxide that can cause damage within the muscle and can also produce other reactive species (Nguyen and Tidball 2003). Obesity, a major public health problem, is characterized by elevated markers of oxidative stress. When people who are obese perform either aerobic or resistance exercise, they produce higher levels of lipid peroxidation products than normal-weight individuals (Vincent, Morgan, and Vincent 2004). Acute feeding of high-fat diets also increases RONS production in rodents. Such a response suggests either an enhanced production or a reduced ability to scavenge free radicals, or both, in the obese state. Aging is also characterized by increased RONS production and oxidative stress, and increased RONS are associated with various diseases of senescence (Pourova et al. 2010).

Rested muscle produces RONS. When muscle becomes more active, formation of RONS increases. Having some RONS in muscle confers a benefit, since they are often important signaling agents that help regulate acute responses to exercise as well as positive adaptations to training in the muscles. Animal studies have demonstrated that inhibiting RONS production during exercise training actually blunts the signaling pathways that regulate adaptations to training, such as increasing mitochondria and aerobic capacity in skeletal muscle (Gomez-Cabrera et al. 2005). Important signaling pathways such as NFkB can help regulate antioxidant adaptations and mitochondrial synthesis consequent to endurance training. The inhibition of RONS production during exercise can blunt the response of these pathways and, consequently, positive training-induced adaptations (Powers et al. 2010). However, a study involving endurance training in humans failed to demonstrate a short-term negative effect of antioxidant supplements on endurance training adaptations (Yfanti et al. 2010).

Dietary antioxidants such as vitamin E and vitamin C have been touted as important for minimizing muscular damage from exercise. Athletes have often been encouraged to supplement their diets with a variety of antioxidants. While eating a diet high in antioxidants would likely have health benefits and could influence longer term effects of oxidative stress such as aging, studies have demonstrated that it is unlikely that large intakes of antioxidants will have significant physiological effects in diminishing exercise-induced muscle damage; instead, they may inhibit positive adaptations to training (McGinley, Shafat, and Donnelly 2009; Gomez-Cabrera et al. 2008). In addition, studies have demonstrated that antioxidant supplements taken during training may also suppress health-promoting benefits such as improved insulin resistance (Ristow et al. 2009).

Unaccustomed exercise and overtraining lead to muscle soreness, inflammation, and damage. This can be reduced by repeated exposure to the activity or through training. Attempts to limit the amount of stress by taking anti-inflammatory medication such as NSAIDs can potentially reduce oxidative stress in muscles that results from infiltration of white blood cells, such as neutrophils and macrophages. However, since the presence of white blood cells, particularly macrophages in muscle following exercise, is obligatory for the activation of muscle satellite cells and their role in stimulating muscle hypertrophy, reducing inflammation may also limit the amount of muscle repair, adaptation, and hypertrophy that would be induced by the training.

MicroRNAs and the Adaptive Response to Exercise Training

MicroRNAs (miRNAs) are a class of short, noncoding RNA molecules that bind to mRNA molecules and play a central role in regulating gene expression through posttranscriptional gene silencing (reviewed in Bushati and Cohen 2007). Most miRNAs are encoded in introns of protein-coding genes and are transcribed by RNA polymerase II as long primary-miRNAs (pri-miRNA) that encode a single miRNA or a cluster of miRNA species. Processing of pri-miRNA species in the nucleus produces stem-loop structures of ∼70 nucleotides, termed precursor-miRNA (pre-miRNA). These pre-miRNAs are transported to the cytoplasm where they are further processed, giving rise to the mature ∼19 to 22 bp of miRNA. The mature miRNA is incorporated into a ribonucleoprotein complex known as the RNA-induced silencing complex (RISC). Generally, miRNAs inhibit protein synthesis by binding (base-pairing) in the 3' untranslated regions of target mRNAs, either repressing translation or bringing about deadenylation and subsequent degradation of mRNA targets. Individual miRNAs can target hundreds of genes, while individual mRNAs can be targeted by multiple miRNAs, making this one of the most complex gene-regulatory processes.

Studies have uncovered a cluster of muscle-specific miRNAs that regulate muscle differentiation and modulate diverse aspects of muscle function (reviewed in van Rooij, Liu, and Olson 2008). The most highly studied are miR-1, miR-133 and miR-206, which are induced during differentiation of myoblasts into myotubes (Callis et al. 2008) and play an important role in muscle mass maintenance. Other miRNA species (miR-23, miR-103, miR-107, and so on) are proposed to play an important role in regulating expression of genes encoding metabolic pathway enzymes in skeletal muscle and other tissues (Wilfred, Wang, and Nelson 2007). Interestingly, studies have shown the potential importance of miRNA regulation in skeletal-muscle adaptations to exercise (reviewed in Roth 2011). For example, miR-1 and miR-133a are downregulated in mouse skeletal muscle during functional overload-induced hypertrophy of the plantaris muscle (McCarthy and Esser 2007). In response to endurance exercise, miR-23, a putative negative regulator of the transcriptional coactivator peroxisome proliferator a coactivator 1 (PGC-1a), was downregulated in mouse skeletal muscle (Safdar et al. 2009). Importantly, downregulation of miR-23 was associated with increased expression of PGC-1a mRNA and protein, along with several downstream targets of PGC-1a signaling.

You are no doubt aware that the ability to increase muscle size in response to resistance and strength training is greater for some people than others. Have you ever wondered why strength training causes large gains in muscle mass in some people (i.e., high responders), whereas others gain very little muscle mass in response to the same training stimulus (i.e., low responders)? This is true even after accounting for differences in age, training status, exercise adherence, and diet. A study by Davidsen and colleagues (2011) helps to shed some light on this issue. In their study, vastus lateralis biopsies were taken from the top and bottom 49 responders, in terms of muscle mass gain, of 56 men who completed a 12-week strength-training program. The expression level of 21 abundant miRNAs was measured to determine whether variation in these miRNAs was able to explain the variation in resistance training-induced gains in muscle mass. They indentified 4 miRNAs that showed uniquely different responses between high responders and low responders. MiR-378, miR-29a, and miR-26a were downregulated in low responders and were unchanged in high responders, while miR-451 was upregulated only in low responders. Therefore, the regulation of protein synthesis by miRNAs may play an important role in explaining the variability in strength training adaptations. However, further research is required to uncover how these miRNAs themselves are regulated and whether they can be targeted for therapeutic interventions.

Compared with endurance exercise, resistance exercise consists of much higher intensity contractions (e.g., usually 70% to 80% of one repetition maximum) repeated at high frequencies (referring again to motoneuron firing frequency) that can only be sustained for short durations due to the fatiguing nature of the exercise. Therefore, the pattern of cytoplasmic [Ca2+] oscillations and changes in [AMP]/[ATP] and ROS production with skeletal muscle contractions is different between endurance and resistance exercise. For example, endurance exercise likely results in extended periods of moderately elevated [Ca2+], while resistance exercise would generate short cycles of significantly higher intracellular [Ca2+] in skeletal muscle (Chin 2010). Therefore, differences in the magnitude and pattern of these primary signals generated by endurance and resistance exercise will result in the activation of different signaling pathways and different gene-expression and protein-synthesis responses in skeletal muscle. A number of studies have shown that resistance exercise results in increased rates of muscle-protein synthesis, about two- to fivefold after exercise for periods up to 48 h before declining to baseline values. Although acute resistance and endurance-type exercise result in a similar global anabolic response in untrained skeletal muscle, increases in muscle mass (i.e., hypertrophy) and strength are significantly greater following chronic resistance training compared with chronic endurance training. This means that chronic resistance training, but not endurance training, increases the rate of muscle-protein synthesis to levels above the rate of protein degradation.

Activation and differentiation of muscle satellite cells into new muscle cells that fuse with existing muscle fibers can also contribute significantly to the hypertrophic response to resistance exercise. Muscle fibers are large multinucleated cells. They are also postmitotic cells in that they no longer have the ability to divide and reproduce themselves. Additionally, in order to significantly increase in size or hypertrophy, muscles have to add more nuclei, since a nucleus is only able to supply mRNA for new protein synthesis to a limited amount of cytoplasm in its vicinity. Hence, a specific ratio of nuclei to cytoplasm must be maintained. In order to do this, skeletal muscles have small specialized satellite cells,or myogenic precursor cells, located at the periphery of their outer membranes. When stimulated by resistance training or by exercise-induced muscle damage, these specialized cells will be activated and induced to create daughter cells or proliferate. These new satellite cells will then fuse with the existing muscle fibers and add their nuclei to the cells to support greater protein synthesis and increase muscle hypertrophy or repair.

It is well known that the primary factor determining the hypertrophic response to contractile activity is the load across the muscle or the mechanical stretch/strain imposed on the muscle fibers, which is higher in resistance exercise than in endurance exercise. Ultimately, these mechanical/force signals are transduced by signaling pathways that regulate transcriptional and translational processes and satellite-cell activation.

For several years, researchers have focused on the role of insulin-like growth factor (IGF)-1 as a primary signaling molecule that mediates skeletal-muscle growth in response to resistance exercise, given that resistance exercise stimulates the secretion of IGF-1. IGF-1 is known to induce muscle hypertrophy by binding to its receptor on the muscle-cell surface and activating the classical growth factor pathway (see figure 3.27). IGF-1 binding to the receptor activates phosphatidylinositol 3-kinase (PI3K), which leads to the activation of Akt, a serine/threonine protein kinase. Akt phosphorylates and inactivates tuberosclerosis complex (TSC2), resulting in the activation of the ras homologous protein enriched in brain (Rheb) and mammalian target of rapamycin (mTOR). mTOR phosphorylates and suppresses the eukaryotic initiation factor 4E binding protein (4E-BP1) to blunt 4E-BP1 inhibition of translation-initiation cap-binding protein eIF4E (see figure 3.21). mTOR also phosphorylates the 70KDa ribosomal S6 protein kinase (p70S6K1), resulting in an increase in protein synthesis.

It is well accepted that mTOR signaling plays a dominant role in the adaptive response to resistance training; however, researchers have questioned the importance of IGF-1 signaling in mediating this response, based on evidence from several studies employing pharmacological and knockout-mouse approaches to systematically manipulate the IGF-1-PI3K-Akt pathway (Philp, Hamilton, and Baar 2011). It appears that the IGF-1 signaling pathway is not required for mTOR activation or increased protein synthesis that is induced by resistance-type exercise. It is possible that mechanical signals working through stretch-activated membrane channels, for example, might be able to activate mTOR and its downstream targets. However, this hypothesis needs to be examined experimentally (Philp, Hamilton, and Baar 2011).

Cellular Damage From RONS

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are sometimes collectively referred to as reactive oxygen and nitrogen species (RONS). RONS, and particularly the highly reactive hydroxyl radical, can react with many molecules in cells, including DNA, proteins, and lipids. The slow buildup of threats to mitochondrial and nuclear DNA over years of oxidative damage is thought to be a factor in aging. The relatively rapid turnover (breakdown and resynthesis) of proteins is also partly due to their susceptibility to oxidative damage and their need to ensure structural integrity to maintain optimal function. The free radical theory of aging was first proposed in the 1950s by the American scientist Denham Harman. It has continued to receive scientific support over the years, and it is now well established as an integral component of the aging process. Polyunsaturated fatty acids, which are important components of the phospholipids found in cell membranes, are particularly susceptible to damage by RONS. It has been known for more than 200 years that exposure of fats to air or oxygen will turn them rancid. This rancidity is caused by peroxidation of the fatty acids by RONS. During peroxidation, RONS such as the hydroxyl radical can react with the phospholipids by abstracting a hydrogen from one of the double bonded hydrogen molecules in the unsaturated fat, and in so doing, forming water. In the following equation, LH represents the lipid and OH* represents the hydroxyl radical, with the * representing the electron that is donated:

LH + OH* → L* + H2O

The lipid radical (L*) can then spontaneously propagate a chain reaction, which can result in peroxidation and breakdown of a number of phospholipids in a cell membrane and damage and disruption of cellular function. Similar damage also results from RONS-induced oxidation of proteins and DNA. As will be discussed in the following section, endogenous antioxidants (e.g., glutathione) and antioxidant vitamins (e.g., vitamin E) can help stop these reactions and prevent peroxidative damage to membranes and other cellular molecules, including proteins and DNA. Limiting oxidative stress may be important in limiting various conditions associated with aging (Pourova et al. 2010)

Protection From Reactive Oxygen and Nitrogen Species

Our bodies produce a number of endogenous oxidants that we have categorized as RONS. Added to this is a host of exogenous substances, such as pollution, ozone, automobile exhaust, solvents, pesticides, and cigarette smoke. Foreign molecules that may pose a hazard to us are generally categorized as xenobiotics. Some xenobiotics are free radicals or can produce RONS. Three approaches to maintaining control over RONS are principal. First, reducing the formation of or exposure to RONS and xenobiotics is an essential step. Second, our endogenous scavenging systems, including antioxidant enzymes and other antioxidant molecules, can scavenge those RONS already present. Finally, mechanisms in cells can upregulate antioxidant defenses in the face of persistent problems (Halliwell and Gutteridge 1999). A failure to maintain a balance between the formation of oxidants (e.g., RONS) and their removal by our various antioxidant systems produces a state known as oxidative stress. Powerful oxidants such as those just discussed can damage proteins, DNA, and the unsaturated fatty acid molecules in membranes. It is no wonder, then, that aging and a variety of diseases, such as cancers, heart disease, neurodegenerative diseases, and type 2 diabetes, have a relationship to oxidative
stress.

Figure 5.20 summarizes a number of important reactions that produce and remove ROS, such as SOD, catalase, and GPX. In addition to specific antioxidant enzymes, the body contains a number of nonprotein antioxidant molecules. For example, the essential nutrients vitamin C, vitamin E, and carotenoids such as b-carotene can by themselves scavenge free radicals. Minerals such as selenium, zinc, manganese, and copper are constituents of antioxidant enzymes. Finally, a host of non-nutrient antioxidants from a wide variety of foods of plant origin are important. The general name phytochemical is applied to those molecules that can function in our bodies as antioxidants. Fruits, vegetables, and grains, as well as common products derived from these such as tea and wine, are the major sources. Flavonoids, polyphenols, and lycopene are common examples of phytochemicals.

Exercise and Oxidative Stress

It is extremely difficult to directly measure free radical formation in a human being. Rather, we use indirect measures to point to increased or decreased ROS formation; from this, we make inferences. From a theoretical perspective, during exercise, we should expect a greater formation of superoxide, and therefore hydrogen peroxide and hydroxyl radicals, simply because of an increased flux through the electron transport chain with the increased need for ATP. Since we can greatly increase oxygen consumption and oxidative phosphorylation during exercise, it has been postulated that exercise can increase the generation of oxygen radicals and the possibility of oxidative damage or stress in muscles and other tissues.

Even though the proportion of oxygen that undergoes one- instead of four-electron reduction should decrease with increased activity of the electron transport chain, the fact that the overall flux may increase 10- to 20-fold above resting levels when we exercise should lead us to suspect an increase in superoxide formation. Bailey and colleagues (2003) were the first to actually measure increased formation of free radicals in venous blood of humans during a single-leg exercise task, using an expensive technique known as electron paramagnetic resonance spectroscopy. In this study, the researchers noted that the outflow of free radical species in the venous blood increased as exercise intensity increased.

The active muscle is not the only source of increased RONS formation during exercise. It has been observed that RONS production by white blood cells increases with exercise. However, the effect of RONS production by white blood cells is increasingly blunted the more trained the person is for the particular exercise activity (Mooren, Lechtermann, and Völker 2004). This is likely a consequence of increased antioxidant enzyme activity in white blood cells with training (Elosua et al. 2003). Some types of exercise, such as intense isometric or eccentric contractions, can create inflammation in muscle. Part of the inflammatory response is caused by neutrophils (white blood cell subset) accumulating in the muscle. As mentioned earlier, these cells produce superoxide that can cause damage within the muscle and can also produce other reactive species (Nguyen and Tidball 2003). Obesity, a major public health problem, is characterized by elevated markers of oxidative stress. When people who are obese perform either aerobic or resistance exercise, they produce higher levels of lipid peroxidation products than normal-weight individuals (Vincent, Morgan, and Vincent 2004). Acute feeding of high-fat diets also increases RONS production in rodents. Such a response suggests either an enhanced production or a reduced ability to scavenge free radicals, or both, in the obese state. Aging is also characterized by increased RONS production and oxidative stress, and increased RONS are associated with various diseases of senescence (Pourova et al. 2010).

Rested muscle produces RONS. When muscle becomes more active, formation of RONS increases. Having some RONS in muscle confers a benefit, since they are often important signaling agents that help regulate acute responses to exercise as well as positive adaptations to training in the muscles. Animal studies have demonstrated that inhibiting RONS production during exercise training actually blunts the signaling pathways that regulate adaptations to training, such as increasing mitochondria and aerobic capacity in skeletal muscle (Gomez-Cabrera et al. 2005). Important signaling pathways such as NFkB can help regulate antioxidant adaptations and mitochondrial synthesis consequent to endurance training. The inhibition of RONS production during exercise can blunt the response of these pathways and, consequently, positive training-induced adaptations (Powers et al. 2010). However, a study involving endurance training in humans failed to demonstrate a short-term negative effect of antioxidant supplements on endurance training adaptations (Yfanti et al. 2010).

Dietary antioxidants such as vitamin E and vitamin C have been touted as important for minimizing muscular damage from exercise. Athletes have often been encouraged to supplement their diets with a variety of antioxidants. While eating a diet high in antioxidants would likely have health benefits and could influence longer term effects of oxidative stress such as aging, studies have demonstrated that it is unlikely that large intakes of antioxidants will have significant physiological effects in diminishing exercise-induced muscle damage; instead, they may inhibit positive adaptations to training (McGinley, Shafat, and Donnelly 2009; Gomez-Cabrera et al. 2008). In addition, studies have demonstrated that antioxidant supplements taken during training may also suppress health-promoting benefits such as improved insulin resistance (Ristow et al. 2009).

Unaccustomed exercise and overtraining lead to muscle soreness, inflammation, and damage. This can be reduced by repeated exposure to the activity or through training. Attempts to limit the amount of stress by taking anti-inflammatory medication such as NSAIDs can potentially reduce oxidative stress in muscles that results from infiltration of white blood cells, such as neutrophils and macrophages. However, since the presence of white blood cells, particularly macrophages in muscle following exercise, is obligatory for the activation of muscle satellite cells and their role in stimulating muscle hypertrophy, reducing inflammation may also limit the amount of muscle repair, adaptation, and hypertrophy that would be induced by the training.

MicroRNAs and the Adaptive Response to Exercise Training

MicroRNAs (miRNAs) are a class of short, noncoding RNA molecules that bind to mRNA molecules and play a central role in regulating gene expression through posttranscriptional gene silencing (reviewed in Bushati and Cohen 2007). Most miRNAs are encoded in introns of protein-coding genes and are transcribed by RNA polymerase II as long primary-miRNAs (pri-miRNA) that encode a single miRNA or a cluster of miRNA species. Processing of pri-miRNA species in the nucleus produces stem-loop structures of ∼70 nucleotides, termed precursor-miRNA (pre-miRNA). These pre-miRNAs are transported to the cytoplasm where they are further processed, giving rise to the mature ∼19 to 22 bp of miRNA. The mature miRNA is incorporated into a ribonucleoprotein complex known as the RNA-induced silencing complex (RISC). Generally, miRNAs inhibit protein synthesis by binding (base-pairing) in the 3' untranslated regions of target mRNAs, either repressing translation or bringing about deadenylation and subsequent degradation of mRNA targets. Individual miRNAs can target hundreds of genes, while individual mRNAs can be targeted by multiple miRNAs, making this one of the most complex gene-regulatory processes.

Studies have uncovered a cluster of muscle-specific miRNAs that regulate muscle differentiation and modulate diverse aspects of muscle function (reviewed in van Rooij, Liu, and Olson 2008). The most highly studied are miR-1, miR-133 and miR-206, which are induced during differentiation of myoblasts into myotubes (Callis et al. 2008) and play an important role in muscle mass maintenance. Other miRNA species (miR-23, miR-103, miR-107, and so on) are proposed to play an important role in regulating expression of genes encoding metabolic pathway enzymes in skeletal muscle and other tissues (Wilfred, Wang, and Nelson 2007). Interestingly, studies have shown the potential importance of miRNA regulation in skeletal-muscle adaptations to exercise (reviewed in Roth 2011). For example, miR-1 and miR-133a are downregulated in mouse skeletal muscle during functional overload-induced hypertrophy of the plantaris muscle (McCarthy and Esser 2007). In response to endurance exercise, miR-23, a putative negative regulator of the transcriptional coactivator peroxisome proliferator a coactivator 1 (PGC-1a), was downregulated in mouse skeletal muscle (Safdar et al. 2009). Importantly, downregulation of miR-23 was associated with increased expression of PGC-1a mRNA and protein, along with several downstream targets of PGC-1a signaling.

You are no doubt aware that the ability to increase muscle size in response to resistance and strength training is greater for some people than others. Have you ever wondered why strength training causes large gains in muscle mass in some people (i.e., high responders), whereas others gain very little muscle mass in response to the same training stimulus (i.e., low responders)? This is true even after accounting for differences in age, training status, exercise adherence, and diet. A study by Davidsen and colleagues (2011) helps to shed some light on this issue. In their study, vastus lateralis biopsies were taken from the top and bottom 49 responders, in terms of muscle mass gain, of 56 men who completed a 12-week strength-training program. The expression level of 21 abundant miRNAs was measured to determine whether variation in these miRNAs was able to explain the variation in resistance training-induced gains in muscle mass. They indentified 4 miRNAs that showed uniquely different responses between high responders and low responders. MiR-378, miR-29a, and miR-26a were downregulated in low responders and were unchanged in high responders, while miR-451 was upregulated only in low responders. Therefore, the regulation of protein synthesis by miRNAs may play an important role in explaining the variability in strength training adaptations. However, further research is required to uncover how these miRNAs themselves are regulated and whether they can be targeted for therapeutic interventions.

Compared with endurance exercise, resistance exercise consists of much higher intensity contractions (e.g., usually 70% to 80% of one repetition maximum) repeated at high frequencies (referring again to motoneuron firing frequency) that can only be sustained for short durations due to the fatiguing nature of the exercise. Therefore, the pattern of cytoplasmic [Ca2+] oscillations and changes in [AMP]/[ATP] and ROS production with skeletal muscle contractions is different between endurance and resistance exercise. For example, endurance exercise likely results in extended periods of moderately elevated [Ca2+], while resistance exercise would generate short cycles of significantly higher intracellular [Ca2+] in skeletal muscle (Chin 2010). Therefore, differences in the magnitude and pattern of these primary signals generated by endurance and resistance exercise will result in the activation of different signaling pathways and different gene-expression and protein-synthesis responses in skeletal muscle. A number of studies have shown that resistance exercise results in increased rates of muscle-protein synthesis, about two- to fivefold after exercise for periods up to 48 h before declining to baseline values. Although acute resistance and endurance-type exercise result in a similar global anabolic response in untrained skeletal muscle, increases in muscle mass (i.e., hypertrophy) and strength are significantly greater following chronic resistance training compared with chronic endurance training. This means that chronic resistance training, but not endurance training, increases the rate of muscle-protein synthesis to levels above the rate of protein degradation.

Activation and differentiation of muscle satellite cells into new muscle cells that fuse with existing muscle fibers can also contribute significantly to the hypertrophic response to resistance exercise. Muscle fibers are large multinucleated cells. They are also postmitotic cells in that they no longer have the ability to divide and reproduce themselves. Additionally, in order to significantly increase in size or hypertrophy, muscles have to add more nuclei, since a nucleus is only able to supply mRNA for new protein synthesis to a limited amount of cytoplasm in its vicinity. Hence, a specific ratio of nuclei to cytoplasm must be maintained. In order to do this, skeletal muscles have small specialized satellite cells,or myogenic precursor cells, located at the periphery of their outer membranes. When stimulated by resistance training or by exercise-induced muscle damage, these specialized cells will be activated and induced to create daughter cells or proliferate. These new satellite cells will then fuse with the existing muscle fibers and add their nuclei to the cells to support greater protein synthesis and increase muscle hypertrophy or repair.

It is well known that the primary factor determining the hypertrophic response to contractile activity is the load across the muscle or the mechanical stretch/strain imposed on the muscle fibers, which is higher in resistance exercise than in endurance exercise. Ultimately, these mechanical/force signals are transduced by signaling pathways that regulate transcriptional and translational processes and satellite-cell activation.

For several years, researchers have focused on the role of insulin-like growth factor (IGF)-1 as a primary signaling molecule that mediates skeletal-muscle growth in response to resistance exercise, given that resistance exercise stimulates the secretion of IGF-1. IGF-1 is known to induce muscle hypertrophy by binding to its receptor on the muscle-cell surface and activating the classical growth factor pathway (see figure 3.27). IGF-1 binding to the receptor activates phosphatidylinositol 3-kinase (PI3K), which leads to the activation of Akt, a serine/threonine protein kinase. Akt phosphorylates and inactivates tuberosclerosis complex (TSC2), resulting in the activation of the ras homologous protein enriched in brain (Rheb) and mammalian target of rapamycin (mTOR). mTOR phosphorylates and suppresses the eukaryotic initiation factor 4E binding protein (4E-BP1) to blunt 4E-BP1 inhibition of translation-initiation cap-binding protein eIF4E (see figure 3.21). mTOR also phosphorylates the 70KDa ribosomal S6 protein kinase (p70S6K1), resulting in an increase in protein synthesis.

It is well accepted that mTOR signaling plays a dominant role in the adaptive response to resistance training; however, researchers have questioned the importance of IGF-1 signaling in mediating this response, based on evidence from several studies employing pharmacological and knockout-mouse approaches to systematically manipulate the IGF-1-PI3K-Akt pathway (Philp, Hamilton, and Baar 2011). It appears that the IGF-1 signaling pathway is not required for mTOR activation or increased protein synthesis that is induced by resistance-type exercise. It is possible that mechanical signals working through stretch-activated membrane channels, for example, might be able to activate mTOR and its downstream targets. However, this hypothesis needs to be examined experimentally (Philp, Hamilton, and Baar 2011).

Cellular Damage From RONS

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are sometimes collectively referred to as reactive oxygen and nitrogen species (RONS). RONS, and particularly the highly reactive hydroxyl radical, can react with many molecules in cells, including DNA, proteins, and lipids. The slow buildup of threats to mitochondrial and nuclear DNA over years of oxidative damage is thought to be a factor in aging. The relatively rapid turnover (breakdown and resynthesis) of proteins is also partly due to their susceptibility to oxidative damage and their need to ensure structural integrity to maintain optimal function. The free radical theory of aging was first proposed in the 1950s by the American scientist Denham Harman. It has continued to receive scientific support over the years, and it is now well established as an integral component of the aging process. Polyunsaturated fatty acids, which are important components of the phospholipids found in cell membranes, are particularly susceptible to damage by RONS. It has been known for more than 200 years that exposure of fats to air or oxygen will turn them rancid. This rancidity is caused by peroxidation of the fatty acids by RONS. During peroxidation, RONS such as the hydroxyl radical can react with the phospholipids by abstracting a hydrogen from one of the double bonded hydrogen molecules in the unsaturated fat, and in so doing, forming water. In the following equation, LH represents the lipid and OH* represents the hydroxyl radical, with the * representing the electron that is donated:

LH + OH* → L* + H2O

The lipid radical (L*) can then spontaneously propagate a chain reaction, which can result in peroxidation and breakdown of a number of phospholipids in a cell membrane and damage and disruption of cellular function. Similar damage also results from RONS-induced oxidation of proteins and DNA. As will be discussed in the following section, endogenous antioxidants (e.g., glutathione) and antioxidant vitamins (e.g., vitamin E) can help stop these reactions and prevent peroxidative damage to membranes and other cellular molecules, including proteins and DNA. Limiting oxidative stress may be important in limiting various conditions associated with aging (Pourova et al. 2010)

Protection From Reactive Oxygen and Nitrogen Species

Our bodies produce a number of endogenous oxidants that we have categorized as RONS. Added to this is a host of exogenous substances, such as pollution, ozone, automobile exhaust, solvents, pesticides, and cigarette smoke. Foreign molecules that may pose a hazard to us are generally categorized as xenobiotics. Some xenobiotics are free radicals or can produce RONS. Three approaches to maintaining control over RONS are principal. First, reducing the formation of or exposure to RONS and xenobiotics is an essential step. Second, our endogenous scavenging systems, including antioxidant enzymes and other antioxidant molecules, can scavenge those RONS already present. Finally, mechanisms in cells can upregulate antioxidant defenses in the face of persistent problems (Halliwell and Gutteridge 1999). A failure to maintain a balance between the formation of oxidants (e.g., RONS) and their removal by our various antioxidant systems produces a state known as oxidative stress. Powerful oxidants such as those just discussed can damage proteins, DNA, and the unsaturated fatty acid molecules in membranes. It is no wonder, then, that aging and a variety of diseases, such as cancers, heart disease, neurodegenerative diseases, and type 2 diabetes, have a relationship to oxidative
stress.

Figure 5.20 summarizes a number of important reactions that produce and remove ROS, such as SOD, catalase, and GPX. In addition to specific antioxidant enzymes, the body contains a number of nonprotein antioxidant molecules. For example, the essential nutrients vitamin C, vitamin E, and carotenoids such as b-carotene can by themselves scavenge free radicals. Minerals such as selenium, zinc, manganese, and copper are constituents of antioxidant enzymes. Finally, a host of non-nutrient antioxidants from a wide variety of foods of plant origin are important. The general name phytochemical is applied to those molecules that can function in our bodies as antioxidants. Fruits, vegetables, and grains, as well as common products derived from these such as tea and wine, are the major sources. Flavonoids, polyphenols, and lycopene are common examples of phytochemicals.

Exercise and Oxidative Stress

It is extremely difficult to directly measure free radical formation in a human being. Rather, we use indirect measures to point to increased or decreased ROS formation; from this, we make inferences. From a theoretical perspective, during exercise, we should expect a greater formation of superoxide, and therefore hydrogen peroxide and hydroxyl radicals, simply because of an increased flux through the electron transport chain with the increased need for ATP. Since we can greatly increase oxygen consumption and oxidative phosphorylation during exercise, it has been postulated that exercise can increase the generation of oxygen radicals and the possibility of oxidative damage or stress in muscles and other tissues.

Even though the proportion of oxygen that undergoes one- instead of four-electron reduction should decrease with increased activity of the electron transport chain, the fact that the overall flux may increase 10- to 20-fold above resting levels when we exercise should lead us to suspect an increase in superoxide formation. Bailey and colleagues (2003) were the first to actually measure increased formation of free radicals in venous blood of humans during a single-leg exercise task, using an expensive technique known as electron paramagnetic resonance spectroscopy. In this study, the researchers noted that the outflow of free radical species in the venous blood increased as exercise intensity increased.

The active muscle is not the only source of increased RONS formation during exercise. It has been observed that RONS production by white blood cells increases with exercise. However, the effect of RONS production by white blood cells is increasingly blunted the more trained the person is for the particular exercise activity (Mooren, Lechtermann, and Völker 2004). This is likely a consequence of increased antioxidant enzyme activity in white blood cells with training (Elosua et al. 2003). Some types of exercise, such as intense isometric or eccentric contractions, can create inflammation in muscle. Part of the inflammatory response is caused by neutrophils (white blood cell subset) accumulating in the muscle. As mentioned earlier, these cells produce superoxide that can cause damage within the muscle and can also produce other reactive species (Nguyen and Tidball 2003). Obesity, a major public health problem, is characterized by elevated markers of oxidative stress. When people who are obese perform either aerobic or resistance exercise, they produce higher levels of lipid peroxidation products than normal-weight individuals (Vincent, Morgan, and Vincent 2004). Acute feeding of high-fat diets also increases RONS production in rodents. Such a response suggests either an enhanced production or a reduced ability to scavenge free radicals, or both, in the obese state. Aging is also characterized by increased RONS production and oxidative stress, and increased RONS are associated with various diseases of senescence (Pourova et al. 2010).

Rested muscle produces RONS. When muscle becomes more active, formation of RONS increases. Having some RONS in muscle confers a benefit, since they are often important signaling agents that help regulate acute responses to exercise as well as positive adaptations to training in the muscles. Animal studies have demonstrated that inhibiting RONS production during exercise training actually blunts the signaling pathways that regulate adaptations to training, such as increasing mitochondria and aerobic capacity in skeletal muscle (Gomez-Cabrera et al. 2005). Important signaling pathways such as NFkB can help regulate antioxidant adaptations and mitochondrial synthesis consequent to endurance training. The inhibition of RONS production during exercise can blunt the response of these pathways and, consequently, positive training-induced adaptations (Powers et al. 2010). However, a study involving endurance training in humans failed to demonstrate a short-term negative effect of antioxidant supplements on endurance training adaptations (Yfanti et al. 2010).

Dietary antioxidants such as vitamin E and vitamin C have been touted as important for minimizing muscular damage from exercise. Athletes have often been encouraged to supplement their diets with a variety of antioxidants. While eating a diet high in antioxidants would likely have health benefits and could influence longer term effects of oxidative stress such as aging, studies have demonstrated that it is unlikely that large intakes of antioxidants will have significant physiological effects in diminishing exercise-induced muscle damage; instead, they may inhibit positive adaptations to training (McGinley, Shafat, and Donnelly 2009; Gomez-Cabrera et al. 2008). In addition, studies have demonstrated that antioxidant supplements taken during training may also suppress health-promoting benefits such as improved insulin resistance (Ristow et al. 2009).

Unaccustomed exercise and overtraining lead to muscle soreness, inflammation, and damage. This can be reduced by repeated exposure to the activity or through training. Attempts to limit the amount of stress by taking anti-inflammatory medication such as NSAIDs can potentially reduce oxidative stress in muscles that results from infiltration of white blood cells, such as neutrophils and macrophages. However, since the presence of white blood cells, particularly macrophages in muscle following exercise, is obligatory for the activation of muscle satellite cells and their role in stimulating muscle hypertrophy, reducing inflammation may also limit the amount of muscle repair, adaptation, and hypertrophy that would be induced by the training.

MicroRNAs and the Adaptive Response to Exercise Training

MicroRNAs (miRNAs) are a class of short, noncoding RNA molecules that bind to mRNA molecules and play a central role in regulating gene expression through posttranscriptional gene silencing (reviewed in Bushati and Cohen 2007). Most miRNAs are encoded in introns of protein-coding genes and are transcribed by RNA polymerase II as long primary-miRNAs (pri-miRNA) that encode a single miRNA or a cluster of miRNA species. Processing of pri-miRNA species in the nucleus produces stem-loop structures of ∼70 nucleotides, termed precursor-miRNA (pre-miRNA). These pre-miRNAs are transported to the cytoplasm where they are further processed, giving rise to the mature ∼19 to 22 bp of miRNA. The mature miRNA is incorporated into a ribonucleoprotein complex known as the RNA-induced silencing complex (RISC). Generally, miRNAs inhibit protein synthesis by binding (base-pairing) in the 3' untranslated regions of target mRNAs, either repressing translation or bringing about deadenylation and subsequent degradation of mRNA targets. Individual miRNAs can target hundreds of genes, while individual mRNAs can be targeted by multiple miRNAs, making this one of the most complex gene-regulatory processes.

Studies have uncovered a cluster of muscle-specific miRNAs that regulate muscle differentiation and modulate diverse aspects of muscle function (reviewed in van Rooij, Liu, and Olson 2008). The most highly studied are miR-1, miR-133 and miR-206, which are induced during differentiation of myoblasts into myotubes (Callis et al. 2008) and play an important role in muscle mass maintenance. Other miRNA species (miR-23, miR-103, miR-107, and so on) are proposed to play an important role in regulating expression of genes encoding metabolic pathway enzymes in skeletal muscle and other tissues (Wilfred, Wang, and Nelson 2007). Interestingly, studies have shown the potential importance of miRNA regulation in skeletal-muscle adaptations to exercise (reviewed in Roth 2011). For example, miR-1 and miR-133a are downregulated in mouse skeletal muscle during functional overload-induced hypertrophy of the plantaris muscle (McCarthy and Esser 2007). In response to endurance exercise, miR-23, a putative negative regulator of the transcriptional coactivator peroxisome proliferator a coactivator 1 (PGC-1a), was downregulated in mouse skeletal muscle (Safdar et al. 2009). Importantly, downregulation of miR-23 was associated with increased expression of PGC-1a mRNA and protein, along with several downstream targets of PGC-1a signaling.

You are no doubt aware that the ability to increase muscle size in response to resistance and strength training is greater for some people than others. Have you ever wondered why strength training causes large gains in muscle mass in some people (i.e., high responders), whereas others gain very little muscle mass in response to the same training stimulus (i.e., low responders)? This is true even after accounting for differences in age, training status, exercise adherence, and diet. A study by Davidsen and colleagues (2011) helps to shed some light on this issue. In their study, vastus lateralis biopsies were taken from the top and bottom 49 responders, in terms of muscle mass gain, of 56 men who completed a 12-week strength-training program. The expression level of 21 abundant miRNAs was measured to determine whether variation in these miRNAs was able to explain the variation in resistance training-induced gains in muscle mass. They indentified 4 miRNAs that showed uniquely different responses between high responders and low responders. MiR-378, miR-29a, and miR-26a were downregulated in low responders and were unchanged in high responders, while miR-451 was upregulated only in low responders. Therefore, the regulation of protein synthesis by miRNAs may play an important role in explaining the variability in strength training adaptations. However, further research is required to uncover how these miRNAs themselves are regulated and whether they can be targeted for therapeutic interventions.

Compared with endurance exercise, resistance exercise consists of much higher intensity contractions (e.g., usually 70% to 80% of one repetition maximum) repeated at high frequencies (referring again to motoneuron firing frequency) that can only be sustained for short durations due to the fatiguing nature of the exercise. Therefore, the pattern of cytoplasmic [Ca2+] oscillations and changes in [AMP]/[ATP] and ROS production with skeletal muscle contractions is different between endurance and resistance exercise. For example, endurance exercise likely results in extended periods of moderately elevated [Ca2+], while resistance exercise would generate short cycles of significantly higher intracellular [Ca2+] in skeletal muscle (Chin 2010). Therefore, differences in the magnitude and pattern of these primary signals generated by endurance and resistance exercise will result in the activation of different signaling pathways and different gene-expression and protein-synthesis responses in skeletal muscle. A number of studies have shown that resistance exercise results in increased rates of muscle-protein synthesis, about two- to fivefold after exercise for periods up to 48 h before declining to baseline values. Although acute resistance and endurance-type exercise result in a similar global anabolic response in untrained skeletal muscle, increases in muscle mass (i.e., hypertrophy) and strength are significantly greater following chronic resistance training compared with chronic endurance training. This means that chronic resistance training, but not endurance training, increases the rate of muscle-protein synthesis to levels above the rate of protein degradation.

Activation and differentiation of muscle satellite cells into new muscle cells that fuse with existing muscle fibers can also contribute significantly to the hypertrophic response to resistance exercise. Muscle fibers are large multinucleated cells. They are also postmitotic cells in that they no longer have the ability to divide and reproduce themselves. Additionally, in order to significantly increase in size or hypertrophy, muscles have to add more nuclei, since a nucleus is only able to supply mRNA for new protein synthesis to a limited amount of cytoplasm in its vicinity. Hence, a specific ratio of nuclei to cytoplasm must be maintained. In order to do this, skeletal muscles have small specialized satellite cells,or myogenic precursor cells, located at the periphery of their outer membranes. When stimulated by resistance training or by exercise-induced muscle damage, these specialized cells will be activated and induced to create daughter cells or proliferate. These new satellite cells will then fuse with the existing muscle fibers and add their nuclei to the cells to support greater protein synthesis and increase muscle hypertrophy or repair.

It is well known that the primary factor determining the hypertrophic response to contractile activity is the load across the muscle or the mechanical stretch/strain imposed on the muscle fibers, which is higher in resistance exercise than in endurance exercise. Ultimately, these mechanical/force signals are transduced by signaling pathways that regulate transcriptional and translational processes and satellite-cell activation.

For several years, researchers have focused on the role of insulin-like growth factor (IGF)-1 as a primary signaling molecule that mediates skeletal-muscle growth in response to resistance exercise, given that resistance exercise stimulates the secretion of IGF-1. IGF-1 is known to induce muscle hypertrophy by binding to its receptor on the muscle-cell surface and activating the classical growth factor pathway (see figure 3.27). IGF-1 binding to the receptor activates phosphatidylinositol 3-kinase (PI3K), which leads to the activation of Akt, a serine/threonine protein kinase. Akt phosphorylates and inactivates tuberosclerosis complex (TSC2), resulting in the activation of the ras homologous protein enriched in brain (Rheb) and mammalian target of rapamycin (mTOR). mTOR phosphorylates and suppresses the eukaryotic initiation factor 4E binding protein (4E-BP1) to blunt 4E-BP1 inhibition of translation-initiation cap-binding protein eIF4E (see figure 3.21). mTOR also phosphorylates the 70KDa ribosomal S6 protein kinase (p70S6K1), resulting in an increase in protein synthesis.

It is well accepted that mTOR signaling plays a dominant role in the adaptive response to resistance training; however, researchers have questioned the importance of IGF-1 signaling in mediating this response, based on evidence from several studies employing pharmacological and knockout-mouse approaches to systematically manipulate the IGF-1-PI3K-Akt pathway (Philp, Hamilton, and Baar 2011). It appears that the IGF-1 signaling pathway is not required for mTOR activation or increased protein synthesis that is induced by resistance-type exercise. It is possible that mechanical signals working through stretch-activated membrane channels, for example, might be able to activate mTOR and its downstream targets. However, this hypothesis needs to be examined experimentally (Philp, Hamilton, and Baar 2011).

Cellular Damage From RONS

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are sometimes collectively referred to as reactive oxygen and nitrogen species (RONS). RONS, and particularly the highly reactive hydroxyl radical, can react with many molecules in cells, including DNA, proteins, and lipids. The slow buildup of threats to mitochondrial and nuclear DNA over years of oxidative damage is thought to be a factor in aging. The relatively rapid turnover (breakdown and resynthesis) of proteins is also partly due to their susceptibility to oxidative damage and their need to ensure structural integrity to maintain optimal function. The free radical theory of aging was first proposed in the 1950s by the American scientist Denham Harman. It has continued to receive scientific support over the years, and it is now well established as an integral component of the aging process. Polyunsaturated fatty acids, which are important components of the phospholipids found in cell membranes, are particularly susceptible to damage by RONS. It has been known for more than 200 years that exposure of fats to air or oxygen will turn them rancid. This rancidity is caused by peroxidation of the fatty acids by RONS. During peroxidation, RONS such as the hydroxyl radical can react with the phospholipids by abstracting a hydrogen from one of the double bonded hydrogen molecules in the unsaturated fat, and in so doing, forming water. In the following equation, LH represents the lipid and OH* represents the hydroxyl radical, with the * representing the electron that is donated:

LH + OH* → L* + H2O

The lipid radical (L*) can then spontaneously propagate a chain reaction, which can result in peroxidation and breakdown of a number of phospholipids in a cell membrane and damage and disruption of cellular function. Similar damage also results from RONS-induced oxidation of proteins and DNA. As will be discussed in the following section, endogenous antioxidants (e.g., glutathione) and antioxidant vitamins (e.g., vitamin E) can help stop these reactions and prevent peroxidative damage to membranes and other cellular molecules, including proteins and DNA. Limiting oxidative stress may be important in limiting various conditions associated with aging (Pourova et al. 2010)

Protection From Reactive Oxygen and Nitrogen Species

Our bodies produce a number of endogenous oxidants that we have categorized as RONS. Added to this is a host of exogenous substances, such as pollution, ozone, automobile exhaust, solvents, pesticides, and cigarette smoke. Foreign molecules that may pose a hazard to us are generally categorized as xenobiotics. Some xenobiotics are free radicals or can produce RONS. Three approaches to maintaining control over RONS are principal. First, reducing the formation of or exposure to RONS and xenobiotics is an essential step. Second, our endogenous scavenging systems, including antioxidant enzymes and other antioxidant molecules, can scavenge those RONS already present. Finally, mechanisms in cells can upregulate antioxidant defenses in the face of persistent problems (Halliwell and Gutteridge 1999). A failure to maintain a balance between the formation of oxidants (e.g., RONS) and their removal by our various antioxidant systems produces a state known as oxidative stress. Powerful oxidants such as those just discussed can damage proteins, DNA, and the unsaturated fatty acid molecules in membranes. It is no wonder, then, that aging and a variety of diseases, such as cancers, heart disease, neurodegenerative diseases, and type 2 diabetes, have a relationship to oxidative
stress.

Figure 5.20 summarizes a number of important reactions that produce and remove ROS, such as SOD, catalase, and GPX. In addition to specific antioxidant enzymes, the body contains a number of nonprotein antioxidant molecules. For example, the essential nutrients vitamin C, vitamin E, and carotenoids such as b-carotene can by themselves scavenge free radicals. Minerals such as selenium, zinc, manganese, and copper are constituents of antioxidant enzymes. Finally, a host of non-nutrient antioxidants from a wide variety of foods of plant origin are important. The general name phytochemical is applied to those molecules that can function in our bodies as antioxidants. Fruits, vegetables, and grains, as well as common products derived from these such as tea and wine, are the major sources. Flavonoids, polyphenols, and lycopene are common examples of phytochemicals.

Exercise and Oxidative Stress

It is extremely difficult to directly measure free radical formation in a human being. Rather, we use indirect measures to point to increased or decreased ROS formation; from this, we make inferences. From a theoretical perspective, during exercise, we should expect a greater formation of superoxide, and therefore hydrogen peroxide and hydroxyl radicals, simply because of an increased flux through the electron transport chain with the increased need for ATP. Since we can greatly increase oxygen consumption and oxidative phosphorylation during exercise, it has been postulated that exercise can increase the generation of oxygen radicals and the possibility of oxidative damage or stress in muscles and other tissues.

Even though the proportion of oxygen that undergoes one- instead of four-electron reduction should decrease with increased activity of the electron transport chain, the fact that the overall flux may increase 10- to 20-fold above resting levels when we exercise should lead us to suspect an increase in superoxide formation. Bailey and colleagues (2003) were the first to actually measure increased formation of free radicals in venous blood of humans during a single-leg exercise task, using an expensive technique known as electron paramagnetic resonance spectroscopy. In this study, the researchers noted that the outflow of free radical species in the venous blood increased as exercise intensity increased.

The active muscle is not the only source of increased RONS formation during exercise. It has been observed that RONS production by white blood cells increases with exercise. However, the effect of RONS production by white blood cells is increasingly blunted the more trained the person is for the particular exercise activity (Mooren, Lechtermann, and Völker 2004). This is likely a consequence of increased antioxidant enzyme activity in white blood cells with training (Elosua et al. 2003). Some types of exercise, such as intense isometric or eccentric contractions, can create inflammation in muscle. Part of the inflammatory response is caused by neutrophils (white blood cell subset) accumulating in the muscle. As mentioned earlier, these cells produce superoxide that can cause damage within the muscle and can also produce other reactive species (Nguyen and Tidball 2003). Obesity, a major public health problem, is characterized by elevated markers of oxidative stress. When people who are obese perform either aerobic or resistance exercise, they produce higher levels of lipid peroxidation products than normal-weight individuals (Vincent, Morgan, and Vincent 2004). Acute feeding of high-fat diets also increases RONS production in rodents. Such a response suggests either an enhanced production or a reduced ability to scavenge free radicals, or both, in the obese state. Aging is also characterized by increased RONS production and oxidative stress, and increased RONS are associated with various diseases of senescence (Pourova et al. 2010).

Rested muscle produces RONS. When muscle becomes more active, formation of RONS increases. Having some RONS in muscle confers a benefit, since they are often important signaling agents that help regulate acute responses to exercise as well as positive adaptations to training in the muscles. Animal studies have demonstrated that inhibiting RONS production during exercise training actually blunts the signaling pathways that regulate adaptations to training, such as increasing mitochondria and aerobic capacity in skeletal muscle (Gomez-Cabrera et al. 2005). Important signaling pathways such as NFkB can help regulate antioxidant adaptations and mitochondrial synthesis consequent to endurance training. The inhibition of RONS production during exercise can blunt the response of these pathways and, consequently, positive training-induced adaptations (Powers et al. 2010). However, a study involving endurance training in humans failed to demonstrate a short-term negative effect of antioxidant supplements on endurance training adaptations (Yfanti et al. 2010).

Dietary antioxidants such as vitamin E and vitamin C have been touted as important for minimizing muscular damage from exercise. Athletes have often been encouraged to supplement their diets with a variety of antioxidants. While eating a diet high in antioxidants would likely have health benefits and could influence longer term effects of oxidative stress such as aging, studies have demonstrated that it is unlikely that large intakes of antioxidants will have significant physiological effects in diminishing exercise-induced muscle damage; instead, they may inhibit positive adaptations to training (McGinley, Shafat, and Donnelly 2009; Gomez-Cabrera et al. 2008). In addition, studies have demonstrated that antioxidant supplements taken during training may also suppress health-promoting benefits such as improved insulin resistance (Ristow et al. 2009).

Unaccustomed exercise and overtraining lead to muscle soreness, inflammation, and damage. This can be reduced by repeated exposure to the activity or through training. Attempts to limit the amount of stress by taking anti-inflammatory medication such as NSAIDs can potentially reduce oxidative stress in muscles that results from infiltration of white blood cells, such as neutrophils and macrophages. However, since the presence of white blood cells, particularly macrophages in muscle following exercise, is obligatory for the activation of muscle satellite cells and their role in stimulating muscle hypertrophy, reducing inflammation may also limit the amount of muscle repair, adaptation, and hypertrophy that would be induced by the training.

MicroRNAs and the Adaptive Response to Exercise Training

MicroRNAs (miRNAs) are a class of short, noncoding RNA molecules that bind to mRNA molecules and play a central role in regulating gene expression through posttranscriptional gene silencing (reviewed in Bushati and Cohen 2007). Most miRNAs are encoded in introns of protein-coding genes and are transcribed by RNA polymerase II as long primary-miRNAs (pri-miRNA) that encode a single miRNA or a cluster of miRNA species. Processing of pri-miRNA species in the nucleus produces stem-loop structures of ∼70 nucleotides, termed precursor-miRNA (pre-miRNA). These pre-miRNAs are transported to the cytoplasm where they are further processed, giving rise to the mature ∼19 to 22 bp of miRNA. The mature miRNA is incorporated into a ribonucleoprotein complex known as the RNA-induced silencing complex (RISC). Generally, miRNAs inhibit protein synthesis by binding (base-pairing) in the 3' untranslated regions of target mRNAs, either repressing translation or bringing about deadenylation and subsequent degradation of mRNA targets. Individual miRNAs can target hundreds of genes, while individual mRNAs can be targeted by multiple miRNAs, making this one of the most complex gene-regulatory processes.

Studies have uncovered a cluster of muscle-specific miRNAs that regulate muscle differentiation and modulate diverse aspects of muscle function (reviewed in van Rooij, Liu, and Olson 2008). The most highly studied are miR-1, miR-133 and miR-206, which are induced during differentiation of myoblasts into myotubes (Callis et al. 2008) and play an important role in muscle mass maintenance. Other miRNA species (miR-23, miR-103, miR-107, and so on) are proposed to play an important role in regulating expression of genes encoding metabolic pathway enzymes in skeletal muscle and other tissues (Wilfred, Wang, and Nelson 2007). Interestingly, studies have shown the potential importance of miRNA regulation in skeletal-muscle adaptations to exercise (reviewed in Roth 2011). For example, miR-1 and miR-133a are downregulated in mouse skeletal muscle during functional overload-induced hypertrophy of the plantaris muscle (McCarthy and Esser 2007). In response to endurance exercise, miR-23, a putative negative regulator of the transcriptional coactivator peroxisome proliferator a coactivator 1 (PGC-1a), was downregulated in mouse skeletal muscle (Safdar et al. 2009). Importantly, downregulation of miR-23 was associated with increased expression of PGC-1a mRNA and protein, along with several downstream targets of PGC-1a signaling.

You are no doubt aware that the ability to increase muscle size in response to resistance and strength training is greater for some people than others. Have you ever wondered why strength training causes large gains in muscle mass in some people (i.e., high responders), whereas others gain very little muscle mass in response to the same training stimulus (i.e., low responders)? This is true even after accounting for differences in age, training status, exercise adherence, and diet. A study by Davidsen and colleagues (2011) helps to shed some light on this issue. In their study, vastus lateralis biopsies were taken from the top and bottom 49 responders, in terms of muscle mass gain, of 56 men who completed a 12-week strength-training program. The expression level of 21 abundant miRNAs was measured to determine whether variation in these miRNAs was able to explain the variation in resistance training-induced gains in muscle mass. They indentified 4 miRNAs that showed uniquely different responses between high responders and low responders. MiR-378, miR-29a, and miR-26a were downregulated in low responders and were unchanged in high responders, while miR-451 was upregulated only in low responders. Therefore, the regulation of protein synthesis by miRNAs may play an important role in explaining the variability in strength training adaptations. However, further research is required to uncover how these miRNAs themselves are regulated and whether they can be targeted for therapeutic interventions.

Compared with endurance exercise, resistance exercise consists of much higher intensity contractions (e.g., usually 70% to 80% of one repetition maximum) repeated at high frequencies (referring again to motoneuron firing frequency) that can only be sustained for short durations due to the fatiguing nature of the exercise. Therefore, the pattern of cytoplasmic [Ca2+] oscillations and changes in [AMP]/[ATP] and ROS production with skeletal muscle contractions is different between endurance and resistance exercise. For example, endurance exercise likely results in extended periods of moderately elevated [Ca2+], while resistance exercise would generate short cycles of significantly higher intracellular [Ca2+] in skeletal muscle (Chin 2010). Therefore, differences in the magnitude and pattern of these primary signals generated by endurance and resistance exercise will result in the activation of different signaling pathways and different gene-expression and protein-synthesis responses in skeletal muscle. A number of studies have shown that resistance exercise results in increased rates of muscle-protein synthesis, about two- to fivefold after exercise for periods up to 48 h before declining to baseline values. Although acute resistance and endurance-type exercise result in a similar global anabolic response in untrained skeletal muscle, increases in muscle mass (i.e., hypertrophy) and strength are significantly greater following chronic resistance training compared with chronic endurance training. This means that chronic resistance training, but not endurance training, increases the rate of muscle-protein synthesis to levels above the rate of protein degradation.

Activation and differentiation of muscle satellite cells into new muscle cells that fuse with existing muscle fibers can also contribute significantly to the hypertrophic response to resistance exercise. Muscle fibers are large multinucleated cells. They are also postmitotic cells in that they no longer have the ability to divide and reproduce themselves. Additionally, in order to significantly increase in size or hypertrophy, muscles have to add more nuclei, since a nucleus is only able to supply mRNA for new protein synthesis to a limited amount of cytoplasm in its vicinity. Hence, a specific ratio of nuclei to cytoplasm must be maintained. In order to do this, skeletal muscles have small specialized satellite cells,or myogenic precursor cells, located at the periphery of their outer membranes. When stimulated by resistance training or by exercise-induced muscle damage, these specialized cells will be activated and induced to create daughter cells or proliferate. These new satellite cells will then fuse with the existing muscle fibers and add their nuclei to the cells to support greater protein synthesis and increase muscle hypertrophy or repair.

It is well known that the primary factor determining the hypertrophic response to contractile activity is the load across the muscle or the mechanical stretch/strain imposed on the muscle fibers, which is higher in resistance exercise than in endurance exercise. Ultimately, these mechanical/force signals are transduced by signaling pathways that regulate transcriptional and translational processes and satellite-cell activation.

For several years, researchers have focused on the role of insulin-like growth factor (IGF)-1 as a primary signaling molecule that mediates skeletal-muscle growth in response to resistance exercise, given that resistance exercise stimulates the secretion of IGF-1. IGF-1 is known to induce muscle hypertrophy by binding to its receptor on the muscle-cell surface and activating the classical growth factor pathway (see figure 3.27). IGF-1 binding to the receptor activates phosphatidylinositol 3-kinase (PI3K), which leads to the activation of Akt, a serine/threonine protein kinase. Akt phosphorylates and inactivates tuberosclerosis complex (TSC2), resulting in the activation of the ras homologous protein enriched in brain (Rheb) and mammalian target of rapamycin (mTOR). mTOR phosphorylates and suppresses the eukaryotic initiation factor 4E binding protein (4E-BP1) to blunt 4E-BP1 inhibition of translation-initiation cap-binding protein eIF4E (see figure 3.21). mTOR also phosphorylates the 70KDa ribosomal S6 protein kinase (p70S6K1), resulting in an increase in protein synthesis.

It is well accepted that mTOR signaling plays a dominant role in the adaptive response to resistance training; however, researchers have questioned the importance of IGF-1 signaling in mediating this response, based on evidence from several studies employing pharmacological and knockout-mouse approaches to systematically manipulate the IGF-1-PI3K-Akt pathway (Philp, Hamilton, and Baar 2011). It appears that the IGF-1 signaling pathway is not required for mTOR activation or increased protein synthesis that is induced by resistance-type exercise. It is possible that mechanical signals working through stretch-activated membrane channels, for example, might be able to activate mTOR and its downstream targets. However, this hypothesis needs to be examined experimentally (Philp, Hamilton, and Baar 2011).

Cellular Damage From RONS

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are sometimes collectively referred to as reactive oxygen and nitrogen species (RONS). RONS, and particularly the highly reactive hydroxyl radical, can react with many molecules in cells, including DNA, proteins, and lipids. The slow buildup of threats to mitochondrial and nuclear DNA over years of oxidative damage is thought to be a factor in aging. The relatively rapid turnover (breakdown and resynthesis) of proteins is also partly due to their susceptibility to oxidative damage and their need to ensure structural integrity to maintain optimal function. The free radical theory of aging was first proposed in the 1950s by the American scientist Denham Harman. It has continued to receive scientific support over the years, and it is now well established as an integral component of the aging process. Polyunsaturated fatty acids, which are important components of the phospholipids found in cell membranes, are particularly susceptible to damage by RONS. It has been known for more than 200 years that exposure of fats to air or oxygen will turn them rancid. This rancidity is caused by peroxidation of the fatty acids by RONS. During peroxidation, RONS such as the hydroxyl radical can react with the phospholipids by abstracting a hydrogen from one of the double bonded hydrogen molecules in the unsaturated fat, and in so doing, forming water. In the following equation, LH represents the lipid and OH* represents the hydroxyl radical, with the * representing the electron that is donated:

LH + OH* → L* + H2O

The lipid radical (L*) can then spontaneously propagate a chain reaction, which can result in peroxidation and breakdown of a number of phospholipids in a cell membrane and damage and disruption of cellular function. Similar damage also results from RONS-induced oxidation of proteins and DNA. As will be discussed in the following section, endogenous antioxidants (e.g., glutathione) and antioxidant vitamins (e.g., vitamin E) can help stop these reactions and prevent peroxidative damage to membranes and other cellular molecules, including proteins and DNA. Limiting oxidative stress may be important in limiting various conditions associated with aging (Pourova et al. 2010)

Protection From Reactive Oxygen and Nitrogen Species

Our bodies produce a number of endogenous oxidants that we have categorized as RONS. Added to this is a host of exogenous substances, such as pollution, ozone, automobile exhaust, solvents, pesticides, and cigarette smoke. Foreign molecules that may pose a hazard to us are generally categorized as xenobiotics. Some xenobiotics are free radicals or can produce RONS. Three approaches to maintaining control over RONS are principal. First, reducing the formation of or exposure to RONS and xenobiotics is an essential step. Second, our endogenous scavenging systems, including antioxidant enzymes and other antioxidant molecules, can scavenge those RONS already present. Finally, mechanisms in cells can upregulate antioxidant defenses in the face of persistent problems (Halliwell and Gutteridge 1999). A failure to maintain a balance between the formation of oxidants (e.g., RONS) and their removal by our various antioxidant systems produces a state known as oxidative stress. Powerful oxidants such as those just discussed can damage proteins, DNA, and the unsaturated fatty acid molecules in membranes. It is no wonder, then, that aging and a variety of diseases, such as cancers, heart disease, neurodegenerative diseases, and type 2 diabetes, have a relationship to oxidative
stress.

Figure 5.20 summarizes a number of important reactions that produce and remove ROS, such as SOD, catalase, and GPX. In addition to specific antioxidant enzymes, the body contains a number of nonprotein antioxidant molecules. For example, the essential nutrients vitamin C, vitamin E, and carotenoids such as b-carotene can by themselves scavenge free radicals. Minerals such as selenium, zinc, manganese, and copper are constituents of antioxidant enzymes. Finally, a host of non-nutrient antioxidants from a wide variety of foods of plant origin are important. The general name phytochemical is applied to those molecules that can function in our bodies as antioxidants. Fruits, vegetables, and grains, as well as common products derived from these such as tea and wine, are the major sources. Flavonoids, polyphenols, and lycopene are common examples of phytochemicals.

Exercise and Oxidative Stress

It is extremely difficult to directly measure free radical formation in a human being. Rather, we use indirect measures to point to increased or decreased ROS formation; from this, we make inferences. From a theoretical perspective, during exercise, we should expect a greater formation of superoxide, and therefore hydrogen peroxide and hydroxyl radicals, simply because of an increased flux through the electron transport chain with the increased need for ATP. Since we can greatly increase oxygen consumption and oxidative phosphorylation during exercise, it has been postulated that exercise can increase the generation of oxygen radicals and the possibility of oxidative damage or stress in muscles and other tissues.

Even though the proportion of oxygen that undergoes one- instead of four-electron reduction should decrease with increased activity of the electron transport chain, the fact that the overall flux may increase 10- to 20-fold above resting levels when we exercise should lead us to suspect an increase in superoxide formation. Bailey and colleagues (2003) were the first to actually measure increased formation of free radicals in venous blood of humans during a single-leg exercise task, using an expensive technique known as electron paramagnetic resonance spectroscopy. In this study, the researchers noted that the outflow of free radical species in the venous blood increased as exercise intensity increased.

The active muscle is not the only source of increased RONS formation during exercise. It has been observed that RONS production by white blood cells increases with exercise. However, the effect of RONS production by white blood cells is increasingly blunted the more trained the person is for the particular exercise activity (Mooren, Lechtermann, and Völker 2004). This is likely a consequence of increased antioxidant enzyme activity in white blood cells with training (Elosua et al. 2003). Some types of exercise, such as intense isometric or eccentric contractions, can create inflammation in muscle. Part of the inflammatory response is caused by neutrophils (white blood cell subset) accumulating in the muscle. As mentioned earlier, these cells produce superoxide that can cause damage within the muscle and can also produce other reactive species (Nguyen and Tidball 2003). Obesity, a major public health problem, is characterized by elevated markers of oxidative stress. When people who are obese perform either aerobic or resistance exercise, they produce higher levels of lipid peroxidation products than normal-weight individuals (Vincent, Morgan, and Vincent 2004). Acute feeding of high-fat diets also increases RONS production in rodents. Such a response suggests either an enhanced production or a reduced ability to scavenge free radicals, or both, in the obese state. Aging is also characterized by increased RONS production and oxidative stress, and increased RONS are associated with various diseases of senescence (Pourova et al. 2010).

Rested muscle produces RONS. When muscle becomes more active, formation of RONS increases. Having some RONS in muscle confers a benefit, since they are often important signaling agents that help regulate acute responses to exercise as well as positive adaptations to training in the muscles. Animal studies have demonstrated that inhibiting RONS production during exercise training actually blunts the signaling pathways that regulate adaptations to training, such as increasing mitochondria and aerobic capacity in skeletal muscle (Gomez-Cabrera et al. 2005). Important signaling pathways such as NFkB can help regulate antioxidant adaptations and mitochondrial synthesis consequent to endurance training. The inhibition of RONS production during exercise can blunt the response of these pathways and, consequently, positive training-induced adaptations (Powers et al. 2010). However, a study involving endurance training in humans failed to demonstrate a short-term negative effect of antioxidant supplements on endurance training adaptations (Yfanti et al. 2010).

Dietary antioxidants such as vitamin E and vitamin C have been touted as important for minimizing muscular damage from exercise. Athletes have often been encouraged to supplement their diets with a variety of antioxidants. While eating a diet high in antioxidants would likely have health benefits and could influence longer term effects of oxidative stress such as aging, studies have demonstrated that it is unlikely that large intakes of antioxidants will have significant physiological effects in diminishing exercise-induced muscle damage; instead, they may inhibit positive adaptations to training (McGinley, Shafat, and Donnelly 2009; Gomez-Cabrera et al. 2008). In addition, studies have demonstrated that antioxidant supplements taken during training may also suppress health-promoting benefits such as improved insulin resistance (Ristow et al. 2009).

Unaccustomed exercise and overtraining lead to muscle soreness, inflammation, and damage. This can be reduced by repeated exposure to the activity or through training. Attempts to limit the amount of stress by taking anti-inflammatory medication such as NSAIDs can potentially reduce oxidative stress in muscles that results from infiltration of white blood cells, such as neutrophils and macrophages. However, since the presence of white blood cells, particularly macrophages in muscle following exercise, is obligatory for the activation of muscle satellite cells and their role in stimulating muscle hypertrophy, reducing inflammation may also limit the amount of muscle repair, adaptation, and hypertrophy that would be induced by the training.

MicroRNAs and the Adaptive Response to Exercise Training

MicroRNAs (miRNAs) are a class of short, noncoding RNA molecules that bind to mRNA molecules and play a central role in regulating gene expression through posttranscriptional gene silencing (reviewed in Bushati and Cohen 2007). Most miRNAs are encoded in introns of protein-coding genes and are transcribed by RNA polymerase II as long primary-miRNAs (pri-miRNA) that encode a single miRNA or a cluster of miRNA species. Processing of pri-miRNA species in the nucleus produces stem-loop structures of ∼70 nucleotides, termed precursor-miRNA (pre-miRNA). These pre-miRNAs are transported to the cytoplasm where they are further processed, giving rise to the mature ∼19 to 22 bp of miRNA. The mature miRNA is incorporated into a ribonucleoprotein complex known as the RNA-induced silencing complex (RISC). Generally, miRNAs inhibit protein synthesis by binding (base-pairing) in the 3' untranslated regions of target mRNAs, either repressing translation or bringing about deadenylation and subsequent degradation of mRNA targets. Individual miRNAs can target hundreds of genes, while individual mRNAs can be targeted by multiple miRNAs, making this one of the most complex gene-regulatory processes.

Studies have uncovered a cluster of muscle-specific miRNAs that regulate muscle differentiation and modulate diverse aspects of muscle function (reviewed in van Rooij, Liu, and Olson 2008). The most highly studied are miR-1, miR-133 and miR-206, which are induced during differentiation of myoblasts into myotubes (Callis et al. 2008) and play an important role in muscle mass maintenance. Other miRNA species (miR-23, miR-103, miR-107, and so on) are proposed to play an important role in regulating expression of genes encoding metabolic pathway enzymes in skeletal muscle and other tissues (Wilfred, Wang, and Nelson 2007). Interestingly, studies have shown the potential importance of miRNA regulation in skeletal-muscle adaptations to exercise (reviewed in Roth 2011). For example, miR-1 and miR-133a are downregulated in mouse skeletal muscle during functional overload-induced hypertrophy of the plantaris muscle (McCarthy and Esser 2007). In response to endurance exercise, miR-23, a putative negative regulator of the transcriptional coactivator peroxisome proliferator a coactivator 1 (PGC-1a), was downregulated in mouse skeletal muscle (Safdar et al. 2009). Importantly, downregulation of miR-23 was associated with increased expression of PGC-1a mRNA and protein, along with several downstream targets of PGC-1a signaling.

You are no doubt aware that the ability to increase muscle size in response to resistance and strength training is greater for some people than others. Have you ever wondered why strength training causes large gains in muscle mass in some people (i.e., high responders), whereas others gain very little muscle mass in response to the same training stimulus (i.e., low responders)? This is true even after accounting for differences in age, training status, exercise adherence, and diet. A study by Davidsen and colleagues (2011) helps to shed some light on this issue. In their study, vastus lateralis biopsies were taken from the top and bottom 49 responders, in terms of muscle mass gain, of 56 men who completed a 12-week strength-training program. The expression level of 21 abundant miRNAs was measured to determine whether variation in these miRNAs was able to explain the variation in resistance training-induced gains in muscle mass. They indentified 4 miRNAs that showed uniquely different responses between high responders and low responders. MiR-378, miR-29a, and miR-26a were downregulated in low responders and were unchanged in high responders, while miR-451 was upregulated only in low responders. Therefore, the regulation of protein synthesis by miRNAs may play an important role in explaining the variability in strength training adaptations. However, further research is required to uncover how these miRNAs themselves are regulated and whether they can be targeted for therapeutic interventions.

Compared with endurance exercise, resistance exercise consists of much higher intensity contractions (e.g., usually 70% to 80% of one repetition maximum) repeated at high frequencies (referring again to motoneuron firing frequency) that can only be sustained for short durations due to the fatiguing nature of the exercise. Therefore, the pattern of cytoplasmic [Ca2+] oscillations and changes in [AMP]/[ATP] and ROS production with skeletal muscle contractions is different between endurance and resistance exercise. For example, endurance exercise likely results in extended periods of moderately elevated [Ca2+], while resistance exercise would generate short cycles of significantly higher intracellular [Ca2+] in skeletal muscle (Chin 2010). Therefore, differences in the magnitude and pattern of these primary signals generated by endurance and resistance exercise will result in the activation of different signaling pathways and different gene-expression and protein-synthesis responses in skeletal muscle. A number of studies have shown that resistance exercise results in increased rates of muscle-protein synthesis, about two- to fivefold after exercise for periods up to 48 h before declining to baseline values. Although acute resistance and endurance-type exercise result in a similar global anabolic response in untrained skeletal muscle, increases in muscle mass (i.e., hypertrophy) and strength are significantly greater following chronic resistance training compared with chronic endurance training. This means that chronic resistance training, but not endurance training, increases the rate of muscle-protein synthesis to levels above the rate of protein degradation.

Activation and differentiation of muscle satellite cells into new muscle cells that fuse with existing muscle fibers can also contribute significantly to the hypertrophic response to resistance exercise. Muscle fibers are large multinucleated cells. They are also postmitotic cells in that they no longer have the ability to divide and reproduce themselves. Additionally, in order to significantly increase in size or hypertrophy, muscles have to add more nuclei, since a nucleus is only able to supply mRNA for new protein synthesis to a limited amount of cytoplasm in its vicinity. Hence, a specific ratio of nuclei to cytoplasm must be maintained. In order to do this, skeletal muscles have small specialized satellite cells,or myogenic precursor cells, located at the periphery of their outer membranes. When stimulated by resistance training or by exercise-induced muscle damage, these specialized cells will be activated and induced to create daughter cells or proliferate. These new satellite cells will then fuse with the existing muscle fibers and add their nuclei to the cells to support greater protein synthesis and increase muscle hypertrophy or repair.

It is well known that the primary factor determining the hypertrophic response to contractile activity is the load across the muscle or the mechanical stretch/strain imposed on the muscle fibers, which is higher in resistance exercise than in endurance exercise. Ultimately, these mechanical/force signals are transduced by signaling pathways that regulate transcriptional and translational processes and satellite-cell activation.

For several years, researchers have focused on the role of insulin-like growth factor (IGF)-1 as a primary signaling molecule that mediates skeletal-muscle growth in response to resistance exercise, given that resistance exercise stimulates the secretion of IGF-1. IGF-1 is known to induce muscle hypertrophy by binding to its receptor on the muscle-cell surface and activating the classical growth factor pathway (see figure 3.27). IGF-1 binding to the receptor activates phosphatidylinositol 3-kinase (PI3K), which leads to the activation of Akt, a serine/threonine protein kinase. Akt phosphorylates and inactivates tuberosclerosis complex (TSC2), resulting in the activation of the ras homologous protein enriched in brain (Rheb) and mammalian target of rapamycin (mTOR). mTOR phosphorylates and suppresses the eukaryotic initiation factor 4E binding protein (4E-BP1) to blunt 4E-BP1 inhibition of translation-initiation cap-binding protein eIF4E (see figure 3.21). mTOR also phosphorylates the 70KDa ribosomal S6 protein kinase (p70S6K1), resulting in an increase in protein synthesis.

It is well accepted that mTOR signaling plays a dominant role in the adaptive response to resistance training; however, researchers have questioned the importance of IGF-1 signaling in mediating this response, based on evidence from several studies employing pharmacological and knockout-mouse approaches to systematically manipulate the IGF-1-PI3K-Akt pathway (Philp, Hamilton, and Baar 2011). It appears that the IGF-1 signaling pathway is not required for mTOR activation or increased protein synthesis that is induced by resistance-type exercise. It is possible that mechanical signals working through stretch-activated membrane channels, for example, might be able to activate mTOR and its downstream targets. However, this hypothesis needs to be examined experimentally (Philp, Hamilton, and Baar 2011).

Cellular Damage From RONS

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are sometimes collectively referred to as reactive oxygen and nitrogen species (RONS). RONS, and particularly the highly reactive hydroxyl radical, can react with many molecules in cells, including DNA, proteins, and lipids. The slow buildup of threats to mitochondrial and nuclear DNA over years of oxidative damage is thought to be a factor in aging. The relatively rapid turnover (breakdown and resynthesis) of proteins is also partly due to their susceptibility to oxidative damage and their need to ensure structural integrity to maintain optimal function. The free radical theory of aging was first proposed in the 1950s by the American scientist Denham Harman. It has continued to receive scientific support over the years, and it is now well established as an integral component of the aging process. Polyunsaturated fatty acids, which are important components of the phospholipids found in cell membranes, are particularly susceptible to damage by RONS. It has been known for more than 200 years that exposure of fats to air or oxygen will turn them rancid. This rancidity is caused by peroxidation of the fatty acids by RONS. During peroxidation, RONS such as the hydroxyl radical can react with the phospholipids by abstracting a hydrogen from one of the double bonded hydrogen molecules in the unsaturated fat, and in so doing, forming water. In the following equation, LH represents the lipid and OH* represents the hydroxyl radical, with the * representing the electron that is donated:

LH + OH* → L* + H2O

The lipid radical (L*) can then spontaneously propagate a chain reaction, which can result in peroxidation and breakdown of a number of phospholipids in a cell membrane and damage and disruption of cellular function. Similar damage also results from RONS-induced oxidation of proteins and DNA. As will be discussed in the following section, endogenous antioxidants (e.g., glutathione) and antioxidant vitamins (e.g., vitamin E) can help stop these reactions and prevent peroxidative damage to membranes and other cellular molecules, including proteins and DNA. Limiting oxidative stress may be important in limiting various conditions associated with aging (Pourova et al. 2010)

Protection From Reactive Oxygen and Nitrogen Species

Our bodies produce a number of endogenous oxidants that we have categorized as RONS. Added to this is a host of exogenous substances, such as pollution, ozone, automobile exhaust, solvents, pesticides, and cigarette smoke. Foreign molecules that may pose a hazard to us are generally categorized as xenobiotics. Some xenobiotics are free radicals or can produce RONS. Three approaches to maintaining control over RONS are principal. First, reducing the formation of or exposure to RONS and xenobiotics is an essential step. Second, our endogenous scavenging systems, including antioxidant enzymes and other antioxidant molecules, can scavenge those RONS already present. Finally, mechanisms in cells can upregulate antioxidant defenses in the face of persistent problems (Halliwell and Gutteridge 1999). A failure to maintain a balance between the formation of oxidants (e.g., RONS) and their removal by our various antioxidant systems produces a state known as oxidative stress. Powerful oxidants such as those just discussed can damage proteins, DNA, and the unsaturated fatty acid molecules in membranes. It is no wonder, then, that aging and a variety of diseases, such as cancers, heart disease, neurodegenerative diseases, and type 2 diabetes, have a relationship to oxidative
stress.

Figure 5.20 summarizes a number of important reactions that produce and remove ROS, such as SOD, catalase, and GPX. In addition to specific antioxidant enzymes, the body contains a number of nonprotein antioxidant molecules. For example, the essential nutrients vitamin C, vitamin E, and carotenoids such as b-carotene can by themselves scavenge free radicals. Minerals such as selenium, zinc, manganese, and copper are constituents of antioxidant enzymes. Finally, a host of non-nutrient antioxidants from a wide variety of foods of plant origin are important. The general name phytochemical is applied to those molecules that can function in our bodies as antioxidants. Fruits, vegetables, and grains, as well as common products derived from these such as tea and wine, are the major sources. Flavonoids, polyphenols, and lycopene are common examples of phytochemicals.

Exercise and Oxidative Stress

It is extremely difficult to directly measure free radical formation in a human being. Rather, we use indirect measures to point to increased or decreased ROS formation; from this, we make inferences. From a theoretical perspective, during exercise, we should expect a greater formation of superoxide, and therefore hydrogen peroxide and hydroxyl radicals, simply because of an increased flux through the electron transport chain with the increased need for ATP. Since we can greatly increase oxygen consumption and oxidative phosphorylation during exercise, it has been postulated that exercise can increase the generation of oxygen radicals and the possibility of oxidative damage or stress in muscles and other tissues.

Even though the proportion of oxygen that undergoes one- instead of four-electron reduction should decrease with increased activity of the electron transport chain, the fact that the overall flux may increase 10- to 20-fold above resting levels when we exercise should lead us to suspect an increase in superoxide formation. Bailey and colleagues (2003) were the first to actually measure increased formation of free radicals in venous blood of humans during a single-leg exercise task, using an expensive technique known as electron paramagnetic resonance spectroscopy. In this study, the researchers noted that the outflow of free radical species in the venous blood increased as exercise intensity increased.

The active muscle is not the only source of increased RONS formation during exercise. It has been observed that RONS production by white blood cells increases with exercise. However, the effect of RONS production by white blood cells is increasingly blunted the more trained the person is for the particular exercise activity (Mooren, Lechtermann, and Völker 2004). This is likely a consequence of increased antioxidant enzyme activity in white blood cells with training (Elosua et al. 2003). Some types of exercise, such as intense isometric or eccentric contractions, can create inflammation in muscle. Part of the inflammatory response is caused by neutrophils (white blood cell subset) accumulating in the muscle. As mentioned earlier, these cells produce superoxide that can cause damage within the muscle and can also produce other reactive species (Nguyen and Tidball 2003). Obesity, a major public health problem, is characterized by elevated markers of oxidative stress. When people who are obese perform either aerobic or resistance exercise, they produce higher levels of lipid peroxidation products than normal-weight individuals (Vincent, Morgan, and Vincent 2004). Acute feeding of high-fat diets also increases RONS production in rodents. Such a response suggests either an enhanced production or a reduced ability to scavenge free radicals, or both, in the obese state. Aging is also characterized by increased RONS production and oxidative stress, and increased RONS are associated with various diseases of senescence (Pourova et al. 2010).

Rested muscle produces RONS. When muscle becomes more active, formation of RONS increases. Having some RONS in muscle confers a benefit, since they are often important signaling agents that help regulate acute responses to exercise as well as positive adaptations to training in the muscles. Animal studies have demonstrated that inhibiting RONS production during exercise training actually blunts the signaling pathways that regulate adaptations to training, such as increasing mitochondria and aerobic capacity in skeletal muscle (Gomez-Cabrera et al. 2005). Important signaling pathways such as NFkB can help regulate antioxidant adaptations and mitochondrial synthesis consequent to endurance training. The inhibition of RONS production during exercise can blunt the response of these pathways and, consequently, positive training-induced adaptations (Powers et al. 2010). However, a study involving endurance training in humans failed to demonstrate a short-term negative effect of antioxidant supplements on endurance training adaptations (Yfanti et al. 2010).

Dietary antioxidants such as vitamin E and vitamin C have been touted as important for minimizing muscular damage from exercise. Athletes have often been encouraged to supplement their diets with a variety of antioxidants. While eating a diet high in antioxidants would likely have health benefits and could influence longer term effects of oxidative stress such as aging, studies have demonstrated that it is unlikely that large intakes of antioxidants will have significant physiological effects in diminishing exercise-induced muscle damage; instead, they may inhibit positive adaptations to training (McGinley, Shafat, and Donnelly 2009; Gomez-Cabrera et al. 2008). In addition, studies have demonstrated that antioxidant supplements taken during training may also suppress health-promoting benefits such as improved insulin resistance (Ristow et al. 2009).

Unaccustomed exercise and overtraining lead to muscle soreness, inflammation, and damage. This can be reduced by repeated exposure to the activity or through training. Attempts to limit the amount of stress by taking anti-inflammatory medication such as NSAIDs can potentially reduce oxidative stress in muscles that results from infiltration of white blood cells, such as neutrophils and macrophages. However, since the presence of white blood cells, particularly macrophages in muscle following exercise, is obligatory for the activation of muscle satellite cells and their role in stimulating muscle hypertrophy, reducing inflammation may also limit the amount of muscle repair, adaptation, and hypertrophy that would be induced by the training.

MicroRNAs and the Adaptive Response to Exercise Training

MicroRNAs (miRNAs) are a class of short, noncoding RNA molecules that bind to mRNA molecules and play a central role in regulating gene expression through posttranscriptional gene silencing (reviewed in Bushati and Cohen 2007). Most miRNAs are encoded in introns of protein-coding genes and are transcribed by RNA polymerase II as long primary-miRNAs (pri-miRNA) that encode a single miRNA or a cluster of miRNA species. Processing of pri-miRNA species in the nucleus produces stem-loop structures of ∼70 nucleotides, termed precursor-miRNA (pre-miRNA). These pre-miRNAs are transported to the cytoplasm where they are further processed, giving rise to the mature ∼19 to 22 bp of miRNA. The mature miRNA is incorporated into a ribonucleoprotein complex known as the RNA-induced silencing complex (RISC). Generally, miRNAs inhibit protein synthesis by binding (base-pairing) in the 3' untranslated regions of target mRNAs, either repressing translation or bringing about deadenylation and subsequent degradation of mRNA targets. Individual miRNAs can target hundreds of genes, while individual mRNAs can be targeted by multiple miRNAs, making this one of the most complex gene-regulatory processes.

Studies have uncovered a cluster of muscle-specific miRNAs that regulate muscle differentiation and modulate diverse aspects of muscle function (reviewed in van Rooij, Liu, and Olson 2008). The most highly studied are miR-1, miR-133 and miR-206, which are induced during differentiation of myoblasts into myotubes (Callis et al. 2008) and play an important role in muscle mass maintenance. Other miRNA species (miR-23, miR-103, miR-107, and so on) are proposed to play an important role in regulating expression of genes encoding metabolic pathway enzymes in skeletal muscle and other tissues (Wilfred, Wang, and Nelson 2007). Interestingly, studies have shown the potential importance of miRNA regulation in skeletal-muscle adaptations to exercise (reviewed in Roth 2011). For example, miR-1 and miR-133a are downregulated in mouse skeletal muscle during functional overload-induced hypertrophy of the plantaris muscle (McCarthy and Esser 2007). In response to endurance exercise, miR-23, a putative negative regulator of the transcriptional coactivator peroxisome proliferator a coactivator 1 (PGC-1a), was downregulated in mouse skeletal muscle (Safdar et al. 2009). Importantly, downregulation of miR-23 was associated with increased expression of PGC-1a mRNA and protein, along with several downstream targets of PGC-1a signaling.

You are no doubt aware that the ability to increase muscle size in response to resistance and strength training is greater for some people than others. Have you ever wondered why strength training causes large gains in muscle mass in some people (i.e., high responders), whereas others gain very little muscle mass in response to the same training stimulus (i.e., low responders)? This is true even after accounting for differences in age, training status, exercise adherence, and diet. A study by Davidsen and colleagues (2011) helps to shed some light on this issue. In their study, vastus lateralis biopsies were taken from the top and bottom 49 responders, in terms of muscle mass gain, of 56 men who completed a 12-week strength-training program. The expression level of 21 abundant miRNAs was measured to determine whether variation in these miRNAs was able to explain the variation in resistance training-induced gains in muscle mass. They indentified 4 miRNAs that showed uniquely different responses between high responders and low responders. MiR-378, miR-29a, and miR-26a were downregulated in low responders and were unchanged in high responders, while miR-451 was upregulated only in low responders. Therefore, the regulation of protein synthesis by miRNAs may play an important role in explaining the variability in strength training adaptations. However, further research is required to uncover how these miRNAs themselves are regulated and whether they can be targeted for therapeutic interventions.

Compared with endurance exercise, resistance exercise consists of much higher intensity contractions (e.g., usually 70% to 80% of one repetition maximum) repeated at high frequencies (referring again to motoneuron firing frequency) that can only be sustained for short durations due to the fatiguing nature of the exercise. Therefore, the pattern of cytoplasmic [Ca2+] oscillations and changes in [AMP]/[ATP] and ROS production with skeletal muscle contractions is different between endurance and resistance exercise. For example, endurance exercise likely results in extended periods of moderately elevated [Ca2+], while resistance exercise would generate short cycles of significantly higher intracellular [Ca2+] in skeletal muscle (Chin 2010). Therefore, differences in the magnitude and pattern of these primary signals generated by endurance and resistance exercise will result in the activation of different signaling pathways and different gene-expression and protein-synthesis responses in skeletal muscle. A number of studies have shown that resistance exercise results in increased rates of muscle-protein synthesis, about two- to fivefold after exercise for periods up to 48 h before declining to baseline values. Although acute resistance and endurance-type exercise result in a similar global anabolic response in untrained skeletal muscle, increases in muscle mass (i.e., hypertrophy) and strength are significantly greater following chronic resistance training compared with chronic endurance training. This means that chronic resistance training, but not endurance training, increases the rate of muscle-protein synthesis to levels above the rate of protein degradation.

Activation and differentiation of muscle satellite cells into new muscle cells that fuse with existing muscle fibers can also contribute significantly to the hypertrophic response to resistance exercise. Muscle fibers are large multinucleated cells. They are also postmitotic cells in that they no longer have the ability to divide and reproduce themselves. Additionally, in order to significantly increase in size or hypertrophy, muscles have to add more nuclei, since a nucleus is only able to supply mRNA for new protein synthesis to a limited amount of cytoplasm in its vicinity. Hence, a specific ratio of nuclei to cytoplasm must be maintained. In order to do this, skeletal muscles have small specialized satellite cells,or myogenic precursor cells, located at the periphery of their outer membranes. When stimulated by resistance training or by exercise-induced muscle damage, these specialized cells will be activated and induced to create daughter cells or proliferate. These new satellite cells will then fuse with the existing muscle fibers and add their nuclei to the cells to support greater protein synthesis and increase muscle hypertrophy or repair.

It is well known that the primary factor determining the hypertrophic response to contractile activity is the load across the muscle or the mechanical stretch/strain imposed on the muscle fibers, which is higher in resistance exercise than in endurance exercise. Ultimately, these mechanical/force signals are transduced by signaling pathways that regulate transcriptional and translational processes and satellite-cell activation.

For several years, researchers have focused on the role of insulin-like growth factor (IGF)-1 as a primary signaling molecule that mediates skeletal-muscle growth in response to resistance exercise, given that resistance exercise stimulates the secretion of IGF-1. IGF-1 is known to induce muscle hypertrophy by binding to its receptor on the muscle-cell surface and activating the classical growth factor pathway (see figure 3.27). IGF-1 binding to the receptor activates phosphatidylinositol 3-kinase (PI3K), which leads to the activation of Akt, a serine/threonine protein kinase. Akt phosphorylates and inactivates tuberosclerosis complex (TSC2), resulting in the activation of the ras homologous protein enriched in brain (Rheb) and mammalian target of rapamycin (mTOR). mTOR phosphorylates and suppresses the eukaryotic initiation factor 4E binding protein (4E-BP1) to blunt 4E-BP1 inhibition of translation-initiation cap-binding protein eIF4E (see figure 3.21). mTOR also phosphorylates the 70KDa ribosomal S6 protein kinase (p70S6K1), resulting in an increase in protein synthesis.

It is well accepted that mTOR signaling plays a dominant role in the adaptive response to resistance training; however, researchers have questioned the importance of IGF-1 signaling in mediating this response, based on evidence from several studies employing pharmacological and knockout-mouse approaches to systematically manipulate the IGF-1-PI3K-Akt pathway (Philp, Hamilton, and Baar 2011). It appears that the IGF-1 signaling pathway is not required for mTOR activation or increased protein synthesis that is induced by resistance-type exercise. It is possible that mechanical signals working through stretch-activated membrane channels, for example, might be able to activate mTOR and its downstream targets. However, this hypothesis needs to be examined experimentally (Philp, Hamilton, and Baar 2011).

Cellular Damage From RONS

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are sometimes collectively referred to as reactive oxygen and nitrogen species (RONS). RONS, and particularly the highly reactive hydroxyl radical, can react with many molecules in cells, including DNA, proteins, and lipids. The slow buildup of threats to mitochondrial and nuclear DNA over years of oxidative damage is thought to be a factor in aging. The relatively rapid turnover (breakdown and resynthesis) of proteins is also partly due to their susceptibility to oxidative damage and their need to ensure structural integrity to maintain optimal function. The free radical theory of aging was first proposed in the 1950s by the American scientist Denham Harman. It has continued to receive scientific support over the years, and it is now well established as an integral component of the aging process. Polyunsaturated fatty acids, which are important components of the phospholipids found in cell membranes, are particularly susceptible to damage by RONS. It has been known for more than 200 years that exposure of fats to air or oxygen will turn them rancid. This rancidity is caused by peroxidation of the fatty acids by RONS. During peroxidation, RONS such as the hydroxyl radical can react with the phospholipids by abstracting a hydrogen from one of the double bonded hydrogen molecules in the unsaturated fat, and in so doing, forming water. In the following equation, LH represents the lipid and OH* represents the hydroxyl radical, with the * representing the electron that is donated:

LH + OH* → L* + H2O

The lipid radical (L*) can then spontaneously propagate a chain reaction, which can result in peroxidation and breakdown of a number of phospholipids in a cell membrane and damage and disruption of cellular function. Similar damage also results from RONS-induced oxidation of proteins and DNA. As will be discussed in the following section, endogenous antioxidants (e.g., glutathione) and antioxidant vitamins (e.g., vitamin E) can help stop these reactions and prevent peroxidative damage to membranes and other cellular molecules, including proteins and DNA. Limiting oxidative stress may be important in limiting various conditions associated with aging (Pourova et al. 2010)

Protection From Reactive Oxygen and Nitrogen Species

Our bodies produce a number of endogenous oxidants that we have categorized as RONS. Added to this is a host of exogenous substances, such as pollution, ozone, automobile exhaust, solvents, pesticides, and cigarette smoke. Foreign molecules that may pose a hazard to us are generally categorized as xenobiotics. Some xenobiotics are free radicals or can produce RONS. Three approaches to maintaining control over RONS are principal. First, reducing the formation of or exposure to RONS and xenobiotics is an essential step. Second, our endogenous scavenging systems, including antioxidant enzymes and other antioxidant molecules, can scavenge those RONS already present. Finally, mechanisms in cells can upregulate antioxidant defenses in the face of persistent problems (Halliwell and Gutteridge 1999). A failure to maintain a balance between the formation of oxidants (e.g., RONS) and their removal by our various antioxidant systems produces a state known as oxidative stress. Powerful oxidants such as those just discussed can damage proteins, DNA, and the unsaturated fatty acid molecules in membranes. It is no wonder, then, that aging and a variety of diseases, such as cancers, heart disease, neurodegenerative diseases, and type 2 diabetes, have a relationship to oxidative
stress.

Figure 5.20 summarizes a number of important reactions that produce and remove ROS, such as SOD, catalase, and GPX. In addition to specific antioxidant enzymes, the body contains a number of nonprotein antioxidant molecules. For example, the essential nutrients vitamin C, vitamin E, and carotenoids such as b-carotene can by themselves scavenge free radicals. Minerals such as selenium, zinc, manganese, and copper are constituents of antioxidant enzymes. Finally, a host of non-nutrient antioxidants from a wide variety of foods of plant origin are important. The general name phytochemical is applied to those molecules that can function in our bodies as antioxidants. Fruits, vegetables, and grains, as well as common products derived from these such as tea and wine, are the major sources. Flavonoids, polyphenols, and lycopene are common examples of phytochemicals.

Exercise and Oxidative Stress

It is extremely difficult to directly measure free radical formation in a human being. Rather, we use indirect measures to point to increased or decreased ROS formation; from this, we make inferences. From a theoretical perspective, during exercise, we should expect a greater formation of superoxide, and therefore hydrogen peroxide and hydroxyl radicals, simply because of an increased flux through the electron transport chain with the increased need for ATP. Since we can greatly increase oxygen consumption and oxidative phosphorylation during exercise, it has been postulated that exercise can increase the generation of oxygen radicals and the possibility of oxidative damage or stress in muscles and other tissues.

Even though the proportion of oxygen that undergoes one- instead of four-electron reduction should decrease with increased activity of the electron transport chain, the fact that the overall flux may increase 10- to 20-fold above resting levels when we exercise should lead us to suspect an increase in superoxide formation. Bailey and colleagues (2003) were the first to actually measure increased formation of free radicals in venous blood of humans during a single-leg exercise task, using an expensive technique known as electron paramagnetic resonance spectroscopy. In this study, the researchers noted that the outflow of free radical species in the venous blood increased as exercise intensity increased.

The active muscle is not the only source of increased RONS formation during exercise. It has been observed that RONS production by white blood cells increases with exercise. However, the effect of RONS production by white blood cells is increasingly blunted the more trained the person is for the particular exercise activity (Mooren, Lechtermann, and Völker 2004). This is likely a consequence of increased antioxidant enzyme activity in white blood cells with training (Elosua et al. 2003). Some types of exercise, such as intense isometric or eccentric contractions, can create inflammation in muscle. Part of the inflammatory response is caused by neutrophils (white blood cell subset) accumulating in the muscle. As mentioned earlier, these cells produce superoxide that can cause damage within the muscle and can also produce other reactive species (Nguyen and Tidball 2003). Obesity, a major public health problem, is characterized by elevated markers of oxidative stress. When people who are obese perform either aerobic or resistance exercise, they produce higher levels of lipid peroxidation products than normal-weight individuals (Vincent, Morgan, and Vincent 2004). Acute feeding of high-fat diets also increases RONS production in rodents. Such a response suggests either an enhanced production or a reduced ability to scavenge free radicals, or both, in the obese state. Aging is also characterized by increased RONS production and oxidative stress, and increased RONS are associated with various diseases of senescence (Pourova et al. 2010).

Rested muscle produces RONS. When muscle becomes more active, formation of RONS increases. Having some RONS in muscle confers a benefit, since they are often important signaling agents that help regulate acute responses to exercise as well as positive adaptations to training in the muscles. Animal studies have demonstrated that inhibiting RONS production during exercise training actually blunts the signaling pathways that regulate adaptations to training, such as increasing mitochondria and aerobic capacity in skeletal muscle (Gomez-Cabrera et al. 2005). Important signaling pathways such as NFkB can help regulate antioxidant adaptations and mitochondrial synthesis consequent to endurance training. The inhibition of RONS production during exercise can blunt the response of these pathways and, consequently, positive training-induced adaptations (Powers et al. 2010). However, a study involving endurance training in humans failed to demonstrate a short-term negative effect of antioxidant supplements on endurance training adaptations (Yfanti et al. 2010).

Dietary antioxidants such as vitamin E and vitamin C have been touted as important for minimizing muscular damage from exercise. Athletes have often been encouraged to supplement their diets with a variety of antioxidants. While eating a diet high in antioxidants would likely have health benefits and could influence longer term effects of oxidative stress such as aging, studies have demonstrated that it is unlikely that large intakes of antioxidants will have significant physiological effects in diminishing exercise-induced muscle damage; instead, they may inhibit positive adaptations to training (McGinley, Shafat, and Donnelly 2009; Gomez-Cabrera et al. 2008). In addition, studies have demonstrated that antioxidant supplements taken during training may also suppress health-promoting benefits such as improved insulin resistance (Ristow et al. 2009).

Unaccustomed exercise and overtraining lead to muscle soreness, inflammation, and damage. This can be reduced by repeated exposure to the activity or through training. Attempts to limit the amount of stress by taking anti-inflammatory medication such as NSAIDs can potentially reduce oxidative stress in muscles that results from infiltration of white blood cells, such as neutrophils and macrophages. However, since the presence of white blood cells, particularly macrophages in muscle following exercise, is obligatory for the activation of muscle satellite cells and their role in stimulating muscle hypertrophy, reducing inflammation may also limit the amount of muscle repair, adaptation, and hypertrophy that would be induced by the training.

MicroRNAs and the Adaptive Response to Exercise Training

MicroRNAs (miRNAs) are a class of short, noncoding RNA molecules that bind to mRNA molecules and play a central role in regulating gene expression through posttranscriptional gene silencing (reviewed in Bushati and Cohen 2007). Most miRNAs are encoded in introns of protein-coding genes and are transcribed by RNA polymerase II as long primary-miRNAs (pri-miRNA) that encode a single miRNA or a cluster of miRNA species. Processing of pri-miRNA species in the nucleus produces stem-loop structures of ∼70 nucleotides, termed precursor-miRNA (pre-miRNA). These pre-miRNAs are transported to the cytoplasm where they are further processed, giving rise to the mature ∼19 to 22 bp of miRNA. The mature miRNA is incorporated into a ribonucleoprotein complex known as the RNA-induced silencing complex (RISC). Generally, miRNAs inhibit protein synthesis by binding (base-pairing) in the 3' untranslated regions of target mRNAs, either repressing translation or bringing about deadenylation and subsequent degradation of mRNA targets. Individual miRNAs can target hundreds of genes, while individual mRNAs can be targeted by multiple miRNAs, making this one of the most complex gene-regulatory processes.

Studies have uncovered a cluster of muscle-specific miRNAs that regulate muscle differentiation and modulate diverse aspects of muscle function (reviewed in van Rooij, Liu, and Olson 2008). The most highly studied are miR-1, miR-133 and miR-206, which are induced during differentiation of myoblasts into myotubes (Callis et al. 2008) and play an important role in muscle mass maintenance. Other miRNA species (miR-23, miR-103, miR-107, and so on) are proposed to play an important role in regulating expression of genes encoding metabolic pathway enzymes in skeletal muscle and other tissues (Wilfred, Wang, and Nelson 2007). Interestingly, studies have shown the potential importance of miRNA regulation in skeletal-muscle adaptations to exercise (reviewed in Roth 2011). For example, miR-1 and miR-133a are downregulated in mouse skeletal muscle during functional overload-induced hypertrophy of the plantaris muscle (McCarthy and Esser 2007). In response to endurance exercise, miR-23, a putative negative regulator of the transcriptional coactivator peroxisome proliferator a coactivator 1 (PGC-1a), was downregulated in mouse skeletal muscle (Safdar et al. 2009). Importantly, downregulation of miR-23 was associated with increased expression of PGC-1a mRNA and protein, along with several downstream targets of PGC-1a signaling.

You are no doubt aware that the ability to increase muscle size in response to resistance and strength training is greater for some people than others. Have you ever wondered why strength training causes large gains in muscle mass in some people (i.e., high responders), whereas others gain very little muscle mass in response to the same training stimulus (i.e., low responders)? This is true even after accounting for differences in age, training status, exercise adherence, and diet. A study by Davidsen and colleagues (2011) helps to shed some light on this issue. In their study, vastus lateralis biopsies were taken from the top and bottom 49 responders, in terms of muscle mass gain, of 56 men who completed a 12-week strength-training program. The expression level of 21 abundant miRNAs was measured to determine whether variation in these miRNAs was able to explain the variation in resistance training-induced gains in muscle mass. They indentified 4 miRNAs that showed uniquely different responses between high responders and low responders. MiR-378, miR-29a, and miR-26a were downregulated in low responders and were unchanged in high responders, while miR-451 was upregulated only in low responders. Therefore, the regulation of protein synthesis by miRNAs may play an important role in explaining the variability in strength training adaptations. However, further research is required to uncover how these miRNAs themselves are regulated and whether they can be targeted for therapeutic interventions.

Compared with endurance exercise, resistance exercise consists of much higher intensity contractions (e.g., usually 70% to 80% of one repetition maximum) repeated at high frequencies (referring again to motoneuron firing frequency) that can only be sustained for short durations due to the fatiguing nature of the exercise. Therefore, the pattern of cytoplasmic [Ca2+] oscillations and changes in [AMP]/[ATP] and ROS production with skeletal muscle contractions is different between endurance and resistance exercise. For example, endurance exercise likely results in extended periods of moderately elevated [Ca2+], while resistance exercise would generate short cycles of significantly higher intracellular [Ca2+] in skeletal muscle (Chin 2010). Therefore, differences in the magnitude and pattern of these primary signals generated by endurance and resistance exercise will result in the activation of different signaling pathways and different gene-expression and protein-synthesis responses in skeletal muscle. A number of studies have shown that resistance exercise results in increased rates of muscle-protein synthesis, about two- to fivefold after exercise for periods up to 48 h before declining to baseline values. Although acute resistance and endurance-type exercise result in a similar global anabolic response in untrained skeletal muscle, increases in muscle mass (i.e., hypertrophy) and strength are significantly greater following chronic resistance training compared with chronic endurance training. This means that chronic resistance training, but not endurance training, increases the rate of muscle-protein synthesis to levels above the rate of protein degradation.

Activation and differentiation of muscle satellite cells into new muscle cells that fuse with existing muscle fibers can also contribute significantly to the hypertrophic response to resistance exercise. Muscle fibers are large multinucleated cells. They are also postmitotic cells in that they no longer have the ability to divide and reproduce themselves. Additionally, in order to significantly increase in size or hypertrophy, muscles have to add more nuclei, since a nucleus is only able to supply mRNA for new protein synthesis to a limited amount of cytoplasm in its vicinity. Hence, a specific ratio of nuclei to cytoplasm must be maintained. In order to do this, skeletal muscles have small specialized satellite cells,or myogenic precursor cells, located at the periphery of their outer membranes. When stimulated by resistance training or by exercise-induced muscle damage, these specialized cells will be activated and induced to create daughter cells or proliferate. These new satellite cells will then fuse with the existing muscle fibers and add their nuclei to the cells to support greater protein synthesis and increase muscle hypertrophy or repair.

It is well known that the primary factor determining the hypertrophic response to contractile activity is the load across the muscle or the mechanical stretch/strain imposed on the muscle fibers, which is higher in resistance exercise than in endurance exercise. Ultimately, these mechanical/force signals are transduced by signaling pathways that regulate transcriptional and translational processes and satellite-cell activation.

For several years, researchers have focused on the role of insulin-like growth factor (IGF)-1 as a primary signaling molecule that mediates skeletal-muscle growth in response to resistance exercise, given that resistance exercise stimulates the secretion of IGF-1. IGF-1 is known to induce muscle hypertrophy by binding to its receptor on the muscle-cell surface and activating the classical growth factor pathway (see figure 3.27). IGF-1 binding to the receptor activates phosphatidylinositol 3-kinase (PI3K), which leads to the activation of Akt, a serine/threonine protein kinase. Akt phosphorylates and inactivates tuberosclerosis complex (TSC2), resulting in the activation of the ras homologous protein enriched in brain (Rheb) and mammalian target of rapamycin (mTOR). mTOR phosphorylates and suppresses the eukaryotic initiation factor 4E binding protein (4E-BP1) to blunt 4E-BP1 inhibition of translation-initiation cap-binding protein eIF4E (see figure 3.21). mTOR also phosphorylates the 70KDa ribosomal S6 protein kinase (p70S6K1), resulting in an increase in protein synthesis.

It is well accepted that mTOR signaling plays a dominant role in the adaptive response to resistance training; however, researchers have questioned the importance of IGF-1 signaling in mediating this response, based on evidence from several studies employing pharmacological and knockout-mouse approaches to systematically manipulate the IGF-1-PI3K-Akt pathway (Philp, Hamilton, and Baar 2011). It appears that the IGF-1 signaling pathway is not required for mTOR activation or increased protein synthesis that is induced by resistance-type exercise. It is possible that mechanical signals working through stretch-activated membrane channels, for example, might be able to activate mTOR and its downstream targets. However, this hypothesis needs to be examined experimentally (Philp, Hamilton, and Baar 2011).

Cellular Damage From RONS

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are sometimes collectively referred to as reactive oxygen and nitrogen species (RONS). RONS, and particularly the highly reactive hydroxyl radical, can react with many molecules in cells, including DNA, proteins, and lipids. The slow buildup of threats to mitochondrial and nuclear DNA over years of oxidative damage is thought to be a factor in aging. The relatively rapid turnover (breakdown and resynthesis) of proteins is also partly due to their susceptibility to oxidative damage and their need to ensure structural integrity to maintain optimal function. The free radical theory of aging was first proposed in the 1950s by the American scientist Denham Harman. It has continued to receive scientific support over the years, and it is now well established as an integral component of the aging process. Polyunsaturated fatty acids, which are important components of the phospholipids found in cell membranes, are particularly susceptible to damage by RONS. It has been known for more than 200 years that exposure of fats to air or oxygen will turn them rancid. This rancidity is caused by peroxidation of the fatty acids by RONS. During peroxidation, RONS such as the hydroxyl radical can react with the phospholipids by abstracting a hydrogen from one of the double bonded hydrogen molecules in the unsaturated fat, and in so doing, forming water. In the following equation, LH represents the lipid and OH* represents the hydroxyl radical, with the * representing the electron that is donated:

LH + OH* → L* + H2O

The lipid radical (L*) can then spontaneously propagate a chain reaction, which can result in peroxidation and breakdown of a number of phospholipids in a cell membrane and damage and disruption of cellular function. Similar damage also results from RONS-induced oxidation of proteins and DNA. As will be discussed in the following section, endogenous antioxidants (e.g., glutathione) and antioxidant vitamins (e.g., vitamin E) can help stop these reactions and prevent peroxidative damage to membranes and other cellular molecules, including proteins and DNA. Limiting oxidative stress may be important in limiting various conditions associated with aging (Pourova et al. 2010)

Protection From Reactive Oxygen and Nitrogen Species

Our bodies produce a number of endogenous oxidants that we have categorized as RONS. Added to this is a host of exogenous substances, such as pollution, ozone, automobile exhaust, solvents, pesticides, and cigarette smoke. Foreign molecules that may pose a hazard to us are generally categorized as xenobiotics. Some xenobiotics are free radicals or can produce RONS. Three approaches to maintaining control over RONS are principal. First, reducing the formation of or exposure to RONS and xenobiotics is an essential step. Second, our endogenous scavenging systems, including antioxidant enzymes and other antioxidant molecules, can scavenge those RONS already present. Finally, mechanisms in cells can upregulate antioxidant defenses in the face of persistent problems (Halliwell and Gutteridge 1999). A failure to maintain a balance between the formation of oxidants (e.g., RONS) and their removal by our various antioxidant systems produces a state known as oxidative stress. Powerful oxidants such as those just discussed can damage proteins, DNA, and the unsaturated fatty acid molecules in membranes. It is no wonder, then, that aging and a variety of diseases, such as cancers, heart disease, neurodegenerative diseases, and type 2 diabetes, have a relationship to oxidative
stress.

Figure 5.20 summarizes a number of important reactions that produce and remove ROS, such as SOD, catalase, and GPX. In addition to specific antioxidant enzymes, the body contains a number of nonprotein antioxidant molecules. For example, the essential nutrients vitamin C, vitamin E, and carotenoids such as b-carotene can by themselves scavenge free radicals. Minerals such as selenium, zinc, manganese, and copper are constituents of antioxidant enzymes. Finally, a host of non-nutrient antioxidants from a wide variety of foods of plant origin are important. The general name phytochemical is applied to those molecules that can function in our bodies as antioxidants. Fruits, vegetables, and grains, as well as common products derived from these such as tea and wine, are the major sources. Flavonoids, polyphenols, and lycopene are common examples of phytochemicals.

Exercise and Oxidative Stress

It is extremely difficult to directly measure free radical formation in a human being. Rather, we use indirect measures to point to increased or decreased ROS formation; from this, we make inferences. From a theoretical perspective, during exercise, we should expect a greater formation of superoxide, and therefore hydrogen peroxide and hydroxyl radicals, simply because of an increased flux through the electron transport chain with the increased need for ATP. Since we can greatly increase oxygen consumption and oxidative phosphorylation during exercise, it has been postulated that exercise can increase the generation of oxygen radicals and the possibility of oxidative damage or stress in muscles and other tissues.

Even though the proportion of oxygen that undergoes one- instead of four-electron reduction should decrease with increased activity of the electron transport chain, the fact that the overall flux may increase 10- to 20-fold above resting levels when we exercise should lead us to suspect an increase in superoxide formation. Bailey and colleagues (2003) were the first to actually measure increased formation of free radicals in venous blood of humans during a single-leg exercise task, using an expensive technique known as electron paramagnetic resonance spectroscopy. In this study, the researchers noted that the outflow of free radical species in the venous blood increased as exercise intensity increased.

The active muscle is not the only source of increased RONS formation during exercise. It has been observed that RONS production by white blood cells increases with exercise. However, the effect of RONS production by white blood cells is increasingly blunted the more trained the person is for the particular exercise activity (Mooren, Lechtermann, and Völker 2004). This is likely a consequence of increased antioxidant enzyme activity in white blood cells with training (Elosua et al. 2003). Some types of exercise, such as intense isometric or eccentric contractions, can create inflammation in muscle. Part of the inflammatory response is caused by neutrophils (white blood cell subset) accumulating in the muscle. As mentioned earlier, these cells produce superoxide that can cause damage within the muscle and can also produce other reactive species (Nguyen and Tidball 2003). Obesity, a major public health problem, is characterized by elevated markers of oxidative stress. When people who are obese perform either aerobic or resistance exercise, they produce higher levels of lipid peroxidation products than normal-weight individuals (Vincent, Morgan, and Vincent 2004). Acute feeding of high-fat diets also increases RONS production in rodents. Such a response suggests either an enhanced production or a reduced ability to scavenge free radicals, or both, in the obese state. Aging is also characterized by increased RONS production and oxidative stress, and increased RONS are associated with various diseases of senescence (Pourova et al. 2010).

Rested muscle produces RONS. When muscle becomes more active, formation of RONS increases. Having some RONS in muscle confers a benefit, since they are often important signaling agents that help regulate acute responses to exercise as well as positive adaptations to training in the muscles. Animal studies have demonstrated that inhibiting RONS production during exercise training actually blunts the signaling pathways that regulate adaptations to training, such as increasing mitochondria and aerobic capacity in skeletal muscle (Gomez-Cabrera et al. 2005). Important signaling pathways such as NFkB can help regulate antioxidant adaptations and mitochondrial synthesis consequent to endurance training. The inhibition of RONS production during exercise can blunt the response of these pathways and, consequently, positive training-induced adaptations (Powers et al. 2010). However, a study involving endurance training in humans failed to demonstrate a short-term negative effect of antioxidant supplements on endurance training adaptations (Yfanti et al. 2010).

Dietary antioxidants such as vitamin E and vitamin C have been touted as important for minimizing muscular damage from exercise. Athletes have often been encouraged to supplement their diets with a variety of antioxidants. While eating a diet high in antioxidants would likely have health benefits and could influence longer term effects of oxidative stress such as aging, studies have demonstrated that it is unlikely that large intakes of antioxidants will have significant physiological effects in diminishing exercise-induced muscle damage; instead, they may inhibit positive adaptations to training (McGinley, Shafat, and Donnelly 2009; Gomez-Cabrera et al. 2008). In addition, studies have demonstrated that antioxidant supplements taken during training may also suppress health-promoting benefits such as improved insulin resistance (Ristow et al. 2009).

Unaccustomed exercise and overtraining lead to muscle soreness, inflammation, and damage. This can be reduced by repeated exposure to the activity or through training. Attempts to limit the amount of stress by taking anti-inflammatory medication such as NSAIDs can potentially reduce oxidative stress in muscles that results from infiltration of white blood cells, such as neutrophils and macrophages. However, since the presence of white blood cells, particularly macrophages in muscle following exercise, is obligatory for the activation of muscle satellite cells and their role in stimulating muscle hypertrophy, reducing inflammation may also limit the amount of muscle repair, adaptation, and hypertrophy that would be induced by the training.

MicroRNAs and the Adaptive Response to Exercise Training

MicroRNAs (miRNAs) are a class of short, noncoding RNA molecules that bind to mRNA molecules and play a central role in regulating gene expression through posttranscriptional gene silencing (reviewed in Bushati and Cohen 2007). Most miRNAs are encoded in introns of protein-coding genes and are transcribed by RNA polymerase II as long primary-miRNAs (pri-miRNA) that encode a single miRNA or a cluster of miRNA species. Processing of pri-miRNA species in the nucleus produces stem-loop structures of ∼70 nucleotides, termed precursor-miRNA (pre-miRNA). These pre-miRNAs are transported to the cytoplasm where they are further processed, giving rise to the mature ∼19 to 22 bp of miRNA. The mature miRNA is incorporated into a ribonucleoprotein complex known as the RNA-induced silencing complex (RISC). Generally, miRNAs inhibit protein synthesis by binding (base-pairing) in the 3' untranslated regions of target mRNAs, either repressing translation or bringing about deadenylation and subsequent degradation of mRNA targets. Individual miRNAs can target hundreds of genes, while individual mRNAs can be targeted by multiple miRNAs, making this one of the most complex gene-regulatory processes.

Studies have uncovered a cluster of muscle-specific miRNAs that regulate muscle differentiation and modulate diverse aspects of muscle function (reviewed in van Rooij, Liu, and Olson 2008). The most highly studied are miR-1, miR-133 and miR-206, which are induced during differentiation of myoblasts into myotubes (Callis et al. 2008) and play an important role in muscle mass maintenance. Other miRNA species (miR-23, miR-103, miR-107, and so on) are proposed to play an important role in regulating expression of genes encoding metabolic pathway enzymes in skeletal muscle and other tissues (Wilfred, Wang, and Nelson 2007). Interestingly, studies have shown the potential importance of miRNA regulation in skeletal-muscle adaptations to exercise (reviewed in Roth 2011). For example, miR-1 and miR-133a are downregulated in mouse skeletal muscle during functional overload-induced hypertrophy of the plantaris muscle (McCarthy and Esser 2007). In response to endurance exercise, miR-23, a putative negative regulator of the transcriptional coactivator peroxisome proliferator a coactivator 1 (PGC-1a), was downregulated in mouse skeletal muscle (Safdar et al. 2009). Importantly, downregulation of miR-23 was associated with increased expression of PGC-1a mRNA and protein, along with several downstream targets of PGC-1a signaling.

You are no doubt aware that the ability to increase muscle size in response to resistance and strength training is greater for some people than others. Have you ever wondered why strength training causes large gains in muscle mass in some people (i.e., high responders), whereas others gain very little muscle mass in response to the same training stimulus (i.e., low responders)? This is true even after accounting for differences in age, training status, exercise adherence, and diet. A study by Davidsen and colleagues (2011) helps to shed some light on this issue. In their study, vastus lateralis biopsies were taken from the top and bottom 49 responders, in terms of muscle mass gain, of 56 men who completed a 12-week strength-training program. The expression level of 21 abundant miRNAs was measured to determine whether variation in these miRNAs was able to explain the variation in resistance training-induced gains in muscle mass. They indentified 4 miRNAs that showed uniquely different responses between high responders and low responders. MiR-378, miR-29a, and miR-26a were downregulated in low responders and were unchanged in high responders, while miR-451 was upregulated only in low responders. Therefore, the regulation of protein synthesis by miRNAs may play an important role in explaining the variability in strength training adaptations. However, further research is required to uncover how these miRNAs themselves are regulated and whether they can be targeted for therapeutic interventions.

Compared with endurance exercise, resistance exercise consists of much higher intensity contractions (e.g., usually 70% to 80% of one repetition maximum) repeated at high frequencies (referring again to motoneuron firing frequency) that can only be sustained for short durations due to the fatiguing nature of the exercise. Therefore, the pattern of cytoplasmic [Ca2+] oscillations and changes in [AMP]/[ATP] and ROS production with skeletal muscle contractions is different between endurance and resistance exercise. For example, endurance exercise likely results in extended periods of moderately elevated [Ca2+], while resistance exercise would generate short cycles of significantly higher intracellular [Ca2+] in skeletal muscle (Chin 2010). Therefore, differences in the magnitude and pattern of these primary signals generated by endurance and resistance exercise will result in the activation of different signaling pathways and different gene-expression and protein-synthesis responses in skeletal muscle. A number of studies have shown that resistance exercise results in increased rates of muscle-protein synthesis, about two- to fivefold after exercise for periods up to 48 h before declining to baseline values. Although acute resistance and endurance-type exercise result in a similar global anabolic response in untrained skeletal muscle, increases in muscle mass (i.e., hypertrophy) and strength are significantly greater following chronic resistance training compared with chronic endurance training. This means that chronic resistance training, but not endurance training, increases the rate of muscle-protein synthesis to levels above the rate of protein degradation.

Activation and differentiation of muscle satellite cells into new muscle cells that fuse with existing muscle fibers can also contribute significantly to the hypertrophic response to resistance exercise. Muscle fibers are large multinucleated cells. They are also postmitotic cells in that they no longer have the ability to divide and reproduce themselves. Additionally, in order to significantly increase in size or hypertrophy, muscles have to add more nuclei, since a nucleus is only able to supply mRNA for new protein synthesis to a limited amount of cytoplasm in its vicinity. Hence, a specific ratio of nuclei to cytoplasm must be maintained. In order to do this, skeletal muscles have small specialized satellite cells,or myogenic precursor cells, located at the periphery of their outer membranes. When stimulated by resistance training or by exercise-induced muscle damage, these specialized cells will be activated and induced to create daughter cells or proliferate. These new satellite cells will then fuse with the existing muscle fibers and add their nuclei to the cells to support greater protein synthesis and increase muscle hypertrophy or repair.

It is well known that the primary factor determining the hypertrophic response to contractile activity is the load across the muscle or the mechanical stretch/strain imposed on the muscle fibers, which is higher in resistance exercise than in endurance exercise. Ultimately, these mechanical/force signals are transduced by signaling pathways that regulate transcriptional and translational processes and satellite-cell activation.

For several years, researchers have focused on the role of insulin-like growth factor (IGF)-1 as a primary signaling molecule that mediates skeletal-muscle growth in response to resistance exercise, given that resistance exercise stimulates the secretion of IGF-1. IGF-1 is known to induce muscle hypertrophy by binding to its receptor on the muscle-cell surface and activating the classical growth factor pathway (see figure 3.27). IGF-1 binding to the receptor activates phosphatidylinositol 3-kinase (PI3K), which leads to the activation of Akt, a serine/threonine protein kinase. Akt phosphorylates and inactivates tuberosclerosis complex (TSC2), resulting in the activation of the ras homologous protein enriched in brain (Rheb) and mammalian target of rapamycin (mTOR). mTOR phosphorylates and suppresses the eukaryotic initiation factor 4E binding protein (4E-BP1) to blunt 4E-BP1 inhibition of translation-initiation cap-binding protein eIF4E (see figure 3.21). mTOR also phosphorylates the 70KDa ribosomal S6 protein kinase (p70S6K1), resulting in an increase in protein synthesis.

It is well accepted that mTOR signaling plays a dominant role in the adaptive response to resistance training; however, researchers have questioned the importance of IGF-1 signaling in mediating this response, based on evidence from several studies employing pharmacological and knockout-mouse approaches to systematically manipulate the IGF-1-PI3K-Akt pathway (Philp, Hamilton, and Baar 2011). It appears that the IGF-1 signaling pathway is not required for mTOR activation or increased protein synthesis that is induced by resistance-type exercise. It is possible that mechanical signals working through stretch-activated membrane channels, for example, might be able to activate mTOR and its downstream targets. However, this hypothesis needs to be examined experimentally (Philp, Hamilton, and Baar 2011).

Cellular Damage From RONS

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are sometimes collectively referred to as reactive oxygen and nitrogen species (RONS). RONS, and particularly the highly reactive hydroxyl radical, can react with many molecules in cells, including DNA, proteins, and lipids. The slow buildup of threats to mitochondrial and nuclear DNA over years of oxidative damage is thought to be a factor in aging. The relatively rapid turnover (breakdown and resynthesis) of proteins is also partly due to their susceptibility to oxidative damage and their need to ensure structural integrity to maintain optimal function. The free radical theory of aging was first proposed in the 1950s by the American scientist Denham Harman. It has continued to receive scientific support over the years, and it is now well established as an integral component of the aging process. Polyunsaturated fatty acids, which are important components of the phospholipids found in cell membranes, are particularly susceptible to damage by RONS. It has been known for more than 200 years that exposure of fats to air or oxygen will turn them rancid. This rancidity is caused by peroxidation of the fatty acids by RONS. During peroxidation, RONS such as the hydroxyl radical can react with the phospholipids by abstracting a hydrogen from one of the double bonded hydrogen molecules in the unsaturated fat, and in so doing, forming water. In the following equation, LH represents the lipid and OH* represents the hydroxyl radical, with the * representing the electron that is donated:

LH + OH* → L* + H2O

The lipid radical (L*) can then spontaneously propagate a chain reaction, which can result in peroxidation and breakdown of a number of phospholipids in a cell membrane and damage and disruption of cellular function. Similar damage also results from RONS-induced oxidation of proteins and DNA. As will be discussed in the following section, endogenous antioxidants (e.g., glutathione) and antioxidant vitamins (e.g., vitamin E) can help stop these reactions and prevent peroxidative damage to membranes and other cellular molecules, including proteins and DNA. Limiting oxidative stress may be important in limiting various conditions associated with aging (Pourova et al. 2010)

Protection From Reactive Oxygen and Nitrogen Species

Our bodies produce a number of endogenous oxidants that we have categorized as RONS. Added to this is a host of exogenous substances, such as pollution, ozone, automobile exhaust, solvents, pesticides, and cigarette smoke. Foreign molecules that may pose a hazard to us are generally categorized as xenobiotics. Some xenobiotics are free radicals or can produce RONS. Three approaches to maintaining control over RONS are principal. First, reducing the formation of or exposure to RONS and xenobiotics is an essential step. Second, our endogenous scavenging systems, including antioxidant enzymes and other antioxidant molecules, can scavenge those RONS already present. Finally, mechanisms in cells can upregulate antioxidant defenses in the face of persistent problems (Halliwell and Gutteridge 1999). A failure to maintain a balance between the formation of oxidants (e.g., RONS) and their removal by our various antioxidant systems produces a state known as oxidative stress. Powerful oxidants such as those just discussed can damage proteins, DNA, and the unsaturated fatty acid molecules in membranes. It is no wonder, then, that aging and a variety of diseases, such as cancers, heart disease, neurodegenerative diseases, and type 2 diabetes, have a relationship to oxidative
stress.

Figure 5.20 summarizes a number of important reactions that produce and remove ROS, such as SOD, catalase, and GPX. In addition to specific antioxidant enzymes, the body contains a number of nonprotein antioxidant molecules. For example, the essential nutrients vitamin C, vitamin E, and carotenoids such as b-carotene can by themselves scavenge free radicals. Minerals such as selenium, zinc, manganese, and copper are constituents of antioxidant enzymes. Finally, a host of non-nutrient antioxidants from a wide variety of foods of plant origin are important. The general name phytochemical is applied to those molecules that can function in our bodies as antioxidants. Fruits, vegetables, and grains, as well as common products derived from these such as tea and wine, are the major sources. Flavonoids, polyphenols, and lycopene are common examples of phytochemicals.

Exercise and Oxidative Stress

It is extremely difficult to directly measure free radical formation in a human being. Rather, we use indirect measures to point to increased or decreased ROS formation; from this, we make inferences. From a theoretical perspective, during exercise, we should expect a greater formation of superoxide, and therefore hydrogen peroxide and hydroxyl radicals, simply because of an increased flux through the electron transport chain with the increased need for ATP. Since we can greatly increase oxygen consumption and oxidative phosphorylation during exercise, it has been postulated that exercise can increase the generation of oxygen radicals and the possibility of oxidative damage or stress in muscles and other tissues.

Even though the proportion of oxygen that undergoes one- instead of four-electron reduction should decrease with increased activity of the electron transport chain, the fact that the overall flux may increase 10- to 20-fold above resting levels when we exercise should lead us to suspect an increase in superoxide formation. Bailey and colleagues (2003) were the first to actually measure increased formation of free radicals in venous blood of humans during a single-leg exercise task, using an expensive technique known as electron paramagnetic resonance spectroscopy. In this study, the researchers noted that the outflow of free radical species in the venous blood increased as exercise intensity increased.

The active muscle is not the only source of increased RONS formation during exercise. It has been observed that RONS production by white blood cells increases with exercise. However, the effect of RONS production by white blood cells is increasingly blunted the more trained the person is for the particular exercise activity (Mooren, Lechtermann, and Völker 2004). This is likely a consequence of increased antioxidant enzyme activity in white blood cells with training (Elosua et al. 2003). Some types of exercise, such as intense isometric or eccentric contractions, can create inflammation in muscle. Part of the inflammatory response is caused by neutrophils (white blood cell subset) accumulating in the muscle. As mentioned earlier, these cells produce superoxide that can cause damage within the muscle and can also produce other reactive species (Nguyen and Tidball 2003). Obesity, a major public health problem, is characterized by elevated markers of oxidative stress. When people who are obese perform either aerobic or resistance exercise, they produce higher levels of lipid peroxidation products than normal-weight individuals (Vincent, Morgan, and Vincent 2004). Acute feeding of high-fat diets also increases RONS production in rodents. Such a response suggests either an enhanced production or a reduced ability to scavenge free radicals, or both, in the obese state. Aging is also characterized by increased RONS production and oxidative stress, and increased RONS are associated with various diseases of senescence (Pourova et al. 2010).

Rested muscle produces RONS. When muscle becomes more active, formation of RONS increases. Having some RONS in muscle confers a benefit, since they are often important signaling agents that help regulate acute responses to exercise as well as positive adaptations to training in the muscles. Animal studies have demonstrated that inhibiting RONS production during exercise training actually blunts the signaling pathways that regulate adaptations to training, such as increasing mitochondria and aerobic capacity in skeletal muscle (Gomez-Cabrera et al. 2005). Important signaling pathways such as NFkB can help regulate antioxidant adaptations and mitochondrial synthesis consequent to endurance training. The inhibition of RONS production during exercise can blunt the response of these pathways and, consequently, positive training-induced adaptations (Powers et al. 2010). However, a study involving endurance training in humans failed to demonstrate a short-term negative effect of antioxidant supplements on endurance training adaptations (Yfanti et al. 2010).

Dietary antioxidants such as vitamin E and vitamin C have been touted as important for minimizing muscular damage from exercise. Athletes have often been encouraged to supplement their diets with a variety of antioxidants. While eating a diet high in antioxidants would likely have health benefits and could influence longer term effects of oxidative stress such as aging, studies have demonstrated that it is unlikely that large intakes of antioxidants will have significant physiological effects in diminishing exercise-induced muscle damage; instead, they may inhibit positive adaptations to training (McGinley, Shafat, and Donnelly 2009; Gomez-Cabrera et al. 2008). In addition, studies have demonstrated that antioxidant supplements taken during training may also suppress health-promoting benefits such as improved insulin resistance (Ristow et al. 2009).

Unaccustomed exercise and overtraining lead to muscle soreness, inflammation, and damage. This can be reduced by repeated exposure to the activity or through training. Attempts to limit the amount of stress by taking anti-inflammatory medication such as NSAIDs can potentially reduce oxidative stress in muscles that results from infiltration of white blood cells, such as neutrophils and macrophages. However, since the presence of white blood cells, particularly macrophages in muscle following exercise, is obligatory for the activation of muscle satellite cells and their role in stimulating muscle hypertrophy, reducing inflammation may also limit the amount of muscle repair, adaptation, and hypertrophy that would be induced by the training.

MicroRNAs and the Adaptive Response to Exercise Training

MicroRNAs (miRNAs) are a class of short, noncoding RNA molecules that bind to mRNA molecules and play a central role in regulating gene expression through posttranscriptional gene silencing (reviewed in Bushati and Cohen 2007). Most miRNAs are encoded in introns of protein-coding genes and are transcribed by RNA polymerase II as long primary-miRNAs (pri-miRNA) that encode a single miRNA or a cluster of miRNA species. Processing of pri-miRNA species in the nucleus produces stem-loop structures of ∼70 nucleotides, termed precursor-miRNA (pre-miRNA). These pre-miRNAs are transported to the cytoplasm where they are further processed, giving rise to the mature ∼19 to 22 bp of miRNA. The mature miRNA is incorporated into a ribonucleoprotein complex known as the RNA-induced silencing complex (RISC). Generally, miRNAs inhibit protein synthesis by binding (base-pairing) in the 3' untranslated regions of target mRNAs, either repressing translation or bringing about deadenylation and subsequent degradation of mRNA targets. Individual miRNAs can target hundreds of genes, while individual mRNAs can be targeted by multiple miRNAs, making this one of the most complex gene-regulatory processes.

Studies have uncovered a cluster of muscle-specific miRNAs that regulate muscle differentiation and modulate diverse aspects of muscle function (reviewed in van Rooij, Liu, and Olson 2008). The most highly studied are miR-1, miR-133 and miR-206, which are induced during differentiation of myoblasts into myotubes (Callis et al. 2008) and play an important role in muscle mass maintenance. Other miRNA species (miR-23, miR-103, miR-107, and so on) are proposed to play an important role in regulating expression of genes encoding metabolic pathway enzymes in skeletal muscle and other tissues (Wilfred, Wang, and Nelson 2007). Interestingly, studies have shown the potential importance of miRNA regulation in skeletal-muscle adaptations to exercise (reviewed in Roth 2011). For example, miR-1 and miR-133a are downregulated in mouse skeletal muscle during functional overload-induced hypertrophy of the plantaris muscle (McCarthy and Esser 2007). In response to endurance exercise, miR-23, a putative negative regulator of the transcriptional coactivator peroxisome proliferator a coactivator 1 (PGC-1a), was downregulated in mouse skeletal muscle (Safdar et al. 2009). Importantly, downregulation of miR-23 was associated with increased expression of PGC-1a mRNA and protein, along with several downstream targets of PGC-1a signaling.

You are no doubt aware that the ability to increase muscle size in response to resistance and strength training is greater for some people than others. Have you ever wondered why strength training causes large gains in muscle mass in some people (i.e., high responders), whereas others gain very little muscle mass in response to the same training stimulus (i.e., low responders)? This is true even after accounting for differences in age, training status, exercise adherence, and diet. A study by Davidsen and colleagues (2011) helps to shed some light on this issue. In their study, vastus lateralis biopsies were taken from the top and bottom 49 responders, in terms of muscle mass gain, of 56 men who completed a 12-week strength-training program. The expression level of 21 abundant miRNAs was measured to determine whether variation in these miRNAs was able to explain the variation in resistance training-induced gains in muscle mass. They indentified 4 miRNAs that showed uniquely different responses between high responders and low responders. MiR-378, miR-29a, and miR-26a were downregulated in low responders and were unchanged in high responders, while miR-451 was upregulated only in low responders. Therefore, the regulation of protein synthesis by miRNAs may play an important role in explaining the variability in strength training adaptations. However, further research is required to uncover how these miRNAs themselves are regulated and whether they can be targeted for therapeutic interventions.